Kenneth P. McNatty

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.

Relationship Between Plasma Prolactin and the Endocrine Microenvironment of the Developing Human Antral Follicle

The aim of the present study was to determine whether any obvious relationships exist between the circulating levels of prolactin at the time of ovariectomy and the endocrine microenvironment and developmental status of antral follicles. The concentrations of prolactin, follicle-stimulating hormone (FSH), and estradiol were measured in peripheral plasma and in antral fluid of follicles collected at varying stages of the menstrual cycle. In addition, the granulosa cells were recovered from each follicle (greater than or equal to 4 mm in diameter) and their numbers were quantitated. When the plasma levels of prolactin ranged from 11 to 100 ng/ml, the antral fluid levels of prolactin were uniformly low (less than 20 ng/ml) and the over-all level of intrafollicular activity remained unchanged. However, when the prolactin concentrations in plasma exceeded 100 ng/ml, the levels of prolactin in antral fluid were significantly elevated. Moreover, the high levels of intrafollicular prolactin were associated with a marked reduction in FSH accumulation and low levels of estradiol in antral fluid. Also, these follicles were severely deficient in granulosa cells. This marked reduction in intrafollicular activity was not associated with any significant changes in the mean levels of estradiol and FSH in peripheral plasma. These findings suggest that hyperprolactinemia is associated with a marked reduction in intraovarian activity and that the extent of this reduction may not be always apparent from the levels of circulating estradiol.

Follicular determinants of corpus luteum function in the human ovary.

The corpus luteum is a direct continuation of preovulatory follicle development. Growth and differentiation of a follicle to the preovulatory stage depends on certain endocrine changes taking place within the follicle itself (5). These endocrine changes markedly influence the viability, the rate of proliferation, and the biosynthetic potential of granulosa cells. Therefore, the sequence of endocrine events within the developing antral follicle is likely to be of considerable importance for the formation and secretory activity of the corpus luteum. If granulosa cells in developing antral follicles are exposed to certian hormones in the wrong sequence, the follicle may be prevented from developing into a normal corpus luteum. For example, if granulosa cells in antral follicles are not exposed to FSH, they are unable to generate high levels of oestrogen (Table 1). In the absence of oestrogen, granulosa cells do not increase in number. The same end result may also arise if the granulosa cells are exposed prematurely to elevated levels of LH or to high levels of prolactin: LH blocks granulosa cell mitoses even if the cells are exposed to oestrogen (1, 21); high levels of prolactin in antral fluid are associated with low levels of oestrogen and a reduced rate of granulosa cell proliferation. In women, it is likely that a fully functional corpus luteum is formed from a follicle that was exposed to the most favourable sequence of endocrine changes during its antral stages of development.

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.

Is resumption of meiosis in the human preovulatory oocyte triggered by a meiosis-inducing substance (MIS) in the follicular fluid?**Supported by Nordisk Insulinfond (A. G. B).

Aspirates from 31 ovarian follicles and 2 corpora lutea of 26 women at different stages of the menstrual cycle were investigated for activity of meiosis-inducing substance (MIS) and meiosis-preventing substance (MPS). The aspirated follicles were classified as dominant (i.e., preovulatory), healthy, or atretic according to their size, steroid hormone content, and stage of the menstrual cycle. To test for MIS and MPS activity, gonads of sexually undifferentiated fetal mice were cultured in media containing either 15% follicular fluid aspirate (test gonads) or 15% human blood serum (control gonads). MIS activity is present in follicular fluid if the test testes contain more meiotic germ cells than the control testes. MPS activity is present if the test ovaries have less meiotic germ cells than their controls. MIS activity was present only in healthy follicles of the late follicular phase (12 of 15 follicles). No MIS activity was seen in healthy or atretic follicles from other phases of the menstrual cycle. The MIS activity is apparently unrelated to the composition of steroids in the follicular fluid. MPS activity was not found in any of the follicles. It is proposed that the preovulatory resumption of meiosis may be triggered by a MIS in the follicular fluid.

Effects of Luteinizing Hormone on Steroidogenesis by Thecal Tissue from Human Ovarian Follicles in Vitro

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.