Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.     

Hormonal regulation of spermatogenesis and spermiogenesis

Normal testicular function is dependent upon hormones acting through endocrine and paracrine pathways both in vivo and in vitro. Sertoli cells provide factors necessary for the successful progression of spermatogonia into spermatozoa. Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone which are the main hormonal regulators of spermatogenesis. Hormones such as testosterone, FSH and luteinizing hormone (LH) are known to influence the germ cell fate. Their removal induces germ cell apoptosis. Proteins of the Bcl-2 family provide one signaling pathway which appears to be essential for male germ cell homeostasis. In addition to paracrine signals, germ cells also depend upon signals derived from Sertoli by direct membrane contact. Somatostatin is a regulatory peptide playing a role in the regulation of the proliferation of the male gametes. Activin A, follistatin and FSH play a role in germ cell maturation during the period when gonocytes resume mitosis to form the spermatogonial stem cells and differentiating germ cell populations. In vitro cultures systems have provided evidence that spermatogonia in advance stage of differentiation have specific regulatory mechanisms that control their fate. This review article provides an overview of the literature concerning the hormonal pathways regulating spermatogenesis.     

Clonogenic culture of normal spermatogonia: in vitro regulation of postnatal germ cell proliferation

The stem cell properties of neonatal germ cells have recently been demonstrated by in vivo transplantation. Regulation of proliferation of these cells, however, is not yet understood, and an in vitro system is needed for directly testing the action of differentiation and proliferation-related factors for germ cells. We developed an in vitro model involving micromanipulation and a single-cell clonogenic assay in which results from independent experiments on spermatogonia and gonocytes have been analyzed and compared. Neonatal germ cells can be distinguished by their large size both in vivo and in vitro in a single-cell suspension. These cells are picked up singly using a micropipette and deposited into a 96-well plate precoated with an extracellular matrix component, e.g., collagen IV. The effect of growth factors or cocultured somatic cells was assayed by counting the percentage of wells containing a colony and comparing this percentage with that of control cultures. Addition of platelet-derived growth factor significantly shifted the modal colony size for gonocytes from >16-64 to >64-128 cells/colony (P < 0.001, chi2) but had no effect on spermatogonia-derived colony size and number. For testis somatic cell underlays, there was a profound inhibition of all colony types, and immunohistochemical staining of testis cell underlays showed inhibin/activinbetaA subunit expression. This finding suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant recovery (P = 0.046) of gonocyte-derived colony numbers but not spermatogonia-derived colonies, which may reflect the functional regulation by these factors observed in vivo. This proliferation assay also highlights many similarities in the regulation of gonocyte and spermatogonia proliferation in vitro, suggesting that proliferation potential is not noticeably affected by the transition of gonocytes to spermatogonia. For example, the average colony cloning efficiency was 80% for gonocytes and 76% for spermatogonia. This technology forms a basis for optimizing growth of neonatal germ cells for applications such as introduction of genetic material into the germ line to produce transgenic mice and to explore gene therapy.     

Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.     

Follicle-stimulating hormone regulates both Sertoli cell and spermatogonial populations in the adult photoinhibited Djungarian hamster testis

The hormones that regulate spermatogonial development are ill defined, in part due to lack of appropriate experimental models. The photoinhibited hamster model provides a rich source of spermatogonia, thus making it an ideal model to study their control. This study aimed to assess the effects of FSH, in the absence of testosterone, on the reinitiation of Sertoli cell and spermatogonial development in the photosensitive adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16L:8D) were exposed to short photoperiods (SD, 8L:16D) for 11 wk, leading to suppression of gonadotropins and regression of testicular function. Groups of 10 animals then received FSH alone or in combination with the antiandrogen, flutamide, for 7 days. Two control groups maintained either under long or short photoperiods were treated with vehicle. Sertoli and germ cell number were then determined using the optical disector (sic) stereological technique. The number of Sertoli cells, type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes were suppressed in SD controls to 66%, 34%, 19%, and 10% (all P < 0.01) of long-day control values, respectively. Later germ cell types were not detected. FSH treatment, with or without flutamide, increased Sertoli cell number (P < 0.01) to normal long-day values. Similarly, FSH treatment in the absence/presence of flutamide increased type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes to approximately 85%, 69%, and 80% (all P < 0.01) of long-day controls, respectively. Our data demonstrate that the reinitiation of spermatogonial maturation in this model is dependent on FSH in the presence of an antiandrogen. Surprisingly, the adult Sertoli cell population in this model is also hormone dependent. This naturally occurring model provides a unique opportunity to understand the mechanisms (apoptotic and/or proliferative) by which FSH regulates Sertoli and germ cell development in the adult animal.     

Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

Background  

Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA.     

Differential expression of inhibin subunits and follistatin, but not of activin receptor type II, during early murine embryonic development.

Activins are known to be potentially important regulators of early developmental processes in amphibians, birds, and mammalians. In this study we report the expression of the inhibin subunits, including those that make up activin, the activin-binding protein follistatin, and activin receptor type II in several in vitro systems that model early murine embryonic development, namely embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and their differentiated derivatives. In addition, we examine the expression pattern of these factors in different stages of the mouse embryo itself. Expression of inhibin alpha and beta A subunits is restricted to certain differentiated cell types, while beta B subunits are expressed in both differentiated and undifferentiated cells. Our results further indicate a change in the expression pattern of inhibin subunits during early development from beta B at the blastocyst stage largely to beta A in postgastrulation embryos. This is similar to the expression pattern at equivalent stages of Xenopus and chick development. Expression of the activin-binding protein follistatin is altered by the induction of differentiation of P19 EC and ES cells by several factors, including retinoic acid. In contrast to the inhibin subunits and follistatin, activin receptor levels are not influenced by differentiation in these cell types. The results of this study demonstrate that the inhibin subunits and follistatin, but not the activin receptor type II, are differentially expressed during early murine development and suggest that the different forms of activin/inhibin are involved in the regulation of different developmental processes.     

Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells

Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.     

Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.     

Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells

Recent reports suggest that activin (the dimer of inhibin beta subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogen-treated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin alpha- and beta A-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin alpha beta dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin alpha-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-alpha by activin, FSH, and activin plus FSH, respectively. TGF beta, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin alpha-subunit cRNA or beta-actin cDNA probes, and inhibin-alpha message levels were normalized with beta-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin alpha-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin alpha-subunit mRNA levels by FSH and activin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine follicle-stimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-microm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below % at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.     

Ultrastructure of germ cell development in the human fetal testis

Summary Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.Supported by a grant from the Ford Foundation and by General Research Support Grant RR055511 from the National Institutes of Health. Technical assistance was provided by Mrs. Lucy A. Conner.     

Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium

The gap junction proteins, connexins (Cx), are present in the testis and among them Cx43 play an essential role in spermatogenesis. By using an in vitro proliferation model of germ cells and Sertoli cells, we tempted here to clarify the role of Cx43 in the control of Sertoli and germ cell proliferation and apoptosis. Cx43 was detected in purified preparations of Sertoli cells and spermatogonia and immunolocalized in both cell types identified by vimentin and c-kit, respectively. Inhibition of gap junction coupling by the gap junction inhibitor α-GA significantly enhanced BrdU incorporation in Sertoli cells and reduced the number of activated caspase-3 positive germ cells. Similarly, inhibitory Cx43 and pan-Cx mimetic inhibitory peptides increased proliferation of Sertoli cells and stimulated survival of germ cells. Cx32 mimetic inhibitory peptide also stimulated Sertoli cell proliferation without altering germ cell proliferation and apoptosis. The present results reveal that Cx43 gap junctions between Sertoli cells participate in the control of Sertoli cell proliferation and that Cx43 gap junctions between Sertoli cells and spermatogonia are indirectly involved in germ cell number increase by controlling germ cell survival rather than germ cell proliferation.     

Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse

Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D(1) is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D(1) in spermatogonial proliferation, in particular during the G(1)/S phase transition. Cyclin D(2) is first expressed at the start of spermatogenesis when gonocytes produce A(1) spermatogonia. In the adult testis, cyclin D(2) is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A(al) spermatogonia differentiate into A(1) spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D(2) during spermatogenesis, cyclin D(2) expression was studied in vitamin A-deficient testis. Cyclin D(2) was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A(1) spermatogonia by administration of retinoic acid. Overall, cyclin D(2) seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A(1) spermatogonia. Cyclin D(3) is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D(3) expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D(3) may control G(1)/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D(1) and D(3) seem to be involved in cell cycle regulation, whereas cyclin D(2) likely has a role in spermatogonial differentiation.