David W. Schomberg

Targeted Disruption of the Estrogen Receptor-α Gene in Female Mice: Characterization of Ovarian Responses and Phenotype in the Adult1

Targeted disruption of the mouse estrogen receptor-α gene (estrogen receptor-α knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ERα-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA ...

Prevention of the Polycystic Ovarian Phenotype and Characterization of Ovulatory Capacity in the Estrogen Receptor-α Knockout Mouse

Ovarian-derived estradiol plays a critical endocrine role in the regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. In turn, several para/autocrine effects of estrogen within the ovary are known, including increased ovarian weight, stimulation of granulosa cell growth, augmentation of FSH action, and attenuation of apoptosis. The estrogen receptor-alpha (ERalpha) is present in all three components of the hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen receptor-beta (ERbeta) is easily detectable in ovarian granulosa cells but is low to absent in the pituitary of the adult mouse. This distinct expression pattern for the two ERs suggests the presence of separate roles for each in the regulation of ovarian function. Herein, we definitively show that a lack of ERalpha in the hypothalamic-pituitary axis of the ERalpha-knockout (alphaERKO) mouse results in chronic elevation of serum LH and is the primary cause of the ovarian phenotype of polycystic follicles and anovulation. Prolonged treatment with a GnRH antagonist reduced serum LH levels and prevented the alphaERKO cystic ovarian phenotype. To investigate a direct role for ERalpha within the ovary, immature alphaERKO females were stimulated to ovulate with exogenous gonadotropins. Ovulatory capacity in the immature alphaERKO female was reduced compared with age-matched wild-type (14.5+/-2.9 vs. 40.6+/-2.6 oocytes/animal, respectively); however, oocytes collected from the alphaERKO were able to undergo successful in vitro fertilization. A similar discrepancy in oocyte yield was observed after superovulation of peripubertal (42 days) wild-type and alphaERKO females. In addition, ovaries from immature superovulated alphaERKO females possessed several ovulatory but unruptured follicles. Investigations of the possible reasons for the reduced number of ovulations in the alphaERKO included ribonuclease protection assays to assess the mRNA levels of several markers of follicular maturation and ovulation, including ERbeta, LH-receptor, cyclin-D2, P450-side chain cleavage enzyme, prostaglandin synthase-2, and progesterone receptor. No marked differences in the expression pattern for these mRNAs during the superovulation regimen were observed in the immature alphaERKO ovary compared with that of the wild-type. Serum progesterone levels just before ovulation were slightly lower in the alphaERKO compared with wild-type. These studies indicate that treatment of alphaERKO females with a GnRH antagonist decreased the serum LH levels to within the wild-type range and concurrently prevented development of the characteristic ovarian phenotype of cystic and hemorrhagic follicles. Furthermore, a lack of functional ERalpha within the ovary had no effect on the regulation of several genes required for follicular maturation and ovulation. However, the reduced numbers of ovulations following the administration of exogenous gonadotropins in the alphaERKO suggests an intraovarian role for ERalpha in follicular development and ovulation.

Modulation of progestin secretion in ovarian cells by 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone): A direct demonstration in monolayer culture

Abstract These studies with a monolayer system of porcine granulosa cells provide a direct demonstration of the ability of androgen to stimulate progestin secretion by ovarian cells. A preferential action of the more potent androgens, dihydrotestosterone and testosterone, was shown but only dihydrotestosterone demonstrated the capacity to stimulate progestin secretion throughout the culture period. Estradiol 17-β markedly depressed progestin synthesis. The results suggest a modulatory role for androgens in the development of full steroidogenic potential by ovarian granulosa cells during follicular development.

INTERACTIONS BETWEEN HORMONES AND GROWTH FACTORS IN THE REGULATION OF GRANULOSA CELL DIFFERENTIATION IN VITRO

Evidence from studies in vitro supports the concept that growth factors modulate endocrine-dependent differentiative processes in follicle development. Based upon results from granulosa cell culture systems, it is suggested that the study of growth factors and their regulatory mechanisms (endocrine, paracrine, autocrine control) could perhaps be generalized to other areas concerned with the regulation of steroid secretion such as placental physiology, the regulation of fetal gonads and puberty, secondary steroid metabolism and steroid-secreting tumors.

2-Methoxyestradiol, an Endogenous Estradiol Metabolite, Differentially Inhibits Granulosa and Endothelial Cell Mitosis: A Potential Follicular Antiangiogenic Regulator

Abstract 2-Methoxyestradiol (2-ME) is an estradiol metabolite with antiangiogenic and antitumor activity. It is formed by granulosa cell (GC) catechol-O-methyltransferase activity and is present in the normal follicle at high concentrations. In this unique microenvironment, it may regulate selected cell types via autocrine and/or paracrine action. To assess the possibility that 2-ME or estradiol might exert differential mitotic and/or apoptotic effects on endothelial cells and GCs, we compared their actions on primary cultures of hormone- and/or growth factor-stimulated porcine GCs (pGCs) as well as two types of endothelial cells, primary cultures of porcine endothelial cells (pECs), and a spontaneously transformed rabbit endothelial vascular cell (REVC) line. The 2-ME, but not estradiol, dose dependently suppressed tritiated thymidine (3H-T) incorporation into epidermal growth factor (EGF)-stimulated REVCs and EGF/insulin (INS)-stimulated pECs. In contrast, 2-ME did not attenuate incorporation in FSH/INS-stimulated pGCs. It reduced incorporation by approximately 50% in EGF/INS-stimulated pGCs, indicating that responsiveness to 2-ME in normal cells can be modulated by hormone and growth factor treatment. Estradiol was not antimitotic to pGCs. As indicated by 4′,6-diamido-2-phenylindole hydrochloride nuclear staining, estradiol was nonapoptotic in either cell type, and 2-ME significantly increased apoptosis of REVCs, but not of pGCs. In a cell migration assay, REVC movement was attenuated by 2-ME, but not by estradiol. In summary, the results show that antimitotic as well as proapoptotic responses to 2-ME vary with cell type and, in the case of pGC antimitotic activity, with the regulatory microenvironment. Thus, they provide a rationale for autocrine and/or paracrine action of 2-ME at its site of production in vivo, and they strongly support the concept of 2-ME as a candidate ovarian angiogenesis inhibitor.

Effect of exogenous dehydroepiandrosterone upon the fetoplacental biosynthesis of estrogens and its effect upon uterine blood flow in the term pregnant ewe

The fetoplacental unit of the ewe is capable of increasing the biosynthesis of estrogens following the exogenous administration of DHEA to the fetus. The maximum concentrations of estrogens appeared approximately 30 minutes after the administration of DHEA. Uterine blood flow in the pregnant ewe increased approximately 90 minutes after the maximum concentrations of estrogens were noted. The administration of DHEA to the nonpregnant, ovariectomized ewe did not elicit estrogen biosynthesis or changes in uterine blood flow. Isotope experiments in the pregnant ewe demonstrated the incorporation of DHEA into urinary estradiol.

The effect of plating density on granulosa cell growth and differentiation in vitro

The extent of FSH-mediated LH/hCG receptor induction and of basal and FSH-stimulated progesterone production by porcine granulosa cells in vitro, in serum-containing medium, is directly related to the plating density. Relative to pre-culture levels, low- and high-density cultures of cells routinely exhibited 1-2- and 10-11-fold increases in [125I]iodo-hCG binding, respectively. Monolayer growth, i.e. cell division, as measured by increases in cell protein or DNA content, was inversely related to plating density. This density-directed inverse relationship between growth and differentiation did not appear to be coupled under the conditions utilized. Whereas monolayer growth was dependent upon the cell surface density, i.e. the number of cells per unit surface area, differentiation was dependent upon cell concentration, i.e. cells per unit volume of medium. Cells plated at low density in medium containing 10% serum exhibited 50% less [125I]iodo-hCG binding than cells in 5% serum (P less than 0.025). Conversely, cells plated at high density exhibited a 14% increase (P less than 0.025) in binding at the higher serum level. Thus, it appears that the extent of differentiation depends upon the capacity of cells to neutralize serum inhibition which in turn is dependent upon the cell concentration. Serum neutralization by granulosa cells is an FSH-dependent process. Conditioned medium derived from insulin-treated, high-density cultures did not facilitate optimum LH/hCG receptor induction in low-density cultures. Conditioned medium from cultures treated with insulin plus FSH, however, facilitated LH/hCG receptor induction in low-density cultures to the same extent as obtained in high-density cultures. The enhancement by FSH-conditioned medium cannot be attributed to residual FSH or to dilution of serum components during the preparation of the conditioned medium. The phenomena of serum-attenuated granulosa cell differentiation in vitro, and of a density-dependent reversal of this process, may have regulatory implications in vivo since follicular fluid contains many serum components and since the granulosa cell complement is an important determinant of follicle maturation.

Whole and Disaturated Lung Phosphatidylcholine in Cortisol-Treated, Intrauterine Growth-Retarded and Twin Control Lambs at Different Gestational Ages

Lambs 116--124 days gestation infused in utero for 75 h with cortisol showed, when compared to twin controls, more mature lung histology and pressure-volume relationships. 32P orthophosphate incorporation into whole lung phosphatidylcholine (PC) was increased in the four cortisol-treated lambs at 116--117 days but not at 121, 123, and 124 days gestation. 14C palmitate incorporation into PC or disaturated phosphatidylcholine (DSPC) was not enhanced at 116--117 days gestation. At 121 days in a cortisol-treated and at 128 days in a growth-retarded lamb fetus not treated with cortisol, a larger quantity of DSPC was present although the incorporation of 14C palmitate into DSPC per milligram DNA was the same. This indicated that the synthesis of DSPC had been initiated in the cortisol-treated and growth-retarded animals prior to the controls and at the time of sacrifice both were incorporating 14C palmitate at a similar rate suggesting similar rates of synthesis.

Steroidal Modulation of Steroid Secretion in vitro : An Experimental Approach to Intra-Follicular Regulatory Mechanisms

Studies in vitro and in vivo indicate that steroid secretion by granulosa cells of the developing follicle is modulated in part by steroid hormones themselves. Androgens stimulate progestin synthesis in vitro by granulosa cells of all developmental stages independently of their role as an estrogen precursor; generally, the action of estradiol results in suppression of progesterone secretion. The nature of the relationship between steroid secretion by granulosa cells and the physiological processes of follicular development or atresia remains to be investigated in greater detail. The cellular mechanism(s) of steroidal modulation of steroid secretion is unknown but apparently does not involve changes in the number of granulosa cell LH receptors per se.

Hyperglycosylated human chorionic gonadotropin does not increase progesterone production by luteinized granulosa cells.

CONTEXT Trophoblast-derived human chorionic gonadotropin (hCG) promotes corpus luteum progesterone (P4) production, and wide ranges of serum P4 levels are noted in various pregnancy outcomes, despite similar hCG concentrations. There are five unique biologically active hCG variants in human pregnancy urine, and previous studies of P4 production in response to hCG have used only preparations containing all isoforms. Understanding exactly which hCG variant is primarily responsible for stimulating corpus luteum steroidogenesis may have great clinical and diagnostic implications, including in the setting of ectopic pregnancy. OBJECTIVE Our objective was to delineate the role of the standard and hyperglycosylated (H)-hCG isoforms in stimulating P4 production by luteinized granulosa cells. DESIGN AND SETTING Cell culture, ELISA, and fluorometric-based protein assays were done at Duke University Medical Center. PATIENTS Patients were anonymous oocyte donors. INTERVENTION Cultured luteinized granulosa cells were treated with 0.25, 0.5, and 1.0 ng/ml total hCG, which contains all isoforms, purified standard hCG (37.1 kDa), and purified H-hCG (42.8 kDa). MAIN OUTCOME MEASURE P4 produced per total cellular protein (nanograms per microgram) was measured via ELISA and fluorometric protein determination kits. RESULTS Both total hCG (P = 0.0003) and purified standard hCG (P < 0.0001) stimulated a dose-dependent increase in P4 production. Purified H-hCG did not change the P4 produced per total cellular protein response (P value not significant). CONCLUSIONS Standard hCG stimulated P4 production by cultured granulosa cells and likely supports corpus luteum function via interactions with the LH/hCG receptor. In contrast, H-hCG did not increase P4 production, which indicates a nonsteroidogenic role for this protein during early gestation.