TY - JOUR
AU - Breier, Bernhard, H.
AB - Abstract Appropriate growth, development, and function of the placenta is central to the success of nutrient partitioning between the mother, placenta, and fetus. Hormones such as placental lactogen (PL) and leptin are produced in the bovine placenta and play an important role in nutrient partitioning and regulation of placental and fetal growth. Nuclear transfer pregnancies are associated with a number of fetal and placental abnormalities, including increased placental growth and macrosomia, and hence represent a unique situation to gain insight into fetoplacental growth regulation. We have examined the expression of bovine PL (bPL) and leptin in placentomes of artificially inseminated (AI), in vitro produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation in the cow. Immunolocalization studies showed that spatial and temporal patterns of expression of bPL and leptin were markedly altered in the placentomes of NT pregnancies compared with AI or IVP controls. Concentrations of bPL in allantoic fluid, as determined by radioimmunoassay (RIA), were significantly higher (P ≤ 0.001) in NT pregnancies (17.9 ± 3.2 ng/ml; mean ± SD) compared with AI (2.03 ± 1.5 ng/ml), but not IVP (23.4 ± 12.8 ng/ml) pregnancies on Day 150 of gestation. In contrast, amniotic fluid levels of bPL were significantly decreased in NT pregnancies at Day 150 gestation. Leptin mRNA expression, as determined by real-time reverse transcription-PCR, was increased 2.4- to 3.0-fold in NT placentomes compared with AI controls at all gestational ages examined. We speculate that the observed dysregulation of expression of bPL and leptin in NT placentomes could contribute to aberrations in cell migration and invasion and subsequently to alterations in placental metabolism and transfer of nutrients to the fetus, thus leading to increased placental and fetal macrosomia in NT pregnancies. Introduction Placental size and functional capacity are considered important determinants of nutrient supply to the fetus [1, 2] and manipulations in nutrient partitioning affect uteroplacental and fetal growth [3]. In pregnant sheep, appropriate growth, development, and function of the placenta are central to the partitioning process and essential for optimal fetal growth and neonatal outcome [4]. Carunclectomy [5], uterine artery ligation [6], and chronic heat stress [7] have all resulted in varying degrees of fetal growth restriction. The partitioning of nutrients between maternal and fetoplacental compartments is orchestrated by a number of endocrine hormones [8, 9], including both placental lactogen (PL) and placental leptin, which are also involved in fetal and placental growth [10, 11]. In cattle, bovine PL (bPL) is a product of trophoblastic binucleate cells (BNCs), which are directly involved in the modification of the uterine epithelium [12, 13]. Bovine PL is detectable in the peripheral circulation following attachment of the trophoblast to the endometrium [14, 15]. In the early to midstages of pregnancy, there is preferential secretion of bPL to the fetal circulation, where it may exert growth-promoting effects during a crucial time when the rate of linear growth of the fetus is maximal [16]. Fetal bPL peaks at midgestation (5– 25 ng/ml) followed by a plateau and/or gradual decline until term [14, 15, 17]. In the latter half of pregnancy, the metabolic actions of bPL in the mother and fetus are thought to play an important role in partitioning of nutrients between mother, fetus, and placenta, thus ensuring the optimal supply of nutrients to the fetus and utilization of the nutrients by fetal tissues [16]. The maternal concentrations of bPL follow a different pattern; it is detectable in the maternal circulation by the fourth month of pregnancy, but circulating concentrations remain low (not usually exceeding 1 ng/ml) throughout gestation [14, 18]. In contrast with bPL, the presence of leptin in the bovine placenta has only recently been demonstrated and evidence suggests that it is an important fetal growth factor [19]. In humans, placental and cord blood leptin levels are significantly decreased in association with fetal growth retardation [10, 20] and there appear to be a number of positive correlations among fetal plasma leptin and various indices of fetal growth in sheep [21]. The function of leptin in the placenta is less clear, but the presence of leptin protein and receptor gene expression in the placenta suggests that leptin could be involved in nutrient partitioning during placental and/or fetal development [22, 23]. In humans, abnormal placental leptin release has been shown to be associated with increased trophoblast proliferation and differentiation in choriocarcinoma cell lines [24] and concentrations of leptin in cord blood are positively correlated with placental size [20]. Furthermore, leptin is proposed to play a functional role in invasive processes of the extravillous cytotrophoblastic cells by virtue of its stimulatory effect on matrix metalloproteinase expression [25, 26]. Leptin may also have regulatory roles in the metabolic adaptation required during pregnancy to ensure optimum fetal growth and development [22, 27]. Leptin has been proposed to regulate processes such as insulin secretion, glucose utilization, glycogen synthesis, and fatty acid metabolism [27]. Other suggested physiological effects of leptin on the placenta include regulation of angiogenesis and hematopoiesis [23], local immunomodulation at the fetomaternal interface [27], and maintenance of pregnancy [20]. Coordinated substrate supply between the fetus, placenta, and endometrium is critical to the successful growth, development, and function of the fetoplacental unit and to the production of hormones that are needed to maintain pregnancy and fetal growth. Nuclear transfer (NT) pregnancies are associated with a number of both fetal and placental abnormalities throughout gestation, including increased placental growth and fetal macrosmia [28, 29]. The present study was performed to investigate the relationship between the placental bPL and leptin hormones and abnormalities of fetal and placental growth in NT pregnancies. Materials and Methods Nuclear Transfer, In Vitro Embryo Production, and Artificial Insemination Investigations were conducted in accordance with the regulations of the New Zealand Animal Welfare Act of 1999. The generation of embryos for transfer to recipients or artificial insemination of the heifers was carried out as described previously [30–32]. The methods used to generate the cloned (NT) embryos are essentially as described previously [30]. In brief, an ovarian follicular cell line (EFC), derived from a 4-yr-old Friesian dairy cow and demonstrated to be totipotent following NT [30], was used in this study. Serum-starved donor cells used for NT were injected underneath the zona pellucida and then electrically fused with cytoplasts. Reconstructed embryos were artificially activated 3–6 h after fusion and cultured in vitro for 7 days in a modified synthetic oviduct fluid (SOF) medium, as described previously [33, 34]. In vitro-matured oocytes from the same batches as used for NT were fertilized with frozen-thawed sperm obtained from the Friesian bull that was the sire of the dairy cow from which the EFC cells were isolated. Zygotes were cultured contemporaneously in the same SOF medium as described for NT. Semen from the same sire was also used to artificially inseminate (AI) 21 Friesian heifers. Recipients for either in vitro-produced (IVP) or NT embryos were Friesian or dairy cross-bred heifers. A total of 19 single IVP and 49 single NT embryos were nonsurgically transferred to each heifer. Tissue and Fluid Collection Maternal blood samples were obtained from the tail vein during ultrasound scanning. A sample of pregnant animals from each group was killed at Days 50, 100, and 150 of gestation and the reproductive tracts collected and transported to the laboratory within 1 h. Caruncles were removed and trimmed of their associated membranes. Samples of amniotic and allantoic fluids were collected at all three gestation stages and fetal blood was collected at Day 150. These were stored at −20°C pending analysis. Both fetal cotyledons, maternal caruncles, and tissue from intact placentomes were snap frozen in liquid nitrogen or fixed in formalin in phosphate-buffered saline (PBS) for subsequent analysis. Immunohistochemistry Two formalin-fixed placentomes from each heifer were processed for sectioning, staining, and subsequent histological and immunohistochemical analysis. Antiserum to bPL (gifted by J.C. Byatt, Monsanto Animal Agriculture Group, St Louis, MO) was raised in rabbits against purified native bPL [35]. Leptin antiserum was raised in rabbits against a synthetic fragment (aa 30–45) of bovine leptin [36] and specificity was confirmed in rat pancreatic tissues as previously described [37]. Bound antisera were localized in placentomes by immunoperoxidase staining employing the avidin-biotin complex method for immunostaining of paraffin-embedded sections (Vector Laboratories, CA). Tissues were embedded in paraffin wax, sectioned at 5 μm, and mounted on chrome alum-coated slides. Tissues were deparaffinized in xylene and rehydrated through graded alcohols. Endogenous peroxidase activity was depleted by incubation with 0.3% hydrogen peroxide (H2O2) in methanol for 30 min at room temperature. Tissues were preincubated for 30 min with normal goat serum diluted 1:200 to block nonspecific binding, then were incubated with the appropriate dilution of antiserum, either bPL (1:10 000) or leptin (1:200) for 24 h at 4°C. After incubation with the antibody, tissues were washed in PBS and incubated with biotinylated goat anti-rabbit IgG for 2 h at room temperature, followed by streptavidin-peroxidase conjugate for 20 min, each at room temperature. After a further PBS wash, the tissues were then incubated in diaminobenzidine until a positive reaction was observed (20–90 sec). The same conditions were applied to all samples across Days 50–150 gestation. Normal rabbit serum and antiserum preabsorbed with excess antigen were used as negative controls. Neutralization of immunoreactivity was defined as a marked reduction in staining intensity in the presence of antigen. Slides were photographed at 400× magnification. A double-blind procedure (n = 2) was used to assess immunohistochemical scores based on staining intensity. Radioimmunoassays A radioimmunoassay (RIA) was established and validated for the measurement of bPL concentrations in serum (maternal and fetal) and in allantoic and amniotic fluid. Primary rabbit anti-bovine antiserum (R3B3) was diluted in assay buffer to an initial working dilution of 1:24 000. This buffer was used for measuring PL in amniotic and allantoic fluid samples. Antibody and tracer were also diluted in this buffer. Nonpregnant cow serum was used as a sample diluent. Following antiserum dilution, 200 μl of the diluted sample, control, or standard (recombinant bPL, Monsanto Animal Agriculture Group, MO) was incubated with 200 μl of primary antibody for 24 h at room temperature. Two hundred microliters of bPL (batch PTR166001; Monsanto) was radioiodinated by the chloromine T oxidation method [38] and diluted to 15 000 counts per minute per 200 μl in assay buffer. Equilibrium conditions were established after 24 h incubation at 4°C. A second antibody (in-house anti-rabbit γ globulin sera raised in sheep) was used to separate bound from free ligand. Standard stock (recombinant bPL) was stored at −20°C at 0.1 μg/μl. Cold recoveries and parallelism were performed on all bovine fluids. Bovine serum (r2 = 0.99; slope = 0.89), amniotic (r2 = 0.92, slope = 0.98), and allantoic fluid (r2 = 0.93, slope = 0.96) samples showed parallel displacement to the standard curve. The limits of detection for fetal and maternal serum, amniotic, and allantoic bPL were 1.0, 1.0, 0.3, and 0.3 ng/ml, respectively. The intra- and interassay coefficients of variation were less than 10%. RIAs for leptin could not be established. Statistical Analysis of RIAs Statistical analyses were carried out using Sigma Stat (Jandel Scientific, San Rafael, CA). Differences between groups were determined by one-way ANOVA with post hoc Tukey tests. Data are shown as mean ± SEM. Statistical significance was accepted at the P < 0.05 level. RNA Extraction and Reverse Transcription Frozen placental samples (AI and NT) at Days 50, 100, and 150 were first homogenized with QIAshreder homogenizer (Qiagen) as described by the manufacturer. Total RNA was prepared using the TRIZol extraction method (Invitrogen, NZ). The RNA was further purified with RNeasy mini Kits (Qiagen). Integrity of the RNA was verified by electrophoresis and ethidium bromide staining by optical density (OD) absorption ratio (OD260 nm/OD280 nm > 1.9). To eliminate residual genomic DNA from the RNA sample, before the reverse transcription (RT) reaction, 1 U of DNaseI (Invitrogen) was added to RNA. RT was performed for 1 μg of total RNA in a 20-μl reaction volume. Briefly, RNA, random hexamer primers, and 10 mM dNTP mix were denatured by incubating at 65°C for 10 min. Then, the reaction mix (5× first-strand buffer, 0.1 mM dithiothreitol, 40 U RNase OUT RNase inhibitor) was added and the mixture incubated at 55°C for 5 min, followed by addition of 15 U thermoscript reverse transcriptase and subsequent incubation at 55°C for 90 min. The reaction was stopped by heating to 85°C for 5 min. Finally, RNA complementary to the cDNA was removed by adding 2 U Escherichia coli ribonuclease H and incubating for 20 min at 37°C. All enzymes were purchased from Invitrogen. RT Reverse Transcription-PCR All primers used for the production of cDNA were derived from bovine sequences using Primer Express 1.5 (Applied Biosystems Ltd., Foster City, CA) software according to the software guidelines. DNA sequences were obtained from the GenBank sequence database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Information regarding the primer sequences, accession numbers, primer length, and product size are summarized in Table 1. Table 1 Forward (F) and reverse (R) bovine primer sequences (5′ → 3′), accession number, primer length, and product size base pairs (bp) of the investigated factors, Housekeeping genes; 18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2 acetylase (H2A), hypoxanthine phosphoribosyltransferase 1 (HPRT1), p actin, a tubulin, and leptin. Primer name GenBank accession no. Primer length Sequence (5′ to 3′) Product size (bp) 18SF AF176811 19 5′-AACGTCTGCCCTATCAACT-3′ 72 18SR 19 5′-AACCTCCGACTTTCGTTCT-3′ GAPDHF U85042 20 5′-TATCATCCCTGCTTCTACTG-3′ 91 GAPDHR 20 5′-CTGTTGAAGTCGCAGGAGAC-3′ H2AF NM174809 23 5′-GACTCCGGAAAGGCCAAGAC-3′ 98 H2AR 20 5′-CGTCCTAGATTTCAGGTGTCGAT-3′ HPRT1F NM000194 27 5′-AACAGGCGACATAAAAGTAATTGGT-3′ 102 HPRT1R 22 5′-CTGCATTGTCTTCCCAGTGTCA-3′ P ACTINF AF176419 19 5′-GCCCTGAGGCTCTCTTCCA-3′ 89 P ACTINR 24 5′-CGTCACACTTCATGATGAAATTGA-3′ a TUBULINF AB098999 21 5′-AATGCCTGCTGGGAGCTCTAC-3′ a TUBULINR 24 5′-CTCACTGAAGAAGGTGTTGAAGGA-3′ 113 LEPTINF U50365 22 5′-CACCAAAACCCTCATCAAGACA-3′ LEPTINR 21 5′-TCCAAACCAGTGACCCTCTGT-3′ 120 Primer name GenBank accession no. Primer length Sequence (5′ to 3′) Product size (bp) 18SF AF176811 19 5′-AACGTCTGCCCTATCAACT-3′ 72 18SR 19 5′-AACCTCCGACTTTCGTTCT-3′ GAPDHF U85042 20 5′-TATCATCCCTGCTTCTACTG-3′ 91 GAPDHR 20 5′-CTGTTGAAGTCGCAGGAGAC-3′ H2AF NM174809 23 5′-GACTCCGGAAAGGCCAAGAC-3′ 98 H2AR 20 5′-CGTCCTAGATTTCAGGTGTCGAT-3′ HPRT1F NM000194 27 5′-AACAGGCGACATAAAAGTAATTGGT-3′ 102 HPRT1R 22 5′-CTGCATTGTCTTCCCAGTGTCA-3′ P ACTINF AF176419 19 5′-GCCCTGAGGCTCTCTTCCA-3′ 89 P ACTINR 24 5′-CGTCACACTTCATGATGAAATTGA-3′ a TUBULINF AB098999 21 5′-AATGCCTGCTGGGAGCTCTAC-3′ a TUBULINR 24 5′-CTCACTGAAGAAGGTGTTGAAGGA-3′ 113 LEPTINF U50365 22 5′-CACCAAAACCCTCATCAAGACA-3′ LEPTINR 21 5′-TCCAAACCAGTGACCCTCTGT-3′ 120 Open in new tab Table 1 Forward (F) and reverse (R) bovine primer sequences (5′ → 3′), accession number, primer length, and product size base pairs (bp) of the investigated factors, Housekeeping genes; 18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2 acetylase (H2A), hypoxanthine phosphoribosyltransferase 1 (HPRT1), p actin, a tubulin, and leptin. Primer name GenBank accession no. Primer length Sequence (5′ to 3′) Product size (bp) 18SF AF176811 19 5′-AACGTCTGCCCTATCAACT-3′ 72 18SR 19 5′-AACCTCCGACTTTCGTTCT-3′ GAPDHF U85042 20 5′-TATCATCCCTGCTTCTACTG-3′ 91 GAPDHR 20 5′-CTGTTGAAGTCGCAGGAGAC-3′ H2AF NM174809 23 5′-GACTCCGGAAAGGCCAAGAC-3′ 98 H2AR 20 5′-CGTCCTAGATTTCAGGTGTCGAT-3′ HPRT1F NM000194 27 5′-AACAGGCGACATAAAAGTAATTGGT-3′ 102 HPRT1R 22 5′-CTGCATTGTCTTCCCAGTGTCA-3′ P ACTINF AF176419 19 5′-GCCCTGAGGCTCTCTTCCA-3′ 89 P ACTINR 24 5′-CGTCACACTTCATGATGAAATTGA-3′ a TUBULINF AB098999 21 5′-AATGCCTGCTGGGAGCTCTAC-3′ a TUBULINR 24 5′-CTCACTGAAGAAGGTGTTGAAGGA-3′ 113 LEPTINF U50365 22 5′-CACCAAAACCCTCATCAAGACA-3′ LEPTINR 21 5′-TCCAAACCAGTGACCCTCTGT-3′ 120 Primer name GenBank accession no. Primer length Sequence (5′ to 3′) Product size (bp) 18SF AF176811 19 5′-AACGTCTGCCCTATCAACT-3′ 72 18SR 19 5′-AACCTCCGACTTTCGTTCT-3′ GAPDHF U85042 20 5′-TATCATCCCTGCTTCTACTG-3′ 91 GAPDHR 20 5′-CTGTTGAAGTCGCAGGAGAC-3′ H2AF NM174809 23 5′-GACTCCGGAAAGGCCAAGAC-3′ 98 H2AR 20 5′-CGTCCTAGATTTCAGGTGTCGAT-3′ HPRT1F NM000194 27 5′-AACAGGCGACATAAAAGTAATTGGT-3′ 102 HPRT1R 22 5′-CTGCATTGTCTTCCCAGTGTCA-3′ P ACTINF AF176419 19 5′-GCCCTGAGGCTCTCTTCCA-3′ 89 P ACTINR 24 5′-CGTCACACTTCATGATGAAATTGA-3′ a TUBULINF AB098999 21 5′-AATGCCTGCTGGGAGCTCTAC-3′ a TUBULINR 24 5′-CTCACTGAAGAAGGTGTTGAAGGA-3′ 113 LEPTINF U50365 22 5′-CACCAAAACCCTCATCAAGACA-3′ LEPTINR 21 5′-TCCAAACCAGTGACCCTCTGT-3′ 120 Open in new tab Real-time RT-PCR (RT-PCR) was used for quantification of relative gene expression using an Applied Biosystems (ABI) Prism 7700 Sequence Detection System thermal cycler (Applied Biosystems Ltd.). The SYBR green PCR Master Mix (Applied Biosystems) was used to perform real-time RT-PCR according to the manufacturer’s instructions. A master mix of the following reaction components was prepared to the indicated end concentrations: 1× SYBR green PCR Master Mix (Invitrogen), 300 mM forward primer, 300 mM reverse primer, 0.9 μl water, 0.1 U uracil DNA glycosylase, and 2 μl of cDNA template. Real-time RT-PCR assays were performed in triplicate and contained two negative controls (RT- and water). The following real-time RT-PCR protocol was used: initial activation of AmpliTaq Gold DNA Polymerase at 95°C for 10 min, followed by 40 cycles of denaturation (95°C for 15 sec), and primer annealing and extension (60°C for 1 min). Following the real-time run, a dissociation analysis curve was performed to screen for nonspecific amplification products. Standard procedures were as follows: temperatures were ramped to 95°C to separate double-stranded DNA (95°C for 15 sec), and the strands reannealed at 60°C for 20 sec. The strands were then melted slowly to 95°C for 20 min. Leptin real-time amplified products were sequenced (ABI, model 3100) at the Centre for Genomics and Proteomics, School of Biological Sciences, University of Auckland; 100% homology to bovine sequences could be confirmed. The specificities of the products amplified by SYBR green PCR were monitored by analyzing the amplification profiles and the corresponding dissociation curves of each amplicon. Dissociation curve analysis confirmed the presence of only one product. Bands visible after electrophoresis in 3% agarose gel and ethidium bromide staining were of the expected size for the PCR product, thus confirming specificity of the desired product (Fig. 1). CT values for each triplicate sample were highly reproducible and did not differ by 0.2 cycles of each other. Fig. 1 Open in new tabDownload slide Gel electrophoresis (3% agarose) of the leptin real-time RT-PCR product derived from bovine placental total RNA showing a band of the expected size (120 base pairs). The RT-control lane showed no band, as expected Fig. 1 Open in new tabDownload slide Gel electrophoresis (3% agarose) of the leptin real-time RT-PCR product derived from bovine placental total RNA showing a band of the expected size (120 base pairs). The RT-control lane showed no band, as expected Data Processing and Analysis PCR efficiencies for each gene were calculated using a linear regression model (LinRegPCR, Department of Anatomy & Embryology, Academic Medical Center, Amsterdam, The Netherlands) as previously described [39]. Relative quantitation of leptin gene expression was performed based on modifications of principles derived from the comparative CT method [40]. To remove any nonspecific variation, six housekeeping genes were evaluated to determine the most stably expressed control genes in placental samples at Days 50, 100, and 150 gestation and the minimum number of genes required to calculate a reliable normalization factor. First, a gene-stability measure was used to determine the expression stability of nonnormalized expression levels of all housekeeping genes using GeNorm VBA applet for Microsoft Excel (Center for Medical Genetics, Ghent, Belgium; http://allserv.rug.ac.be/∼jvdesomp/genorm/). From these calculations, four housekeeping genes were chosen. Having determined the most stable housekeeping genes to use, a normalization factor, based on the expression levels of the best performing housekeeping genes, was calculated using the geometric mean (as determined by GeNorm). To assess the validity of the established gene stability, the gene-specific variation for each housekeeping gene was determined as the variation coefficient of the expression levels after normalization for all samples at Days 50, 100, and 150 gestation. The generated normalization factors were used to normalize expression of leptin in AI and NT placentas as discussed by Vandesompele [41]. Standard deviations were calculated based on the error propagation rules for independent variables [41]. Expression levels of leptin in AI and NT placentas were expressed relative to a randomly chosen AI control placenta. Results Immunolocalization of bPL The bPL was localized predominantly to the fetal trophoblastic tissue of the bovine placentome at Days 50, 100, and 150 of gestation in AI, IVP, and NT pregnancies (Fig. 2). Intense staining was restricted to BNCs, often found in close association with the microvillous boundary that separates the fetal trophoblast from the uterine epithelium. Immunostaining with bPL antibodies produced intense brown staining of the BNCs in all tissues examined at Days 50, 100, and 150 (Fig. 2, A–G). BNC staining was significantly more intense in NT placentomes compared with AI and IVP (not shown) at all three gestational stages examined. The intercrypt columns and maternal epithelium were immunonegative for bovine PL. No staining was detected in negative controls (Fig. 2G). Fig. 2 Open in new tabDownload slide Immunolocalization of bPL in the AI placentomes at Days 50 (A), 100 (C), and 150 (E) gestation and in the NT placentome at Days 50 (B), 100 (D), and 150 (F) gestation. A representative negative control is illustrated (G). Arrows indicate binucleate cells (BNC). ME, Maternal epithelium; CV, chorionic villi; IC, intercrypt column. Magnification ×400 Fig. 2 Open in new tabDownload slide Immunolocalization of bPL in the AI placentomes at Days 50 (A), 100 (C), and 150 (E) gestation and in the NT placentome at Days 50 (B), 100 (D), and 150 (F) gestation. A representative negative control is illustrated (G). Arrows indicate binucleate cells (BNC). ME, Maternal epithelium; CV, chorionic villi; IC, intercrypt column. Magnification ×400 Immunolocalization of Leptin Leptin immunostaining was only present in the NT placentomes on Days 50 and 100 (Fig. 3). AI and IVP (not shown) placentomes were immunonegative for leptin at all stages of gestation examined. In the NT placentome, leptin expression was localized to both the fetal trophoectoderm and the maternal tissues only at Days 50 (Fig. 3B) and 100 of gestation (Fig. 3D). Images of AI immunostaining at Days 50 (Fig. 3A) and 100 (Fig. 3C) are included for comparison. Specifically, intense immunoreactive leptin staining was observed in the chorionic villi and maternal epithelium of NT placentomes, while stromal and intercrypt regions were immunonegative for leptin staining. There was no visible staining in either fetal or maternal tissues at Day 150 gestation in AI, IVP, or NT placentomes. Negative controls showed no staining (Fig. 3E). Fig. 3 Open in new tabDownload slide Immunolocalization of bovine leptin in AI placentomes at Days 50 (A) and 100 (C) and in the NT placentome at Days 50 (B) and 100 (D) gestation. A representative negative control is illustrated (E). ME, Maternal epithelium; CV, chorionic villi; IC, intercrypt column; S, stroma. Magnification ×400 Fig. 3 Open in new tabDownload slide Immunolocalization of bovine leptin in AI placentomes at Days 50 (A) and 100 (C) and in the NT placentome at Days 50 (B) and 100 (D) gestation. A representative negative control is illustrated (E). ME, Maternal epithelium; CV, chorionic villi; IC, intercrypt column; S, stroma. Magnification ×400 Placental Lactogen Measurements No differences in fetal serum bPL concentrations were found at Day 150 gestation between AI/IVP or NT pregnancies (Table 2). Maternal serum concentrations of bPL were either at or below the minimum detectable level of 1.0 ng/ml in all pregnancies at the three gestational stages examined. Table 2 Bovine PL concentrations in fetal serum and fluids (amniotic and allantoic) as determined by RIA.* Gestational age AI IVP NT Fetal serum (ng/ml)  150 50.7 ± 9.5 (4) 34.6 ± 8.6 (3) 37.2 ± 6.9 (8) Amniotic fluid (ng/ml)  50 ND ND ND  100 2.7 ± 0.1 (3) 4.2 ± 14 (4) 2.6 ± 0.7 (6)  50 2.8 ± 0.6 (4)a 1.0 ± 0.9 (3)b 1.7 ± 0.1 (7)c Allantoic fluid (ng/ml)  50 66.7 ± 1.6 (3) 16.3 ± 1.9 (3) 47.6 ± 1.1 (10)  100 18.1 ± 6.7 (4) 9.6 ± 3.0 (3) 26.3 ± 11.9 (4)  150 2.0 ± 1.5 (3)a 23.4 ± 12.5 (3)b 17.9 ± 3.2 (6)c Gestational age AI IVP NT Fetal serum (ng/ml)  150 50.7 ± 9.5 (4) 34.6 ± 8.6 (3) 37.2 ± 6.9 (8) Amniotic fluid (ng/ml)  50 ND ND ND  100 2.7 ± 0.1 (3) 4.2 ± 14 (4) 2.6 ± 0.7 (6)  50 2.8 ± 0.6 (4)a 1.0 ± 0.9 (3)b 1.7 ± 0.1 (7)c Allantoic fluid (ng/ml)  50 66.7 ± 1.6 (3) 16.3 ± 1.9 (3) 47.6 ± 1.1 (10)  100 18.1 ± 6.7 (4) 9.6 ± 3.0 (3) 26.3 ± 11.9 (4)  150 2.0 ± 1.5 (3)a 23.4 ± 12.5 (3)b 17.9 ± 3.2 (6)c * Number of samples in parentheses (n). ND = no data available. a,b,c Within rows, ab, not significant; ac, P < 0.001; bc, not significant. Mean ± SEM. Open in new tab Table 2 Bovine PL concentrations in fetal serum and fluids (amniotic and allantoic) as determined by RIA.* Gestational age AI IVP NT Fetal serum (ng/ml)  150 50.7 ± 9.5 (4) 34.6 ± 8.6 (3) 37.2 ± 6.9 (8) Amniotic fluid (ng/ml)  50 ND ND ND  100 2.7 ± 0.1 (3) 4.2 ± 14 (4) 2.6 ± 0.7 (6)  50 2.8 ± 0.6 (4)a 1.0 ± 0.9 (3)b 1.7 ± 0.1 (7)c Allantoic fluid (ng/ml)  50 66.7 ± 1.6 (3) 16.3 ± 1.9 (3) 47.6 ± 1.1 (10)  100 18.1 ± 6.7 (4) 9.6 ± 3.0 (3) 26.3 ± 11.9 (4)  150 2.0 ± 1.5 (3)a 23.4 ± 12.5 (3)b 17.9 ± 3.2 (6)c Gestational age AI IVP NT Fetal serum (ng/ml)  150 50.7 ± 9.5 (4) 34.6 ± 8.6 (3) 37.2 ± 6.9 (8) Amniotic fluid (ng/ml)  50 ND ND ND  100 2.7 ± 0.1 (3) 4.2 ± 14 (4) 2.6 ± 0.7 (6)  50 2.8 ± 0.6 (4)a 1.0 ± 0.9 (3)b 1.7 ± 0.1 (7)c Allantoic fluid (ng/ml)  50 66.7 ± 1.6 (3) 16.3 ± 1.9 (3) 47.6 ± 1.1 (10)  100 18.1 ± 6.7 (4) 9.6 ± 3.0 (3) 26.3 ± 11.9 (4)  150 2.0 ± 1.5 (3)a 23.4 ± 12.5 (3)b 17.9 ± 3.2 (6)c * Number of samples in parentheses (n). ND = no data available. a,b,c Within rows, ab, not significant; ac, P < 0.001; bc, not significant. Mean ± SEM. Open in new tab Amniotic fluid concentrations of bPL were also at the minimum detectable limit at Day 50. There were no significant differences in amniotic bPL concentrations at Day 100 among AI, IVP, or NT pregnancies, although concentrations appeared slightly higher in IVP pregnancies compared with AI and NT groups (Table 2). At Day 150, amniotic bPL concentrations were significantly lower (P < 0.05) in IVP versus AI and NT versus AI pregnancies (P < 0.001) but not IVP versus NT pregnancies. Concentrations of bPL in amniotic fluids were similar from Days 100 NS 150 in AI pregnancies (1.03-fold difference) but decreased in NT and IVP pregnancies (35.7% and 76.4%, respectively). No significant differences in allantoic bPL concentrations were observed at Days 50 or 100 gestation between AI, IVP, and NT pregnancies. However, at Day 150, NT pregnancies had significantly higher concentrations (P < 0.001) of allantoic bPL compared with AI pregnancies (Table 2). Mean concentrations of bPL in allantoic fluid were considerably higher compared with amniotic fluid levels in AI, IVP, and NT pregnancies at all three stages of gestation examined. There was also an apparent effect of stage of gestation on allantoic bPL concentrations in all treatment groups. Allantoic bPL concentrations were high at Day 50 and decreased 72.9%, 41.3%, and 44.7% by Day 100 in AI, IVP, and NT pregnancies, respectively. Concentrations continued to decline, 88.7% and 33.1% from Day 100 to Day 150 in AI and NT pregnancies, respectively, but increased 40.9% in IVP pregnancies. Small sample number and large intersample variability may account for the increased concentrations observed in the IVP group. Leptin Expression Leptin mRNA expression levels are illustrated in Figure 4. Normalized (AI) placenta was used as a control. Leptin expression was high in both AI and NT placentas throughout the gestational ages examined, as indicated by emergence of the amplified product on the cycle threshold (AI ∼ CT 23 and NT ∼ CT 21). Expression levels of leptin (relative units: mean ± SD) were significantly increased in NT versus AI control placentas at Day 50 (3.57 ± 0.50, 1.19 ± 0.32), Day 100 (3.53 ± 0.78, 1.19 ± 0.44) and Day 150 (3.46 ± 1.06, 1.43 ± 0.35) respectively (P < 0.05 for each). This resulted in relative increases in leptin expression of 3.0-, 3.0-, and 2.4-fold in NT versus AI placentomes at Days 50, 100, and 150 gestation, respectively. Fig. 4 Open in new tabDownload slide Leptin mRNA expression relative to (AI) control ± SD in individual AI and NT placentas at Days 50 (A), 100 (B), and 150 (C) gestation. Grey bars represent an individual control AI placenta, white bars represent individual AI placentas, and black bars represent individual NT placentas Fig. 4 Open in new tabDownload slide Leptin mRNA expression relative to (AI) control ± SD in individual AI and NT placentas at Days 50 (A), 100 (B), and 150 (C) gestation. Grey bars represent an individual control AI placenta, white bars represent individual AI placentas, and black bars represent individual NT placentas Discussion Immunohistochemical findings presented in this study indicate that there is a marked increase in the expression of bPL protein in bovine placentomes at Days 50–150 gestation and in leptin protein expression at Days 50 and 100 gestation in NT-derived pregnancies compared with their AI/IVP counterparts. Allantoic and amniotic bPL levels are also altered in NT versus AI/IVP pregnancies at Day 150. Furthermore, leptin mRNA expression was significantly increased in NT versus AI placentomes at all stages of gestation examined. We speculate that abnormal growth, development, and function of NT placentomes results in an altered functional capacity for synthesis and secretion of bPL and leptin, leading to interference with nutrient partitioning and disturbances in fetal growth regulation. Recently, we showed that cotyledonary burr formation and vascularization of NT cotyledons was similar to AI/IVP controls at Day 50 [32]. In addition, there were no significant differences in fetal weights or crown-rump lengths among treatment groups. Although one NT fetus at Day 50 appeared slightly anemic and cotyledon-caruncle attachment was poor, fetal development was similar between AI/IVP versus NT pregnancies. At Day 100, however, caruncle numbers were significantly reduced and caruncle and fetal weights significantly increased. It was suggested that the absence of any differences in cotyledonary burr formation and vascularization among AI, NT, and IVP pregnancies at Day 50 was an indication that a high proportion of NT fetal cotyledons fail to interact with maternal caruncles to form placentomes at Day 100 [32]. Aberrations of the anchoring chorionic villi and decreased collagen fibers in NT pregnancies has previously been reported [42]. Plausibly, impaired apposition between fetal and maternal units would affect cell migration and exocytosis of secretory granules in NT pregnancies. In humans, the effectiveness of trophoblast migration/invasion into the maternal spiral arteries is critical to a well-functioning placenta, with failure of this process being associated with pregnancy disorders such as preeclampsia [43, 44]. It is interesting that abnormal trophoblast proliferation and invasion has been associated with increased protein expression and abnormal release of leptin and PL in preeclampsia [43]. Leptin is expressed in the trophoectoderm and uterine epithelial cells at the fetal/maternal interface. Leptin also serves as a mitogen for a number of different cell types [45–47]; thus, leptin may serve as a mitogen for the placenta. Recently, we reported increased fetal, maternal, and BNC numbers in NT placentomes [31], and in the current study, increased expression of bPL and leptin protein in NT placentomes coincided with the previously reported increases in cell numbers observed in these pregnancies. Because bPL and leptin are detectable in the peripheral circulation following attachment of the trophoblast to the endometrium [14, 15], disturbances in attachment could result in a lower efficiency of secretion into the maternal tissues and, as such, higher retention of these proteins in NT pregnancies. This could explain the observed differences in fetal fluid bPL levels in NT versus AI/IVP pregnancies. Although the precise cause of the increased cell numbers in NT placentomes is unknown, BNC production is apparently under indirect control of the fetus [48]; our findings may thus indicate disturbances in fetal growth-regulatory signals [49]. However, it should also be noted that posttranscriptional regulation of PL and leptin expression has been reported [50–52], and this could also provide a possible explanation for the presence of high protein levels. Leptin has direct metabolic effects on several tissues, resulting in increased glucose utilization and lipolysis [53]. Previous studies have shown that factors such as maternal glucose levels and fetal hyperinsulinemia can contribute to fetal macrosomia through stimulating increased fetal and placental leptin production, and hence increased fetal growth in tissues expressing the leptin receptor [53, 54]. The fetal sheep kidney and liver are highly sensitive to nutrient availability [3], and we have previously described increased allometric growth and fat accumulation on livers and kidney of NT fetuses [32]. PL has also been shown to alter maternal metabolism to increase the amount of glucose available to the fetus [49], although a large fetus could also produce signals that result in increased bPL production. Active constraint in the delivery of nutrients to the fetus is necessary for the prevention of fetal overgrowth [2, 44]. Physiological studies of cloned calves susceptible to large offspring syndrome have described correlations between increased plasma insulin and glucagon and fetal size, suggesting that such differences in energy supply and utilization in utero may promote fetal growth [55]. Placental transport capacity and diffusion areas are considered primary contributors to nutrient availability and NT placentomes are significantly larger than AI or IVP placentomes [32, 56], and therefore have a larger surface area for exchange of nutrients. Therefore, presumably there is an increased capacity for transfer of nutrients across the placenta to the fetus in NT pregnancies. Tissue regeneration and nutrition of the fetus also require high rates of cellular turnover [57] and cell numbers are significantly increased in NT placentomes [31]. Furthermore, PL and leptin are positively correlated with placental and fetal weight in ruminants [56, 58] and we have shown that expression of these proteins was significantly increased in NT placentomes. In cattle, cell-specific rates of cellular turnover also reflect placental maturation [57]. As shown recently, NT placentomes at Days 100 and 150 of gestation resembled those of later gestational periods, suggesting that NT placentome growth was advanced for gestation [32]. This may suggest that increased rates of cell turnover may give rise to increased placentome growth, perhaps in response to altered endocrine status. In summary, we have shown that, in NT pregnancies, a condition of altered fetoplacental growth and function and disturbances in placental bPL and leptin expression and production are apparent, which may be the result of inadequate placentation. This could in turn contribute to aberrations in cell migration and invasion and alterations in placental metabolism and transfer of nutrients to the fetus, possibly by lifting constraints on nutrient availability and thus leading to growth deregulation in NT fetuses. 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TI - Altered Placental Lactogen and Leptin Expression in Placentomes from Bovine Nuclear Transfer Pregnancies
JO - Biology of Reproduction
DO - 10.1095/biolreprod.104.032201
DA - 2004-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/altered-placental-lactogen-and-leptin-expression-in-placentomes-from-29sl8kUaoO
SP - 1862
VL - 71
IS - 6
DP - DeepDyve
ER -