Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus.

Female mice lacking the follistatin gene but expressing a human follistatin-315 transgene (tghFST315) have reproductive abnormalities (reduced follicles, no corpora lutea and ovarian-uterine inflammation). We hypothesised that the absence of follistatin-288 causes the abnormal reproductive tract via both developmental abnormalities and abnormal ovarian activity. We characterised the morphology of oviducts and uteri in wild type (WT), tghFST315 and follistatin-knockout mice expressing human follistatin-288 (tghFST288). The oviducts and uteri were examined in postnatal Day-0 and adult mice (WT and tghFST315 only) using histology and immunohistochemistry. Adult WT and tghFST315 mice were ovariectomised and treated with vehicle, oestradiol-17ß (100ng injection, dissection 24h later) or progesterone (1mg×three daily injections, dissection 24h later). No differences were observed in the oviducts or uteri at birth, but abnormalities developed by adulthood. Oviducts of tghFST315 mice failed to coil, the myometrium was disorganised, endometrial gland number was reduced and oviducts and uteri contained abundant leukocytes. After ovariectomy, tghFST315 mice had altered uterine cell proliferation, and inflammation was maintained and exacerbated by oestrogen. These studies show that follistatin is crucial to postnatal oviductal-uterine development and function. Further studies differentiating the role of ovarian versus oviductal-uterine follistatin in reproductive tract function at different developmental stages are warranted.

Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus.

Follistatin, an inhibitor of activin A, has key regulatory roles in the female reproductive tract. Follistatin has two splice variants: FST288, largely associated with cell surfaces, and FST315, the predominant circulating form. The mechanism regulating uterine expression of these variants is unknown. Quantitative RT-PCR was used to measure expression of follistatin splice variants (Fst288, Fst315), the activin bA subunit (Inhba) and the inhibin a subunit (Inha) in uterine tissues during early pregnancy (days 1­4, preimplantation) and in response to exogenous 17b-oestradiol (single s.c. injection) and progesterone (three daily s.c. injections) in ovariectomized mice. Uterine Fst288, Fst315 and Inhba expression increased during early pregnancy, with greater increases in Fst315 relative to Fst288 suggesting differential regulation of these variants. Fst288, Fst315, Inhba and Inha all increased in response to progesterone treatment. Fst288, but not Fst315, mRNA decreased in response to 17b-oestradiol treatment, whereas Inhba increased. A comparison of the absolute concentrations of uterine follistatin mRNA using crossing thresholds indicated that both variants were more highly expressed in early pregnancy in contrast to the hormone treatment models. It is concluded that progesterone regulates uterine expression of both follistatin variants, as well as activin A, during early pregnancy in the mouse uterus

A missense mutation in the transcription factor Foxo3a causes teratomas and oocyte abnormalities in mice.

An N-ethyl-N-nitrosourea random mutation screen was used to identify recessive modifiers of gene silencing in the mouse using an epigenetically sensitive reporter transgene. One of the mutant lines, MommeR1, was identified as a suppressor of variegation and it showed female-specific age-associated infertility in homozygotes. Linkage analysis identified a region on chromosome 10, containing the Foxo3a gene, previously shown to play a critical role in female gametogenesis. Foxo3a is a transcription factor with roles in cell cycle control, apoptosis, neural and hematopoietic cell differentiation, and DNA repair. Sequencing of the Foxo3a gene in MommeR1 mice revealed a point mutation that causes an amino acid substitution in the highly conserved Forkhead DNA-binding domain. In vitro transcription assays showed that the point mutation causes loss of FOXO3a transactivation activity. Compound heterozygotes made with Foxo3a-null mice (carrying the targeted deletion of exon 2) displayed complementation with respect to both the activation of the reporter transgene and defects in folliculogenesis similar to those seen in MommeR1 homozygotes, supporting the conclusion that this is the causative mutation. Approximately one in six female MommeR1 homozygotes develop teratomas, a phenotype not reported in Foxo3a-null mice. Ovulated oocytes from MommeR1 homozygotes display a number of abnormalities. The MommeR1 mice provide a novel platform to investigate teratocarcinogenesis and link Foxo3a with parthenogenesis and ovarian cancer. The finding of Foxo3a as a modifier of epigenetic reprogramming is discussed.

Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights.

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented.

Polymorphisms in the human cysteine-rich secretory protein 2 (CRISP2) gene in Australian men.

BACKGROUND: Cysteine-rich secretory protein 2 (CRISP2) is localized to the human sperm acrosome and tail. It can regulate ryanodine receptors Ca(2+) gating and binds to mitogen-activated protein kinase kinase kinase 11 in the acrosome and gametogenetin 1 (GGN1) in the tail. METHODS AND RESULTS: In order to test the hypothesis that CRISP2 variations contribute to male infertility, we screened coding and flanking intronic regions in 92 infertile men with asthenozoo- and/or teratozoospermia and 176 control men using denaturing HPLC and sequencing. There were 21 polymorphisms identified, including 13 unreported variations. Three SNPs resulted in amino acid substitutions: L59V, M176I and C196R. All were only present in a heterozygous state and found in fertile men. However, the C196R polymorphism was of particular interest as it resulted in the loss of a strictly conserved cysteine involved in intramolecular disulphide bonding. Screening of an additional 637 infertile men identified 23 heterozygous C196R men to give an overall frequency of 3.6%, compared with 3.4% in control men. The functional significance of the C196R polymorphism was defined using a yeast two-hybrid assay. The C196R substitution resulted in the loss of CRISP2-GGN1 binding. CONCLUSIONS: Although none of the many polymorphisms identified herein showed a significant association with male infertility, functional studies suggested that the C196R polymorphism may compromise CRISP2 function.

Structural heterogeneity of the axonemes of respiratory cilia and sperm flagella in normal men.

The ultrastructure of normal human cilia and flagella was examined and quantitatively assessed to determine the normal variations in the structure of the axoneme. Ciliated respiratory epithelial cells and spermatozoa from 10 normal, nonsmoking male volunteers who had normal semen parameters were fixed for electron microscopy. Tannic acid and MgSO4 were included during fixation to enhance, in particular, axonemal components. In 75 axonemal cross sections per sample, the number of outer doublet and central singlet microtubules, outer and inner dynein arms, and radial spokes were recorded. Statistical analysis of the results showed a marked reduction, from the expected value of nine, in the numbers of inner dynein arms (mean +/- SE, cilia, 5.31 +/- 0.13; sperm, 5.38 +/- 0.16) and radial spokes (cilia, 4.95 +/- 0.22; sperm, 5.80 +/- 0.19). The ideal axoneme with all its structural components was seen in only 0.13% of cilia and 0.80% of sperm tails. Significantly more doublet microtubules (P less than 0.05) and less central microtubules (P less than 0.01) and radial spokes (P less than 0.01) were seen in cilia than in sperm tail axonemes. Between subjects there was little variation in the mean number of a structure seen per axoneme. However, within each sample, the variation was considerably higher, particularly for the inner and outer dynein arms and radial spokes. The doublet microtubules had significantly greater standard deviations in the sperm tails compared with the cilia (P less than 0.01), and furthermore, a significantly greater number of sperm tails compared with cilia showed the incorrect number of doublet microtubules (P less than 0.02). In one semen sample, with normal semen analysis, 20% of the sperm tails showed incorrect numbers of doublet microtubules, ranging from 12 + 2 to 5 + 2 compared with only 1.3% in cilia from this subject. This study has demonstrated that the ideal axoneme is rarely seen even in normal samples, probably because of the technical difficulties in resolution and visualization, and stresses the need for thorough documentation of axonemal ultrastructure. This work provides a normal data base for comparison with patients who have chronic respiratory disease and suspected infertility.

Relative roles of follicle-stimulating hormone and luteinizing hormone in the control of inhibin secretion in normal men.

The glycoprotein hormone inhibin is produced by the Sertoli cells of the testis under the influence of follicle-stimulating hormone (FSH) and is postulated in turn to inhibit FSH secretion. Luteinizing hormone (LH) is not recognized to have an important role in the control of inhibin secretion in any species. To determine the relative roles of FSH and LH in the control of inhibin secretion in man, we examined the effects of selective FSH and LH replacement on serum inhibin levels in normal men whose endogenous gonadotropins were suppressed by testosterone (T). After a 3-mo control period, nine men received 200 mg T enanthate i.m. weekly for 3-9 mo. During T treatment, serum LH and FSH levels were markedly suppressed and serum inhibin levels fell to 40% of control values. While continuing T, 3-5 mo of treatment with purified hFSH (n = 4) or hLH (n = 4) increased the respective serum gonadotropin level into the upper normal range and significantly increased inhibin levels back to 64 and 55% of control values, respectively. Supraphysiological LH replacement with high doses of human chorionic gonadotropin (n = 3) returned serum inhibin levels to 63% of control values. In no case did inhibin levels return fully to control levels. In conclusion, serum inhibin levels fell during gonadotropin suppression and were partially and approximately equally restored by either FSH or LH treatment. FSH presumably acts directly on the Sertoli cell to increase inhibin secretion whereas LH may act via increases in intratesticular T levels and/or other factor(s).

Activin and follistatin interactions in the male reproductive tract: activin expression and morphological abnormalities in mice lacking follistatin 288.

Activin A is an important regulator of testicular and epididymal development and function, as well as inflammation and immunity. In the adult murine reproductive tract, activin A mRNA (Inhba) expression levels are highest in the caput epididymis and decrease progressively towards the distal vas deferens. The activin-binding protein, follistatin (FST), shows the opposite expression pattern, with exceptionally high levels of the Fst288 mRNA variant in the vas deferens. This unique pattern of expression suggests that activin A and follistatin, in particular FST288, play region-specific roles in regulating the epididymis and vas deferens. The cellular distribution of activin and follistatin and structural organization of the male reproductive tract was examined in wild-type and transgenic (TghFST315) mice lacking FST288. Compared to wild-type littermates, TghFST315 mice showed a 50% reduction in serum follistatin and a significant elevation of both activin A and B. Testicular, epididymal and seminal vesicle weights were reduced, but intra-testicular testosterone was normal. A decrease in the epididymal duct diameter in the corpus and thickening of the peritubular smooth muscle in the cauda, together with increased coiling of the proximal vas deferens, were observed in TghFST315 mice. No immune cell infiltrates were detected. Immunohistochemistry indicated that epithelial cells are the main source of activins and follistatin in the epididymis and vas deferens. Activin A, but not activin B, was also localized to sperm heads in the lumen of the epididymis and vas deferens. Expression of Inhba and another immunoregulatory gene, indoleamine-2,3-dioxygenase (Ido-1), was increased approximately twofold in the TghFST315 caput epididymis, but several other genes associated with immunoregulation, inflammation or fibrosis were unaffected. Our novel data indicate that disruption of follistatin expression has significant effects on the testis and epididymis, and suggest an association between activin A and indoleamine-2,3-dioxygenase in the caput epididymis, with implications for the epididymal immunoenvironment.

Regulated production of activin A and inhibin B throughout the cycle of the seminiferous epithelium in the rat.

Production and regulation of activin A and inhibin B during the cycle of the seminiferous epithelium were investigated in adult rats. Immunohistochemistry localised the activin beta(A)-subunit to the Sertoli cell cytoplasm, with much weaker expression in spermatocytes and spermatids. Both activin A and inhibin B, measured by ELISA were secreted by, seminiferous tubule fragments over 72 h in culture. Activin A was secreted in a cyclic manner with peak secretion from tubules isolated at stage VIII. Tubules collected during stage VI produced the least activin A. Inhibin B secretion was highest from stage IX-I tubules and lowest from stage VII tubules. Addition of interleukin-1beta (IL-1beta) had relatively little effect on activin A or inhibin B secretion in culture. In contrast, the peak secretion of activin A by stage VIII tubules was blocked by co-incubation with an excess of human recombinant IL-1 receptor antagonist, whereas inhibin B secretion increased slightly. Dibutyryl cAMP stimulated activin A secretion by late stage VII and VIII tubules and stimulated inhibin B across all stages. These data indicate that activin A and inhibin B are cyclically regulated within the seminiferous epithelium, with endogenous IL-1 (presumably IL-1alpha produced by the Sertoli cells), responsible for a peak of activin A production subsequent to sperm release at stage VIII. These data provide direct evidence that production of activin A and inhibin B by the Sertoli cell is locally modulated by IL-1alpha , in addition to FSH/cAMP, under the influence of the developing spermatogenic cells.

Activins in reproductive biology and beyond.

BACKGROUND: Activins are members of the pleiotrophic family of the transforming growth factor-beta (TGF-ß) superfamily of cytokines, initially isolated for their capacity to induce the release of FSH from pituitary extracts. Subsequent research has demonstrated that activins are involved in multiple biological functions including the control of inflammation, fibrosis, developmental biology and tumourigenesis. This review summarizes the current knowledge on the roles of activin in reproductive and developmental biology. It also discusses interesting advances in the field of modulating the bioactivity of activins as a therapeutic target, which would undoubtedly be beneficial for patients with reproductive pathology. METHODS: A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies in the English language which have contributed to the advancement of the field of activin biology, since its initial isolation in 1987 until July 2015. 'Activin', 'testis', 'ovary', 'embryonic development' and 'therapeutic targets' were used as the keywords in combination with other search phrases relevant to the topic of activin biology. RESULTS: Activins, which are dimers of inhibin ß subunits, act via a classical TGF-ß signalling pathway. The bioactivity of activin is regulated by two endogenous inhibitors, inhibin and follistatin. Activin is a major regulator of testicular and ovarian development. In the ovary, activin A promotes oocyte maturation and regulates granulosa cell steroidogenesis. It is also essential in endometrial repair following menstruation, decidualization and maintaining pregnancy. Dysregulation of the activin-follistatin-inhibin system leads to disorders of female reproduction and pregnancy, including polycystic ovary syndrome, ectopic pregnancy, miscarriage, fetal growth restriction, gestational diabetes, pre-eclampsia and pre-term birth. Moreover, a rise in serum activin A, accompanied by elevated FSH, is characteristic of female reproductive aging. In the male, activin A is an autocrine and paracrine modulator of germ cell development and Sertoli cell proliferation. Disruption of normal activin signalling is characteristic of many tumours affecting reproductive organs, including endometrial carcinoma, cervical cancer, testicular and ovarian cancer as well as prostate cancer. While activin A and B aid the progression of many tumours of the reproductive organs, activin C acts as a tumour suppressor. Activins are important in embryonic induction, morphogenesis of branched glandular organs, development of limbs and nervous system, craniofacial and dental development and morphogenesis of the Wolffian duct. CONCLUSIONS: The field of activin biology has advanced considerably since its initial discovery as an FSH stimulating agent. Now, activin is well known as a growth factor and cytokine that regulates many aspects of reproductive biology, developmental biology and also inflammation and immunological mechanisms. Current research provides evidence for novel roles of activins in maintaining the structure and function of reproductive and other organ systems. The fact that activin A is elevated both locally as well as systemically in major disorders of the reproductive system makes it an important biomarker. Given the established role of activin A as a pro-inflammatory and pro-fibrotic agent, studies of its involvement in disorders of reproduction resulting from these processes should be examined. Follistatin, as a key regulator of the biological actions of activin, should be evaluated as a therapeutic agent in conditions where activin A overexpression is established as a contributing factor.

Regulation of activin A and inhibin B secretion by inflammatory mediators in adult rat Sertoli cell cultures.

The regulation of Sertoli cell activin A and inhibin B secretion during inflammation was investigated in vitro. Adult rat Sertoli cells were incubated with the inflammatory mediators, lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), IL-6 and the IL-1 receptor antagonist (IL-1ra) over 48 h in culture. Activin A, inhibin B and IL-1alpha were measured in the culture medium by specific two-site ELISAs. Both IL-1beta- and LPS-stimulated activin A and inhibited inhibin B secretion. LPS also stimulated the production of IL-1alpha in the cultures. In contrast to IL-1beta, IL-6 had no effect on activin A, although it did have a significant inhibitory effect on inhibin B secretion. Ovine follicle-stimulating hormone (FSH) and the cAMP analogue dibutyryl cAMP opposed the actions of IL-1 and LPS by suppressing activin A and IL-1alpha secretion and by stimulating inhibin B. Blocking IL-1 activity in the cultures by addition of an excess of IL-1ra completely prevented the response of activin A to exogenous IL-1beta, and reduced the response to LPS by 50%. In the presence of IL-1ra, basal secretion of inhibin B was increased, but IL-1ra was unable to reverse the suppression of inhibin B by LPS. These data indicate the importance of both IL-1 isoforms in regulating secretion of activin A and inhibin B by mature Sertoli cells during inflammation. The data also establish that inflammation exerts its effects on activin A and inhibin B secretion via other pathways in addition to those mediated by IL-1, and that hormonal stimulation by FSH and cAMP moderates the Sertoli cell response to inflammation. Interference with the complex interactions between these cytokines and hormones may contribute to the disruption of reproductive function that can accompany infection and illness in men.

Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro.

In several biological systems, the inhibin beta(A) homodimer activin A is stimulated by, and in turn, inhibits the action of interleukin (IL)-1 (both IL-1alpha and IL-1beta) and IL-6. The possibility that a similar regulatory relationship operates within the testis was investigated. Sertoli cells from immature (20-day-old) rats were cultured with human IL-1alpha or IL-1beta, human IL-6 and/or ovine FSH or dibutyryl cAMP. Activin A and the inhibin dimers, inhibin A and inhibin B, were measured by specific ELISA. Immunoreactive inhibin (ir-inhibin) was measured by RIA. Activin/inhibin subunit mRNA expression was measured by quantitative real-time PCR. Both IL-1 isoforms, but not IL-6, stimulated activin A secretion through increased synthesis of beta(A)-subunit mRNA. IL-1 also stimulated activin A secretion by testicular peritubular cells. In contrast to the effect on activin A, IL-1 suppressed inhibin beta(B)-subunit and, to a lesser extent, alpha-subunit mRNA expression, thereby reducing basal and FSH-stimulated inhibin B secretion by the Sertoli cells. Conversely, FSH inhibited basal activin A secretion and antagonised the stimulatory effects of IL-1. Dibutyryl cAMP partially inhibited the action of IL-1 on activin A secretion, but had no significant effect on basal activin A secretion. Secretion of inhibin A was low in all treatment groups. These data demonstrate that IL-1 and FSH/cAMP exert a reciprocal regulation of activin A and inhibin B synthesis and release by the Sertoli cell, and suggest a role for activin A as a potential feedback regulator of IL-1 and IL-6 activity in the testis during normal spermatogenesis and in inflammation.

Hormonal control of spermatogenesis.

FSH and testosterone (T) secretion are essential for the successful completion of spermatogenesis. Because there are no receptors for FSH or testosterone on germ cells, there are intermediate steps in this action, the nature of which are unknown. However, as the Sertoli cell contains receptors for both FSH and T, it is likely that these hormones exert their influence on germ cells by modulating Sertoli cell function. Both FSH and T exert synergistic actions on germ cells, but T has a specific action on the later stages of spermatid maturation. FSH, by its ability to stimulate Sertoli cell mitosis during testicular development, can influence the spermatogenic capacity of the adult testis.

The involvement of Leydig cells in the regulation of inhibin secretion by the testis.

The stimulation of Leydig cells by the administration of a single injection of 100 IU human CG (hCG) to adult male rats caused a significant biphasic stimulation of serum testosterone levels at 2 h and 3 days after injection. Serum immunoreactive (IR)-inhibin levels were elevated by 6 h and peaked at 24 h after hCG, then progressively declined thereafter. The removal of Leydig cells in vivo by the injection of the cytotoxic drug ethane dimethane sulfonate (EDS) causes a significant decrease in serum testosterone levels within 4 days, which is sustained 1 and 2 weeks after EDS. Serum IR-inhibin levels, however, rise significantly 2 and 4 weeks after injection of EDS. An injection of 100 IU hCG, 4 days after EDS (when no Leydig cells were present in vivo), failed to provoke an elevation of either testosterone or IR-inhibin levels in serum. But 2 or 4 weeks after administration of EDS, as a new population of Leydig cells develops in the interstitium, an injection of 100 IU hCG provokes a significant increase in serum testosterone and IR-inhibin levels. The possibility that the failure of IR-inhibin levels to rise after EDS and hCG treatment could be due to changes in the seminiferous epithelium, caused by testosterone deprivation induced by Leydig cell destruction after EDS, was examined by administering high doses of testosterone known to maintain spermatogenesis. Under such conditions, hCG failed to induce a rise of IR-inhibin after EDS treatment had destroyed the Leydig cells. These data strongly support the concept that the Leydig cells are involved in the regulation of IR-inhibin secretion in vivo through factors other than testosterone.

Changes in testicular inhibin after a single episode of heating of rat testes.

Brief heating (43 C for 15 min) of the scrota of adult rats was used to induce reversible spermatogenic damage to the testes. In these animals the changes in testicular inhibin content and an index of inhibin production rate, measured after efferent duct ligation, were examined and correlated with serum gonadotropin levels. The effect of heating was not evident until after 1 week when testis weight, inhibin content, and inhibin production rate were significantly reduced and both serum FSH and LH were elevated. By 2 weeks, the maximal effects were observed, and, thereafter, all parameters gradually returned to control values (FSH: by 6 weeks; testis and epididymal weight, inhibin content, inhibin production rate, and seminiferous tubule fluid production: by 17 weeks). Throughout the study, serum testosterone levels showed no significant changes. Significant inverse correlations were found between serum FSH levels and inhibin content (r = -0.502, P less than 0.001) or inhibin production rate (r = -0.533, P less than 0.001), and these were taken as supportive evidence for the hypothesis that inhibin is involved in the feedback control of pituitary FSH secretion. Although serum LH levels were also negatively correlated to the corresponding inhibin content (r = -0.669, P less than 0.001) or inhibin production rate (r = -0.420, P less than 0.001), recent findings of Leydig cell dysfunction in these animals led us to relate the transient rise in LH to the altered state of Leydig cell function.

Alterations in steroidogenesis and human chorionic gonadotropin binding in the cryptorchid rat testis.

One month after the induction of cryptorchidism in adult rats, serum levels of LH and FSH were significantly elevated in comparison with sham-operated controls, whereas serum levels of testosterone remained low to normal. Testis weight in cryptorchid rats was reduced by over 66%, and once the extratubular fluid was removed by decapsulation, the reduction in weight was 78%. The basal production of testosterone, pregnenolone, and estradiol in vitro by testes from cryptorchid rats was similar to controls, whereas significantly less androstenedione was produced. Testicular stimulation in vitro with a high dose of hCG (360 pM) resulted in significantly greater production of testosterone, pregnenolone, and estradiol by cryptorchid than by control rat tissue. The in vitro binding of [125I]hCG per testis was decreased in the cryptorchid state to 40% of control values, probably as a result of down-regulation of LH receptors due to the 4-fold elevation of serum LH levels in the cryptorchid rats.

Morphological and functional characterization of interstitial cells from mouse testes fractionated on Percoll density gradients.

Cell fractions were obtained by separation on Percoll density gradients after dissociation of mature mouse testes by mechanical or collagenase dispersion, and the ultrastructure, hCG receptor properties, and hCG-stimulated testosterone (T) production of these fractions were compared. Gradients were fractionated according to specific gravity, and all cell types were quantitated using morphometric techniques. Three peaks of specific [125I]iodo-hCG binding corresponding to densities of 1.0667 g/cm3 (fractions 2-3), 1.045 g/cm3 (fractions 6-7), and 1.0365-1.0215 g/cm3 (fraction 9) were obtained after collagenase dispersion, but the second peak of binding (fractions 6-7) was not observed after mechanical dispersion. Morphometric studies were performed by light microscopy on the cells present in the fractions corresponding to the first and second hCG binding peaks obtained by either mechanical or collagenase dispersion; the third representing germ cells and membranous debris was not studied further. Regardless of the method of preparation, morphologically intact Leydig cells represented 60-80% and 7-10% of the cells in fractions 2-3 and 6-7 that were associated with the first and second peaks of hCG binding Leydig cells obtained from fractions 6-7 contained greater numbers of lipid inclusions than Leydig cells from fractions 2-3. Mechanically dispersed Leydig cells exhibited similar numbers of hCG receptors in the dense and light Leydig cells, but hCG stimulated T production per Leydig cell was significantly greater in the dense Leydig cells containing few lipid inclusions. T production by dense and light collagenase-dispersed Leydig cells was not significantly different. The second hCG binding peak in collagenase-dispersed cell fractions 6-7 was associated with an increase in the number of an indeterminate connective cell type released from the testes by collagenase treatment, whose ultrastructure and limited hCG-binding capacity suggested that they may represent Leydig cell precursors. It is concluded that the identification and quantitation of different cell types in isolated testicular cell fractions is, therefore, of fundamental importance in the interpretation of receptor and secretory capacities of enriched preparations of Leydig cells.

The effects of chronic human chorionic gonadotropin treatment on Leydig cell function.

The purpose of this study was to examine the effect of chronic daily hCG treatment on interstitial cell function in the rat, as judged by plasma testosterone levels, the testicular binding of labeled hCG, and the capacity of the testis to respond to gonadotropin stimulation by the production of testosterone in vitro. TWenty-four hours after the first injection of 100 IU hCG there was a significant decline in hCG binding to testis homogenates and an inability to respond to hCG stimulation in vitro, After 7 days of daily injections of 10 IU or 100 IU hCG, the loss of hCG binding was maintained. However, despite the marked decline in hCG binding, there was an enhanced testosterone response to hCG stimulation in vitro, and plasma testosterone levels were significantly elevated. With continued injections of hCG for 14 or 21 days, the testes remained hyperresponsive to hCG stimulation in vitro, but hCG binding returned to control levels, and plasma testosterone concentrations declined and were not statistically different from controls. The latter changes probably result from the formation of specific hCG antibodies (Kd at 4 C, 7.8 +/- 4.5 X 10(-10) M) that were detected in plasma from rats treated for 14 or mopre days with hCG. The formation and levels of the hCG antibodies in these animals were sufficient to neutralize the effects of the exogenous hCG, thereby returning plasma testosterone levels to normal and restoring the complement of hCG receptors.

The isolation of activin from ovine amniotic fluid.

During a study of the levels of inhibin and follistatin in ovine amniotic fluid, we noted that although detectable levels of immunoactive inhibin and follistatin were found throughout gestation, the addition of amniotic fluid to a rat anterior pituitary cell culture resulted in a stimulation, rather than the expected suppression, of FSH concentrations. These data suggested the possibility that activin was present in amniotic fluid. We, therefore, set out to isolate the molecules responsible for this activin-like activity and determine their structure. Amniotic fluid, collected from pregnant sheep between 120-140 days gestation, was used as starting material in the purification and diluted in parallel to a human activin-A standard in the activin RIA employed to monitor the purification. A total pool of 7.4 liters amniotic fluid was processed by dye affinity chromatography, hydrophobic interactive chromatography, gel filtration, and a series of reverse phase HPLC steps. Polyacrylamide gel electrophoresis of fractions from the final HPLC step, which showed both activin immunoactivity and bioactivity, revealed a band with a mol wt of 25.3 kilodaltons (kDa), which reduced to 15.8 kDa, and a minor band of 45 kDa, which reduced to 25 kDa. NH2-terminal amino acid sequences of several active fractions from the same region were identical to the known sequence of ovine activin-A. The identification of immunoactive activin, follistatin, and inhibin in amniotic fluid raises the question of the sites of production of these proteins and their interactions and role in fetal physiology.

The negative feedback effects of testicular steroids are predominantly at the hypothalamus in the ram.

This study aimed to delineate the hypothalamic and/or pituitary actions of testosterone and its primary metabolites 5 alpha-dihydrotestosterone and estradiol (E) in adult castrated rams (wethers) during the breeding season. In Exp 1, wethers were treated for a week with twice daily injections (im) of peanut oil, 8, 16 or 32 mg/day testosterone propionate (TP) or dihydrotestosterone benzoate (DHTB) or an sc silastic implant containing 1 or 3 cm E. TP decreased plasma LH concentrations, increased (P less than 0.05) LH interpulse interval, did not have consistent effects on LH pulse amplitude, and had minimal effects on plasma FSH concentrations. DHTB decreased LH and FSH concentrations and increased (P less than 0.05) LH interpulse interval. E reduced (P less than 0.05) plasma LH and FSH concentrations and increased LH interpulse interval but had no effects on LH pulse amplitude. In Exp 2, hypothalamo-pituitary disconnected wethers given 125 ng GnRH every 2 h, were treated with either peanut oil, 32 mg/day TP or DHTB or 3 cm E. None of the treatments affected plasma LH or FSH concentrations or LH pulse amplitude. Exp 3 investigated the effects on GnRH of treatment of wethers either with peanut oil or TP. TP reduced GnRH concentrations (P less than 0.05) and pulse amplitude (P less than 0.01) and increased interpulse interval (P less than 0.05). These data provide evidence that, during the breeding season, the principal site of negative feedback of testicular steroids in the ram is the hypothalamus, resulting in decreased GnRH secretion; feedback effects at the pituitary are minimal.