Sheep Testis Research Articles

Transcriptome analysis of the Tan sheep testes: Differential expression of antioxidant enzyme-related genes and proteins in response to dietary vitamin E supplementation

Gene-chip technology was employed to study the effect of dietary vitamin E on gene expression in sheep testes based on our previous research. Thirty-five male Tan sheep (20–30days after weaning) with similar body weight were randomly allocated into five groups and supplemented 0, 20, 100, 200 and 2000IUsheep−1day−1 vitamin E (treatments denoted as E0, E20, E100, E200, and E2000, respectively) for 120days. At the end of the study the sheep were slaughtered and the testis samples were immediately collected and stored in liquid nitrogen. Differences in gene expression between different treated groups were identified. Based on GO enrichment analysis and the KEGG database to evaluate the gene expression data we found that vitamin E might affect genes in the testes by modulating the oxidation level, by affecting the expression of various receptors and transcription factors in biological pathways, and by regulating the expression of metabolism-associated genes. The effect of vitamin E supplementation on the expression of oxidative enzyme-related genes was detected by quantitative real-time PCR (qRT-PCR) and Western blot. The results show that dietary vitamin E, at various doses, can significantly increase (P<0.05) the mRNA and protein expression of Glutathione peroxidase 3 and Glutathione S-transferase alpha 1. In addition, the results of qRT-PCR of the antioxidant enzyme genes were consistent with those obtained using the gene chip microarray analysis. In summary, the dietary vitamin E treatment altered the expression of a number of genes in sheep testes. The increase in the mRNA and protein levels of antioxidant enzyme genes, coupled with the elevation in the activity of the antioxidant enzymes were primarily responsible for the improved reproductive performance promoted by dietary vitamin E.

CDH1, a Novel Surface Marker of Spermatogonial Stem Cells in Sheep Testis

Spermatogonial stem cells (SSCs) are unique stem cells in adult body that can transmit genetic information to the next generation. They have self-renewal potential and can continuously support spermatogenesis throughout life of a male animal. However, the SSC population is extremely small, isolation and purification of the SSCs is challenging, especially for livestock animals. It has been confirmed that CDH1 (cadherin-1, also known as E-cadherin) can be expressed in undifferentiated SSCs of mouse and rats, but it has not been verified in sheep. Here, CDH1 was found as a novel surface marker for sheep SSCs. In this paper, sheep anti-CDH1 polyclonal antibodies were prepared and its activity was checked. Using the obtained antibodies and immunohistochemistry analysis, we confirmed that CDH1 can be expressed by SSCs in sheep testis.

Cloning and expression of MyoG gene from Hu sheep and identification of its myogenic specificity

This experiment was conducted to explore the biological functions of myogenin (MyoG) gene. MyoG gene was cloned from genome of Hu sheep by overlap extension PCR. Then, pEGFP-C1-MyoG and pcDNA3.0-MyoG fusion expression vectors was constructed and pEGFP-C1-MyoG vector had been transfected into NIH-3T3 cells by liposomes-mediated method, and MyoG was detected in vitro by RT-PCR,western blotting and its subcellular localization by EGFP marker. pcDNA3.0-MyoG was transfected into goat embryonic fibroblasts (GEF) cells in order to detect the myogenic function of MyoG in vitro. Then pEGFP-C1-MyoG plasmid was injected into the testes of sheep and goat, respectively, to produce the transgenic generation. The results showed that the length of MyoG coding region of Hu sheep was 675bp, encoding 224 amino acids. Compared with goat, cattle, pig and rat, the sequence homology of sheep MyoG cDNA was 99.26, 97.04, 92.00, and 87.70%, respectively. The bioinformatics prediction showed that MyoG protein contained a typical bHLH structure, but without a short signal peptide, revealing that MyoG protein might be a non-secretory protein. The result of RT-PCR and western blotting demonstrated that MyoG could be expressed successfully in the transfected cells in vitro and the MyoG protein was located in nucleus. The positive transfected GEF cells with pcDNA3.0-MyoG were found to express desmin protein. The positive rates of transgenic sheep and transgenic goat were 7.1 and 7.4% in F1 generation, respectively. Conclusively, MyoG cDNA from Hu sheep had been cloned successfully. The subcellular localization and myogenic activity of MyoG were exactly detected on the basis of multiple biological analyses, which expanded our understanding of the biological function of MyoG.

Foetal and post‐natal exposure of sheep to sewage sludge chemicals disrupts sperm production in adulthood in a subset of animals

Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to ‘real life’, environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.

Molecular Cloning of Sheep and Cashmere Goat Pdia3 and Localization in Sheep Testis

Protein disulphide isomerase family A3 (PDIA3) is a member of the protein disulphide isomerase (PDI) family and is multifunctional in many processes. Recently, it has been confirmed as a sperm membrane component and is implicated in sperm-egg fusion under which the molecular mechanism is still obscure. Protein disulphide isomerase family A3 cDNA has been cloned in several mammals; however, goat and sheep counterparts have not been identified. To facilitate the studies on the potential function of PDIA3 protein in sperm-egg fusion in sheep and goat, we cloned the cDNA encoding for Pdia3 of sheep (Ovis aries) and cashmere goat (Capra hircus) and studied its transcript and protein localization in sheep testis. The cloned sheep and cashmere goat Pdia3 cDNA are 1660 bp and 1591 bp, respectively. Bioinformatics analysis showed that sheep and goat Pdia3 cDNA both have a coding region of 1518 bp with the same nucleotides encoding the same 505 amino acids. The predicted peptide, with two typical motifs of Trp-Cys-Gly-His-Cys-Lys (WCGHCK) which is a hallmark of the PDI family, has high homology to that of bovine (99.21%), human (95.05%), rat (89.50%) and mouse (90.89%). Protein disulphide isomerase family A3 protein was observed in cells of various stages of spermatogenesis, from the primary spermatocyte to spermatozoa phases, as well as in the Leydig cells. It was observed in the entire sheep spermatozoa and mostly at the equatorial segment and the forepart of the flagellum. The Pdia3 mRNA was detected over the seminiferous epithelium in all stages of spermatogenesis.

Effect of Vitamin E supplementation on the enzymatic activity of selected markers in Aohan fine-wool sheep testis

The aim of this study was to evaluate the effects of dietary Vitamin E supplementation on the testicular ‘marker’ enzyme activity and Vitamin E content in Aohan fine-wool sheep. Thirty male Aohan fine-wool sheep (5 months of age) with similar body weight were selected from the Aohan fine-wool sheep-breeding farm of Inner Mongolia Autonomous Region, China. The sheep were randomly divided into five groups and supplemented with 0, 20, 200, 1000 or 2400 IU sheep −1 d −1 Vitamin E for 12 months. Three sheep in each group were slaughtered at 17 months to collect a testis sample for testicular marker enzyme analysis. The results showed that, compared to Control, supplementing the diet with Vitamin E at 200 IU sheep −1 d −1 significantly increased the content of Vitamin E in testis and improved the activity of testicular mitochondrial ATPase ( P < 0.01), lactate dehydrogenase (LDH) ( P < 0.01), sorbitol dehydrogenase (SDH) ( P < 0.01), and alkaline phosphatase (ALP) ( P < 0.05). The present study demonstrated that supplementing Vitamin E can have a positive role in improving testicular marker enzyme activity and that the optimum range of dose appeared to be 100–200 IU sheep −1 d −1.

Cloning of Sheep &lt;I&gt;tnp2&lt;/I&gt; Gene and Transcription Analysis of Round Spermatid Cells in the in vitro Co-culture System

The transition protein-2 gene (tnp2) have been reported as the round spermatid-specific marker gene. However, the DNA sequence of tnp2 gene in Ovis aries have not been reported. In order to study the sheep round spermatid-specific marker gene, we designed primers according to the reported Bos taurus tnp2 gene cDNA sequences in the GenBank. The partial CDS was amplified by RT-PCR. The PCR fragment was inserted into the T-A cloning vector pMD19-T. The partial nucleotide sequences of Mongolia sheep tnp2 gene were compared with the counterpart sequences of Bos taurus, and the nucleotide homology was 95.3%. Using the primers designed according to the tnp2 cDNA sequence, the tnp2 gene transcription expression in the co-cultured cells derived from four-months old sheep testis was assessed by RT-PCR. The results demonstrate that the round spermatid cells are generated in the co-culture system until ten weeks in vitro. The cDNA cloning and sequencing lay down a foundation for further study on spermatogenesis of sheep.

Transduction and Transplantation of Spermatogonia into the Testis of Ram Lambs through the Extra-testicular Rete

Spermatogonial transplantation will provide a new way to study spermatogenesis in domestic animals, disseminate male genetics and produce transgenic animals, if efficiency can be improved. We evaluated a 'surgical' method for transplanting donor cells into testes of ram lambs, where the head of the epididymis is reflected, and a catheter introduced into the extra-testicular rete testis. We also tested transduction of ram spermatogonia with a lentiviral (LV) vector as a means to identify permanent colonization, and introduce genes into donor cells. Eight ram lambs, 11- to 13-week olds, were the recipients: in five, spermatogonia were injected into one testis, and the contralateral testis was an un-manipulated control: in two, spermatogonia were injected into one testis and the contralateral was sham-injected: in one, both testes were injected. Six lambs received spermatogonia labelled with a cell-tracking dye and these were collected 1 or 2 weeks after transplantation; three lambs received spermatogonia transduced with a LV vector driving the expression of enhanced Green Fluorescence Protein and these were collected after 2 months. Donor cells were detected by immunohistochemistry in tubules of seven of nine recipient testes. Approximately 22% of tubule cross-sections contained donor cells immediately after transplantation, and 0.2% contained virally transduced cells 2 months after transplantation. The onset of spermatogenesis was delayed, and there were lesions in both injected and sham-injected testes. Despite the effects of the surgery, elongated spermatids were present in one recipient testis 2 months after surgery. The results suggest that, after modifying the surgical and transduction techniques, this approach will be a means to produce good colonization by donor spermatogonia in sheep testes.

Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates

Recovery of germ cells could be an option for preservation of the genetic pool of endangered animals. In immature males, xenografting of testis tissue provides the opportunity to recover sperm from these animals. In adult animals, xenografting has been less successful, but de novo morphogenesis of functional testis tissue from dissociated testis cells could be an alternative. To assess the potential use of these techniques in endangered bovid species, the domestic sheep was used as a model. Testes from 2-week-old lambs were grafted as tissue fragments or cell suspensions into nude mice. Grafts were recovered at 4, 8, 12 and 16 weeks post grafting. For isolated cells, two additional time points at 35 and 40 weeks after grafting were added. In addition, to analyse the possible effect of social stress among mice within a group on the development of the grafts, testis tissue grafts were recovered 13 weeks post grafting from mice housed individually and in groups. Complete spermatogenesis occurred in sheep testis xenografts at 12 weeks, similar to the situation in situ. Isolated sheep testis cells were able to reorganize and form functional testicular tissue de novo. Housing mice individually or in groups did not have any effect on the development of xenografts. Xenografting of testis tissue might be useful to obtain sperm from immature endangered ungulates that die prematurely. Testis tissue de novo morphogenesis from isolated cells could open interesting options to recover germ cells from mature males with impaired spermatogenesis.

438. PLZF is a spermatogonia stem cell-specific marker in the sheep testis: application to enrichment of ovine spermatogonial stem cell

Identification and isolation of spermatogonial stem cells (SSCs) are prerequisite for long-term culture, genetic manipulation, and transplantation research. The promyelocytic leukemia zinc-finger (PLZF) has been identified as a spermatogonia stem cell marker in rodent and other species, however its expression in sheep testis has not been reported yet. In this study, we validated an antibody that specifically binds to spermatogonia stem cell in sheep testis, thus demonstrated that PLZF is a spermatogonia stem cell marker and can be used for its identification. Testes from 12 Merino rams were selected to represent four stages of testis development at testis weights of 3–5 g (neonatal), 30 g (peripubertal), 50 g (prepubertal) and 100 g (mature). Three testes sections from 4 different developmental stage were stained with PLZF antibody and 25 individual tubules in each section were counted. In the sections, the percentage of PLZF positive cells/per tubule was increased nearly 2-fold from neonatal (6. 4 ± 0. 4%) to peripubertal (1 2.2 ± 2.8%), and then the percentage begin to decline in prepubertal (4.6 ± 0. 7%) and mature testes (3.1 ± 0.6%). A single cell suspension of testicular cells was generated by a two step enzymic digestion (n = 4) and spermatogonia stem cells were enriched by overnight differential plating with 0.2% gelatine coated flask. The percentages of spermatogonia stem cells in the single cell suspensions were assessed by PLZF antibody staining of smears. Compared with the initial isolation (3.1 ± 0.6%), spermatogonia were enriched 11-fold in overnight differential plating (34.0 ± 5.7,%) (P &lt; 0.05). These data provide the basis for future studies aimed at refining conditions of spermatogonial stem cell culture and manipulation before male germ stem cell transplantation in sheep.

Leptin inhibits basal but not gonadotrophin-stimulated testosterone production in the immature mouse and sheep testis

The mechanisms whereby leptin regulates testosterone secretion are complex and are likely to involve actions at different levels of the hypothalamus-pituitary-gonadal axis. In the present study, the effect of leptin on testicular steroidogenesis at different developmental stages in mice and sheep was investigated. Testosterone data from testicular slice and Leydig cells of immature and adult mice testes demonstrated that the action of leptin in the regulation of steroidogenesis appears to be dependent on the developmental stage of the testis. Leptin biphasically modulates basal testosterone production in immature testicular slice cultures: at relatively low concentrations (6.25-12.5 ng mL(-1)) leptin exerts a significant inhibitory effect, but has less of an effect at very low (1.25 ng mL(-1)) or high concentrations (25 ng mL(-1)). However, leptin failed to modulate basal testosterone levels in Leydig cell preparations. In contrast with immature testes, leptin was unable to regulate either basal or human chorionic gonadotrophin (10 IU mL(-1))-stimulated testosterone production in adult testicular slices or Leydig cell cultures. The age- and concentration-dependent regulation pattern was confirmed using sheep testicular slice culture. Leptin (1.56-25 ng mL(-1)) significantly inhibited basal testosterone production in the testis from birth to Day 21, but had no effect on Day 27 or older testes. However, the plasma and testicular concentrations of leptin and testosterone data in the ram indicate that such a regulatory effect of leptin on testis steroidogenesis in vitro is unable to efficiently influence testosterone concentrations in vivo. This does not exclude the possibility of a non-competitive mechanism of interaction between leptin and luteinising hormone to regulate testosterone production. Thus, we hypothesise that leptin is not an important independent regulator of testosterone concentration in the normal physiological state. The physiological significance and mechanism of leptin regulation of basal testosterone production are not known; further studies are required to elucidate these important issues.

Tumor Cells Enhance Their Own CD44 Cleavage and Motility by Generating Hyaluronan Fragments

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.

Dynamic expression of the prion‐like protein Doppel in ovine testicular tissue

Transgenic knockout of the gene encoding the prion-like protein Doppel (Dpl) leads to male infertility in mice. The precise role of Dpl in male fertility is still unclear, but sperm from Dpl-deficient mice appear to be unable to undergo the normal acrosome reaction that is necessary to penetrate the zona pellucida of the ovum. We have investigated the expression pattern and some biochemical properties of Dpl in sheep testicular tissue and spermatozoa. Neither the Dpl protein nor its mRNA was detected in pre-pubertal sheep testis. This was in contrast to the findings in adult rams where both Dpl mRNA and protein were present. The molecular mass and glycosylation pattern of sheep Dpl were similar to that of mice Dpl. The Dpl protein was detected in the seminiferous epithelium during the two final (7 and 8) and the two initial (1 and 2) stages of the spermatogenic cycle in a characteristic pattern. In stage 8, an intense brim of granular Dpl-immunoreactivity associated with maturation phase spermatids was observed, while after the release of spermatozoa in stages 1 and 2, the Dpl-staining was disseminated more diffusely in the epithelium, reaching the basal lamina. From stage 3 to stage 6, Dpl-immunoreactivity could not be detected, indicating that the Dpl protein had disappeared between stages 2 and 3. Dpl was not detected on ejaculated spermatozoa. These patterns of staining indicate that Dpl is enriched in residual bodies, which are phagocytosed and destroyed by Sertoli cells after release of sperm into the lumen of the seminiferous tubule.

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep

BackgroundThe gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA).MethodsAntibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference.ResultsIn adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01).ConclusionEvidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep.

Effects of scrotal heating on sperm surface protein PH-20 expression in sheep.

Sperm surface protein PH-20 expression was studied during spermatogenesis in pubertal and adult sheep, using molecular and histological methods. The effects of 24 hr of insulation raising scrotal temperatures to 39 degrees C on PH-20 expression in ejaculated sheep sperm were also determined. A 282 nt cDNA fragment of ovine PH-20 was identified in total RNA extracts of sheep testes, which exhibited 76% identity at the nucleotide level with the equivalent region of the human sequence. Ovine PH-20 mRNA and immunoreactivity were identified only in adult ram testis and not in peri-pubertal ram testis tubules lacking round spermatids, nor in adult sheep brain, pituitary, heart, spleen, lung, liver, kidney, epididymis, or ovary. Ovine PH-20 protein was distributed predominantly on the postacrosomal membrane and was also present on the anterior membrane of the sperm head in fresh, unheated sheep semen. Scrotal heating caused a significant, transient decrease in the percentage of PH-20 immunoreactive sperm, but did not change the pattern of PH-20 staining on the sperm head. The results strongly suggest that ovine PH-20 is postmeiotically expressed in haploid germ cells in sheep testis and is arrayed on the membrane of the mature ovine spermatozoon. Scrotal heating appears to have few effects on PH-20 expression and distribution on ejaculated sperm.

Prolactin actions in the sheep testis: a test of the priming hypothesis.

This study investigated whether prolactin (PRL) plays a priming role in the testis during the nonmating season and thereby facilitates gonadal reactivation. Sexually inactive Soay rams under long days were treated as follows: 1) group C (control) received vehicle, 2) group B received bromocriptine to suppress PRL secretion, 3) group B + PRL received bromocriptine + ovine PRL to reinstate physiological levels of PRL (n = 5/group). Treatments were for 10 wk. The photoperiod was then switched to short days to reactivate the reproductive axis. Testis diameter and sex skin coloration were recorded, and routine blood samples were collected to measure concentrations of FSH, inhibin A, and testosterone (T). At the end of the treatments, blood samples were collected every 10 min for 10 h to monitor LH pulses and the T-response to exogenous LH, and a testis biopsy was collected to assess spermatogenic activity (bromodeoxyuridine [BrDU] method) and expression of PRL receptor (reverse transcription-polymerase chain reaction and immunocytochemistry). There were no significant differences between groups in spermatogenesis (BrDU index) or steroidogenesis (T-response), and no difference in the time taken to achieve full testicular redevelopment under short days. Testis diameter and inhibin A were marginally increased in group B + PRL. Overall, this thorough experiment provides minimal support for the priming hypothesis.

Prolactin receptor expression in the testis of the ram: localisation, functional activation and the influence of gonadotrophins

The present study investigated the pattern and site of expression of the prolactin receptor gene in the testis of the seasonally breeding Soay sheep. In experiment 1, Northern blot analysis confirmed expression of the prolactin receptor gene in the testis which was encoded by RNA transcripts of ≈3.6, 11.2, 12.6, and 14.1 kb. In situ hybridisation localised expression of the receptor within the interstitial and seminiferous tubule compartments of the testis and immunohistochemistry localised expression of the receptor to Leydig cells and to pachytene spermatocytes, round and elongating spermatids. In experiment 2, phosphorylation of Jak2, Stat1 and Stat5 proteins in response to prolactin was investigated by Western blotting following incubation of testicular samples with 100 ng/ml ovine prolactin. Jak2 and Stat1 phosphorylation were induced by prolactin within 10 min and Stat5 within 30 min. In experiment 3, intact and hypothalamo-pituitary disconnected (HPD) rams were transferred from a short to a long day photoperiod regimen for a period of 8 weeks. By week 8, testicular diameter had declined in intact rams (52.71±1.06 cm vs. 48.00±0.49 cm for weeks 1 and 8 after transfer respectively, P<0.01, n=3) and increased in HPD rams (27.00±0.45 cm and 29.66±0.99 cm for weeks 1 and 8 after transfer respectively, P<0.05, n=3). RT-PCR using RNA extracted from intact and HPD rams confirmed expression of the prolactin receptor in the testis of both groups. Immunohistochemistry localised prolactin receptor expression in Leydig cells and in pachytene spermatocytes, round and elongating spermatids of intact sheep testis. In HPD rams, prolactin receptor expression was localised in Leydig cells and germ cells which were arrested predominantly at the pachytene spermatocyte stage. These data demonstrate expression of functional prolactin receptors in the testis of Soay rams. The site and pattern of expression of the receptor gene suggest a role for prolactin in the regulation of steroidogenesis and spermatogenesis.

Alternative splicing converts the G-protein coupled follitropin receptor gene into a growth factor type I receptor: implications for pleiotropic actions of the hormone.

Pituitary follitropin (FSH) has pleiotropic actions on gonads, but it is not certain if all these events are mediated by a single receptor. A single gene for the FSH receptor undergoes extensive alternate splicing generating multiple transcripts, and several of these have been cloned and characterized from the sheep testis. In this study we have investigated the expression in HEK (human embryonic kidney) 293 cells of a cloned cDNA encoding the first eight exons of the FSH receptor along with a carboxyterminal extension that contributed a hypothetical single transmembrane domain. This cDNA, which lacked the conventional seven transmembrane motif of the full-length 695 residue wild-type receptor protein, was also efficiently expressed on the cell surface and exhibited high affinity and specificity for FSH binding. LH did not compete for FSH binding indicating that these structures contained all the motifs necessary for specific hormone recognition. Following hormone binding and affinity crosslinking the deduced M(r) of the expressed receptor was compatible with dimer formation. The expression of these altered FSH receptors on the cell surface was confirmed by immunohistochemistry, which revealed punctate labeling in a pattern comparable to that shown by cells transfected by wild-type receptor cDNA. Addition of FSH stimulated 3H-thymidine incorporation in transfected cells in a biphasic manner. By performing RT-PCR we could show that similar altered receptor transcripts were present in both the ovary and testis. Our results reveal for the first time that the seven transmembrane structure of FSH-receptor is not absolutely necessary for cell surface expression and hormone binding provided other compensating motifs are present in the protein structure for membrane insertion. Some of these features are typical of growth factor receptors. Our investigations also demonstrate that alternate splicing of the FSH receptor gene provides a mechanism for creating receptor diversity and suggest that multiple receptors could be involved in regulation of hormone action.

A novel endozepine-like peptide (ELP) is exclusively expressed in male germ cells

A cDNA clone encoding a novel endozepine-like peptide (ELP) was isolated from mouse testes, sequenced, and its mRNA expression characterized by northern and in situ hybridization. ELP mRNA was found exclusively in the late spermatid stages of spermatogenesis in the testes of sexually mature mice and in no other tissue or cell type examined. It was also expressed in rat, bovine, porcine and sheep testes. Mouse ELP-encoding cDNA was used to construct expression vectors for the production of ELP in bacteria, and the purified bacterial protein used to raise polyclonal antibodies in rats. These antibodies identified the predicted endogenous ELP in extracts of mouse testis and epididymis and in no other tissue. Immunohistochemistry confirmed that the ELP antigen was present only in late spermatids and spermatozoa, particularly within the cytoplasmic droplet which is retained by the mature spermatozoa during their transit into the epididymis. We conclude that ELP is an intracytoplasmic peptide exclusively expressed in post-meiotic spermatozoa and which may be involved in the energy metabolism of the mature sperm.

Expression of mRNA and immunocytochemical localization of inhibin alpha- and inhibin beta A-subunits in the fetal sheep testis.

In order to investigate the ontogeny of gonadal inhibin production in the male fetal sheep, testes were collected from male fetuses at days 70, 100, 130 and 140 of gestation (term = 145 days). The expression and localization of inhibin alpha- and inhibin beta A-subunit mRNA and protein were evaluated using in situ hybridization and immunocytochemistry. The expression of inhibin alpha-subunit mRNA was localized within the seminiferous cords of the developing fetal testis and progressively increased with gestational age. Immunostaining corresponding to immunoreactive inhibin alpha-subunit was detected in Sertoli cells within the seminiferous cords at days 100, 130 and 140 of gestation. In addition, immunostaining was detectable in a small proportion of Leydig cells. No expression of inhibin beta A-subunit mRNA or immunoreactivity was detected in any testicular tissue at any stage of gestation. These data show that the Sertoli cells of the developing fetal sheep testis have the capacity to produce inhibin alpha-subunit by day 100 of gestation and that production increases during late gestation.