Suppression of serum follicle-stimulating hormone by follicular fluid in the maximally estrogenized, ovariectomized mouse.

Serum levels of FSH in ovariectomized female mice are only partially suppressed by estradiol regardless of the dosage administered. Conversely, within the limits of detection, serum FSH in intact females is totally suppressed by estradiol. Thus, the present experiments used the maximally estrogenized, ovariectomized female as a test system for evaluating other nonestrogenic factors which could depress serum FSH. It was found that a transplanted ovary lowered serum FSH dramatically under such conditions. Of the large number of steroid and pituitary hormones tested, however, only large doses of testosterone propionate yielded any further suppression of serum FSH (24%). Charcoal-extracted follicular fluid (porcine) markedly lowered serum FSH concentrations (50% suppression), while a charcoal-extracted saline homogenate of PMS-treated mouse ovaries also was active in this regard (26% suppression). Finally, charcoal-extracted porcine follicular fluid was administered to nonestrogenized, ovariectomized females, where it both depressed serum FSH and markedly elevated serum LH concentrations. These studies, when viewed in toto, support a contention that follicular fluid contains a nonsteroidal factor which is capable of acting additively with estradiol to regulate FSH secretion. The question of whether this factor also regulates LH secretion in a reciprocal fashion in the mouse requires further work.

Contribution of granulosa cells and follicular fluid to ovarian estrogen secretion in rhesus monkey in vivo.

In order to ascertain which ovarian cell type within the follicle is the source of preovulatory estrogen secretion in vivo, ovarian venous, as well as peripheral venous, blood was collected prior to, 5 min, 30 min, and 120 min after the removal of follicular fluid and granulosa cells from 17 monkeys. In addition, estrogen, progesterone, and progestins were measured in the peripheral blood, ovarian venous blood, and follicular fluid of the follicle-containing and contralateral ovary in 24 monkeys, in order to prove that the preovulatory follicle is the principal source of estrogen. Estradiol was the principal estrogen and was secreted in larger amounts by the ovary with the large preovulatory follicle (7-10 mm in diameter) compared with the contralateral ovary. In 15 experiments ovarian venous estrogen (3934 +/- 798 pg/ml, mean +/- SE) in the vein draining the large follicle-containing ovary was usually 5-15-fold higher than peripheral plasma estrogen levels which were 307 +/- 31 pg/ml. The contralateral ovary secreted a small amount of estrogen (654 +/- 162 pg/ml). Follicular fluid contained large amounts of estrogen (2754 +/- 695 ng/ml) with levels which did not always correlate well with peripheral plasma or ovarian venous estrogen. Ovaries containing non-preovulatory or recently ovulated follicles secreted less estrogen. The removal of granulosa cells and follicular fluid from the preovulatory follicle led to no significant decrease (P greater than 0.5) in ovarian venous secretion of estrogen after a 5, 30, or 120 min time interval. This would indicate that, within the time constraints of this experiments, the follicular fluid and granulosa cells contribute relatively little to ovarian venous estrogen and that thecal cells of the large preovulatory follicle alone can secrete more of the estrogen into the ovarian vein.

An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro.

Isolated oocytes will resume meiosis spontaneously in vitro whereas follicle-enclosed oocytes will remain in the dictyate stage when cultured unless they have been exposed to gonadotropins in vivo or in vitro. To examine the source of the follicular inhibitory influence, porcine oocytes have been cultured alone, with hemisections of follicle wall, granulosa cells, or with follicular fluid. Oocytes isolated from medium-sized (3-5 min) follicles resumed meiosis when cultured; 77.5 plus or minus 3.4 percent matured beyond the dictyate stage. When oocytes were cultured in the presence of follicle wall hemisections of medium and large (6-12 mm) follicles, the percentage of maturing oocytes was significantly reduced. The maturation of oocytes cultured in a medium containing 50 percent follicular fluid from small or large follicles was significantly inhibited. Resumption of meiosis was completely inhibited by co-culture of isolated oocytes with 10-7 granulosa cells from small, medium or large follicles. Addition of serially diminishing amounts of granulosa cells from 10-7 to 10-4 cells reduced the inhibitory influence. It is concluded that the granulosa cells are responsible for the maintenance of the oocytes in the dictyate stage within the follicle. The granulosa cells appear to exert their inhibitory influence upon meiosis by secretion of a chemical message into follicular fluid.

Suppression of serum follicle stimulating hormone in intact and acutely ovariecomized rats by porcine follicular fluid.

Sham ovariectomy (SOV) or ovariectomy (OVX) was performed at 0800h of metastrus; porcine serum (PS) or porcine follicular fluid (PFF) was injected intraperitoneally at 0830h and/or 1130h, with terminal blood samples collected at 1700h. Fluid from medium and large follicles in porcine ovaries, charcoal extracted to remove steroids, significantly reduced serum FSH, in a dose-dependent manner. Futhermore, the SOV rats were more sensitive to PFF with respect to serum FSH lowering than the OVX rats, although when enough PFF was injected, both groups showed the same level of suppression. Since serum LH was very low, the possible effect of PFF on this hormone could not be assessed.

Role of the carbohydrate residues of human chorionic gonadotropin in binding and stimulation of adenosine 3',5'-monophosphate accumulation by porcine granulosa cells.

Removal of the sugar residues internal to sialic acid from the hCG molecule markedly diminished the ability of hCG to stimulate cAMP accumulation by porcine granulosa cells during a 30-min incubation period. At the same time, removal of sugars internal to sialic acid enabled the resulting hCG derivatives to become competitive inhibitors of hCG stimulation of cAMP accumulation. This contrasts with the finding that removal of sugars internal to sialic acid residues has a lesser effect on the ability of hCG to inhibit the binding of human [125I]iodo-CG to granulosa cells, provided the hCG derivatives were added before the tracer. It would, therefore, seem that hCG with sugars internal to sialic acid removed is able to bind to the receptor, but that, once bound, it is unable to activate adenylate cyclase. Under these conditions, it can also act as a competitive inhibitor of hCG stimulation of cAMP accumulation.

Stimulatory effect of luteinizing hormone upon relaxin secretion by cultured porcine preovulatory granulosa cells.

Relaxin, a peptide hormone capable of causing connective tissue alterations, is produced by the corpus luteum and traditionally considered a hormone of pregnancy. We have cultured granulosa cells from preovulatory porcine follicles and have found that these non-pregnancy associated cells secrete relaxin, and that luteinizing hormone, which stimulates ovulation, enhances relaxin secretion by cells from large preovulatory follicles. These results suggest that relaxin secreted prior to ovulation may have a local ovarian effect, perhaps facilitating ovulation.

Inhibitory effect of human follicular fluid upon the maturation of porcine oocytes in culture.

Human follicular fluid (hFFl) was harvested from follicles (5-15 mm) of human ovaries obtained at laparotomy. Addition of native hFFl, or a low molecular weight fraction of it, to the culture medium was found to inhibit the spontaneous maturation of cumulus-enclosed porcine oocytes. It was also found that hFFl inhibited progesterone secretion by the cumulus cells. The results extend earlier observations in other mammalian species, indicating that in the human, also, a specific oocyte maturation inhibitor is responsible for keeping the oocyte in meiotic arrest.

Levels of inhibin-F activity and steroids in human follicular fluid from normal women and women with polycystic ovarian disease.

To examine inhibin-F activity (FSH-suppressing activity) in human follicular fluid of polycystic ovary (PCO) patients, 13 follicles from 5 documented PCO patients and an additional 31 follicles from normal women in the follicular phase of the menstrual cycle were sampled, and inhibin-F activity was measured in rat anterior pituitary cell cultures. Inhibin-F activity was measured in follicular fluid after stripping steroids from the fluids using treatment with dextran and activated charcoal. Estrogen, progesterone, and delta 4-androstenedione in the follicular fluid were also determined by RIA. Estrogen and progesterone levels in follicular fluid from PCO follicles 3.9 +/- 0.34 mm in diameter were comparable with those in follicular fluid obtained from viable follicles (which had a delta 4-androstenedione to estrogen ratio of 10 or less) from normal women. delta 4-Androstenedione levels in PCO follicles were higher (P less than 0.01) than those in viable and atretic follicles of normal women. Inhibin-F levels in PCO follicles were comparable to those in viable follicles, but significantly greater (P less than 0.01) than levels in atretic follicles of normal women. If inhibin-F levels in both atretic and viable follicles of normal women were pooled, the levels were less (P less than 0.05) compared to the level in PCO follicular fluid. As an additional control, follicular fluid was collected from 90 follicles of normal women throughout the menstrual cycle, and follicular size was determined as well as inhibin-F and steroid content. Small follicles less than 8 mm; (comparable in size to the PCO follicles examined) obtained at each stage represented 79%, 24%, 0%, and 94% of the total follicles obtained in the early to midfollicular, late follicular, preovulatory, and luteal phases of the cycle, respectively. Inhibin-F activity in the fluids of these follicles was less than that in PCO follicular fluid. The average inhibin content of all of the small normal follicles was 197 +/- 34 U/10 microliter, which was significantly less (P less than 0.05) than the level in PCO follicles (332 +/- 13 U/10 microliters). These data represent the first observation of inhibin-F activity in PCO follicular fluid and suggest the possibility of involvement of inhibin-F in bringing about low or normal basal levels of FSH in the presence of elevated basal LH levels often observed in PCO patients.

Comparison of the stimulatory effects of ovine, porcine and human follicle-stimulating hormone and of ovine and human luteinizing hormone on the accumulation of cyclic AMP by porcine granulosa cells.

The effects of ovine, porcine and human FSH, and ovine and human LH on the accumulation of cyclic AMP by porcine granulosa cells obtained from follicles at various stages of maturation were investigated. During incubation periods of 15 min, 10 micrograms ovine FSH pretreated with antiserum to LH or 10 micrograms human FSH resulted in an 11- to 18-fold, five- to ninefold, and less than a twofold increase in intracellular accumulation of cyclic AMP by granulosa cells from small (1--2 mm), medium (3--5 mm) and large (6--12 mm) follicles respectively. Similar patterns of response occurred with addition of porcine FSH. After incubation for 30 and 60 min with ovine, porcine or human FSH, significant accumulation of cyclic AMP in the incubation medium occurred with cells obtained from small and medium-sized follicles. After 60 min of incubation with FSH the accumulation of cyclic AMP in the incubation medium exceeded the intracellular cyclic AMP levels in granulosa cells from small and medium-sized follicles. During incubation periods of 15 min, 1.0 micrograms ovine LH resulted in less than a twofold, a fourfold and greater than a tenfold increase in intracellular accumulation of cyclic AMP by granulosa cells from small, medium and large follicles respectively. Addition of human LH brought about a similar response. Incubation periods of 30 and 60 min with 1.0 micrograms ovine or human LH resulted in significant accumulation of cyclic AMP in the incubation medium by granulosa cells from large follicles; cyclic AMP content in the incubation medium was greater after 60 min compared with 30 min of incubation. It was concluded that ovine FSH pretreated with an antiserum to LH had similar effects on cyclic AMP levels as did purified human and porcine FSH, and that the stimulatory effects of the less pure ovine FSH were probably not due to an impurity in the FSH preparation. Porcine granulosa cells obtained from small follicles should be suitable as an in-vitro FSH bioassay while granulosa cells obtained from large follicles should be suitable as an in-vitro LH bioassay.

Suppression of pituitary secretion of follicle-stimulating hormone by porcine follicular fluid during pro-oestrus and oestrus in the rat: effects on gonadotrophin and steroid secretion, follicular development and ovulation during the following cycle.

In this study, we have examined whether the suppression of raised plasma FSH concentrations at pro-oestrus and/or oestrus by porcine follicular fluid (PFF) affected the development of follicles for ovulation in the next cycle. Adult, 4-day-cyclic rats were injected with PFF or pig serum at various hours of pro-oestrus and oestrus. Plasma FSH levels were suppressed following PFF treatment at any time of pro-oestrus and oestrus. Furthermore, this suppression was always followed by a 'rebound' increase in plasma FSH. In contrast, plasma LH concentrations were unaffected by PFF treatment and neither gonadotrophin was altered by treatment with pig serum. When rats treated with PFF or pig serum were allowed to complete one additional cycle, plasma LH and FSH concentrations at the pro-oestrus and oestrus after treatment were not significantly different among groups regardless of treatment or time of treatment. All ovaries of rats treated with PFF or pig serum on the next pro-oestrus morning before ovulation were histologically similar. Furthermore, all animals ovulated a normal complement of ova at the next oestrus regardless of whether preovulatory, secondary or both increases of plasma FSH had been blocked by PFF treatment during the previous cycle. However, animals given PFF during the preceding cycle, plasma oestradiol and progesterone concentrations were significantly altered on the morning and afternoon of pro-oestrus respectively. These results suggest that increased plasma FSH concentrations at pro-oestrus and oestrus may not be essential for folliculogenesis and ovulation in the subsequent cycle. Alternatively, the 'rebound' of FSH on day 1 of dioestrus after the suppression of both phases of FSH secretion at pro-oestrus and oestrus may be sufficient to provide ovulatory follicles for the next pro-oestrous day.

Inhibitory action of porcine follicular fluid upon granulosa cell luteinization in vitro: assay and influence of follicular maturation.

Culture medium 199 supplemented with follicular fluid from 1-2 mm antral porcine follicles inhibited spontaneous luteinization of granulosa cells from preovulatory porcine follicles in vitro. Three characteristics of luteinization were inhibited: morphological transformation, progesterone secretion, and accumulation of cyclic AMP in response to LH. The last was inhibited more effectively by culture media containing 50% follicular fluid than by media containing 20% follicular fluid. The inhibitory actions of the follicular fluid were not altered by charcoal or petroleum ether extraction. Follicular fluid from large follicles (6-12 mm) did not exhibit any of these inhibitory actions. These observations may indicate the presence of a luteinization inhibitor in the fluid of small follicles which (1) is lost by the time the follicle reaches the preovulatory stage, or (2) is overcome by a stimulatory agent which may accumulate as the follicle grows.

Porcine granulosa and cumulus cell properties. LH/hCG receptors, ability to secrete progesterone and ability to respond to LH.

In order to compare the properties of isolated cumulus and granulosa cells, granulosa cells and cumulus cells surrounding oocytes were harvested from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) porcine antral follicles and the number of LH/hCG receptors was measured by the binding of [125I]hCG. The ability of the cells to secrete progesterone in culture was examined in the presence and absence of hCG and LH. In 3 separate experiments of 1-h incubations at 37 degrees C using cells harvested from medium-sized follicles, granulosa cells bound 10--15-fold more iodinated hCG than an equivalent number of cumulus cells. During a 2-day culture period, cumulus cells secreted less progesterone than granulosa cells from medium- and large-sized antral follicles (p less than 0.01). The potential of both cumulus and granulosa cells to secrete progesterone in culture increased as the follicle progressed from small to large size. Also, the ability of the oocyte to mature in culture increased with antral follicle size. Concurrently the ability of cumulus-oocyte complexes to form monolayers in culture decreased as the follicle matured. Cumulus and granulosa cells harvested from small- and medium-sized follicles responded similarly to LH and hCG with a stimulation in progesterone secretion after 2-6 days in culture.

Mechanism of action of luteinizing hormone and follicle-stimulating hormone on the ovary in vitro.

The mechanism of action of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) upon various cell types of the mammalian ovary is reviewed. Emphasis is placed upon in vitro studies using organ and cell culture as well as short-term incubations. FSH and LH actions upon the following ovarian functions are discussed: steroidogenesis and metabolism of the ovary as a whole and of the isolated follicle and its component cell types, the granulosa and thecal cells, as well as folliculogenesis and follicular growth, oocyte maturation, follicular rupture, and corpus luteum maintenance and steroidogenesis. The roles of gonadotropin receptors, AMP, prostaglandins, protein kinase, and protein synthesis in these LH and FSH actions are discussed. Intra-ovarian regulation of LH and FSH action is reviewed, including a discussion of the possible roles of follicular fluid inhibitors upon oocyte maturation and granulosa cell luteinization.

Effect of follicle-stimulating hormone upon estrogen and progesterone secretion by cultures of monkey granulosa cells recovered during the periovulatory period.

For determination of how exposure of the monkey follicle to the preovulatory luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge alters its responsiveness to FSH in terms of estrogen and progesterone secretory ability, monkey thecal tissue and granulosa cells were harvested prior to and during the midcycle LH/FSH surge and cultured for 8 days with testosterone and with and without 100 ng human FSH. The addition of FSH enhanced estrogen secretion in culture (6.8 and 7 times on the average after 6 and 8 days, respectively; P less than 0.05) by granulosa cells if they were harvested prior to, but not during, the midcycle LH/FSH surge. In contrast, the FSH could stimulate granulosa cell progesterone secretion if the cells were harvested both prior to (60- to 100-fold stimulation) and during the midcycle LH/FSH surge (10- to 60-fold stimulation; P less than 0.05). It can be concluded that exposure of the preovulatory monkey follicle to the midcycle LH/FSH surge alters its responsiveness to FSH in terms of estrogen secretion.

Inhibin activity and steroid hormone levels in ovarian extracts and ovarian vein plasma of female monkeys during postnatal development.

Inhibin activity, follicle-stimulating hormone (FSH)-suppressing substance, estrogen, progesterone, and androstenedione were measured in charcoal-treated ovarian tissue and ovarian venous and peripheral blood of eight rhesus monkeys ranging from 12 to 48 months of age. All of the monkeys demonstrated inhibin activity in ovarian tissue, which, if expressed per milligram protein, was relatively constant throughout development. However, if the activity was expressed per ovary, the amount of ovarian FSH-suppressing substance increased between 26 and 48 months; it was present in detectable amounts in ovarian venous blood only in one 26-month-old monkey. Detectable levels of estrogen were present in ovarian venous blood of the 26-month-old and the 48-month-old monkeys but not in the younger monkeys. It is possible that the secretion of inhibin activity may be in part responsible for low levels of serum FSH observed prior to puberty, because it has been observed by others that bilateral ovariectomy in the prepubertal monkey can result in a rise in FSH and that administration of charcoal-treated ovarian follicular fluid can suppress serum FSH in castrated prepubertal and adult rhesus monkeys.

Follicular fluid inhibin activity and steroid levels in ovarian tissue obtained at autopsy from human infants from 18 to 200 days of age.

The ovaries of 25 human infants from 18 to 200 days of age were obtained at autopsy, and their follicular fluid was subjected to measurement of inhibin activity, estrogen, progesterone, androstenedione, and testosterone. Significant inhibin activity was present in all samples of follicular fluid (charcoal-treated) (138 +/- 19 U/10 microliter follicular fluid; 10,545 +/- 2758 U/ovary). There was a tendency for greater inhibin activity, follicular volume, and estrogen in infants from 18 to 59 days than in older infants. There was a significant positive correlation between follicular fluid volume, estrogen, and androstenedione, compared with follicular fluid inhibin content per ovary. It is possible that elevated serum follicle-stimulating hormone and luteinizing hormone observed early in life stimulates follicle growth, inhibin, and estrogen production. As a result of elevated inhibin and estrogen, the gonadotropins may be inhibited, which may cause a decline in follicular activity after 4 to 6 months.

Stimulatory effects of follicle-stimulating hormone and luteinizing hormone upon secretion of progesterone and inhibin activity by cultured infant human ovarian granulosa cells.

Ovarian tissue obtained from three human infants (60, 120, and 210 days of age) was separated into cell types and cultured. Granulosa cells from two of three subjects were viable and grew in culture. The cells had the potential to secrete low levels of progesterone and responded to luteinizing hormone and follicle-stimulating hormone added in culture with greatly enhanced ability to secrete progesterone. Granulosa cells could also secrete inhibin activity in culture and responded to luteinizing hormone and follicle-stimulating hormone with enhanced inhibin secretion. The granulosa cells also had the potential to secrete estrogen in the presence of testosterone. Serum levels of gonadotropin in the human infant are elevated for a period between 1 and 4 months; yet only follicular growth, not luteinization, occurs. It can be concluded that infant human granulosa cells, like adult human granulosa cells, have the potential of responding in vitro to gonadotropin.

Inhibin activity of preovulatory follicles of gonadotropin-treated and untreated women.

In order to determine whether treatment of endocrinologically normal women with human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) led to alternations in follicular fluid inhibin activity and steroids, both inhibin activity and steroid levels were measured in follicular fluid of 10 follicles of 9 women treated with hMG/hCG and in follicular fluid of 15 preovulatory follicles of 15 untreated women. The women were given 150 IU hMG daily from day 3 until the serum estrogen levels were greater than 300 pg/ml, at which time hMG was discontinued and 10,000 IU hCG was given 50 hours later. Follicular fluid was aspirated from all visible follicles 36 hours after hCG. Women treated with hMG/hCG had multiple (two to five) follicles containing 1.5 to 4.5 ml of follicular fluid and a mature oocyte and elevated serum estrogen (400 to 800 pg/ml) on the day after hCG treatment. At the time of follicular fluid recovery, there was more inhibin activity (204 +/- 14 U/10 ml) in the hMG/hCG-stimulated follicles than in unstimulated follicles of a control group of women (65 +/- 14, mean +/- standard error, P less than 0.05). Follicular fluid from follicles of hMG/hCG-treated women had lower estrogen and progesterone levels, compared with normal preovulatory women. These data show that treatment of patients with hMG/hCG is associated with elevated follicular fluid inhibin activity.

Demonstration of a gradient in inhibin activity, estrogen, progesterone, and delta 4-androstenedione in follicular fluid, ovarian vein blood, and peripheral blood of normal women.

Ovarian vein serum from 3 subjects during the late follicular phase of the menstrual cycle had detectable inhibin activity, whereas ovarian vein serum of 12 other subjects during the early follicular phase and luteal phase had no detectable inhibin activity in a rat anterior pituitary cell culture assay. Subjects having detectable inhibin activity (102 +/- 47 U/100 microliters) had 1257 +/- 582 U/100 microliters inhibin activity in FF, whereas subjects having no detectable inhibin activity had FF levels of 711 +/- 203 U/100 microliters of inhibin activity. Estrogen levels of FF and ovarian vein serum of the group having detectable inhibin activity in ovarian vein serum were 282 +/- 239 ng/ml and 4.8 +/- 1.77 ng/ml, respectively. The estrogen content of FF and ovarian vein blood of the group having nondetectable inhibin activity in ovarian vein blood was 127 +/0 45 ng/ml and 3.03 +/- 0.6 ng/ml, respectively.

The effect of porcine follicular fluid on ovulation, mating and pregnancy in the rat.

We have examined the effects of charcoal-treated, greater than 10,000 MW porcine follicular fluid (PFF retentate) treatment on ovulation, cyclicity, mating and pregnancy in the rat. Rats treated i.p. with PFF retentate twice on the day of metestrus and once on the day of diestrus exhibited a dose-related reduction in the mean number of ova when oviducts were checked on the afternoon of expected estrus. However, oviducts checked on the morning of expected metestrus contained a normal number of ova. To study the effects of PFF retentate on cyclicity, mating and subsequent pregnancy, rats were treated (0.2 ml, i.p.) two, three or four times a day beginning on the day of metestrus until proestrus cytology was exhibited. PFF retentate treatment resulted in a dose-related delay in the appearance of proestrus cytology and a reduction in the number of embryonic implants following cohabitation with males on the night of proestrus. However, PFF retentate had no effect on the number of implants, when given post-coitally. These results show that administration of PFF retentate to female rats delays ovulation, alters the estrous cycle and reduces the mean number of embryonic implants in a dose-related manner.