Elevated ovarian expression and serum concentration of alpha inhibin in the luteal phase during follicular development in the squirrel monkey (Saimiri boliviensis) compared to the human.

The goal of the present investigation was to determine in the squirrel monkey the source and pattern of inhibin, a hormone known to effect reproductive steroid levels via pituitary and ovarian mechanisms. Since this seasonally polyestrous species is known to have elevated serum levels of reproductive steroids compared to other primates, the levels of ovarian alpha subunit mRNA expression and serum total alpha inhibin, estradiol, progesterone, and luteinizing hormone were measured and compared to human levels. Expression of the alpha subunit was robust in monkey luteal tissue compared to expression in human luteal tissue. Squirrel monkey serum inhibin peaked 4 days after the luteinizing hormone surge and correlated with progesterone changes. These luteal serum levels of inhibin were greater than 12 times higher than the human levels yet bio-LH activities were less than in the human during the luteal phase. Inhibin concentrations during the nonbreeding season were generally half the levels measured in the breeding season and undetectable in ovariectomized animals. However, exogenous FSH stimulation induced a marked rise in inhibin, which correlated with an estradiol rise. In conclusion, abundant alpha inhibin subunit expression in the luteal ovary of the squirrel monkey and loss of serum delectability in ovariectomized animals indicates that the principle source of inhibin in the squirrel monkey is the ovary. Elevated serum inhibin levels during the luteal phase concurrent with ovulatory-size follicular development is unique among species studied thus far. Possible simultaneous inhibin production from both follicular and luteal tissue may be responsible for the exceptionally high inhibin levels.

Immunoreactive and bioactive LH release from pituitaries of intact or castrated male rats: effect of in vitro GnRH and KCl administration.

The in vitro immunoreactive (i-LH) and bioactive (b-LH) LH release from hemipituitaries of intact adult male rats (INT) or rats castrated 7 days earlier (CAS) was studied. Hemipituitaries were incubated for 30 min (time 1) plus an additional 30 min (time 2) with GnRH (10 nM) and/or KCl (50 mM), according to one of the following protocols: media alone (C), KCl+KCl (K/K), GnRH+GnRH (G/G), KCl+GnRH (K/G), GnRH+KCl (G/K). All of the hemipituitaries were further incubated in media alone for 120 min (time 3). I-LH, b-LH and i-FSH were assayed on the media. In both models, the highest bioactive:immunoactive (b/i) ratio was noted during time 1; however, CAS always secreted more b-LH than INT at any given time of the study. In INT, GnRH--but not KCl--administration during time 2 resulted in blunted i-LH. During the same time, the b/i ratios decreased in all groups but G/K. LH secretion recovered during time 3 in all groups. In CAS, i-LH levels comparable to those of time 1 were sustained by either stimulus during time 2, while the b/i ratios were markedly decreased. LH secretion recovered in the K/K group during time 3. These results suggest that: 1) promptly releasable pools of b-LH are available in both models; 2) CAS always secrete more b-LH; 3) in INT, desensitization occurs involving parallel changes in both i-LH and b-LH, while changes in b-LH rather than i-LH are noted in CAS; 4) prolonged KCl administration might play a role in new gonadotropin synthesis and/or release.

Recombinant human inhibin-A administered early in the menstrual cycle alters concurrent pituitary and follicular, plus subsequent luteal, function in rhesus monkeys.

Inhibin, a suppressor of pituitary FSH secretion in nonprimate species, may also act in the ovary to regulate follicular development. To examine whether inhibin has similar actions in primates, female rhesus monkeys (n = 3/treatment), exhibiting regular menstrual cycles, received sc injections of either vehicle or 60 micrograms/kg recombinant human inhibin-A at 0800 and 1600 h for 5 days beginning at menses. The vehicle-treated monkeys displayed menstrual cycles of normal length, with the follicular (11.3 +/- 2.5 days, mean +/- SE) and luteal (16.3 +/- 2.5 days) phases demarcated by midcycle peaks in serum estradiol (E) and bioactive LH. After the first inhibin injection, levels of immunoreactive inhibin A peaked at 10 ng/mL within 1 h and returned to baseline (< 0.1 ng/mL) before the second injection 8 h later. Although serum E and LH did not change, bioactive FSH decreased (to 66% of pretreatment levels, P < 0.05) within 8 h. Within 1 day, circulating bioactive FSH was less (P < 0.05) in inhibin-treated monkeys, compared with controls. By 2-3 days, serum E levels were also markedly (P < 0.05) reduced in inhibin-treated animals, whereas bioactive LH rose 3-fold (P < 0.05). After inhibin treatment, the midcycle rises in serum E and LH were delayed; hence, the follicular phase was prolonged (15.0 +/- 2.6 days, P < 0.05), compared with controls. Although the patterns and levels of serum LH circulating during the subsequent luteal phase seemed comparable in both groups, mean progesterone levels were suppressed to 2-3 ng/mL (P < 0.05) during the midluteal phase in inhibin-treated monkeys. However, the length of the luteal phase in inhibin-treated cycles (13.0 +/- 2.6 days) was not significantly altered. We conclude that exogenous inhibin rapidly diminishes pituitary FSH secretion in female monkeys during the early follicular phase of the menstrual cycle. This action, and/or other actions directly on the ovary, leads to subsequent effects on follicular steroidogenesis and pituitary LH secretion that culminate in an aberrant ovarian cycle characterized by an insufficient luteal phase. The study identifies, for the first time, possible activities and roles of inhibin during the ovarian cycle in primates.

Circulating immunoreactive and bioactive follicle stimulating hormone concentrations in anovulatory infertile women and during gonadotrophin induction of ovulation using a decremental dose regimen.

Our purpose was to determine whether decreased follicle stimulating hormone (FSH) activity, either systemic or at the follicular level, is involved in impaired follicle growth associated with normogonadotrophic anovulation. To differentiate between the possible levels of disturbance, bioactive (BIO-FSH; using the in-vitro rat granulosa cell aromatase bioassay) and immunoreactive (IRMA-FSH) FSH serum concentrations of three groups of subjects were compared: (i) 172 normogonadotrophic anovulatory infertile women during baseline conditions, (ii) 22 clomiphene-resistant polycystic ovary syndrome patients undergoing ovulation induction by exogenous gonadotrophins using a decremental dose regimen, and (iii) nine regularly cycling controls. BIO-FSH [13.2 (range 0.8-29.5) IU/l] and IRMA-FSH [4.4 (range 1.2-9.3) IU/l] concentrations in anovulatory women during baseline conditions were significantly lower than maximum concentrations reached during the follicular phase in controls [18.7 (13.2-23.4) and 6.4 (5.7-10.0) IU/l respectively], but were not significantly different from initial concentrations in controls [10.4 (7.2-19.6) and 4.8 (2.8-8.2) IU/l respectively]. Moreover, concentrations of IRMA-FSH and BIO-FSH were negatively correlated (r = -0.25, P = 0.01, and r = -0.24, P = 0.02 respectively) with the interval between last vaginal bleeding and blood sampling. Maximum concentrations of IRMA-FSH [7.6 (3.9-10.9) IU/l] during ovulation induction by gonadotrophins were not significantly different from maximum [6.4 (5.7-10.0) IU/l] concentrations in controls, whereas maximum BIO-FSH concentrations [13.5 (8.7-17.4) versus 18.7 (13.2-23.4) IU/l] were significantly lower. Our findings suggest that (i) circulating FSH does not reach concentrations that are sufficient to induce normal follicle development in anovulatory women during baseline conditions, and (ii) the FSH threshold for ovarian stimulation of this patient group is not different from normal.

A technique for sorting rat gonadotropes using anti-LH or anti-FSH antibodies covalently attached to magnetic beads.

A technique for sorting live LH- and FSH-secreting cells was developed. After enzymatic dispersion, a suspension of pituitary cells from male rats castrated 7 days earlier was incubated in potassium chloride (KCI 50 mmol/l) for 30 min and gonadotropin outflow was provoked. Then, considering either LH or FSH as temporary surface markers, we positively selected the secreting cells by means of antibodies toward either LH (anti-LH beads) or FSH (anti-FSH beads) covalently attached to magnetic beads. The spontaneous secretion of LH and FSH overnight and the release induced by KCI the following morning were calculated. A population enriched in gonadotropes (16% of the total) able to secrete both gonadotropins was selected by means of anti-LH beads; this released 7 times as much LH as non-selected cells. A similar population (14% of the total) was selected by means of anti-FSH-coated beads; this produced 3.3 times as much LH as non-selected cells. In some experiments, the cells not previously sorted with anti-LH-coated beads were further incubated in the presence of anti-FSH beads, in an attempt to isolate a population secreting only FSH. A limited number of cells were sorted (6% of the total cells), able to produce both gonadotropins, but with a lower LH/FSH ratio. Similarly, those cells excluded by the selection with anti-FSH beads were further incubated with anti-LH beads, with a view to obtaining only-LH-secreting cells. However, both gonadotropins were still secreted by these cells (8% of the total), which had the highest LH/FSH ratio. In conclusion, fractions from castrated male rats that are enriched in gonadotropes contain cells that secrete both gonadotropins in vitro. The secretion of LH is prevalent. However, differences in the LH/FSH ratio between the populations sorted and changes from spontaneous to stimulated release are observed. This suggests that some gonadotropes might 'specialize' in releasing LH and others in releasing both LH and FSH.

Delayed puberty in males with beta-thalassemia major: pulsatile gonadotropin-releasing hormone administration induces changes in gonadotropin isoform profiles and an increase in sex steroids.

Patients with beta-thalassemia major often have pubertal delay, the etiology of which has not been fully elucidated. We investigated the pituitary-gonadal response to short-term subcutaneous pulsatile gonadotropin-releasing hormone (GnRH) administration (150 ng/kg body weight every 120 min for 7 days) in five young males (aged 13.6-19.0 years) affected by beta-thalassemia major and presenting signs of delayed puberty. Immunoreactive and bioactive gonadotropin levels were determined and their isoform profiles were examined, before and after GnRH treatment, in a pool of samples collected every 15 min for 240 min. Testosterone, androstenedione, 17-hydroxyprogesterone, dehydroepiandrosterone and 17 beta-estradiol were measured as markers of gonadal function on days 0, 1, 3, 5 and 7 of treatment. Five patients (aged 16.9-26.8 years) with confirmed diagnosis of idiopathic hypogonadotropic hypogonadism who were starting pulsatile GnRH therapy were also studied in the same protocol. Increased sex steroid levels were observed in both groups as a result of treatment. On day 7, the thalassemic patients had increased bioactive luteinizing hormone (LH) and follide-stimulating hormone (FSH), although immunoreactive LH and FSH were comparable to day 0. Moreover, fewer acidic and more basic immunoreactive and bioactive isoforms were noted in LH profiles on day 7. Similar results were observed in hypogonadal patients, who also had increased immunoreactive LH and FSH values. We suggest that the early stage of delayed puberty in thalassemia might be characterized by a neuroendocrine dysfunction resulting in an impaired hypothalamic GnRH release, which is inadequate for a proper pituitary stimulation. Pulsatile GnRH treatment seems to re-establish partially the correct pituitary-gonadal function.

Gonadotropin requirements of the developing follicle.

OBJECTIVE: To evaluate follicular FSH and LH requirements during suppression of endogenous gonadotropins with the GnRH antagonist Nal-Glu and whether LH-like activity could be supplied by administering subcutaneous hCG. DESIGN: Randomized clinical trial. PARTICIPANTS: Thirty-two normally cycling females in the late follicular phase (dominant follicle mean diameter > or = 14 mm). INTERVENTION: Twelve normal women were randomized to receive 150 IU IM FSH with or without 75 IU SC hCG; 11 subjects were randomized to receive 225 IU FSH with or without 50 IU SC hCG; 9 women received 150 or 225 IU IM hMG. Subjects returned the next day for repeat blood sample and ultrasound. RESULTS: Continued follicular maturation, as evidenced by rising E2 levels, correlated with serum immunoactive and bioactive FSH levels and was unrelated to bioactive LH-hCG. Two hundred twenty-five international units of exogenous FSH consistently supported follicular maturation. There was a similar increase in mean follicular diameter in women with an E2 rise versus those with a plateau or fall. In subjects receiving SC mini-dose hCG, serum bioactive LH-hCG levels were increased significantly and were similar to levels before Nal-Glu. CONCLUSIONS: During administration of a GnRH-a, the maturing follicle appears to require only FSH support. In markedly hypogonadotropic women, mini-dose hCG may be a more practical alternative to recombinant LH to promote normal follicle maturation.

Effects of corticosterone and testosterone on pituitary gonadotropin content, secretion, bioactivity and messenger RNA levels in the presence or absence of GnRH in male rats.

The effects of corticosterone (B) and testosterone (T) on pituitary and serum bioactive and immunoreactive gonadotropins and on gonadotropin hormone subunit messenger RNA levels were compared in the absence of GnRH. Male rats were implanted with pellets of either cholesterol, B or T. At implantation, 2 and 4 days later half of each group received GnRH antagonist and animals were killed 5 days after implantation. As expected, GnRH antagonist lowered bioactive and immunoreactive serum FSH and LH, pituitary FSH, LHß and FSHß mRNA. B treatment alone lowered bioactive and immunoreactive serum FSH and immunoreactive serum LH. B reversed the antagonist effect on bioactive and immunoreactive pituitary FSH and FSHß mRNA. T alone lowered bioactive and immunoreactive serum FSH and LH levels. T reversed the antagonist effect on bioactive and immunoreactive pituitary FSH. T lowered bioactive and immunoreactive pituitary LH and LHß mRNA and partially reversed the antagonist effect on FSHß mRNA. The data suggest that either B or T enhance FSH synthesis by acting directly at the gonadotrope, but that B does not affect LH variables to the same extent as T. The results suggest that in stressed animals, when T levels are reduced, B can substitute for T in sustaining FSH synthesis.

Systemic and intraluteal infusion of inhibin A or activin A in rhesus monkeys during the luteal phase of the menstrual cycle.

The endocrine or local actions of inhibin-related peptides synthesized by the primate corpus luteum (CL) remain undefined. This in vivo study was designed to determine whether exogenous inhibin or activin modulates pituitary gonadotropin secretion and the functional life span of the CL during the luteal phase of the menstrual cycle. Beginning at midluteal phase of the cycle, either vehicle or 1 microgram/h of recombinant human inhibin A or activin A (n = 3-6 per treatment group) was infused into rhesus monkeys via the jugular vein (i.e., peripheral infusion) or directly into the CL (i.e., intraluteal infusion) by means of an osmotic minipump for 7-14 days. Daily samples of saphenous venous serum were assayed for estradiol (E) and progesterone (P) content by RIA, and for FSH and LH levels by bioassay. Intraluteal infusion of inhibin or activin did not alter circulating P levels or the length of the luteal phase compared to those values in vehicle-infused controls. Likewise, LH levels were not different between the three groups. However, FSH levels declined progressively during inhibin infusion to 26% of pretreatment levels (p < 0.05), whereas FSH levels in vehicle-infused controls were unchanged for several days and then rose (p < 0.05) to peak levels around menses. FSH levels did not change significantly during activin infusion into the CL. Although similar results were obtained in monkeys receiving peripheral or intraluteal infusions of inhibin, events following the peripheral infusion of activin were markedly different from those during intraluteal administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Follistatin decreases activin-stimulated FSH secretion with no effect on GnRH-stimulated FSH secretion in prepubertal male monkeys.

Follistatin is an activin-binding glycoprotein that decreases FSH secretion in vitro and in vivo in rats. The mechanism by which follistatin acts is unclear, but it has been suggested that it may bind endogenous activin and neutralize its effects. In this study, we wished to test the ability of follistatin to suppress FSH secretion in vivo in primates whose FSH secretion has been stimulated by activin or by GnRH. Six prepubertal male monkeys were injected intravenously with human recombinant follistatin at the dose of 90 micrograms/kg or 180 micrograms/kg plus activin (90 micrograms/kg) or GnRH (10 micrograms/kg). Frequent blood samples were drawn for 12 hours following each injection. Bio FSH and LH levels were measured in those samples. GnRH and activin each stimulated FSH bioactivity. Both doses of follistatin significantly inhibited the activin-induced increase in FSH (p < 0.05). The GnRH-induced increase in FSH was not affected by follistatin. LH levels were not affected by follistatin in any of the studies. These data suggest that follistatin can suppress the activin-induced increase in FSH in primates and is consistent with the hypothesis that follistatin can block the physiological effects of endogenous activin in primates. This effect is likely to be due to the binding of follistatin to activin either in the peripheral circulation or at the pituitary level.

The direct pituitary effect of testosterone to inhibit gonadotropin secretion in men is partially mediated by aromatization to estradiol.

In men, administration of exogenous testosterone (T) exerts direct negative feedback effects at the pituitary as well as at the hypothalamic level. This study was undertaken to determine whether T itself causes the inhibitory effects on the pituitary, or whether conversion to estradiol (E2) or dihydrotestosterone (DHT) is required. We assessed the biological activity of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as immunoactivity. Blood samples were drawn before, during, and after a continuous, 72-hour i.v. infusion of T (15 mg/day), E2 (90 micrograms/day), or DHT (500 micrograms/day). Each of these doses is twice the daily production rate of the steroid. Each man received each of the three steroid infusions. We studied four men, ages 23-35, with idiopathic hypothalamic hypogonadism (IHH), who were treated with pulsatile gonadotropin releasing hormone (GnRH) until their gonadotropins reached the normal range. Serum levels of T, E2, DHT, and levels of immunologically active and biologically active LH and FSH were measured. We found that administration of each steroid increased serum levels of the infused steroid to the upper physiologic range. Administration of T or E2 resulted in decreased mean levels of biologically and immunologically active LH and FSH; administration of DHT did not alter gonadotropin secretion. These data suggest that some of the direct effect of T at the pituitary level in men is mediated by E2, whereas peripherally formed DHT may not play an important role in this process.

Corticosterone in vivo increases pituitary follicle-stimulating hormone (FSH)-beta messenger ribonucleic acid content and serum FSH bioactivity selectively in female rats.

Experimental objectives were to determine: 1) if the native glucocorticoid, corticosterone (B), can selectively increase pituitary FSH and FSH beta messenger RNA (mRNA) in the presence or absence of a GnRH signal; and 2) if B affects the biological activity of the gonadotropins. Metestrous female rats were implanted with cholesterol or B. Each implant group received 100 micrograms GnRH antagonist or control injections every 48 h beginning at the time of implantation, and were killed 5 days later. B significantly increased bioactive serum FSH, with or without GnRH antagonist. GnRH antagonist decreased bioactive serum FSH. Immunoreactive serum FSH was not affected by any treatment. B did not affect bioactive serum LH, but GnRH antagonist significantly suppressed bioactive serum LH. Immunoreactive serum LH was significantly lowered by either B or GnRH antagonist. Neither bioactive nor immunoreactive pituitary FSH or LH content were affected by B, GnRH antagonist, or combined treatments, and no treatment affected alpha or LH beta mRNA. B significantly increased FSH beta mRNA specifically, in the presence or absence of GnRH antagonist. These results demonstrate that corticosterone can increase biological activity of secreted FSH and increase FSH beta mRNA in the absence of a GnRH signal, suggesting a direct effect on the anterior pituitary gland.

Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness.

In order to study the effects of follicle-stimulating hormone (FSH) on differentiation of granulosa cells, a well-defined and validated in-vitro culture system is indispensable. In this study, pooled follicular aspirates were stimulated in vitro with FSH and luteinizing hormone (LH) for 2, 4 and 6 days, either immediately after plating or after 7 days of preincubation. Cultures were assayed for progesterone and oestradiol production. Fresh cells displayed very high basal progesterone production which could be stimulated with LH but not FSH. After preincubation, addition of LH and FSH resulted in dose-dependent increases of progesterone and oestradiol. When cultured on human fibronectin-coated wells, similar basal but higher progesterone concentrations after stimulation were observed. In comparison with serum-free media, addition of Serum-Plus resulted in higher basal and stimulated progesterone concentration, possibly due to the presence of serum factors. This study demonstrates firstly that after 7 days preincubation, cultures gained responsiveness to FSH but remained responsive to LH during 4 days of stimulation. This suggests a persisting differentiated cell population in vitro. Secondly, the use of human fibronectin extracellular matrix and serum promotes steroid production, either due to factors promoting cell growth and function or to availability of steroid precursors. Therefore one has to be cautious with interpretation of data obtained from this widely used culture system, employing highly differentiated cells obtained after ovarian stimulation for in-vitro fertilization for study of local regulation of granulosa cell function.

Human recombinant activin-A alters pituitary luteinizing hormone and follicle-stimulating hormone secretion, follicular development, and steroidogenesis, during the menstrual cycle in rhesus monkeys.

Activin, a stimulator of pituitary FSH secretion in nonprimate species, may also act in the ovary to modulate follicular development. To examine whether activin has similar actions in primates, female rhesus monkeys (n = 3/treatment) exhibiting regular menstrual cycles received sc injections of either vehicle or 60 micrograms/kg recombinant human activin-A at 0800 and 1600 h for 1 (acute) or 7 (chronic) days beginning in the early follicular phase. The vehicle-treated monkeys displayed menstrual cycles of normal length, with the follicular (11.3 +/- 1.3 days, mean +/- SE) and luteal (16.6 +/- 1.8 days) phases demarcated by midcycle peaks in serum estradiol (E) and bioactive LH. After the first activin injection, levels of human activin A peaked at 90 ng/mL within 1 h and returned to baseline before the second injection 8 h later. Although serum E and FSH levels did not change, LH increased (273%, P < 0.05) within 8 h. Acute activin treatment increased (P < 0.05) serum E within 24 h to levels (1290 +/- 330 pmol/L) typically observed at midcycle. With chronic treatment, serum E peaked on day 2 (2580 +/- 338 pmol/L; P < 0.05), then declined and rose to a second peak (1680 +/- 279 pmol/L) on day 5. During chronic activin treatment, LH levels peaked on day 2 (603 +/- 270 ng/mL; P < 0.05 compared to day 0, 15 +/- 7 ng/mL) whereas FSH increased progressively until day 5 (937 +/- 320 ng/mL; P < 0.05 compared to day 0, 169 +/- 59 ng/mL). After acute or chronic activin, the expected midcycle rises in serum E and gonadotropins were delayed to greater than or equal to day 20 (n = 4) or did not occur before menses (n = 2). Although an enlarged ovary with one greater than or equal to 4-mm follicle was observed by laparoscopy during the late follicular phase in vehicle-treated monkeys, medium-to-large follicles were not visible on ovaries during chronic activin treatment or later at the expected midcycle interval in activin-treated monkeys. Similar hormonal and ovarian events were obtained after activin treatment of amenorrheic monkeys having serum FSH, LH, and E levels that were comparable to those at menses in spontaneous menstrual cycles. Thus, exogenous activin stimulates pituitary LH and FSH secretion and ovarian estrogen secretion during the early follicular phase in intact monkeys. However, acute or chronic activin treatment did not promote complete follicular development and disrupted subsequent events in the menstrual cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Validation of an improved in vitro bioassay to measure LH in diverse species.

Cultured Leydig tumor cells (MA-10) respond to luteinizing hormone (LH) by synthesizing and secreting progesterone (P). The specificity of the response to LH prompted us to develop this system for use as a simple and rapid in vitro bioassay for LH. The aims of this study were to (1) improve sensitivity and reproducibility, and (2) optimize the assay for use in diverse animal species. A minimum sensitivity was observed at 0.05 mlU/well of LH with 0.5 x 10(4) cells/well for 1.5 hours. At higher concentrations of LH, shorter incubation periods also significantly stimulated P production. Addition of human LH standard or serum samples resulted in a dose-dependent increase in P production. Parallel dose-dependent curves were observed with LH preparations from mammalian, avian, and amphibian species. In conclusion, these findings demonstrate that (1) the assay is rapid, sensitive, and reproducible; (2) serum LH levels analyzed using this assay and the mouse Leydig cell bioassay are comparable; (3) shorter incubation times suggest the implementation of this assay for rapid qualitative determination of LH surges; and (4) the assay can be used for the analysis of samples from diverse species, especially those lacking radioimmunoassays. Therefore, this assay system allows for the simple and rapid measurement of circulating bioactive LH levels in humans and diverse animal species, and should provide insight regarding the role of bioactive LH in physiological and pathological conditions.

Apparent absence of negative feedback in middle-aged persistent-estrous rats following luteinizing hormone-releasing hormone agonist treatment: relation to plasma inhibin and 17 beta-estradiol.

Reproductive aging in female rats is associated with a transition from regular estrous cyclicity to an anovulatory condition described as persistent estrous (PE). This PE condition is characterized by continued follicular development with elevated circulating levels of estrogen and FSH. In an attempt to investigate further the age-related changes in neuroendocrine function of PE rats, we have developed a model through which the return of hypothalamic-pituitary and ovarian function can be assessed following the withdrawal of chronic LHRH agonist suppression. Subsequent to withdrawal of continuous (2.5 micrograms/h for 12 days) LHRH agonist [DTrp6, Pro9-NHEt]-LHRH (LHRH-AG) treatment, circulating FSH concentrations in PE rats increase and remain elevated with an apparent absence of ovarian negative feedback, and these rats fail to return to estrous cyclicity. In the present studies, estrogen administration induced significant decreases in FSH secretion in PE rats following withdrawal of LHRH-AG treatment and ovariectomy (OVX), suggesting that the negative feedback response to estrogen is maintained in PE females. However, progesterone administration 2 days later failed to elicit a positive feedback response of gonadotropin secretion in PE females prior to LHRH-AG treatment, serum inhibin and 17 beta-estradiol (E2) concentrations were similar in middle-aged PE rats and young cyclic females on proestrus, while FSH levels were significantly greater in PE rats. After withdrawal of LHRH-AG treatment, plasma FSH concentrations remained elevated in PE rats as compared to young rats despite similar increases in E2. However, increases in plasma inhibin were delayed and significantly attenuated in PE rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of immunoradiometric and bioactive follicle stimulating hormone secretion and clearance in young and elderly men during treatment with tamoxifen or flutamide.

The secretion and clearance of immunoactive and bioactive follicle-stimulating hormone (FSH) in healthy young men (N = 10) and elderly men (N = 7) during blockade of endogenous sex steroid hormones with tamoxifen, an antiestrogen, and flutamide, an antiandrogen, was investigated. To this end, subjects underwent blood sampling basally every 10 minutes for 24 hours, and then received 2 consecutive intravenous pulses of synthetic gonadotropin releasing hormone (GnRH; 10 micrograms and 100 micrograms) every 2 hours. This paradigm was repeated on two subsequent visits, in which subjects received either flutamide HCl, a specific nonsteroidal competitive antagonist of the androgen receptor (750 mg daily for 3 days), or tamoxifen, a selective antagonist of the estrogen receptor (20 mg daily for 9 days). Serum immunoactive FSH concentrations were measured in each sample by immunoradiometric assay (IRMA). Serum bioactive FSH concentrations were determined by an in vitro bioassay (rat granulosa cell aromatase system) on 24-hour serum pools. Deconvolution analysis was used to analyze both the FSH IRMA 24-hour time series and FSH release after GnRH. Comparisons between young and elderly men of the basal state showed significantly increased 24-hour mean serum immunoactive and bioactive FSH concentrations and significantly decreased free testosterone concentrations in elderly men. By deconvolution analysis, elderly men had a significant decrease in FSH secretory burst duration, and an increase in FSH half-life and FSH secretory burst amplitude compared with younger men. In response to sex steroid receptor blockade in young men, there was a significant increase in mean serum bioactive FSH concentrations during antiandrogen treatment, but not during antiestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation in the release of bioactive versus immunoactive gonadotropins from cultured rat pituitary cells by inhibin and androgens.

Using in vitro bioassays and radioimmunoassays, the authors examined the interactions between purified 32kD ovine inhibin, testosterone, and dihydrotestosterone on basal and GnRH-stimulated secretion of gonadotropins in primary rat pituitary cell cultures. This study demonstrates that inhibin and androgens: 1) differentially regulate gonadotropin levels, 2) cause changes in the amounts of bioactive and immunoactive FSH and LH released. In conclusion, differences in bio- and immuno-FSH and LH secretion suggest that the endocrine milieu results in not only quantitative but qualitative changes in the secreted gonadotropin isoforms.

The significance of a single serum LH measurement in women with cycle disturbances: discrepancies between immunoreactive and bioactive hormone estimates.

OBJECTIVE: We evaluated the significance of single serum LH estimates (as assessed by radiometric assay (IRMA) and Leydig cell in-vitro bioassay (BIO)) for the diagnosis of polycystic ovary syndrome (PCOS) in women with infertility and cycle abnormalities. DESIGN: Hormonal and clinical comparisons between subgroups were made based on classification according to (a) rigid clinical and endocrine (excluding LH) characteristics of PCOS, (b) elevated IRMA-LH concentrations, (c) BIO-LH levels. In addition, androgen modulation of LH biopotency was studied in these patients. PATIENTS: Ninety-nine women presenting at our infertility Unit with oligo/amenorrhoea. MEASUREMENTS AND RESULTS: Of the total study group, 35 women were diagnosed positive as PCOS and 42 showed elevated IRMA-LH levels. Only 51% (n = 18) of PCOS patients showed elevated IRMA-LH levels, and in PCOS significantly higher levels of BIO-LH, androstenedione, oestrone, and BIO/IRMA-LH ratios were found as compared to non-PCOS patients. In the group with elevated IRMA-LH only 43% (n = 18) of subjects were diagnosed as PCOS, and no difference in BIO/IRMA-LH ratios was found. With increasing BIO-LH levels the probability of PCOS rises sharply (P < 0.001), whereas this probability is of only marginal significance (P < 0.06) for IRMA-LH. In the total study group a correlation is observed between serum testosterone (T) levels and IRMA-LH (r = 0.47), and BIO-LH (r = 0.51) concentrations. This correlation is absent comparing serum T and BIO/IRMA-LH ratios (r = 0.15). CONCLUSIONS: Results presented in this study indicate that (1) women with infertility and oligo/amenorrhoea classified based on signs of PCOS or IRMA-LH levels, exhibit different clinical and endocrine characteristics, (2) only 51% of PCOS women exhibit elevated IRMA-LH concentrations, and only 43% of women with elevated IRMA-LH were diagnosed as PCOS, (3) IRMA-LH levels are a poor predictor of PCOS, whereas the predictive value of BIO-LH is better, (4) elevated BIO/IRMA-LH ratios in PCOS are dependent on alterations in BIO-LH, rather than IRMA-LH concentrations, and (5) no correlation was observed between serum T levels and BIO/IRMA-LH ratios.

Onset and characteristics of the midcycle surge in bioactive and immunoactive luteinizing hormone secretion in normal women: influence of physiological variations in periovulatory ovarian steroid hormone secretion.

Limited studies in nonhuman primates suggest that the midcycle LH surge is characterized by distinctly different patterns of bioactive (LH-BIO) and immunoactive (LH-RIA) LH secretion. To further examine the patterns of midcycle LH-BIO and LH-RIA secretion and explore the influence of physiological variations in steroid hormone feedback on LH surge dimensions we studied seven normal ovulatory women over the periovulatory interval. In each, blood samples were obtained every 3 h and transvaginal ultrasonography was performed every 12 h over a 5-7 day interval at midcycle. Serum levels of LH-RIA, FSH, estradiol (E2), progesterone (P4), and 17-hydroxyprogesterone were determined by RIA; LH-BIO was estimated using a mouse leydig cell bioassay. Hormone data were standardized to the time of surge onset in LH-RIA (time zero), defined as a 100% increase above a 6-point running mean baseline value; surge cessation was defined as a decline to below baseline concentration. Mean LH-RIA surge duration was 54.0 +/- 4.0 h. LH-BIO surge onset was simultaneous with that of LH-RIA and coincident with the peak in E2 levels (mean data). Mean P4 and 17-hydroxyprogesterone rose in a parallel, phasic manner, an abrupt increase in slope occurred between -6 h and +30 h but an acute rise in P4 was not consistently observed among individuals. The surge onset to follicle rupture interval (mean 37.6 +/- 4.2 h) positively correlated with peak LH-RIA (r = 0.76, P less than 0.05), surge amplitude (r = 0.74, P less than 0.05) and surge onset to peak interval (r = 0.87, P less than 0.02), but not surge duration. There were no significant relationships between E2 or P4 (mean, peak, integrated, slope) and surge amplitude or duration (LH-RIA, FSH), peak value, or surge onset to peak interval (LH-RIA, LH-BIO, FSH). These data suggest that in women, 1) onset of the midcycle surge in LH-RIA and LH-BIO is simultaneous, and 2) surge characteristics are not influenced by physiological variations in steroid hormone secretion that occur beyond the thresholds required for surge initiation.