Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion

The effects of superovulatory treatment (follicle stimulating hormone [FSH] versus human menopausal gonadotropin [HMG]) and of route of administration (intramuscular versus intravenous) of prostaglandin F2a (PGF2a) on hormonal profiles were determined in 32 Angus x Hereford heifers for breeding and subsequent embryo collection and transfer. Heifers were superstimulated either with FSH (total of 26 milligrams) or HMG (total of 1,050 international units) beginning on days 9 to 12 of an estrous cycle and PGF2a (40 milligrams) was administered at 60 and 72 hours after the beginning of superovulatory treatments. Heifers were artificially inseminated three times at 12-hour intervals beginning 48 hours after PGF2a treatment. Blood serum samples were collected immediately before treatments began and at frequent intervals until embryo collection 288 hours later. Concentrations of luteinizing hormone (LH) and FSH were not affected by hormone treatments, route of PGF2a injection, or interactions between them. Estradiol-17ß (E2-17ß) levels were higher in HMG- than in FSH-treated heifers 60 hours after gonadotropin treatment. Peak concentration of E2-17ß occurred earlier in HMGthan in FSH-treated heifers and earlier in heifers injected with PGF2a intramuscularly than those injected intravenously. Progesterone concentrations were not influenced by treatment or route of PGF2a administration. The progesterone:E2-17ß ratio was higher in FSH- than in HMG-treated heifers 24 hours after the LH peak. The high steroid hormone concentrations in superovulated beef heifers before and after ovulation may lead to asynchrony between stages of embryonic development, a situation that may interfere with the pregnancy outcome of superovulated embryos in recipient animals.

A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH- but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive in vivo assay for FSH- and LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.

Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R −/− mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R −/− mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor.

Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors.

The frequency of follicle stimulating hormone receptor gene polymorphisms in Iranian infertile men with azoospermia

Background: Azoospermia is the medical condition of a man not having any measurable level of sperm in his semen. Follicle stimulating hormone (FSH) is a member of the glycoprotein hormone family that plays an important role in human reproduction because of its essential role in normal spermatogenesis. Various Single Nucleotide Polymorphisms (SNPs) have been reported within FSH receptor (FSHR) gene that may affect the receptor function. Objective: The present study aimed to investigate the correlation between two FSHR SNPs at positions A919G, A2039G, and susceptibility to azoospermia in a group of Iranian azoospermic men. The association between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Materials and Methods: This case control study was performed on 212 men with azoospermia (126 non-obstructive and 86 obstructive) and 200 healthy Iranian men. Two FSHR gene SNPs were genotyped using PCR-RFLP method. The relationship between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Results: Statistical analysis indicated that at A919G position, AA genotype and A allele were more frequent in obstructive azoospermia cases compared to non- obstructive or normal men (p=0.001). Regarding A2039G polymorphisms, no significant difference was observed between both azoospermia groups and the controls. The mean level of serum FSH was higher in the non-obstructive men compared to the obstructive patients (23.8 versus 13.8, respectively, p= 0.04). Conclusion: The results of the present study indicated that the genetic polymorphisms in the FSHR gene might increase the susceptibility to azoospermia in Iranian men.

Association of follicle stimulating hormone receptor promoter with ovarian response in IVF-ET patients

Background: Poor ovarian response phenomenon has been observed in some of the in vitro fertilization-embryo transfer patients. Some investigations found that follicle stimulating hormone receptor (FSHR) gene plays a role in the process, but no direct evidence shows the correlation between genotypes of FSHR and ovarian response. Objective: Exploring the molecular mechanism behind the mutation of FSHR promoter association with ovarian granulosa cells and poor ovarian response. Materials and Methods: This cross sectional study was performed using 158 women undergoing the controlled short program ovarian stimulation for IVF treatment. The 263 bp DNA fragments before the follicle stimulating hormone (FSH) receptor 5' initiation site were sequenced in the patients under IVF cycle, 70 of which had poor ovarian response and 88 showed normal ovarian responses. Results: With a mutation rate of 40%, 63 in 158 cases showed a 29th site G→A point mutation; among the mutated cases, the mutation rate of the poor ovarian responders was significantly higher than the normal group (60% versus 23.9%; χ2=21.450, p<0.001). Besides, the variability was also obvious in antral follicle count, and ovum pick-ups. The estradiol peak values and the number of mature eggs between the two groups had significant difference. However, there was no obvious variability (t=0.457, p=0.324) in the basic FSH values between the two groups (normal group, 7.2±2.3 U/L; mutation group, 7.1±2.0 U/L). Conclusion: The activity of FSHR promoter is significantly affected by the 29th site G→A mutation that will weaken promoter activity and result in poor response to FSH.

Role of luteinizing hormone in follicle deviation based on manipulating progesterone concentrations in mares

The effects of several doses of progesterone on FSH and LH concentrations were used to study the role of the gonadotropins on deviation in growth rates of the two largest follicles during the establishment of follicle dominance. Progesterone was given to pony mares at a daily dose rate of 0 mg (controls), 30 mg (low dose), 100 mg (intermediate dose), and 300 mg (high dose). All follicles ≥ 6 mm were ablated at Day 10 (Day 0 = ovulation) to initiate a new follicular wave; prostaglandin F(2α) was given to induce luteolysis, and progesterone was given from Days 10 to 24. The low dose did not significantly alter any of the ovarian or gonadotropin end points. The high dose reduced (P < 0.05) the ablation-induced FSH concentrations on Day 11. Maximum diameter of the largest follicle (17.2 ± 0.6 mm) and the second- largest follicle (15.5 ± 0.9 mm) in the high-dose group was less (P < 0.04) than the diameter of the second-largest follicle in the controls (20.0 ± 1.0 mm) at the beginning of deviation (Day 16.7 ± 0.4). Thus, the growth of the two largest follicles was reduced by the high dose, presumably through depression of FSH, so that the follicles did not attain a diameter characteristic of deviation in the controls. The intermediate dose did not affect FSH concentrations. However, the LH concentrations increased in the control, low, and intermediate groups, but then decreased (P < 0.05) in the intermediate group to pretreatment levels. The LH decrease in the intermediate group occurred 2 days before deviation in the controls. The maximum diameter of the largest follicle was less (P < 0.0001) in the intermediate group (27.3 ± 1.8 mm) than in the controls (38.9 ± 1.5 mm), but the maximum diameter of the second-largest follicle was not different between the two groups (19.0 ± 1.1 vs. 20.3 ± 1.0 mm). Thus, the onset of deviation, as assessed by the second-largest follicle, was not delayed by the decrease in LH. Diameter of the largest follicle by Day 18 in the intermediate group (23.1 ± 1.6 mm) was less (P < 0.05) than in the controls (28.0 ± 1.0 mm). These results suggest that circulating LH was not involved in the initiation of dominance (inhibition of other follicles by the largest follicle) but was required for the continued growth of the largest follicle after or concurrently with its initial expression of dominance.

The ability of prolactin to change the sensitivity of the pituitary of the lactating rat to luteinising hormone releasing hormonein vitro

The ability of prolactin to influence the responsiveness of the lactating rat pituitary to luteinising hormone releasing hormone has been examinedin vitro. The pituitary responsivenessin vivo to luteinising hormone releasing hormone decreased as a function of increase in the lactational stimulus. Prolactin inhibited the spontaneousin vitro release of luteinising hormone and follicle stimulating hormone to a small extent, from the pituitary of lactating rats with the suckling stimulus. However, it significantly inhibited the release of these two hormones from luteinising hormone releasing hormone-stimulated pituitaries. The responsiveness of pituitaries of rats deprived of their litter 24 h earlier, to luteinising hormone releasing hormone was also inhibited by prolactin, although minimal. It was concluded that prolactin could be influencing the functioning of the pituitary of the lactating rat by (a) partially suppressing the spontaneous release of gonadotropin and (b) inhibiting the responsiveness of the pituitary to luteinising hormone releasing hormone.

The Hinge Region of Human Thyroid-Stimulating Hormone (TSH) Receptor Operates as a Tunable Switch between Hormone Binding and Receptor Activation

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the alpha-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.

The antibodies against the computationally designed mimic of the glycoprotein hormone receptor transmembrane domain provide insights into receptor activation and suppress the constitutively activated receptor Mutants

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Stable serum levels of anti-Müllerian hormone during the menstrual cycle: A prospective study in normo-ovulatory women

Background: Anti-Müllerian hormone (AMH), secreted by the granulosa cells of preantral and small antral follicles, has been described as a potential marker of the ovarian reserve. The aim of this prospective study is to investigate the variations of AMH during the menstrual cycle in a young selected population of normo-ovulatory women and to analyse the correlation with other cyclic hormones. Methods: Twenty healthy volunteers from 19 to 35 years old, with regular menstrual cycles (26-31 days), normal ovulation (day 10-16), normal hormonal profile and normal body mass index (18-26 kg/m2) were recruited. AMH, inhibin B, LH, FSH, estradiol and progesterone were measured on days 3, 7, 10, 11, 12, 13, 14, 15, 16, 18, 21 and 25 of a spontaneous cycle. Results: AMH serum levels, either expressed by cycleday or aligned according to the ovulation day, did not show any significant variations during the menstrual cycle. Conclusions: No significant fluctuation of the AMH level during the menstrual cycle was observed. Therefore, this hormone is particularly interesting for clinical evaluation of the ovarian reserve as it may be used at any time during the cycle. © The Author 2007.

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.(C) 2003 Elsevier Science B.V. All rights reserved.