Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters of the Offspring: Male-Preferential Impact and Involvement of Kiss1-Mediated Pathways

Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters... Abstract Obesity and its comorbidities are reaching epidemic proportions worldwide. Maternal obesity is known to predispose the offspring to metabolic disorders, independently of genetic inheritance. This intergenerational transmission has also been suggested for paternal obesity, with a potential negative impact on the metabolic and, eventually, reproductive health of the offspring, likely via epigenetic changes in spermatozoa. However, the neuroendocrine component of such phenomenon and whether paternal obesity sensitizes the offspring to the disturbances induced by high-fat diet (HFD) remain poorly defined. We report in this work the metabolic and reproductive impact of HFD in the offspring from obese fathers, with attention to potential sex differences and alterations of hypothalamic Kiss1 system. Lean and obese male rats were mated with lean virgin female rats; male and female offspring were fed HFD from weaning onward and analyzed at adulthood. The increases in body weight and leptin levels, but not glucose intolerance, induced by HFD were significantly augmented in the male, but not female, offspring from obese fathers. Paternal obesity caused a decrease in luteinizing hormone (LH) levels and exacerbated the drop in circulating testosterone and gene expression of its key biosynthetic enzymes caused by HFD in the male offspring. LH responses to central kisspeptin-10 administration were also suppressed in HFD males from obese fathers. In contrast, paternal obesity did not significantly alter gonadotropin levels in the female offspring fed HFD, although these females displayed reduced LH responses to kisspeptin-10. Our findings suggest that HFD-induced metabolic and reproductive disturbances are exacerbated by paternal obesity preferentially in males, whereas kisspeptin effects are affected in both sexes. Obesity rates have rapidly increased over the last 30 years worldwide. According to the World Health Organization, obesity prevalence has more than doubled since 1980, with 39% of the global population being overweight and 13% obese in 2014 (1). Obesity is associated with a constellation of health problems such as type 2 diabetes, dyslipidemia, hypertension, cardiovascular disease, and infertility (2), whose prevalence has paralleled the obesity epidemic during the last decades. It is widely recognized that inadequate dietary habits and sedentary lifestyle are the main factors responsible for the increased incidence of obesity and related comorbidities. However, the exponential increase in obesity prevalence is difficult to be explained solely by a chronic imbalance between food intake and energy expenditure, suggesting that other factors may influence susceptibility to obesity and its associated health problems. Compelling evidence suggests that the origins of obesity may be traced back to the fetal/neonatal period, in which the maternal nutritional and metabolic status is known to play an important role in developmental programming (3, 4). According to this concept, adaptive responses induced by nutritional challenges during early stages of development may have permanent effects on the metabolic health of the developing offspring, which may result in inappropriate responses to obesogenic insults in later life. In line with this hypothesis, we have recently reported that overnutrition during lactation predisposes male and female offspring to obesity and reproductive disturbances in adulthood when exposed to high-fat diet (HFD) (5, 6). Recent findings have demonstrated that maternal nutritional environment is relevant not only during gestation and lactation, but also during the periconception stage (7). This influence has been surfaced in preclinical models, by using embryo transfer techniques, in which fertilized eggs from mothers fed an HFD at periconception were transferred to subrogate control mothers; this resulted in growth retardation and abnormalities in brain development in the offspring (7). These observations indicate that nongenetic, intergenerational mechanisms influence offspring development and may determine disease risk later in life. Interestingly, this nongenetic intergenerational transmission of adverse phenotypes has also been described from fathers to offspring. Thus, rodent studies have shown that paternal exposure to HFD at periconception impairs the metabolic health of subsequent generations, regardless of the presence of diabetes in the male founders (8–11). In addition to such detrimental effect on the metabolic health of the offspring, diet-induced paternal obesity in mice has been putatively linked to sperm disturbances in subsequent generations (9, 12, 13). These findings suggest that paternal nutritional status prior to fertilization may trigger the intergenerational transmission of metabolic and reproductive traits to descendants and may predispose offspring to obesity and fertility problems. Although the underlying mechanisms for this paternal programming of the offspring phenotype remain unknown, a growing body of evidence indicates that epigenetic processes in the sperm, such as altered DNA methylation, histone modification, and changes in small noncoding RNA levels (namely, microRNAs), may be potential mediators of this intergenerational effect (14–16). Because fathers and children usually share similar dietary habits, and therefore children from obese fathers may be likely exposed to an obesogenic environment, an interesting question that arises is whether paternal obesity at periconception may sensitize the offspring to the deleterious effects of HFD. A recent study has shown that the metabolic and sperm derangements induced by paternal obesity may be exacerbated by HFD in male F1 offspring (17). Yet, the metabolic and gonadotropic effect of paternal obesity in the female offspring fed HFD, as well as its impact on the neuroendocrine reproductive axis in the HFD-fed male offspring, remains virtually unexplored to date. Reproduction is very sensitive to metabolic status (18–20). The neuroendocrine reproductive [aka, hypothalamic-pituitary-gonadal (HPG)] axis is governed by a population of hypothalamic neurons that express and release gonadotropin-releasing hormone (GnRH) (19). This neuropeptide is secreted in a pulsatile fashion and stimulates gonadotropin release from the pituitary, which in turn promotes gonadal maturation and function (19). Metabolic challenges perturb the pattern of GnRH secretion and, consequently, may compromise fertility. However, a wealth of data strongly suggests that GnRH neurons are devoid of functional receptors for the major metabolic cues that modulate their activity, as appears to be the case for leptin and, possibly, insulin (18, 19, 21–26); yet, direct insulin signaling in GnRH neurons might contribute to transmit part of the metabolic impact of obesity on reproductive function (27). In any event, the consensus view is that a substantial component of the metabolic regulation of GnRH neurons is carried out by a network of afferent neurons that express such receptors and convey this information to GnRH neurons in a direct or indirect manner. Among those afferents, Kiss1 neurons are considered essential players in the metabolic control of reproduction (19). In conditions of negative energy balance, Kiss1 neurons are inhibited to repress the activity of the reproductive axis. In addition, we recently reported that in conditions of energy excess, such as in obesity, the hypothalamic Kiss1 system is also suppressed in males and females (5, 6). Yet, whether paternal obesity may impact the Kiss1 system in the progeny is unknown and warrants specific investigation. We report in this work a comprehensive set of studies addressing the impact of paternal obesity on metabolic and reproductive health parameters of the male and female offspring, especially as it pertains to its influence upon responses to HFD, with a particular interest on the neuroendocrine perturbations, sex differences, and potential alterations of the hypothalamic Kiss1 system. Materials and Methods Animals and diets Wistar rats bred in the vivarium of the University of Córdoba were used. Rats were given ad libitum access either to a control diet (CD), D12450B (10% of calories from fat, 20% from protein, and 70% from carbohydrate; Research Diets, Inc. New Brunswick, NJ), or an HFD, D12451 (45% of calories from fat, 20% from protein, and 35% from carbohydrate; Research Diets). The animals were maintained at 22°C under constant conditions of light (14 hours) with free access to water. In all experiments, the animals were housed and handled under strictly similar conditions, allowing the integral analysis of data. Experimental procedures were approved by Córdoba University Ethical Committee and conducted in accordance with European Union guidelines. Experimental design Control and extremely obese male rats (F0) were mated with adult virgin lean chow-fed dams (Charles River, Barcelona, Spain) to generate F1 offspring. Control founder rats were fed a CD for 3 months, whereas obese F0 males were obtained by inducing overnutrition during lactation (small litter size, four pups per dam), followed by HFD from weaning onward for 9 months. Of note, these paternal groups were generated in the context of a large-scale study conducted by our group addressing the impact of sequential obesogenic insults on metabolic and reproductive function in male rats (5). A summary of the most relevant metabolic and reproductive parameters analyzed in control and obese F0 fathers is provided in Supplemental Table 1. To obtain the F1 generation, every F0 male was housed together with a 12-week-old virgin female for 8 consecutive days (two estrous cycles), with free access to chow diet and water. Females were monitored for regular vaginal cycling before initiation of mating, and only females showing at least four consecutive regular, 4-day cycles were used. During the mating period, vaginal smears were obtained and analyzed daily to monitor estrous cycle in the females. Pregnancy was evidenced by the presence of sperm in vaginal smears in the morning of the estrous phase in all F0 females. Of note, despite the confirmation of the presence of sperm in vaginal smears in all females mated with extremely obese males, the proportion of pregnant dams was markedly reduced as compared with control F0 males (Fig. 1A), indicative of impaired fertility in obese males. After the mating period, F0 males were returned back to their initial cages with the original diet, whereas F0 females continued on chow diet during gestation and lactation. Figure 1. View largeDownload slide Overall design of the transgenerational study. (A) Table illustrating the details on the mating process, including number of mated dams, pregnancy rates, and number of offspring obtained. (B) Schematic representation of the experimental design. Lean and extremely obese F0 fathers were mated with lean virgin mothers. F1 male and female offspring from lean fathers were fed on CD or HFD, whereas F1 pups from obese fathers received exclusively an HFD until they were euthanized (PND-120). Figure 1. View largeDownload slide Overall design of the transgenerational study. (A) Table illustrating the details on the mating process, including number of mated dams, pregnancy rates, and number of offspring obtained. (B) Schematic representation of the experimental design. Lean and extremely obese F0 fathers were mated with lean virgin mothers. F1 male and female offspring from lean fathers were fed on CD or HFD, whereas F1 pups from obese fathers received exclusively an HFD until they were euthanized (PND-120). After birth, F1 male and female offspring were cross-fostered and reared in litters of normal size (8 to 13 pups per dam). As previously mentioned, fertility was compromised in obese F0 fathers, which restricted the availability of pups from obese fathers in the study. Because of this limitation, only one group of male (n = 13) and female (n = 8) offspring from obese fathers was obtained (Fig. 1A), from two different litters. Due to the use of F0 males in the context of the large-scale, independent studies mentioned previously (5), we were unable to enlarge the size of the progeny of obese fathers enrolled in the current analyses. The offspring were weaned from mothers at PND-23. From weaning onward, F1 male and female offspring from control lean fathers were fed either a CD (control) or an HFD ad libitum (control + HFD), whereas F1 offspring from obese fathers were maintained exclusively on HFD (paternal obesity + HFD); an independent group of paternal obesity + CD could not be generated due to the operational limitations indicated previously. A schematic representation of the experimental design is provided in Fig. 1B. Blood parameter measurements After euthanasia by decapitation at PND-120, blood samples were collected, immediately placed on ice, and centrifuged, and plasma samples were stored at −20°C until analyzed. Of important note, female rats were euthanized at the same stage of the estrous cycle, namely diestrus, as monitored by regular vaginal smearing for at least three consecutive cycles before sampling, to minimize the potential influence that ovarian cycle-dependent fluctuations may have on the endpoints analyzed. Plasma levels of leptin and insulin were quantified using commercial radioimmunoassay (RIA) kits from Linco Research (St. Charles, MO). Circulating glucose levels were determined by using a handheld glucometer (ACCU-CHECK Aviva; Roche Diagnostics, Basel, Switzerland) after an overnight fast. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured using RIA kits from the National Hormone Peptide Program (Torrance, CA), with reference preparations LH-RP-3 and FSH-RP-2 and intra-assay and interassay coefficients of variation below 8% and 10%, respectively. Testosterone (T) and estrogen levels were assessed by using specific RIA kits, as previously reported (5, 6). In detail, serum T levels were assayed using a RIA kit from MP Biomedicals (Santa Ana, CA). The sensitivity of the assay was 0.1 ng/mL, and the intra-assay and interassay coefficients of variation were <5% and <8%, respectively. Circulating estrogen levels were assayed using a commercial ultrasensitive RIA kit from Beckman Coulter (Brea, CA). The sensitivity of the assay was 2.2 pg/mL, and the intra-assay and interassay coefficients of variation were ≤8.9% and ≤12.2%, respectively. Kisspeptin stimulation tests The LH-releasing ability of an intracerebroventricular low dose of Kp-10 (Phoenix Pharmaceuticals, Belmont, CA) was monitored. To allow delivery of Kp-10 into the lateral cerebral ventricle, rats were implanted with intracerebroventricular cannulas, as described previously (28–30). Before initiation of the functional tests, estrous cycles were monitored in the female groups, and tests were performed at the same cycle stage (diestrus) to exclude potential variations in LH responses to Kp-10 due to the phase of the cycle, as documented by previous references (31). Doses of 50 pmol/rat were used to evoke submaximal stimulation of the gonadotropic axis, allowing discrimination of subtle changes in sensitivity. Blood was collected before (0) and at 15, 60, and 120 minutes after Kp-10 administration. In addition to individual hormonal levels, area under the curve (AUC) responses were calculated over the 120-minute period. Note that to allow complete washout and recovery of the experimental animals before subsequent metabolic testing and euthanasia, Kp-10 tests were conducted around PND-100. Oral glucose tolerance tests and insulin tolerance tests For the determination of glucose tolerance, rats were subjected to oral glucose tolerance tests (OGTTs) around PND-110. Rats were fasted overnight and subsequently received an oral bolus of glucose [1 g/kg body weight (BW)] by gastric gavage. Glucose levels were determined in blood before (0) and at 20, 60, and 120 minutes postadministration. After complete recovery and washout, insulin sensitivity was assessed in overnight fasted rats following an intraperitoneal injection of 1 U insulin (Sigma-Aldrich, St. Louis, MO) per kg body weight; insulin tolerance tests (ITTs) were performed around PND-115. Blood glucose levels were measured before (0) and at 15 and 30 minutes after insulin administration. Insulin sensitivity index (KITT) was calculated to assess peripheral insulin sensitivity, as reported elsewhere (32). All glucose concentrations were measured using a handheld glucometer (ACCU-CHECK Aviva; Roche Diagnostics). Steroidogenic enzyme gene expression analyses by quantitative polymerase chain reaction Testicular and ovarian samples were dissected immediately after euthanasia of the animals, frozen in liquid N2, and stored at −80°C until RNA isolation. Tissues were processed, and total RNA was isolated using TRIsure (BIOLINE, London, UK), following the manufacturer’s instructions. Next, a fixed amount of RNA (2 μg) was transcribed into complementary DNA (cDNA) by the action of the reverse transcriptase (Bio-Rad, Hercules, CA); real-time reverse transcription polymerase chain reaction (PCR) was performed employing the iCycler iQ real-time PCR system (Bio-Rad). Testicular and ovarian expression of StAR, P450scc, and 17β-HSD was measured by using specific primer pairs previously designed and validated in our group (5). To analyze aromatase expression in the ovary, the following primer sequences were used: forward, 5′-TGT TGC TTC TCA TCG CAG AGT ATC C-3′; reverse, 5′-GGC TGA TAC CGC AGG CTC TCG-3′. As internal control, expression of the ribosomal protein S11 was measured by using specific primer sets (5). Calculation of relative expression levels of the targets was conducted based on the cycle threshold method, as described elsewhere (33). Kiss1 riboprobe synthesis For detection of Kiss1 messenger RNA (mRNA) by in situ hybridization, a specific riboprobe for Kiss1 rat mRNA, spanning 83 to 371 nucleotides of the cDNA sequence (GenBank NM_181692.1), was generated. First, a DNA template was synthesized by PCR using specific primers for Kiss1 cDNA amplification carrying at their 5′-end sequences for synthetic promoters for bacteriophage-encoded DNA-dependent RNA polymerases (T7 and T3). Primer sequences were as follows: forward primer T3-Kiss sense (5′-CAG AGA TGC AAT TAA CCC TCA CTA AAG GGA GAT GGT GAA CCC TGA ACC CAC A-3′); reverse primer T7-Kiss as (5′-CCA AGC CTT CTA ATA CGA CTC ACT ATA GGG AGA ACC TGC CTC CTG CCG TAG CG-3′). For PCRs, Go-Taq flexi DNA polymerase (Promega Biotech, Madison, WI) was used, following the recommendations of the manufacturer. Reactions were performed in an iCycler (Bio-Rad Laboratories) using the following protocol: cDNA was denatured for 5 minutes at 95°C, and then four cycles were performed at 94°C for 1 minute, 54°C for 2 minutes, and 72°C for 30 seconds, followed by 35 cycles at 94°C for 1 minute, 65°C for 1 minute, and 72°C for 30 seconds. A final extension at 72°C for 5 minutes was included. After electrophoresis on a 2% agarose (w/v) gel, a single DNA fragment was obtained of the expected size and gel purified with a QiaQuick gel extraction kit (Qiagen, Hilden, Germany). For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μL): 250 μCi [33P]-UTP (Perkin Elmer, Waltham, MA), 0.5 μg template, 2 μL ribonucleoside triphosphates (rNTPs: 5 mM rATP, rCPT, and rGTP), 1 μL RNasin Ribonuclease Inhibitor (Promega), 4 μL transcription buffer, and 2 μL T7 RNA polymerase (Promega). After 120 minutes of incubation at 37°C, another 1 μL T7 RNA was added to the mix, and the reaction was maintained for an additional 60 minutes at 37°C. At the end, the residual DNA was digested with 2 U DNase (Promega), and the reaction was terminated by addition of 3 μL 0.5 M EDTA, pH 8.0. Diethyl pyrocarbonate water was added to a final volume of 50 μL, and the labeled riboprobe was purified using Illustra ProbeQuant G-50 Micro Columns (GE Healthcare, Chalfont, UK). For synthesis of the sense Kiss1 riboprobe, the same procedure was applied, using T3 RNA polymerase (Promega). In situ hybridization Expression of Kiss1 mRNA within the hypothalamus was assessed by in situ hybridization (ISH), with attention on the arcuate (ARC) and the anteroventral periventricular (AVPV) nuclei, as major sites of Kiss1 expression in the brain (19). Considering sex differences in the pattern of hypothalamic expression of Kiss1 in rodents, with males having abundant expression only in the ARC (19), we first assayed ARC Kiss1 mRNA levels in the male offspring from control fathers, fed with CD or HFD, and in the HFD offspring from obese fathers. In a second set of analyses, we assessed ARC and AVPV Kiss1 expression in the same experimental groups in the female offspring; specimens (n = 5/group) for ISH analysis were randomly assigned within each group. From each brain, five sets of coronal plane sections 20 μm thick were thaw mounted in Super-Frost Plus slides (Thermo Fisher Scientific, Waltham, MA). Standard procedures of tissue collection were applied, starting in a fixed coordinate in the rostral hypothalamic area up to the ARC, as regions in which Kiss1 neurons are abundantly located (19, 34). The samples were stored at −80°C until ISH analyses. A specific radiolabeled Kiss1 antisense riboprobe was generated using T7 RNA polymerase (Bio-Rad Laboratories) and Kiss1 cDNA template, as described previously. A single set of sections was used for ISH (adjacent sections 100 μm apart). In brief, tissue sections were as follows: 1) fixed in 4% paraformaldehyde for 15 minutes; 2) stabilized with 0.1 M phosphate buffer (pH 7.4) at room temperature for 10 minutes; 3) treated with saline triethanolamine and acetic anhydride to prevent nonspecific binding of probes; 4) washed in 2× saline sodium citrate (SSC) buffer for 3 minutes; 5) dehydrated in increasing concentrations of ethanol; 6) delipidated with chloroform; and 7) air dried at room temperature for 1 hour. After these steps, hybridization with Kiss1 riboprobe was performed for 16 hours at 55°C. The hybridization buffer contained (for 40 ml) the following: 25 ml deionized formamide, 10 ml dextran sulfate 50%, 3 ml NaCl 5 M, 0.4 ml Tris base 1 M (pH 8), 0.08 ml 0.5 M EDTA (pH 8), 1× Denhardt solution, and RNase-free water up to 40 ml; Kiss1 riboprobe was added to the hybridization buffer to a final concentration of 0.03 pmol/mL along with yeast transfer RNA. After hybridization, slides were as follows: 1) washed with 4× SSC for 30 minutes; 2) incubated in RNase-A buffer (Roche Biochemical) at 37°C (32 μg/mL) for 1 hour; 3) equilibrated with 2× SSC for 30 minutes; 4) washed in 0.1× SSC for 1 hour at 65°C; 5) dehydrated in increasing ethanol series; and 6) air dried at room temperature for 1 hour. Finally, slides were dipped in Kodak Autoradiography Emulsion type NTB (Eastman Kodak, Rochester, NY) and exposed for 1 week at 4°C in the dark. After this, the sections were developed and fixed following the manufacturer instructions (Eastman Kodak): 1) 4 minutes in Kodak Developer D-19; 2) 10 seconds in distilled water; 3) 5 minutes in Kodak Fixer; and 4) 5 minutes in distilled water. For mounting, the sections were previously dehydrated and rinsed with Sub-X Clearing Medium (Leica Bio-systems, Nussloch, Germany). Then slices were coverslipped with Sub-X mounting medium (Leica). For analysis, 50 to 60 sections from each animal (9 to 10 slides; 6 sections/slide) were evaluated. Five animals per group were included in the analysis. Slides were read under dark-field illumination with custom-designed software enabled to count the total number of cells (grain clusters). Cells were counted as Kiss1 mRNA positive when the number of silver grains in a cluster exceeded that of background. Statistical analyses Metabolic and hormonal determinations were conducted in duplicate, with a minimal total number of at least eight samples per group. All results are presented as mean ± standard error of the mean. Statistical analyses were performed on data distributed in a normal pattern using Student t tests or analysis of variance, the latter for statistical comparisons of the three experimental groups. When significant differences were found in analysis of variance, data were further analyzed through post hoc comparison, using Student-Newman-Keuls tests to identify simple effects. The significance level was set at P ≤ 0.05 (Prism 5.0; GraphPad Software). Results Paternal obesity increases susceptibility to the metabolic impact of HFD in the offspring In the male offspring of lean fathers, the detrimental effects of HFD on BW were in general mild and only manifested as significantly increased BW at PND-120, whereas in females this increased BW gain was evident already at PND-70. Strikingly, males from obese fathers fed on HFD exhibited increased BW already at PND-50, whereas in females this increase became detectable around PND-40. Accordingly, the persistent elevation of BW caused by HFD in the male offspring from obese fathers was significantly higher than that of control HFD males (Fig. 2A), reaching the highest levels at PND-120 (Fig. 2B). HFD-fed females born from obese fathers were initially heavier also than HFD females from control fathers (Fig. 2D). However, the amplification of BW gain induced by paternal obesity in HFD females was more modest and transient (Fig. 2A and 2D). Thus, BW curves from both female groups fed on HFD converged after PND-80, with no differences in terminal BW being detected at PND-120 (Fig. 2E). The pronounced increase of BW in HFD-fed males from obese fathers was accompanied at PND-120 by a rise in circulating leptin levels, as index of increased adiposity (Fig. 2C). In contrast, no significant alterations were detected at this age in basal leptin levels between HFD-fed females from control and obese fathers (Fig. 2F). Figure 2. View largeDownload slide Paternal obesity exacerbates the effects of HFD on body weight and adiposity in males. Effects of HFD on (A and D) body weight progression, (B and E) terminal body weight, and (C and F) circulating leptin levels in F1 male and female offspring from lean and obese fathers (control + HFD and paternal obesity + HFD, respectively). For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are shown; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 to 13 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. *P < 0.05; **P < 0.01; ***P < 0.001 vs the corresponding control + HFD group. Figure 2. View largeDownload slide Paternal obesity exacerbates the effects of HFD on body weight and adiposity in males. Effects of HFD on (A and D) body weight progression, (B and E) terminal body weight, and (C and F) circulating leptin levels in F1 male and female offspring from lean and obese fathers (control + HFD and paternal obesity + HFD, respectively). For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are shown; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 to 13 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. *P < 0.05; **P < 0.01; ***P < 0.001 vs the corresponding control + HFD group. To evaluate the impact of paternal obesity on glucose homeostasis in the offspring after HFD, OGTTs were performed in adulthood. As expected, HFD perturbed the glycemic profile in both males and females from control F0 (lean) founders, as revealed by increased basal glucose levels and impaired responses during OGTT, defined by significantly elevated glucose concentrations at 60 and 120 minutes after the oral glucose bolus in males, and at 120 minutes in females (Fig. 3). HFD also had a detrimental impact on glucose tolerance in males from obese fathers. Yet, the overall impact of paternal obesity plus HFD, as reflected by basal glucose levels and integral AUC values during OGTT, was of similar magnitude than in the control + HFD group (Fig. 3). Intriguingly, despite similarly elevated basal glucose levels, HFD-fed females from obese fathers exhibited a trend for improved glycemic profile after glucose oral challenge, as compared with control + HFD females. This was reflected by the fact that neither individual glucose levels following the oral bolus nor the integral AUC glucose responses during the OGTT were statistically different from those of the group fed with CD (Fig. 3). Figure 3. View largeDownload slide Paternal obesity does not exacerbate the impact of HFD on glucose tolerance in male and female offspring. Effects of HFD on glucose tolerance in F1 male and female offspring from lean and obese fathers are shown. The animals were subjected to OGTT. They received an oral bolus of glucose (1 g/kg BW), and blood glucose levels were measured at 0 (before) and 20, 60, and 120 minutes after glucose administration. Integral glucose levels estimated as AUC by the trapezoidal rule are also shown. For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are presented; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. No significant differences between control + HFD and paternal obesity + HFD groups were detected. Figure 3. View largeDownload slide Paternal obesity does not exacerbate the impact of HFD on glucose tolerance in male and female offspring. Effects of HFD on glucose tolerance in F1 male and female offspring from lean and obese fathers are shown. The animals were subjected to OGTT. They received an oral bolus of glucose (1 g/kg BW), and blood glucose levels were measured at 0 (before) and 20, 60, and 120 minutes after glucose administration. Integral glucose levels estimated as AUC by the trapezoidal rule are also shown. For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are presented; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. No significant differences between control + HFD and paternal obesity + HFD groups were detected. HFD impaired basal glucose levels in males and females irrespective of the metabolic status of the father, with significantly elevated basal glycemia values vs the corresponding control (lean fathers + CD) group (Fig. 4). No significant alterations in basal insulin levels were detected in the HFD-fed male and female offspring from both paternal (lean and obese) groups, as compared with the groups receiving CD. HFD-fed males from control and obese founders exhibited decreased insulin sensitivity as reflected by reduced KITT, with no further impairment caused by paternal obesity (Fig. 4A). In contrast, HFD females from obese fathers were moderately less insulin resistant than females from lean fathers, irrespective of being fed control or HFD (Fig. 4B). Figure 4. View largeDownload slide Paternal obesity does not aggravate the effects of HFD on basal glycemia and insulin sensitivity in male and female offspring. Effects of HFD on basal circulating glucose and insulin levels and insulin sensitivity, assessed by KITT calculation, in F1 (A) male and (B) female offspring from lean and obese fathers. KITT was calculated after performing an ITT. The animals were fasted overnight and received an intraperitoneal injection of insulin, and glucose levels were recorded at 0 (before) and 20, 60, and 120 minutes after insulin administration. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs corresponding control groups; *P < 0.05 vs the corresponding control + HFD group. Figure 4. View largeDownload slide Paternal obesity does not aggravate the effects of HFD on basal glycemia and insulin sensitivity in male and female offspring. Effects of HFD on basal circulating glucose and insulin levels and insulin sensitivity, assessed by KITT calculation, in F1 (A) male and (B) female offspring from lean and obese fathers. KITT was calculated after performing an ITT. The animals were fasted overnight and received an intraperitoneal injection of insulin, and glucose levels were recorded at 0 (before) and 20, 60, and 120 minutes after insulin administration. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs corresponding control groups; *P < 0.05 vs the corresponding control + HFD group. Paternal obesity increases susceptibility to the reproductive impact of HFD in the offspring Reproductive analyses in terminal blood samples of PND-120 males revealed that HFD increased circulating LH levels in the offspring from both paternal groups compared with the CD-fed group. Yet, the rise in LH levels was significantly attenuated in HFD-fed males from obese fathers (Fig. 5A). Conversely, HFD did not cause alterations in basal LH levels in females from none of the two paternal groups (Fig. 5D). HFD did not modify basal FSH concentrations in males regardless of the father's metabolic status (Fig. 5B and 5E), but tended to reduce FSH levels in females from obese founders vs HFD-fed controls, although this decline did not reach statistical significance (P = 0.06). Figure 5. View largeDownload slide Paternal obesity perturbs basal and stimulated gonadotropin secretion in the offspring fed HFD. The effects of HFD on (A and D) basal LH levels, (B and E) basal FSH levels, and (C and F) LH responses to kisspeptin-10 are shown in F1 male and female offspring from lean and obese fathers. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are shown. To ease comparison among groups, mean control values are displayed also in the histogram graphs as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD group. Figure 5. View largeDownload slide Paternal obesity perturbs basal and stimulated gonadotropin secretion in the offspring fed HFD. The effects of HFD on (A and D) basal LH levels, (B and E) basal FSH levels, and (C and F) LH responses to kisspeptin-10 are shown in F1 male and female offspring from lean and obese fathers. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are shown. To ease comparison among groups, mean control values are displayed also in the histogram graphs as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD group. Given the prominent role of kisspeptin in the central control of the HPG axis, the impact of HFD on the LH-releasing ability of a low dose of Kp-10 was assessed in the offspring from control and obese fathers. Admittedly, when compared with data in Fig. 5A, results from these analyses revealed some degree of variability in the impact of HFD on basal LH levels in the offspring from lean fathers, as basal LH concentrations were not significantly different between control- and HFD-fed males. Notwithstanding, this analysis fully confirmed that basal LH levels in the male offspring of obese fathers, when fed on HFD, were significantly lower than in the equivalent HFD group from lean fathers (Fig. 5C). In the male offspring from control fathers, HFD caused a reduction in peak LH responses at 15 minutes after Kp-10 injection and a significant 20% drop in global LH responses (AUC) to Kp-10 administration, suggesting a central impairment of gonadotropin responses caused by HFD. Notably, in males from obese fathers, LH responses to kisspeptin were further deteriorated in the presence of HFD, with a significant global 40% reduction in integral (AUC) responses vs the CD-fed group (Fig. 5C). In contrast to males, HFD failed to alter the integral LH responses to Kp-10 injection in females born from control fathers. In clear contrast, HFD significantly suppressed LH responses to kisspeptin in the female offspring from obese fathers (Fig. 5F). HFD also had a detrimental impact on basal T concentrations in males, in keeping with previous reports. Namely, HFD caused a 50% reduction in mean T levels in the offspring from lean founders vs the CD-fed group; yet, due to inherent variability in T levels and the limited group size, this difference did not reach statistical significance (P = 0.09). This effect was clearly exacerbated in the offspring from obese founders, which displayed an additional, significant 50% reduction in circulating T levels as compared with control HFD-fed males (Fig. 6A). Similar trends were detected for testicular expression of key steroidogenic enzymes. The expression of StAR was downregulated by HFD in the offspring from both paternal groups; yet, the magnitude of the decline was significantly greater in the offspring from obese fathers (Fig. 6A). In addition, HFD significantly reduced P450scc and 17β-HSD mRNA expression levels in the offspring from obese founders compared with the HFD-fed offspring of lean fathers. Figure 6. View largeDownload slide Paternal obesity exacerbates the magnitude of HFD-induced hypogonadism in F1 males. (A) The effects of HFD on circulating T levels and gene expression of key enzymes involved in testicular steroidogenesis in F1 males from lean and obese fathers are shown. In addition, the impact of HFD on serum estradiol and T levels, as well as the ovarian gene expression of steroidogenic enzymes, in F1 females from lean and obese fathers is presented in (B). For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD groups. Figure 6. View largeDownload slide Paternal obesity exacerbates the magnitude of HFD-induced hypogonadism in F1 males. (A) The effects of HFD on circulating T levels and gene expression of key enzymes involved in testicular steroidogenesis in F1 males from lean and obese fathers are shown. In addition, the impact of HFD on serum estradiol and T levels, as well as the ovarian gene expression of steroidogenic enzymes, in F1 females from lean and obese fathers is presented in (B). For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD groups. In females, the impact of HFD on circulating sex hormone levels was less obvious than in males, as HFD failed to cause any significant suppression of estradiol levels, regardless of the metabolic status of the father, even when paternal obesity and HFD were combined (P = 0.10). Likewise, the low circulating T levels were not affected by HFD in adult female rats, irrespective of paternal obesity (Fig. 6B). In contrast to males, StAR, P450scc, and 17β-HSD expression levels in the ovary were upregulated by HFD in the female offspring from obese founders (Fig. 6B). Furthermore, HFD significantly increased ovarian aromatase expression in the offspring from obese fathers vs females born from control fathers (Fig. 6B). Changes in hypothalamic expression of Kiss1 after HFD exposure were also explored in the offspring from control and obese fathers by ISH. In general, HFD had a similar impact on hypothalamic Kiss1 mRNA levels in the ARC in males, independently of the father’s metabolic status. Thus, HFD caused a significant >50% drop in ARC Kiss1 expression in the male progeny of lean fathers, with no significant differences being detected between the offspring from lean and control fathers (Fig. 7A). Conversely, HFD did not significantly modify Kiss1 expression in the AVPV and the ARC of the female offspring from lean or obese fathers. Yet, a nonsignificant trend (P = 0.10) for increased Kiss1 expression was observed in the ARC of HFD-fed females from lean fathers, which was completely abrogated in the offspring of obese founders, as Kiss1 levels in the ARC of HFD-fed females from obese fathers were strictly similar to those of the CD-fed group (Fig. 7B). Figure 7. View largeDownload slide HFD inhibits Kiss1 expression in males, but paternal obesity does not aggravate this effect. A schematic representation of the location of the ARC and the AVPV nuclei, as areas of prominent expression of Kiss1 mRNA, is shown from male and female brains (left panels). Representative ISH photomicrographs are presented of Kiss1 mRNA-expressing neurons in the anterior region of the ARC (males and females) and AVPV (females) of the HFD-fed offspring from lean (control + HFD) and obese (paternal obesity + HFD) fathers. For comparative purposes, representative images of CD-fed F1 rats from lean fathers (control) are presented. In addition to micrographs, quantification of Kiss1 mRNA expression is displayed, as estimated by grain density in the areas subjected to analysis in control and HFD (A) male and (B) female offspring from both paternal groups (right panels). For comparative purposes, quantitative values of CD-fed F1 rats from lean fathers are represented. To ease comparison among groups, mean control values are displayed also as dotted lines. Quantitative data are presented as mean ± standard error of the mean, n = 5 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05 vs the corresponding control groups. 3V, third ventricle; aca, anterior commissure, anterior part; AVPe, anteroventral periventricular nucleus; DM, dorsomedial hypothalamic nucleus; LH, lateral hypothalamic area; LV, lateral ventricle; Opt, optic tract; Pe, periventricular hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus. Figure 7. View largeDownload slide HFD inhibits Kiss1 expression in males, but paternal obesity does not aggravate this effect. A schematic representation of the location of the ARC and the AVPV nuclei, as areas of prominent expression of Kiss1 mRNA, is shown from male and female brains (left panels). Representative ISH photomicrographs are presented of Kiss1 mRNA-expressing neurons in the anterior region of the ARC (males and females) and AVPV (females) of the HFD-fed offspring from lean (control + HFD) and obese (paternal obesity + HFD) fathers. For comparative purposes, representative images of CD-fed F1 rats from lean fathers (control) are presented. In addition to micrographs, quantification of Kiss1 mRNA expression is displayed, as estimated by grain density in the areas subjected to analysis in control and HFD (A) male and (B) female offspring from both paternal groups (right panels). For comparative purposes, quantitative values of CD-fed F1 rats from lean fathers are represented. To ease comparison among groups, mean control values are displayed also as dotted lines. Quantitative data are presented as mean ± standard error of the mean, n = 5 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05 vs the corresponding control groups. 3V, third ventricle; aca, anterior commissure, anterior part; AVPe, anteroventral periventricular nucleus; DM, dorsomedial hypothalamic nucleus; LH, lateral hypothalamic area; LV, lateral ventricle; Opt, optic tract; Pe, periventricular hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus. Discussion The escalating prevalence of obesity and its increasing burden on population health (1, 2) urges for a deeper and more integral understanding of its constellation of comorbidities, which clearly go beyond metabolic and cardiovascular complications, and seemingly include a wide array of conditions, ranging from increased risk of cancer to various muscle-skeletal diseases and infertility (2, 35, 36). Worryingly, the detrimental effects of obesity might be transmitted to the progeny via genetic (e.g., by accumulating DNA damage in gametes) and epigenetic mechanisms (37). Although the intergenerational impact of maternal obesity, by perturbing in utero microenvironment, has been long recognized, evidence has accumulated recently on the possibility that paternal obesity may have also a deleterious influence on the health of the offspring, mainly via perturbation of sperm genetic content and/or epigenetic components (37). Yet, most of the limited information available on this phenomenon has focused on the metabolic consequences in (mainly the female) offspring. Among other bodily functions, reproduction appears to be sensitive to the actual and intergenerational impact of male obesity (35, 38). In both preclinical (rodent) models and humans, obese males have been shown to display central hypogonadism, i.e., failure of the upper components of the HPG axis that results in low circulating T levels (5, 39), as well as an array of sperm alterations (37), which can hamper fertility (40). In good agreement, in our experimental setting, obese fathers, which were >40% heavier that lean controls and had significantly elevated leptin, insulin, and glucose levels, displayed also a marked state of hypogonadotropic hypogonadism, defined by concomitantly lower LH and T levels. Expectedly, this resulted in a substantial decline in fecundity, so that only 20% of the virgin females mated with obese fathers became pregnant vs the 100% fertility rate seen in lean fathers. This attested the substantial impact of actual obesity on male fertility; yet, this also imposed limitations in the design of our study, as it markedly reduced the size of the male and female offspring from obese fathers, and therefore prevented us from generating additional experimental groups (such as male and female offspring from obese fathers fed a CD from weaning onward), which might have allowed us a more incisive testing of the putative interplay between intergenerational transmission and actual nutritional status. In this context, we decided to prioritize the analysis of the impact of paternal obesity as potential sensitizing condition for the deleterious consequences of HFD on metabolic and reproductive parameters of the offspring, as a means to explore the importance of environmental/nutritional interventions in the progeny of obese fathers, a phenomenon of obvious translational implications. Paternal obesity had an unambiguous impact on metabolic and reproductive health parameters of the offspring, and tended to exacerbate the detrimental effects of HFD on both sets of markers. Strikingly, however, this impact displayed a distinct sex difference, with males being clearly more sensitive than females. This was illustrated by the fact that HFD-fed males, but not females, born from obese fathers had higher BW gain and serum leptin levels, together with lower circulating LH and T, decreased LH responses to kisspeptin-10, and suppressed testicular gene expression of key steroidogenic enzymes, when compared with HFD males from lean fathers. In contrast, females on HFD from obese fathers had similar metabolic and reproductive parameters than HFD females from lean fathers, except for reduced LH responses to kisspeptin-10. Such a preferential male phenotype caused by paternal obesity is in line with previous experimental and clinical data. Thus, a recent preclinical study reported that male F1 descendants from obese yellow Avy/a male mice, mated to congenic lean dams, exhibited increased adiposity when exposed to a western diet as compared with male F1 offspring from lean fathers fed on the same obesogenic diet; in contrast, no differences in metabolic parameters were found in F1 females from lean and obese fathers fed on western diet (11). In the same line, clinical findings suggest that paternal body mass index impacts growth of the male, but not female offspring (41). Yet, this male-preferential impact is partially in contrast with some previous reports, suggesting that the female progeny seems to be more sensitive to the programming effects of paternal obesity on metabolic and pancreatic dysfunction (42, 43). Although experimental reasons, e.g., differences in the models of paternal obesity in terms of initiation and duration of the obesogenic insults, may account for part of the discrepancy between studies, it is worth noting that, in our current study, paternal obesity had a negligible impact on glucose homeostasis, so that males fed on HFD displayed equally elevated basal glucose and insulin levels, and showed similar indices of glucose intolerance (in OGTT) and insulin resistance (in ITT), irrespective of the nutritional status of the fathers. In females, the regimen of HFD exposure from weaning to PND-120 caused a milder impact on basic indices of glucose homeostasis, with a modest increase in basal glucose levels, which was similar in the offspring of obese and lean fathers, and partially perturbed OGTT responses, especially in HFD females from lean fathers. Curiously enough, HFD females from obese fathers tended to have normalized (rather than worsen) OGTT responses and modestly, but significantly improved insulin sensitivity. The latter is an intriguing finding of as yet unclear basis, which might be related to differences in the time course of the impact of HFD on glucose homeostasis and/or the patterns of fat deposition caused by paternal obesity, as some subcutaneous fat depots have even been reported to partially protect against development of insulin resistance (44, 45). Although the lack of experimental groups of females from obese fathers fed on CD prevented us from a direct comparison with previous studies in other preclinical models of paternal obesity, altogether our current dataset clearly documents that males are more sensitive than females to the detrimental effects of HFD on glucose homeostasis, in line with previous rodent studies (46, 47), and that such deleterious effects are not exacerbated by paternal obesity. In addition to its documented impact on embryo implantation and pregnancy health (48), the studies produced to date addressing the consequences of paternal obesity on the reproductive health of the progeny have been restricted to its effects on gamete (mainly sperm) quality (37). However, it is well known that obese individuals have important derangements at upper levels of the HPG axis (5, 6, 39). Indeed, recent evidence form preclinical models has documented that obesogenic insults evoke central hypogonadism in male and female rats due, in part, to alterations of the hypothalamic Kiss1 system (5, 6). Yet, the putative intergenerational impact of paternal obesity on such neuroendocrine components had not been explored to date. Our data document that paternal obesity not only aggravates the state of hypogonadism caused by HFD in the male progeny, but also dampens both basal and stimulated LH secretory activity after HFD. The integral analysis of the dynamic changes in 1) hypothalamic (ARC) Kiss1 expression, 2) LH responses to kisspeptin-10, 3) basal gonadotropin and T levels, as well as 4) testicular expression of key steroidogenic genes caused by HFD reveals a complex interaction, with suppressed central (e.g., Kiss1 expression; LH responses to Kp-10) and peripheral (e.g., T levels; testicular gene expression) components, together with putative compensatory responses (e.g., a trend to increased LH levels, which nonetheless was not consistent in all analyses). Admittedly, part of the defective LH responses to exogenous Kp-10 observed in our study may derive from suppressed pituitary responsiveness to endogenous GnRH, released in response to kisspeptin (19), a possibility that we cannot rule out, as the limited availability of experimental animals prevented us from conducting parallel GnRH stimulatory tests. Of note, however, previous studies from our group in male rats fed on HFD, not addressing the influence of paternal obesity, clearly documented that the deleterious impact of HFD-induced obesity on LH responses to kisspeptin cannot be solely explained by a suppression of pituitary responsiveness to GnRH (5). In any event, it is clear that paternal obesity, at least the time window explored in this work (PND-120), negatively affected most parameters, because basal and stimulated LH levels, T concentrations, and testicular expression of StAR, P450scc, and 17β-HSD were significantly lower in the progeny of obese founders than in equivalent HFD-fed males from lean fathers. Moreover, considering the well-known negative feedback effects of T on ARC Kiss1 expression in males (49), the fact that such aggravation of the state of hypogonadism in HFD-fed males from obese fathers was not accompanied by a compensatory increase in ARC Kiss1 levels further attests the impairment of the central Kiss1 system caused by paternal obesity. Anyhow, the significant drop of ARC Kiss1 expression caused by HFD in males from lean fathers could not be explained either as a reaction to changes in circulating T levels, therefore supporting the notion that obesity has a primary impact at central levels to suppress the male reproductive axis, in line with previous references (5, 50). Additional studies in equivalent experimental groups after gonadectomy, with or without sex steroid replacement, as a means to control for the actual influence of changes in circulating gonadal hormones, may help to further clarify this issue. As it was the case for metabolic indices, the reproductive profiles were markedly different in the female offspring. Not only HFD failed to cause major alterations of the HPG axis in females, but also paternal obesity did not aggravate the major gonadotropic indices, apart from a nonsignificant decline in circulating estradiol and a decrease in LH responses to Kp-10. In fact, Kiss1 expression in the AVPV and ARC was not significantly different across the experimental groups, and steroidogenic enzyme expression was even enhanced in HFD females from obese fathers. Whether this represents a compensatory response in face of the incipient failure of the HPG axis suggested by defective LH responses to kisspeptin is a tenable possibility that needs further investigation. In any event, this strikingly different phenotype between sexes further documents that the deleterious intergenerational influence of paternal obesity preferentially occurs in the adult male. This does not preclude that paternal obesity may affect the impact of HFD on other reproductive parameters in the female, such as puberty onset, a possibility that merits independent investigation. In sum, our study adds to previous literature supporting an impact of the metabolic/nutritional state of the father on the health status of the progeny. Notably, such an influence displays a clear sex difference, with males being in general more sensitive than females, and involves changes in susceptibility to the detrimental effects of HFD on body weight gain and adiposity (as reflected by leptin), but not glucose homeostasis. In addition, paternal obesity has a discernible impact on the HPG axis of the male progeny, so that it predisposes to development of central hypogonadism and perturbation of hypothalamic Kiss1 system after HFD exposure. Admittedly, our study does not rule out the possibility of additional impacts of HFD and paternal obesity on other reproductive indices. In any event, based on our present preclinical findings, early-onset nutritional interventions, avoiding dietary fat excess preferentially in males born from obese fathers, should be considered to attenuate the risk of appearance of metabolic and reproductive disorders later in life. Abbreviations: ARC arcuate AUC area under the curve AVPV anteroventral periventricular BW body weight CD control diet cDNA complementary DNA FSH follicle-stimulating hormone GnRH gonadotropin-releasing hormone HFD high-fat diet HPG hypothalamic-pituitary-gonadal ISH in situ hybridization ITT insulin tolerance test KITT insulin sensitivity index LH luteinizing hormone mRNA messenger RNA OGTT oral glucose tolerance test PCR polymerase chain reaction RIA radioimmunoassay SSC saline sodium citrate T testosterone. Acknowledgments Financial Support: This work was supported by Grants BFU2011-025021 and BFU2014-57581-P (Ministerio de Economía y Competitividad, Spain, cofunded with European Union funds from FEDER Program), Project PIE14-00005 (Flexi-Met, Instituto de Salud Carlos III, Ministerio de Sanidad, Spain), Projects P08-CVI-03788 and P12-FQM-01943 (Junta de Andalucía, Spain), and European Union Research Contract DEER FP7-ENV-2007-1. CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of Instituto de Salud Carlos III. Disclosure Summary: The authors have nothing to disclose. References 1. World Health Organization Updates. 2016 obesity and overweight fact sheet. Available at: http://www.who.int/mediacentre/factsheets/fs311/en/. Accessed June 5, 2016. 2. Kyrou I, Randeva HS, Weickert MO. Clinical problems caused by obesity. 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Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters of the Offspring: Male-Preferential Impact and Involvement of Kiss1-Mediated Pathways

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Oxford University Press
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-00705
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Abstract

Abstract Obesity and its comorbidities are reaching epidemic proportions worldwide. Maternal obesity is known to predispose the offspring to metabolic disorders, independently of genetic inheritance. This intergenerational transmission has also been suggested for paternal obesity, with a potential negative impact on the metabolic and, eventually, reproductive health of the offspring, likely via epigenetic changes in spermatozoa. However, the neuroendocrine component of such phenomenon and whether paternal obesity sensitizes the offspring to the disturbances induced by high-fat diet (HFD) remain poorly defined. We report in this work the metabolic and reproductive impact of HFD in the offspring from obese fathers, with attention to potential sex differences and alterations of hypothalamic Kiss1 system. Lean and obese male rats were mated with lean virgin female rats; male and female offspring were fed HFD from weaning onward and analyzed at adulthood. The increases in body weight and leptin levels, but not glucose intolerance, induced by HFD were significantly augmented in the male, but not female, offspring from obese fathers. Paternal obesity caused a decrease in luteinizing hormone (LH) levels and exacerbated the drop in circulating testosterone and gene expression of its key biosynthetic enzymes caused by HFD in the male offspring. LH responses to central kisspeptin-10 administration were also suppressed in HFD males from obese fathers. In contrast, paternal obesity did not significantly alter gonadotropin levels in the female offspring fed HFD, although these females displayed reduced LH responses to kisspeptin-10. Our findings suggest that HFD-induced metabolic and reproductive disturbances are exacerbated by paternal obesity preferentially in males, whereas kisspeptin effects are affected in both sexes. Obesity rates have rapidly increased over the last 30 years worldwide. According to the World Health Organization, obesity prevalence has more than doubled since 1980, with 39% of the global population being overweight and 13% obese in 2014 (1). Obesity is associated with a constellation of health problems such as type 2 diabetes, dyslipidemia, hypertension, cardiovascular disease, and infertility (2), whose prevalence has paralleled the obesity epidemic during the last decades. It is widely recognized that inadequate dietary habits and sedentary lifestyle are the main factors responsible for the increased incidence of obesity and related comorbidities. However, the exponential increase in obesity prevalence is difficult to be explained solely by a chronic imbalance between food intake and energy expenditure, suggesting that other factors may influence susceptibility to obesity and its associated health problems. Compelling evidence suggests that the origins of obesity may be traced back to the fetal/neonatal period, in which the maternal nutritional and metabolic status is known to play an important role in developmental programming (3, 4). According to this concept, adaptive responses induced by nutritional challenges during early stages of development may have permanent effects on the metabolic health of the developing offspring, which may result in inappropriate responses to obesogenic insults in later life. In line with this hypothesis, we have recently reported that overnutrition during lactation predisposes male and female offspring to obesity and reproductive disturbances in adulthood when exposed to high-fat diet (HFD) (5, 6). Recent findings have demonstrated that maternal nutritional environment is relevant not only during gestation and lactation, but also during the periconception stage (7). This influence has been surfaced in preclinical models, by using embryo transfer techniques, in which fertilized eggs from mothers fed an HFD at periconception were transferred to subrogate control mothers; this resulted in growth retardation and abnormalities in brain development in the offspring (7). These observations indicate that nongenetic, intergenerational mechanisms influence offspring development and may determine disease risk later in life. Interestingly, this nongenetic intergenerational transmission of adverse phenotypes has also been described from fathers to offspring. Thus, rodent studies have shown that paternal exposure to HFD at periconception impairs the metabolic health of subsequent generations, regardless of the presence of diabetes in the male founders (8–11). In addition to such detrimental effect on the metabolic health of the offspring, diet-induced paternal obesity in mice has been putatively linked to sperm disturbances in subsequent generations (9, 12, 13). These findings suggest that paternal nutritional status prior to fertilization may trigger the intergenerational transmission of metabolic and reproductive traits to descendants and may predispose offspring to obesity and fertility problems. Although the underlying mechanisms for this paternal programming of the offspring phenotype remain unknown, a growing body of evidence indicates that epigenetic processes in the sperm, such as altered DNA methylation, histone modification, and changes in small noncoding RNA levels (namely, microRNAs), may be potential mediators of this intergenerational effect (14–16). Because fathers and children usually share similar dietary habits, and therefore children from obese fathers may be likely exposed to an obesogenic environment, an interesting question that arises is whether paternal obesity at periconception may sensitize the offspring to the deleterious effects of HFD. A recent study has shown that the metabolic and sperm derangements induced by paternal obesity may be exacerbated by HFD in male F1 offspring (17). Yet, the metabolic and gonadotropic effect of paternal obesity in the female offspring fed HFD, as well as its impact on the neuroendocrine reproductive axis in the HFD-fed male offspring, remains virtually unexplored to date. Reproduction is very sensitive to metabolic status (18–20). The neuroendocrine reproductive [aka, hypothalamic-pituitary-gonadal (HPG)] axis is governed by a population of hypothalamic neurons that express and release gonadotropin-releasing hormone (GnRH) (19). This neuropeptide is secreted in a pulsatile fashion and stimulates gonadotropin release from the pituitary, which in turn promotes gonadal maturation and function (19). Metabolic challenges perturb the pattern of GnRH secretion and, consequently, may compromise fertility. However, a wealth of data strongly suggests that GnRH neurons are devoid of functional receptors for the major metabolic cues that modulate their activity, as appears to be the case for leptin and, possibly, insulin (18, 19, 21–26); yet, direct insulin signaling in GnRH neurons might contribute to transmit part of the metabolic impact of obesity on reproductive function (27). In any event, the consensus view is that a substantial component of the metabolic regulation of GnRH neurons is carried out by a network of afferent neurons that express such receptors and convey this information to GnRH neurons in a direct or indirect manner. Among those afferents, Kiss1 neurons are considered essential players in the metabolic control of reproduction (19). In conditions of negative energy balance, Kiss1 neurons are inhibited to repress the activity of the reproductive axis. In addition, we recently reported that in conditions of energy excess, such as in obesity, the hypothalamic Kiss1 system is also suppressed in males and females (5, 6). Yet, whether paternal obesity may impact the Kiss1 system in the progeny is unknown and warrants specific investigation. We report in this work a comprehensive set of studies addressing the impact of paternal obesity on metabolic and reproductive health parameters of the male and female offspring, especially as it pertains to its influence upon responses to HFD, with a particular interest on the neuroendocrine perturbations, sex differences, and potential alterations of the hypothalamic Kiss1 system. Materials and Methods Animals and diets Wistar rats bred in the vivarium of the University of Córdoba were used. Rats were given ad libitum access either to a control diet (CD), D12450B (10% of calories from fat, 20% from protein, and 70% from carbohydrate; Research Diets, Inc. New Brunswick, NJ), or an HFD, D12451 (45% of calories from fat, 20% from protein, and 35% from carbohydrate; Research Diets). The animals were maintained at 22°C under constant conditions of light (14 hours) with free access to water. In all experiments, the animals were housed and handled under strictly similar conditions, allowing the integral analysis of data. Experimental procedures were approved by Córdoba University Ethical Committee and conducted in accordance with European Union guidelines. Experimental design Control and extremely obese male rats (F0) were mated with adult virgin lean chow-fed dams (Charles River, Barcelona, Spain) to generate F1 offspring. Control founder rats were fed a CD for 3 months, whereas obese F0 males were obtained by inducing overnutrition during lactation (small litter size, four pups per dam), followed by HFD from weaning onward for 9 months. Of note, these paternal groups were generated in the context of a large-scale study conducted by our group addressing the impact of sequential obesogenic insults on metabolic and reproductive function in male rats (5). A summary of the most relevant metabolic and reproductive parameters analyzed in control and obese F0 fathers is provided in Supplemental Table 1. To obtain the F1 generation, every F0 male was housed together with a 12-week-old virgin female for 8 consecutive days (two estrous cycles), with free access to chow diet and water. Females were monitored for regular vaginal cycling before initiation of mating, and only females showing at least four consecutive regular, 4-day cycles were used. During the mating period, vaginal smears were obtained and analyzed daily to monitor estrous cycle in the females. Pregnancy was evidenced by the presence of sperm in vaginal smears in the morning of the estrous phase in all F0 females. Of note, despite the confirmation of the presence of sperm in vaginal smears in all females mated with extremely obese males, the proportion of pregnant dams was markedly reduced as compared with control F0 males (Fig. 1A), indicative of impaired fertility in obese males. After the mating period, F0 males were returned back to their initial cages with the original diet, whereas F0 females continued on chow diet during gestation and lactation. Figure 1. View largeDownload slide Overall design of the transgenerational study. (A) Table illustrating the details on the mating process, including number of mated dams, pregnancy rates, and number of offspring obtained. (B) Schematic representation of the experimental design. Lean and extremely obese F0 fathers were mated with lean virgin mothers. F1 male and female offspring from lean fathers were fed on CD or HFD, whereas F1 pups from obese fathers received exclusively an HFD until they were euthanized (PND-120). Figure 1. View largeDownload slide Overall design of the transgenerational study. (A) Table illustrating the details on the mating process, including number of mated dams, pregnancy rates, and number of offspring obtained. (B) Schematic representation of the experimental design. Lean and extremely obese F0 fathers were mated with lean virgin mothers. F1 male and female offspring from lean fathers were fed on CD or HFD, whereas F1 pups from obese fathers received exclusively an HFD until they were euthanized (PND-120). After birth, F1 male and female offspring were cross-fostered and reared in litters of normal size (8 to 13 pups per dam). As previously mentioned, fertility was compromised in obese F0 fathers, which restricted the availability of pups from obese fathers in the study. Because of this limitation, only one group of male (n = 13) and female (n = 8) offspring from obese fathers was obtained (Fig. 1A), from two different litters. Due to the use of F0 males in the context of the large-scale, independent studies mentioned previously (5), we were unable to enlarge the size of the progeny of obese fathers enrolled in the current analyses. The offspring were weaned from mothers at PND-23. From weaning onward, F1 male and female offspring from control lean fathers were fed either a CD (control) or an HFD ad libitum (control + HFD), whereas F1 offspring from obese fathers were maintained exclusively on HFD (paternal obesity + HFD); an independent group of paternal obesity + CD could not be generated due to the operational limitations indicated previously. A schematic representation of the experimental design is provided in Fig. 1B. Blood parameter measurements After euthanasia by decapitation at PND-120, blood samples were collected, immediately placed on ice, and centrifuged, and plasma samples were stored at −20°C until analyzed. Of important note, female rats were euthanized at the same stage of the estrous cycle, namely diestrus, as monitored by regular vaginal smearing for at least three consecutive cycles before sampling, to minimize the potential influence that ovarian cycle-dependent fluctuations may have on the endpoints analyzed. Plasma levels of leptin and insulin were quantified using commercial radioimmunoassay (RIA) kits from Linco Research (St. Charles, MO). Circulating glucose levels were determined by using a handheld glucometer (ACCU-CHECK Aviva; Roche Diagnostics, Basel, Switzerland) after an overnight fast. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured using RIA kits from the National Hormone Peptide Program (Torrance, CA), with reference preparations LH-RP-3 and FSH-RP-2 and intra-assay and interassay coefficients of variation below 8% and 10%, respectively. Testosterone (T) and estrogen levels were assessed by using specific RIA kits, as previously reported (5, 6). In detail, serum T levels were assayed using a RIA kit from MP Biomedicals (Santa Ana, CA). The sensitivity of the assay was 0.1 ng/mL, and the intra-assay and interassay coefficients of variation were <5% and <8%, respectively. Circulating estrogen levels were assayed using a commercial ultrasensitive RIA kit from Beckman Coulter (Brea, CA). The sensitivity of the assay was 2.2 pg/mL, and the intra-assay and interassay coefficients of variation were ≤8.9% and ≤12.2%, respectively. Kisspeptin stimulation tests The LH-releasing ability of an intracerebroventricular low dose of Kp-10 (Phoenix Pharmaceuticals, Belmont, CA) was monitored. To allow delivery of Kp-10 into the lateral cerebral ventricle, rats were implanted with intracerebroventricular cannulas, as described previously (28–30). Before initiation of the functional tests, estrous cycles were monitored in the female groups, and tests were performed at the same cycle stage (diestrus) to exclude potential variations in LH responses to Kp-10 due to the phase of the cycle, as documented by previous references (31). Doses of 50 pmol/rat were used to evoke submaximal stimulation of the gonadotropic axis, allowing discrimination of subtle changes in sensitivity. Blood was collected before (0) and at 15, 60, and 120 minutes after Kp-10 administration. In addition to individual hormonal levels, area under the curve (AUC) responses were calculated over the 120-minute period. Note that to allow complete washout and recovery of the experimental animals before subsequent metabolic testing and euthanasia, Kp-10 tests were conducted around PND-100. Oral glucose tolerance tests and insulin tolerance tests For the determination of glucose tolerance, rats were subjected to oral glucose tolerance tests (OGTTs) around PND-110. Rats were fasted overnight and subsequently received an oral bolus of glucose [1 g/kg body weight (BW)] by gastric gavage. Glucose levels were determined in blood before (0) and at 20, 60, and 120 minutes postadministration. After complete recovery and washout, insulin sensitivity was assessed in overnight fasted rats following an intraperitoneal injection of 1 U insulin (Sigma-Aldrich, St. Louis, MO) per kg body weight; insulin tolerance tests (ITTs) were performed around PND-115. Blood glucose levels were measured before (0) and at 15 and 30 minutes after insulin administration. Insulin sensitivity index (KITT) was calculated to assess peripheral insulin sensitivity, as reported elsewhere (32). All glucose concentrations were measured using a handheld glucometer (ACCU-CHECK Aviva; Roche Diagnostics). Steroidogenic enzyme gene expression analyses by quantitative polymerase chain reaction Testicular and ovarian samples were dissected immediately after euthanasia of the animals, frozen in liquid N2, and stored at −80°C until RNA isolation. Tissues were processed, and total RNA was isolated using TRIsure (BIOLINE, London, UK), following the manufacturer’s instructions. Next, a fixed amount of RNA (2 μg) was transcribed into complementary DNA (cDNA) by the action of the reverse transcriptase (Bio-Rad, Hercules, CA); real-time reverse transcription polymerase chain reaction (PCR) was performed employing the iCycler iQ real-time PCR system (Bio-Rad). Testicular and ovarian expression of StAR, P450scc, and 17β-HSD was measured by using specific primer pairs previously designed and validated in our group (5). To analyze aromatase expression in the ovary, the following primer sequences were used: forward, 5′-TGT TGC TTC TCA TCG CAG AGT ATC C-3′; reverse, 5′-GGC TGA TAC CGC AGG CTC TCG-3′. As internal control, expression of the ribosomal protein S11 was measured by using specific primer sets (5). Calculation of relative expression levels of the targets was conducted based on the cycle threshold method, as described elsewhere (33). Kiss1 riboprobe synthesis For detection of Kiss1 messenger RNA (mRNA) by in situ hybridization, a specific riboprobe for Kiss1 rat mRNA, spanning 83 to 371 nucleotides of the cDNA sequence (GenBank NM_181692.1), was generated. First, a DNA template was synthesized by PCR using specific primers for Kiss1 cDNA amplification carrying at their 5′-end sequences for synthetic promoters for bacteriophage-encoded DNA-dependent RNA polymerases (T7 and T3). Primer sequences were as follows: forward primer T3-Kiss sense (5′-CAG AGA TGC AAT TAA CCC TCA CTA AAG GGA GAT GGT GAA CCC TGA ACC CAC A-3′); reverse primer T7-Kiss as (5′-CCA AGC CTT CTA ATA CGA CTC ACT ATA GGG AGA ACC TGC CTC CTG CCG TAG CG-3′). For PCRs, Go-Taq flexi DNA polymerase (Promega Biotech, Madison, WI) was used, following the recommendations of the manufacturer. Reactions were performed in an iCycler (Bio-Rad Laboratories) using the following protocol: cDNA was denatured for 5 minutes at 95°C, and then four cycles were performed at 94°C for 1 minute, 54°C for 2 minutes, and 72°C for 30 seconds, followed by 35 cycles at 94°C for 1 minute, 65°C for 1 minute, and 72°C for 30 seconds. A final extension at 72°C for 5 minutes was included. After electrophoresis on a 2% agarose (w/v) gel, a single DNA fragment was obtained of the expected size and gel purified with a QiaQuick gel extraction kit (Qiagen, Hilden, Germany). For the generation of the antisense Kiss1 riboprobe, the product of the PCR was used as template for the transcription reaction, as follows (final volume of 20 μL): 250 μCi [33P]-UTP (Perkin Elmer, Waltham, MA), 0.5 μg template, 2 μL ribonucleoside triphosphates (rNTPs: 5 mM rATP, rCPT, and rGTP), 1 μL RNasin Ribonuclease Inhibitor (Promega), 4 μL transcription buffer, and 2 μL T7 RNA polymerase (Promega). After 120 minutes of incubation at 37°C, another 1 μL T7 RNA was added to the mix, and the reaction was maintained for an additional 60 minutes at 37°C. At the end, the residual DNA was digested with 2 U DNase (Promega), and the reaction was terminated by addition of 3 μL 0.5 M EDTA, pH 8.0. Diethyl pyrocarbonate water was added to a final volume of 50 μL, and the labeled riboprobe was purified using Illustra ProbeQuant G-50 Micro Columns (GE Healthcare, Chalfont, UK). For synthesis of the sense Kiss1 riboprobe, the same procedure was applied, using T3 RNA polymerase (Promega). In situ hybridization Expression of Kiss1 mRNA within the hypothalamus was assessed by in situ hybridization (ISH), with attention on the arcuate (ARC) and the anteroventral periventricular (AVPV) nuclei, as major sites of Kiss1 expression in the brain (19). Considering sex differences in the pattern of hypothalamic expression of Kiss1 in rodents, with males having abundant expression only in the ARC (19), we first assayed ARC Kiss1 mRNA levels in the male offspring from control fathers, fed with CD or HFD, and in the HFD offspring from obese fathers. In a second set of analyses, we assessed ARC and AVPV Kiss1 expression in the same experimental groups in the female offspring; specimens (n = 5/group) for ISH analysis were randomly assigned within each group. From each brain, five sets of coronal plane sections 20 μm thick were thaw mounted in Super-Frost Plus slides (Thermo Fisher Scientific, Waltham, MA). Standard procedures of tissue collection were applied, starting in a fixed coordinate in the rostral hypothalamic area up to the ARC, as regions in which Kiss1 neurons are abundantly located (19, 34). The samples were stored at −80°C until ISH analyses. A specific radiolabeled Kiss1 antisense riboprobe was generated using T7 RNA polymerase (Bio-Rad Laboratories) and Kiss1 cDNA template, as described previously. A single set of sections was used for ISH (adjacent sections 100 μm apart). In brief, tissue sections were as follows: 1) fixed in 4% paraformaldehyde for 15 minutes; 2) stabilized with 0.1 M phosphate buffer (pH 7.4) at room temperature for 10 minutes; 3) treated with saline triethanolamine and acetic anhydride to prevent nonspecific binding of probes; 4) washed in 2× saline sodium citrate (SSC) buffer for 3 minutes; 5) dehydrated in increasing concentrations of ethanol; 6) delipidated with chloroform; and 7) air dried at room temperature for 1 hour. After these steps, hybridization with Kiss1 riboprobe was performed for 16 hours at 55°C. The hybridization buffer contained (for 40 ml) the following: 25 ml deionized formamide, 10 ml dextran sulfate 50%, 3 ml NaCl 5 M, 0.4 ml Tris base 1 M (pH 8), 0.08 ml 0.5 M EDTA (pH 8), 1× Denhardt solution, and RNase-free water up to 40 ml; Kiss1 riboprobe was added to the hybridization buffer to a final concentration of 0.03 pmol/mL along with yeast transfer RNA. After hybridization, slides were as follows: 1) washed with 4× SSC for 30 minutes; 2) incubated in RNase-A buffer (Roche Biochemical) at 37°C (32 μg/mL) for 1 hour; 3) equilibrated with 2× SSC for 30 minutes; 4) washed in 0.1× SSC for 1 hour at 65°C; 5) dehydrated in increasing ethanol series; and 6) air dried at room temperature for 1 hour. Finally, slides were dipped in Kodak Autoradiography Emulsion type NTB (Eastman Kodak, Rochester, NY) and exposed for 1 week at 4°C in the dark. After this, the sections were developed and fixed following the manufacturer instructions (Eastman Kodak): 1) 4 minutes in Kodak Developer D-19; 2) 10 seconds in distilled water; 3) 5 minutes in Kodak Fixer; and 4) 5 minutes in distilled water. For mounting, the sections were previously dehydrated and rinsed with Sub-X Clearing Medium (Leica Bio-systems, Nussloch, Germany). Then slices were coverslipped with Sub-X mounting medium (Leica). For analysis, 50 to 60 sections from each animal (9 to 10 slides; 6 sections/slide) were evaluated. Five animals per group were included in the analysis. Slides were read under dark-field illumination with custom-designed software enabled to count the total number of cells (grain clusters). Cells were counted as Kiss1 mRNA positive when the number of silver grains in a cluster exceeded that of background. Statistical analyses Metabolic and hormonal determinations were conducted in duplicate, with a minimal total number of at least eight samples per group. All results are presented as mean ± standard error of the mean. Statistical analyses were performed on data distributed in a normal pattern using Student t tests or analysis of variance, the latter for statistical comparisons of the three experimental groups. When significant differences were found in analysis of variance, data were further analyzed through post hoc comparison, using Student-Newman-Keuls tests to identify simple effects. The significance level was set at P ≤ 0.05 (Prism 5.0; GraphPad Software). Results Paternal obesity increases susceptibility to the metabolic impact of HFD in the offspring In the male offspring of lean fathers, the detrimental effects of HFD on BW were in general mild and only manifested as significantly increased BW at PND-120, whereas in females this increased BW gain was evident already at PND-70. Strikingly, males from obese fathers fed on HFD exhibited increased BW already at PND-50, whereas in females this increase became detectable around PND-40. Accordingly, the persistent elevation of BW caused by HFD in the male offspring from obese fathers was significantly higher than that of control HFD males (Fig. 2A), reaching the highest levels at PND-120 (Fig. 2B). HFD-fed females born from obese fathers were initially heavier also than HFD females from control fathers (Fig. 2D). However, the amplification of BW gain induced by paternal obesity in HFD females was more modest and transient (Fig. 2A and 2D). Thus, BW curves from both female groups fed on HFD converged after PND-80, with no differences in terminal BW being detected at PND-120 (Fig. 2E). The pronounced increase of BW in HFD-fed males from obese fathers was accompanied at PND-120 by a rise in circulating leptin levels, as index of increased adiposity (Fig. 2C). In contrast, no significant alterations were detected at this age in basal leptin levels between HFD-fed females from control and obese fathers (Fig. 2F). Figure 2. View largeDownload slide Paternal obesity exacerbates the effects of HFD on body weight and adiposity in males. Effects of HFD on (A and D) body weight progression, (B and E) terminal body weight, and (C and F) circulating leptin levels in F1 male and female offspring from lean and obese fathers (control + HFD and paternal obesity + HFD, respectively). For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are shown; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 to 13 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. *P < 0.05; **P < 0.01; ***P < 0.001 vs the corresponding control + HFD group. Figure 2. View largeDownload slide Paternal obesity exacerbates the effects of HFD on body weight and adiposity in males. Effects of HFD on (A and D) body weight progression, (B and E) terminal body weight, and (C and F) circulating leptin levels in F1 male and female offspring from lean and obese fathers (control + HFD and paternal obesity + HFD, respectively). For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are shown; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 to 13 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. *P < 0.05; **P < 0.01; ***P < 0.001 vs the corresponding control + HFD group. To evaluate the impact of paternal obesity on glucose homeostasis in the offspring after HFD, OGTTs were performed in adulthood. As expected, HFD perturbed the glycemic profile in both males and females from control F0 (lean) founders, as revealed by increased basal glucose levels and impaired responses during OGTT, defined by significantly elevated glucose concentrations at 60 and 120 minutes after the oral glucose bolus in males, and at 120 minutes in females (Fig. 3). HFD also had a detrimental impact on glucose tolerance in males from obese fathers. Yet, the overall impact of paternal obesity plus HFD, as reflected by basal glucose levels and integral AUC values during OGTT, was of similar magnitude than in the control + HFD group (Fig. 3). Intriguingly, despite similarly elevated basal glucose levels, HFD-fed females from obese fathers exhibited a trend for improved glycemic profile after glucose oral challenge, as compared with control + HFD females. This was reflected by the fact that neither individual glucose levels following the oral bolus nor the integral AUC glucose responses during the OGTT were statistically different from those of the group fed with CD (Fig. 3). Figure 3. View largeDownload slide Paternal obesity does not exacerbate the impact of HFD on glucose tolerance in male and female offspring. Effects of HFD on glucose tolerance in F1 male and female offspring from lean and obese fathers are shown. The animals were subjected to OGTT. They received an oral bolus of glucose (1 g/kg BW), and blood glucose levels were measured at 0 (before) and 20, 60, and 120 minutes after glucose administration. Integral glucose levels estimated as AUC by the trapezoidal rule are also shown. For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are presented; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. No significant differences between control + HFD and paternal obesity + HFD groups were detected. Figure 3. View largeDownload slide Paternal obesity does not exacerbate the impact of HFD on glucose tolerance in male and female offspring. Effects of HFD on glucose tolerance in F1 male and female offspring from lean and obese fathers are shown. The animals were subjected to OGTT. They received an oral bolus of glucose (1 g/kg BW), and blood glucose levels were measured at 0 (before) and 20, 60, and 120 minutes after glucose administration. Integral glucose levels estimated as AUC by the trapezoidal rule are also shown. For comparative purposes, groups of CD-fed F1 rats from lean fathers (control) are presented; to ease comparison in histogram graphs, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group. No significant differences between control + HFD and paternal obesity + HFD groups were detected. HFD impaired basal glucose levels in males and females irrespective of the metabolic status of the father, with significantly elevated basal glycemia values vs the corresponding control (lean fathers + CD) group (Fig. 4). No significant alterations in basal insulin levels were detected in the HFD-fed male and female offspring from both paternal (lean and obese) groups, as compared with the groups receiving CD. HFD-fed males from control and obese founders exhibited decreased insulin sensitivity as reflected by reduced KITT, with no further impairment caused by paternal obesity (Fig. 4A). In contrast, HFD females from obese fathers were moderately less insulin resistant than females from lean fathers, irrespective of being fed control or HFD (Fig. 4B). Figure 4. View largeDownload slide Paternal obesity does not aggravate the effects of HFD on basal glycemia and insulin sensitivity in male and female offspring. Effects of HFD on basal circulating glucose and insulin levels and insulin sensitivity, assessed by KITT calculation, in F1 (A) male and (B) female offspring from lean and obese fathers. KITT was calculated after performing an ITT. The animals were fasted overnight and received an intraperitoneal injection of insulin, and glucose levels were recorded at 0 (before) and 20, 60, and 120 minutes after insulin administration. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs corresponding control groups; *P < 0.05 vs the corresponding control + HFD group. Figure 4. View largeDownload slide Paternal obesity does not aggravate the effects of HFD on basal glycemia and insulin sensitivity in male and female offspring. Effects of HFD on basal circulating glucose and insulin levels and insulin sensitivity, assessed by KITT calculation, in F1 (A) male and (B) female offspring from lean and obese fathers. KITT was calculated after performing an ITT. The animals were fasted overnight and received an intraperitoneal injection of insulin, and glucose levels were recorded at 0 (before) and 20, 60, and 120 minutes after insulin administration. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs corresponding control groups; *P < 0.05 vs the corresponding control + HFD group. Paternal obesity increases susceptibility to the reproductive impact of HFD in the offspring Reproductive analyses in terminal blood samples of PND-120 males revealed that HFD increased circulating LH levels in the offspring from both paternal groups compared with the CD-fed group. Yet, the rise in LH levels was significantly attenuated in HFD-fed males from obese fathers (Fig. 5A). Conversely, HFD did not cause alterations in basal LH levels in females from none of the two paternal groups (Fig. 5D). HFD did not modify basal FSH concentrations in males regardless of the father's metabolic status (Fig. 5B and 5E), but tended to reduce FSH levels in females from obese founders vs HFD-fed controls, although this decline did not reach statistical significance (P = 0.06). Figure 5. View largeDownload slide Paternal obesity perturbs basal and stimulated gonadotropin secretion in the offspring fed HFD. The effects of HFD on (A and D) basal LH levels, (B and E) basal FSH levels, and (C and F) LH responses to kisspeptin-10 are shown in F1 male and female offspring from lean and obese fathers. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are shown. To ease comparison among groups, mean control values are displayed also in the histogram graphs as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD group. Figure 5. View largeDownload slide Paternal obesity perturbs basal and stimulated gonadotropin secretion in the offspring fed HFD. The effects of HFD on (A and D) basal LH levels, (B and E) basal FSH levels, and (C and F) LH responses to kisspeptin-10 are shown in F1 male and female offspring from lean and obese fathers. For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are shown. To ease comparison among groups, mean control values are displayed also in the histogram graphs as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD group. Given the prominent role of kisspeptin in the central control of the HPG axis, the impact of HFD on the LH-releasing ability of a low dose of Kp-10 was assessed in the offspring from control and obese fathers. Admittedly, when compared with data in Fig. 5A, results from these analyses revealed some degree of variability in the impact of HFD on basal LH levels in the offspring from lean fathers, as basal LH concentrations were not significantly different between control- and HFD-fed males. Notwithstanding, this analysis fully confirmed that basal LH levels in the male offspring of obese fathers, when fed on HFD, were significantly lower than in the equivalent HFD group from lean fathers (Fig. 5C). In the male offspring from control fathers, HFD caused a reduction in peak LH responses at 15 minutes after Kp-10 injection and a significant 20% drop in global LH responses (AUC) to Kp-10 administration, suggesting a central impairment of gonadotropin responses caused by HFD. Notably, in males from obese fathers, LH responses to kisspeptin were further deteriorated in the presence of HFD, with a significant global 40% reduction in integral (AUC) responses vs the CD-fed group (Fig. 5C). In contrast to males, HFD failed to alter the integral LH responses to Kp-10 injection in females born from control fathers. In clear contrast, HFD significantly suppressed LH responses to kisspeptin in the female offspring from obese fathers (Fig. 5F). HFD also had a detrimental impact on basal T concentrations in males, in keeping with previous reports. Namely, HFD caused a 50% reduction in mean T levels in the offspring from lean founders vs the CD-fed group; yet, due to inherent variability in T levels and the limited group size, this difference did not reach statistical significance (P = 0.09). This effect was clearly exacerbated in the offspring from obese founders, which displayed an additional, significant 50% reduction in circulating T levels as compared with control HFD-fed males (Fig. 6A). Similar trends were detected for testicular expression of key steroidogenic enzymes. The expression of StAR was downregulated by HFD in the offspring from both paternal groups; yet, the magnitude of the decline was significantly greater in the offspring from obese fathers (Fig. 6A). In addition, HFD significantly reduced P450scc and 17β-HSD mRNA expression levels in the offspring from obese founders compared with the HFD-fed offspring of lean fathers. Figure 6. View largeDownload slide Paternal obesity exacerbates the magnitude of HFD-induced hypogonadism in F1 males. (A) The effects of HFD on circulating T levels and gene expression of key enzymes involved in testicular steroidogenesis in F1 males from lean and obese fathers are shown. In addition, the impact of HFD on serum estradiol and T levels, as well as the ovarian gene expression of steroidogenic enzymes, in F1 females from lean and obese fathers is presented in (B). For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD groups. Figure 6. View largeDownload slide Paternal obesity exacerbates the magnitude of HFD-induced hypogonadism in F1 males. (A) The effects of HFD on circulating T levels and gene expression of key enzymes involved in testicular steroidogenesis in F1 males from lean and obese fathers are shown. In addition, the impact of HFD on serum estradiol and T levels, as well as the ovarian gene expression of steroidogenic enzymes, in F1 females from lean and obese fathers is presented in (B). For comparative purposes, data from groups of CD-fed F1 rats from lean fathers (control) are presented. To ease comparison among groups, mean control values are displayed also as dotted lines. Data are presented as mean ± standard error of the mean, n = 8 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05; ##P < 0.01; ###P < 0.001 vs the corresponding control group; *P < 0.05; **P < 0.01 vs the corresponding control + HFD groups. In females, the impact of HFD on circulating sex hormone levels was less obvious than in males, as HFD failed to cause any significant suppression of estradiol levels, regardless of the metabolic status of the father, even when paternal obesity and HFD were combined (P = 0.10). Likewise, the low circulating T levels were not affected by HFD in adult female rats, irrespective of paternal obesity (Fig. 6B). In contrast to males, StAR, P450scc, and 17β-HSD expression levels in the ovary were upregulated by HFD in the female offspring from obese founders (Fig. 6B). Furthermore, HFD significantly increased ovarian aromatase expression in the offspring from obese fathers vs females born from control fathers (Fig. 6B). Changes in hypothalamic expression of Kiss1 after HFD exposure were also explored in the offspring from control and obese fathers by ISH. In general, HFD had a similar impact on hypothalamic Kiss1 mRNA levels in the ARC in males, independently of the father’s metabolic status. Thus, HFD caused a significant >50% drop in ARC Kiss1 expression in the male progeny of lean fathers, with no significant differences being detected between the offspring from lean and control fathers (Fig. 7A). Conversely, HFD did not significantly modify Kiss1 expression in the AVPV and the ARC of the female offspring from lean or obese fathers. Yet, a nonsignificant trend (P = 0.10) for increased Kiss1 expression was observed in the ARC of HFD-fed females from lean fathers, which was completely abrogated in the offspring of obese founders, as Kiss1 levels in the ARC of HFD-fed females from obese fathers were strictly similar to those of the CD-fed group (Fig. 7B). Figure 7. View largeDownload slide HFD inhibits Kiss1 expression in males, but paternal obesity does not aggravate this effect. A schematic representation of the location of the ARC and the AVPV nuclei, as areas of prominent expression of Kiss1 mRNA, is shown from male and female brains (left panels). Representative ISH photomicrographs are presented of Kiss1 mRNA-expressing neurons in the anterior region of the ARC (males and females) and AVPV (females) of the HFD-fed offspring from lean (control + HFD) and obese (paternal obesity + HFD) fathers. For comparative purposes, representative images of CD-fed F1 rats from lean fathers (control) are presented. In addition to micrographs, quantification of Kiss1 mRNA expression is displayed, as estimated by grain density in the areas subjected to analysis in control and HFD (A) male and (B) female offspring from both paternal groups (right panels). For comparative purposes, quantitative values of CD-fed F1 rats from lean fathers are represented. To ease comparison among groups, mean control values are displayed also as dotted lines. Quantitative data are presented as mean ± standard error of the mean, n = 5 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05 vs the corresponding control groups. 3V, third ventricle; aca, anterior commissure, anterior part; AVPe, anteroventral periventricular nucleus; DM, dorsomedial hypothalamic nucleus; LH, lateral hypothalamic area; LV, lateral ventricle; Opt, optic tract; Pe, periventricular hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus. Figure 7. View largeDownload slide HFD inhibits Kiss1 expression in males, but paternal obesity does not aggravate this effect. A schematic representation of the location of the ARC and the AVPV nuclei, as areas of prominent expression of Kiss1 mRNA, is shown from male and female brains (left panels). Representative ISH photomicrographs are presented of Kiss1 mRNA-expressing neurons in the anterior region of the ARC (males and females) and AVPV (females) of the HFD-fed offspring from lean (control + HFD) and obese (paternal obesity + HFD) fathers. For comparative purposes, representative images of CD-fed F1 rats from lean fathers (control) are presented. In addition to micrographs, quantification of Kiss1 mRNA expression is displayed, as estimated by grain density in the areas subjected to analysis in control and HFD (A) male and (B) female offspring from both paternal groups (right panels). For comparative purposes, quantitative values of CD-fed F1 rats from lean fathers are represented. To ease comparison among groups, mean control values are displayed also as dotted lines. Quantitative data are presented as mean ± standard error of the mean, n = 5 per group. Statistically significant differences were assessed by analysis of variance, followed by Student-Newman-Keuls tests. #P < 0.05 vs the corresponding control groups. 3V, third ventricle; aca, anterior commissure, anterior part; AVPe, anteroventral periventricular nucleus; DM, dorsomedial hypothalamic nucleus; LH, lateral hypothalamic area; LV, lateral ventricle; Opt, optic tract; Pe, periventricular hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus. Discussion The escalating prevalence of obesity and its increasing burden on population health (1, 2) urges for a deeper and more integral understanding of its constellation of comorbidities, which clearly go beyond metabolic and cardiovascular complications, and seemingly include a wide array of conditions, ranging from increased risk of cancer to various muscle-skeletal diseases and infertility (2, 35, 36). Worryingly, the detrimental effects of obesity might be transmitted to the progeny via genetic (e.g., by accumulating DNA damage in gametes) and epigenetic mechanisms (37). Although the intergenerational impact of maternal obesity, by perturbing in utero microenvironment, has been long recognized, evidence has accumulated recently on the possibility that paternal obesity may have also a deleterious influence on the health of the offspring, mainly via perturbation of sperm genetic content and/or epigenetic components (37). Yet, most of the limited information available on this phenomenon has focused on the metabolic consequences in (mainly the female) offspring. Among other bodily functions, reproduction appears to be sensitive to the actual and intergenerational impact of male obesity (35, 38). In both preclinical (rodent) models and humans, obese males have been shown to display central hypogonadism, i.e., failure of the upper components of the HPG axis that results in low circulating T levels (5, 39), as well as an array of sperm alterations (37), which can hamper fertility (40). In good agreement, in our experimental setting, obese fathers, which were >40% heavier that lean controls and had significantly elevated leptin, insulin, and glucose levels, displayed also a marked state of hypogonadotropic hypogonadism, defined by concomitantly lower LH and T levels. Expectedly, this resulted in a substantial decline in fecundity, so that only 20% of the virgin females mated with obese fathers became pregnant vs the 100% fertility rate seen in lean fathers. This attested the substantial impact of actual obesity on male fertility; yet, this also imposed limitations in the design of our study, as it markedly reduced the size of the male and female offspring from obese fathers, and therefore prevented us from generating additional experimental groups (such as male and female offspring from obese fathers fed a CD from weaning onward), which might have allowed us a more incisive testing of the putative interplay between intergenerational transmission and actual nutritional status. In this context, we decided to prioritize the analysis of the impact of paternal obesity as potential sensitizing condition for the deleterious consequences of HFD on metabolic and reproductive parameters of the offspring, as a means to explore the importance of environmental/nutritional interventions in the progeny of obese fathers, a phenomenon of obvious translational implications. Paternal obesity had an unambiguous impact on metabolic and reproductive health parameters of the offspring, and tended to exacerbate the detrimental effects of HFD on both sets of markers. Strikingly, however, this impact displayed a distinct sex difference, with males being clearly more sensitive than females. This was illustrated by the fact that HFD-fed males, but not females, born from obese fathers had higher BW gain and serum leptin levels, together with lower circulating LH and T, decreased LH responses to kisspeptin-10, and suppressed testicular gene expression of key steroidogenic enzymes, when compared with HFD males from lean fathers. In contrast, females on HFD from obese fathers had similar metabolic and reproductive parameters than HFD females from lean fathers, except for reduced LH responses to kisspeptin-10. Such a preferential male phenotype caused by paternal obesity is in line with previous experimental and clinical data. Thus, a recent preclinical study reported that male F1 descendants from obese yellow Avy/a male mice, mated to congenic lean dams, exhibited increased adiposity when exposed to a western diet as compared with male F1 offspring from lean fathers fed on the same obesogenic diet; in contrast, no differences in metabolic parameters were found in F1 females from lean and obese fathers fed on western diet (11). In the same line, clinical findings suggest that paternal body mass index impacts growth of the male, but not female offspring (41). Yet, this male-preferential impact is partially in contrast with some previous reports, suggesting that the female progeny seems to be more sensitive to the programming effects of paternal obesity on metabolic and pancreatic dysfunction (42, 43). Although experimental reasons, e.g., differences in the models of paternal obesity in terms of initiation and duration of the obesogenic insults, may account for part of the discrepancy between studies, it is worth noting that, in our current study, paternal obesity had a negligible impact on glucose homeostasis, so that males fed on HFD displayed equally elevated basal glucose and insulin levels, and showed similar indices of glucose intolerance (in OGTT) and insulin resistance (in ITT), irrespective of the nutritional status of the fathers. In females, the regimen of HFD exposure from weaning to PND-120 caused a milder impact on basic indices of glucose homeostasis, with a modest increase in basal glucose levels, which was similar in the offspring of obese and lean fathers, and partially perturbed OGTT responses, especially in HFD females from lean fathers. Curiously enough, HFD females from obese fathers tended to have normalized (rather than worsen) OGTT responses and modestly, but significantly improved insulin sensitivity. The latter is an intriguing finding of as yet unclear basis, which might be related to differences in the time course of the impact of HFD on glucose homeostasis and/or the patterns of fat deposition caused by paternal obesity, as some subcutaneous fat depots have even been reported to partially protect against development of insulin resistance (44, 45). Although the lack of experimental groups of females from obese fathers fed on CD prevented us from a direct comparison with previous studies in other preclinical models of paternal obesity, altogether our current dataset clearly documents that males are more sensitive than females to the detrimental effects of HFD on glucose homeostasis, in line with previous rodent studies (46, 47), and that such deleterious effects are not exacerbated by paternal obesity. In addition to its documented impact on embryo implantation and pregnancy health (48), the studies produced to date addressing the consequences of paternal obesity on the reproductive health of the progeny have been restricted to its effects on gamete (mainly sperm) quality (37). However, it is well known that obese individuals have important derangements at upper levels of the HPG axis (5, 6, 39). Indeed, recent evidence form preclinical models has documented that obesogenic insults evoke central hypogonadism in male and female rats due, in part, to alterations of the hypothalamic Kiss1 system (5, 6). Yet, the putative intergenerational impact of paternal obesity on such neuroendocrine components had not been explored to date. Our data document that paternal obesity not only aggravates the state of hypogonadism caused by HFD in the male progeny, but also dampens both basal and stimulated LH secretory activity after HFD. The integral analysis of the dynamic changes in 1) hypothalamic (ARC) Kiss1 expression, 2) LH responses to kisspeptin-10, 3) basal gonadotropin and T levels, as well as 4) testicular expression of key steroidogenic genes caused by HFD reveals a complex interaction, with suppressed central (e.g., Kiss1 expression; LH responses to Kp-10) and peripheral (e.g., T levels; testicular gene expression) components, together with putative compensatory responses (e.g., a trend to increased LH levels, which nonetheless was not consistent in all analyses). Admittedly, part of the defective LH responses to exogenous Kp-10 observed in our study may derive from suppressed pituitary responsiveness to endogenous GnRH, released in response to kisspeptin (19), a possibility that we cannot rule out, as the limited availability of experimental animals prevented us from conducting parallel GnRH stimulatory tests. Of note, however, previous studies from our group in male rats fed on HFD, not addressing the influence of paternal obesity, clearly documented that the deleterious impact of HFD-induced obesity on LH responses to kisspeptin cannot be solely explained by a suppression of pituitary responsiveness to GnRH (5). In any event, it is clear that paternal obesity, at least the time window explored in this work (PND-120), negatively affected most parameters, because basal and stimulated LH levels, T concentrations, and testicular expression of StAR, P450scc, and 17β-HSD were significantly lower in the progeny of obese founders than in equivalent HFD-fed males from lean fathers. Moreover, considering the well-known negative feedback effects of T on ARC Kiss1 expression in males (49), the fact that such aggravation of the state of hypogonadism in HFD-fed males from obese fathers was not accompanied by a compensatory increase in ARC Kiss1 levels further attests the impairment of the central Kiss1 system caused by paternal obesity. Anyhow, the significant drop of ARC Kiss1 expression caused by HFD in males from lean fathers could not be explained either as a reaction to changes in circulating T levels, therefore supporting the notion that obesity has a primary impact at central levels to suppress the male reproductive axis, in line with previous references (5, 50). Additional studies in equivalent experimental groups after gonadectomy, with or without sex steroid replacement, as a means to control for the actual influence of changes in circulating gonadal hormones, may help to further clarify this issue. As it was the case for metabolic indices, the reproductive profiles were markedly different in the female offspring. Not only HFD failed to cause major alterations of the HPG axis in females, but also paternal obesity did not aggravate the major gonadotropic indices, apart from a nonsignificant decline in circulating estradiol and a decrease in LH responses to Kp-10. In fact, Kiss1 expression in the AVPV and ARC was not significantly different across the experimental groups, and steroidogenic enzyme expression was even enhanced in HFD females from obese fathers. Whether this represents a compensatory response in face of the incipient failure of the HPG axis suggested by defective LH responses to kisspeptin is a tenable possibility that needs further investigation. In any event, this strikingly different phenotype between sexes further documents that the deleterious intergenerational influence of paternal obesity preferentially occurs in the adult male. This does not preclude that paternal obesity may affect the impact of HFD on other reproductive parameters in the female, such as puberty onset, a possibility that merits independent investigation. In sum, our study adds to previous literature supporting an impact of the metabolic/nutritional state of the father on the health status of the progeny. Notably, such an influence displays a clear sex difference, with males being in general more sensitive than females, and involves changes in susceptibility to the detrimental effects of HFD on body weight gain and adiposity (as reflected by leptin), but not glucose homeostasis. In addition, paternal obesity has a discernible impact on the HPG axis of the male progeny, so that it predisposes to development of central hypogonadism and perturbation of hypothalamic Kiss1 system after HFD exposure. Admittedly, our study does not rule out the possibility of additional impacts of HFD and paternal obesity on other reproductive indices. In any event, based on our present preclinical findings, early-onset nutritional interventions, avoiding dietary fat excess preferentially in males born from obese fathers, should be considered to attenuate the risk of appearance of metabolic and reproductive disorders later in life. Abbreviations: ARC arcuate AUC area under the curve AVPV anteroventral periventricular BW body weight CD control diet cDNA complementary DNA FSH follicle-stimulating hormone GnRH gonadotropin-releasing hormone HFD high-fat diet HPG hypothalamic-pituitary-gonadal ISH in situ hybridization ITT insulin tolerance test KITT insulin sensitivity index LH luteinizing hormone mRNA messenger RNA OGTT oral glucose tolerance test PCR polymerase chain reaction RIA radioimmunoassay SSC saline sodium citrate T testosterone. Acknowledgments Financial Support: This work was supported by Grants BFU2011-025021 and BFU2014-57581-P (Ministerio de Economía y Competitividad, Spain, cofunded with European Union funds from FEDER Program), Project PIE14-00005 (Flexi-Met, Instituto de Salud Carlos III, Ministerio de Sanidad, Spain), Projects P08-CVI-03788 and P12-FQM-01943 (Junta de Andalucía, Spain), and European Union Research Contract DEER FP7-ENV-2007-1. CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of Instituto de Salud Carlos III. Disclosure Summary: The authors have nothing to disclose. References 1. World Health Organization Updates. 2016 obesity and overweight fact sheet. Available at: http://www.who.int/mediacentre/factsheets/fs311/en/. Accessed June 5, 2016. 2. Kyrou I, Randeva HS, Weickert MO. Clinical problems caused by obesity. 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EndocrinologyOxford University Press

Published: Feb 1, 2018

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