Increase In FSH Secretion Research Articles

The Effect of Bone Morphogenetic Protein 2 (BMP-2)/Estrogen Composite Nanoparticles on the Differentiation Function of Osteoporotic Bone Marrow Mesenchymal Stem Cells (BMSCs)

The menopausal hormone abnormal changes such as estrogen deficiency and increased FSH secretion in female patients in old age may cause osteoporosis which is plagued by patients. The pathogenesis of osteoporosis is not yet fully understood. BMP in the transforming growth factor-β superfamily is a key member in the process of bone growth and development, among which BMP-2 exerts critical roles. Impaired osteogenic differentiation of bone marrow mesenchymal stem cells (BMSC) contributes to the progress of osteoporosis. BMSC plays an indispensable role in treating osteoporosis and can develop into different directions through induction. As the regenerative medicine nanotechnology has become a new medical method, it is believed that BMSC can be used to treat osteoporosis and other related diseases. Our study analyzed the effects of BMP-2/estrogen composite nanoparticles on the proliferation and differentiation of osteoporotic BMSC cells to provide a reliable reference for the future treatment. Our results showed that BMP-2/estrogen composite nanoparticles promoted BMSC cell proliferation, increased ALP activity, decreased apoptosis rate, increased the expression of Col-1, Runx2 and Osterix, upregulated the osteogenic marker BMP-2. As confirmed by Alizarin Red staining, it could differentiate into osteoblasts and the content of Trap was decreased. In conclusion, our study confirms that BMP-2/estrogen composite nanoparticles can promote BMSC cell proliferation, osteogenic differentiation, and inhibit osteoclast differentiation, thereby providing new treatments and theoretical reference basis for treating osteoporosis.

SAT-222 Hormone Responses Associated with Letrozole Ovulation Induction in Anovulatory Women with PCOS

: The aromatase inhibitor (AI) letrozole has been shown to be the most effective oral medication for ovulation induction in anovulatory women with PCOS. It has been established that AIs block the conversion of androgens to estrogen, leading to reduction of serum estrogen levels. However, the mechanism by which letrozole induces ovulation is unknown. It has been suggested that the mechanism of AIs in ovulation induction may involve increased FSH secretion in response to reduced estrogen levels. To examine the mechanism by which AIs induce ovulation, we performed a pilot interventional study. Ten anovulatory women with PCOS, aged 24-34 years were enrolled. Each subject underwent baseline serum hormone assessment and transvaginal ultrasound. Women were excluded if they had hypogonadotropic hypogonadism, or ultrasound evidence of a dominant follicle or corpus luteum. Each subject underwent ovulation induction with letrozole 2.5 mg/day for 5 days. Repeat transvaginal ultrasound was done three days after completing letrozole. Ovulation was defined by (1) presence of >=1 ovarian follicle >=16 mm on follow up ultrasound and (2) positive home ovulation predictor test. Serum hormone levels were assessed in all subjects at baseline and at two, four, and seven days after letrozole initiation. Mean serum hormone levels were compared using paired Student’s t test with statistical significance set at p<0.05. Three of 10 subjects ovulated after completing letrozole. In all subjects, the baseline levels (mean ± SD) of serum FSH, LH, estradiol, and androstenedione were 5.4 ± 2.2 mIU/mL, 12.1 mIU/mL, 86.2 ± 32.1pg/mL, and 3.2 ± 1.5 ng/mL, respectively. A significant reduction in serum estradiol was noted on day two compared to baseline (-35.2 pg/mL). There was a significant increase in mean serum FSH (+1.8 mIU/mL) and LH (+4.7 mIU/mL) on day two after starting letrozole. Significant increases in serum androstenedione were observed on day two (+0.8 ng/mL) and day four (+1.5 ng/mL) relative to baseline values. The study was not sufficiently powered to detect differences in serum hormone levels between women that did and did not ovulate in response to letrozole administration. These data suggest that the mechanism of ovulation induction by AI may involve a transient increase in serum gonadotropins resulting from interrupted estrogen negative feedback. Sources of Research Support: NIH Grants T32 HD007203, K12 HD001259, and P50 HD012303.Key Words: Polycystic Ovary Syndrome; Infertility; Anovulation

Pituitary Hormone Secretion Profiles in IGSF1 Deficiency Syndrome

Background: Loss-of-function mutations in immunoglobulin superfamily member 1 (IGSF1) cause an X-linked syndrome of central hypothyroidism, macroorchidism, delayed pubertal testosterone rise, variable prolactin deficiency and variable partial GH deficiency in childhood. The clinical features and gene expression pattern suggest a pivotal role for IGSF1 in the pituitary, but detailed knowledge on pituitary hormone secretion in this syndrome is lacking. We therefore aimed to study the 24-hour pituitary hormone secretion in male patients with IGSF1 deficiency. Methods: We collected blood samples every 10 min for 24 h in eight adult male IGSF1-deficient patients and measured circulating TSH, prolactin and gonadotropins. Deconvolution, modified cosinor and approximate entropy analyses were applied to quantify secretion rates, diurnal rhythmicity and regularity of hormone release. Results were compared to healthy controls matched for age and body mass index. Results: Compared to healthy controls, IGSF1-deficient patients showed decreased pulsatile secretion of TSH with decreased disorderliness and reduced diurnal variation. Basal and pulsatile secretion of FSH was increased by over 200%, while LH secretion did not differ from healthy controls. We observed a bimodal distribution of prolactin secretion, i.e. severe deficiency in three and increased basal and total secretion in the other five patients. Conclusion: The altered TSH secretion pattern is consistent with the previously hypothesized defect in thyrotropin-releasing hormone signaling in IGSF1 deficiency. However, the phenotype is more extensive and includes increased FSH secretion without altered LH secretion as well as either undetectable or increased prolactin secretion.

Growth Differentiation Factor 9 (GDF9) Forms an Incoherent Feed-forward Loop Modulating Follicle-stimulating Hormone β-Subunit (FSHβ) Gene Expression

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus and regulates follicle-stimulating hormone β-subunit (FSHβ) gene expression in pituitary gonadotropes in a frequency-sensitive manner. The mechanisms underlying its preferential and paradoxical induction of FSHβ by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a GnRH-suppressed autocrine inducer of FSHβ gene expression. GDF9 gene transcription and expression were preferentially decreased by high frequency GnRH pulses. GnRH regulation of GDF9 was concentration-dependent and involved ERK and PKA. GDF9 knockdown or immunoneutralization reduced FSHβ mRNA expression. Conversely, exogenous GDF9 induced FSHβ expression in immortalized gonadotropes and in mouse primary pituitary cells. GDF9 exposure increased FSH secretion in rat primary pituitary cells. GDF9 induced Smad2/3 phosphorylation, which was impeded by ALK5 knockdown and by activin receptor-like kinase (ALK) receptor inhibitor SB-505124, which also suppressed FSHβ expression. Smad2/3 knockdown indicated that FSHβ induction by GDF9 involved Smad2 and Smad3. FSHβ mRNA induction by GDF9 and GnRH was synergistic. We hypothesized that GDF9 contributes to a regulatory loop that tunes the GnRH frequency-response characteristics of the FSHβ gene. To test this, we determined the effects of GDF9 knockdown on FSHβ induction at different GnRH pulse frequencies using a parallel perifusion system. Reduction of GDF9 shifted the characteristic pattern of GnRH pulse frequency sensitivity. These results identify GDF9 as contributing to an incoherent feed-forward loop, comprising both intracellular and secreted components, that regulates FSHβ expression in response to activation of cell surface GnRH receptors.

Divergent Roles of the CRH Receptors in the Control of Gonadotropin Secretion Induced by Acute Restraint Stress at Proestrus

CRH has been implicated as a mediator of stress-induced effects on the hypothalamus-pituitary-gonad axis, acting via CRH receptors in various brain regions. We investigated whether the effects of restraint stress on the secretion of gonadotropins on the morning of proestrus are mediated by the CRH-R1 or CRH-R2 receptors in the oval subdivision of the anterolateral BST, the central amygdala, the locus coeruleus (LC), or the A1 and A2 neuron groups in the medulla. At proestrus morning, rats were injected with antalarmin (a CRH-R1 antagonist), asstressin2-B (a CRH-R2 antagonist) or vehicles. Thirty minutes after the injection, the animals were placed into restraints for 30 min, and blood was sampled for 2 h. At the end of the experiment, the brains were removed for immunofluorescence analyses. Restraint stress increased the levels of FSH and LH. Antalarmin blocked the stress-induced increases in FSH and LH secretion, but astressin2-B only blocked the increase in FSH secretion. LC showed intense stress-induced neuronal activity. FOS/tyrosine-hydroxylase coexpression in LC was reduced by antalarmin, but not astressin2-B. The CRH-R1 receptor, more than CRH-R2 receptor, appears to be essential for the stimulation of the hypothalamus-pituitary-gonad axis by acute stress; this response is likely mediated in part by noradrenergic neurons in the LC. We postulate that the stress-induced facilitation of reproductive function is mediated, at least in part, by CRH action through CRH-R1 on noradrenaline neurons residing in the LC that trigger GnRH discharge and gonadotropin secretion.

Effects of Androgens on Serum Concentrations of Gonadotropins and Ovarian Steroids in Gilts1

To examine how androgens affect endocrine events associated with increased ovulation rate, gilts were injected with androgen receptor agonists, an antagonist, or a combination of both. Blood samples were collected hourly from Day 13 to estrus (Day 0 = onset of estrus) coincident with gilts (n = 6 per treatment) receiving daily treatments of vehicle (corn oil), 10 mg of testosterone, 10 mg of 5 alpha-dihydrotestosterone (dihydrotestosterone), 1.5 g of flutamide (an androgen receptor antagonist), testosterone plus flutamide, or dihydrotestosterone plus flutamide. Treatment of gilts with testosterone or dihydrotestosterone alone increased (P < 0.05) concentrations of FSH in serum, and these effects were blocked by cotreatment with flutamide. Estradiol-17beta and androstenedione concentrations in serum were increased (P < 0.05) at 2 h after injection of testosterone or testosterone plus flutamide but not after dihydrotestosterone treatment, probably because of the role of testosterone as a substrate for estradiol-17beta and androstenedione synthesis. There were no effects of the six treatments on serum concentrations of progesterone during luteolysis, but treating gilts with testosterone shortened (P < 0.05) the proestrous period. Total embryonic loss by Day 11 in gilts treated with dihydrotestosterone was reversed when gilts were cotreated with dihydrotestosterone plus flutamide. Results of this experiment indicated that androgen actions both increased FSH secretion and reduced embryonic survival by a mechanism(s) dependent on the androgen receptor.

Activator Protein-1 and Smad Proteins Synergistically Regulate Human Follicle-Stimulating Hormone β-Promoter Activity

GnRH1 stimulates the synthesis and secretion of FSH and LH from the anterior pituitary gland. The molecular mechanisms through which GnRH1 produces these effects in humans have not been determined. Here, we examined transcriptional regulation of the human FSHbeta (FSHB) subunit using reporter assays in immortalized murine gonadotrope cells. GnRH1 dose and time dependently stimulated FSHB promoter activity, with peak stimulation occurring at 8 h. GnRH1 rapidly stimulated various MAPK cascades, though the ERK1/2 and p38 pathways appeared to be most critical for FSHB induction. Indeed, constitutively active forms of both Raf1 kinase and MAP2K6 (MKK6) were sufficient to stimulate reporter activity. GnRH1 stimulated activator protein-1 (AP-1) (FosB, c-fos, JunB, and cJun) synthesis and complex formation, the latter of which bound to a conserved cis-element within -120 bp of the transcription start site. A second, lower affinity, site was mapped more proximally. Mutations of both cis-elements diminished GnRH1-stimulated promoter activity, though disruption of the higher affinity site had a more dramatic effect. A dominant-negative Fos protein dose dependently inhibited GnRH1-stimulated FSHB transcription, confirming a role for endogenous AP-1 proteins. MAPK kinase 1 (MEK1) and p38 inhibitors significantly attenuated GnRH1-stimulated c-fos, FosB, and JunB synthesis, suggesting a mechanism whereby the ERK1/2 and p38 signaling pathways regulate FSHB transcription. Activins and inhibins potently regulate FSH synthesis in rodents, but their roles in FSH regulation in humans are less clear. Activin A, though weak on its own, synergized with GnRH1 to stimulate human FSHB promoter activity. In contrast, activin A partially inhibited GnRH1-stimulated LHbeta subunit (LHB) transcription. The GnRH1 and activin A signaling pathways appear to converge at the level of the high-affinity AP-1 site. Fos and Jun proteins synergistically regulate reporter activity through this element, and their effects are potentiated by coexpression of either Smad2 or Smad3, effectors in the activin signaling cascade. In summary, GnRH1 and activin A synergistically regulate human FSHB subunit transcription. The combined actions of AP-1 and Smad proteins acting through a conserved AP-1 element provide a candidate mechanism for this effect. The ability of activins to potentiate selectively the effects of GnRH1 on FSHB expression suggests a model for preferential increases in FSH secretion at the luteal-follicular transition of the menstrual cycle.

A review of the effects of supplementary nutrition in the ewe on the concentrations of reproductive and metabolic hormones and the mechanisms that regulate folliculogenesis and ovulation rate

This paper discusses the phenomenon of nutritional flushing in ewes whereby increased nutrition stimulates folliculogenesis and ovulation rate. In addition the paper reviews recent findings on the effects of increased levels of nutrition on the blood concentrations of reproductive and metabolic hormones in the ewe and some of the intraovarian changes that take place in response to nutritional stimulation. Finally, in the paper, we propose a model of the physiological mechanism for the nutritional stimulation of folliculogenesis and we review how closely the model fits recent published and unpublished evidence examining the mechanism of flushing. Nutritional stimulation alters the blood concentrations of some metabolic hormones. By using short-term models of nutritional flushing, we have shown that as the blood concentrations of insulin and leptin increase that of growth hormone decreases while that of IGF-I appears unaffected by the nutritional flushing. Nutritional flushing also alters the blood concentrations of some reproductive hormones. Again, using the same model, we have shown that there is a transient increase in FSH and a decrease in oestradiol concentrations in the blood. The changes in oestradiol are particularly evident in the follicular phase of the oestrous cycle. In the ovary, the effect of nutrition is to stimulate folliculogenesis. These changes are associated with intra-follicular alterations in the insulin-glucose, IGF and leptin metabolic systems. The stimulation of these intra-follicular systems leads to a suppression in follicular oestradiol production. The consequence of these direct actions on the follicle is a reduced negative feedback to the hypothalamic-pituitary system and increased FSH secretion that leads to a stimulation of folliculogenesis.

Adenohypophyseal function in bitches treated with medroxyprogesterone acetate

The aim of this study was to investigate the effects of treatment with medroxyprogesterone acetate (MPA) on canine adenohypophyseal function. Five Beagle bitches were treated with MPA (10 mg/kg, every 4 weeks) and their adenohypophyseal function was assessed in a combined adenohypophyseal function test. Four hypophysiotropic hormones (CRH, GHRH, GnRH, and TRH) were administered before and 2, 5, 8, and 11 months after the start of MPA treatment, and blood samples for determination of the plasma concentrations of ACTH, cortisol, GH, IGF-1, LH, FSH, prolactin, α-MSH, and TSH were collected at −15, 0, 5, 10, 20, 30, and 45 min after suprapituitary stimulation. MPA successfully prevented the occurrence of estrus, ovulation, and a subsequent luteal phase. MPA treatment did not affect basal and GnRH-induced plasma LH concentrations. The basal plasma FSH concentration was significantly higher at 2 months after the start of MPA treatment than before or at 5, 8, and 11 months after the start of treatment. The maximal FSH increment and the AUC for FSH after suprapituitary stimulation were significantly higher before treatment than at 5, 8, and 11 months of MPA treatment. Differences in mean basal plasma GH concentrations before and during treatment were not significant, but MPA treatment resulted in significantly elevated basal plasma IGF-1 concentrations at 8 and 11 months. MPA treatment did not affect basal and stimulated plasma ACTH concentrations, with the exception of a decreased AUC for ACTH at 11 months. In contrast, the maximal cortisol increment and the AUC for cortisol after suprapituitary stimulation were significantly lower during MPA treatment than prior to treatment. MPA treatment did not affect basal plasma concentrations of prolactin, TSH, and α-MSH, with the exception of slightly increased basal plasma TSH concentrations at 8 months of treatment. MPA treatment did not affect TRH-induced plasma concentrations of prolactin and TSH. In conclusion, the effects of chronic MPA treatment on adenohypophyseal function included increased FSH secretion, unaffected LH secretion, activation of the mammary GH-induced IGF-I secretion, slightly activated TSH secretion, suppression of the hypothalamic–pituitary–adrenocortical axis, and unaffected secretion of prolactin and α-MSH.

Passive Immunoneutralization of Endogenous Inhibin Increases Ovulation Rate in Miniature Shiba Goats

We reported previously that passive immunization against inhibin enhances follicular growth and increases the ovulation rate. However, the ovulation rate was not comparable to the number of follicles. Therefore, the aim of this study was to attempt to increase the ovulation rate by increasing the interval between inhibin immunization and PGF2alpha injection. Five miniature Shiba goats were treated with 10 ml inhibin antiserum (inhibin-AS) developed against [Tyro30]-inhibin alpha (1-30). A control group (n=5) was treated with normal goat serum. All animals were injected intramuscularly with 125 microg PGF2alpha 72 h after treatment to induce estrus and ovulation. Blood samples were collected for hormonal assay and the ovulation rate was determined by laparotomy. In contrast to the control group, there was a significant increase in plasma concentrations of FSH in the immunized group. After luteolysis, plasma concentrations of estradiol-17beta increased markedly to a preovulatory peak about 2 folds higher (P<0.01) than that of controls. In addition, the ovulation rate was greater in the immunized group (14.4 +/- 2.2) than in the control group (2.2 +/- 0.6), and the mean number of follicles > or = 4 mm in diameter was 10.0 +/- 0.8 in the inhibin-AS group compared with 2.4 +/- 0.3 in control group. The present results demonstrate that immunoneutralization of endogenous inhibin increased FSH secretions in miniature shiba goats. The increased FSH secretion enhanced follicular growth and increased the ovulation rate. Additionally, increasing the interval between inhibin-AS and PGF2alpha injections (to 72 h) resulted in a greater ovulation rate compared with the previous protocol (48 h). Therefore, inhibin-AS treatment proved to be an effective alternative to exogenous gonadotropin methods for induction of superovulation in goats.

Follistatin suppresses steroid-enhanced follicle-stimulating hormone release in vitro in rats.

Previous in vitro and in vivo studies from our laboratory showed that progesterone (P(4)), corticosterone (B), and testosterone (T) increase intracellular content and release of FSH in the anterior pituitary. Activin (Act) and inhibin (Inh) are structurally related proteins with antagonistic actions, as Act stimulates and Inh inhibits FSH secretion from the anterior pituitary. Together with follistatin (FS), a protein that bioneutralizes Act, they form an autocrine-paracrine loop in the anterior pituitary that tightly regulates FSH secretion. The objective of the present study was to test the hypothesis that P(4), B, and T modulate this autocrine-paracrine loop to favor increased FSH secretion. If Act were to mediate steroid-induced FSH release, FS would be expected to block these effects. To test this interaction, cell cultures were prepared from anterior pituitaries of male and female rats, and treated with Act, B, P(4), or T in the absence or presence of FS. Act, B, P(4), and T increased FSH release; FS suppressed both basal and Act- and steroid-stimulated FSH release to approximately 50% below basal levels. Cell cultures from anterior pituitary of female rats were used to compare the interaction of incremental concentrations of FS on dose-related Act- and P(4)-stimulated FSH release. With increasing concentrations of Act, the FS-induced suppression of FSH release was attenuated and eventually abolished; in contrast, maximally stimulatory concentrations of P(4) did not fully overcome the FS-induced suppression of FSH release. The effects of P(4), B, and Act in the presence and absence of estradiol on steady-state mRNA levels of FSHbeta, Actbeta(B), and FS were determined in primary pituitary cell cultures from metestrous female rats by reverse transcription-polymerase chain reaction. Whereas Act, P(4), B increased FSHbeta mRNA levels, only Act raised the level of FS mRNA, and neither steroid increased Actbeta(B) mRNA. The results support the hypothesis that endogenous Act is a common mediator of the action of P(4), B, and T in the rat primary anterior pituitary cell culture. We conclude that the stimulation of FSH release and intracellular content in the gonadotroph by P(4), B, and T is mediated, in part, by Act and involves modulation of a tightly regulated Act/FS autocrine-paracrine loop.

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.

A role of pituitary adenylate cyclase activating polypeptide (PACAP) as a regulator of paracrine interactions between folliculo-stellate cells and gonadotropes through the control of activin-follistatin interactions.

Pituitary folliculo-stellate (FS) cells were able to modify the effect of activin-A on gonadotropes through the paracrine factor, follistatin. The present study was aimed to examine whether a hypothalamic peptide, pituitary adenylate cyclase activating polypeptide (PACAP), could be a regulator of this paracrine interaction. Co-culture of FS cell-originated cell line TtT/GF cells with rat anterior pituitary cells showed faint inhibitory effect on the stimulatory action of activin-A on FSH secretion. When PACAP was added to the culture during the co-culture period, however, the presence of TtT/GF cells caused significant suppression of the effect of activin-A on FSH secretion. Conditioned-media (CM) from TtT/GF cells, obtained by incubation of TtT/GF cells in the presence or absence of PACAP, were next added to the cultures of anterior pituitary cells alone. CM from TtT/GF cells without PACAP treatment revealed slight, but not significant, suppressive effect on activin-induced increases in FSH secretion and the percentage of FSH cells. Meanwhile, CM from PACAP-treated TtT/GF cells attenuated both effects of activin-A. Furthermore, the inhibitory effect of the CM was neutralized when follistatin antibody was present in the culture. These results suggest that PACAP is able to regulate the paracrine action of FS cells on pituitary gonadotropes. Besides expressing direct actions on pituitary endocrine cells, PACAP may have roles as a regulator of cell-to-cell interactions within the pituitary gland.

Activin subunit, follistatin, and activin receptor gene expression in the prepubertal female rat pituitary.

In the prepubertal female rat, a transient and selective increase in FSH secretion and mRNA expression by the pituitary gland occurs toward the end of the second postnatal week of life. To begin to investigate the possibility that activin may play a role in up-regulating FSH during this time, we have studied the ontogeny of the expression of the activin ss subunits, follistatin, and activin receptor subtypes in the prepubertal female rat pituitary. The levels of expression of ssA, ssB, and follistatin mRNAs were determined in the pituitary gland on postnatal days (PND) 8, 10, 12, 15, and 21 by semiquantitative reverse transcription-polymerase chain reaction. All values were compared to those of adult females killed on diestrus. mRNA levels of subunit ssA were significantly (p < 0.05) elevated on all postnatal days examined; ssB mRNA levels were elevated above adult levels only on PND 10 (p < 0.05). Follistatin mRNA was high on PND 8 (p < 0.05) and then decreased to adult levels. The level and distribution of activin receptor type II subtype mRNAs were determined by in situ hybridization. Activin receptor type II (Act RII) mRNA expression was diffusely expressed throughout all areas of the pituitary. Activin receptor type IIB (Act RIIB), on the other hand, was highly expressed by a subset of anterior pituitary cells. In situ hybridization for activin receptor subtype mRNAs was combined with immunocytochemistry to detect FSH-containing cells. We determined that in the infantile female pituitary, Act RII mRNA was generally not expressed in FSH-immunoreactive cells, while Act RIIB mRNA was expressed in FSH-immunoreactive cells. Act RII mRNA was lower on PND 10 and 15 when compared to PND 21 (p < 0.05), whereas Act RIIB mRNA expression did not change with age. These data suggest that the essential components of the activin regulatory system are present in the infantile female pituitary gland and thus may be involved in the differential regulation of FSH at this time.

Effects of active immunization of sheep against an amino terminal peptide of the inhibin alpha C subunit on intrafollicular levels of activin A, inhibin A and follistatin.

Active immunization of ewes against inhibin (IMM) consistently increases ovulation rate but this response is not always accompanied by the expected rise in plasma FSH. Inhibin-related molecules also have local auto/paracrine effects within the ovary and the ovulatory response to IMM could be due to neutralization of one of these effects, independent of changing FSH levels. To investigate this, ovaries were collected from long-term IMM (n = 6) and control (CON; n = 8) ewes killed 48 h after progestagen withdrawal (late follicular phase) and all follicles > or = 3 mm were recovered to determine intrafollicular levels of inhibin A, activin A and follistatin by specific two-site immunoassay and oestradiol and testosterone by radioimmunoassay. Blood samples were collected to assess plasma FSH, oestradiol and inhibin antibody titres. Although plasma FSH levels were similar in IMM and CON ewes, IMM ewes had approximately 3-fold more follicles > or = 3 mm (P < 0.0001) and approximately 3-fold more oestrogenic follicle (P < 0.001) than CON ewes. Compared with CON ewes, follicles from IMM ewes had much higher concentrations of activin A (approximately 6-fold; P < 0.001) and inhibin A (approximately 3-fold; P < 0.001) but only slightly more follistatin (approximately 1.4-fold; not significant). The activin A:follistatin ratio in follicles from IMM ewes (approximately 1:1) was significantly higher (P < 0.001) than in follicles from CON ewes (approximately 0.3:1). Levels of inhibin antibody measured in follicular fluid (FF) from IMM ewes were similar to plasma levels. Given that activin A has been shown previously to up-regulate FSH receptors and aromatase activity in rat granulosa cells, the increase in intrafollicular activin A, unaccompanied by a rise in the concentration of its binding protein (follistatin), could explain how long-term IMM enhances follicle development and ovulation rate without necessarily promoting a sustained increase in FSH secretion.

Contributions of endogenous inhibin and estradiol to the regulation of follicle-stimulating hormone and luteinizing hormone secretion in the pregnant rat.

To examine the contributions of endogenous inhibin and estradiol to the regulation of FSH and LH secretion in the pregnant rat, some rats were passively immunized against inhibin and/or estradiol, and others were ovariectomized, on Days 5, 10, 15, and 20 of pregnancy. Ovarian and uterine venous blood was collected separately to confirm the sources of inhibin and steroid hormones during pregnancy. Immunoreactivity of inhibin in the placenta was also examined by RIA. Levels of inhibin in ovarian venous plasma were significantly higher than those in peripheral plasma during pregnancy. No difference was observed between the levels of inhibin in uterine venous plasma and peripheral plasma. No immunoreactivity of inhibin was detected in placental homogenate from rats at Days 10, 15, and 20. FSH secretion significantly increased after immunoneutralization of inhibin during pregnancy. A marked increase in FSH secretion was noted on Days 5 and 20, and the smallest increase was observed on Day 15. Administration of estradiol antiserum (AS) alone did not induce a significant increase in FSH secretion on any day of pregnancy. However, a synergistic effect of estradiol AS and inhibin AS was observed on Day 20. On Days 5, 10, and 20, administration of inhibin AS or estradiol AS induced a significant increase in LH secretion. A synergistic effect of inhibin AS and estradiol AS on LH secretion was observed on Day 5. On Days 5 and 10, significantly high LH secretion was noted in ovariectomized rats as compared with that in rats treated with both inhibin AS and estradiol AS, indicating that other ovarian hormones such as progesterone may be involved in the suppression of LH secretion in these stages of pregnancy. These data indicate that both inhibin and estradiol, predominantly secreted from the ovary, are involved in the regulation of gonadotropin secretion during pregnancy as during the estrous cycle in the rat.

Effects of passive immunization against inhibin-peptide on secretion of follicle-stimulating hormone and ovulation rate in ewes carrying the Booroola fecundity gene.

Objectives of the study were to determine whether 1) inhibin negative feedback regulation of FSH secretion is diminished in ewes carrying a copy of the Booroola fecundity (FecB) gene and 2) differential FSH secretion is obligatory for expression of gene-specific differences in ovulation rate (OR). The approach was to compare FSH and ovulatory responses to passive immunoneutralization of inhibin in ewes with and without a copy of the FecB gene. Twenty-eight 2- to 3-yr-old ewes were assigned within genotype to antibody (alpha-IF-Ab) or control groups. Genotypes consisted of 3/4 Rambouillet x 1/4 Booroola ewes with one copy of the FecB gene (FecB+; 57 kg) and 3/4 Rambouillet x 1/4 Booroola ewes without the FecB gene (++; 59 kg). Estrus was synchronized during the breeding season using progesterone-releasing pessaries (CIDR-G). Pessaries were removed at 0 h. A single injection of alpha-IF-Ab or control solution was given at -48 h. Alpha-IF-Ab had been developed against a synthetic inhibin fragment matching the N-terminal region of ovine inhibin's alpha subunit. For injection, alpha-IF-Ab had been precipitated from ovine immune sera and concentrated. Blood samples were collected at 6-h intervals from -48 to 48 h, and laparoscopy was performed 14 days after CIDR-G withdrawal. All ewes exhibited estrus and ovulated. Genotype and alpha-IF-Ab treatment were without effect on intervals to estrus. Both factors affected OR (p < or = 0.001). Mean OR in control ++ and FecB+ ewes were 1.6 and 2.7, respectively; mean OR in alpha-IF-Ab-treated ++ and FecB+ ewes were 2.5 and 4.6, respectively. Following injection of alpha-IF-Ab, FSH concentrations increased within 6 h, peaked 12-18 h later, and then declined. Magnitude of FSH increases was similar in ++ and FecB+ ewes (70% and 85% over control values, respectively). Results demonstrate that 1) inhibin negative feedback regulation of FSH secretion is not a site of FecB gene action and 2) the mechanism by which the FecB gene increases OR does not necessarily involve increased FSH secretion during the period of preovulatory follicular development.

A quantitative analysis of testosterone action on FSH secretion from individual pituitary cells using the cell immunoblot assay.

We investigated the effects of testosterone on FSH secretion from male rat anterior pituitary cells in culture at the single cell level. Anterior pituitary cells cultured with or without 10 ng/ml testosterone for 72 h were mono-dispersed and subjected to cell immunoblot assays for FSH. Cell blots specific for FSH were quantified by means of a microscopic image analyzer. The number of FSH-secreting cells detected as immunoreactive cells blots on the transfer membrane represented 4.1% of total pituitary cells applied on the membrane. The amount of FSH secreted by single cells varied from < 20 to > 8,000 fg/cell/h. The number of FSH-secreting cells was not changed by the addition of 10 ng/ml testosterone into the culture medium. Testosterone administration increased the mean FSH secretion by 64% after 3 h incubation, resulting in a shift to the right in the frequency distribution of FSH secretion from single cells. The total amount of FSH, namely the sum of FSH secreted by each FSH-secreting cel, was increased by 92% by the addition of testosterone. However, mean amounts of FSH secretion by the top ten cells of the largest secretor subgroup (> 5 pg/cell/3 h) were not different between control and testosterone-treated groups. The present study analyzed, for the first time, FSH secretion from rat anterior pituitary cells at the single cell level. The results suggest that stimulation by testosterone of FSH secretion in vitro is not due to an increase in the number of FSH-secreting cells but to an increase in FSH secretion from each cell.

Ovine anterior pituitary production of follistatin in vitro.

Rat pituitary cells express messenger RNA for the activin-binding protein, follistatin (FS), and rat and bovine pituitary cell cultures secrete FS into the medium. In the present study, a previously validated, heterologous RIA for ovine FS was employed to investigate FS synthesis, secretion, and regulation in cultures of ovine anterior pituitary cells. The validity of the RIA was confirmed by the finding that FS immunoreactivity in ovine pituitary cell culture-conditioned medium diluted in parallel with purified bovine FS, and fractionation of the conditioned medium resulted in the coelution of activin-binding activity with the FS immunoactivity. The concentration of endogenous ovine FS achieved in the culture medium (0.08-0.6 nM) was in the range over which bovine FS suppresses FSH secretion in these cultures (IC50 = 0.5 nM). To characterize the relationship between endogenous FS and FSH secretion, dispersed ovine pituitary cells were preincubated with 10% fetal bovine serum for 2 days, then cultured between days 2-5 in the presence of a chemically defined serum substitute. Under these conditions, FS was continuously secreted at a rate of 12.1 +/- 1.8 ng/10(6) cells.day (mean +/- SEM; n = 18), whereas FSH was secreted at 64 +/- 13 ng/10(6) cells.day (n = 7). The secretion of FS and FSH changed in a reciprocal way as culture conditions were altered either by maintaining exposure of the cells to fetal bovine serum or by plating the cells at a 6- to 10-fold higher seeding density. Under the latter circumstance, for instance, FS secretion during the 3-day test period decreased to 47 +/- 14% (n = 10) and FSH secretion increased to 137 +/- 6% (n = 6) of the respective values in cultures of dispersed cells. FS secretion was increased nearly 3-fold (P < 0.05) in a dose-dependent manner by continuous exposure of ovine pituitary cells between days 2-5 to recombinant human activin A (1-10 nM), which concomitantly increased FSH secretion. Recombinant human inhibin A (0.003-10 nM); the synthetic glucocorticoids, RU28362 and dexamethasone (each 1-100 nM); the sex steroids, testosterone (1-100 nM), 17 beta-estradiol (0.001-5 nM), and progesterone (4-2500 nM); and the vitamin A derivative, retinoic acid (0.3-32 microM), each inhibited FSH secretion from these cultures, but only the last agent significantly (P < 0.05) increased FS secretion. Inhibin prevented the stimulation of FSH secretion by activin A without affecting its stimulation of FS secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of reproductive deficiency in male rats treated neonatally with a gonadotrophin-releasing hormone antagonist

It is well known that males injected neonatally with oestradiol or antiserum or antagonists (ANT) against gonadotrophin-releasing hormone (GnRH) show multiple reproductive disorders. In the present work, in males treated neonatally with GnRH-ANT, we have analysed: (1) whether the impairment of reproductive function can be blocked by simultaneous treatment with gonadotrophins, (2) the possible differences in the effects of GnRH-ANT injected before or after the proliferation of Sertoli cells which takes place between days 1 and 15 of age, and (3) the mechanism(s) for the increased FSH secretion observed in adulthood. Experimental designs included: administration of GnRH-ANT between days 1 and 16 or 15 and 30 of age, simultaneous administration of gonadotrophins and GnRH-ANT to neonatal males, and measurement of FSH secretion after orchidectomy or specific destruction of Leydig cells with ethylene dimethane sulphonate (EDS) in adult males treated neonatally with GnRH-ANT. The principal new data presented in our studies are the following: (1) delayed puberty was observed not only in males injected neonatally with GnRH-ANT, but also in those injected with gonadotrophins or with GnRH-ANT and gonadotrophins, (2) the decreased fertility and increased FSH secretion observed in adult males treated neonatally with GnRH-ANT were normalized by simultaneous administration of GnRH-ANT and gonadotrophins, and (3) the increased FSH secretion in adult males treated neonatally with GnRH-ANT remained after EDS or orchidectomy, suggesting that mechanisms other than decreased inhibin secretion were involved in the increased secretion of FSH.