Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis

In men, inhibin B is the circulating isoform involved in the regulation of follicle stimulating hormone (FSH) secretion. Within the testis, inhibin B may have a role in Sertoli and germ cell interactions, thus secretion into seminal plasma may reflect seminiferous tubule function. Using specific immunoassays, inhibin B was present in seminal plasma in fertile men (n = 105) and in unselected men attending an infertility clinic (n = 174) with a wide range in concentration from undetectable (<15 pg/ml) up to 54,100 pg/ml (geometric mean 280 pg/ml). There was a highly significant correlation between seminal plasma inhibin B concentration and sperm concentration (r = 0.46, P < 0.001), but no correlation with percentages of spermatozoa with progressive motility or normal morphology. Inhibin A and isoforms containing pro and alphaC immunoreactivity were not detectable. In post-vasectomy seminal plasma samples (18 of 20) inhibin B was undetectable, indicating that the testis is the predominant source. In unselected men attending an infertility clinic, inhibin B was undetectable in 17% (present in remainder; maximum concentration 26,200 pg/ml; mean 263 pg/ml), with a highly significant correlation between seminal plasma inhibin B and sperm concentration (r = 0.55, P < 0.0001). In men with oligo/ azoospermia (sperm concentration <20 x 10(6)/ml), seminal plasma inhibin B concentrations were lower in those with elevated plasma FSH concentrations (mean values 42 and 205 pg/ml, P < 0.05). Inhibin alpha and betaB subunits were localized predominantly in Sertoli and Leydig cells, using immunohistochemistry. We conclude that inhibin B of testicular origin is present in normal human seminal plasma, but with a very wide range in concentration, and may reflect the functional state of the seminiferous epithelium.      

Inhibin

Inhibin has been defined as a gonadal hormone that exerts a specific negative feedback action on the secretion of FSH from the gonadotropic cells of the pituitary gland. The existence of inhibin was postulated as early as 1923 (250). However, only after reliable and sensitive bioassay systems for the detection and estimation of inhibin had been developed and an ample source of inhibin was found in the form of ovarian follicular fluid has progress been made in the isolation and characterization of the hormone. It is apparent now that inhibin, which itself consists of a dimer of two different subunits, alpha and beta, is a member of a much larger family of (glyco)protein hormones and growth factors, which also includes Müllerian-inhibiting substance, transforming growth factor-beta, erythroid differentiation factor, an insect protein that plays an important role in differentiation, and the dimer of two inhibin beta-subunits, activin. The latter substance was reported to counteract the effects of inhibin in pituitary cells. Interactions between these regulatory substances will certainly be a field of major interest in the near future. Inhibin molecules, similar to those in the gonads, have been detected in placental tissue as well. The postulate of inhibin in seminal plasma, prostate, and gastric juice is apparently due to detection of substances that differ from gonadal inhibin. Most likely these substances, the amino acid sequences of which have been elucidated, have a biological function that differs from the suppression of peripheral levels of FSH in vivo. Inhibin is produced in the Sertoli cells in the testis and in the granulosa cells in the ovary. The production of inhibin is stimulated by FSH, but presently a lot of controversy exists about other factors that might play a role in the regulation of the production of inhibin. Because of the lack of reliable methods for estimation of peripheral levels of inhibin in humans and experimental animals, almost all evidence for the physiological importance of inhibin in the regulation of reproductive processes is derived from circumstantial evidence. From these indirect results, it appears likely that inhibin plays an important role in the feedback regulation of peripheral concentrations of FSH during the period in which Sertoli cells and granulosa cells, the target cells for FSH, divide, i.e., during puberty in male animals and during the development of ovarian follicles in female animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation and tissue remodelling, we hypothesized that activin A is involved in the paracrine regulation of decidualization. We examined the expression of activin receptors (ActRs) by semi-quantitative and real-time RT-PCR. mRNA for all ActR subtypes (Ia, Ib, IIa and IIb) was detected in endometrium, with maximal expression in the early secretory phase and in early pregnancy. ActR protein was localized exclusively to stromal and endothelial cells. This expression pattern was confirmed by in-situ hybridization. Activin bioavailability is locally regulated by its binding protein, follistatin, and also by the antagonist, inhibin. Inhibin competition for ActRII binding is enhanced by the binding protein, betaglycan. Follistatin and betaglycan were also detected in the endometrium, localized to stromal and epithelial cells. This co-expression of activin subunits, receptors and binding proteins indicates that stromal cells are capable of responding to activin, and that there is tight local regulation of activin action within the endometrium. As activin production is up-regulated in decidual cells, this provides further evidence for an involvement of activins during stromal cell decidualization.     

Localisation cellulaire de la sécrétion de l’inhibine B testiculaire

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an α and βB subunit. In adult testis, the cellular site of production of these subunits is still controversial: Leydig cells, Sertoli cells and/or germ cells. The immunohistological localization (monoclonal antibodies anti α and anti βB) of both sub-units and the expression patterns of their mRNA (in situ hybridization with RNA probes) were examined in adult testicular biopsies with normal spermatogenesis or spermatogenetic arrest. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin α subunit and expressed inhibin α subunit mRNA. Conversely, germ cells expressed the βB peptide (located from pachytene spermatocytes to round spermatids) and the βB subunit mRNA (located from spermatogonia to round spermatids). These results agree with the recent opinion that inhibin B is possibly a joint product of Sertoli cells and germ cells in adult men and it may be used as a serum marker of spermatogenesis.     

水通道蛋白1在生后小鼠精囊腺与前列腺发育过程中的表达与分布

目的 探讨水通道蛋白1(AQP1)在雄性小鼠出生后不同发育阶段精囊腺和前列腺的表达与分布.方法 应用RT-PCR、免疫印迹和免疫组织化学染色方法,检测出生后15d、35d、70d 小鼠精囊腺、前列腺AQP1 mRNA、蛋白表达水平及细胞定位.结果 RT-PCR与免疫印迹结果均显示,出生后70d小鼠精囊腺、前列腺AQP1 mRNA及蛋白表达量较出生后15d显著增强(P<0.05);免疫组织化学染色显示,AQP1蛋白仅表达于出生后15d、35d小鼠精囊腺平滑肌细胞、前列腺腺泡上皮细胞基膜及间质细胞,而此时期精囊腺上皮细胞AQP1蛋白表达呈阴性;出生后70d,AQP1蛋白强烈表达于精囊腺和前列腺上皮细胞.结论 AQP1 mRNA及蛋白在雄性小鼠精囊腺、前列腺的表达与分布随年龄增长而改变,此变化可能与雄性附属性腺发育相关.     

Infant feeding with soy formula milk: effects on puberty progression, reproductive function and testicular cell numbers in marmoset monkeys in adulthood

BACKGROUND: This marmoset study addresses concerns about feeding human male infants with soy formula milk (SFM). METHODS: From age 4 to 5 days, seven male co-twin sets were fed standard formula milk (SMA) or SFM for 5-6 weeks; blood samples were subsequently collected at 10-week intervals. Testes from co-twins killed at 120-138 weeks were fixed for cell counts. RESULTS: SFM- and SMA-fed twins showed normal weight gain; puberty started and progressed normally, based on blood testosterone measurements. Body weight, organ weights (prostate, seminal vesicles, pituitary, thymus and spleen) and penis length were comparable in co-twins. All SMA- and 6/7 SFM-fed males were fertile. Unexpectedly, testis weight (P = 0.041), Sertoli (P = 0.025) and Leydig cell (P = 0.026) numbers per testis were consistently increased in SFM-fed co-twins; the increase in Leydig cell numbers was most marked in males with consistently low-normal testosterone levels. Seminiferous epithelium volume per tubule showed a less consistent, non-significant increase in SFM-fed males; raised germ cell numbers per testis, probably due to increased Sertoli cells, conceivably resulted in larger testes. Average lumen size, although greater in SFM-fed group, was inconsistent between co-twins and the difference was not significant. CONCLUSIONS: Infant feeding with SFM has no gross adverse reproductive effects in male marmosets, though it alters testis size and cell composition, and there is consistent, if indirect, evidence for possible 'compensated Leydig cell failure'. Similar and perhaps larger changes likely occur in adult men who were fed SFM as infants.     

Follistatin and activin A in extra-embryonic coelomic and amniotic fluids and maternal serum in early pregnancy

Follistatin is a specific binding protein which controls bioavailability of activins and inhibins which have an important role in fetal development. In the first trimester of pregnancy bioactive dimeric inhibins are found at high concentrations in the extra- embryonic coelomic fluid, but the distribution of follistatin and activins is not known. We have used recently developed immunoassays for follistatin, activin A and activin AB to determine their presence in the intrauterine compartments during early pregnancy. Follistatin was present in highest concentrations in the extra-embryonic coelomic fluid (11.72 +/- 1.70 ng/ml; median +/- SEM), with less in maternal serum (6.35 +/- 4.58) and lowest amounts in amniotic fluid (0.97 +/- 0.52). Follistatin concentrations in extra-embryonic coelomic fluid were highly correlated with both dimeric inhibin isoforms. Activin A was present in only barely detectable amounts in some samples of extra- embryonic coelomic fluid (41% of samples) and maternal serum (26%) and was undetectable in all amniotic fluid samples. Activin AB was undetectable in all compartments. The presence of follistatin in the amniotic and extra-embryonic coelomic fluids may regulate the availability of bioactive activins and inhibins which are released into the intrauterine compartments during the development of the fetus and placenta in early pregnancy.      

Distribution and spatiotemporal relationship of activin a and follistatin in mouse decidual and placental tissue

Problem Cells responsible for the synthesis of follistatin and activin A in the pregnant mouse endometrium have not been characterized. Method of study Immunocytochemistry was used to determine the distribution of follistatin and activin A in the pregnant mouse uterus. Results Follistatin was detected in the endometrium prior to decidualization and embryo implantation. Follistatin was not seen in fully decidualized cells, being restricted to non-decidualized fibroblasts and cells in the process of decidualization. In contrast, activin A was detected exclusively in mature antimesometrial decidual cells during involution. After day eleven of pregnancy, both substances were identified in the extracellular matrix of the spongiotrophoblast. Conclusion As previously described for decidual prolactin-related protein and the proteoglycan perlecan, follistatin and activin A were detected in the extracellular matrix of the spongiotrophoblast, suggesting that this region acts as reservoir for these growth factors in the mouse placenta.     

Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes

The expression and localization of mRNA's for tissue plasminogen activator (tPA), urokinase PA (uPA), uPA receptor (uPAR) and inhibin subunits, alpha, beta A and beta B in monkey testes was investigated. Using in-situ hybridization with digoxigenin-labelled cRNA probes (dig- cRNA), we demonstrated that tPA and plasminogen activator inhibitor type 1 (PAI-1) were expressed in testes of both immature and mature rhesus monkeys. tPA mRNA was localized predominantly in Sertoli cells. Expression level was low in immature testis, increased dramatically in the adult and varied with seminiferous cycle. PAI-1 mRNA was localized mainly in germ cells except late spermatids. uPA mRNA was expressed stage-specifically in Sertoli cells of adult testis. uPA receptor mRNA was localized in germ cells of mature testis but not in spermatogonia or late spermatids. Assayed by fibrin overlay technique, PA activity in conditioned media of purified Sertoli cells (Sc) was negligible, PA activity in media obtained from co-cultured Sertoli and Leydig cells (LS), however, was significantly increased, although Leydig cells alone were not capable of producing any PA activity. Addition of follicle stimulating hormone (FSH) to the incubation medium remarkably increased PA secretion in both Sc and LS cultures. Human chronic gonadotrophin (HCG) had no significant effect on PA activity in the Sc culture but dramatically stimulated PA activity in the co-culture system. Dihydrotestosterone (DHT) did not mimic the effect of HCG. PAI-1 activity was secreted mainly by germ cells and did not differ between the two culture systems. FSH and forskolin inhibited PAI-1 secretion. Inhibin alpha, beta A and beta B subunit mRNAs were localized in Sertoli cells of adult monkey testes, with no obvious difference in the expression levels. These data suggest that PA/PAI-1 and other related factors are expressed in rhesus monkey testis under the control of various hormones, seminiferous cycle and cell-cell interactions through paracrine or autocrine regulation. Locally generated fibrinolysis may play an important role in the process of spermatogenesis.      

Changes in serum inhibin, activin and follistatin concentrations during puberty in girls

Serum concentrations of inhibin A, inhibin B, activin A and follistatin were determined using two-site enzyme-linked immunosorbent assays (ELISA) during pubertal ovarian development in 28 girls and five follicular phase women. Blood obtained every 15 to 20 min overnight was pooled for peptide determination. Serum inhibin A concentrations increased in mid puberty, exhibiting positive correlations with bone age (r = 0.527, P = 0.0016) and oestradiol concentrations (r = 0.581, P = 0.0005). Inhibin B concentrations peaked in mid puberty and declined thereafter, but remained greater than concentrations seen in prepubertal girls, and correlating positively with oestradiol (r = 0.362, P = 0.046) and follicle stimulating hormone (FSH) concentrations (r = 0.369, P = 0.038). Total activin A concentrations did not vary significantly across pubertal stages. Total follistatin concentrations, determined by radioimmunoassay, decreased with advancing puberty, exhibiting negative correlations with bone age (r = -0.634, P = 0.0001) and oestradiol concentration (r = -0.687, P = 0.0001). Follistatin concentrations determined by an ELISA specific for follistatin 288 were greatest in mid-pubertal girls, but concentrations in late puberty were less than those in early puberty. The free follistatin assay indicated that all circulating follistatin was activin-bound. These results suggest that significant changes in serum concentrations of FSH-regulatory peptides accompany the onset of puberty.     

Role of the prostate in the regulation of pituitary secretion of follicle stimulating hormone

The secretion of Follicle Stimulating Hormone by the pituitary gland is not regulated entirely by the negative feedback from gonadal steroids. There is ample evidence that the secretion of FSH is regulated by a non-steroidal hormone of gonadal origin termed "inhibin". Using a radioimmunoassay procedure, immunoreactive inhibin-like material was quantified in tissue and biological fluids to study its role in reproductive pathophysiology. High levels of immunoreactive inhibin were observed in human prostate as compared to testis. Biologically active as well as immunoreactive inhibin-like material (ILM) was obtained from human prostate, and was similar to that obtained from human seminal plasma under identical conditions. In patients with benign prostatic hypertrophy, high inhibin levels were observed which reduced after prostatectomy. The presence of immunoreactive inhibin in urine as well as semen of vasectomized and gonadectomized subjects indicates that prostate also secretes inhibin-like material. The specific receptors for inhibin as well as biosynthesis of inhibin in human prostate have been reported. Elevation of serum FSH levels following castration of adult male rats was further enhanced in animals where castration as well as prostatectomy was carried out. All these results demonstrate that apart from the testis, the prostate also secretes ILM and further suggest that the prostate along with testis, may play an important role in the regulation of pituitary secretion of FSH.     

The distribution of plasminogen activator in the male genital tract

The distribution of fibrinolytic activity in the tissues of the male genital tract was studied by a histological technique. Preparations made from testis, epididymis, vas deferens, seminal vesicle, prostate, bulbo-urethral gland, and urethra showed that most activity was related to the blood vessels. However, inconsistent fibrinolytic activity related to epithelium was found in all parts of the genital tract. This epithelial activity was least in the testis, greater in the seminal vesicle and prostate gland, and was greatest in the bulbo-urethral gland and terminal urethra. No fibrinolytic activity could be demonstrated in relation to spermatozoa.     

Serum activin A and follistatin concentrations during human pregnancy: a cross-sectional and longitudinal study

Activin A, a dimer of the betaA-subunit of inhibin, has been shown to have multiple biological activities and sites of production. Follistatin is a high-affinity binding protein for activin, which neutralizes its activity. This study provides the first data, using a cross-sectional design, on the measurement of both these proteins in the maternal circulation of a large cohort of women (6-39 weeks of gestation, n = 2-20 women/time point) during normal pregnancies, and confirms that similar patterns are seen in nine women studied longitudinally during pregnancy. The concentrations of total activin A were measured using a specific two-site enzyme-linked immunosorbent assay (ELISA), and a new radioimmunoassay for measuring total follistatin in serum utilizing dissociating reagents to eliminate the interference of activin is described. At 38-39 weeks gestation, both activin A and follistatin concentrations rose to a peak (4.59 +/- 0.54 ng/ml and 72.7 +/- 3.31 ng/ml, respectively). The activin A and follistatin concentrations were highly correlated both in the cross-sectional study (P <0.0001) and in individual women in the longitudinal study (P <0.05-0.0001). Concentrations of follistatin showed a greater increase in the second trimester of pregnancy relative to activin A concentrations. The parallel increase in the secretion of these two proteins throughout pregnancy probably reflects feto-placental secretion.     

Activin C Antagonizes Activin A in Vitro and Overexpression Leads to Pathologies in Vivo

Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-β_C subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-β_C. Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LβT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-β promoter. Transgenic mice that overexpress activin-βC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-β_C antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-βC immunoreactivity. This study provides evidence that activin-β_C is an antagonist of activin A and supplies an impetus to examine its role in development and disease.     

Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma

BACKGROUND: Activin A is a member of the transforming growth factor-beta superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin. OBJECTIVE: To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2-driven mucosal inflammation in a murine model of allergic asthma. METHODS: Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin-sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes. RESULTS: Follistatin was released concurrently with activin A suggesting it acts as an endogenous regulator: peak BAL concentrations coincided with maximal airway eosinophilia, and frequency of IL-4, IL-5 and IL-13 producing cells in mediastinal lymph nodes but induction lagged behind the onset of inflammation. Follistatin and activin A immunoreactivity were lost in airway epithelial cells in parallel with goblet cell metaplasia. Exogenous follistatin inhibited the allergen-specific Th2 immune response in mediastinal lymph nodes and mucus production in the lung. CONCLUSION: Follistatin is preformed in the normal lung and released in concert with activin A suggesting it serves as an endogenous regulator. Disturbance of the fine balance between activin A and its endogenous inhibitor follistatin may be a determinant of the severity of allergic inflammation or tissue phenotypic shift in asthma.     

Can vasectomy reduce the incidence of prostatic tumor?

Results from our laboratory have revealed that seminal plasma concentration of acid phosphatase, maltase, prolactin, citric acid, zinc and magnesium which are the secretory products of the prostate gland, decreased significantly in vasectomized men compared to those in controls namely normal fertile men. Further, it was observed that the decrease in prostatic function was not related to the time since vasectomy. Considering these two facts together, we propose that vasectomy may lead to decrease in the incidence of prostatic tumors — a disease that claims nearly 22,000 lives each year in the United States alone.     

Genetic engineering to study testicular tumorigenesis

In humans, Sertoli cell tumors account for approximately 4% of all testicular tumors, and 20% of these are malignant. The mechanisms underlying Sertoli cell tumorigenesis remain largely unknown.Using gene knockout technology, we previously generated mutant mice lacking the alpha subunit of inhibin dimers. The inhibin alpha-null male mice develop testicular Sertoli cell tumors with 100% penetrance. These tumors develop as early as 4 weeks of age and cause a cachexia-like wasting syndrome. Castrated inhibin alpha knockout mice develop sex steroidogenic adrenal cortical tumors. These studies have identified inhibins as secreted tumor suppressors with specificity for the gonads and adrenal glands. It had been suggested that endocrine factors play roles in Sertoli cell tumorigenesis by altering cell cycle machinery of the Sertoli cells. To test the potential of these factors to function as modifiers of Sertoli cell tumorigenesis, we have employed a genetic intercross strategy, breeding inhibin a mutant mice with mutant mice deficient in endocrine signaling factors including gonadotropin releasing hormone (hypogonadal, hpg mice), follicle stimulating hormone, anti-Miillerian hormone (AMH), activin receptor type II, or androgen receptor (testicular feminization, tfm mice), or mice overexpressing follistatin. We are also investigating the effects of loss of critical cell cycle regulators, such as cyclin dependent kinase inhibitor p27, on Sertoli cell tumorigenesis in inhibin alpha knockout males. These studies clearly demonstrate the roles of these factors as modifiers of the Sertoli cell tumorigenesis. Activin signaling through activin receptor type II is responsible for the cachexia-like syndrome observed in the inhibin a knockout mice with tumors. The gonadotropin hormones are essential for testicular tumor development, but elevated FSH levels are not sufficient to cause Sertoli cell tumors. Absence of FSH, lack of androgen receptor, or overexpression of follistatin slows the tumor growth and minimizes the cachexia symptoms, thus prolonging the life span of these double mutant mice. In contrast, absence of AMH or p27 causes earlier onset and more aggressive development of testicular tumor, with an earlier death of double mutant mice. We are currently investigating roles of estrogen signaling pathways, and other cell cycle regulators, in tumor development in the inhibin alpha knockout mice by generating mice with double or triple mutations. Genetic engineering in mouse models provides a powerful tool to study the mechanisms of testicular tumorigenesis and define the important genetic modifiers in vivo.