Long-term effects of early-life exposure to environmental oestrogens on ovarian function: role of epigenetics.

Oestrogens play an important role in development and function of the brain and reproductive tract. Accordingly, it is considered that developmental exposure to environmental oestrogens can disrupt neural and reproductive tract development, potentially resulting in long-term alterations in neurobehaviour and reproductive function. Many chemicals have been shown to have oestrogenic activity, whereas others affect oestrogen production and turnover, resulting in the disruption of oestrogen signalling pathways. However, these mechanisms and the concentrations required to induce these effects cannot account for the myriad adverse effects of environmental toxicants on oestrogen-sensitive target tissues. Hence, alternative mechanisms are assumed to underlie the adverse effects documented in experimental animal models and thus could be important to human health. In this review, the epigenetic regulation of gene expression is explored as a potential target of environmental toxicants including oestrogenic chemicals. We suggest that toxicant-induced changes in epigenetic signatures are important mechanisms underlying the disruption of ovarian follicular development. In addition, we discuss how exposure to environmental oestrogens during early life can alter gene expression through effects on epigenetic control potentially leading to permanent changes in ovarian physiology.

Epigenetic mechanisms in the actions of endocrine-disrupting chemicals: gonadal effects and role in female reproduction.

There is a heightened interest and concern among scientists, clinicians and regulatory agencies as well as the general public, regarding the effects of environmental endocrine-disrupting chemicals (EDCs). In this review, we identify the main epigenetic mechanisms and describe key ovarian processes that are vulnerable to the epigenetic actions of EDCs. We also provide an overview of the human epidemiological evidence documenting the detrimental effects of several common environmental EDCs on female reproduction. We then focus on experimental evidence demonstrating the epigenetic effects of these EDCs in the ovary and female reproductive system, with an emphasis on methoxychlor, an organochlorine pesticide. We conclude the review by describing several critical issues in studying epigenetic effects of EDCs in the ovary, including transgenerational epigenetic effects.

Multi-View Active Appearance Models: application to X-ray LV angiography and cardiac MRI.

This paper describes a Multi-View Active Appearance Model (AAM) for coherent segmentation of multiple cardiac views. Cootes' AAM framework was adapted by considering shapes and intensities from multiple views, while eliminating trivial difference in object pose in different views. This way, the coherence in organ shape and intensities between different views is modeled, and utilized during image search. The method is validated in two substantially different and novel applications: segmentation of combined end-diastolic and end-systolic left ventricular X-ray angiograms, and simultaneous segmentation of a combination of four chamber, two chamber and short-axis cardiac MR views.

Oxytocin stimulates secretion of prostaglandin F(2alpha) from endometrial cells of swine in the presence of progesterone.

Oxytocin (OT) stimulates endometrial secretion of prostaglandin (PG) F(2 alpha) during corpus luteum regression in swine but there is differential responsiveness to OT among endometrial cell types. To determine if progesterone influenced responsiveness of luminal epithelial, glandular epithelial, and stromal cells to 100 nM OT during luteolysis in swine, cells were isolated from endometrium of 15 gilts by differential enzymatic digestion and sieve filtration on day 16 postestrus and cultured continuously in the presence of 0, 10 or 100 nM progesterone. For phospholipase C (PLC) activity and PGF(2 alpha) secretion, stromal cells were most responsive to OT (P<0.01) in the absence of progesterone, whereas luminal epithelial cells were unresponsive and glandular epithelial cells displayed an intermediate response to OT (P<0.09). Progesterone enhanced PLC activity linearly in glandular epithelial cells (P<0.05) and influenced it quadratically in stromal cells (P=0.05). The effect of OT and progesterone on PLC activity in luminal epithelial cells was not significant, and progesterone did not increase PLC activity in response to OT in any cell type. Culture in the presence of progesterone, enhanced PGF(2 alpha) secretion in response to OT in luminal epithelial cells (P<0.05) but not in glandular epithelial or stromal cells. Progesterone also increased overall PGF(2 alpha) release from glandular epithelial (P<0.05) and stromal cells (P<0.06) across both levels of OT treatment. These results indicate that progesterone enhanced PGF(2 alpha) secretion from luminal epithelial cells in response to OT and increased basal PGF(2 alpha) release from glandular epithelial and stromal cells.

Localization of connective tissue growth factor in human uterine tissues.

Connective tissue growth factor (CTGF) is a recently described heparin-binding mitogen for fibroblasts and smooth muscle cells. The aim of this study was to investigate the production of CTGF by human uterine tissues using immunohistochemical and Northern blotting analyses. For immunohistochemistry, formalin-fixed human proliferative (n = 5), early secretory (n = 5; days 15-19), mid-secretory (n = 5; days 20-23), late secretory (n = 5; days 24-28) endometrial, and decidual (n = 5) tissues were stained using a highly specific affinity-purified polyclonal antibody raised against residues 81-94 of human CTGF. Myometrial (n = 5) and leiomyoma (n = 5) tissues were also used for CTGF immunochemistry. In proliferative endometrium, epithelial and vascular endothelial cells showed strong CTGF immunoreactivity, whereas stromal cells were negative or only weakly positive for the CTGF protein. Throughout the entire secretory stage, CTGF was detected in epithelial and endothelial cells of endometrium. Stromal cells showed strong immunoreactivity to CTGF only in oedematous areas for early and mid-secretory endometrium, and in decidualized regions of late secretory endometrium. During pregnancy, the decidual, epithelial and endothelial cells of the endometrium were all immunoreactive to CTGF. In myometrial and leiomyoma samples, CTGF immunoreactivity was found only in the endothelial cells. Northern blotting of mRNA from normal uterus (n = 2) or leiomyoma (n = 6) using a 320 bp human CTGF cDNA probe revealed a single 2.4 kb transcript. This study is the first to demonstrate CTGF gene expression and localization of its encoded protein in human uterine tissues. The cell- and cycle-specific localization of CTGF support a role for this molecule in regulating aspects of uterine cell growth, migration, and/or matrix production during the menstrual cycle and pregnancy.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F2alpha secretion by luminal epithelial, glandular epithelial and stromal cells from pig endometrium. II. Responses of cyclic, pregnant and pseudopregnant pigs on days 12 and 16 post oestrus.

In pigs, the exact mechanism for the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine mode during pregnancy recognition is not known. The objective of this study was to examine whether this shift involved a change in the responsiveness of luminal epithelial, glandular epithelial and stromal cells to 0 or 100 nM oxytocin. Luminal epithelial cells, glandular epithelial cells and stromal cells were isolated from cyclic, pregnant or oestrogen-induced pseudopregnant gilts on Day 12 (Experiment 1) or Day 16 (Experiment 2) post oestrus (oestrus = Day 0). For cells obtained on Day 12, oxytocin stimulated PGF2alpha secretion by stromal cells (P<0.01) similarly for each reproductive status, whereas oxytocin stimulated PGF2alpha secretion from luminal and glandular epithelial cells (P<0.05) from pregnant and pseudopregnant gilts but not from cyclic gilts. For both concentrations of oxytocin, mean PGF2alpha secretion was less (P<0.05) from stromal cells of pregnant than cyclic gilts. For cells obtained on Day 16, oxytocin stimulated PGF2alpha release from stromal cells of cyclic gilts but not from stromal cells of pregnant gilts. Mean PGF2alpha secretion also was less (P<0.05) from stromal cells of pregnant gilts than cyclic gilts. Oxytocin tended to stimulate PGF2alpha release (P<0.07) from glandular epithelial cells of cyclic but not pregnant or pseudopregnant gilts. Luminal epithelial cells from all reproductive statuses were similarly unresponsive to oxytocin. In conclusion, the increased PGF2alpha secretory response to oxytocin of luminal and glandular epithelial cells from pregnant gilts on Day 12, combined with the decreased response of stromal cells from pregnant gilts on Days 12 and 16, may contribute, in part, to the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine direction during early pregnancy in pigs.

Intracellular free calcium in response to oxytocin in pig endometrial cells.

Intracellular free calcium concentration ([Ca2+]i) in response to oxytocin (OT) was studied in stromal, glandular epithelial and luminal epithelial cells obtained from the endometrium of gilts 16 days post-estrus. The amplitude of increased [Ca2+]i in response to 100 nM OT was greatest in stromal cells, intermediate in glandular epithelial cells and not evident in luminal epithelial cells. During continuous OT administration, stromal cells responded initially with a synchronous spike of [Ca2+]i that did not require extracellular Ca2+ and then displayed spontaneous asynchronous [Ca2+]i spikes that required extracellular Ca2+. Each cell possessed its own characteristic response. Increasing concentrations of OT induced an increasing percentage of stromal cells responding, with some cells having nearly equal [Ca2+]i responses at all concentrations and others having graded [Ca2+]i responses as the concentration of OT increased. These results are consistent with the proposed mechanism of OT action in pig endometrium involving activation of phosphoinositide-Ca2+ signaling pathway.

Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs.

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.

Effect of human chorionic gonadotropin on cytokine production from human endometrial cells in vitro.

PROBLEM: To examine whether human chorionic gonadotropin (hCG) is involved in the regulation of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and leukemia inhibitory factor (LIF) secretion from cultured human endometrial cells. METHOD OF STUDY: A mixed population of endometrial cells from six in vitro fertilization/embryo transfer patients was cultured and incubated with various doses of hCG (0, 1, 10, 50, 100, and 500 IU/ml) for 24 hr. IL-6, TNF-alpha, and LIF levels in the culture medium were measured with enzyme-linked immunosorbent assay. RESULTS: IL-6 and TNF-alpha levels were stimulated by hCG in a dose-dependent manner. Stimulation of IL-6 and TNF-alpha levels by 500 IU/ml of hCG increased their production by 3.7- and 2.8-fold, respectively (P < 0.05). Stimulation of IL-6 by 100 IU/ml of hCG was also significant (P < 0.05). However, there was no significant effect of hCG on LIF secretion by endometrial cells (P = 0.31). CONCLUSIONS: hCG is involved in the regulation of endometrial cytokine production from human endometrial cells in vitro. This finding supports the recently emerging notion that hCG could have important local roles within the uterus besides its well-known luteotrophic role on the corpus luteum for maintenance of pregnancy.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F secretion by luminal epithelial, glandular epithelial, and stromal cells from pig endometrium. I. Response of cyclic pigs on day 16 postestrus.

Oxytocin (OT) is the physiological stimulus for pulsatile release of endometrial prostaglandin (PG) F2alpha during luteolysis in domestic ungulates, and the cellular mechanism for this appears to involve phosphoinositide (PI) hydrolysis. To determine which endometrial cell type(s) was responsive to OT during luteolysis in swine, luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) were isolated from endometrium by differential enzymatic digestion and sieve filtration on Day 16 postestrus and cultured. For PI hydrolysis in experiment 1, SC were most responsive to 100 nM OT (p < 0.001), whereas LEC were least responsive and GEC had an intermediate response (p < 0.001). For PGF secretion in experiment 2, the response to OT was greatest for SC, least for LEC, and intermediate for GEC. In experiment 3, 100 nM OT increased PI hydrolysis in SC within 30 min (p < 0.05) and in GEC within 60 min (p < 0.05) but did not increase PI hydrolysis in LEC. In experiment 4, PI hydrolysis in SC was increased (p < 0.05) by 33-333 nM OT but was not increased by

Characterization of 16- to 20-kilodalton (kDa) connective tissue growth factors (CTGFs) and demonstration of proteolytic activity for 38-kDa CTGF in pig uterine luminal flushings.

Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor ss. Although the primary translational product of the pig CTGF gene is predicted to be of approximate Mr 38 000, pig uterine luminal flushings (ULF) contained 10- to 20-kDa CTGF proteins that were heparin-binding and mitogenic, whereas 38-kDa CTGF was not apparent. The N-termini of two microheterogeneous forms of 16-kDa CTGF, as well as 18-kDa and 20-kDa forms of CTGF, commenced at, respectively, Cys199, Ala197, Asp186, and Asp186 and did not correspond to intron-exon boundaries in the CTGF gene. Northern blotting revealed a single porcine (p) CTGF transcript of 2.4 kilobases in endometrium from Day 10 to 16 cycling or pregnant pigs. Ten- to twenty-kilodalton pCTGF proteins in ULF were stable for 48 h at 37 degreesC whereas native 38-kDa pCTGF was degraded within 10 min under the same conditions. CTGF-degrading activity in pig ULF was heat-sensitive and concentration- and time-dependent. Ten- to twenty-kilodalton CTGF levels in ULF peaked on Day 16 of the cycle and on Day 12 of pregnancy and were highly correlated with the levels of proteolytic activity for 38-kDa CTGF. Collectively these data suggest that bioactive 10- to 20-kDa CTGF proteins are generated in utero through limited proteolysis of the 38-kDa CTGF primary translational product.

Presence of leukemia inhibitory factor and interleukin-12 in human follicular fluid during follicular growth.

PROBLEM: Cytokines have been shown to be present in human follicular fluid and have regulatory functions on follicular maturation. The presence of leukemia inhibitory factor (LIF) and interleukin (IL)-12 in human follicular fluid obtained at different stages of maturation was investigated. METHOD OF STUDY: Follicular fluids and granulosa cells were obtained from preovulatory and immature follicles. Follicular fluids from both groups were assayed for IL-12 and LIF by enzyme-linked immunosorbent assay. Granulosa cells from preovulatory and immature follicles were treated with human chorionic gonadotropin (hCG) in vitro and subsequent LIF and IL-12 production were measured. RESULTS: The average concentration of LIF was significantly higher in preovulatory follicles (7.6 +/- 1.3 pg/ml, n = 24) than in immature follicles (2.0 +/- 1.3 pg/ml, n = 6). The concentration of IL-12 was significantly higher in follicular fluid obtained from immature follicles (10.9 +/- 5.0 pg/ml) than in preovulatory follicles (1.3 +/- 0.4 pg/ml). hCG only stimulated LIF production from mature granulosa cells; it had no effect on IL-12 production. CONCLUSIONS: IL-12 and LIF are present in follicular fluid and their levels are regulated differently during follicular maturation. hCG stimulates LIF production from granulosa cells in vitro.

Partial purification and characterization of two non-FSH steroid-modulating factors in rat thymic epithelial cell-conditioned medium (TCM).

Previously, we have reported that unknown factor(s) in rat thymic epithelial cell-conditioned medium (TCM) stimulates basal and follicle-stimulating hormone (FSH)-induced steroid hormone production and aromatase enzyme activity in cultured rat granulosa cells. Here we report the partial purification and characterization of two of these activities. Thymic epithelial cells were prepared from immature female rats and used for TCM production. Lyophilized aliquots of TCM were reconstituted with distilled water at 25% of the original volume, applied to a gel filtration column, and column fractions were tested for their stimulation of steroidogenesis in granulosa cells prepared from immature diethylstilbestrol-treated rats. Two distinct biologically active regions were identified that corresponded to apparent molecular weights of approximately 22,000 and less than 1,000. The < 1 kDa activity ("TCM-1") stimulated (P < 0.01) basal production of progestins [progesterone and 20 alpha-hydroxypregn-4-en-4-one (20 alpha-OH-progesterone)] and estrogen, and also induced dramatic morphological changes on the rat granulosa cells. In contrast, the approximately 22 kDa activity ("TCM-22") stimulated (P < 0.01) only basal progestins, and had no effect (P < 0.05) on basal estrogen production or morphology of the cultured rat granulosa cells. In the presence of 100 ng/ml FSH, TCM-1 stimulated (P < 0.01) estradiol and progesterone production, whereas TCM-22 stimulated (P < 0.01) progesterone, but inhibited (P < 0.01) estradiol production. When both activities were assayed together, they were synergistic in stimulating (P < 0.01) basal progesterone production, but TCM-22 antagonized (P < 0.01) TCM-1-induced estradiol production. The biologic and physico-chemical characteristics of TCM-1 and TCM-22 were distinct from one another, as well as from FSH. When subjected to C8 reverse-phase HPLC. TCM-1 retained its characteristic biologic properties and was eluted (54% acetonitrile) as A214-absorbing moiety with a peak retention time of 92-93 minutes. The elution of TCM-22 was not correlated with an identifiable protein peak. These results suggest that ovarian steroid production may be modified by non-FSH factors produced by thymic epithelial cells although amino acid sequencing of TCM-1 was unsuccessful. This highlights a potential role of the thymus gland in regulating ovarian function.

Endometrial responsiveness to oxytocin during diestrus and early pregnancy in pigs is not controlled solely by changes in oxytocin receptor population density.

These studies were performed to test the hypotheses that: 1) endometrial responsiveness to oxytocin (OT) in pig endometrium is associated with changes in OT receptor (OTr) population density resulting from corresponding regulation of OTr gene transcription, 2) endometrial responsiveness to OT is controlled solely through a mechanism involving changes in OTr population density, and 3) OTr population density and endometrial responsiveness to OT differ between diestrus and early pregnancy in pigs. In experiment 1, OTr population density and dissociation constant (Kd) in cyclic pigs were constant on Days 10-16 but increased (p < 0.05) between Days 10 and 12 of pregnancy before decreasing (p < 0.05) through Day 16. OT induced phosphoinositide (PI) hydrolysis and prostaglandin (PG) F2 alpha secretion in cyclic pigs only on Day 16 (p < 0.05), and during pregnancy only on Day 12 (p < 0.05). Activation of G protein by aluminum fluoride (AIF4-) treatment maximally stimulated (p < 0.05) PI hydrolysis and PGF2 alpha secretion in cyclic pigs on all days, indicating that downstream from the OTr, the PGF2 alpha secretory pathway was fully functional. During pregnancy, PI hydrolysis and PGF2 alpha secretion in response to AIF4- decreased (p < 0.01) on Days 14 compared to Days 10 and 12, and AIF4- did not stimulate PGF2 alpha release on Day 16. In experiment 2, abundance of OTr mRNA in cyclic pigs decreased between Days 0 and 5 before increasing between Days 5 and 12 (p < 0.05), but it was higher (p < 0.05) on Days 10-15 of pregnancy than on equivalent days in cyclic gilts. These results indicate that control of PGF2 alpha secretion in cyclic pigs appeared to occur primarily at the level of OTr coupling to G protein because changes in OTr number were not associated with increased sensitivity to OT or G-protein activation by AIF4-. During pregnancy, control was exerted at multiple levels, which included the OTr, G protein, phospholipase C, and subsequent aspects of the secretory pathway. The present study also indicated that endometrium was responsive to OT during luteolysis in cyclic pigs but not during corpus luteum maintenance in pregnant pigs.

The role of phosphoinositide-derived second messengers in oxytocin-stimulated prostaglandin F2 alpha release from endometrium of pigs.

The mechanism for prostaglandin (PG) F2 alpha release from pig endometrium after oxytocin (OT) treatment is unknown. OT may rapidly stimulate inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) formation, consistent with the concept of rapid activation of a second-messenger system. In support of this hypothesis, endometrial IP3 levels were increased (P < 0.05) within 0.5 min after treatment with 0.1 microM OT. In contrast, increased DAG formation was not detected after treatment with OT. However, similar to the stimulation of endometrial PGF2 alpha secretion observed after OT treatment (P < 0.001), PGF2 alpha release was increased (P < 0.01) after treatment with phorbol-12-myristate-13-acetate (PMA), which mimics DAG activation of protein kinase C. Further, stimulation of endometrial PGF2 alpha secretion did not result from cell death induced by PMA or OT because lactate dehydrogenase, a cytosolic marker of cellular integrity, did not leak into the medium after PMA or OT treatment. In contrast, 0.5% saponin (positive control for cell death and concomitant release of lactate dehydrogenase) increased PGF2 alpha secretion (P < 0.05) and lactate dehydrogenase release (P < 0.001). These results indicate that OT induces endometrial IP3 production in a rapid manner indicative of a second-messenger system. The finding that increased DAG was not also detected after OT treatment may reflect rapid metabolism or compartmentalized production of DAG involved in the second-messenger stimulation of phospholipase C. The high background of DAG used in the biosynthesis of cellular lipids would obscure the rather small spatially localized changes in DAG levels resulting from the activation of phospholipase C. The finding that DAG was present at approximately 10 to 20-fold higher levels than IP3 in resting cells was consistent with this conclusion.

Regulation of cumulus cell steroidogenesis by the porcine oocyte and preliminary characterization of oocyte-produced factor(s).

This study was designed to investigate whether porcine oocytes produce a factor(s) that influences cumulus and mural granulosa cell steroid production and to characterize the biochemical nature and mode of action of a such factor(s). Porcine cumulus-oocyte complexes (COC) were collected from 2-5 mm follicles and cultured either intact or after oocytectomy for 48 h. Steroid levels were then measured in the culture media. Conditioned media, obtained by culturing denuded oocytes for 48 h, were subjected to heat treatment of charcoal extraction and utilized to culture intact and oocytectomized COC. FSH-stimulated progesterone, 20 alpha-OH-progesterone, and estradiol were significantly higher in oocytectomized vs. intact COC cultures. Denuded oocytes cultured with granulosa cells significantly inhibited progesterone production compared to control. Also, media conditioned with different numbers of denuded oocytes (0 to 300) significantly inhibited progesterone production by oocytectomized COC in a manner dependent on oocyte number. Charcoal extraction, but not heat treatment, significantly removed the inhibitory effect of the conditioned media on progesterone production by oocytectomized COC. Increased progesterone production by oocytectomized COC was not accompanied by a similar increase in cAMP formation. Heptanol, a gap junction blocker, did not alter progesterone production by intact COC. In conclusion, porcine oocytes secrete a factor(s) that inhibits cumulus and mural granulosa cell steroidogenesis. This factor(s) is heat stable but extractable by charcoal. The factor(s) appears not to be transferred to somatic cells via gap junctions, and its effect is downstream of cAMP formation.

Characterization of the stimulatory actions of thymic factor(s) on basal and gonadotropin-induced steroidogenesis in cultured rat granulosa cells.

Recently, there have been numerous reports that demonstrate the importance of the thymus gland in reproductive physiology. Previously, we have reported that thymic factors (TFs) which are present in thymic cell culture-conditioned medium (TCM) could stimulate basal progesterone and estradiol production from cultured rat granulosa cells. The current study attempts to characterize the stimulatory actions of TFs on both basal and FSH induced steroidogenesis. Thymic epithelial cells from immature female rats were isolated and used for production of TCM. Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TFs stimulated both basal and FSH-induced progesterone secretions 80 and 17 times, respectively, as compared to the control media. The effects of TFs on basal and FSH-induced 20 alpha-hydroxyprogesterone secretion were comparable to those on progesterone production (40x and 10x, respectively). In addition, TCM stimulated basal and FSH-induced estradiol secretion approximately 4 and 2.5 times, respectively, compared to control. Stimulation of aromatase enzyme activity followed a similar trend as estradiol secretion, and TCM stimulated basal and FSH-stimulated aromatase enzyme activity approximately 15 and 3 times, respectively compared to control. Thus, these results indicate that the observed increases in progesterone and estradiol secretions in TCM-treated rat granulosa cells are likely to be due to elevated activities of specific steroidogenic enzymes. Measurements of total cell protein and DNA synthesis indicate that enhanced steroidogenesis in TCM-treated cells is not due to increased cell growth and/or proliferation. Rather, the enhanced steroidogenesis is probably due to an increased steroid biosynthetic capability of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Gossypol inhibits aromatase activity in cultured porcine granulosa cells.

The present study investigated whether gossypol inhibited aromatase activity in cultured porcine granulosa cells. Aromatase activity was assayed by measuring (3)H-H(2)O released from [1beta-(3)H]-androstenedione. First, immature porcine granulosa cells were cultured with various doses of follicle stimulating hormone (FSH, 1 to 1000 ng/ml) for 1 to 5 d to determine optimal culture conditions for aromatase activity assay. Second, porcine granulosa cells were cultured with or without FSH in the presence or absence of gossypol. Gossypol, at 4 muM, significantly inhibited FSH-induced aromatase activity while showing no effect on basal aromatase activity. Gossypol did not inhibit cell proliferation during cell culture. These results suggest that gossypol inhibits aromatase activity by interfering with FSH induction of aromatase in cultured porcine granulosa cells.

Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells.

Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occurred at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occurred when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-alpha 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function.