The mechanism of follicle growth. II. The mode of action of the follicle-stimulating hormone (FSH) on follicle growth (author's transl)

The mode of action of the follicle-stimulating hormone (FSH) on ovarian follicle growth was studied in hypophysectomized rats using the histologic, autoradiographic and histochemical techniques. The follicle growth was stimulated by the administration of both FSH and estrogen. The histologic finding of the follicle growth induced by the two hormones was different. Namely, after the administration of FSH, the theca layer was thick, but after the administration of estrogen, it was thin. 3H-thymidine and 3H-leucine were used to investigate cell division in a growing follicle. The uptake of 3H-thymidine and 3H-leucine by the theca layer was enhanced remarkably by FSH. On the other hand, the uptake of 3H-thymidine by the granulosa layer was enhanced by FSH or estrogen, while the grain count of granulosa cells was increased only by the administration of estrogen. Moreover, the administration of FSH resulted in an increase of the enzyme activity of glucose-6-phosphate dehydrogenase (G-6-PD), DELTA5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) and alkaline phosphatase (ALPase) in the theca layer. Furthermore, the administration of FSH caused an increase in the serum estradiol and estriol of rats, whereas the administration of estrogen did not. It seems possible, therefore, that FSH stimulated proliferation of theca cells and produced estrogen. The results suggest that the estrogen produced by the theca cells might stimulate proliferation of granulosa cells; consequently, follicle growth might be induced.     

In vivo regulation of steroidogenesis by ovine gonadotropins in male and female mudpuppies,Necturus maculosus Rafinesque

Using steroid radioimmunoassay the in vivo steroidogenic responses of male and female mudpuppies (Necturus maculosus) to single injections of ovine FSH and LH were investigated. In males, the major effect of LH was to stimulate testosterone and 5α-dihydrotestosterone secretion within 2 hr of injection, with a lesser effect on estrogen (estrone and estradiol-17β) secretion. In contrast, FSH primarily stimulated secretion of estrogens. The effects of both FSH and LH on steroid secretion in males were dose related. Castration of males reduced plasma androgen, and estrogen to very low or nondetectable levels and abolished the steroidogenic response to LH. Interrenalectomy prevented a postcastration rise in the progesterone level. In females, FSH did not stimulate steroid secretion but LH increased plasma androgens. Homologous pituitary extracts also stimulated plasma levels of all gonadal steroids measured.     

LHRH - modulator of progesterone and estradiol secretion by theca and granulosa cells on porcine follicle in mono- and co-culture

Progesterone (P4) and estradiol (E2) secretion by granulosa and theca cells cultured alone or in co-culture under the influence of LHRH was studied. Follicular cells were separated from the follicles of different size: small (1-3 mm), middle (4-6 mm) and large (7-10 mm). The cells were cultured in medium M199 containing 100 ng LH/ml for 30 h. LHRH in a dose of 10[-8] M was added to LH treated cultures after 24 h in culture. Then the cultures were incubated with this hormone during subsequent 6 h. LHRH significantly increased LH-stimulated E2 secretion by granulosa cells (GC) harvested from small and middle follicles, but not from large ones. LHRH had no effect on P4 secretion by granulosa cells alone from small and middle follicles, but it significantly decreased P4 secretion in cultures of granulosa cells harvested from large follicles. In cultures of theca layers (T) alone, decreased secretion of E2 was observed from small follicles only. LHRH had no effect on P4 secretion by theca cell from large follicles cultured alone. However, it markedly increased P4 secretion by T cells from middle follicles and decreased that by theca cells from small follicles. In co-culture of granulosa and theca cells resembling an in vivo follicle, the addition of LHRH to LH stimulated cells harvested from small and middle follicles caused an increase of E2 and decrease of P4 secretion. On the other hand, in co-cultures of cells from large preovulatory follicles, both E2 and P4 secretion was suppressed. It may be concluded that the diverse effects of LHRH (either inhibitory or stimulatory) depend on the degree of follicular maturation.     

Prolactin binding analysis and immunohistochemical localization of prolactin receptor in porcine ovarian cells

In the present study we searched for prolactin receptor (PRL-R) in porcine ovarian theca tissue (Tc) of small, medium and large follicles, as well as in early corpus luteum (ECL). The objectives of this investigation were: 1) comparison of the direct effect of PRL action on progesterone (P4) and estradiol (E2) secretion from Tc and ECL cells in culture with adequate effects caused by luteinizing hormone (LH). 2) detection of the presence and distribution of PRL-R in thecal tissue of porcine follicles and in ECL. Tissues were cultured as monolayers either in control M199 medium with calf serum or in medium either with PRL (100 ng/ml) or with LH (100 ng/ml). After 2 days in vitro cultured media were assayed for steroid concentrations by radioimmunoassays. Content and distribution of PRL-R were evaluated by Scatchard analysis and by an immunohistochemical assay. Separated theca layers as well as fragments of ECL were excised on dry ice, homogenized, and incubated with [125I]-PRL. PRL stimulated P4 secretion from Tc 10-fold versus controls. LH stimulated P4 secretion only 2.5-fold. E2 secretion was stimulated by PRL 2.7-fold and by LH 2.4-fold. LH enhanced P4 secretion from ECL cells by 18% while PRL increased P4 secretion by as much as 73%. Femtomol amounts of PRL-R protein were detected in theca tissues of medium and large follicles and also in ECL, which was in accordance with immunohistochemical results. The results showed for the first time the presence of PRL-R in porcine Tc and ECL.     

Follicle-stimulating hormone-dependent estrogen secretion by rat Sertoli cells in vitro: modulation by calcium.

We have investigated the role of Ca2+ in the control of FSH-induced estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Exogenous Ca2+ (4-8 mmol/l) inhibited FSH-stimulated E2 secretion such that, with 8 mmol/l Ca2+ and FSH (8 IU/l) E2 secretion decreased from 2091 +/- 322 to 1480 +/- 84 pmol/l (p less than 0.002), whilst chelation of Ca2+ in the culture medium with EGTA (3 mmol/l) increased E2 secretion from 360 +/- 45 to 1242 +/- 133 pmol/l) in the absence of FSH. Further, EGTA (3 mmol/l) markedly potentiated FSH (8 IU/l), forskolin (1 mumol/l) and dibutyryl cAMP (1 mmol/l)-stimulated E2 secretion. Addition of the Ca2+ ionophores, ionomycin (2-5 mumol/l) and A23187 (2 mumol/l), inhibited FSH (8 IU/l)-stimulated E2 secretion by greater than 80%. The effect of ionomycin was totally reversible, whereas that of A23187 was irreversible. Ionomycin (5 mumol/l) had no effect on EGTA-induced E2 secretion in the absence of FSH, but reduced EGTA-provoked E2 secretion by 59% in the presence of FSH (8 IU/l). Similarly, forskolin- and dibutyryl cAMP-provoked E2 production was inhibited 46-50% by ionomycin (5 mumol/l). We conclude that FSH-induced E2 secretion from immature rat Sertoli cells is modulated by intra- and extracellular Ca2+.     

Steroid secretion by ovarian cells of the Japanese quail (Coturnix coturnix japonica)

Mixed cell preparations (theca plus granulosa) prepared from the hierarchy of follicles of quails ovaries were incubated under defined conditions with or without the addition of ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), theophylline, cycloheximide, or dibutyryl cyclic adenine monophosphate (db cAMP); or in the presence of androstenedione or testosterone as aromatizable substrate. Steroids secreted into the medium during the 4-hr incubation period were assayed by radioimmunoassay. Cells from the largest follicles (F1) secreted predominantly progesterone, were stimulated by LH and db cAMP, and the response was potentiated by theophylline, but FSH had no stimulatory effect. The F1 cells showed increasing basal and LH-stimulated responses between 18 and 12 hr before the next expected oviposition. Cells from the smaller follicles (F3 and F4) secreted predominantly estrogens, and were stimulated by FSH but not by db cAMP and only to a small extent by theophylline. Addition of androstenedione (10(-7) M) or testosterone (10(-7) M) enhanced estrogen secretion, which was further raised by the simultaneous addition of FSH. These results confirm previous reports on the sites of steroid secretion within quail follicles and suggest that while the action of LH on the cells from F1 follicles may be mediated in part through the adenylate cyclase system, the action of FSH on the smaller follicles may be substantially independent of cAMP.     

The role of gonadotropins and testosterone in progesterone production by human ovarian granulosa cells

Granulosa and theca cells obtained from patients were isolated and cultivated in a chemically defined medium containing gonadotropins and/or testosterone. Progesterone secretion by granulosa cells was consistently stimulated (2-40-fold) in all 5 patients by the addition of follicle-stimulating hormone (FSH, 0.25 micrograms/ml). In the presence of testosterone (0.5 micro M) alone, progesterone production was stimulated (2-8-fold) in 4 out of the 5 patients and cells of one patient showed a greater response to testosterone than to FSH alone. In 2 of the 5 patients, it was also noted that FSH and testosterone acted in a synergistic manner to stimulate the production of progesterone by granulosa cells. On the other hand, human chorionic gonadotropin (hCG, 1.0 IU/ml) alone failed to exert any significant effect. None of the treatments examined altered the production of progesterone by theca cells. These results suggest a role for FSH and testosterone in regulating progesterone biosynthesis by granulosa cells of the human ovary during follicular development.     

Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Steroidogenic activities of follicle-stimulating hormone in the ovary of Japanese eel, Anguilla japonica

To clarify the physiological functions of follicle-stimulating hormone (FSH) during oogenesis in Japanese eel, Anguilla japonica, the steroidogenic activities of recombinant Japanese eel FSH (rjeFSH) were assessed in the eel ovary. Female eel were injected with salmon pituitary homogenate to enhance the ovarian development, and the ovaries at different developmental stages were subjected to steroidogenic bioassay. These ovaries could be classified into three types according to oocyte growth and development of ovarian follicular cells. The type-A ovary possessed poorly developed follicular cells around pre- or early vitellogenic oocytes, and rjeFSH did not induce sex steroid secretion. Testosterone (T) secretion was stimulated by rjeFSH in the type-B ovary with developed theca cells and undeveloped granulosa cells around early to mid-vitellogenic oocytes, whereas estradiol-17beta (E2) secretion was not enhanced. The rjeFSH stimulated both T and E2 secretion in a dose-dependent manner from the type-C ovary with fully developed theca and granulosa cells around mid-vitellogenic oocytes. Salmon GTH fraction (sGTH) and a membrane permeable cAMP analogue, 8-bromo-cAMP (8-Br-cAMP) also enhanced T and E2 secretion from the type-C ovary. Human chorionic gonadotropin (hCG) similarly enhanced T secretion, but failed to stimulate E2 secretion from the type-C ovary, suggesting different effects on steroidogenic activities between eel FSH and hCG in eel ovary. There was a positive correlation between the oocyte diameter and E2 secretion from eel ovaries stimulated by rjeFSH. These results suggest that aromatase activity is accelerated by eel FSH in the granulosa cells, which develop following theca cell development in this species.     

Escape from chronic estrogenic suppression of ovarian function in the adult rhesus monkey: evidence for changing sensitivity of gonadotropin secretion to estrogen inhibition

Regularly menstruating female rhesus monkeys were implanted sc with Silastic tubing packed with estrone. The implants were replaced every 6-8 months to maintain the serum estrone and estradiol concentrations constantly elevated 1.5-2.5-fold, i.e. in the midfollicular phase range, for 4 yr. Increasing the estrone and estradiol concentrations initially led to anovulation which persisted for 6 to 15 months after which three of four of the estrogen-treated animals resumed ovulatory cycles. These animals then waxed and waned between anovulation and ovulatory cycles for the remainder of the study period. The resumption of ovulatory cycles were attributed to an escape of the central nervous system-pituitary axis from the suppressive effect of the elevated estrogen concentrations, since serum estradiol concentrations did not change. During anovulatory and ovulatory months the basal LH concentration was not significantly increased in the estrogen-treated monkeys compared to the control animals, but it was significantly reduced during anovulatory months compared to ovulatory months. Furthermore, LH secretion in response to a bolus of GnRH was attenuated in the monkeys chronically exposed to the acyclic elevation of blood estrogen levels. As the sensitivity to estrogen decreased, sufficient basal concentrations of FSH and LH were achieved to support ovulatory cycles with associated midcycle surges of LH and FSH secretion and apparently normal luteal patterns of progesterone secretion. Since the daily FSH concentrations of these ovulatory cycles of the estrone-treated animals were significantly lower than that of ovulatory cycles of the control monkeys, the ovulatory cycles of the estrogen animals may not have been normal, but this was not directly documented. These studies suggest that elevated basal estrogen levels do not lead to elevated basal LH secretion in the female primate; and also in the intact adult primate the central nervous system-pituitary axis has the potential to change its sensitivity to the negative feedback effects of estrogen on gonadotropin secretion.     

Stimulation of aromatase activity in the fetal rat gonads by cAMP and FSH

The gonads from 17- to 21-day-old fetal rats were cultured in vitro in the presence of [3H]testosterone and in the presence or absence of cAMP or FSH, and estrone and estradiol formed were measured by double isotopic dilution and recrystallization to constant specific activity. Estrogen synthesis by testes was stimulated by both cAMP and FSH as early as at 18 days of gestation. FSH did not enhance aromatase activity in ovaries, although cAMP did. It is remarkable that FSH controls estrogen synthesis in the testis earlier than in the ovary.     

Reduced serum inhibin concentrations during ovulatory cycles of estrogen-treated rhesus monkeys: an indicator of FSH bioactivity

Female rhesus monkeys treated with exogenous estrone initially were anovulatory. Although estrone and estradiol concentrations were maintained 1.5- to 2.5-fold elevated, i.e. in the midfollicular range, ovulatory cycles resumed in three of four animals after 6-15 months of anovulation. During the ovulatory cycles the serum bio LH concentrations were the same in estrone-treated animals as during ovulatory cycles of control monkeys, but the daily basal serum FSH concentrations detectable by RIA were significantly reduced during the ovulatory cycles of the estrone-treated animals compared to the cycles of the controls. In the present study serum inhibin concentrations were measured to determine whether or not they were increased and the cause of the selective decrease in FSH concentrations in the estrogen-treated monkeys. Serum LH, FSH, progesterone, and inhibin concentrations were measured by RIA in blood samples collected during the third year of continuous estrogen treatment. The lack of an effect of elevated estrogen on LH concentrations and a significant estrogen-induced decrease in serum FSH concentrations during ovulatory cycles was confirmed (FSH, control: 5.6 +/- 0.68 ng/ml; estrogen-treated: 2.5 +/- 0.09 ng/ml; P = 0.01). There was also a significant decrease in the serum inhibin concentrations detectable by RIA during the follicular phase of the estrone-treated monkeys compared to the follicular phase of the control animals (119 +/- 17 vs. 462 +/- 105 microliter eq/ml; P = 0.04). These results indicate that the lower serum concentrations of FSH in the estrogen-treated monkeys were not a result of an increase in ovarian secretion of inhibin. The lower inhibin concentrations suggest that FSH bioactivity, as well as immunoreactive FSH, is significantly reduced during the ovulatory cycles of the estrone-treated monkeys. Even though the estrogen treatment decreased the FSH bioactivity, sufficient FSH secretion occurs in the presence of the elevated estrone and estradiol concentrations to induce and support apparently normal ovulatory cycles.     

Insulin-like growth factor-I (IGF-I) system during follicle development in the bovine ovary: relationship among IGF-I, type 1 IGF receptor (IGFR-1) and pregnancy-associated plasma protein-A (PAPP-A)

Insulin-like growth factor-I (IGF-I) system that is exerted mainly through the type 1 IGF receptor (IGFR-1) and releasing of free IGF-I is regulated by the proteases of IGF-binding proteins (IGFBPs), an important factor in follicle development of bovine ovary. The aims of the present study were to examine the mRNA expressions of IGF-I, IGFR-1 and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells and theca tissues during bovine follicular development and the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of these genes in cultured bovine granulosa cells. Follicles were classified into four groups such as small follicle (SF), estrogen inactive dominant follicle (EID), estrogen active dominant follicle (EAD) and preovulatory follicle (POF). The concentration of free IGF-I in follicular fluid of POF was significantly higher than those in EID, whereas the total IGF-I in follicular fluid did not change at all developmental stages. The expression of IGF-I mRNA was not detected in the granulosa cells at all at any developmental stages but the expression was detected in the theca tissues. The amount of IGFR-1 mRNA in granulosa cell showed the constant level at all developmental stages except EID. The expressions of IGFR-1 and PAPP-A in cultured bovine granulosa cells were stimulated with FSH but not with E2. The PAPP-A mRNA expression was stimulated by FSH in presence of 1 ng/ml E2. These results indicate that IGF-I in follicular fluid is mainly derived from the circulation and that FSH is an inducer for the expression of IGFR-1 and PAPP-A genes in granulosa cells. Therefore, we suggest that PAPP-A stimulated with FSH play a crucial role for IGF-I system in bovine follicular development.     

Secretion of Active and Inactive Renin by Rabbit Kidney Cortex Slices: Effects of Verapamil,Flunarizine and A23187

Secretion control for active and inactive (acid-activatable) renin was investigated using a rabbit kidney cortex slice preparation. In complete Krebs-Ringer bicarbonate buffer (pH 7.4) the calcium-channel-blocking drugs verapamil (50 μM) and flunarizine (50 μM) had no effect on active renin release but increased inactive renin secretion. In Ca-free or Ca-depleted (+EGTA) buffers secretion of both active and inactive renin was increased. Neither verapamil nor flunarizine under these conditions had any additional effect on active renin secretion but inactive renin release was stimulated by verapamil and inhibited by flunarizine. Although having no effect in the presence of Ca⁺⁺ ions, the ionophore A23187 (17 μM) selectively abolished the secretion of inactive renin in the absence of calcium. The mechanisms regulating the secretion of the two forms of renin are clearly not identical. Differential release of active and inactive renin by the kidney may contribute to the overall control of the renin-angiotensin system     

Expression and regulation of kit ligand in the ovary of the hen

The Kit system, composed of Kit ligand (KL) and its tyrosine kinase receptor, cKit, has been well characterized in mammals. Studies have shown that it is involved in signaling between the oocyte and somatic cells during the process of follicle maturation. We characterized KL mRNA expression during follicle maturation in the domestic hen, examined regulation of KL and a possible function of the Kit system. KL mRNA expression was assessed using quantitative PCR (n=4 replicates) in follicles of various sizes (1, 3, 5, 6-12mm, F1). Expression of KL mRNA decreased significantly (p<0.01) with follicle development and was highest in <1mm follicles, which contained the theca as well as granulosa layers, with high levels also found in the granulosa layer of 3mm follicles and ovarian stroma. To study regulation of KL mRNA, granulosa cells from 6-8mm follicles (n=4 replicates) were plated in M199 plus 0.1% BSA in the presence of various treatments including: oocyte conditioned medium (OCM), Vitamin D(3), FSH, estradiol, progesterone and testosterone. OCM caused a dose-related increase (p<0.05) in expression of KL mRNA; Vitamin D(3) increased and FSH decreased expression of KL mRNA. cKit was detected (at the expected size) in the theca layer of 3-5mm follicles and in a lysate of whole <1mm follicles. Culture of granulosa cells in the presence of OCM resulted in a decrease of P4 secretion, an effect blocked by pre-incubation of OCM with cKit antibody. Although OCM caused a dose-related increase in E2 secretion from theca, this was not blocked by cKit antibody.     

A novel mechanism for the induction of aromatase in ovarian cells in vitro: role of transforming growth factor alpha-induced protein tyrosine kinase

In the developing follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the primary stimulators of steroidogenesis in granulosa and theca cells. The steroidogenic actions of both these gonadotropins are largely if not exclusively mediated through cAMP. Previous studies have shown that EGF and TGF alpha do not affect basal estrogen production but attenuate FSH-stimulated estrogen production in vitro in rat granulosa cells. Here we present evidence that TGF alpha stimulates basal estrogen production in prepubertal porcine ovarian granulosa and theca cells in culture under defined conditions. In granulosa cells, TGF alpha is more potent than FSH in stimulating estrogen production. LH does not stimulate estrogen production in prepubertal porcine theca cells but rather attenuates that stimulated by TGF alpha. Treatment of follicular cells with genistein (inhibitor of protein tyrosine kinase) attenuates TGF alpha-induced estrogen biosynthesis suggesting that the action of TGF alpha is mediated through protein tyrosine kinase. These studies provide evidence for an alternative cAMP-independent and TGF alpha-induced tyrosine kinase-dependent mechanism for the induction of ovarian aromatase.     

Estrone/17β‐estradiol conversion to,and tumor necrosis factor inhibition by,estrogen metabolites in synovial cells of patients with rheumatoid arthritis and patients with osteoarthritis

Objective

The role of estrogens in rheumatoid arthritis (RA) is debated since both proinflammatory and antiinflammatory effects have been reported. Important evidence of the dual role of estrogens is conversion to various proinflammatory or antiinflammatory metabolites. This study was undertaken to examine the downstream conversion of estrogens in synovial cells from patients with RA or osteoarthritis (OA).

Methods

We studied serum levels of estrone, estrone sulfate, and estrone sulfate membrane transporters, intracellular interconversion of estrone and 17β‐estradiol, and conversion of estrone/17β‐estradiol to various estrogen metabolites in RA and OA synovial cells. The effect of estrogen metabolites on tumor necrosis factor (TNF) secretion was also studied in RA and OA synovial cells.

Results

Serum levels of estrone sulfate were similar in healthy controls and RA patients. Estrone sulfate transporters were present in synovial tissue. Interconversion of estrone and 17β‐estradiol and the expression of converting enzymes of the cytochrome P450 family were similar in RA and OA cells. Using estrone and 17β‐estradiol as substrates, RA and OA synovial cells produced 16α‐, 4‐, and 2‐hydroxylated estrogens and their 4‐ and 2‐methylation products. The levels of 16α‐hydroxylated estrone/17β‐estradiol (16αOH‐estrone/16αOH‐17β‐estradiol) were higher than the levels of all other estrogen metabolites. RA synovial cells produced more 16αOH‐estrone than did OA synovial cells. Importantly, the 16αOH estrogens did not inhibit TNF secretion, whereas all other estrogen metabolites had marked inhibitory effects.

Conclusion

Our findings indicate that precursor estrogens are converted to proinflammatory metabolites, particularly in RA synovial cells. RA synovial cells mainly produce the proproliferative 16αOH‐estrone, which, in addition to 16αOH‐17β‐estradiol, is one of the only 2 estrogens studied that does not inhibit TNF secretion. A preponderance of 16α‐hydroxylated estrogens is an unfavorable sign in synovial inflammation.

     

Sex hormones in amenorrheic women with alcoholic liver disease

Urinary excretion of estrogens and plasma concentrations of estrone, estradiol, LH, FSH, PRL, progesterone, testosterone, and sex hormone binding globulin were measured in nine chronic alcoholic women with cirrhosis or alcoholic fatty liver. They were aged 24-40 yr and all had secondary amenorrhea which had lasted for at least 3 months. The response of pituitary gonadotropin secretion to administration of LHRH and estradiol benzoate and of PRL secretion to TRH were also investigated. Urinary excretion of estrogens in the alcoholic women with liver disease was similar to that in normal postmenopausal women and less than half that in normal women of the same age in the midfollicular phase of the menstrual cycle. Plasma estradiol levels in the alcoholic women were lower than in the menstruating women but higher than in the postmenopausal women, whereas their plasma estrone levels were higher than in the menstruating women. Plasma concentrations of progesterone and testosterone in the alcoholic women did not differ from those in the postmenopausal women but were lower than in the menstruating women. In spite of the relative estrogen deficiency plasma LH and FSH levels were not elevated in the alcoholic women. The responses of LH and FSH to LHRH were similar in the patients and in the menstruating women. Intramuscular administration of estradiol benzoate did not increase plasma LH and FSH concentrations in the alcoholic women. Hyperprolactinemia was not found and there were no differences in the PRL responses to TRH between the patients and the control groups. In conclusion, disturbed regulation of gonadotropin secretion is an important factor in the genesis of estrogen deficiency and amenorrhea in alcoholic women with liver disease, although ovarian function may also be directly impaired.     

Divergent effects of prolactin on estrogen and progesterone production by granulosa cells of rat Graafian follicles

The effect of PRL on ovarian steroidogenesis was studied in cultured granulosa cells isolated from follicles of mature cycling rats on the morning of proestrus. Ovine PRL 10-1000 ng/ml) inhibited estradiol production but stimulated progesterone biosynthesis in a dose-dependent manner. The effect of PRL was most prominent after 4 days of culture: 1000 ng/ml PRL suppressed estradiol production by 80% but increased progesterone synthesis by 290%, whereas the lower dose of 10 ng/ml inhibited estrogen secretion by 20% without altering progesterone synthesis. The divergent effect of PRL was not shown to be species specific, since ovine, rat and human PRL had similar effects. Using increasing concentrations of androstenedione (the aromatase substrate), estrogen secretion remained suppressed and progesterone production was stimulated by PRL. FSH stimulated both estrogen and progesterone production. The FSH-induced increased in estrogen production was inhibited by concomitant treatment with PRL. In contrast, PRL and FSH had an additive action in stimulating progesterone production. Although LH alone had no effect on steroidogenesis, concomitant treatment with LH and PRL resulted in a stimulation of progesterone production that was additive. This study demonstrates that PRL acts directly on granulosa cells of Graafian follicles of adult cycling rats to stimulate the secretion of progesterone and to suppress estradiol production.     

LH stimulation of estrogen secretion by cultured rat granulosa cells

The direct effect of LH on estrogen secretion by rat granulosa cells was investigated. Ovarian granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were primed with FSH for 2 days in vitro to induce LH receptors. After the FSH priming, the granulosa cells were washed, and recultured for 4 additional days in media containing aromatase substrate (10(-7) M androstenedione) and purified FSH or LH. After the incubations, estrogen (E), progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in the media were measured by RIA. When granulosa cells from hypophysectomized DES-treated rats were cultured for 6 days with FSH and androstenedione, the production of E, P and 20 alpha-OH-P was stimulated to a maximum of 100-, 200- and 270-fold, respectively, above that of control levels. In contrast, LH did not increase steroidogenesis in these cells. Following 2 days of FSH priming in vitro, however, the cultured granulosa cells exhibited marked increases (400-600%) in E, P and 20 alpha-OH-P production in response to LH treatment over a 4-day incubation period. This stimulatory effect of LH on estrogen and progestin production was dose-related; the minimum and maximum effective doses of LH for steroid production were 3 and 30 ng/ml, respectively, and the ED50 was calculated to be 6 ng/ml of LH. As with LH, FSH also stimulated steroidogenesis in a dose-related manner and the apparent ED50 of FSH on steroidogenesis was 45 ng/ml. To investigate whether LH can also stimulate aromatase activity in granulosa cells primed with FSH in vivo, immature hypophysectomized DES-treated rats were injected for 2 days with FSH after which the granulosa cells were isolated and cultured for 4 days in medium containing 10(-7) M androstenedione and LH or FSH. Both LH and FSH stimulated E, P and 20 alpha-OH-P production, and the maximum steroidogenic responses of LH and FSH were similar to those observed in cultured granulosa cells primed with FSH in vitro. THese results have demonstrated that LH is effective in stimulating both estrogen and progestin secretion in rat granulosa cells pretreated with FSH. This suggests an important role of LH in the direct control of both aromatization and luteinization in the granulosa cell.