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PubMed1998

A naturally occurring basically charged human follicle-stimulating hormone (FSH) variant inhibits FSH-induced androgen aromatization and tissue-type plasminogen activator enzyme activity in vitro.

It is well known that deglycosylation of gonadotropins by enzymatic or chemical procedures or by deletion of sites for N-linked glycosylation produces antagonistic analogs which are able to interact strongly with the receptor and to inhibit binding of the wild-type hormone. In the present study, we analyzed the antagonistic properties of a naturally occurring basic follicle-stimulating hormone (FSH) charge isoform obtained after high-resolution chromatofocusing of human anterior pituitary glycoprotein extracts. Coincubation of increasing amounts of this isoform with a highly purified human pituitary FSH preparation or with recombinant human FSH at doses equivalent to their corresponding ED50 for estradiol and tissue-type plasminogen activator (tPA) production, inhibited FSH-induced estrogen production and tPA enzyme activity by cultured rat granulosa cells in a dose-dependent manner. These inhibitory effects were apparently exerted at steps following 3',5'-cyclic adenosine monophosphate (cAMP) formation and did not involve activation of the protein kinase C pathway since: (a) at low doses, this basic FSH isoform moderately increased FSH-induced cAMP production by cultured rat granulosa cells; (b) coincubation of the antagonist isoform with dibutyryl cAMP completely inhibited the effects of this cAMP analog on estrogen and tPA production; (c) the isoform was able to stimulate production of cAMP in a human fetal cell line expressing the recombinant human FSH receptor, and (d) the inhibitory effects of the isoform were not affected by staurosporine, a protein kinase C inhibitor. The effects of this isoform upon dibutyryl cAMP-induced estrogen and tPA production were blocked by the addition of a highly specific antibody directed against human FSH, further demonstrating that the antagonistic effects observed were due to FSH-like molecules. In contrast to the inhibitory effects exhibited by this basic FSH isoform, a more acidic FSH charge variant consistently acted as an agonist of pituitary and recombinant FSH on both estrogen production and induction of tPA enzyme activity. These results indicate that the anterior pituitary gland normally produces FSH isoforms which act as either agonists or antagonists of FSH at the target cell level.

Androgens↗
PubMed1997

Neuroendocrine control of follicle-stimulating hormone (FSH) secretion. I. Direct evidence for separate episodic and basal components of FSH secretion.

Continuous sampling of hypophyseal portal blood from unrestrained sheep is providing an unprecedented means for measuring and defining the characteristics of the secretory profile of GnRH. With this method, GnRH has been shown to be released in discrete pulses lasting 5-8 min, with the amplitude of some pulses exceeding 50-fold. Although the relationship between these pulses and the accompanying pulses of LH measured in the jugular vein are unambiguous, the relationship of GnRH pulses to the release of FSH has not been well defined due to the longer clearance of FSH. In previous studies we have shown that hypophyseal portal blood, in addition to serving as a source material for hypothalamic secretions, provides a means to define secretory patterns of pituitary hormones. Because of this we hypothesized that the GnRH-FSH secretory relationship would be easier to define in hypophyseal portal than in jugular vein blood before the secretory products are subjected to dispersion and clearance in circulation. To test this possibility, we monitored hormonal patterns in blood collected at 5-min intervals for 6-12 h from the peripheral and hypophyseal portal circulation of six ovariectomized ewes from a previous study. In contrast to the nonpulsatile pattern of FSH in the peripheral blood, 93% of the GnRH pulses were associated with essentially coincident, discrete pulses of FSH in the portal plasma. Of potentially even greater interest, additional episodes of FSH release were clearly discernible between the GnRH-associated pulses of FSH. As concentrations of peripheral plasma FSH did not reach those in hypophyseal portal plasma, the inter-GnRH episodes of FSH secretion could not result from contaminating peripheral blood. In addition to the episodic mode of secretion, substantial amounts of FSH were found between FSH pulses. This basal component of FSH appeared to be the dominant mode of secretion rather than pulses. The results of this study not only confirm that GnRH pulses lead to pulsatile release of FSH, they also suggest that some other mechanism or factor may be controlling the non-GnRH-associated episodes as well as the basal components of FSH secretion.

Animals↗
PubMed1981

Age dependent changes in FSH responsive adenylyl cyclase and FSH binding in rat testes.

FSH responsive adenylyl cyclase (AC) activities were examined in both homogenates and membrane particles from 9-71 days old rat testes, and FSH binding was determined in membrane particles from the same animals. FSH responsive AC was measured in the presence of either GTP (0.04 mM) or the non-metabolizable GTP analogue, GMP-P(NH)P (0.04 mM). From days 9 and 11 there was a gradual decrease in both basal and FSH stimulated AC activities and a very similar decrease in FSH binding. The relative stimulation by FSH, however, was fairly constant (2-3 fold stimulation). The excellent correlation between specific FSH binding and FSH responsive AC activities during sexual maturation does not support the notion that uncoupling of the FSH receptors from the AC is a primary reason for the attenuated FSH response during pubertal development. The age dependent decrease in both basal and FSH stimulated AC activities during sexual maturation is a reflection of the relative increase in germ cells (containing no FSH receptors and FSH responsive AC). The LH/hCG responsive AC in membrane particles from the same ages showed an age dependent increase in both absolute activities and relative responses, probably indicating the increased number of Leydig cells in the developing testis.

Adenylyl Cyclases↗
PubMed1992

Effects of activin and follicle-stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells.

The aim of this study was to investigate the actions of both activin and FSH-suppressing protein (FSP)/follistatin either alone or in combination on FSH receptor number and on the responsiveness of granulosa cells to FSH and LH. Granulosa cells were harvested from diethylstilbestrol-treated immature Sprague-Dawley rats and cultured 48 h in serum-free medium with or without treatment. Activin treatment alone (3-100 ng/ml) resulted in a 4-fold increase in FSH receptor number with no change in binding affinity. This effect of activin was inhibited 31% by FSP (100 ng/ml) treatment which alone had no effect on FSH receptor number. Treatment with activin (100 ng/ml) prevented FSH-induced down-regulation of FSH receptor number, whereas at lower concentrations (3-30 ng/ml) activin enhanced down-regulation of FSH receptor number by 20% (P less than 0.05). In contrast, FSP alone prevented FSH-induced down-regulation by increasing FSH receptor number up to 40-50%. Pretreatment of granulosa cells with activin, but not FSP, for 24 h increased the responsiveness of cells to FSH (20 ng/ml) and LH (40 ng/ml) shown by increases in aromatase activity, progesterone, and immunoreactive inhibin production over and above control in a manner which depended upon activin doses. We conclude that 1) activin enhancement of FSH action on rat granulosa cells may be mediated in part via regulation of FSH receptor number, and 2) the effects of FSP on granulosa cells are likely to be due to its activin binding properties.

Activins↗
PubMed1993

Effect of an activin A on follicle-stimulating hormone (FSH) receptor messenger ribonucleic acid levels and FSH receptor expressions in cultured rat granulosa cells.

Activin, a dimer of beta-subunits of inhibin, has been found to induce FSH receptor on cultured rat granulosa cells. The effect of activin on FSH receptor messenger RNA (mRNA) levels has not been elucidated. To study the effect of activin on FSH receptor mRNA levels, we used a specific complementary RNA probe to evaluate changes in FSH receptor transcripts in cultured rat granulosa cells. Granulosa cells obtained from immature diethylstilbestrol-treated rats contained two predominant FSH receptor mRNA transcripts (5.5 and 2.4 kilobases). Compared to the control, the treatment of granulosa cells with activin (100 ng/ml) increased FSH receptor mRNA in a time-dependent manner with a maximum of about a 4-fold increase at 24 h. FSH receptor mRNA markedly decreased after 48 h and maintained a level comparable to that found in the control. The FSH receptor expression was also increased by activin. Scatchard analysis of the binding of rat FSH to granulosa cells showed that the increase in FSH binding after activin treatment was due to an increase in the receptor number and not the affinity of binding. Treatment of granulosa cells for 24 h with activin (20-300 ng/ml) increased FSH receptor mRNA in a dose-dependent manner to a maximum of about a 4-fold increase at a concentration of 100-300 ng/ml. We analyzed rat type II activin receptor mRNA transcripts in cultured rat granulosa cells with a specific complementary RNA probe to study the action of activin on granulosa cells. Granulosa cells contained two predominant rat type II activin receptor mRNA transcripts (6.0 and 3.0 kilobases). Furthermore, we measured intracellular cAMP production by activin to examine the mechanism by which activin acts on granulosa cells. In result, activin alone did not increase intracellular cAMP accumulation. In conclusion, this study demonstrates that the effect of activin A on the induction of FSH receptor expression is associated with a change in FSH receptor mRNA levels, suggesting that modulation of follicle development occurs.

Activins↗
PubMed1993

Functional properties of polyclonal antibodies raised against the N-terminus region (residues 9-30) of the follicle-stimulating hormone (FSH) receptor: significance of this receptor region in FSH recognition and signal transduction.

In this study, we raised polyclonal antibodies in rabbits against a synthetic peptide corresponding to a unique region of the FSH receptor, residues 9-30, with no sequence homology to receptors for LH and TSH, and examined their characteristics relevant to receptor function. Binding of [125I]human (h) FSH to membrane-bound receptors was inhibited in a concentration-dependent manner by the anti-FSH receptor-(9-30) peptide antibody. Preimmune serum had no effect. Lineweaver-Burke plot analysis of [125I]hFSH binding to membrane receptors in the presence or absence of antireceptor peptide antibody indicated that the antibody effectively competed with FSH at a hormone-binding site on the receptor. Also, antireceptor peptide antibody, but not preimmune serum, inhibited the ability of FSH to stimulate the conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulation of estradiol synthesis by Sertoli cells caused by cholera toxin or forskolin (which are known to act through the Gs-protein and catalytic unit of adenylate cyclase, respectively) was not inhibited by antireceptor peptide antibody. Indirect immunofluorescence staining of cultured rat Sertoli cells showed binding of antibody to plasma membrane receptor. No fluorescent staining of receptor was observed when cells were incubated with preimmune serum or antireceptor peptide antibody in the presence of excess receptor-(9-30) peptide or hFSH. These results were consistent with specific labeling of membrane-bound FSH receptors by anti-receptor-(9-30) peptide antibody. When detergent-solubilized membrane preparations from rat Sertoli cells were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and then subjected to Western blot analysis, antireceptor peptide antibody, but not preimmune rabbit serum, specifically recognized intact FSH receptor. Although the antireceptor peptide antibody occupied the N-terminus 9-30 epitope region in the FSH receptor, it did not induce postbinding events, such as receptor patching (aggregation), as shown by indirect immunofluorescence staining of rat Sertoli cells and the estradiol response. In contrast, a polyclonal antibody against the FSH holoreceptor capable of interacting with multiple epitopes on the receptor could induce FSH-like effects, such as receptor patching and estradiol response in Sertoli cells. In conclusion, antibody raised against the N-terminus region (9-30) of the FSH receptor recognized intact FSH receptor, inhibited FSH binding, and behaved as an antagonist, suggesting that this N-terminus epitope region of the receptor is involved in hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2002

Role of FSH, numbers of FSH receptors and testosterone in the regulation of inhibin secretion during the seasonal testicular cycle of adult rams.

The regulation of inhibin secretion has not been elucidated fully in male ruminants. The aim of this study was to determine the relative importance of FSH and testosterone concentrations, and FSH receptors, in the control of secretion of immunoactive inhibin in rams. In Expt 1, temporal changes in hormone concentrations and testicular FSH binding were determined for two groups of rams (n = 4) kept under opposite, alternating 4 month periods of long (16 h light:8 h dark) and short (8 h light:16 h dark) days. Testicular biopsies (1-2 g) were collected when the testes were regressed, redeveloping, redeveloped and regressing. In Expt 2, separate groups of rams (n = 4) kept under natural photoperiod (latitude 45 degrees 48 minutes N) were designated as controls or passively immunized (for 3 weeks) with sufficient oestradiol antiserum to increase testosterone secretion without altering LH and FSH; this was done when the testes were regressed (non-breeding season) and redeveloped (breeding season). In both groups of rams (Expt 1), 'seasonal' increases in FSH concentrations began a few weeks earlier than did increases in inhibin concentrations. FSH reached maximum concentrations during testicular recrudescence, whereas numbers of FSH receptors in the testis and circulatory inhibin concentrations did not reach peak values until the testes were fully developed. Numbers of FSH receptors per testis, but not FSH concentration, were positively correlated (r = 0.65) with inhibin concentrations across the four stages of the testicular cycle. Near the end of testicular recrudescence early in the breeding season (Expt 2), relatively high FSH concentration was associated with increased abundance of FSH receptor mRNA (90%) and number of receptors (45%) in the testis and increased inhibin concentrations (50%), compared with when the testes were regressed. Moderate, physiological increases in testosterone secretion in immunized rams did not affect inhibin in either season. These results indicate that: (i) FSH stimulation of immunoactive inhibin secretion by Sertoli cells as testes recrudesce is via increases in secretion (early) and cognate receptors (late); (ii) FSH upregulates the synthesis of its own receptor late in recrudescence; and (iii) the positive correlation (r = 0.70) observed between circulatory testosterone and immunoactive inhibin does not reflect a causal relationship.

Analysis of Variance↗
PubMed1998

Characteristics of consecutive in vitro fertilization cycles among patients treated with follicle-stimulating hormone (FSH) and human menopausal gonadotropin versus FSH alone.

OBJECTIVE: To compare the endocrine responses of patients who first received hMG plus FSH, then were treated in a subsequent cycle with FSH alone. DESIGN: Retrospective study. SETTING: An academic research environment. PATIENT(S): Ninety-six women with pituitary down-regulation who underwent two sequential IVF treatments, the first with combined hMG and FSH and the second with FSH alone. MAIN OUTCOME MEASURE(S): Duration of stimulation, serum estradiol level on the day of hCG administration, amount of gonadotropin used, number of oocytes retrieved, number of oocytes fertilized, and selected preembryo morphologic features. RESULT(S): No difference in the mean duration of stimulation was observed between the treatment cycles among patients who received hMG and FSH (11.9 days) followed by FSH alone (11.7 days). The mean number of oocytes retrieved, the mean number of oocytes fertilized, the percentage of preembryo fragmentation, and the preembryo cell number at transfer did not differ significantly between the stimulation protocols. The cumulative amount of gonadotropin used during stimulation was slightly greater in the cycles stimulated with FSH alone, but this difference was not significant (29.4 ampules of hMG plus FSH versus 31.8 ampules of FSH alone). Serum estradiol levels measured on the day of hCG administration during stimulation with hMG and FSH (1,382 pg/mL) were higher than those measured during stimulation with FSH alone (1,149 pg/mL). CONCLUSION(S): Follicular response and preembryo quality were not significantly different when patients were treated first with hMG and FSH and then with FSH alone in a subsequent cycle. Similarities in ovarian response and preembryo characteristics, as well as differences in estradiol patterns seen in each stimulation setting, should be anticipated when patients receive these protocols.

Adult↗
PubMed1999

Urinary follicle-stimulating hormone (FSH) versus recombinant FSH in clomiphene citrate-resistant, normogonadotropic,chronic anovulation: a prospective randomized study.

OBJECTIVE: To compare the efficacy and safety of urinary FSH and recombinant FSH for ovulation induction in patients with clomiphene citrate-resistant, normogonadotropic, chronic anovulation. DESIGN: Prospective, randomized trial. SETTING: University-based infertility clinic. PATIENT(S): Fifty-one women. INTERVENTION(S): Subjects were randomized to receive either urinary FSH (35 patients, 64 cycles) or recombinant FSH (16 patients, 32 cycles). A maximum of three cycles using the low-dose step-up protocol with the same gonadotropin were undertaken. MAIN OUTCOME MEASURE(S): Cumulative ovulation and pregnancy rates after three cycles, total gonadotropin dose, duration of stimulation, and single follicle development rate. RESULT(S): Cumulative ovulation rates were 89.3% and 93.1% for the urinary FSH and recombinant FSH groups, respectively. The threshold and total doses of FSH and the duration of stimulation were similar between the two groups. Significantly more single follicle development was noted in the recombinant FSH group. The respective clinical pregnancy rates per noncanceled cycles in the urinary FSH and recombinant FSH groups were 23.2% and 27.6%. There were three sets of twins in the urinary FSH group. No case of ovarian hyperstimulation syndrome was encountered. CONCLUSION(S): Urinary FSH and recombinant FSH are both efficient and safe for inducing ovulation with the low-dose step-up protocol.

Adult↗
PubMed2002

FSH and ovarian response: spontaneous recovery of pituitary-ovarian activity during the pill-free period vs. exogenous recombinant FSH during high-dose combined oral contraceptives.

OBJECTIVE: Compare spontaneous recovery of pituitary-ovarian activity during the pill-free period following the correct use of low-dose oral contraceptives and subsequent ovarian function during the administration of exogenous recombinant FSH (recFSH) after switching to continued Lyndiol (2.5 mg lynestrenol + 0.05 mg ethinyl-oestradiol) medication. DESIGN: Prospective, randomized, group-comparative, single-centre study. Following the monitoring of the pill-free period (week 1) and subsequent treatment with Lyndiol (for a total of 5 weeks), all subjects were randomly allocated to one of four groups receiving daily FSH injections for 1 week [75, 150, 225 IU recFSH or 150 IU purified urinary FSH (uFSH)] during the fourth week of Lyndiol use. PATIENTS: Thirty-six healthy volunteers aged 18-39 years, prestudy oral contraceptive use for at least 3 months, cycle length between 24 and 35 days. MEASUREMENTS: Serum FSH, LH and oestradiol (E2) concentrations as well as transvaginal ultrasound assessment of the number and diameter of follicles > 2 mm were used to monitor pituitary ovarian function. RESULTS: At the start of the pill-free period following the prestudy contraceptive medication, 67% of the women presented with LH and FSH levels < 1 IU/l and only one follicle > 10 mm was observed. Initial levels of LH and FSH correlated (P < 0.05) with the extent of pituitary-ovarian activity during the pill-free period. At the end of the pill-free period a follicle > 10 mm had emerged in one subject only. During the first 3 days of Lyndiol use, seven women (19%) eventually showed at least one follicle > 10 mm. During combined exogenous FSH and Lyndiol administration, LH levels remained completely suppressed (< or = 0.5 IU/l) in all women studied. FSH levels and number and size of follicles increased with increasing doses of exogenous FSH in a dose-dependent manner. E2 levels remained low in all groups (< 150 pmol/l). During the week following FSH administration, FSH levels and E2 levels decreased gradually while the number of follicles > 10 mm still increased. CONCLUSIONS: We have confirmed that dominant follicles > 10 mm are present at the end of the pill-free period and during the first days after resumption of pill intake. Once follicles > 10 mm arose at the end of the pill-free period, continued use of Lyndiol did not reduce follicle diameters. One week of Lyndiol reduces pituitary-ovarian activity to levels observed after 3 weeks of low-dose pills. FSH administration during Lyndiol resulted in dose-dependent follicle growth despite extremely low LH levels. E2 secretion (56 +/- 51 pmol/l) occurred to a limited and variable extent along with extremely low serum LH concentrations. Recovery of pituitary-ovarian activity at the end of the pill-free period is comparable to FSH levels and follicle dynamics following 7 days of 75-150 IU/l recFSH.

Adolescent↗
PubMed2005

Repeated testing of basal FSH levels has no predictive value for IVF outcome in women with elevated basal FSH.

BACKGROUND: It is a common practice to repeatedly test the level of basal FSH early in the cycle and to start IVF treatment only when the FSH level is below a certain threshold value. This is based on the idea that these women will respond better to ovarian stimulation when the basal FSH level is lower at the start of the cycle. The aim of this study is to assess the value of this practice. METHODS: Between January 1995 and January 2003, 39 women were identified. These women underwent two IVF treatment cycles within a 12 month period. The basal FSH level prior to each of these cycles was known to have changed. The treatment cycles were divided into cycles with a high basal FSH (> or =10 IU/l) and cycles with a low basal FSH (<10 IU/l). RESULTS: The 39 women underwent a total of 78 treatment cycles (in the first cycle 20 had elevated level of FSH and 19 had low FSH and vice versa in the second cycle). Therefore, there were 39 cycles with high FSH and 39 cycles with low FSH. There was obviously no live birth in the first treatment cycle, hence the reason for the patient undergoing another treatment cycle within 12 months of the first one. In the high FSH group, six became pregnant [pregnancy rate (PR) = 15.4%] and five delivered [live birth rate (LBR) = 12.8%]. In the low FSH group, three became pregnant (PR = 7.7%) and two delivered (LBR = 5.1%). The difference in PR and LBR, however, was not significant. Neither were there significant differences between the two groups with regard to the number of oocytes collected, oocytes fertilized, embryos transferred or miscarriage rate. CONCLUSION: The results of this study reveal that women who are poor responders or with reduced ovarian reserve have a poor outcome and repeatedly testing them will add no value. Cycling women with a history of elevated FSH should be offered treatment without further delay. Delaying treatment for these women could be counterproductive, as they may have to wait for many months, during which time they are getting older and closer to their menopause.

Adult↗
PubMed2005

The effect of the manipulation of follicle-stimulating hormone (FSH)-peak characteristics on follicular wave dynamics in sheep: does an ovarian-independent endogenous rhythm in FSH secretion exist?

We designed three experiments to investigate the relationship between FSH peaks and ovarian follicular waves and to examine whether an endogenous rhythm of FSH peaks exists in sheep. In experiment 1, anestrous ewes were treated with ovine FSH (oFSH) or vehicle (6 ewes per group) at the expected time of an endogenous FSH peak, to double the FSH-peak amplitude in treated ewes. In experiment 2, anestrous ewes were treated with either oFSH or vehicle (6 ewes per group) at the expected time of two consecutive interpeak nadirs, such that the treated ewes had 5 FSH peaks in the time frame of 3 FSH peaks in control ewes. In experiment 3, to measure FSH concentrations, daily blood samples were collected from 5 cyclic ewes for a control period during the estrous cycle and then for three 17-day periods after ovariectomy. Daily blood samples were collected from another group of 8 ovariectomized ewes that were treated with estradiol-releasing implants and intravaginal progestogen sponges. Doubling the FSH-peak amplitude did not alter the characteristics of the following follicular wave. Increasing the frequency of FSH peaks stimulated the emergence of additional follicular waves, but did not alter the rhythmic occurrence of FSH peaks and follicular wave emergence. Endogenous follicular waves in oFSH-treated ewes emerged and grew in the presence of the growing largest follicle of the induced follicular waves. Finally, based on the observation of serum FSH concentrations in ovariectomized ewes, it appears that there exists an endogenous rhythm for peaks in daily serum FSH concentrations, which is, at least in part, independent of regulation by ovarian follicular growth patterns.

Animals↗
PubMed2005

Mixed protocols: multiple ratios of FSH and LH bioactivity using highly purified, human-derived FSH (BRAVELLE) and highly purified hMG (MENOPUR) are unaltered by mixing together in the same syringe.

BACKGROUND: The use of mixed or blended protocols, that utilize both FSH and hMG, for controlled ovarian hyperstimulation is increasing in use. To reduce the number of injections a patient must administer, many physicians instruct their patients to mix their FSH and hMG together to be given as a single injection. Therefore, the goal of this study was to definitively determine if the FSH and LH bioactivities of highly purified, human-derived FSH (Bravelle) and highly purified hMG (Menopur) were altered by reconstituting in 0.9% saline and mixing in the same syringe. METHODS: Bravelle and Menopur were reconstituted in 0.9% saline and mixed in a Becton Dickinson plastic syringe. The FSH and LH bioactivities of the products were determined after injecting female and male rats, respectively, with Bravelle, Menopur, or a mixture of Bravelle and Menopur. Ratios of FSH:LH activity tested were 150:75 IU (1 vial Bravelle: 1 vial Menopur), 300:75 IU (3 vials Bravelle: 1 vial Menopur) or 300:225 IU (1 vial Bravelle: 3 vials of Menopur). RESULTS: There were no statistically significant changes in either FSH or LH bioactivity that occurred after mixing Bravelle with Menopur in the same syringe. The theoretical vs. actual FSH bioactivity for Bravelle and Menopur were 75 vs. 76.58 IU/mL and 75 vs. 76.0 IU/mL, respectively. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle: 1 vial Menopur), 300:75 IU (3 vials Bravelle: 1 vial Menopur) or 300:225 IU (1 vial Bravelle: 3 vials of Menopur) tested, the theoretical vs. actual FSH bioactivities were 150 vs. 156.86 IU/mL, 300 vs. 308.69 IU/mL and 300 vs. 306.58 IU/mL, respectively. The theoretical vs. actual LH bioactivity for Menopur in the above mentioned ratios tested were 75 vs. 77.50 IU/mL. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle: 1 vial Menopur), 300:75 IU (3 vials Bravelle: 1 vial Menopur) or 300:225 IU (1 vial Bravelle: 3 vials of Menopur), the theoretical vs. actual LH bioactivities were 75 vs. 78.38 IU/mL, 75 vs. 78.63 IU/mL and 225 vs. 233.48 IU/mL, respectively. CONCLUSION: Mixing human-derived FSH (Bravelle) with highly purified hMG (Menopur) in the same diluent, 0.9% NaCL, does not alter the FSH or LH bioactivity of either gonadotropin preparation.

Animals↗
PubMed2001

Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression.

We have recently reported that oocyte-derived bone morphogenetic protein-15 (BMP-15) can directly modulate follicle-stimulating hormone (FSH) action in rat granulosa cells. Here, we investigate underlying mechanisms of this BMP-15 effect. Treatment with BMP-15 alone exerted no significant effect on the basal expression of mRNAs encoding steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, P450 aromatase, 3beta-hydroxysteroid dehydrogenase, luteinization hormone receptor, and inhibin/activin subunits. However, BMP-15 markedly inhibited the FSH-induced increases in these messages. In striking contrast, BMP-15 did not change the forskolin-induced levels of these transcripts. Thus, the inhibitory effect of BMP-15 on FSH action must be upstream of cAMP signaling. We next examined changes in FSH receptor mRNA expression. Interestingly, BMP-15 severely reduced the levels of FSH receptor mRNA in both basal and FSH-stimulated cells. To determine whether this effect was at the level of FSH function, we investigated the effect of BMP-15 on FSH bioactivity. Consistent with the mRNA data, BMP-15 inhibited the biological response of FSH, but not that of forskolin. Based on these results, we propose that BMP-15 is an important determinant of FSH action through its ability to inhibit FSH receptor expression. Because FSH plays an essential role in follicle growth and development, our findings could have new implications for understanding how oocyte growth factors contribute to folliculogenesis.

Animals↗
PubMed1988

Effects of photoperiod and temperature on the binding of follicle-stimulating hormone (FSH) to testicular preparations and plasma FSH concentration in the Djungarian hamster, Phodopus sungorus.

The effects of artificial photoperiod and temperature on testicular FSH binding and plasma FSH levels were studied in adult male Djungarian hamsters. In Exp I, hamsters were transferred to long day (LD) photoperiods (16-h light, 8-h dark) after 8 weeks of adaptation in short day (SD) photoperiods (8-h light, 16-h dark), but the ambient temperature was maintained at 25 C throughout the experiments. A marked increase in the total FSH binding per two testes occurred between 10 and 47 days after transfer to LD, a change that was accompanied by testicular growth. Binding of FSH per testicular weight decreased during the same period. Scatchard plot analyses of the parameters indicated that LD decreased both the concentration of FSH binding sites and the equilibrium dissociation constant (Kd). Plasma FSH levels increased between 10 and 47 days after transfer to LD. In contrast, when hamsters reared under LD for 12 weeks were transferred to SD (Exp II), FSH binding per unit weight basis increased 19 weeks after transfer to SD, but the total binding per two testes decreased markedly. In Exp III, sexually mature male hamsters were subjected to different ambient temperature and photoperiods. There were no significant effects of different ambient temperatures on the testicular weight and testicular FSH binding in animals exposed for 8 weeks to either LD or SD. However, plasma FSH levels of hamsters maintained at 25 C under LD was significantly higher than FSH levels at 7 C under LD. A similar effect of temperature on plasma FSH levels was observed in hamsters under SD. The present study indicates that photoperiod is a more important environmental factor than temperature for the regulation of FSH receptor in the Djungarian hamster.

Animals↗
PubMed1994

In vitro bioassay for human serum follicle-stimulating hormone (FSH) based on L cells transfected with recombinant rat FSH receptor: validation of a model system.

FSH plays a central role in normal reproductive function, i.e. control of follicular maturation in the female and initiation and maintenance of spermatogenesis in the male. The effects of FSH are mediated by its interaction with a specific receptor that belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors. Due to the microheterogeneity of gonadotropins, measurement of immunoreactivity does not necessarily reflect their bioactivity. Mutations in gonadotropin beta-subunits, which affect bioreactivity and/or immunoreactivity of gonadotropins, have been described as causes of infertility, thus highlighting the need for rapid and convenient methods to measure bioactivity. To establish a model system for recombinant in vitro bioassays for FSH that would obviate the use of live animals, we developed a strategy for efficient expression of the rat FSH receptor (FSHR) in L cells. A cell line, FSHR 7/12, was developed that bound [125I]FSH with high affinity (Kd 1.42 nM) and responded to human FSH with an increase in cAMP accumulation. Untreated human serum was found to have an unspecific inhibitory effect on cAMP formation. This effect could be thoroughly avoided by mild heating (10 min at 56 C) of serum samples before addition to cells without detectable loss of FSH immunoactivity or bioactivity. Studies on the hormone-sensitive adenylyl cyclase system of transformed FSHR 7/12 cells and of the parental Ltk- cells showed that the cellular response to FSH was highly specific. Using a parallel line assay design, FSHR 7/12 cells were used to validate a novel recombinant in vitro bioassay relying on intracellular cAMP accumulation as a readout system. Up to 10% of serum could be added to the incubation buffer without leading to nonparallelism to the standard curve. When 70 serum samples of male patients attending an infertility clinic were analyzed, the novel assay system displayed high sensitivity and a close correlation (r > 0.8; P < 0.01) to the established rat Sertoli cell aromatase bioassay and to a highly specific fluoroimmunoassay. When sera of 25 normal menstruating women were analyzed for FSH bioactivity at different stages of the menstrual cycle, a midcycle FSH peak followed by a decline in the late luteal phase could be discerned. The analysis of 26 serum samples of postmenopausal women revealed a close correlation between FSH values obtained by the novel in vitro bioassay and by a fluoroimmunoassay (r = 0.90; P < 0.01). Thus, the present in vitro bioassay represents a sensitive, rapid, and convenient model system to measure bioactive FSH in human serum.

Animals↗
PubMed1997

Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms.

Under physiological conditions, FSH is secreted into the circulation as a complex mixture of several isoforms that vary in the degree of glycosylation. Although it is well established that the glycosylation of FSH is important for the serum half-life of the hormone and coupling of the receptor to adenylate cyclase, little is known concerning how physiologically occurring glycosylation patterns of this hormone affect receptor signaling. In this study, we have examined the biological activity of deglycosylated human FSH (DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as compared with that of purified human pituitary FSH (phFSH) using a Chinese hamster ovarian cell line stably expressing the hFSH receptor (3D2 cells). Differentially glycosylated forms of FSH did not bind to the FSH receptor in the same manner as phFSH. Although all hormones showed similar potency in competing for [125I]phFSH binding to the hFSH receptor, competition curves for deglycosylated and insect cell-produced FSH were steeper. Similarly, glycosylation of FSH had a profound effect on bioactivity of the hormone. Purified hFSH produced a sigmoidal dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH also elicited biphasic effects on steroidogenesis in primary cultures of rat granulosa cells. The cellular response to BV-hFSH was dependent on the degree of receptor-transducer activation. BV-hFSH bioactivity was strictly inhibitory when combined with the ED80 of phFSH. Lower concentrations of phFSH resulted in a gradual shift from inhibition to a biphasic activity in the presence of the ED20 of phFSH. Biphasic responses to BV-hFSH were attributed to activation of different G protein subtypes, since treatment of 3D2 cells with cholera toxin or pertussis toxin differentially blocked the two phases of BV-hFSH bioactivity. These data suggest that alternative glycosylation of FSH leads to a functionally altered form of the hormone. Functionally different hormones appear to convey distinct signals that are transduced by the receptor-transduction system as either stimulatory or inhibitory intracellular events via promiscuous, glycosylation-dependent G protein coupling. Promiscuity in signaling of the FSH receptor, in turn, may represent a potentially novel mechanism for FSH action, whereby the gonad may respond in diverse ways to complex hormonal signals such as those presented by circulating FSH isoforms.

Animals↗
PubMed1993

Synergistic effects of steroids with FSH on folliculogenesis, steroidogenesis and FSH- and hCG-receptors in hypophysectomized mice.

Injection of ovine FSH (4 micrograms day-1) for 4 days into hypophysectomized mice does not restore folliculogenesis to normal cyclic values. This may be due to insufficient production of oestradiol. The present study was designed to determine whether FSH- and LH-induced oestradiol was critical for growth and differentiation of follicles at all stages. Twelve days after hypophysectomy, mice were injected s.c. with 10, 50 or 250 micrograms oestradiol cyclopentylpropionate daily with or without ovine FSH (4 micrograms day-1) for 1-4 days. One ovary from each animal was used for histology. From the second ovary, follicles were isolated at different stages and incubated with [3H]thymidine for 3 h to determine the rate of DNA synthesis. Incubation medium and serum were used for steroid determinations. After oestradiol treatment alone, there was a dose-dependent response in serum oestradiol, but ovarian and uterine weights did not increase further with the increasing doses of oestradiol administered. This finding was consistent with an increase in the number of preantral follicles and small antral follicles but excluding the development of preovulatory follicles. Treatment with 10 and 50 micrograms of oestradiol cyclopentylpropionate did not prevent antral follicles from undergoing atresia. The higher dose (250 micrograms day-1) of oestradiol cyclopentylpropionate delayed atresia of antral follicles and maintained more large healthy antral follicles. After concurrent injection of oestradiol and FSH, ovarian weight was 2-3 times greater than with either FSH or oestradiol alone; the number of follicles and follicular DNA synthesis at all stages of development increased without any signs of atresia; the in vitro accumulation of oestradiol also increased. Oestradiol alone induced FSH receptors in granulosa cells, but did not induce hCG receptors in any ovarian compartment; FSH alone induced FSH and hCG receptors in granulosa cells but not in thecal-interstitial tissues, whereas, oestradiol plus FSH enhanced FSH receptors in granulosa cells and LH/hCG receptors in granulosa and thecal-interstitial tissues. The synergistic effect of oestradiol with FSH was mimicked by the same dose of diethylstilboestrol, testosterone or dihydrotestosterone, but the latter steroids increased only the number of antral follicles, presumably because of their shorter half-lives. These results indicate that in mice oestradiol stimulates the growth of preantral and antral follicles and delays follicular atresia; oestrogens and androgens act synergistically with FSH to enhance follicular proliferation and differentiation, and prevent follicles from undergoing atresia.

Animals↗