Charles L. Chaffin

Modulation of ovarian follicle maturation and effects on apoptotic cell death in holtzman rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) in utero and lactationally

Recent reports have described the reproduction-modulating and endocrine-disrupting effects following exposure to toxic substances such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Herein, we set out (1) to determine whether TCDD exposure exerts detrimental effects on follicle maturation in the Holtzman rat ovary and (2) to determine whether the effects of TCDD are mediated in part via apoptotic cell death. In certain species, dioxin exposure is correlated with reduced fecundity, reduced ovulatory rate, an increased incidence of endometriosis, and various reproductive cancers. Although some of the effects of TCDD are mediated via the hypothalamic-pituitary axis, direct effects on the ovary have also been observed. In the present study, an oral dose of 1 microgram TCDD/kg maternal body weight was administered on Day 15 of gestation. Female pups were sacrificed on Postnatal Day 21/22, and the ovaries were excised, fixed for histologic analysis, and analyzed in a double-blind paradigm. The analysis included a count and measurement and classification of preantral and antral follicles throughout the entire ovary. The contralateral ovary from each animal was analyzed for DNA fragmentation indicative of apoptotic cell death. The results indicate that TCDD treatment significantly reduced the number of antral follicles in the size classes 50,000 to 74,999 microns2 and > 100,000 microns2. We also observed a reduction in the number of preantral follicles less than 50,000 microns2. No difference was observed in the degree of apoptotic cell death in antral (50,000 to > 100,000 microns2) and preantral follicles (50,000 microns2 to > 75,000 microns2) between TCDD-treated and control-treated tissues. These data support the hypothesis that TCDD results in a diminution in the number of antral and preantral follicles of certain size classes in animals exposed during critical periods of development, but that apoptosis does not appear to be the underlying mechanism in these particular follicles. This does not preclude apoptosis occurring in pools of smaller precursor follicles.

Expression of Estrogen Receptor α and β in the Rhesus Monkey Corpus Luteum during the Menstrual Cycle: Regulation by Luteinizing Hormone and Progesterone1

There are conflicting reports on the presence or absence of estrogen receptor (ER) in the primate corpus luteum, and the discovery of a second type of estrogen receptor, ERβ, adds an additional level of complexity. To reevaluate ER expression in the primate luteal tissue, we used semiquantitative RT-PCR based assays and Western blotting to assess ERα and β messenger RNA (mRNA) and protein levels in corpora lutea (n = 3/stage) obtained from adult female rhesus monkeys at early (days 3–5), mid (days 6–8), mid-late (days 10–12), and late (days 14–16) luteal phase of the natural menstrual cycle. ERα mRNA levels did not vary across the stages of the luteal phase, and ERα protein was not consistently detected in luteal tissues. However, ERβ mRNA and protein levels were detectable in early and mid luteal phases and increased (P < 0.05) to peak levels at mid-late luteal phase before declining by late luteal phase. To determine if ERβ mRNA expression in the corpus luteum is regulated by LH, monkeys received the Gn...

Gonadotropin and steroid regulation of steroid receptor and aryl hydrocarbon receptor messenger ribonucleic acid in macaque granulosa cells during the periovulatory interval.

Although steroids play a local role(s) in ovulation and luteinization of the primate follicle, the dynamics of steroid receptor expression during the 36- to 38-h periovulatory interval has yet to be elucidated. The present study examines the regulation of messenger RNAs (mRNAs) for progesterone (PR), androgen (AR), and estrogen (ERα, ERβ) receptors as well as the aryl hydrocarbon receptor (AhR) in macaque granulosa cells during controlled ovarian stimulation cycles before (0 h) and after (up to 36 h) administration of the ovulatory hCG bolus with or without steroid depletion and progestin replacement. All steroid receptor mRNAs were detected in granulosa cells before the ovulatory stimulus, as determined by RT-PCR. PR mRNA increased (P < 0.05) by 12 h after hCG; 24 and 36 h after hCG, levels were intermediate between 0–12 h. PR mRNA was reduced by steroid depletion throughout the periovulatory interval (P < 0.05); however, progestin replacement returned PR mRNA to control levels at 12 h. AR mRNA increased...

Progesterone Promotes Oocyte Maturation, but Not Ovulation, in Nonhuman Primate Follicles Without a Gonadotropin Surge

Abstract During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% ± 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% ± 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% ± 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 ± 9) and DHT- (36 ± 15) but not vehicle- (4 ± 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.

Gonadotropin and Steroid Control of Granulosa Cell Proliferation During the Periovulatory Interval in Rhesus Monkeys

Abstract Progesterone produced in response to the midcycle gonadotropin surge is essential for ovulation and luteinization of the primate follicle. Because cell-cycle arrest is associated with the initiation of luteinization, this study was designed to determine the dynamics and regulation of granulosa cell proliferation by gonadotropin and progesterone during the periovulatory interval in the primate follicle. Granulosa cells or ovaries were obtained from macaques undergoing controlled ovarian stimulation either before (0 h) or as long as 36 h following the administration of an ovulatory hCG bolus with or without a 3β-hydroxysteroid dehydrogenase inhibitor with or without a nonmetabolizable progestin. The percentage of cells staining positive for Ki-67, a nuclear marker for cell proliferation, decreased (P < 0.05) within 12 h of hCG administration in a steroid-independent manner. Levels of cyclin D2 and E mRNA did not decline during the periovulatory interval; however, cyclin B1 mRNA was reduced significantly by 12 h. Steroid depletion increased (P < 0.05) cyclin B1 mRNA at both 12 and 36 h post-hCG and was reversible by progestin replacement at 36 h. The cyclin-dependent kinase inhibitor p21Cip1 was transiently increased 12 h post-hCG, whereas p27Kip1 mRNA levels increased at 36 h in a steroid-independent fashion. These data suggest that a gonadotropin bolus inhibits mitosis in granulosa cells early (12 h) in the periovulatory interval, whereas progesterone may play a later, antiproliferative role in luteinized cells of primates.

Local role of progesterone in the ovary during the periovulatory interval

An intraovarian role for steroids in periovulatory events was postulated as far back as the early 1970s, when Rondell hypothesized that steroids facilitate the actions of gonadotropins on follicle rupture [1]. Subsequent research indicated that progesterone (P) may be one of the mediators of periovulatory events [2,3] and Rothchild [4] provided an elegant treatise formulating the theory that progesterone was a local luteotropin promoting the development of the corpus luteum. However, it was not until after 1988, when investigators discovered that P receptors (PR) were expressed in the luteinizing granulosa cells of the ovulatory follicle in several species (monkey [5]; human [6]; rabbit [7]; rat [8,9]; chicken [10]), that the possibility of progesterone acting in a universal manner to regulate ovarian events and ultimately cyclicity, received attention. Since then, investigators have performed a variety of in vivo and in vitro studies in a number of species to examine the role of progesterone in periovulatory events. These studies generally attempted to characterize progesterone’s role by preventing progesterone action via one of three approaches: (1) administration of a PR antagonist, (2) inhibiting progesterone synthesis, or (3) studying the PR null (PR-/-) mutant mouse. While each approach has its advantages and disadvantages, collectively they provide unequivocal evidence that progesterone is an essential mediator of ovulation in all mammalian species examined to date [2,3,11–13]. Futhermore, there are suggestions, albeit less evidence, that progesterone also performs important roles in the development and/or function of the corpus luteum, particularly in species such as the monkey and cow that have a long functional luteal phase during the ovarian cycle. With recent breakthroughs in analyses of gene expression, investigators are beginning to identify progesterone-regulated genes in the ovary. The aim of this review is to highlight recent findings that demonstrate progesterone actions, and progesterone-regulated gene activity, that are associated with and perhaps critical for ovarian events, including follicle rupture and formation of the corpus luteum.

Estrous cycle-dependent changes in the expression of aromatic hydrocarbon receptor (AHR) and AHR-nuclear translocator (ARNT) mRNAs in the rat ovary and liver.

The aromatic hydrocarbon receptor (AHR) and AHR nuclear translocator protein (ARNT) mediate the toxic effects of a wide variety of halogenated and polycyclic aromatic hydrocarbons. While it can be assumed that AHR has an endogenous function, its role in reproduction is currently undefined. The present study seeks to examine the regulation of AHR and ARNT mRNAs in liver and ovarian tissues across the rat estrous cycle. Message for hepatic AHR was increased significantly on the morning of proestrus, and decreased dramatically by the evening of proestrus; while hepatic ARNT mRNA was significantly decreased between diestrus and the morning of proestrus, and between the evening of proestrus and the morning of estrus. Ovarian AHR mRNA was unchanged from diestrus to proestrus, and was decreased on the evening of proestrus. Changes in the expression of ARNT mRNA mirrored changes in the liver. To assess interaction between the AHR- and estrogen-receptor (ER)-signaling pathways and to test the hypothesis that estrogen regulates AHR mRNA, 25-day-old female rats were injected with either 17beta-estradiol, the ER antagonist ICI 182 780, or with vehicle, and hepatic AHR mRNA was measured. Treatment with estrogen or the estrogen antagonist did not alter the abundance of AHR mRNA in the liver. These data suggest that while estrogen may not be the key regulator of AHR mRNA expression, a factor associated with the rat reproductive cycle may be important in regulating the expression of both the AHR and ARNT genes in the ovary and liver.

Follicle growth, ovulation, and luteal formation in primates and rodents: a comparative perspective.

Ovarian function has a great deal of functional overlap between species; antral follicles grow in response to FSH, ovulation involves proteolysis, and the steroidogenic pathway is largely the same. However, embedded in these similarities are important differences that reflect the evolutionary and natural history of species and may focus future research into these critical areas. This review compares ovarian function of rats and mice with primates, focusing on estradiol and follicle growth, steroidogenesis and rupture during the periovulatory interval, and the formation of a functional corpus luteum, drawing the conclusion that careful comparison of species yields more functional information about both than studying them in isolation.

Mineralocorticoid Synthesis During the Periovulatory Interval in Macaques

Abstract Ovulation and luteal formation in primates are associated with the sustained synthesis of progesterone. The observed high intrafollicular concentrations of progesterone during the periovulatory interval raise the possibility that this steroid serves as a precursor for mineralocorticoids. The aim of this study was to determine if mineralocorticoids are synthesized by the luteinizing macaque follicle during controlled ovarian stimulation cycles in which follicular fluid and granulosa cell aspirates were obtained before or after an ovulatory hCG bolus. Follicular fluid concentrations of progesterone and 17alpha-hydroxyprogesterone increased within 3 h of an ovulatory hCG bolus. Their respective metabolites, 11-deoxycorticosterone (DOC) and 11-deoxycortisol, were not detectable before an ovulatory stimulus and increased starting at 6 h after hCG, while corticosterone and aldosterone were undetectable. Cortisol was present before and after hCG administration and had increased 2-fold at 24 h after an ovulatory stimulus. The expression of 21-hydroxylase (CYP21A2) mRNA increased within 3 h of hCG administration, while 11beta-hydroxylase-1 (CYP11B1) and 11beta-hydroxylase-2 (CYP11B2) mRNAs were not detectable. 11beta-Hydroxysteroid dehydrogenase-1 (HSD11B1) mRNA had increased at 12 h after hCG administration, and 11beta-hydroxysteroid dehydrogenase-2 (HSD11B2) had decreased by 3 h after hCG administration. Mineralocorticoid receptor mRNA levels did not change following hCG administration, while glucocorticoid receptor mRNA levels increased in response to an ovulatory stimulus. Treatment of granulosa cells with the mineralocorticoid receptor antagonist spironolactone blocked hCG-induced progesterone synthesis in vitro. These data indicate that macaque granulosa cells can synthesize mineralocorticoids in response to an ovulatory stimulus and that the mineralocorticoid receptor plays a key role in steroid synthesis associated with luteinization of macaque granulosa cells.

Estrogen receptor and aromatic hydrocarbon receptor in the primate ovary.

We have previously shown by immunocytochemistry and autoradiography the presence of estrogen receptors (ER) in rhesus monkey ovary. Intense chromogen staining showed specific binding for ER in nuclei of germinal epithelium and granulosa cells of antral follicles; and radiolabeled ligand bound specifically to functional corpora lutea (CL). Although it is accepted that the germinal epithelium of the primate ovary contains ER, some controversy still persists regarding the intraovarian localization of this molecule. In addition, no data exist that localize the aromatic hydrocarbon (dioxin) receptor (AHR), which is known to modulate ER, to the primate ovary. In the present study, we show the presence of ER using Western blot analysis, and ER capable of binding DNA within intraovarian compartments in two species of the genusMacaca (rhesus macaque,Macaca mulatta and stumptail macaque,Macaca arctoides); extend these findings to human ovarian granulosa cells (GC) using Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and gel mobility-shift analysis; and localize the AHR to intraovarian compartments of the macaque ovary by Western blots and gel-shift assays. These experiments strongly suggest that estrogens can exert effects on follicle development directly at the ovary, and provide the first direct evidence that AHR-mediated toxicity may be manifested at the ovary to induce possible antifertility effects.