Diane M. Duffy

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...

Androgen receptor mRNA expression in the rhesus monkey ovary.

Immunocytochemical detection of androgen receptors (ARs) in several compartments of the macaque ovary, including the germinal epithelium, follicle, and corpus luteum, suggests a role for androgens in modulating ovarian function via the classical receptor-mediated pathway. To examine AR mRNA expression in the rhesus monkey ovary, total RNA was isolated from whole ovaries, the germinal epithelium-enriched cortical and medullary compartments of the ovary, and corpora lutea from early (d 3–5), mid (d 6–8), midlate (d 10–12), and late (d 13–15) stages of the luteal phase of the menstrual cycle. RNA was also obtained from luteinized granulosa cells from monkeys receiving gonadotropin treatment to stimulate the development of multiple ovarian follicles. After reverse transcription of total RNA using oligo-dT as a primer, polymerase chain reaction (PCR) was used to amplify a unique 329 bp segment of the monkey AR hormone-binding region. Reverse transcriptase (RT)-PCR products of the expected size were detected in all ovarian and control tissues. Sequence analysis of the AR cDNA from the macaque ovary revealed 99% nucleotide homology and 100% predicted amino acid homology to the cDNA for the hormone-binding region of human AR. Northern analysis demonstrated the presence of a major AR mRNA species at 9.5 kb in corpus luteum, luteinized granulosa cells, and prostate, with additional bands detected in the corpus luteum and prostate at 7.9 and 3.4 kb, respectively. A sensitive RNase protection assay was used to examine AR mRNA levels in ovarian tissues and showed AR mRNA expression throughout the life-span of the corpus luteum. Thus, detection of AR mRNA in the primate ovary, including the periovulatory follicle and corpus luteum, supports the concept that these tissues are targets for receptor-mediated androgen action during the menstrual cycle.

EP3 Receptor Isoforms are Differentially Expressed in Subpopulations of Primate Granulosa Cells and Couple to Unique G-Proteins

Prostaglandin E2 (PGE2) produced within the ovarian follicle is necessary for ovulation. PGE2 is recognized by four distinct G-protein-coupled receptors. Among them, PTGER3 (also known as EP3) is unique in that mRNA splicing generates multiple isoforms. Each isoform has a distinct amino acid composition in the C-terminal region, which is involved in G-protein coupling. To determine whether monkey EP3 isoforms couple to different G-proteins, each EP3 isoform was expressed in Chinese hamster ovary cells, and intracellular signals were examined after stimulation with the EP3 agonist sulprostone. Stimulation of EP3 isoform 5 (EP3-5) reduced cAMP in a pertussis toxin (PTX)-sensitive manner, indicating involvement of Gαi. Stimulation of EP3-9 increased cAMP, which was reduced by the general G-protein inhibitor GDP-β-S, and also increased intracellular calcium, which was reduced by PTX and GDP-β-S. So, EP3-9 likely couples to both Gαs and a PTX-sensitive G-protein to regulate intracellular signals. Stimulation of EP3-14 increased cAMP, which was further increased by PTX, so EP3-14 likely regulates cAMP via multiple G-proteins. Granulosa cell expression of all EP3 isoforms increased in response to an ovulatory dose of human chorionic gonadotropin. Two EP3 isoforms were differentially expressed in functional subpopulations of granulosa cells. EP3-5 was low in granulosa cells at the follicle apex while EP3-9 was high in cumulus granulosa cells. Differential expression of EP3 isoforms may yield different intracellular responses to PGE2 in granulosa cell subpopulations, contributing to the different roles played by granulosa cell subpopulations in the process of ovulation.

Vascular endothelial growth factors C and D may promote angiogenesis in the primate ovulatory follicle

ABSTRACT Angiogenesis in the ovary occurs rapidly as the ovarian follicle transforms into a mature corpus luteum. Granulosa cells produce vascular endothelial growth factor A (VEGFA) in response to the ovulatory gonadotropin surge. VEGFA is established as a key mediator of angiogenesis in the primate ovulatory follicle. To determine if additional VEGF family members may be involved in angiogenesis within the ovulatory follicle, cynomolgus monkeys (Macaca fascicularis) received gonadotropins to stimulate multiple follicular development, and human chorionic gonadotropin (hCG) substituted for the luteinizing hormone surge to initiate ovulatory events. Granulosa cells of monkey ovulatory follicles contained mRNA and protein for VEGFC and VEGFD before and after hCG administration. VEGFC and VEGFD were detected in monkey follicular fluid and granulosa cell-conditioned culture media, suggesting that granulosa cells of ovulatory follicles secrete both VEGFC and VEGFD. To determine if these VEGF family members can stimulate angiogenic events, monkey ovarian microvascular endothelial cells (mOMECs) were obtained from monkey ovulatory follicles and treated in vitrowith VEGFC and VEGFD. Angiogenic events aremediated via three VEGF receptors; mOMECs express all three VEGF receptors in vivo and in vitro. Exposure of mOMECs to VEGFC increased phosphorylation of AKT, while VEGFD treatment increased phosphorylation of both AKT and CREB. VEGFC and VEGFD increased mOMEC migration and the formation of endothelial cell sprouts in vitro. However, only VEGFD increased mOMEC proliferation. These findings suggest that VEGFC and VEGFD may work in conjunction with VEGFA to stimulate early events in angiogenesis of the primate ovulatory follicle. Summary Sentence VEGFC and VEGFD produced by granulosa cells of the ovulatory folliclemay contribute to follicular angiogenesis and ovulation.

Intraovarian Control of Ovulation: Lessons from Steroid Ablation/Replacement in Monkeys

An important action of estrogen secreted by the maturing follicle is to trigger the release of surge levels of the gonadotropic hormones, follicle stimulating hormone (FSH), and luteinizing hormone (LH) from the pituitary. The gonadotropin surge initiates a cascade of events within the follicle that promotes oocyte maturation, ovulation, and conversion of the follicle wall into a new endocrine structure, the corpus luteum. The latter process, termed luteinization, involves major changes in the structure—function of the tissue, including development of the molecular and cellular components required to produce large quantities of the steroid hormone progesterone. As the periovulatory interval proceeds, the luteinizing follicle switches from primarily an estrogen-secreting to a progesterone-secreting tissue. In some series, the corpus luteum is devoid of estrogen synthetic capacity, whereas in others (e. g., Old World macaques and women) significant production of estrogen (and its bioactive precursor, androgen) remains. Thus, the periovulatory follicle experiences remarkable changes in the steroid milieu as androgen and estrogen initially rise and then decline, and appreciable levels of progestins are achieved well before follicle rupture (1, 2).