Activin B is produced early in antral follicular development and suppresses thecal androgen production.

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (ßA, ßB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin ßB mRNA levels decreased as follicle diameter increased from 1-3 to >6  mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin ßA mRNA at 3-4  mm, and TCs more inhibin α mRNA at >4  mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1-3  mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin ßA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).

Heterogeneous Subgreenschist Deformation in an Exhumed Sediment-Poor Mélange.

Many described subduction complexes (or mélanges) exhumed from seismogenic depths comprise thick, turbidite-dominated sequences with deformed zones containing clasts or boudins of more competent sandstone and/or basalt. In contrast, many active subduction zones have a relatively small thickness of sedimentary inputs (<2 km), turbidite sequences are commonly accreted rather than subducted, and the role of pelagic sediments and basalt (lavas and hyaloclastites) in the deforming zone near the plate interface at <20 km depth is poorly understood. Field investigation of Neoproterozoic oceanic sequences accreted in the Gwna Complex, Anglesey, UK, reveals repeated lenticular slices of variably sampled ocean plate stratigraphy (OPS) bounded by thin mélange-bearing shear zones. Mélange matrix material is derived from adjacent OPS lithologies and is either dominantly illitic, likely derived from altered siliciclastic sediment, or chloritic, likely derived from altered volcanics. In the illitic mélange, mutually cross-cutting phyllosilicate foliation and variably deformed chlorite-quartz-calcite veins suggest ductile creep was cyclically punctuated by transient, localized fluid pulses. Chlorite thermometry indicates the veins formed at 260 ± 10°C. In the chloritic mélange, recrystallized through-going calcite veins are deformed to shear strains of 4-5 within a foliated chlorite matrix, suggesting calcite veins in subducting volcanics may localize deformation in the seismogenic zone. Shear stress-strain rate curves constructed using existing empirical relationships in a simplified shear zone geometry predict that slip velocities varied depending on pore fluid pressure; models predict slow slip velocities preferentially by frictional sliding in chlorite, at pore fluid pressures greater than hydrostatic but less than lithostatic.

Structural and composition of the N2 neuraminidase of influenza virus. Effect of carbohydrate content on the validity of molecular weight estimations.

Neuraminidase was isolated by proteolysis of the X7-(F1) (HON2) strain of influenza virus, and purified by gel filtration. The molecule contained a total of 46% (w/w) carbohydrate. The Mr was estimated as 152 500 (sedimentation diffusion) and 147 000 (sedimentation equilibrium). In 6 M guanidine-HCl the molecular weight was halved to 66 000 (sedimentation equilibrium). After irreversible reduction and blocking of sulphydryl groups the molecular weight was halved again to 33 500 (sedimentation equilibrium). These results confirm the tetrameric model of neuraminidase structure. They also provide strong evidence that the tetramer is composed of two disulphide linked dimers, themselves associated by non-covalent linkages. Theoretical considerations based on this model predict that assembly of the molecule must be accompanied by allosteric conformational changes in the subunits. The high carbohydrate content was thought to explain the discrepancy between the molecular weight values for the neuraminidase polypeptide obtained by different methods, and also the exceptional resistance of the molecule to digestion by proteolytic enzymes.

betaA- and betaC-activin, follistatin, activin receptor mRNA and betaC-activin peptide expression during rat liver regeneration.

The mRNA expression of two activin growth factor subunits (betaA- and betaC-activin), activin receptor subunits (ActRIIA, ActRIIB) and the activin-binding protein follistatin, and peptide expression of betaA-activin and betaC-activin subunits, were examined in regenerating rat liver after partial hepatectomy (PHx). Liver samples were collected from adult, male Sprague-Dawley rats, 12-240 h (n=3-5 rats per time point) after PHx or from sham-operated controls at the same time points. Hepatocyte mitosis and apoptosis were assessed histologically and by in situ cell death detection. RT and PCR were used to assess relative gene expression. betaA- and betaC-activin peptide immunoreactivity was assessed in liver and serum samples by western blotting, whereas cellular expression was investigated by immunohistochemistry, using specific monoclonal antibodies. betaA- and betaC-activin mRNA dropped to < 50% of sham control values 12 h after PHx and remained at this level until 168 h post-PHx, when betaA-activin expression increased to three times sham control values and betaC-activin mRNA returned to pre-PHx levels. A peak in follistatin expression was observed 24-48 h post-PHx, coincident with an increase in hepatocyte mitosis. No changes were observed in ActRIIA mRNA, whereas ActRIIB expression paralleled that of betaA-activin mRNA. betaC-activin immunoreactive homo- and heterodimers were observed in regenerating liver and serum. Mitotic hepatocytes frequently contained betaC-activin immunoreactivity, whereas apoptotic hepatocytes were often immunoreactive for betaA-activin. We conclude that betaA- and betaC-activin subunit proteins are autocrine growth regulators in regenerating liver and when expressed independently lead to hepatocyte apoptosis or mitosis in a subset of hepatocytes.

Oocyte-expressed genes affecting ovulation rate.

From examination of inherited patterns of ovulation rate in sheep, several breeds have been identified with point mutations in two growth factor genes (BMP15 and GDF9) and a related receptor (ALK6) that are expressed in oocytes. Five different point mutations have been identified in the BMP15 gene, one in GDF9 and one in ALK6. Animals heterozygous for these mutations or heterozygous for two of these mutations or homozygous for the ALK6 mutation have higher ovulation rates (i.e. +0.6-10) than their wild-type contemporaries. Animals homozygous for the BMP15 or GDF9 mutations are sterile due to arrested follicular development from the primary stage of growth. The BMP15 and GDF9 mutations are thought to result in reduced levels of mature protein or altered binding to cell-surface receptors. In sheep, GDF9 mRNA is present in germ cells before and after ovarian follicular formation as well as throughout follicular growth, whereas BMP15 mRNA is found in oocytes only from the primary stage of growth. Also ALK6 together with related cell-surface receptors such as ALK5 and BMPRII mRNA are present in oocytes at most, if not all, stages of follicular growth. Both GDF9 and BMP15 proteins are present in follicular fluid indicating that they are secreted products. Immunisation of sheep with GDF9 or BMP15 peptides shows that both growth factors are essential for follicular development, ovulation and/or corpus luteum formation. In animals with the ALK6 mutation, ovarian follicles undergo precocious maturation leading to three to seven follicles ovulating at smaller diameters without any increase above wild-types in the ovarian secretions of steroid or inhibin. One important consequence of the ALK6 mutation appears to be a decreased ability of some BMPs to inhibit differentiation of follicular cells. Current findings in sheep suggest that BMP15, GDF9 and ALK6 are targets for new methods of fertility regulation in some mammals.

The secretion and effect of inhibin A, activin A and follistatin on first-trimester trophoblasts in vitro.

OBJECTIVE: The objectives of this study were to investigate the effect of activin A and follistatin on first-trimester cytotrophoblast invasion in culture and to study the secretion of inhibin A, activin A and follistatin by these cells in vitro. DESIGN AND METHODS: Cytotrophoblasts were isolated from human placental chorionic villous tissue obtained from 6-8, 8-10 and 10-12 weeks gestation. Cells were cultured for 3 days on cell-culture inserts coated with gelatine for invasion studies and in 24-well culture plates for secretion studies. The effects of activin A (10 ng/ml), follistatin (100 ng/ml), interleukin 1beta (IL-1beta; 10 ng/ml) and epidermal growth factor (EGF; 10 ng/ml) on cytotrophoblast invasion were investigated using a non-radioactive invasion assay. Secretion of inhibin A, activin A and follistatin in the presence of EGF, IL-1beta, activin A and follistatin were measured using in-house ELISAs. RESULTS AND CONCLUSION: Activin A, follistatin and EGF had a significant stimulatory effect on cytotrophoblast invasion from 6-10 weeks gestation. IL-1beta had a significant stimulatory effect at 8-10 weeks and a significant inhibitory effect on invasion at 10-12 weeks gestation. Follistatin also had a significant inhibitory effect on invasion at 10-12 weeks gestation. In the secretion study, activin A secretion at 8-10 weeks was significantly stimulated by IL-1beta and EGF. At 10-12 weeks, follistatin and EGF had a significant inhibitory effect on activin A secretion. Follistatin secretion was significantly increased in the presence of IL-1beta at 6-8 weeks gestation. Inhibin A secretion was not significantly altered by EGF, IL-1beta, activin A and follistatin. These results show that activin A promotes invasion of first-trimester cytotrophoblasts until 10 weeks gestation. There is a difference in the control of secretion of these proteins dependent on the gestation, suggesting that there is a tight regulation in the function of first-trimester trophoblasts depending on the gestational age.

Monoclonal antibodies reactive with myelin basic protein.

New monoclonal antibodies (MAbs) to myelin basic protein (BP) reveal epitopes to be in sequences 22-34, 75-82, 83-96, 118-131 and 125-131. Comparison of these results with those previously reported suggest that almost every sequence of about 10 amino acid residues may be sufficiently antigenic to make a single MAb but that certain regions are immunodominant, strong enough to make practically the same MAb repeatedly. One of these new MAbs (clone 3) has especially interesting reactivity, sharply limited to residues 75-82 in bovine and porcine BP: Lys-Ala-Gln-His-Gly-Arg-Pro. Whales presumably have the same sequence, since their BPs are fully reactive with clone 3 MAb, but all other species of BP, with known sequences of BP, have at least two changes in this sequence. Deletion of Lys75 (as in a tryptic peptide of porcine BP) reduces reactivity with the MAb about 10-fold, whereas substitution of Ala76 by Ser (as in all other species of BP) and either deletion of Gln77 (as in human, monkey and rabbit BP) or His78 (as in the guinea pig and rat BP) or substitution of Pro82 by Thr (as in human, monkey, rat and mouse BP) eliminates reactivity. We speculate that woodchuck and prairie dog BPs in this region closely resemble chicken BP, which has about 2% of the original reactivity. However, squirrel BP is unique, probably having only one of the changes in this region of BP, since it possesses 10-20 times the reactivity of chicken BP but still only 20-50% of the original reactivity with clone 3 MAb, a degree of reactivity not seen with any other species of BP.

Dimeric inhibin A and B production are differentially regulated by hormones and local factors in rat granulosa cells.

In the present study, we have examined the role of hormones and growth factors in regulating dimeric inhibin production in immature rat granulosa cells. Purified granulosa cells from estrogen-primed immature rats were cultured under defined conditions. Inhibins A and B in the culture media were measured using a two-site enzyme-linked immunosorbent assay specific for each dimer. Under basal conditions, granulosa cells produced 14-fold more inhibin A than inhibin B (inhibin A, 2.0; inhibin B, 0.14 ng/ml, measured against human standards; average A/B apparent ratio, 14). Addition of increasing doses of FSH elicited dose-dependent increases in both inhibins, the effects being more pronounced on inhibin A than on inhibin B (9.4- and 4.1-fold increases, respectively; average A/B ratio, 34). Estradiol, when added alone, stimulated inhibin A production 3- to 6-fold, whereas minor changes were observed in inhibin B production. Insulin-like growth factor-I produced a similar stimulation of both inhibins (3-fold stimulation over control). This growth factor, however, induced a marked dissociation in the sensitivity of inhibins A and B to FSH stimulation, with maximal stimulation of inhibin B observed at comparatively lower concentrations of the gonadotropin. Transforming growth factor-beta (TGF-beta, 5 ng/ml) had a more marked stimulatory effect on inhibin B than on inhibin A production (7- to 14-fold vs. 2- to 5-fold for inhibin B and A, respectively). A more pronounced differential stimulation of inhibin B was also exerted by another member of the TGF-beta superfamily, activin A (A/B ratio, 0.66). This preferential stimulation of inhibin B by TGF-beta and activin A was amplified in the presence of FSH. Coculture of rat granulosa cells with freshly isolated bovine oocytes was also associated with a marked stimulation of inhibin B production (100-fold increase) and a comparatively lower stimulation of inhibin A (10-fold increase; A/B ratio, 1). The discrepancy between the proportion of inhibin dimers in serum (A/B ratio, 0.13) and those produced by untreated granulosa cells may suggest that intraovarian factors, such as TGF-beta, activin A, or oocyte-derived factor(s), are responsible for the shift of the ratio toward the predominance of inhibin B.

Activin A release into the circulation is an early event in systemic inflammation and precedes the release of follistatin.

Recent evidence suggests a role for activin A, and its binding protein, follistatin, in inflammatory pathways. However, whether activin is released systemically during inflammation is not known. In this study, a release of activin A into the circulation occurred in sheep within 1 hour of injection of lipopolysaccharide. This rapid peak in activin A preceded the release of the key inflammatory cytokines, tumor necrosis factor-alpha and interleukin-6. Follistatin release into the circulation occurred some 4 hours after the peak of activin A and continued out to 24 hours from lipopolysaccharide treatment. These data are the first to document a circulatory response of activin A to an inflammatory stimulus, and together with previous findings, suggest that activin A may have both pro- and anti-inflammatory actions in regulating cytokine-driven pathways.

Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner.

Inhibin A and inhibin B are related dimeric protein hormones and endocrine regulators of the reproductive axis. Specifically, inhibin inhibits FSH secretion from the anterior pituitary. The inhibins are synthesized by the gonads and are themselves modulated by FSH. Although the activity of these ligands has been well characterized, the circulating concentrations of dimeric inhibin A and dimeric inhibin B have not previously been reported for the rat. Our group examined the serum concentration of inhibin A and inhibin B in normally cycling female rats, male rats, and in gonadectomized animals. Both inhibin isoforms are detected in intact female rat serum. Interestingly, inhibin B, but not inhibin A, is detected in intact male rat serum. Neither inhibin isoform is detected in long-term castrate female or male rats. In normally cycling female rats, inhibin A was low on the morning of metestrus and rose steadily to a peak on proestrus. In contrast, inhibin B was elevated on the mornings of metestrus, diestrus, and proestrus. Both ligands persisted in the serum until proestrus evening. Serum inhibins then declined beginning at 2100 h (inhibin A) or 1800 h (inhibin B) on proestrus, and the concentrations reached a nadir on the morning of estrus (0600 h). The nadir coincided with the peak of the secondary FSH surge. Both inhibins rebounded later on the morning of estrus. The results of this study demonstrate that dimeric, ovarian-derived inhibin A and inhibin B circulate in the female rat. The inverse relationship of the inhibins during the secondary FSH surge is consistent with the hypothesis that they participate in the regulation of reproductive cyclicity. The differing patterns of inhibin A and inhibin B during the period of follicular development on metestrus and diestrus suggest different follicle sources or regulation of these molecules during this period. We further demonstrate that inhibin B is the dominant form of FSH regulating protein in the male rat.

Differential changes in inhibin A, activin A, and total alpha-subunit levels in granulosa and thecal layers of developing preovulatory follicles in the chicken.

Accumulating evidence implicates inhibins and activins as endocrine and local regulators of follicular development in mammals, and it was recently confirmed that inhibin/activin alpha and betaA genes are also expressed in the avian ovary. To investigate the potential involvement of these proteins in the chicken ovary, thecal and granulosa layers of the four largest follicles (F1-F4) and the most recent postovulatory follicle were collected from hens (10/group) killed 4, 12, and 20 h before the expected time of F1 ovulation. Inhibin A and activin A concentrations of tissue extracts (expressed per mg DNA) were measured using validated two-site enzyme-linked immunosorbent assays; total immunoreactive inhibin alpha-subunit (ir-alpha) was also measured by heterologous RIA (Monash assay). Inhibin A and ir-alpha were largely confined to the granulosa layer, whereas activin A was much more abundant in the thecal layer. Granulosa inhibin A contents were similar in F4 and F3, but increased approximately 40-fold from F3-F1 (P < 0.0001). As such, the F1 granulosa layer was by far the richest source of inhibin A in the chicken ovary, but contained very little activin A. Total ir-alpha in granulosa was much more abundant than inhibin A and increased only 3-fold from F4-F1 (P < 0.001). Activin A in both granulosa and theca showed little variation between F1 and F4 follicles (by ANOVA, P > 0.05). The inhibin A content of F1 granulosa was maximal 12 h before ovulation and had fallen approximately 6-fold (P < 0.0001) within 8 h, suggesting an inhibitory effect of the preovulatory LH surge on the F1 capacity to synthesize inhibin A. Inhibin A, activin A, and ir-alpha were all less in the postovulatory follicle compared with F1 before ovulation (P < 0.0001). In conclusion, application of the present two-site enzyme-linked immunosorbent assays to the chicken ovary revealed 1) divergent tissue distribution of inhibin A and activin A within preovulatory follicles, and 2) differential regulation of granulosa cell production of inhibin A and activin A dimers during preovulatory follicular development. These findings of dynamic changes in inhibin A, activin A, and total ir-alpha support the hypothesis that these proteins subserve regulatory roles during preovulatory follicular development in the hen.

Differential localization of inhibin subunit proteins in the ovine testis during fetal gonadal development.

Inhibins and activins are dimeric proteins that are involved in cell proliferation, apoptosis, and differentiation in a number of systems and have previously been detected in fetal testes of many species. This study used immunohistochemistry to examine the localization of inhibin alpha-, betaA-, and betaB- subunits during ovine testicular development from days 40-135 of gestation. Localization of inhibin betaA- and betaB-subunit messenger RNAs was confirmed by in situ hybridization. The results showed that there was differential localization of inhibin alpha-, betaA-, and betaB-subunits to specific cells in the ovine fetal testis from 40 days of gestation. All three inhibin subunits were present in Sertoli cells throughout gestation, whereas the rete epithelium and gonocytes did not express inhibin alpha-subunit. These data suggest that the fetal Sertoli cells have the capacity to produce all forms of inhibins and activins, i.e. inhibin A and B, and activins A, AB, and B, whereas the rete testis epithelial cells can only synthesize activin A. In the interstitium, the fetal Leydig cells expressed all three inhibin subunits, but this was restricted to the period between 40 and 90 days of gestation. Thereafter, inhibin alpha-subunit immunoreactivity was not observed in fetal Leydig cells, which suggests that only activin ligands are produced by Leydig cells during late gestation. Collectively, the data demonstrate that fetal ovine testes have the potential to produce the full repertoire of inhibins and activins from very early in testicular differentiation. The distinct and restricted localization of the various subunits to specific cells suggests that specific dimeric proteins have particular roles in the development and function of the fetal testis.

Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels.

This study investigated whether neonatal exposure of male rats to estrogenic compounds altered pubertal spermatogenesis (days 18 and 25) and whether the changes observed resulted in long-term changes in testis size, mating, or fertility (days 90-100). Rats were treated neonatally with a range of doses (0.01-10 microg) of diethylstilbestrol (DES; administered on alternate days from days 2-12), a high dose of octylphenol (OP; 2 mg administered daily from days 2-12) or bisphenol A (Bis-A; 0.5 mg administered daily from days 2-12), or vehicle, while maintained on a standard soy-containing diet. The effect on the same parameters of rearing control animals on a soy-free diet was also assessed as was the effect of administering such animals genistein (4 mg/kg/day daily from days 2-18). Testis weight, seminiferous tubule lumen formation, the germ cell apoptotic index (apoptotic/viable germ cell nuclear volume), and spermatocyte nuclear volume per unit Sertoli cell nuclear volume were used to characterize pubertal spermatogenesis. Compared with (soy-fed) controls, DES administration caused dose-dependent retardation of pubertal spermatogenesis on day 18, as evidenced by decreases in testis weight, lumen formation, and spermatocyte nuclear volume per unit Sertoli cell and elevation of the germ cell apoptotic index. However, the two lowest doses of DES (0.1 and 0.01 microg) significantly increased spermatocyte nuclear volume per unit Sertoli cell. Similarly, treatment with either OP or Bis-A significantly advanced this and some of the other aspects of pubertal spermatogenesis. Maintenance of control animals on a soy-free diet also significantly advanced lumen formation and spermatocyte nuclear volume per unit Sertoli cell compared with controls fed a soy-containing diet. Administration of genistein reversed the stimulatory effects of a soy-free diet and significantly retarded most measures of pubertal spermatogenesis. In general, plasma FSH levels in the treatment groups changed in parallel to the spermatogenic changes (reduced when pubertal spermatogenesis retarded, increased when pubertal spermatogenesis advanced). By day 25, although the changes in FSH levels largely persisted, all of the stimulatory effects on spermatogenesis seen on day 18 in the various treatment groups were no longer evident. In adulthood, testis weight was decreased dose dependently in rats treated neonatally with DES, but only the lowest dose group (0.01 microg) showed evidence of mating (3 of 6) and normal fertility (3 litters). Animals treated neonatally with OP or Bis-A had normal or increased (Bis-A) testis weights and exhibited reasonably normal mating/fertility. Animals fed a soy-free diet had significantly larger testes than controls fed a soy-containing diet, and this difference was confirmed in a much larger study of more than 24 litters, which also showed a significant decrease in plasma FSH levels and a significant increase in body weight in the males kept on a soy-free diet. Neonatal treatment with genistein did not alter adult testis weight, and although most males exhibited normal mating and fertility, a minority did not mate or were infertile. It is concluded that 1) neonatal exposure of rats to low levels of estrogens can advance the first wave of spermatogenesis at puberty, although it is unclear whether this is due to direct effects of the estrogen or to associated elevation of FSH levels; 2) the effect of high doses of OP and Bis-A on these processes is essentially benign; and 3) the presence or absence of soy or genistein in the diet has significant short-term (pubertal spermatogenesis) and long-term (body weight, testis size, FSH levels, and possibly mating) effects on males.

Testicular expression of inhibin and activin subunits and follistatin in the rat and human fetus and neonate and during postnatal development in the rat.

Inhibins, activins, and follistatins are all believed to play roles in the regulation of FSH secretion by the pituitary and in the paracrine regulation of testis function. Previous studies have resulted in conflicting data on the pattern of expression of the inhibin/activin subunits, and little information on expression of follistatin during fetal/neonatal life. We have made use of new, highly specific monoclonal antibodies and fixed tissue sections from fetal, neonatal, and adult rats, and limited amounts of fetal and neonatal human testis, to undertake a detailed immunocytochemical study of the pattern of expression of these regulatory proteins. In the rat, positive immunostaining for the alpha-subunit of inhibin (alpha) was first detectable on day 14.5 post coitum (p.c.), the first day on which the testis could be morphologically distinguished from the ovary. During fetal life, the alpha-immunostaining was most prominent in the fetal Leydig cells. In Sertoli cells, alpha-immunostaining was slightly stronger on days 14.5 and 15.5 p.c. compared with 16.5-20.5. After birth, alpha-immunostaining remained intense in fetal Leydig cells but declined following their replacement with their adult-type counterparts; in contrast, alpha-subunit increased in Sertoli cells immediately after birth. Immunostaining with antibodies specific to betaB-subunit showed a similar pattern to that of the alpha-subunit, except that positive immunostaining was first detectable on day 16.5 p.c., 2 days later than immunostaining for the alpha-subunit. The pattern of betaB-immunostaining in postnatal samples paralleled that of the alpha-subunit. Immunostaining using antibodies against the betaA-subunit did not produce any significant reaction product in any sample. Follistatin was undetectable in the fetal rat testis but appeared in the Leydig cells immediately after birth and its expression remained intense throughout postnatal development and in adult testis. No evidence was obtained for expression of either the inhibin/activin subunits or follistatin in the germ cells, peritubular myoid cells, or other interstitial cells in any of the sections examined. In the human fetal testis, both alpha- and betaB-subunits were immunodetectable at 16, 18, and 24 weeks gestation in Sertoli and Leydig cells, with stronger immunostaining in Sertoli cells at 24 weeks. Postnatally at 4 months, immunoexpression of the betaB-subunit was no longer detectable, whereas the alpha-immunostaining became weaker but was still present in both Sertoli and Leydig cells. No positive immunostaining for betaA-subunit or follistatin was detectable at any time point studied. In conclusion, we have shown that, in the rat testis, the majority of inhibin alpha-subunit and inhibin/activin betaB-subunit is immunolocalized to the fetal-type Leydig cells during fetal/neonatal life but, following birth, immunoexpression in the Sertoli cells of both subunits increases markedly while follistatin is immunodetectable only postnatally.

Expression of activin A and follistatin core proteins by human prostate tumor cell lines.

Activin and follistatin (FS) messenger RNA and protein are expressed and localized to human prostate tissue from men with high grade cancer and to human prostate tumor cell lines LNCaP, DU145, and PC3. Although activin A induces apoptosis and inhibits cell proliferation in LNCaP cells, PC3 cells are insensitive to the effect of exogenous addition of activin A. The results of this study show that activin A and FS are produced and can be measured by specific enzyme-linked immunosorbent assays in PC3 cells and media but are not detectable in LNCaP cells. Over 10 days in culture, the production of activin A by PC3 cells declines and is inversely correlated (r = -0.779) to FS288 production, which steadily increases and is significantly elevated compared with Day 1 of culture. The presence of FS288 and FS315 proteins was confirmed by immunocytochemistry and showed that only PC3 cells produced the FS288 isoform. Western blotting of PC3 cell media confirmed the presence of the FS288 isoform. Blockade of FS288 activity with a neutralizing antibody rendered PC3 cells responsive to activin A, as measured by inhibition of proliferation. Collectively, these results suggest that PC3 tumor cells are insensitive to activin A because they produce measurable amounts of activin ligand and FS288 protein, which is capable of blocking the autocrine response of these cells to activin A.

Permanent effects of neonatal estrogen exposure in rats on reproductive hormone levels, Sertoli cell number, and the efficiency of spermatogenesis in adulthood.

This study aimed to identify the mechanism(s) for impairment of spermatogenesis in adulthood in rats treated neonatally with estrogens. Rats were treated (days 2-12) with 10, 1, or 0.1 microg diethylstilbestrol (DES), 10 microg ethinyl estradiol (EE), 10 mg/kg of a GnRH antagonist (GnRHa), or vehicle and killed in adulthood. DES/EE caused dose-dependent reductions in testis weight, total germ cell volume per testis, and Sertoli cell volume per testis. Sertoli cell number at 18 days of age in DES-treated rats was reduced dose dependently. GnRHa treatment caused changes in these parameters similar to those in rats treated with 10 microg DES. Plasma FSH levels were elevated (P < 0.001) to similar levels in all treatment groups regardless of differences in Sertoli cell number and levels of inhibin B; the latter reflected Sertoli cell number, but levels were disproportionately reduced in animals treated with high doses of DES/EE. Neonatal estrogen treatment, but not GnRHa, caused dose-dependent reductions (40-80%) in plasma testosterone levels in adulthood, but did not alter LH levels. Preliminary evidence suggests that the decrease in testosterone levels in estrogen-treated rats is not due to reduced Leydig cell volume per testis. GnRHa-treated rats exhibited a significant increase in germ cell volume per Sertoli cell and a reduction in germ cell apoptosis, probably because of the raised FSH levels. Despite similar raised FSH levels, rats treated with DES (10 or 1 microg) or EE (10 microg) had reduced germ cell volume/Sertoli cell and increased germ cell apoptosis, especially when compared with GnRHa-treated animals. The latter changes were associated with an increase in lumen size per testis, indicative of impaired fluid resorption from the efferent ducts, resulting in fluid accumulation in the testis. Rats treated neonatally with 0.1 microg DES showed reduced germ cell apoptosis comparable to that in GnRHa-treated animals. The changes in apoptotic rate among treatment groups occurred across all stages of the spermatogenic cycle. It is concluded that 1) neonatal estrogen treatment results in dose-dependent alterations in Sertoli cell numbers, germ cell volume, efficiency of spermatogenesis, and germ cell apoptosis in adulthood; 2) the relatively poor spermatogenesis in estrogen-treated animals is most likely due to altered testis fluid dynamics and/or altered Sertoli cell function; 3) as indicated by FSH (LH) and testosterone levels, the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than the Sertoli cells to reprogramming by estrogens neonatally; and 4) elevated FSH levels in adulthood may improve the efficiency of spermatogenesis.

Inhibin A and activin A in the first trimester of human pregnancy.

Recent studies show that high concentrations of inhibin A and activin A are present in the maternal serum throughout human pregnancy. The aim of this study was to determine whether the corpus luteum produces significant quantities of inhibin A and activin A during the first trimester of pregnancy. This prospective study examined two groups of women who had blood samples taken from 5-12 weeks gestation. One group consisted of 14 women with donor egg pregnancies (8 singletons and 6 multiples) who did not have corpora lutea, and the other group consisted 5 women with spontaneous pregnancies who had corpora lutea. Inhibin A and activin A were measured at weekly intervals using specific enzyme immunoassays. All pregnancies progressed to term, with healthy babies being delivered. Maternal serum concentrations of inhibin A significantly increased throughout the study period in the donor egg pregnancies (P < 0.001) and the control pregnancies (P < 0.001). Circulating concentrations of activin A also increased significantly in both the spontaneous and donor egg pregnancies (P < 0.001) during the study period. However, the concentrations of inhibin A and activin A in the first trimester of human pregnancy were not significantly different in the women with or without corpora lutea, suggesting a fetoplacental origin. Multiple donor egg pregnancies were found to have higher concentrations of inhibin A (P < 0.001) and activin A (P < 0.05) compared with singleton donor egg pregnancies, which also supports a placental source.

Follicle-stimulating hormone-inhibin B interactions during the follicular phase of the primate menstrual cycle revealed by gonadotropin-releasing hormone antagonist and antiestrogen treatment.

The aim was to determine the pattern of inhibin A and inhibin B secretion during the ovulatory cycle of the macaque and to explore the effects of manipulating follicular phase FSH on inhibin B secretion by: 1) blocking the early follicular phase rise in FSH with GnRH antagonist treatment; 2) administering FSH in GnRH antagonist-treated animals; and 3) preventing the midfollicular phase decline in FSH by a specific antiestrogen. Treatment with GnRH antagonist, starting on day 25 of the cycle, abolished the early follicular phase rise in FSH and the associated increase in inhibin B. The same treatment, followed by exogenous FSH, restored the secretion of inhibin B. Treatment with antiestrogen, commencing during the midfollicular phase, induced a supraphysiological rise in FSH, followed by a marked stimulation of inhibin B and estradiol secretion. Despite continued antiestrogen treatment, FSH secretion declined before peak values of inhibin B and estradiol were attained, implying a potential endocrine role for inhibin B, in addition to estradiol, in the negative feedback regulation of FSH. These results show that follicular phase FSH is the major stimulus for inhibin B secretion.

Both inhibin A and B respond to exogenous follicle-stimulating hormone in the follicular phase of the human menstrual cycle.

To ascertain whether changes in the concentrations of the dimeric inhibins A and/or B (INH-A and INH-B) contributed to the previously described dose-dependent increase in immunoreactive inhibin (INH) in response to FSH during the follicular phase of the human menstrual cycle, both dimers were measured by specific two-site assays in stored serum samples from regularly cycling normal volunteers who had received saline as a control (n = 5) or FSH [100 IU (n = 6) or 200 IU (n = 5)] between days 3-5 of the menstrual cycle. Both INH-A and INH-B showed a dose-dependent increase in response to administered FSH; INH-A rose from 13.5 to 35.9 ng/L (P < 0.01), and INH-B rose from 77.8 to 205 ng/L (P < 0.05) at 36 h after 200 IU FSH. Highly significant correlations were observed between INH and each of the specific inhibin dimers (A: r = 0.79, P < 0.001; B: r = 0.76, P < 0.001), and the responses of the two dimers were also highly correlated (r = 0.59, P < 0.001). The response of each inhibin was also highly correlated with the response of serum estradiol (A: r = 0.45, P < 0.001; B: r = 0.40, P < 0.001). When analyzed by ANOVA, the INH response of INH-B was significantly above the control value at 36 h after treatment with both 100 and 200 IU FSH, whereas the response of INH-A was significant only at 200 IU. It is concluded that the concentrations of both dimeric INH-A and INH-B are stimulated by increases in FSH within the physiological range in the follicular phase of the human menstrual cycle and that both contribute to the previously observed rise in INH.

Endothelial cells and peripheral blood mononuclear cells are a potential source of extraplacental activin a in preeclampsia.

An excessive systemic inflammatory response, involving endothelial cells and leukocytes, underlies the maternal symptoms of preeclampsia. Activin A is raised in preeclampsia, suggesting a possible involvement in its pathophysiology. The placenta is the main source of activin A in normal pregnancy. We investigated whether peripheral blood mononuclear cells (PBMCs) and endothelium, activated by proinflammatory stimuli, were a potential source of activin A in preeclampsia. Both endotoxin and TNFalpha stimulated activin A secretion by PBMCs from nonpregnant, preeclamptic, and matched normal pregnant women (P < 0.05). Pregnancy increased the responsiveness of PBMCs to endotoxin (P < 0.05), whereas only the preeclamptic group were significantly more responsive to TNFalpha (P < 0.05). Human umbilical vein endothelial cells secreted activin A spontaneously and in response to TNFalpha (P < 0.05), but recombinant IL-1beta and IL-6 had no significant effect over the 72-h culture period. Inhibin A and follistatin were undetectable (<2 pg/ml and < 20 pg/ml, respectively) in PBMCs and human umbilical vein endothelial cell culture media. These data suggest that PBMCs and endothelium, activated by TNFalpha, could be extraplacental sources of activin A in preeclampsia. The pathological significance of increased activin A in preeclampsia is unknown, although it may have a role in the mechanisms underlying endothelium dysfunction.