Effect of inhibin A on proliferation of porcine granulosa cells in vitro.

Inhibins regulate folliculogenesis, gametogenesis and hormone secretion via endocrine, paracrine and autocrine manners, and play roles in encouraging or suppressing proliferation of cells. In order to investigate the effects of inhibin A on proliferation and apoptosis of porcine granulosa cells (GCs), GCs were isolated from ovarian follicles (3-6 mm), treated with inhibin A at different concentrations. The cell viability, mitochondrial membrane potential (MMP), expression of proliferation-related genes and cell cycle were detected. Inhibin α subunit (INHA) gene was silenced to detect the effect of down-regulation of inhibin on expression of proliferation-related genes in GCs. The results showed that cell viability was associated with inhibin A concentration and significantly enhanced by inhibin A at high doses (50 and 100 ng/mL, P < 0.05) compared to control group (0 ng/mL). Meanwhile, the MMP boosted after treated with 100 ng/mL inhibin A for 48 h. Expression of proliferating cell nuclear antigen (PCNA) (200 ng/mL) and CyclinB1 (100 and 200 ng/mL) was promoted while Caspase-3(100 and 200 ng/mL) and BAX (200 ng/mL) was inhibited in dose dependent manner after the cells were incubated with inhibin A for 24 h (P < 0.05) compared to control group, thereby the transition from G1 phase to S phase was promoted and the number of S phase cells was increased. After silencing the INHA gene expression, expression of Caspase-3 was enhanced and CyclinB1 was inhibited (P < 0.05) compared to Ri-negative group. All the results pointed to the conclusion that inhibin A promotes the proliferation of GCs while inhibits apoptosis.

Oral vaccination with inhibin DNA delivered using attenuated Salmonella choleraesuis for improving reproductive traits in mice.

The objective of this study was to examine the efficacy and safety of a novel inhibin vaccine containing inhibin α (1-32) fragments in mice. A recombinant plasmid pVAX-asd-IS was constructed by inserting recombinant inhibin α (1-32) and the hepatitis B surface antigen S into the plasmid in which the asd gene, rather than the kanamycin gene, was a selection marker. Ninety Kuming mice were divided into six groups consisting of 15 mice each. First group was (C1) injected with 200 µl of PBS, second (C2) received 1 × 10(10) CFU of crp(-) /asd(-) C500/pVAX-asd and served as vector control, third did not receive any treatment (C3), while fourth, fifth, and sixth group received 1 × 10(10) , 1 × 10(9) , 1 × 10(8) CFU of the recombinant inhibin vaccine crp(-) /asd(-) C500/pVAX-asd-IS (group T1, T2, T3), respectively. Western blotting demonstrated that recombinant expressed inhibin protein possessed immune function and that this plasmid could replicate for up to 40 generations stably. Vaccination with this strain at a dose of 1 × 10(10) CFU/200 µl per mouse induced high anti-inhibin antibody levels, significantly increased large-follicle production in T1 group (p < 0.05) and average litter size (p > 0.05) compared with control groups. Integration studies showed no evidence of inhibin fusion gene integrated into mice's genome 2-month after immunization. These results suggest that the vaccine described in the present study may provide a safe method to improve reproductive traits in animals. A trend towards increased litter size and significant increase in large follicle population depict that this vaccine may have direct application in large animal industry.

The inhibin B (InhB) response to the testicular toxicants mono-2-ethylhexyl phthalate (MEHP), 1,3 dinitrobenzene (DNB), or carbendazim (CBZ) following short-term repeat dosing in the male rat.

OBJECTIVE: The objective of this study was to evaluate the utility of plasma Inhibin B (InhB) as a biomarker of testicular injury in adult rats following short-term exposure to the known Sertoli cell toxicants mono-2-ethylhexyl phthalate (MEHP), 1,3 dinitrobenzene (DNB), or carbendazim (CBZ). METHODS: Following oral gavage administration of the compounds for 2 or 7 days, the rats were evaluated for clinical signs, body weight, food consumption, organ weights, plasma hormone levels, and gross and microscopic pathology. RESULTS: MEHP, DNB, and CBZ produced a range of testicular toxicity characterized by minimal exfoliation of germ cells as demonstrated by increased cellular debris in the epididymis (MEHP) to more severe and dose/duration responsive Sertoli cell vacuolation, germ cell degeneration, and multinucleated giant cells of germ cell origin (DNB and CBZ). The slight to moderate Sertoli and germinal cell injuries did not correlate with significant changes in plasma InhB levels following 2- or 7-day exposures. However, more severe injury to germinal epithelium following up to 7 days of exposure, but not after 2 days of exposure, correlated with decreased plasma InhB levels and less consistently with increases in plasma follicle stimulating hormone. CONCLUSION: In conclusion, under the conditions of these studies, changes in InhB were not an effective early onset marker of testicular toxicity or an effective marker for slight to moderate levels of acute injury, and only reflected more severe disruption of spermatogenesis. Changes in plasma InhB and follicle stimulating hormone were poorly correlated except in some instances of moderate to marked testicular toxicity.

Immunization against inhibin enhances both embryo quantity and quality in Holstein heifers after superovulation and insemination with sex-sorted semen.

The objective was to investigate the feasibility of improving embryo yield in superovulated cows following insemination with sex-sorted semen by prior immunization against inhibin. Twenty-eight heifers were allocated into three groups: High (n=10), Low (n=10), and Control (n=8). The High group received one primary (1mg) and two booster (0.5mg) vaccinations (28-d intervals) with a recombinant inhibin alpha-subunit in 1 mL of white oil adjuvant, whereas the Low group received half that dose, and the Control group received only adjuvant. After the last immunization, all heifers underwent a standard superovulation treatment (decreasing doses of pFSH for 4d), followed by two AI with 2 x 10(6) sex-sorted semen after the onset of estrus. Inhibin-immunized heifers had higher (P<0.01) plasma antibody titres, and an earlier onset of estrus (P<0.05) than Control heifers. The total number of embryo/ova, transferable, and grade 1 embryos in the High group (15.4+/-1.9, 5.7+/-0.7, and 3.8+/-1.0, respectively) was significantly greater than that of the Control group (9.1+/-1.2, 3.1+/-0.5, and 0.6+/-0.2), but was intermediate (P>0.05) in the Low group (13.0+/-2.3, 4.4+/-0.7, and 1.2+/-0.3). There were no significant differences among groups in number of unfertilized ova and degenerated embryos. The High group also had higher (P>0.05) plasma progesterone concentrations on the day of embryo collection. In conclusion, immunization against inhibin improved both embryo quantity and quality following superovulation and insemination with sex-sorted semen.

Autoimmune targeted disruption of the pituitary-ovarian axis causes premature ovarian failure.

Premature ovarian failure (POF) is characterized by amenorrhea and high serum levels of follicle-stimulating hormone (FSH). POF causes female infertility and represents a substantial women's health risk affecting 1% of women by age 40. Although ovarian autoimmunity has been associated with POF, the identity of ovarian Ags recognized is unknown. In this study, we show that autoimmune-targeted disruption of the pituitary-ovarian axis leads to POF. Immunization of SWXJ female mice with the p215-234 peptide derived from mouse inhibin-alpha activates CD4(+) T cells and induces experimental autoimmune oophoritis with a unique biphasic phenotype characterized by an early stage of enhanced fertility followed by a delayed stage of POF. Affected mice show high serum levels of inhibin-alpha-neutralizing Abs that prevent inhibin-mediated down-regulation of activin-induced pituitary FSH release. The loss of activin/FSH down-regulation leads to prolonged metestrus-diestrus, superovulation, increased numbers of mature follicles, increased offspring, accelerated depletion of primordial follicles, and ultimately premature infertility. Thus, inhibin-alpha-targeted experimental autoimmune oophoritis is initiated by CD4(+) Th1 T cells that stimulate B cells to produce inhibin-alpha-neutralizing Abs directly capable of mediating POF and transferring disease into naive recipients. Our inhibin-alpha autoimmune model of POF shows how premature infertility may develop in the context of elevated FSH levels thereby closely mimicking the hallmark features of human POF.

Regulation of activin mRNA and Smad2 phosphorylation by antidepressant treatment in the rat brain: effects in behavioral models.

Activin is a member of the transforming growth factor-beta family that is involved in cell differentiation, hormone secretion, and regulation of neuron survival. The cellular responses to activin are mediated by phosphorylation of a downstream target, Smad2. The current study examines the influence of chronic electroconvulsive seizures (ECSs), as well as chemical antidepressants, on the expression of activin betaA and the phosphorylation of Smad2 in the rat hippocampus and frontal cortex. Chronic ECSs (10 d) resulted in a significant increase in activin betaA mRNA expression and Smad2 phosphorylation in both the hippocampus and frontal cortex. Chronic fluoxetine did not influence activin betaA expression, but fluoxetine as well as desipramine did increase Smad2 phosphorylation in the frontal cortex. The functional significance of increased activin was further tested by examining the effects of activin infusions into the hippocampus on a behavioral model of depression, the forced swim test (FST). A single bilateral infusion of activin A or activin B into the dentate gyrus of the hippocampus produced an antidepressant-like effect in the FST that was comparable in magnitude with fluoxetine. In contrast, infusion of the activin antagonist inhibin A did not influence behavior but blocked the effect of activin A. The results suggest that regulation of activin and Smad signaling may contribute to the actions of antidepressant treatment and may represent novel targets for antidepressant drug development.

Active immunization against inhibin improves superovulatory response to exogenous FSH in cattle.

The effect of active immunization against inhibin on the response to superovulatory treatment by porcine FSH (pFSH) was investigated in cattle. Japanese black cows were sc injected with 1 mg of porcine inhibin alpha-subunit fragment (1-26) conjugated with rabbit serum albumin (inhibin-immunized group; n=14) or rabbit serum albumin alone (control group; n=12) in Freund's complete adjuvant. Booster injections (half the amount of the primary injection) were given 35 and 70 days after the primary injection. All cows were superovulated three times with pFSH. Three days after each injection of the antigen, a progesterone-releasing intravaginal device (CIDR-B) was inserted vaginally into all animals and left in place for 10 days. Forty-eight hours before CIDR-B removal, all animals were sc injected with 30 mg pFSH dissolved in 40% polyvinylpyrrolidone, and im injected with 750 microg of PGF2alpha at CIDR-B removal. Cows were artificially inseminated twice during estrus, and ova or embryos were collected 7 or 8 days after estrus. The number of corpora lutea, the number of ova or embryos and the number of transferable embryos in inhibin-immunized cows (12.1+/-1.2, 11.1+/-1.3 and 6.2+/-1.0, respectively) were significantly greater than those in the controls (8.2+/-1.0, 5.7+/-1.1 and 3.1+/-0.7, respectively). These results indicate that active immunization against inhibin enhanced ovarian response to the usual superovulatory treatment in cattle. Therefore, immunization against inhibin may be a useful approach for improving the response to superovulation in cattle.

Effects of re-immunization of heifers against inhibin on hormonal profiles and ovulation rate.

To study the effect of re-immunization against inhibin on ovarian response and hormonal profiles, Japanese beef heifers (n = 5) were re-immunized three times with inhibin vaccine (recombinant ovine inhibin alpha-subunit in oil emulsion, 125 microg ml(-1)) one year after the primary immunization. Control heifers (n = 5) were injected with placebo (Montanide: Marcol adjuvant alone). Oestrous cycles were synchronized by using prostaglandin F2alpha (PGF2alpha) and ovarian response was monitored daily by ultrasonography. Blood samples were collected by jugular venipuncture for assessment of hormonal levels and inhibin antibody titres. In contrast to controls, inhibin re-immunized heifers generated antibodies against inhibin rapidly reaching a peak level 9 days after the first booster injection. The mean concentrations of FSH in re-immunized cows increased significantly in comparison with controls. In addition, there was a significant increase in oestradiol-17beta and progesterone levels in re-immunized cows compared with controls. Inhibin re-immunized heifers had a significant increase in small (> or =4 < 7 mm), medium (> or =7 < 10 mm) and large (> or =10 mm in diameter) sized follicles. Moreover, the mean ovulation rate was 5.0 +/- 1.1 after the third booster injection in re-immunized heifers compared with control heifers (single ovulation). These results clearly demonstrate that re-immunization of inhibin can be used to enhance ovarian follicular development and ovulation rate. Furthermore, the great number of follicles is a potential source of oocytes that could be harvested for in vitro fertilization and embryo transfer programmes.

Tumor de células granulares de piel mamaria. Estudio morfológico de dos casos mostrando inmunorreactividad frente alfa-inhibina / Granular cell tumor of the mammary skin. Morphological study of two cases showing immunoreactivity against alfa-inhibin

Introducción: El tumor de células granulares (TCG) es una lesión infrecuente en la mama, representando solo el 6-8 por ciento de los TCG, comprometiendo a menudo a la piel mamaria. Pacientes y métodos: Se revisa la casuística de TCG mamarios del Hospital Universitario La Fe de Valencia en un período de 19 años, con un total de 8000 especímenes biópsicos mamarios con 2200 diagnósticos de cáncer, realizando de forma retrospectiva un estudio morfológico (óptico, inmunohistoquímico y ultraestructural) a partir de material incluido en parafina. Resultados: Se detectan dos casos de TCG con afectación de piel mamaria con distinta presentación clínica (TCG como nódulo tumoral único y TCG como lesión tumoral múltiple en relación a una cicatriz de mastectomía previa). En ambas observaciones se comprobó, junto a otros datos ya conocidos (PAS + citoplásmica granular diastasa resistente, positividad a S-100, VT, CD68 y ultraestructura con numerosos cuerpos densos lisosomiales) la presencia de una reactividad inmunohistoquímica frente a alfa-inhibina. Conclusiones: Los TCG de glándula mamaria muestran reactividad inmunohistoquímica frente a alfa-inhibina, aspecto éste que amplia la aplicabilidad de este anticuerpo, sumándose al panel existente de inmunomarcadores en el diagnóstico de los TCG (AU)

Ovarian influence on gonadotropin and prolactin release in mated rabbits.

In 17beta-estradiol (E)-treated ovariectomized (OVX) rabbits, the coitus-induced luteinizing hormone (LH) surge is only one fourth that in ovarian-intact rabbits. In this study, we determined the pattern of the coitus-induced gonadotropin release, i.e., LH and follicle-stimulating hormone (FSH), in OVX + E animals without or with continuous 3-wk treatment of 20-alphahydroxypregn-4-en-3-one (20alphaP). For positive and negative experimental controls, ovarian-intact rabbits were either mated or sham mated, respectively. The pituitary hormones prolactin (PRL) and growth hormone (GH) were measured to serve as collateral controls for gonadotropins. The addition of continuous 20alphaP in OVX + E does fail to stimulate a coitus-induced LH surge equal in magnitude and duration to the LH surge in ovarian-intact rabbits. Postcoital levels of FSH were greater in OVX + E + 20alphaP animals than those in OVX + E rabbits. Coitus induced a PRL surge in ovarian-intact and OVX + steroid-treated females, but not in mated males, thereby suggesting a gender difference in this neuroendocrine circuit. Neither coitus nor steroids altered plasma GH values in female or male animals. We conclude that chronic administration of neither E nor E + 20alphaP can restore full-scale gonadotropin surges in OVX rabbits, whereas replacement of one or both of these steroids is sufficient for a coitus-induced PRL surge. Moreover, the presented observation that activin stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release suggests a possible involvement of ovarian proteins in the production of a full-scale coitus-induced GnRH/LH surge.

Involvement of distinct signaling pathways in activin-induced increases in FSH secretion and enlargement of FSH cell population in the rat pituitary.

Activin-A induces increases in FSH secretion, as well as the number of immunoreactive FSH cells, in cultured rat pituitary cells. In this study, we examined whether mechanisms involved in these two actions of activin-A are identical or not, with respect to the involvement of cellular proliferation and of Ca2+-dependent signaling pathways. Treatment with activin-A (25 ng/ml) for 48 h caused increases in the number of cultured rat anterior pituitary cells that incorporated BrdU, a thymidine analog. The stimulatory effects of activin-A on FSH secretion and on the percentage of immunoreactive FSH cells were, however, not inhibited by the presence of the mitotic inhibitor cytosine arabinoside. On the other hand, the stimulatory effect of activin-A on the percentage of immunoreactive FSH cells was completely blocked in the presence of the Ca2+/calmodulin kinase inhibitor KN-62 or the L-type Ca2+ channel blocker nicardipine. Neither of these inhibitors, however, revealed significant influence on the effect of activin-A on FSH secretion. These results suggest that activin-A exhibits its dual effect on FSH cells without causing cellular proliferation. Furthermore, activin-A appears to induce increases in FSH secretion and enlargement of FSH cell population through distinct intracellular signaling pathways, the former through Ca2+-independent and the latter through Ca2+-dependent mechanisms.

Activin increases bone mass and mechanical strength of lumbar vertebrae in aged ovariectomized rats.

Activin is a member of the transforming growth factor-beta superfamily and is thought to be involved in the regulation of bone formation due to its presence in bone tissue and its osteogenic activity both in vitro and in vivo. We recently found that systemic administration of activin increased both tibial bone mass and mechanical strength in young growing rats. The present study investigated the effects of activin in aged ovariectomized (ovx) rats. Twelve-month-old Fischer rats were ovariectomized and maintained for 10 months. Recombinant human activin A (activin) or human parathyroid hormone 1-34 (PTH) was administered intramuscularly three times a week for 12 weeks. Activin (1 and 5 microg/kg) markedly increased lumbar vertebral bone mineral content and bone mineral density. Activin also increased the mechanical strength of the vertebral body, which was highly correlated to the bone mineral density of the vertebral body. The maximal response in bone mass and strength was observed at 1 microg/kg of activin, which was approximately equal to that induced by PTH at 40 microg/kg. Peripheral quantitative computed tomography revealed that activin enlarged the cross-sectional size of the vertebrae without changing the foramen area, indicating its effects on cortical shells. Histomorphometric analysis of cancellous bone of vertebral body in similar experiment showed that activin (3 microg/kg) increased bone volume and the mineralizing surface, although its effects were less than PTH. The present results indicate that low doses of activin are effective against vertebral bone loss in aged ovx rats.

Activin A stimulates insulin secretion in cultured human pancreatic islets.

Activin A is a dimeric glycoprotein showing a high sequence homology with transforming growth factor-beta (TGF-beta) and playing autocrine/paracrine actions in reproductive tissues. However, since the synthesis of activin is ubiquitous it may have a role in regulating cell growth and differentiation in several tissues. Previous studies showed that activin A is expressed by insulin-positive B cells of human pancreatic islets, and women with gestational diabetes have higher serum activin A levels than healthy pregnant women at the same gestational age. The present study aimed to evaluate the effect of activin A on insulin secretion from cultured human pancreatic islets. With this purpose human pancreatic islets were incubated with varying concentrations of activin A (0.1 to 10.0 nM). In absence of glucose, activin A did not modify insulin secretion at the different concentrations used. In absence of activin A, 8.3 mM and 16.7 mM glucose significantly increased insulin secretion, with a dose-dependent pattern. In presence of a non stimulatory concentration of glucose (3.3 mM), activin A significantly increased insulin secretion starting from low concentration (0.1 nM). Furthermore, the addition of activin A to 8.3 mM and 16.7 mM glucose induced an additional effect of the dose-dependent glucose-mediated insulin secretion (p<0.001). The present data could support a role for activin A in human endocrine pancreas in modulating insulin response to different glucose concentrations.

More than 95% reversal of left-right axis induced by right-sided hypodermic microinjection of activin into Xenopus neurula embryos.

In recent years, genes that show left-right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula-neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus neurula embryos. After right-side injection of 10-250 pg activin protein, both early neurulae just after gastrulation movement (stage 13-14) and late neurulae just before neural tube closure (stage 17-18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1-50 pg) was microinjected into both sides of neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the neurula stage. Activin not only acts on the neurulae to cancel the original L-R specification up to the late neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of neurulae have equal potential for L-R differentiation.

Activin A is an essential cofactor for osteoclast induction.

Recently, receptor activator of NF-kappaB ligand (RANKL) was shown to be necessary for osteoclast formation. We now report that activin A, a cytokine enriched in bone matrix and secreted by osteoblasts and osteoclasts, powerfully synergized with RANKL for induction of osteoclast-like cells (OCL) from bone marrow precursors depleted of stromal cells. Moreover, OCL formation in RANKL was virtually abolished by soluble type II A activin receptors (ActR-II(A)), suggesting that activin A is essential for OCL formation. Activin A was most effective when precursors were exposed to RANKL and activin A simultaneously: resistance to OCL-induction that occurs when precursors are pre-incubated in M-CSF was reduced. Incubation on bone matrix also enhanced the sensitivity of precursors to OCL-induction by RANKL; and this was prevented by soluble ActR-II(A). Thus, activin A in bone matrix, or released from osteoblastic or other cells, enhances the osteoclast-forming potential of precursors and synergizes with RANKL in inducing osteoclastic differentiation.

Activin A and follistatin influence expression of somatostatin in the ciliary ganglion in vivo.

An important developmental question concerns whether neurotransmitter phenotype is an inherent property of neurons or is influenced by target tissues. This issue can be addressed in the avian ciliary ganglion (CG) which contains two cholinergic populations, ciliary and choroid neurons, that differentially express the peptide cotransmitter, somatostatin. The present study tests the hypothesis that differences in the level of expression of activin A and its endogenous inhibitor follistatin in CG neuron target tissues are responsible for selective expression of somatostatin in choroid neurons. Intraocular injection of activin A or follistatin (300 ng injected at E10/E11) in cultured embryos resulted in a 39% increase or a 23% decrease, respectively, in somatostatin-positive neurons relative to controls. Chorioallantoic membrane application of follistatin (1 microgram daily from E7 to E13) reduced somatostatin positive neurons by 54%. Neuron number, size, and target tissue morphology were unaffected by these treatments. Together with our previous studies, these data suggest that activin A and follistatin are target-derived molecules that regulate neuropeptide phenotype in the ciliary ganglion.

Activin A and follistatin regulate developmental competence of In vitro-produced bovine embryos.

The effects of activin A and/or follistatin on the development of bovine embryos were investigated. Presumptive zygotes matured and fertilized in vitro were cultured in a chemically defined medium (modified synthetic oviduct fluid medium; mSOF). Addition of 1-100 ng/ml of activin A to mSOF significantly increased the percentage of zygotes that developed to morulae and blastocysts (48-54% and 31-41%, respectively) compared with no addition (41% and 25%, respectively). In contrast, addition of 1-100 ng/ml follistatin significantly reduced the percentage of zygotes developing to morulae and blastocysts (29-31% and 17-20%, respectively) compared with no addition (41% and 28%, respectively). In a culture with 10 ng/ml of activin A, supplementation with the same concentration of follistatin neutralized the positive effect of activin A, while supplementation with 100 ng/ml of follistatin reduced the percentage of zygotes that developed. The total cell numbers in morulae and blastocysts were not affected by the addition of activin A and/or follistatin. The development-enhancing effects of activin A and the development-impeding effects of follistatin were observed when embryos were exposed to activin A or follistatin at a concentration of 10 ng/ml prior to the 9- to 16-cell stage. These results suggest that activin A and follistatin may affect bovine embryos until the third cell cycle and may play important roles in regulation of the developmental competence of bovine embryos.

Activin-induced inhibin alpha-subunit production by rat granulosa cells requires endogenous insulin-like growth factor-I.

Inhibin-alpha subunit (Inh-alpha) gene expression is important for granulosa cell (GC) differentiation and prevention of ovarian tumorigenesis. Studies on Inh-alpha regulation have implicated activin and insulin-like growth factor-I (IGF-I) in the mechanisms of expression. Here we present evidence that endogenously produced IGF-I plays an obligatory role in activin-induced Inh-alpha production. Primary cultures of rat GC were incubated with increasing concentrations of various regulatory molecules, and the levels of Inh-alpha protein and its mRNA were measured in conditioned medium and cells, respectively. Recombinant activin A stimulated Inh-alpha expression, and the effects were dose- and time-dependent. The receptor tyrosine kinase inhibitor tyrphostin A23 caused a dose-dependent inhibition of activin-dependent Inh-alpha expression, whereas the inactive isomer, A63, had no effect. The stimulatory effect of activin was also blocked in a dose-dependent manner by added IGF binding protein-4 or -5, and the effects were reversed by IGF-I. Moreover, increasing concentrations of an anti-IGF-I antibody had a similar inhibitory effect on activin-stimulated Inh-alpha expression. Collectively, these results suggest, for the first time, that endogenously produced IGF-I is required for activin stimulation of Inh-alpha expression in cultured rat GC.

Expression of activin and follistatin in the rat hypothalamus: anatomical association with gonadotropin-releasing hormone neurons and possible role of central activin in the regulation of luteinizing hormone release.

The central role of activin in the regulation of the reproductive axis remains largely unexplored. Evidence suggests that activin may play a role in control-linggonadotropin-releasing hormone (GnRH) release. We assessed potential neuroanatomical associations between activin- and GnRH-neuronal systems via examination of the distribution of activin betaA-subunit and activin binding protein (follistatin) protein and mRNA signals relative to GnRH neurons in the adult rat brain. Activin betaA-subunit-immunostained fibers were distributed throughout the hypothalamus and GnRH-positive perikarya, and fibers were in close association with betaA-subunit-immunoreactive fibers. Follistatin mRNA-expressing cells were also identified throughout the hypothalamus with GnRH fibers often observed juxtaposed to follistatin cell bodies. Colocalization of either the betaA-subunit or follistatin within GnRH neurons was not detected. The functional significance of central activin in the regulation of the reproductive axis was also demonstrated. The intracerebroventricular infusion of rh-activin A significantly increased luteinizing hormone, but not follicule-stimulating hormone, serum levels in adult male rats. Taken together, the present results support an interaction between activin and GnRH neuronal systems in the rat hypothalamus, and suggest activin may act within the brain to regulate the reproductive axis.

Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone- and serum-free medium.

The aim of this study was to investigate whether inhibin A and/or activin A play a role in the acquisition of oocyte competence during the final stages of oogenesis. The particular goal was to establish whether inhibin A and activin A exert development-enhancing effects during in vitro maturation in serum-free media and whether such effects are related to changes in the kinetics of meiotic resumption and/or fertilization rates. Cumulus-oocyte-complexes (COCs) were matured in two control media (Medium 199 [M199] with hormones and serum, hormone-serum control; M199 + 0.6% BSA, BSA-control) and nine treatment media (M199 with 0.6% BSA containing 100, 10, and 1 ng/ml of recombinant human inhibin A, recombinant human activin A, and the combination of the two). Oocytes were fertilized and cultured using standard procedures. Cleavage was assessed at 54 h and blastocyst development at 8 days after in vitro fertilization. Kinetics of oocyte maturation and the fertilization rates were evaluated after fixing and staining (Hoechst 33342) of oocytes at 8, 16, and 22 h after onset of in vitro maturation or of presumptive zygotes at 12 h after in vitro fertilization, respectively. Although there was no effect on cleavage rates, inhibin A and activin A significantly enhanced postcleavage development at concentrations of 10 ng/ml (57.7 +/- 7.5% and 56.6 +/- 11.7%, respectively) and 100 ng/ml (50.6 +/- 18.6% and 56.4 +/- 4.0%, respectively) compared to that in the BSA-control group (24.6 +/- 3.2%). Whereas inhibin A- and activin A-treated oocytes showed development-enhancing effects similar to those in the hormone-serum controls, these groups differed with regard to the kinetics of meiotic resumption. Likewise, the enhanced development of the hormone-serum control and the inhibin A/activin A-treated oocytes was not related to increased fertilization rates relative to the BSA-control. These results suggest that inhibin A and activin A may play important roles during the final stages of oogenesis and that recombinant inhibins and activins are useful compounds for the development of a serum-free culture system for in vitro maturation of oocytes from cattle and possibly other mammalian species.