Exogenous leptin affects sperm parameters and impairs blood testis barrier integrity in adult male mice

Exogenous leptin affects sperm parameters and impairs blood testis barrier integrity in adult... Background: Serum leptin levels are augmented in obese infertile men and in men with azoospermia. They also correlate inversely with sperm concentration, motility and normal forms. The mechanisms underlying the adverse effects of excess leptin on male reproductive function remain unclear. The present study aimed to evaluate the effects of exogenous leptin on sperm parameters in mice and to explore the underlying mechanisms. Methods: We treated normal adult male mice with saline, 0.1, 0.5 or 3 mg/kg leptin daily for 2 weeks. After treatment, serum leptin levels, serum testosterone levels, sperm parameters and testicular cell apoptosis were evaluated. Blood testis barrier integrity and the expression of tight junction-associated proteins in testes were also assessed. We further verified the direct effects of leptin on tight junction-associated proteins in Sertoli cells and the possible leptin signaling pathways involved in this process. Results: After treatment, there were no significant differences in body weights, reproductive organ weights, serum leptin levels and serum testosterone levels between leptin-treated mice and control mice. Administration of 3 mg/kg leptin reduced sperm concentration, motility and progressive motility while increasing the percentage of abnormal sperm and testicular cell apoptosis. Mice treated with 3 mg/kg leptin also had impaired blood testis barrier integrity, which was related to decreased tight junction-associated proteins in testes. Leptin directly reduced tight junction- associated proteins in Sertoli cells, JAK2/STAT, PI3K and ERK pathways were suggested to be involved in this process. Conclusions: Exogenous leptin negatively affects sperm parameters and impairs blood testis barrier integrity in mice. Leptin reduced tight junction-associated proteins in Sertoli cells, indicating that leptin has a direct role in impairing blood testis barrier integrity. Given the function of blood testis barrier in maintaining normal spermatogenesis, leptin- induced blood testis barrier impairment may be one of the mechanisms contributing to male subfertility and infertility. Keywords: Obesity, Leptin, Male infertility, Mice, Sertoli cell, Blood testis barrier Background neuroendocrine function that has been shown in both Leptin is a 16 kDa peptide product of the ob gene and is humans and rodents [3]. Leptin is able to restore fertility secreted by the adipose tissue [1]. It binds to leptin in ob/ob mice which are leptin deficient, obese and infer- receptors (OB-R) to mediate several signaling pathways, tile, indicating that leptin serves as a permissive signal to including Janus kinase 2/signal transducers and activa- the reproductive system [4, 5]. Certainly, there is in- tors of transcription (JAK2/STAT), extracellular signal- creasing evidence that leptin participates in many events regulated kinase (ERK) and phosphoinositide 3-kinase in reproduction [1]. (PI3K) [2]. Leptin has a role in energy homeostasis, glu- Serum leptin levels are higher in most obese people cose and lipid metabolism, and immune and and in rodents that have ingested the high-fat diet for a long-term [6, 7]. Obese men also have higher seminal * Correspondence: hulian02@163.com; lhgyx@hotmail.com leptin levels which are associated with increased serum Family Planning Research Institute/Center of Reproductive Medicine, Tongji leptin levels [8]. Body mass index (BMI) has positive Medical College, Huazhong University of Science and Technology, Wuhan correlations with serum leptin levels; both BMI and 430030, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 2 of 11 serum leptin levels correlate positively with abnormal studies, we supposed that leptin might affect male sperm morphology, and correlate negatively with sperm reproduction by impairing BTB integrity. concentration and motility [9, 10]. This supports the In this study, we administered different doses of leptin concept that serum leptin mediates a link between obes- or same volume of saline as a control to adult male mice ity and male infertility [10]. Moreover, serum leptin for 2 weeks. We examined the effects of exogenous lep- levels are also increased in azoospermic men compared tin on serum leptin levels, serum testosterone levels, with normozoospermic fertile men [11]. This elevation is sperm parameters and testicular cell apoptosis, as well not gonadotropin dependent, indicating that leptin as BTB integrity and TJ-associated proteins. To evaluate has a direct effect on testis function, especially on whether leptin had a direct effect on TJ-associated spermatogenesis [11]. Animal studies have provided proteins, we treated TM4 cells (a mouse Sertoli cell line) evidence that leptin negatively affects male with leptin and further investigated the possible leptin- reproduction. Hyperleptinemia has been found to in- mediated signaling pathways involved in this process. hibit testicular steroidogenesis and halt testicular maturation in rodents [12, 13]. Methods Administration of exogenous leptin decreased sperm Animals and treatments count and increased the percentage of abnormal sperm Seven-week-old male C57BL/6 mice were purchased in nonobese rodents, suggesting that leptin plays a role from Hubei Research Center of Laboratory Animals. in the negative correlations between BMI and sperm Mice were kept under a 12 h light and 12 h darkness quantity and quality [14]. In nonobese rodents, some cycle at 24 °C and allowed to adapt for 1 week before studies have also shown that exogenous leptin can in- the experiments. At the age of 8 weeks, mice received crease the percentage of abnormal sperm and the DNA daily intraperitoneal injections with 0.1, 0.5 or 3 mg/kg fragmentation level while decreasing sperm count and leptin (recombinant mouse leptin, Prospec, Israel) dis- motility, histone to protamine transition during sperm- solved in saline or same volume of saline as a control for atogenesis, and the ability to generate offspring [15–19]. 2 weeks. The weights of mice and food in each cage Leptin may exhibit a direct effect on testicular tissues or were measured every 2 days. spermatozoa leading to abnormal sperm parameters Mice were sacrificed by exsanguination under [14]. It may also induce reactive oxygen species (ROS) anesthesia the day after treatment ended. Reproductive production and hormone profile modulation to affect organs including testes and epididymides were weighed male fertility [15]. However, additional research is immediately and used for further experiments. Blood needed to further clarify the mechanisms of leptin’s samples were collected and stored at room temperature negative effects on male reproductive function. for 1 h to clot, before centrifuging at 3000 rpm for Leptin secreted by visceral adipose tissue has been 15 min to obtain serum for ELISA. reported to increase the permeability of the intestinal All animal experiments were approved by the Tongji epithelial barrier by reducing the expression of tight Medical College Committee on the Use and Care of Ani- junction (TJ)-associated proteins such as zona mals and were conducted according to the Committee’s occludens-1 (ZO-1), zona occludens-3 (ZO-3), claudin 5 guidelines. and occludin [20–22]. In addition to be the primary structure of the intestinal epithelial barrier, TJ is also a Cell culture and treatment vital structure of the blood testis barrier (BTB). The TM4 cells were obtained from ATCC and stored in Fam- BTB is comprised of coexisting TJ, basal ectoplasmic ily Planning Research Institute of Tongji Medical Col- specialization, gap junction and desmosome [23]. TJ in lege. TM4 cells were cultured in DMEM/F12 the BTB has two main functions, restricting the passage supplemented with 2.5% fetal bovine serum and 5% of molecules and dividing the seminiferous epithelium equine serum at 37 °C and 5% CO . into basal and apical compartments [24]. In mice, the To detect the direct effects of leptin on TJ-associated contribution of occludin and claudins to BTB integrity proteins in TM4 cells, cells were seeded at a density of are determined by deletion of occludin gene or genes for 1×10 /ml in 6-well dishes, cultured with low-serum transcription factors that are upstream regulators of medium containing 0, 10 or 100 nM leptin for 48 h and claudins [25]. The BTB creates a specialized microenvir- then harvested for further experiments. Inhibitors of lep- onment that is necessary for germ cells development tin signaling mediators were employed to determine the and movement [24]. Damage to the BTB can cause germ possible leptin-mediated signaling pathways in vitro. cell loss, reduced sperm count, male infertility or subfer- Cells were pretreated with low-serum medium contain- tility [23, 26–28]. As leptin impairs TJ integrity in the in- ing 10 μM AG490, LY294002 or U0126 (the inhibitors testinal epithelium, and because the impact of leptin on of JAK2, PI3K and ERK, respectively) (MCE, USA) dis- BTB integrity has not been addressed in previous solved in dimethyl sulfoxide (DMSO) for 4 h (the final Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 3 of 11 concentration of DMSO was 0.1%). The inhibitors were 30 μl of biotin solution into the testes. After 30 min, removed, and cells were washed with pre-warmed phos- mice were euthanized, and their testes were immediately phate buffered saline (PBS). Low-serum medium removed and frozen. Sections (5 μm) from frozen testes containing 100 nM leptin was then added to cells. After were blocked with 5% albumin from bovine serum in cultivation for 48 h, cells were harvested for further ex- PBS containing 0.1% Triton X-100 for 1 h, and then in- periments. The concentration of AG490, LY294002 and cubated with streptavidin conjugate-Alexa Fluor 568 (1: U0126 was chosen according to earlier studies [29–31]. 3000, Invitrogen, USA) for 30 min at room temperature. Finally, sections were stained with DAPI, mounted in Measurement of serum leptin and testosterone levels glycerin and observed using a fluorescent microscope. Mouse serum leptin and testosterone levels were At least 30 seminiferous tubules from three non- measured using commercial ELISA kits from Boster consecutive testis sections from each mouse were Biological Technology (Wuhan, China) and Cusabio examined. (Wuhan, China), respectively. The measurements were processed according to the manufacturer’s protocols. Western blot Western blot was used to detect the expression of TJ- Assessment of sperm parameters associated proteins both in testes and in TM4 cells, and Cauda epididymides from each mouse were dissected in was performed according to the standard procedure. 1 ml pre-warmed Ham’ s F10 buffer (Sigma-Aldrich, Antibodies against claudin 5 (Invitrogen, USA), occludin USA) and incubated at 37 °C for 15 min to allow sperm- (Proteintech, USA), ZO-1 (Proteintech, USA) and β- atozoa to swim out. Sperm concentration, motility and Actin (Proteintech, USA) were used with the details progressive motility were determined according to the given in Additional file 1: Table S1. Immunopositive 5th WHO laboratory manual guidelines [32]. For the bands were detected using the enhanced chemilumines- detection of sperm with abnormal morphology, sperm cence (ECL) (Beyotime, Beijing, China). β-Actin served suspensions were smeared on glass slides, allowed to as the loading control. The densitometric analysis was dry, and then fixed and stained using a Diff-Quick kit performed using ImageJ software. (Phygene, Fuzhou, China) according to the manufac- turer’s protocol. The slides were viewed under a light Immunofluorescence microscope. At least 200 spermatozoa from each sample Immunofluorescence was used to detect the expression were assessed. Abnormalities in sperm morphology and localization of TJ-associated proteins in sections including head, tail and head-neck connection abnor- (5 μm) from frozen testes, and was conducted according malities were determined according to Ward et al. [33]. to the standard procedure. Antibodies against claudin 5 (Invitrogen, USA), occludin (Proteintech, USA) and ZO- Apoptosis of testicular cells 1 (Proteintech, USA) were used with the details given in TUNEL assay was conducted to detect testicular cell Additional file 1: Table S1. After incubated with Alexa apoptosis. Sections (5 μm) from frozen testes were Fluor 488-conjugated secondary antibodies (1:200, Pro- deproteinized using proteinase K for 25 min at 37 °C. teintech, USA), sections were stained with DAPI, After blocking with 0.1% Triton X-100 for 20 min at mounted in glycerin and observed by a fluorescent room temperature, sections were incubated with TUNEL microscope. working solutions (Roche, Germany) in the dark at 37 °C for 1 h and then stained with DAPI and mounted in gly- RNA isolation and PCR cerin. Sections were observed using a fluorescent micro- Total RNA in TM4 cells and testes was extracted using scope, and TUNEL positive nuclei which indicated TRIzol reagent (Invitrogen, USA), and was then apoptosis were counted in at least 40 seminiferous tu- reversed-transcribed into cDNA using a PrimeScript RT bules from three non-consecutive testis sections from reagent kit (TAKARA, Japan). To detect leptin receptor each mouse. mRNA in TM4 cells, synthesized cell cDNA was sub- jected to PCR using Premix Taq (TAKARA, Japan). PCR Biotin tracer experiment products were run in 1.5% agarose gel electrophoresis The biotin tracer experiment was used to determine (120 V, 30 min) and visualized using an imaging system BTB integrity according to the method of Meng et al. (Bio-Rad, USA). To determine the expression of ste- [34], with a minor modification. EZ-Link Sulfo-NHS- roidogenic genes (Sf-1, Star and Cyp11a1) and androgen LC-Biotin (Thermo Scientific, USA) was freshly diluted receptor in testes, synthesized testicular cDNA was sub- in PBS containing 1 mM CaCl at a final concentration jected to real time quantitative PCR using SYBR ® Pre- of 10 mg/ml. Mice were anesthetized, and their testes mix Ex Taq II (TAKARA, Japan). The primer sequences were exposed. A 30G needle was used to gently inject were listed in Additional file 2: Table S2. Primers for Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 4 of 11 leptin receptor and Sf-1 have been reported by El- (Cyp11a1) were significantly downregulated in mice treated Hefnawy et al. [35] and Woods et al. [36], respectively. with a relatively high dose of leptin (3 mg/kg) compared with control mice (p < 0.05) (Additional file 3:Figure S1 E). Statistical analysis Throughout the experiment, there were no significant All data were analyzed using SPSS (ver.21) software. Differ- differences in body weights, food intake or reproductive ences between groups were determined using Kruskal- organ weights between the leptin-treated groups and the Wallis test, one-way ANOVA followed by Dunnett test, control group (Additional file 3: Figure S1 A–D). chi-square test or Student’s t test. Data were expressed as mean ± SD, and differences were considered significant Leptin administration altered sperm parameters and when p < 0.05. Graphs were made using Graphpad Prism 7. increased testicular cell apoptosis Sperm concentration in the 3 mg/kg leptin-treated Results group decreased by 50.90% compared with the control Leptin administration did not significantly alter serum group (3.26 ± 1.27 vs. 6.41 ± 2.06 × 10 /ml, p < 0.05) (Fig. leptin and testosterone levels in mice 1c). Sperm motility was 68.38 ± 1.87% in the control After 2 weeks of leptin administration, serum leptin and group but was lower at 58.57 ± 6.24% in the 0.5 mg/kg testosterone levels in the leptin-treated groups showed leptin-treated group and 56.60 ± 6.32% in the 3 mg/kg no significant differences compared with the control leptin-treated group (both p < 0.05) (Fig. 1d). Sperm pro- group (Fig. 1a and b). However, the expression of tes- gressive motility in the 0.5 (24.71 ± 7.49%) and 3 mg/kg ticular steroidogenic genes such as steroidogenic factor (20.93 ± 4.43%) leptin-treated groups also decreased 1(Sf-1), steroidogenic acute regulatory protein (Star) significantly compared with the control group (40.84 ± 4. and cytochrome P450 family 11 subfamily A member 1 55%) (both p < 0.05) (Fig. 1e). The 0.5 and 3 mg/kg ab 10000 1.0 0.8 0.6 0.4 0.2 0.0 Control 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) cd 10 80 0 0 Control 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) ef 50 100 40 80 30 60 20 40 10 20 0 0 Control 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) Fig. 1 Serum leptin, serum testosterone and sperm parameters in mice. a serum leptin. b serum testosterone. Data are expressed as mean ± SD, n =5. c sperm concentration. d sperm motility. e sperm progressive motility. f percentage of abnormal sperm (sperm with abnormal morphology). Data are expressed as mean ± SD, n = 8. * versus control, p < 0.05 Serum leptin(pg/ml) Progressive motility(%) Sperm concentration(×10 /ml) Abnormal sperm(%) Serum testosterone(ng/ml) Motility(%) Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 5 of 11 leptin-treated groups both had higher proportions of 3 mg/kg leptin-treated mice (Fig. 3c). In addition, an- spermatozoa with abnormal morphology, which were 1.25- drogen receptor (AR) is reported to be an upstream fold (65.31 ± 6.51%) and 1.38-fold (72.05 ± 5.30%) com- factor affecting BTB integrity. We found that testicu- pared with the control group (51.80 ± 8.01%), respectively lar AR expression in 3 mg/kg leptin-treated mice (both p < 0.05) (Fig. 1f). Administration of 0.1 mg/kg leptin decreased significantly compared with control mice did not alter sperm parameters significantly. (p < 0.05) (Additional file 3:FigureS1F). TUNEL was conducted to detect whether leptin treatment induced testicular cell apoptosis. The number Leptin directly reduced TJ-associated proteins in TM4 cells of TUNEL positive nuclei (indicating apoptotic cells) per To identify whether leptin could directly reduce TJ- seminiferous tubule increased significantly in the associated proteins in Sertoli cells in vitro, we treated 3 mg/kg leptin-treated group compared with the con- TM4 cells, an OB-R expressing mouse Sertoli cell line trol group (p < 0.05), and it seemed that apoptosis (Fig. 4a), with 0 (control), 10 or 100 nM leptin for 48 h. mainly occurred in germ cells in seminiferous tubules. We found that the expression of claudin 5, occludin and The number of apoptotic cells per seminiferous tu- ZO-1 decreased significantly in cells treated with bule in the control, 0.1 and 0.5 mg/kg leptin-treated 100 nM leptin compared with control cells (p < 0.05). groups were similar (Fig. 2a and b). The presence of 10 nM leptin showed no significant in- fluence on the expression of TJ-associated proteins in TM4 cells (Fig. 4b and c). Leptin administration impaired BTB integrity We used a biotin tracer to assess if BTB integrity was af- fected in leptin-treated mice. Biotin passed through the Leptin’s effect on TJ-associated proteins in TM4 cells was BTB and accumulated visibly in the adluminal compart- attenuated by leptin signaling pathway inhibitors ments of most seminiferous tubules in 3 mg/kg leptin- We further investigated the requirement of leptin- treated mice, indicating impaired BTB integrity in these mediated signaling pathways for reducing TJ-associated mice. In contrast, biotin was restricted to the interstitial and proteins in TM4 cells. Various inhibitors of leptin signal- seminiferous tubule-basal compartments in control mice, as ing mediators were used in this study: AG490, LY294002 well as in 0.1 and 0.5 mg/kg leptin-treated mice (Fig. 2c). and U0126 (the inhibitors of JAK2, PI3K and ERK, re- Interestingly, we also observed that seminiferous tubules spectively). The decreased expression of claudin 5, at stage VIII, of which the BTB undergoes restructuring to occludin and ZO-1 in TM4 cells induced by 100 nM allow the transit of preleptotene spermatocytes, more leptin was reversed in various degrees when cells were often have the biotin in the adluminal compartments pretreated with inhibitors. Leptin’s effect on claudin 5 compared with seminiferous tubules at other stages. The was significantly reduced by LY294002, and U0126 was proportion of seminiferous tubules that have the biotin in the most effective inhibitor to abolish leptin’s effect on the adluminal compartments in all observed seminiferous occludin and ZO-1 (both p < 0.05) (Fig. 4d and e). The tubules at stage VIII and other stages were 66.67% and 39. results indicated that JAK2/STAT, PI3K and ERK path- 77%, respectively (χ = 10.323, p < 0.05) (Fig. 2d). ways were involved in leptin-induced decline in TJ- associated proteins in TM4 cells. Leptin administration reduced TJ-associated proteins in testes Discussion We determined whether impaired BTB integrity was re- Leptin is a well-known protein secreted by adipose tissue lated to decreased expression of TJ-associated proteins, that maintains normal reproductive function. This is as TJ restricts the passage of molecules at this barrier. proven by administering leptin to ob/ob mice to restore Western blot results demonstrated that the expression fertility [4, 5]. However, leptin seems to have adverse of testicular claudin 5, occludin and ZO-1 in 3 mg/kg impacts on male fertility when serum leptin levels are leptin-treated mice, which had impaired BTB integrity, higher than normal and in nonobese rodents given ex- decreased significantly compared with control mice (p < ogenous leptin. The present study highlighted the effects 0.05) (Fig. 3a and b). In control mice, immunofluores- of exogenous leptin on sperm parameters and the role of cence showed that claudin 5, occludin and ZO-1 were leptin in damaging BTB integrity, which could be a located at the basal compartments of seminiferous tu- mechanism for leptin-related male subfertility and bules, consistent with their expression locations at infertility. BTB area, and claudin 5 was simultaneously It is evident that leptin treatment restores repro- expressed in germ cells and in vascular endothelium. ductive function in ob/ob mice [4, 5, 37]. However, However, the immunofluorescent stains of these the effects of leptin treatment on normal rodents are proteins at BTB area became thin and irregular in negative [14–19]. In our study, sperm concentration, Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 6 of 11 8 * Control 0.1 0.5 3 Leptin(mg/kg) Fig. 2 Evaluation of testicular cell apoptosis and BTB integrity. a the number of TUNEL positive nuclei per seminiferous tubule. Data are expressed as mean ± SD, n = 3. * versus control, p < 0.05. b TUNEL positive nuclei (green) which indicated apoptosis were mainly localized in germ cells in seminiferous tubules. Cell nuclei were stained with DAPI (blue). c biotin (red) only passed through the BTB and accumulated in the adluminal compartments of seminiferous tubules in 3 mg/kg leptin-treated mice. Cell nuclei were stained with DAPI (blue). d proportion of seminiferous tubules that have the biotin in the adluminal compartments in all observed seminiferous tubules at stage VIII and other stages. n =4. χ = 10.323, p < 0.05 TUNEL nuclei per tubule Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 7 of 11 Claudin 5 Occludin ZO-1 1.5 1.5 1.5 1.0 1.0 1.0 * * 0.5 0.5 0.5 0.0 0.0 0.0 Saline 0.1 0.5 3 Saline 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) Leptin(mg/kg) Fig. 3 Expression and localization of TJ-associated proteins in testes. a western blot analysis of claudin 5, occludin and ZO-1 in testes. b densitometric analysis for immunopositive bands of claudin 5, occludin and ZO-1 in testes. Data are expressed as mean ± SD, n = 6. * versus control, p <0.05. c immunofluorescence showed the expression and localization of claudin 5, occludin and ZO-1 (green) in testes motility and progressive motility decreased whereas to protect spermatogenic cells from toxicants, and the percentage of abnormal sperm and the number of from being recognized and attacked by the immune apoptotic testicular cells increased in 3 mg/kg leptin- system [23]. Exposure to some environmental toxi- treated mice. Using a biotin tracer, we showed that cants can induce injury to the BTB and elicit subse- these mice also had impaired BTB integrity. Haron et quent damage as germ cell loss, reduced sperm al. suggested that decreased sperm count and in- count, male infertility or subfertility [23, 26–28]. Im- creased abnormal spermatozoa in leptin-treated ro- paired BTB might alter the microenvironment for dentswerelikelydueto adirecteffect of leptin on spermatogenesis in 3 mg/kg leptin-treated mice lead- spermatozoa or testicular tissues [14]; Abbasihormozi ing to germ cell apoptosis and compromised sperm et al. proposed that exogenous leptin suppressed male quantity and quality. The biotin tracer assay also fertility by sperm ROS production or hormone showed that biotin was more often observed in the modulation [15]. Here, we showed that altered sperm adluminal compartments of seminiferous tubules at parameters in normal mice exposed to exogenous stage VIII. Although the BTB disassembles and recon- leptin had a relationship with impaired BTB integrity. structs to facilitate the transit of preleptotene sper- The BTB acts as a physical and immunological barrier matocytes into the apical compartments at this stage, Protein abundance relative to control Protein abundance relative to control Protein expression relative to control Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 8 of 11 ab Claudin 5 Occludin ZO-1 1.5 1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0.0 Control 10 100 Control 10 100 Control 10 100 Leptin(nM) Leptin(nM) Leptin(nM) de Control 1.5 100nM Leptin AG490 1.0 LY294002 U0126 * * 0.5 0.0 Claudin 5 Occludin ZO-1 Fig. 4 Leptin directly reduced the expression of TJ-associated proteins in vitro, and inhibitors of leptin signaling mediators abolished leptin’s effect to different degrees. a detection of OB-R in TM4 cells, the bands of 471 bp and 281 bp corresponded to OB-R and β-Actin, respectively. b western blot analysis of claudin 5, occludin and ZO-1 in TM4 cells after treated with 0 (control), 10 or 100 nM leptin for 48 h. c densitometric analysis for immunopositive bands of claudin 5, occludin and ZO-1 in TM4 cells. d western blot analysis of claudin 5, occludin and ZO-1 in TM4 cells, cells were treated with 100 nM leptin or pre-treated with different inhibitors following a 100 nM leptin treatment. e densitometric analysis for immunopositive bands of claudin 5, occludin and ZO-1 in inhibitor assay. Data are expressed as mean ± SD, n = 5. * versus control, versus 100 nM leptin, p < 0.05 it still holds intact function due to its distinctive associated proteins. As expected, in 3 mg/kg leptin- structure under normal condition [23]. The results of treated mice, which had impaired BTB integrity, the biotin tracer assay suggested that leptin might inter- expression of claudin 5, occludin and ZO-1 decreased fere with the reconstruction process of the BTB at significantly and the immunofluorescent stains of these stage VIII,and thus theBTBwasmoreoftenim- proteins became thin and irregular at BTB area. Further paired at this stage. studies are needed to fully understand the mechanisms The BTB is formed largely by TJ between Sertoli cells, underlying leptin’s disruption to the BTB as other junc- which serves as a barrier and boundary in the seminifer- tions in the BTB have not yet been evaluated. Fan et al. ous epithelium. We hypothesized that impaired BTB determined AR expression using testicular proteins in integrity was associated with decreased expression of TJ- diet-induced obese mice and suggested AR as an Protein expression relative to control Protein expression relative to control Protein abundance relative to control Protein expression relative to control Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 9 of 11 upstream factor affecting BTB integrity [38]. The de- The relationship between leptin treatment and testoster- creased testicular AR gene expression in 3 mg/kg leptin- one has been investigated in many studies. In ob/ob mice, treated mice suggested that it could contribute to indu- leptin treatment increases intratesticular testosterone via cing BTB impairment. On the other hand, we could not improved Leydig cell function [37]. However, serum tes- rule out the possibility that leptin directly reduced TJ- tosterone is negatively correlated with serum leptin in associated proteins in Sertoli cells. Leptin carries out its humans and rodents [45–47]. In vitro experiments also biological effects through OB-R which is expressed in rat show that leptin can directly reduce testosterone secretion Sertoli cells and human Sertoli cells [39, 40]. Since leptin and the expression of steroidogenic genes [12, 39, 48]. In can modulate the nutritional support for spermatogenesis normal rats, leptin treatment does not change serum tes- by altering the metabolic behavior of human Sertoli cells tosterone significantly [14, 18], although parenchymal tes- [40], we treated TM4 cells with low (10 nM) and high tosterone decreases by about 49% compared with control concentration (100 nM) of leptin to test the direct effect rats [18]. Our study showed that 3 mg/kg leptin treatment of leptin on TJ-associated proteins. We first confirmed the repressed testicular steroidogenesis genes expression in presence of OB-R, which allows leptin to interact with vivo but did not produce a significant decrease in serum TM4 cells. We then found that 100 nM leptin reduced the testosterone levels. The conflicting findings observed in expression of claudin 5, occludin and ZO-1 in TM4 cells. various studies are most likely due to different experimen- Taken together, our in vitro experiments confirmed that tal objectives and variable study designs such as the differ- leptin alone directly reduced TJ-associated proteins, which ent doses of leptin used and experiment durations. In our could contribute to BTB impairment in vivo. study, altered sperm parameters and impaired BTB integ- Leptin uses JAK2/STAT3 as its principle signaling rity observed in leptin-treated mice were unlikely to be re- pathway [41], and it also activates ERK and PI3K path- lated to the alterations in serum testosterone levels since ways [2]. Inhibition of JAK2, ERK and PI3K reversed the decreases were not statistically different. Instead, it leptin-induced decline in TJ-associated proteins in TM4 seemed to be leptin that exerted critical and direct effects cells to different extents (Fig. 4e). However, AG490, an on male reproductive tissues. inhibitor of JAK2, was not the most effective inhibitor to rescue the decrease of TJ-associated proteins in TM4 Conclusions cells. Activation of JAK2/STAT3, along with the activa- The present study shows that exogenous leptin exhibits tion of PI3K and ERK, is involved in leptin-induced TJ significant adverse effects on sperm parameters, induces dysfunction in intestinal cells [20]. When SUMO-2/3 spe- testicular cell apoptosis, and possibly suppresses testicu- cific protease (SENP3) is knocked down, it compromises lar steroidogenesis. Exogenous leptin impairs BTB integ- the activation of STAT3, resulting in TJ dysfunction in rity in vivo, which is likely to be a result of decreased Sertoli cells [42]. In addition, a high level of leptin has also TJ-associated proteins. We have further verified that been found to upregulate the expression of suppressor of leptin can directly reduce TJ-associated proteins in Ser- cytokine signaling 3 (SOCS3), which can inhibit STAT3 toli cells in vitro, and identified that JAK2/STAT, PI3K phosphorylation [12]. The role of the JAK2/STAT3 path- and ERK pathways may be involved in this process. way in leptin-induced decline in TJ-associated proteins re- Given the pivotal role of BTB integrity in maintaining an quired further investigation. In this study, JAK2/STAT, appropriate microenvironment for normal spermatogen- PI3K and ERK pathways were suggested to be involved in esis, BTB impairment may cause male subfertility and leptin-induced decline in TJ-associated proteins. infertility. We have proposed a mechanism for leptin’s Leptin treatment causes body weight loss and in- adverse effects on male reproductive function, which will creases reproductive organ weights in ob/ob mice [4]. help to have a deeper insight into subfertility and infer- However, leptin treatment hardly alters body weights or tility in the context of obesity and azoospermia. reproductive organ weights in normal rodents [14, 16– 19, 43], also shown in this study, indicating that the ef- Additional files fect on sperm parameters after leptin administration is unlikely due to leptin resistance [14]. Previous studies Additional file 1: Table 1. Antibodies used for western blot and immunofluorescence in this study. (XLSX 8 kb) have reported that leptin treatment has no influence on Additional file 2: Table 2. Primers used for PCR in this study. (XLSX 9 kb) serum leptin levels in normal rodents [14, 18]. Although Additional file 3: Figure S1. Body weights, food intake, reproductive circulating leptin levels at 1 h after administration of organ weights, the expression of testicular steroidogenic genes and the 3 mg/kg leptin show a 170-fold increase in fasted mice expression of testicular AR and OB-R in mice. A, body weights. B, food and a 13-fold increase in fed mice, the half-life of mouse intake. C, testis weights. D, epididymis weights. Data are expressed as mean ± SD, n = 8. E, testicular steroidogenic genes expression. F, testicular leptin is found to be 40.2 min [44]. This could explain AR and OB-R expression. Data are expressed as mean ± SD, n = 5. * versus why serum leptin levels in our leptin-treated mice were control, p < 0.05. (TIF 1202 kb) not significantly different compared with control mice. Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 10 of 11 Abbreviations 9. Einollahi N, Dashti N, Emamgholipour S, Zarebavani M, Sedighi-Gilani MA, AR: Androgen receptor; BMI: Body mass index; BTB: Blood testis barrier; Choobineh H. Evidence for alteration in serum concentrations of leptin in Cyp11a1: Cytochrome P450 family 11 subfamily A member 1; ERK: Extracellular infertile men categorized based on BMI. Clin Lab. 2016;59:2361–6. signal-regulated kinase; JAK2: Janus kinase 2; OB-R: Leptin receptor; 10. Hofny ER, Ali ME, Abdel-Hafez HZ, Eel-D K, Mohamed EE, Abd El-Azeem HG, PI3K: Phosphoinositide 3-kinase; ROS: Reactive oxygen species; Sf- Mostafa T. Semen parameters and hormonal profile in obese fertile and 1: Steroidogenic factor 1; SOCS3: Suppressor of cytokine signaling 3; infertile males. Fertil Steril. 2010;94(2):581–4. Star: Steroidogenic acute regulatory protein; STAT3: Signal transducer 11. Steinman N, Gamzu R, Yogev L, Botchan A, Schreiber L, Yavetz H. Serum and activator of transcription 3; TJ: Tight junction; TUNEL: TdT-mediated dUTP leptin concentrations are higher in azoospermic than in normozoospermic Nick End Labeling; ZO-1: Zona occludens-1; ZO-3: Zona occludens-3 men. Fertil Steril. 2001;75(4):821–2. 12. Yuan M, Huang G, Li J, Zhang J, Li F, Li K, et al. Hyperleptinemia directly affects Acknowledgments testicular maturation at different sexual stages in mice, and suppressor of We would like to thank Liling Wang, Na Fang, and Yuanyuan Li of the Family cytokine signaling 3 is involved in this process. Reprod Biol Endocrinol. 2014;12:15. Planning Research Institute/Center of Reproductive Medicine, Tongji Medical 13. Giovambattista A, Suescun MO, Nessralla CC, França LR, Spinedi E, Calandra College, HUST, for their excellent technical assistance. RS. Modulatory effects of leptin on leydig cell function of normal and hyperleptinemia rats. Neuroendocrinology. 2003;78(5):270–9. Funding 14. Haron MN, D'Souza UJ, Jaafar H, Zakaria R, Singh HJ. Exogenous leptin This study was supported by National Key Research and Development Program of administration decreases sperm count and increases the fraction of China 2017YFC1002001, Integrated Innovative Team for Major Human Diseases abnormal sperm in adult rats. Fertil Steril. 2010;93(1):322–4. Program of Tongji Medical College, HUST; the Fundamental Research Funds for 15. Abbasihormozi S, Shahverdi A, Kouhkan A, Cheraghi J, Akhlaghi AA, the Central Universities,2015MS130. Kheimeh A. Relationship of leptin administration with production of reactive oxygen species, sperm DNA fragmentation, sperm parameters and Availability of data and materials hormone profile in the adult rat. Arch Gynecol Obstet. 2013;287(6):1241–9. The datasets used and/or analysed during the current study are available from 16. Almabhouh FA, Osman K, Ibrahim SF, Gupalo S, Gnanou J, Ibrahim E, Singh the corresponding author on reasonable request. HJ. Melatonin ameliorates the adverse effects of leptin on sperm. Asian J Androl. 2016;19(6):647–54. Authors’ contributions 17. Almabhouh FA, Singh HJ. Adverse effects of leptin on histone- to- All authors participated in study design. WXT and ZXK performed experiments and protamine transition during spermatogenesis are prevented by melatonin in data analysis. WXT wrote the manuscript. HL and LHG revised and commented the Sprague- Dawley rats. Andrologia. 2018;50(1):e12814. manuscript. All authors read and approved the final version of the manuscript. 18. Fernandez CD, Fernandes GS, Favareto AP, Perobelli JE, Sanabria M, Kempinas WD. Decreased implantation number after in utero artificial Ethics approval and consent to participate insemination can reflect an impairment of fertility in adult male rats after All animal experiments were approved by the Tongji Medical College exogenous leptin exposure. Reprod Sci. 2017;24(2):234–41. Committee on the Use and Care of Animals and were conducted according 19. Almabhouh FA, Osman K, Siti Fatimah I, Sergey G, Gnanou J, Singh HJ. Effects to the Committee’s guidelines. of leptin on sperm count and morphology in Sprague-Dawley rats and their reversibility following a 6-week recovery period. Andrologia. 2015;47(7):751–8. Competing interests 20. Kim CY, Kim KH. Curcumin prevents leptin-induced tight junction The authors declare that they have no competing interests. dysfunction in intestinal Caco-2 BBe cells. J Nutr Biochem. 2014;25(1):26–35. 21. Le Dréan G, Haure-Mirande V, Ferrier L, Bonnet C, Hulin P, de Coppet P, Segain JP. Visceral adipose tissue and leptin increase colonic epithelial tight Publisher’sNote junction permeability via a RhoA-ROCK-dependent pathway. FASEB J. Springer Nature remains neutral with regard to jurisdictional claims in 2014;28(3):1059–70. published maps and institutional affiliations. 22. Le Dréan G, Segain JP. 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Alterations in the spermatic function generated by obesity in rats. Acta Histochem. 2011;113(2):214–20. 48. Landry DA, Sormany F, Haché J, Roumaud P, Martin LJ. Steroidogenic genes expressions are repressed by high levels of leptin and the JAK/STAT signaling pathway in MA-10 Leydig cells. Mol Cell Biochem. 2017;433(1–2):79–95. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Reproductive Biology and Endocrinology Springer Journals

Exogenous leptin affects sperm parameters and impairs blood testis barrier integrity in adult male mice

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Abstract

Background: Serum leptin levels are augmented in obese infertile men and in men with azoospermia. They also correlate inversely with sperm concentration, motility and normal forms. The mechanisms underlying the adverse effects of excess leptin on male reproductive function remain unclear. The present study aimed to evaluate the effects of exogenous leptin on sperm parameters in mice and to explore the underlying mechanisms. Methods: We treated normal adult male mice with saline, 0.1, 0.5 or 3 mg/kg leptin daily for 2 weeks. After treatment, serum leptin levels, serum testosterone levels, sperm parameters and testicular cell apoptosis were evaluated. Blood testis barrier integrity and the expression of tight junction-associated proteins in testes were also assessed. We further verified the direct effects of leptin on tight junction-associated proteins in Sertoli cells and the possible leptin signaling pathways involved in this process. Results: After treatment, there were no significant differences in body weights, reproductive organ weights, serum leptin levels and serum testosterone levels between leptin-treated mice and control mice. Administration of 3 mg/kg leptin reduced sperm concentration, motility and progressive motility while increasing the percentage of abnormal sperm and testicular cell apoptosis. Mice treated with 3 mg/kg leptin also had impaired blood testis barrier integrity, which was related to decreased tight junction-associated proteins in testes. Leptin directly reduced tight junction- associated proteins in Sertoli cells, JAK2/STAT, PI3K and ERK pathways were suggested to be involved in this process. Conclusions: Exogenous leptin negatively affects sperm parameters and impairs blood testis barrier integrity in mice. Leptin reduced tight junction-associated proteins in Sertoli cells, indicating that leptin has a direct role in impairing blood testis barrier integrity. Given the function of blood testis barrier in maintaining normal spermatogenesis, leptin- induced blood testis barrier impairment may be one of the mechanisms contributing to male subfertility and infertility. Keywords: Obesity, Leptin, Male infertility, Mice, Sertoli cell, Blood testis barrier Background neuroendocrine function that has been shown in both Leptin is a 16 kDa peptide product of the ob gene and is humans and rodents [3]. Leptin is able to restore fertility secreted by the adipose tissue [1]. It binds to leptin in ob/ob mice which are leptin deficient, obese and infer- receptors (OB-R) to mediate several signaling pathways, tile, indicating that leptin serves as a permissive signal to including Janus kinase 2/signal transducers and activa- the reproductive system [4, 5]. Certainly, there is in- tors of transcription (JAK2/STAT), extracellular signal- creasing evidence that leptin participates in many events regulated kinase (ERK) and phosphoinositide 3-kinase in reproduction [1]. (PI3K) [2]. Leptin has a role in energy homeostasis, glu- Serum leptin levels are higher in most obese people cose and lipid metabolism, and immune and and in rodents that have ingested the high-fat diet for a long-term [6, 7]. Obese men also have higher seminal * Correspondence: hulian02@163.com; lhgyx@hotmail.com leptin levels which are associated with increased serum Family Planning Research Institute/Center of Reproductive Medicine, Tongji leptin levels [8]. Body mass index (BMI) has positive Medical College, Huazhong University of Science and Technology, Wuhan correlations with serum leptin levels; both BMI and 430030, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 2 of 11 serum leptin levels correlate positively with abnormal studies, we supposed that leptin might affect male sperm morphology, and correlate negatively with sperm reproduction by impairing BTB integrity. concentration and motility [9, 10]. This supports the In this study, we administered different doses of leptin concept that serum leptin mediates a link between obes- or same volume of saline as a control to adult male mice ity and male infertility [10]. Moreover, serum leptin for 2 weeks. We examined the effects of exogenous lep- levels are also increased in azoospermic men compared tin on serum leptin levels, serum testosterone levels, with normozoospermic fertile men [11]. This elevation is sperm parameters and testicular cell apoptosis, as well not gonadotropin dependent, indicating that leptin as BTB integrity and TJ-associated proteins. To evaluate has a direct effect on testis function, especially on whether leptin had a direct effect on TJ-associated spermatogenesis [11]. Animal studies have provided proteins, we treated TM4 cells (a mouse Sertoli cell line) evidence that leptin negatively affects male with leptin and further investigated the possible leptin- reproduction. Hyperleptinemia has been found to in- mediated signaling pathways involved in this process. hibit testicular steroidogenesis and halt testicular maturation in rodents [12, 13]. Methods Administration of exogenous leptin decreased sperm Animals and treatments count and increased the percentage of abnormal sperm Seven-week-old male C57BL/6 mice were purchased in nonobese rodents, suggesting that leptin plays a role from Hubei Research Center of Laboratory Animals. in the negative correlations between BMI and sperm Mice were kept under a 12 h light and 12 h darkness quantity and quality [14]. In nonobese rodents, some cycle at 24 °C and allowed to adapt for 1 week before studies have also shown that exogenous leptin can in- the experiments. At the age of 8 weeks, mice received crease the percentage of abnormal sperm and the DNA daily intraperitoneal injections with 0.1, 0.5 or 3 mg/kg fragmentation level while decreasing sperm count and leptin (recombinant mouse leptin, Prospec, Israel) dis- motility, histone to protamine transition during sperm- solved in saline or same volume of saline as a control for atogenesis, and the ability to generate offspring [15–19]. 2 weeks. The weights of mice and food in each cage Leptin may exhibit a direct effect on testicular tissues or were measured every 2 days. spermatozoa leading to abnormal sperm parameters Mice were sacrificed by exsanguination under [14]. It may also induce reactive oxygen species (ROS) anesthesia the day after treatment ended. Reproductive production and hormone profile modulation to affect organs including testes and epididymides were weighed male fertility [15]. However, additional research is immediately and used for further experiments. Blood needed to further clarify the mechanisms of leptin’s samples were collected and stored at room temperature negative effects on male reproductive function. for 1 h to clot, before centrifuging at 3000 rpm for Leptin secreted by visceral adipose tissue has been 15 min to obtain serum for ELISA. reported to increase the permeability of the intestinal All animal experiments were approved by the Tongji epithelial barrier by reducing the expression of tight Medical College Committee on the Use and Care of Ani- junction (TJ)-associated proteins such as zona mals and were conducted according to the Committee’s occludens-1 (ZO-1), zona occludens-3 (ZO-3), claudin 5 guidelines. and occludin [20–22]. In addition to be the primary structure of the intestinal epithelial barrier, TJ is also a Cell culture and treatment vital structure of the blood testis barrier (BTB). The TM4 cells were obtained from ATCC and stored in Fam- BTB is comprised of coexisting TJ, basal ectoplasmic ily Planning Research Institute of Tongji Medical Col- specialization, gap junction and desmosome [23]. TJ in lege. TM4 cells were cultured in DMEM/F12 the BTB has two main functions, restricting the passage supplemented with 2.5% fetal bovine serum and 5% of molecules and dividing the seminiferous epithelium equine serum at 37 °C and 5% CO . into basal and apical compartments [24]. In mice, the To detect the direct effects of leptin on TJ-associated contribution of occludin and claudins to BTB integrity proteins in TM4 cells, cells were seeded at a density of are determined by deletion of occludin gene or genes for 1×10 /ml in 6-well dishes, cultured with low-serum transcription factors that are upstream regulators of medium containing 0, 10 or 100 nM leptin for 48 h and claudins [25]. The BTB creates a specialized microenvir- then harvested for further experiments. Inhibitors of lep- onment that is necessary for germ cells development tin signaling mediators were employed to determine the and movement [24]. Damage to the BTB can cause germ possible leptin-mediated signaling pathways in vitro. cell loss, reduced sperm count, male infertility or subfer- Cells were pretreated with low-serum medium contain- tility [23, 26–28]. As leptin impairs TJ integrity in the in- ing 10 μM AG490, LY294002 or U0126 (the inhibitors testinal epithelium, and because the impact of leptin on of JAK2, PI3K and ERK, respectively) (MCE, USA) dis- BTB integrity has not been addressed in previous solved in dimethyl sulfoxide (DMSO) for 4 h (the final Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 3 of 11 concentration of DMSO was 0.1%). The inhibitors were 30 μl of biotin solution into the testes. After 30 min, removed, and cells were washed with pre-warmed phos- mice were euthanized, and their testes were immediately phate buffered saline (PBS). Low-serum medium removed and frozen. Sections (5 μm) from frozen testes containing 100 nM leptin was then added to cells. After were blocked with 5% albumin from bovine serum in cultivation for 48 h, cells were harvested for further ex- PBS containing 0.1% Triton X-100 for 1 h, and then in- periments. The concentration of AG490, LY294002 and cubated with streptavidin conjugate-Alexa Fluor 568 (1: U0126 was chosen according to earlier studies [29–31]. 3000, Invitrogen, USA) for 30 min at room temperature. Finally, sections were stained with DAPI, mounted in Measurement of serum leptin and testosterone levels glycerin and observed using a fluorescent microscope. Mouse serum leptin and testosterone levels were At least 30 seminiferous tubules from three non- measured using commercial ELISA kits from Boster consecutive testis sections from each mouse were Biological Technology (Wuhan, China) and Cusabio examined. (Wuhan, China), respectively. The measurements were processed according to the manufacturer’s protocols. Western blot Western blot was used to detect the expression of TJ- Assessment of sperm parameters associated proteins both in testes and in TM4 cells, and Cauda epididymides from each mouse were dissected in was performed according to the standard procedure. 1 ml pre-warmed Ham’ s F10 buffer (Sigma-Aldrich, Antibodies against claudin 5 (Invitrogen, USA), occludin USA) and incubated at 37 °C for 15 min to allow sperm- (Proteintech, USA), ZO-1 (Proteintech, USA) and β- atozoa to swim out. Sperm concentration, motility and Actin (Proteintech, USA) were used with the details progressive motility were determined according to the given in Additional file 1: Table S1. Immunopositive 5th WHO laboratory manual guidelines [32]. For the bands were detected using the enhanced chemilumines- detection of sperm with abnormal morphology, sperm cence (ECL) (Beyotime, Beijing, China). β-Actin served suspensions were smeared on glass slides, allowed to as the loading control. The densitometric analysis was dry, and then fixed and stained using a Diff-Quick kit performed using ImageJ software. (Phygene, Fuzhou, China) according to the manufac- turer’s protocol. The slides were viewed under a light Immunofluorescence microscope. At least 200 spermatozoa from each sample Immunofluorescence was used to detect the expression were assessed. Abnormalities in sperm morphology and localization of TJ-associated proteins in sections including head, tail and head-neck connection abnor- (5 μm) from frozen testes, and was conducted according malities were determined according to Ward et al. [33]. to the standard procedure. Antibodies against claudin 5 (Invitrogen, USA), occludin (Proteintech, USA) and ZO- Apoptosis of testicular cells 1 (Proteintech, USA) were used with the details given in TUNEL assay was conducted to detect testicular cell Additional file 1: Table S1. After incubated with Alexa apoptosis. Sections (5 μm) from frozen testes were Fluor 488-conjugated secondary antibodies (1:200, Pro- deproteinized using proteinase K for 25 min at 37 °C. teintech, USA), sections were stained with DAPI, After blocking with 0.1% Triton X-100 for 20 min at mounted in glycerin and observed by a fluorescent room temperature, sections were incubated with TUNEL microscope. working solutions (Roche, Germany) in the dark at 37 °C for 1 h and then stained with DAPI and mounted in gly- RNA isolation and PCR cerin. Sections were observed using a fluorescent micro- Total RNA in TM4 cells and testes was extracted using scope, and TUNEL positive nuclei which indicated TRIzol reagent (Invitrogen, USA), and was then apoptosis were counted in at least 40 seminiferous tu- reversed-transcribed into cDNA using a PrimeScript RT bules from three non-consecutive testis sections from reagent kit (TAKARA, Japan). To detect leptin receptor each mouse. mRNA in TM4 cells, synthesized cell cDNA was sub- jected to PCR using Premix Taq (TAKARA, Japan). PCR Biotin tracer experiment products were run in 1.5% agarose gel electrophoresis The biotin tracer experiment was used to determine (120 V, 30 min) and visualized using an imaging system BTB integrity according to the method of Meng et al. (Bio-Rad, USA). To determine the expression of ste- [34], with a minor modification. EZ-Link Sulfo-NHS- roidogenic genes (Sf-1, Star and Cyp11a1) and androgen LC-Biotin (Thermo Scientific, USA) was freshly diluted receptor in testes, synthesized testicular cDNA was sub- in PBS containing 1 mM CaCl at a final concentration jected to real time quantitative PCR using SYBR ® Pre- of 10 mg/ml. Mice were anesthetized, and their testes mix Ex Taq II (TAKARA, Japan). The primer sequences were exposed. A 30G needle was used to gently inject were listed in Additional file 2: Table S2. Primers for Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 4 of 11 leptin receptor and Sf-1 have been reported by El- (Cyp11a1) were significantly downregulated in mice treated Hefnawy et al. [35] and Woods et al. [36], respectively. with a relatively high dose of leptin (3 mg/kg) compared with control mice (p < 0.05) (Additional file 3:Figure S1 E). Statistical analysis Throughout the experiment, there were no significant All data were analyzed using SPSS (ver.21) software. Differ- differences in body weights, food intake or reproductive ences between groups were determined using Kruskal- organ weights between the leptin-treated groups and the Wallis test, one-way ANOVA followed by Dunnett test, control group (Additional file 3: Figure S1 A–D). chi-square test or Student’s t test. Data were expressed as mean ± SD, and differences were considered significant Leptin administration altered sperm parameters and when p < 0.05. Graphs were made using Graphpad Prism 7. increased testicular cell apoptosis Sperm concentration in the 3 mg/kg leptin-treated Results group decreased by 50.90% compared with the control Leptin administration did not significantly alter serum group (3.26 ± 1.27 vs. 6.41 ± 2.06 × 10 /ml, p < 0.05) (Fig. leptin and testosterone levels in mice 1c). Sperm motility was 68.38 ± 1.87% in the control After 2 weeks of leptin administration, serum leptin and group but was lower at 58.57 ± 6.24% in the 0.5 mg/kg testosterone levels in the leptin-treated groups showed leptin-treated group and 56.60 ± 6.32% in the 3 mg/kg no significant differences compared with the control leptin-treated group (both p < 0.05) (Fig. 1d). Sperm pro- group (Fig. 1a and b). However, the expression of tes- gressive motility in the 0.5 (24.71 ± 7.49%) and 3 mg/kg ticular steroidogenic genes such as steroidogenic factor (20.93 ± 4.43%) leptin-treated groups also decreased 1(Sf-1), steroidogenic acute regulatory protein (Star) significantly compared with the control group (40.84 ± 4. and cytochrome P450 family 11 subfamily A member 1 55%) (both p < 0.05) (Fig. 1e). The 0.5 and 3 mg/kg ab 10000 1.0 0.8 0.6 0.4 0.2 0.0 Control 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) cd 10 80 0 0 Control 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) ef 50 100 40 80 30 60 20 40 10 20 0 0 Control 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) Fig. 1 Serum leptin, serum testosterone and sperm parameters in mice. a serum leptin. b serum testosterone. Data are expressed as mean ± SD, n =5. c sperm concentration. d sperm motility. e sperm progressive motility. f percentage of abnormal sperm (sperm with abnormal morphology). Data are expressed as mean ± SD, n = 8. * versus control, p < 0.05 Serum leptin(pg/ml) Progressive motility(%) Sperm concentration(×10 /ml) Abnormal sperm(%) Serum testosterone(ng/ml) Motility(%) Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 5 of 11 leptin-treated groups both had higher proportions of 3 mg/kg leptin-treated mice (Fig. 3c). In addition, an- spermatozoa with abnormal morphology, which were 1.25- drogen receptor (AR) is reported to be an upstream fold (65.31 ± 6.51%) and 1.38-fold (72.05 ± 5.30%) com- factor affecting BTB integrity. We found that testicu- pared with the control group (51.80 ± 8.01%), respectively lar AR expression in 3 mg/kg leptin-treated mice (both p < 0.05) (Fig. 1f). Administration of 0.1 mg/kg leptin decreased significantly compared with control mice did not alter sperm parameters significantly. (p < 0.05) (Additional file 3:FigureS1F). TUNEL was conducted to detect whether leptin treatment induced testicular cell apoptosis. The number Leptin directly reduced TJ-associated proteins in TM4 cells of TUNEL positive nuclei (indicating apoptotic cells) per To identify whether leptin could directly reduce TJ- seminiferous tubule increased significantly in the associated proteins in Sertoli cells in vitro, we treated 3 mg/kg leptin-treated group compared with the con- TM4 cells, an OB-R expressing mouse Sertoli cell line trol group (p < 0.05), and it seemed that apoptosis (Fig. 4a), with 0 (control), 10 or 100 nM leptin for 48 h. mainly occurred in germ cells in seminiferous tubules. We found that the expression of claudin 5, occludin and The number of apoptotic cells per seminiferous tu- ZO-1 decreased significantly in cells treated with bule in the control, 0.1 and 0.5 mg/kg leptin-treated 100 nM leptin compared with control cells (p < 0.05). groups were similar (Fig. 2a and b). The presence of 10 nM leptin showed no significant in- fluence on the expression of TJ-associated proteins in TM4 cells (Fig. 4b and c). Leptin administration impaired BTB integrity We used a biotin tracer to assess if BTB integrity was af- fected in leptin-treated mice. Biotin passed through the Leptin’s effect on TJ-associated proteins in TM4 cells was BTB and accumulated visibly in the adluminal compart- attenuated by leptin signaling pathway inhibitors ments of most seminiferous tubules in 3 mg/kg leptin- We further investigated the requirement of leptin- treated mice, indicating impaired BTB integrity in these mediated signaling pathways for reducing TJ-associated mice. In contrast, biotin was restricted to the interstitial and proteins in TM4 cells. Various inhibitors of leptin signal- seminiferous tubule-basal compartments in control mice, as ing mediators were used in this study: AG490, LY294002 well as in 0.1 and 0.5 mg/kg leptin-treated mice (Fig. 2c). and U0126 (the inhibitors of JAK2, PI3K and ERK, re- Interestingly, we also observed that seminiferous tubules spectively). The decreased expression of claudin 5, at stage VIII, of which the BTB undergoes restructuring to occludin and ZO-1 in TM4 cells induced by 100 nM allow the transit of preleptotene spermatocytes, more leptin was reversed in various degrees when cells were often have the biotin in the adluminal compartments pretreated with inhibitors. Leptin’s effect on claudin 5 compared with seminiferous tubules at other stages. The was significantly reduced by LY294002, and U0126 was proportion of seminiferous tubules that have the biotin in the most effective inhibitor to abolish leptin’s effect on the adluminal compartments in all observed seminiferous occludin and ZO-1 (both p < 0.05) (Fig. 4d and e). The tubules at stage VIII and other stages were 66.67% and 39. results indicated that JAK2/STAT, PI3K and ERK path- 77%, respectively (χ = 10.323, p < 0.05) (Fig. 2d). ways were involved in leptin-induced decline in TJ- associated proteins in TM4 cells. Leptin administration reduced TJ-associated proteins in testes Discussion We determined whether impaired BTB integrity was re- Leptin is a well-known protein secreted by adipose tissue lated to decreased expression of TJ-associated proteins, that maintains normal reproductive function. This is as TJ restricts the passage of molecules at this barrier. proven by administering leptin to ob/ob mice to restore Western blot results demonstrated that the expression fertility [4, 5]. However, leptin seems to have adverse of testicular claudin 5, occludin and ZO-1 in 3 mg/kg impacts on male fertility when serum leptin levels are leptin-treated mice, which had impaired BTB integrity, higher than normal and in nonobese rodents given ex- decreased significantly compared with control mice (p < ogenous leptin. The present study highlighted the effects 0.05) (Fig. 3a and b). In control mice, immunofluores- of exogenous leptin on sperm parameters and the role of cence showed that claudin 5, occludin and ZO-1 were leptin in damaging BTB integrity, which could be a located at the basal compartments of seminiferous tu- mechanism for leptin-related male subfertility and bules, consistent with their expression locations at infertility. BTB area, and claudin 5 was simultaneously It is evident that leptin treatment restores repro- expressed in germ cells and in vascular endothelium. ductive function in ob/ob mice [4, 5, 37]. However, However, the immunofluorescent stains of these the effects of leptin treatment on normal rodents are proteins at BTB area became thin and irregular in negative [14–19]. In our study, sperm concentration, Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 6 of 11 8 * Control 0.1 0.5 3 Leptin(mg/kg) Fig. 2 Evaluation of testicular cell apoptosis and BTB integrity. a the number of TUNEL positive nuclei per seminiferous tubule. Data are expressed as mean ± SD, n = 3. * versus control, p < 0.05. b TUNEL positive nuclei (green) which indicated apoptosis were mainly localized in germ cells in seminiferous tubules. Cell nuclei were stained with DAPI (blue). c biotin (red) only passed through the BTB and accumulated in the adluminal compartments of seminiferous tubules in 3 mg/kg leptin-treated mice. Cell nuclei were stained with DAPI (blue). d proportion of seminiferous tubules that have the biotin in the adluminal compartments in all observed seminiferous tubules at stage VIII and other stages. n =4. χ = 10.323, p < 0.05 TUNEL nuclei per tubule Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 7 of 11 Claudin 5 Occludin ZO-1 1.5 1.5 1.5 1.0 1.0 1.0 * * 0.5 0.5 0.5 0.0 0.0 0.0 Saline 0.1 0.5 3 Saline 0.1 0.5 3 Control 0.1 0.5 3 Leptin(mg/kg) Leptin(mg/kg) Leptin(mg/kg) Fig. 3 Expression and localization of TJ-associated proteins in testes. a western blot analysis of claudin 5, occludin and ZO-1 in testes. b densitometric analysis for immunopositive bands of claudin 5, occludin and ZO-1 in testes. Data are expressed as mean ± SD, n = 6. * versus control, p <0.05. c immunofluorescence showed the expression and localization of claudin 5, occludin and ZO-1 (green) in testes motility and progressive motility decreased whereas to protect spermatogenic cells from toxicants, and the percentage of abnormal sperm and the number of from being recognized and attacked by the immune apoptotic testicular cells increased in 3 mg/kg leptin- system [23]. Exposure to some environmental toxi- treated mice. Using a biotin tracer, we showed that cants can induce injury to the BTB and elicit subse- these mice also had impaired BTB integrity. Haron et quent damage as germ cell loss, reduced sperm al. suggested that decreased sperm count and in- count, male infertility or subfertility [23, 26–28]. Im- creased abnormal spermatozoa in leptin-treated ro- paired BTB might alter the microenvironment for dentswerelikelydueto adirecteffect of leptin on spermatogenesis in 3 mg/kg leptin-treated mice lead- spermatozoa or testicular tissues [14]; Abbasihormozi ing to germ cell apoptosis and compromised sperm et al. proposed that exogenous leptin suppressed male quantity and quality. The biotin tracer assay also fertility by sperm ROS production or hormone showed that biotin was more often observed in the modulation [15]. Here, we showed that altered sperm adluminal compartments of seminiferous tubules at parameters in normal mice exposed to exogenous stage VIII. Although the BTB disassembles and recon- leptin had a relationship with impaired BTB integrity. structs to facilitate the transit of preleptotene sper- The BTB acts as a physical and immunological barrier matocytes into the apical compartments at this stage, Protein abundance relative to control Protein abundance relative to control Protein expression relative to control Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 8 of 11 ab Claudin 5 Occludin ZO-1 1.5 1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0.0 Control 10 100 Control 10 100 Control 10 100 Leptin(nM) Leptin(nM) Leptin(nM) de Control 1.5 100nM Leptin AG490 1.0 LY294002 U0126 * * 0.5 0.0 Claudin 5 Occludin ZO-1 Fig. 4 Leptin directly reduced the expression of TJ-associated proteins in vitro, and inhibitors of leptin signaling mediators abolished leptin’s effect to different degrees. a detection of OB-R in TM4 cells, the bands of 471 bp and 281 bp corresponded to OB-R and β-Actin, respectively. b western blot analysis of claudin 5, occludin and ZO-1 in TM4 cells after treated with 0 (control), 10 or 100 nM leptin for 48 h. c densitometric analysis for immunopositive bands of claudin 5, occludin and ZO-1 in TM4 cells. d western blot analysis of claudin 5, occludin and ZO-1 in TM4 cells, cells were treated with 100 nM leptin or pre-treated with different inhibitors following a 100 nM leptin treatment. e densitometric analysis for immunopositive bands of claudin 5, occludin and ZO-1 in inhibitor assay. Data are expressed as mean ± SD, n = 5. * versus control, versus 100 nM leptin, p < 0.05 it still holds intact function due to its distinctive associated proteins. As expected, in 3 mg/kg leptin- structure under normal condition [23]. The results of treated mice, which had impaired BTB integrity, the biotin tracer assay suggested that leptin might inter- expression of claudin 5, occludin and ZO-1 decreased fere with the reconstruction process of the BTB at significantly and the immunofluorescent stains of these stage VIII,and thus theBTBwasmoreoftenim- proteins became thin and irregular at BTB area. Further paired at this stage. studies are needed to fully understand the mechanisms The BTB is formed largely by TJ between Sertoli cells, underlying leptin’s disruption to the BTB as other junc- which serves as a barrier and boundary in the seminifer- tions in the BTB have not yet been evaluated. Fan et al. ous epithelium. We hypothesized that impaired BTB determined AR expression using testicular proteins in integrity was associated with decreased expression of TJ- diet-induced obese mice and suggested AR as an Protein expression relative to control Protein expression relative to control Protein abundance relative to control Protein expression relative to control Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 9 of 11 upstream factor affecting BTB integrity [38]. The de- The relationship between leptin treatment and testoster- creased testicular AR gene expression in 3 mg/kg leptin- one has been investigated in many studies. In ob/ob mice, treated mice suggested that it could contribute to indu- leptin treatment increases intratesticular testosterone via cing BTB impairment. On the other hand, we could not improved Leydig cell function [37]. However, serum tes- rule out the possibility that leptin directly reduced TJ- tosterone is negatively correlated with serum leptin in associated proteins in Sertoli cells. Leptin carries out its humans and rodents [45–47]. In vitro experiments also biological effects through OB-R which is expressed in rat show that leptin can directly reduce testosterone secretion Sertoli cells and human Sertoli cells [39, 40]. Since leptin and the expression of steroidogenic genes [12, 39, 48]. In can modulate the nutritional support for spermatogenesis normal rats, leptin treatment does not change serum tes- by altering the metabolic behavior of human Sertoli cells tosterone significantly [14, 18], although parenchymal tes- [40], we treated TM4 cells with low (10 nM) and high tosterone decreases by about 49% compared with control concentration (100 nM) of leptin to test the direct effect rats [18]. Our study showed that 3 mg/kg leptin treatment of leptin on TJ-associated proteins. We first confirmed the repressed testicular steroidogenesis genes expression in presence of OB-R, which allows leptin to interact with vivo but did not produce a significant decrease in serum TM4 cells. We then found that 100 nM leptin reduced the testosterone levels. The conflicting findings observed in expression of claudin 5, occludin and ZO-1 in TM4 cells. various studies are most likely due to different experimen- Taken together, our in vitro experiments confirmed that tal objectives and variable study designs such as the differ- leptin alone directly reduced TJ-associated proteins, which ent doses of leptin used and experiment durations. In our could contribute to BTB impairment in vivo. study, altered sperm parameters and impaired BTB integ- Leptin uses JAK2/STAT3 as its principle signaling rity observed in leptin-treated mice were unlikely to be re- pathway [41], and it also activates ERK and PI3K path- lated to the alterations in serum testosterone levels since ways [2]. Inhibition of JAK2, ERK and PI3K reversed the decreases were not statistically different. Instead, it leptin-induced decline in TJ-associated proteins in TM4 seemed to be leptin that exerted critical and direct effects cells to different extents (Fig. 4e). However, AG490, an on male reproductive tissues. inhibitor of JAK2, was not the most effective inhibitor to rescue the decrease of TJ-associated proteins in TM4 Conclusions cells. Activation of JAK2/STAT3, along with the activa- The present study shows that exogenous leptin exhibits tion of PI3K and ERK, is involved in leptin-induced TJ significant adverse effects on sperm parameters, induces dysfunction in intestinal cells [20]. When SUMO-2/3 spe- testicular cell apoptosis, and possibly suppresses testicu- cific protease (SENP3) is knocked down, it compromises lar steroidogenesis. Exogenous leptin impairs BTB integ- the activation of STAT3, resulting in TJ dysfunction in rity in vivo, which is likely to be a result of decreased Sertoli cells [42]. In addition, a high level of leptin has also TJ-associated proteins. We have further verified that been found to upregulate the expression of suppressor of leptin can directly reduce TJ-associated proteins in Ser- cytokine signaling 3 (SOCS3), which can inhibit STAT3 toli cells in vitro, and identified that JAK2/STAT, PI3K phosphorylation [12]. The role of the JAK2/STAT3 path- and ERK pathways may be involved in this process. way in leptin-induced decline in TJ-associated proteins re- Given the pivotal role of BTB integrity in maintaining an quired further investigation. In this study, JAK2/STAT, appropriate microenvironment for normal spermatogen- PI3K and ERK pathways were suggested to be involved in esis, BTB impairment may cause male subfertility and leptin-induced decline in TJ-associated proteins. infertility. We have proposed a mechanism for leptin’s Leptin treatment causes body weight loss and in- adverse effects on male reproductive function, which will creases reproductive organ weights in ob/ob mice [4]. help to have a deeper insight into subfertility and infer- However, leptin treatment hardly alters body weights or tility in the context of obesity and azoospermia. reproductive organ weights in normal rodents [14, 16– 19, 43], also shown in this study, indicating that the ef- Additional files fect on sperm parameters after leptin administration is unlikely due to leptin resistance [14]. Previous studies Additional file 1: Table 1. Antibodies used for western blot and immunofluorescence in this study. (XLSX 8 kb) have reported that leptin treatment has no influence on Additional file 2: Table 2. Primers used for PCR in this study. (XLSX 9 kb) serum leptin levels in normal rodents [14, 18]. Although Additional file 3: Figure S1. Body weights, food intake, reproductive circulating leptin levels at 1 h after administration of organ weights, the expression of testicular steroidogenic genes and the 3 mg/kg leptin show a 170-fold increase in fasted mice expression of testicular AR and OB-R in mice. A, body weights. B, food and a 13-fold increase in fed mice, the half-life of mouse intake. C, testis weights. D, epididymis weights. Data are expressed as mean ± SD, n = 8. E, testicular steroidogenic genes expression. F, testicular leptin is found to be 40.2 min [44]. This could explain AR and OB-R expression. Data are expressed as mean ± SD, n = 5. * versus why serum leptin levels in our leptin-treated mice were control, p < 0.05. (TIF 1202 kb) not significantly different compared with control mice. Wang et al. Reproductive Biology and Endocrinology (2018) 16:55 Page 10 of 11 Abbreviations 9. Einollahi N, Dashti N, Emamgholipour S, Zarebavani M, Sedighi-Gilani MA, AR: Androgen receptor; BMI: Body mass index; BTB: Blood testis barrier; Choobineh H. Evidence for alteration in serum concentrations of leptin in Cyp11a1: Cytochrome P450 family 11 subfamily A member 1; ERK: Extracellular infertile men categorized based on BMI. Clin Lab. 2016;59:2361–6. signal-regulated kinase; JAK2: Janus kinase 2; OB-R: Leptin receptor; 10. Hofny ER, Ali ME, Abdel-Hafez HZ, Eel-D K, Mohamed EE, Abd El-Azeem HG, PI3K: Phosphoinositide 3-kinase; ROS: Reactive oxygen species; Sf- Mostafa T. Semen parameters and hormonal profile in obese fertile and 1: Steroidogenic factor 1; SOCS3: Suppressor of cytokine signaling 3; infertile males. Fertil Steril. 2010;94(2):581–4. Star: Steroidogenic acute regulatory protein; STAT3: Signal transducer 11. Steinman N, Gamzu R, Yogev L, Botchan A, Schreiber L, Yavetz H. Serum and activator of transcription 3; TJ: Tight junction; TUNEL: TdT-mediated dUTP leptin concentrations are higher in azoospermic than in normozoospermic Nick End Labeling; ZO-1: Zona occludens-1; ZO-3: Zona occludens-3 men. Fertil Steril. 2001;75(4):821–2. 12. Yuan M, Huang G, Li J, Zhang J, Li F, Li K, et al. Hyperleptinemia directly affects Acknowledgments testicular maturation at different sexual stages in mice, and suppressor of We would like to thank Liling Wang, Na Fang, and Yuanyuan Li of the Family cytokine signaling 3 is involved in this process. Reprod Biol Endocrinol. 2014;12:15. Planning Research Institute/Center of Reproductive Medicine, Tongji Medical 13. Giovambattista A, Suescun MO, Nessralla CC, França LR, Spinedi E, Calandra College, HUST, for their excellent technical assistance. RS. Modulatory effects of leptin on leydig cell function of normal and hyperleptinemia rats. Neuroendocrinology. 2003;78(5):270–9. Funding 14. Haron MN, D'Souza UJ, Jaafar H, Zakaria R, Singh HJ. Exogenous leptin This study was supported by National Key Research and Development Program of administration decreases sperm count and increases the fraction of China 2017YFC1002001, Integrated Innovative Team for Major Human Diseases abnormal sperm in adult rats. Fertil Steril. 2010;93(1):322–4. Program of Tongji Medical College, HUST; the Fundamental Research Funds for 15. Abbasihormozi S, Shahverdi A, Kouhkan A, Cheraghi J, Akhlaghi AA, the Central Universities,2015MS130. Kheimeh A. Relationship of leptin administration with production of reactive oxygen species, sperm DNA fragmentation, sperm parameters and Availability of data and materials hormone profile in the adult rat. Arch Gynecol Obstet. 2013;287(6):1241–9. The datasets used and/or analysed during the current study are available from 16. Almabhouh FA, Osman K, Ibrahim SF, Gupalo S, Gnanou J, Ibrahim E, Singh the corresponding author on reasonable request. HJ. Melatonin ameliorates the adverse effects of leptin on sperm. Asian J Androl. 2016;19(6):647–54. Authors’ contributions 17. Almabhouh FA, Singh HJ. Adverse effects of leptin on histone- to- All authors participated in study design. WXT and ZXK performed experiments and protamine transition during spermatogenesis are prevented by melatonin in data analysis. WXT wrote the manuscript. HL and LHG revised and commented the Sprague- Dawley rats. Andrologia. 2018;50(1):e12814. manuscript. All authors read and approved the final version of the manuscript. 18. Fernandez CD, Fernandes GS, Favareto AP, Perobelli JE, Sanabria M, Kempinas WD. Decreased implantation number after in utero artificial Ethics approval and consent to participate insemination can reflect an impairment of fertility in adult male rats after All animal experiments were approved by the Tongji Medical College exogenous leptin exposure. Reprod Sci. 2017;24(2):234–41. 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Reproductive Biology and EndocrinologySpringer Journals

Published: May 31, 2018

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