Anastasia Makris

Insulin Stimulates Androgen Accumulation in Incubations of Human Ovarian Stroma and Theca

&NA; The effects of insulin on ovarian steroidogenesis were examined in four‐day incubations of minced stroma and theca obtained from a woman with hyperandrogenism, insulin resistance, and acanthosis nigricans, and from a normally cycling woman. In incubations of theca obtained from the patient with hyperandrogenism, insulin resistance, and acanthosis nigricans, lutenizing hormone (LH) (25 ng/ mL) alone stimulated androstenedione, testosterone, progesterone, and estradiol accumulation. Insulin (500 ng/mL) alone stimulated androstenedione and testosterone accumulation, but not progesterone or estradiol accumulation. In incubations of stroma obtained from the hyperandrogenism, insulin resistance, and acanthosis nigricans patient, LH (25 ng/mL) alone stimulated androstenedione and testosterone accumulation, but not dihydrotestosterone accumulation. Insulin (500 ng/mL) alone stimulated androstenedione, testosterone, and dihydrotestosterone accumulation. In incubations of stroma from the normally cycling woman, LH plus insulin acted synergistically to stimulate androstenedione accumulation. These results suggest that insulin may be a regulator of steroid biosynthesis in the thecal and stromal compartments of the human ovary. (Obstet Gynecol 64:73S, 1984)

Effects of insulin on steroidogenesis in cultured porcine ovarian theca

The effects of insulin on porcine thecal steroidogenesis were examined in long-term cultures of hyaluronidase-collagenase dispersed thecal cells. The thecal cultures made significant amounts of progesterone (P) and androstenedione (delta 4 A). Testosterone, dihydrotestosterone, estrone, and estradiol could not be detected in the media. Luteinizing hormone (LH) alone significantly increased P and delta 4 A accumulation. Insulin alone increased P accumulation on days 2 to 4 of culture. Insulin alone did not stimulate delta 4 A accumulation. Insulin plus LH resulted in a significantly greater accumulation of P and delta 4 A than LH alone. These results suggest that insulin may be a regulator of ovarian thecal steroidogenesis.

Danazol Inhibits Steroidogenesis

Danazol was found to inhibit multiple enzymes of steroidogenesis directly in the pregnant mare serum (PMS)-treated hamster ovary and the rat testis and adrenal in vitro. In the PMS-treated hamster ovary, danazol inhibited 17alpha-hydroxylase, 17,20-lyase, and 3beta-hydroxysteroid dehydrogenase. In the rat testis, danazol inhibited 17alpha-hydroxylase, 17,20-lyase, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase. In the rat adrenal, danazol inhibited 3beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11beta-hydroxylase. Two hours after a subcutaneous injection of 5 mg/kg of danazol to adult male rats, serum luteinizing hormone levels were significantly increased and serum testosterone levels were significantly suppressed. These findings suggest that in the rodent one of danazols major pharmacologic effects is the direct inhibition of steroidogenesis.

Metabolism of androstenedione by human ovarian tissues in vitro with particular reference to reductase and aromatase activity

The ability of granulosa and theca cells of the human ovarian follicle at different stages of development, as well as stromal and luteal tissues from human ovaries to metabolize androstenedione (delta 4) to testosterone (T), dihydrotestosterone (DHT), estrone (E1) and estradiol (E2) with or without exposure to additional amounts of folicle-stimulating hormone was investigated by in vitro experiments. The results show that all the aforementioned ovarian tissues metabolized delta 4 to DHT. Indeed, with the exception of estrogen-secreting granulosa cells from large antral follicle (greater than 10 mm diameter) and possibly also luteal tissue from mid-luteal phase ovaries, the various ovarian tissues preferentially metabolized delta 4 to DHT instead of E (E1 + E2). Although thecal tissue is a major source of delta 4 in human ovaries it is concluded that the granulosa cells do not interact with the theca for the synthesis of E as the follicle enlarges from 1 to 10 mm in diameter. Indeed, excessive thecal delta 4 during this growth phase probably inhibits normal follicular development. However, as the follicle enlarges beyond 10 mm in diameter, and as the granulosa cells begin to preferentially metabolize delta 4 to E, the two cell-types of the follicle may increasingly interact to enhance the follicular output of E.

Aromatase activity of isolated and recombined hamster granulosa cells and theca.

The major synthesis of estrogen by the follicle is postulated to require both theca and granulosa cells. Theca in this scheme provide androgens to the major aromatizing site in the follicle, and the granulosa cell. One aspect of this theory was tested here. We investigated the comparative ability of isolated granulosa and theca, alone and in recombination to aromatize androgen in vitro. We found that the granulosa aromatize [14C]substrate more efficiently than do theca, and compare with the recombined system in their ability to aromatize [14C]androgen. The data therefore substantiates one aspect of the theory regarding the nature of the synergism, i.e., that the granulosa cells, at least in vitro, are the major site of aromatization of the preovulatory follicle.

Effects of luteinizing hormone on steroidogenesis by thecal tissue from human ovarian follicles in vitro

Abstract The steroidogenic responsiveness of human thecal tissue to different doses of LH was investigated in vitro in relation to the health of the follicle and to the responsiveness of stromal tissue. The results show that small incremental increases in LH, over a low range of concentrations (1 to 10 ng/ml), markedly increased the thecal output of androstenedione from healthy and/or atretic follicles. Theca from healthy follicles were also stimulated to increase their output of progesterone and estradiol in response to small increases in LH whereas theca from atretic follicles produced more variable amounts of progesterone and were unable to generate estradiol. In contrast, relatively high concentrations of LH (50 ng/ml) reduced the total steroid output from the theca of both healthy and atretic follicles while ‘switching on’ a low level of steroidogenesis in stromal tissue. These data suggest that the steroidogenic response of thecal tissue is related to the mass of tissue (i.e., the size of the follicle), the health of the follicle and the amount of LH to which it is exposed.

The production of progesterone, androgens and oestrogens by human granulosa cells in vitro and in vivo.

Abstract The production of progesterone, androgens and oestrogens by human granulosa cells from healthy and atretic follicles was assessed both in vitro and in vivo. Follicles were determined as healthy or atretic from a knowledge of the follicle diameter, the granulosa-cell number, the status of the oocyte and the concentrations of follicle-stimulating hormone, androstenedione and oestradiol in antral fluid. Irrespective of whether the cells were from healthy or atretic follicles, the membrana granulosa were found by direct measurement to be capable of producing progesterone, androstenedione, testosterone, dihydrotestosterone, oestrone and oestradiol in vitro. By indirect measurement these cells were also found to have a similar capacity to produce these steroids in vivo. The relative amounts of the individual steroids produced in vitro varied according to whether the follicle, from which the cells were harvested, was healthy or atretic. Granulosa cells from healthy follicles produced large amounts of oestradiol (> 1 pg/cell/48 h), smaller amounts of androstenedione (⩽ 0.05 pg/cell/48 h) and variable amounts of progesterone (0.7 ± 0.3 pg/cell/48 h). In contrast, as the follicle degenerated, the granulosa cells lost their capacity to produce oestradiol, oestrone and progesterone but maintained their capacity to synthesize the androgens. It is concluded that Steroidogenesis by the human follicle is not rigidly compartmentalized between the theca and granulosa cell-types. Also, it is concluded that the theca cells alone do not determine the level of steroidogenic activity in either developing or atretic follicles. Furthermore, it is suggested that the granulosa cells have a major influence in determining the level of steroid in antral fluid.

Steroidogenesis by the human oocyte-cumulus cell complex in vitro

The ability of the human oocyte-cumulus cell complex to synthesize progesterone, androgens and estrogens and to modify its endocrine environment in vitro was investigated. Germinal-vesicle stage oocytes with adhering layers of cumulus cells were recovered from human ovaries and maintained for 40--50 h in vitro in a culture medium with or without antral fluid. The results show that oocyte-cumulus (O-C) cell complexes were capable of synthesizing progesterone, androgens and estrogens. Oocytes with the capacity of resuming meiosis in vitro were part of an O-C complex producing significantly more progesterone than those O-C complexes containing oocytes incapable of resuming meiosis. Irrespective of the stage of oocyte maturation at the end of culture, testosterone and estrone were respectively the major androgen and estrogen produced. It is concluded that the oocyte-cumulus compartment of the antral follicle is a steroidogenically competent unit and that it has the capacity to modify the endocrine microenvironment of the follicle.

Significance of Angiogenic and Growth Factors in Ovarian Follicular Development

There are many events taking place during follicular development which cannot be explained by the presently known hormones and their inter-relationships as now understood (di Zerga and Hodgen, 1981). With the two-cell model of ovarian steroid secretion, luteinizing hormone (LH) stimulates androgen production by the theca and follicle stimulating hormone (FSH) induces increased aromatase conversion of that androgen to estrogen by the granulosa cells. The FSH and estrogen locally induce LH receptors in the granulosa cell in anticipation of ovulation and formation of the corpus luteum (Hsueh et al., 1984). After much searching for the elusive follicular inhibin it has now been shown that FSH also stimulates granulosa production of inhibin (McLachlan et al., 1986) which along with estrogen provide the feedback from the follicle to the hypothalamic- pituitary unit (Tsonis and Sharpe, 1986). In spontaneous ovulators, estrogen feedback can trigger the LH surge which ultimately results in ovulation. The follicle destined to ovulate can be characterized by a microenvironent of characteristic follicular fluid steroid and gonadotropin concentrations and by an optimal granulosa cell number which distinguish the ripe follicles and their oocytes from those destined for atresia (McNatty et al., 1979).

The source of follicular androgens in the hamster follicle

The comparative ability of granulosa cells and theca of the hamster preovulatory follicle to produce androgens in vitro from endogenous and exogenous substrates was assessed. The results indicate that theca are the major source of follicular androstenedione, but that the granulosa cells may be the major source of follicular testosterone. Theca and granulosa cells accumulate comparable amounts of dihydrotestosterone from exogenous androstenedione and testosterone and both may be a significant source of follicular DHT. LH stimulates the conversion of progesterone and 17 alpha-OH progesterone to androstenedione, testosterone and DHT in theca. LH does not stimulate the conversion of androstenedione to testosterone or DHT, and that of testosterone to DHT in either granulosa cells or theca. FSH, in granulosa cells but not in theca, stimulates the conversion of adrostenedione to testosterone but it has no effect in DHT accumulation from exogenous testosterone.