Dose-response relationship of luteinizing hormone to luteinizing hormone--releasing hormone in man.

In previous clinical studies with highly purified porcine luteinizing hormone-releasing hormone (LH-RH), administration of the somewhat arbitrarily chosen doses of 700-1500 mug resulted in increased serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The present study determined the minimum effective dose as well as the relationship of the response of serum LH and FSH to the dose of LH-RH administered. Three normal men received i.v. injections of 1.1-810 mug of LH-RH. A dose of 10 mug of LH-RH caused a statistically significant elevation in serum LH. 30 mug of LH-RH significantly increased serum FSH levels. A highly significant linear trend was observed in the log dose-response curve. The results indicate that both LH and FSH release occurs in man with doses of LH-RH much lower than previously used and that a linear log dose-response relationship can be obtained.

Alteration in the hypothalamic-pituitary-ovarian axis in depressed women.

BACKGROUND: Stress and corticotropin-releasing hormone inhibit the reproductive axis. We hypothesized that reproductive axis hormone secretion, particularly luteinizing hormone secretion, is inhibited in women with depression, similar to what has been observed to be caused by stress in numerous species. METHODS: Blood samples were collected every 10 minutes for 12 hours in 25 untreated premenopausal women with depression and 25 nondepressed women who were matched by age and menstrual cycle day. Samples were assayed for luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone. RESULTS: The mean plasma estradiol level was 30% lower in the follicular phase in women with depression than in their matched controls: 191 + 136 vs 261 + 169 pmol/L (52 + 37 vs 71 + 46 pg/mL). The half-life of luteinizing hormone was significantly shorter in women with depression than in their matched controls during both the follicular (22% shorter) and luteal (15% shorter) phases. CONCLUSIONS: The blood levels of reproductive hormones were mostly normal in women with depression, but the blood level of estradiol was significantly lower. Estradiol is known to affect a number of neurotransmitter systems in the brain. Arch Gen Psychiatry. 2000;57:1157-1162.

Changes in expression of connexin 43 gap junction messenger ribonucleic acid and protein during ovarian follicular growth.

Within the ovarian follicle, communication between granulosa cells and the oocyte via gap junction channels may be crucial for the determination of whether a given follicle continues its growth and development or becomes atretic. Numerous gap junction channels exist between granulosa cells and between the oocyte and granulosa cells. This study addresses the potential hormonal regulation of the gap junction gene during the processes of follicular growth, ovulation, and atresia. Because the ovary is an exceptionally heterogeneous tissue with numerous follicles at different stages of development, in situ hybridization and immunocytochemistry were used to localize gap junction gene expression precisely to specific cells in the ovary. The results demonstrated that only the granulosa cells of healthy, developing, preantral and antral follicles express large amounts of connexin 43 (cx43) gap junction messenger RNA (mRNA) and protein. Theca cells contained negligible levels of cx43 gap junction mRNA or protein. Very little cx43 gap junction mRNA or protein was detected in follicles undergoing atresia or in the corpora lutea. Additionally, during the night of proestrus, the level of cx43 gap junction mRNA and protein seen in the granulosa cells of the large preovulatory follicles dramatically decreased after the ovulatory surge of LH. This decrease was seen only in the preovulatory follicles and not in less-developed follicles, thereby demonstrating a differential response of the follicles to the preovulatory hormonal stimuli. Therefore, the expression of the gap junction gene may be hormonally regulated during follicular growth and development, in preovulatory follicles in preparation for ovulation, and during the process of atresia.

Lack of difference between retinoic acid and retinol in stimulating progesterone production by luteinizing granulosa cells in vitro.

Receptors for retinoids in the immature rat ovary and the effects of retinol and retinoic acid on luteinizing granulosa cells were studied. Radioreceptor assay demonstrated the presence of specific cellular retinol-binding protein and cellular retinoic acid-binding protein in the ovaries of rats injected with PMSG alone or PMSG and hCG. In addition, when luteinizing granulosa cell from PMSG/hCG-injected immature rats were cultured with or without retinoic acid, the morphology, viability, number of cells in culture, and progesterone (P) accumulation were not affected by up to 10 microM retinoic acid. Beyond 10 microM, the cells began to round up, which was associated with a decrease in cell viability. Surprisingly, the deleterious concentrations of retinoic acid increased progesterone accumulation significantly higher than the medium control value. This increase in progesterone, however, was not accompanied by an increase in cAMP. When cells preincubated for 2 days with 1 microM of either retinoic acid or retinol were subsequently incubated in retinoid-free medium containing various substrates for steroidogenesis, the following results were obtained. Basal progesterone and its accumulation in response to human low density lipoprotein were significantly higher in cells preincubated with retinoids than in the control cells. However, no difference was seen in the degree of stimulation between retinol and retinoic acid pretreatments. Both 25-hydroxycholesterol, a substrate for side-chain cleavage enzyme, and pregnenolone, a substrate for 3 beta-hydroxysteroid dehydrogenase, significantly stimulated the accumulation of progesterone in cells preincubated with retinoids over the control value. Again, no appreciable difference was observed between retinol and retinoic acid pretreatments. Our results suggest that receptors for retinoids are present in gonadotropin-primed immature rat ovaries, retinoids increase luteal cell progesterone accumulation, and no difference exists between retinol and retinoic acid in their ability to increase the accumulation of progesterone by these cells.

In situ hybridization of luteinizing hormone/human chorionic gonadotropin receptor messenger ribonucleic acid during hormone-induced down-regulation and the subsequent recovery in rat corpus luteum.

Previous studies have shown that injection of a pharmacological dose of hCG to rats primed with PMSG/hCG results in a loss of hCG binding in the luteinized ovary, which is closely coupled with the loss of LH/hCG receptor (LH/hCG-R) messenger RNA (mRNA). The time course of down-regulation of the receptor mRNA reveals that mRNA totally disappears 24 h after the hormone injection, but fully recovers by 72 h. The purpose of this study was to determine by in situ hybridization whether the recovery of the receptor mRNA occurs in preexisting or newly formed corpora lutea. Twenty-one-day-old female rats were treated with 50 IU PMSG, followed 56 h later by a single injection of hCG. On day 5 after the hCG injection, one group of rats was treated with a desensitizing dose of hCG, and a control group received saline. The ovaries were collected 6, 12, 24, 48, 72, and 96 h after the treatments and were processed for in situ hybridization using 35S antisense RNA synthesized from a 750-mer LH/hCG-R complementary DNA. In control ovaries, heavily labeled LH/hCG-R mRNA-containing cells were observed in the numerous corpora lutea. Ribonuclease pretreatment of the sections eliminated the signal, and no specific hybridization was observed when sense strand probe was used. No hybridization to the granulosa cells was seen. Some hybridization occurred to the theca interna, but the intensity was lower than that in the corpora lutea. Time-course studies showed a marked decline in the hCG-R mRNA signals in corpora lutea as early as 6 h after hCG injection, with a maximum loss of receptor mRNA by 24 h. After 48 h, hCG-R mRNA reappeared in preexisting corpora lutea, with the intensity of the hybridization signal equaling that in corpora lutea of controls. New corpora lutea could not be identified at any time after injection of the down-regulating dose of hCG. As down-regulated receptor mRNA recovered in preexisting, not new, corpora lutea, hormone-induced loss of luteal cell receptors would appear to be reversible.

Use of newly designed microperifusion system with amperometric sensors for near-continuous on-line monitoring of hormone secretion. I. correlation with the luteinizing hormone secretory response to gonadotropin-releasing hormone.

LH is rapidly secreted from the anterior pituitary gland in response to episodic release of GnRH. To understand better the detailed dynamics of this secretory process and related time- and dose-dependent effects, we developed a microperifusion system able to preserve resolution of dynamic secretory responses by minimizing dispersion and with capabilities for automation and continuous on-line monitoring. To monitor secretion at a frequency greater than that feasible with discrete samples, we determined whether LH or another molecule cosecreted by anterior pituitary cells could be monitored on a near-continuous basis using amperometric electrochemical sensors operated under conditions of cyclic voltammetry. The developed culture system incorporated a 32-mu l cell chamber in a controllable constant environment. Miniature sensors were positioned immediately adjacent to the cells to permit differential measurements of the input and effluent streams of medium. With the system, square wave pulses of electrochemically active, biologically inert molecules, e.g. ascorbate and phenol red, showed similar redox profiles before and after the cells, with minimal dispersion. Enzymatically dispersed ovine anterior pituitary cells were cultured on SoloHill glass beads for 4 days, loaded into the chamber, and perifused with DMEM containing 10% FCS for 2 h. After stabilization, the medium was switched to protein-free, HEPES-buffered HBSS with L-glutamine and allowed to flow for a 1-h washout period, followed by GnRH challenges of varying concentrations in a random order. Perifusate was collected in six-drop fractions at approximately 30-sec intervals. Although LH, its individual subunits, and FSH could not be detected amperometrically, GnRH stimulation of the cells simultaneously induced a several log order dose-dependent secretion of both an electrochemically detectable molecule and LH, as measured by RIA. The secretory profiles of the amperometrically detected signal and immunoactive LH were very similar. Dose-response relationships of the amperometric signal and LH to a wide range of GnRH were similar. The responses of both secreted LH and the amperometric signaling molecule(s) to GnRH were triphasic; an initial peak of activity was observed within 20-40 sec, a lower plateau level was observed for the duration of the 4-min GnRH stimulation, and a gradual return to baseline followed. The cells then maintained a constant level of GnRH-independent basal secretion. These results indicate that it is feasible to monitor the complex dynamics of endocrine and cellular responsiveness to secretagogues from endocrine cells/tissues continuously in real-time.

Dynamics of gonadotropin-releasing hormone release during a pulse.

This study examined the nature of the GnRH signal that travels down the pituitary portal vessels and causes an LH pulse. Individual GnRH pulses were described in terms of abruptness of increase and decrease, amplitude, duration, and amount of GnRH released. Pituitary portal blood was obtained at 30-sec intervals for 2.5 or 5 h from five short-term ovariectomized ewes. Jugular blood was sampled every 10 min for LH. We examined 13 GnRH pulss; each produced an LH pulse. The contour of most GnRH pulses approximated a square wave. The rising edge of the GnRH pulse was very abrupt; GnRH secretion increased as much as 50-fold within 1 min. The mean peak amount of GnRH collected during pulses (24 pg/min, range 2-66) was 70-fold greater than the interpulse baseline (0.2-0.5 pg/min). The release period was sustained an average of 5.5 min; thereafter, GnRH fell to prepulse levels within 3 min. Overall, the larger and more prolonged pulses of GnRH were associated with higher amplitude LH pulses. To assess the distortion of the GnRH signal by the collection procedure, samples were obtained in vitro using the same technique during application of 4- and 7-min square wave GnRH pulses by means of a syringe pump. Signals were carried as square-waves through the sampling operation with minimal distoration, with the exception that amplitude decreased during the collection procedure. Our findings indicate the square-wave pulses observed in vivo are an accurate description of the dynamics of GnRH release during a pulse in short-term overiectomized ewes.

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.

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG.

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and the resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.

Progesterone metabolism by the ovary of the pregnant rat: discrepancies in the catabolic regulation model.

The intraovarian site of 20 alpha-hydroxysteroid dehydrogenase activity (20 alpha-OH-SDH) was determined biochemically by measuring enzyme activity in homogenates of the whole ovary, or of isolated ovarian compartments, during the last third segment of pregnancy in the rat. In agreement, with previously reported histochemical evidence, an increase in 20 alpha-OH-SDH activity was observed in isolated corpora lutea, but not in the non-luteal compartment of the ovary. Enzyme activity in corpora lutea was low between days 16 and 22 of pregnancy, but increased markedly (4-6 fold) on day 23. Between days 17 and 20 of pregnancy, serum concentrations of progesterone declined from 130 +/- 3 to 80 +/- 3 ng/ml, while 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) concentrations declined from 34 +/- 3 to 18.5 +/- 3 ng/ml. Only later, between days 20 and 22 of pregnancy, was a significant decline in serum progesterone concentrations associated with an increase in serum 20 alpha-OH-P concentrations (50 +/- 15 ng/ml at 0800 h and 128.5 +/- 15 ng/ml at 1400 h on day 22). Thus the decline in progesterone concentration late in pregnancy can be explained only partially by conversion of progesterone to 20 alpha-OH-P. Further, a dissociation between changes in enzyme activity and in serum concentrations of 20 alpha-OH-P was also observed. A marked increase in serum levels of 20 alpha-OH-P on day 22 preceded any increase in enzyme activity by at least 10 h, and continued increases in enzyme activity on day 23 were not associated with any steady increase in peripheral 20 alpha-OH-P levels. We conclude from these observations that luteal regression is a more complex phenomenon than the regulation of a single enzyme, 20 alpha-hydroxysteroid dehydrogenase, and may involve regulation of both the synthesis and degradation of progesterone.

Effect of prolactin on LH receptor in rat luteal cells.

This investigation was undertaken to determine whether prolactin might act as a luteotropic hormone in the rat by increasing the receptor for LH. Immature female rats were treated with 5 SC injections of oFSH beginning at 0900 h on day 25; luteinization was induced with a single SC injection of oLH at 1500 h on day 27. In this model, LH-receptor activity, assessed by specific binding of [125I]iodohCG to ovarian membrane fractions, increased steadily (greater than 7-fold) from day 27 to day 33; specific [125I]iodoprolactin binding rose greater than 3-fold between days 27 and 31. Serum progesterone measured at 1500 h rose from greater than 10 ng/ml on day 27 to greater than 130 ng/ml on day 34. 2-Bromo-alpha-ergocryptine (CB-154, [EC]; 35 mug) given 2 X/day beginning at the time of LH injection, blocked nocturnal increases in serum prolactin measured at 0200 h on days 29, 30, and 31, and resulted in reduced binding of [125I]iodohCG on days 31 and 33; serum progesterone was similarly reduced on days 31 and 34 in EC-treated rats. Simultaneous treatment with EC and prolactin completely reversed the effects of EC with regard to both binding of [125A]iodohCG and serum progesterone. Changes in ovarian uptake of [125I]iodohCG in vivo were similar to those of the in vitro experiments described above. EC and/or prolactin treatment did not affect binding of [125I]iodoprolactin. In summary, the data indicate that LH in the absence of prolactin can induce the formation of corpora lutea which have an increased number of prolactin receptors but which have a few LH receptors and a reduced capacity to produce progesterone. If prolactin is present during the early luteinization process, corpora lutea develop an increased capacity to bind LH and to produce progesterone. It is possible that the increase in LH-receptor and progesterone production occur as independent events both mediated by the luteotropic action of prolactin. Alternatively, LH receptor might be limiting for LH stimulation of progesterone production.

Regulation of gonadotropin receptors by luteinizing hormone in granulosa cells.

In association with luteinization, LH induces a decrease in the content of receptors for FSH and LH and an increase in that for PRL. To elucidate if the mechanism by which LH regulates its own receptors involved occupancy of sites and/or loss of receptors the effects of a luteinizing dose of LH were examined in the preovulatory follicles of immature hypophysectomized rats primed with estradiol and FSH. The measurable LH receptor content declined by 82% 24 h after LH administration. Serum concentration of the hormone declined by 24 h to 1.4% of the concentration measured 2 h after LH administration. Administration of iodinated LH to demonstrate occupancy of sites in vivo, resulted in a decline in the amount of hormone bound in vivo, over a period of time. This decline in occupancy paralleled the decrease in the number of available sites as measured in vitro. Furthermore, a large dose of highly purified hFSH administered in lieu of LH induced luteinization and an associated loss of gonadotropin receptors. These results indicate that luteinizing doses of LH and FSH induce a loss in gonadotropin receptors by mechanisms other than occupancy.

Differential processing of the two subunits of human choriogonadotropin by granulosa cells. II. In vivo studies.

Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.

Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone.

The effects of estradiol, FSH and LH on ovarian follicular development and granulosa cell differentiation were examined in the immature rat hypophysectomized on day 24 of age. Administration of estradiol to hypophysectomized rats for 4 days stimulated the growth of large preantral follicles with a concomitant 1.5-fold increase in FSH receptor content and a 4-fold decrease in LH receptor content in the granulosa cells. When highly purified hFSH was administered alone, receptor content for FSH increased progressively for 4 days while receptor for LH remained essentially unchanged. However, when rats were pretreated with estradiol, the response of follicles to FSH was markedly enhanced as indicated by the appearance of large, antral follicles and elevated receptor content for both FSH and LH. Receptor content for FSH increased markedly in response to hFSH following only one day of estradiol pretreatment, while receptor content for LH increased most rapidly in response to hFSH after 3 days of estradiol pretreatment. LH administered to rats possessing large preovulatory follicles caused luteinization of granulosa cells and a marked decline in receptor content for both gonadotropins within 24 h. Receptor content remained low even 48 h after LH administration when granulosa cells were fully luteinized. These results indicated that follicular development and granulosa cell differentiation are dependent on steroid-protein hormone regulation of hormone specific receptors.

Regulation of human gonadotropins. X. Episodic fluctuation of LH during the menstrual cycle.

PIP: In order to determine the episodic fluctuation of LH (luteinizing hormone) during the menstrual cycle, the University of Michigan (Ann Arbor, 1971) subjected 49 samples, obtained at consecutive hourly intervals from seven women (ages 21-28) at different stages of the menstrual cycle, to radioimmunoassay. In each case, hourly analysis indicated that serum concentrations of LH, and possibly FSH (follicle stimulating hormone), are maintained as a series of peaks of varying magnitude. The length of the sampling interval prevented an exact determination of the frequency of the peaks which appeared to occur at approximately 2 1/2 hour intervals. The largest LH peaks were recorded during the ascending and descending phases of the major peak of LH at midcycle and during the early luteal phase of the cycle. The follicular phase produced the smallest LH peaks. These observations support the hypothesis that serum concentrations of LH and FSH are maintained by brief periods of release followed by periods of little or no release. Corresponding changes in LH and FSH suggest that they are released simultaneously and may be contained within the same cell. To maintain blood concentrations, the relative amount of simultaneously released LH should be greater than that of FSH since the latter has a longer half-life. Additional factors may be involved, since widely different ratios of LH/FSH can be observed at various phases of the menstrual cycle. It is suggested that accurate analysis LH and FSH concentrations requires sampling at least at half-hour intervals as is necessary for ACTH.^ieng

Serum follistatin levels in women: evidence against an endocrine function of ovarian follistatin.

Follistatin is a monomeric protein first identified in and isolated from ovarian follicular fluid. Evidence that follistatin might be an ovarian endocrine hormone functioning in a negative feedback fashion to modulate pituitary FSH production is based primarily on in vitro experiments. To examine the possible role of follistatin as an endocrine agent in vivo, we sought to relate circulating levels of follistatin to ovarian activity in women. Therefore, we developed a specific and sensitive homologous RIA using antiserum generated against recombinant human follistatin for the measurement of total follistatin in the presence or absence of activin. Follistatin was measured quantitatively (106 +/- 6% recovery) using calibration standards ranging from 0.4-25 ng/tube and up to 400 microL/tube serum. Furthermore, all of the endogenous follistatin measured in human serum could be removed by adsorption to activin-coated plates. Using this homologous RIA, human follicular fluid (100-600 ng/mL; n = 75) contained 3-150 times more follistatin than serum (4-35 ng/mL), an observation consistent with the notion that serum follistatin originates from the gonad. However, further studies of follistatin levels across the normal menstrual cycle (mean +/- SE, 8.09 +/- 0.73; n = 72 daily samples from 4 women), in pregnant women (17.49 +/- 1.34; n = 8), in daily samples from 20 women undergoing ovarian stimulation by exogenous FSH (9.90 +/- 0.62; n = 119), in postmenopausal women including two ovariectomized individuals (9.57 +/- 0.43; n = 8), and in GnRH-deficient women (9.85 +/- 0.50; n = 6) failed to support the hypothesis that serum levels of follistatin reflect ovarian activity in women. Levels of follistatin measured in serum collected across normal menstrual cycles did not fluctuate. However, the roughly nanomolar concentrations of follistatin measured suggest a physiological role for this protein. Follistatin at nanomolar concentrations may be capable of binding and inactivating circulating activin and perhaps in this way limiting the biological activity of activin to local autocrine or paracrine mechanisms. Measurement of peripheral levels of follistatin apparently represents only a first, albeit crucial, step in the study of the physiological significance of this protein in human reproduction.

Circulating concentrations of dimeric inhibin A and B in the male rhesus monkey (Macaca mulatta).

The purpose of this study was to determine the relative concentrations of inhibin A and B in peripheral serum of the adult male rhesus monkey and to examine the testicular contribution to these circulating forms of inhibin. In addition, inhibin B concentrations were also determined in peripheral sera of neonatal and juvenile males and in spermatic vein blood of adults. Immunoradiometric assays specific for the measurement of inhibin A and B were used. These assays also provided an opportunity to reexamine the physiological significance of a replacement infusion of recombinant human (rh)-inhibin A previously employed to study the role of this hormone in regulating FSH secretion in the monkey. In intact adults, the mean (+/-SE) serum concentration of inhibin B was 1008 +/- 184 pg/mL. In contrast, circulating inhibin A concentrations were very low (< 46 pg/mL). Inhibin B was consistently detected in neonatal monkey serum (275 +/- 57 pg/mL), and concentrations of this inhibin dimer increased throughout postnatal development, reaching maximum values in adulthood. Circulating inhibin A concentrations in neonatal and juvenile monkeys were undetectable (< 7 pg/mL). Both forms of inhibin were generally undetectable in castrate sera. The ratio of inhibin B concentrations in testicular venous blood to those in the peripheral circulation was 1.4:1. These findings indicate that, in the male monkey, inhibin B is the principal form of circulating dimeric inhibin, and that this hormone is derived exclusively from the testis. The elevated levels of circulating inhibin B in the juvenile male monkey suggest that, during this phase of development, testicular inhibin B secretion is relatively gonadotropin independent. Additionally, we found that the concentration of circulating inhibin A in castrate animals that had earlier received an iv infusion of rh-inhibin A (832 ng/h/kg BW) was 9881 +/- 2135 pg/mL, indicating that this mode of inhibin replacement may not have been entirely physiological.

A two-site monoclonal antibody immunoradiometric assay for human follistatin: secretion by a human ovarian teratocarcinoma-derived cell line (PA-1).

The follistatin/activin/inhibin system increasingly appears to have important growth and differentiating effects in a variety of cell types, including cancer. We have developed a two-site immunoradiometric assay for measurement of human follistatin using two monoclonal antibodies against recombinant human follistatin. This cloned protein donor assay is sensitive (0.5 ng/mL), specific for free human follistatin, and precise (<5% within assay coefficient of variation). Using this assay, native human follistatin could be measured in human pituitary extracts, follicular fluid, and granulosa-luteal cell-conditioned medium. To identify and characterize human follistatin secreted by ovarian cancer cells, we screened five human ovarian carcinoma cell lines currently available from the American Type Culture Collection (Rockville, MD). One of these, a cell line derived from a teratocarcinoma (designated PA-1, American Type Culture Collection, CRL1572), secreted large (3 microg/10(6) cells per 24 h) quantities of immunoreactive follistatin constituitively. Increasing volumes of conditioned medium from these cultured cells generated response curves parallel to those of recombinant human follistatin 288 reference protein, human follicular fluid, or culture medium from human granulosa-luteal cells. Secretion of follistatin by PA-1 cells was time and cell-number dependent with 297.9 +/- 15.2, 654 +/- 29.8, and 940 +/- 49.1 ng follistatin secreted over 24 h by 1 x 10(5), 2 x 10(5), and 3 x 10(5) cells, respectively. Western and ligand blot analysis revealed that the immunoreactive follistatin secreted by PA-1 cells and isolated by sulfate-cellufine chromatography was identical to the molecular weight variants (32,000 and 35,000 Mr) of recombinant human follistatin 288. PA-1 cell-conditioned medium suppressed basal secretion of FSH by cultured rat anterior pituitary cells in a dose-dependent fashion. This follistatin bioactivity was completely removed by adsorption with either solid-phase monoclonal antifollistatin or a dextran-sulfate chromatography gel. Because activin suppressed the proliferation of PA-1 cells, secretion of bioactive follistatin may represent an autocrine mechanism opposing activin to maintain the rapid growth rate of PA-1 cells. These observations demonstrate that the ovarian teratocarcinoma cell line, PA-1, secretes considerable amounts of human follistatin that is biologically active, capable of binding human activin, and antigenically similar to recombinant human follistatin 288. The monoclonal antibodies and two-site assay reported herein should be useful in assessing the regulation of follistatin secretion and as a diagnostic tool, especially if follistatin measurements prove to be a marker for some ovarian cancers.

A two-site chemiluminescent assay for activin-free follistatin reveals that most follistatin circulating in men and normal cycling women is in an activin-bound state.

Follistatin (FS) is a monomeric protein that binds and regulates the bioavailability of activin. Previously, we found circulating levels of total FS to be similar in men and cycling women. Because relative amounts of activin-bound and free FS are important considerations in determining activin bioavailability, we asked here whether the relative proportions of these two changed during different physiologic states. For this, we developed a two-site, solid-phase, immunochemiluminescent assay for free FS. The assay recognizes the 288 or 315 amino acid variants of human FS and has a detectable limit of 1 ng/mL. Inhibin, transforming growth factor-beta, or alpha-2-macroglobulin do not cross-react or interfere in this assay. Preincubation of FS with activin results in dose-dependent loss of immunoreactivity, confirming specificity of the assay for free FS. Human follicular fluid, pituitary extract, and serum with added FS dilute parallel with the recombinant human FS-288 standard. Recovery of recombinant human FS-288 from serum is quantitative. Using this assay, we found circulating concentrations of free FS to be at or below the detection limit of the assay throughout the menstrual cycle. Comparison of circulating total and free FS levels in postmenopausal or cycling women and normal men suggested that at least 90% is activin-bound. In contrast, measurable quantities of free FS were found in follicular fluid and pituitary extracts. The results of this study, showing that most circulating FS is normally activin-bound, argue against an endocrine role for FS and suggest that a major role of circulating FS is to bind and neutralize the bioactivity of circulating activin. The roles of FS as a local autocrine or paracrine regulator of activin in target tissues, where FS exists in free form, or as an endocrine regulator in human pathophysiology, warrants further investigation.

Developmental changes in the ovarian follicular basal lamina detected by immunofluorescence and electron microscopy.

Alterations in the basal lamina (BL) of developing follicles were studied by immunofluorescent microscopy using antibodies against type IV collagen, laminin, and fibronectin, and by electron microscopy. Ovarian development was induced in immature rats by sequential administration of estradiol, follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). A continuous BL was observed in healthy follicles treated with estradiol and FSH. As determined by immunofluorescence, laminin, type IV collagen, and fibronectin were restricted to the BL and the theca but not to the granulosa. When follicles were allowed to undergo atresia or induced to ovulate with hCG, the BL became fragmented. This was confirmed by electron microscopy of healthy, atretic, and luteinizing follicles which showed that in healthy follicles the BL was continuous, whereas in both atretic and luteinizing follicles, it was fragmented. Atresia was also associated with the penetration of thecal cells into the follicles. These observations indicate that the intact BL present in healthy follicles undergo extensive changes during atresia and ovulation.