Regulation of baboon fetal ovarian folliculogenesis by estrogen.

Although it is well established that formation of the pool of follicles available for ovarian function and fertility in adulthood in human and non human primates occurs in utero, our understanding of the regulation of fetal ovarian development is incomplete. Our laboratories have been instrumental in establishing the baboon as a model for the study of human reproductive endocrinology and showed that estrogen plays a central integrative role in regulating fetal-placental development. Therefore, we adapted our baboon model to study the role of estrogen on fetal ovarian development. Estrogen receptors alpha and beta were expressed in pregranulosa cells and interfollicular nests of the baboon fetal ovary. In baboons in which estrogen levels had been suppressed by administration of an aromatase inhibitor throughout the second half of gestation, fetal ovarian follicle numbers were reduced by 50%, whereas the number of interfollicular nests comprised of oocytes and pregranulosa cells was increased. The decrease in follicles in estrogen-deprived animals was associated with a marked upregulation of expression of alpha-inhibin, but not activins or activin receptors and signaling molecules. Moreover, the majority of the follicles formed in ovaries of estrogen-depleted fetuses appeared unhealthy and contained oocytes with a marked reduction/depletion in microvilli, structures essential for uptake of substrates from surrounding granulosa cells. We propose that estrogen regulates fetal ovarian folliculogenesis and formation of healthy oocytes by controlling the intraovarian activin:inhibin ratio and the development of oocyte microvilli. These findings demonstrate a need for translational research studies of the impact of impairment of estrogen action/availability on reproductive function in adulthood.

Role of RIHP and renal tubular sodium transporters in volume retention of pregnant rats.

BACKGROUND: Normal pregnancy is characterized by sodium and water conservation and an increase in plasma volume that is required for an uncomplicated pregnancy. Renal interstitial hydrostatic pressure (RIHP) is significantly decreased in pregnant rats. This decrease in RIHP may play an important role in the sodium and water retention that characterizes normal pregnancy. Paradoxically this enhanced renal sodium and water reabsorption appear to conflict with the consistent findings of a general decrease in abundance of renal tubular sodium transporters during normal pregnancy. The objective of this review is to examine the apparent discrepancy between the increases in renal tubular sodium and water reabsorption, facilitated by decreases in RIHP, and the seemingly discordant decreases in abundance of renal tubular transporters during normal pregnancy in rats. METHODS: Western blots and immunohistochemistry were used to evaluate abundance and localization of renal tubular transporters. RIHP was measured directly and continuously via a polyethylene (PE) matrix that was implanted in the left kidney of rats at the age of 11 to 16 weeks. RESULTS: Average basal RIHP and fractional excretion of sodium (FENa) were found to be significantly lower (P < .05) in midterm pregnant (MP; n = 18) and late-term pregnant (LP; n = 20) rats compared with nonpregnant (NP; n = 16) rats (3.5 +/- 0.3 mm Hg and 1.46 +/- 0.24% for MP; 3.3 +/- 0.1 mm Hg and 1.41 +/- 0.21% for LP; and 7.6 +/- 0.6 mm Hg and 3.67 +/- 0.24% for NP). Cortical Na+-K+-ATPase and Na-Pi2a cotransporter (Na-Pi) protein expression tend to decline with pregnancy. Also cortical Na+-H+ exchanger-1 (NHE-1) protein expression declines steadily during the course of pregnancy from MP to LP compared with that in NP rats, and cortical Na+-H+ exchanger-3 (NHE-3) protein expression is significantly lower in MP and LP compared with NP rats. CONCLUSIONS: We propose that during normal uncomplicated pregnancy, simultaneous decreases in RIHP and in net abundance of renal tubular sodium transporters occur. The effects of decreased RIHP exceed those of the reduction in net abundance, and presumably activity, of renal tubular transporters resulting in an enhanced net sodium and water retention during pregnancy.

Regulation of oocyte microvilli development in the baboon fetal ovary by estrogen.

We recently showed that the number of primordial follicles was reduced by 50% in ovaries of near-term fetal baboons deprived of estrogen in utero and restored to normal in animals supplemented with estrogen. Oocytes are avascular and rely on surrounding granulosa cells for nutrients, a process facilitated by microvilli on the oocyte surface. However, our understanding of oocyte microvillus development in the primate fetal ovary is incomplete. Thus, we determined whether estrogen regulates formation of oocyte microvilli in utero. Fetal ovaries were obtained on d 165 gestation (term = d 184) from baboons untreated (n = 3) or treated on d 100-165 with aromatase inhibitor CGS 20267 (estrogen suppressed by 95%; n = 5) or CGS 20267 and estradiol (n = 4). Follicles with intact (homogeneous cytoplasm) or nonintact (cytoplasm vacuolated) oocytes were quantified and the number/height of oocyte microvilli determined by electron microscopy. In untreated baboons, the mean (+/-se) number of follicles/0.08 mm(2) with an intact oocyte (11.5 +/- 0.5) was decreased (P < 0.05) by 70% in fetal ovaries of estrogen-suppressed baboons (3.4 +/- 0.2) and restored (P < 0.05) by CGS 20267 and estradiol (11.2 +/- 1.2). In estrogen-deprived fetuses, the number of microvilli/intact oocyte (23 +/- 3) was 56% lower (P < 0.01) than normal (52 +/- 5) and restored by CGS 20267 and estrogen (62 +/- 4). Moreover, in intact oocytes of estrogen-suppressed baboons, height (nm) of microvilli (105 +/- 11) was 54-62% lower (P < 0.01) than in intact oocytes of fetal ovaries of untreated (228 +/- 13) or estrogen-treated (274 +/- 17) baboons. In estrogen-replete baboons, the number of microvilli in intact oocytes was 2-fold greater (P < 0.01) than in nonintact oocytes. However, in estrogen-deprived baboons, no microvilli were detected in nonintact oocytes and the number of microvilli in intact oocytes was similar to that in nonintact oocytes of untreated fetuses. We conclude that development of microvilli in oocytes of primordial follicles in the primate fetal ovary is regulated by estrogen. Collectively, these results and those of our previous studies indicate that estrogen regulates fetal ovarian folliculogenesis and development of follicles with oocytes composed of microvilli critical for nutrient uptake and presumably long-term survival.

Regulation of expression of microvillus membrane proteins by estrogen in baboon fetal ovarian oocytes.

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estrogen. Ezrin (total and phosphorylated) and SLC9A3R1 expression was localized to the surface of oocytes at mid and late gestation in estrogen-replete baboons and to the cytoplasm in late gestation after estrogen suppression. These results are the first to show that the fetal baboon oocyte expressed ezrin, SLC9A3R1, and alpha-actinin, and that these proteins were localized to the oocyte surface consistent with their role in microvilli development in epithelial cells. The current study also showed that the developmental increase in oocyte expression of alpha-actinin is regulated by estrogen and correlated with the estrogen-dependent increase in oocyte microvilli demonstrated previously. Therefore, we propose that development of oocyte microvilli requires expression of alpha-actinin and that expression of alpha-actinin and localization of ezrin-phosphate and SLC9A3R1 to the oocyte membrane are regulated by estrogen.

Oocytes of baboon fetal primordial ovarian follicles express estrogen receptor beta mRNA.

In fetal ovaries of estrogen-suppressed baboons, we have previously shown that follicle numbers were 50% lower than in estrogen-replete animals and contained oocytes with a reduced number of microvilli. In the baboon fetal ovary, although estrogen receptor (ER)alpha and beta have been detected by immunocytochemistry in granulosa cells, it is not known whether oocytes express ER. Because the actions of estrogen are mediated by interaction with cell-specific receptors, the current study determined whether ERalpha/beta mRNA were expressed in oocytes of baboon fetal ovaries obtained on day 165 (term = day 184) of gestation. Oocyte nuclei and cytoplasm from primordial follicles were isolated by laser capture microdissection and ERalpha, ERbeta, GATA-4 (granulosa cell specific marker) mRNAs, and 18S rRNA determined by RT-PCR and products verified by sequencing. ERbeta mRNA was expressed in oocytes of 5 of 5 fetuses. In contrast, fetal oocytes did not express ERalpha mRNA. Although 18S rRNA was expressed in all oocytes, GATA-4 mRNA was not detected in oocytes and only detected in granulosa cells confirming purity of oocytes sampled. We conclude that oocytes of the fetal baboon ovary express ERbeta mRNA, thereby providing a mechanism by which estrogen regulates oocyte function, e.g. microvillus development.

Developmental regulation of baboon fetal ovarian maturation by estrogen.

Ovarian function in adult human and nonhuman primates is dependent on events that take place during fetal development, including the envelopment of oocytes by granulosa (i.e., folliculogenesis). However, our understanding of fetal ovarian folliculogenesis is incomplete. During baboon pregnancy, placental production and secretion of estradiol into the fetus increases with advancing gestation, and the fetal ovary expresses estrogen receptors alpha and beta in mesenchymal-epithelial cells (i.e., pregranulosa) as early as midgestation. Therefore, the current study determined whether estrogen regulates fetal ovarian follicular development. Pregnant baboons were untreated or treated with the aromatase inhibitor CGS 20267, or with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100-164 (term = Day 184). On Day 165, baboon fetuses were delivered by cesarean section and the number of total follicles and interfollicular nests consisting of oocytes and mesenchymal-epithelial cells in areas (0.33 mm(2)) of the outer and inner cortices of each fetal ovary were quantified using image analysis. Maternal and umbilical serum estradiol levels were decreased by >95% with CGS 20267. Treatment with CGS 20267 and estrogen restored maternal estradiol to normal and fetal estradiol to 30% of normal. Although fetal ovarian weight was unaltered, the mean number of follicles +/- SEM/0.33 mm(2) in the inner (59.0 +/- 1.7) and outer (95.3 +/- 2.4) cortical regions of fetal ovaries in untreated animals was 35%-50% lower (P < 0.01) in estrogen-depleted baboons (25.9 +/- 1.4, inner cortex; 62.5 +/- 2.7, outer cortex) and was restored to normal by treatment with CGS 20267 and estrogen. In contrast, the number of interfollicular nests was 2-fold greater (P < 0.01) in fetal ovaries of estrogen-suppressed animals, a change that was prevented by treatment with estrogen. In summary, fetal ovarian follicular development was significantly altered in baboons in which estrogen was depleted during the second half of gestation and restored to normal by estradiol. We propose that estrogen plays an integral role in regulating, and perhaps programming, primate fetal ovarian development.

Developmental regulation of follicle-stimulating hormone receptor messenger RNA expression in the baboon fetal ovary.

In the adult ovary, pituitary FSH via interaction with its receptor (FSHR) is required for follicular maturation and granulosa cell development. In humans and nonhuman primates, the pool of follicles available for adult ovarian function is established in utero. However, our understanding of the ontogeny and developmental regulation of FSHR in the ovary of the primate fetus is incomplete. Our goal was to determine whether the baboon fetal ovary expresses the full-length FSHR mRNA transcript and whether levels are developmentally regulated. Fetal ovaries were obtained at mid (Day 100) and late (Day 165) gestation (term = Day 184) from untreated baboons and on Day 165 from baboons in which fetal estrogen levels were either decreased by >95% by treatment with the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100-164. The full-length 2088-base pair FSHR mRNA transcript was expressed in ovaries of adult and fetal baboons untreated or treated with CGS 20267 or CGS 20267 and estrogen. Mean (+/-SEM) FSHR mRNA levels (ratio of FSHR mRNA:18S rRNA), quantified by reverse transcription polymerase chain reaction, were increased (P < 0.05) 2-fold between mid (0.34 +/- 0.06) and late gestation (0.76 +/- 0.07), an increase prevented (P < 0.05) in estrogen-depleted baboons (0.44 +/- 0.10) and partially restored by treatment with CGS 20267 and estrogen (0.58 +/- 0.16). We previously showed that the number of follicles/0.33 mm2 in fetal ovaries of untreated baboons in late gestation was reduced 50% by treatment with CGS 20267 and restored to normal in baboons treated with CGS 20267 and estrogen. Thus, when corrected for the number of follicles/0.33 mm2, FSHR mRNA levels were similar in baboon fetal ovaries untreated (0.010 +/- 0.001) or treated with CGS 20267 (0.009 +/- 0.002) or CGS 20267 and estrogen (0.007 +/- 0.003). We conclude that estrogen plays a major role in regulating ovarian FSHR mRNA expression in the primate fetus, and that the developmental increase in FSHR mRNA levels reflects the estrogen-dependent increase in folliculogenesis (i.e., increased number of granulosa cells and oocytes).

Expression of estrogen receptors alpha and beta in the baboon fetal ovary.

In adult mammals, estrogen regulates ovarian function, and estrogen receptor (ER) is expressed in granulosa cells of antral follicles of the adult baboon ovary. Because the foundation of adult ovarian function is established in utero, the present study determined whether ERalpha and/or ERbeta were expressed in fetal ovaries obtained on Days 100 (n = 3) and 165-181 (n = 5) of baboon gestation (term = Day 184). On Day 100, ERalpha protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERbeta protein was also detected by immunocytochemistry on Day 100 of gestation and was abundantly expressed in mesenchymal-epithelial cells in germ cell cords, lightly expressed in the germ cells, but was not detected in the surface epithelium. On Days 165-180 of gestation, ERalpha expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ERalpha expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERbeta expression was most intense in granulosa cells, especially in flattened granulosa cells, was weaker in mesenchymal-epithelial cells and nests of cells between follicles, and was absent in the surface epithelium. Using an antibody to the carboxy terminal of human ERbeta, ERbeta protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa on Day 100 and primarily 55 kDa on Day 180. The mRNAs for ERalpha and ERbeta were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ERalpha and ERbeta mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate.

Up-regulation of alpha-inhibin expression in the fetal ovary of estrogen-suppressed baboons is associated with impaired fetal ovarian folliculogenesis.

We recently demonstrated that the number of primordial follicles was significantly reduced in the ovaries of near-term baboon fetuses deprived of estrogen in utero and restored to normal in animals administered estradiol. Although the baboon fetal ovary expressed estrogen receptors alpha and beta, the mechanism(s) of estrogen action remains to be determined. It is well established that inhibin and activins function as autocrine/paracrine factors that impact adult ovarian function. However, our understanding of the expression of these factors in the primate fetal ovary is incomplete. Therefore, we determined the expression of alpha-inhibin, activin beta(A), activin beta(B), and activin receptors in fetal ovaries obtained at mid and late gestation from untreated baboons and at late gestation from animals in which fetal estrogen levels were reduced by >95% by maternal administration of the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 and estradiol benzoate. Immunocytochemical expression of alpha-inhibin was minimal to nondetectable in fetal ovaries from untreated baboons. In contrast, in baboons depleted of estrogen, alpha-inhibin was abundantly expressed in pregranulosa cells of interfollicular nests and granulosa cells of primordial follicles. Thus, the number (mean +/- SEM) per 0.08 mm2 of fetal ovarian cells expressing alpha-inhibin, determined by image analysis, was similar at mid and late gestation and increased approximately 8-fold (P < 0.01) near term in baboons treated with CGS 20267 and was restored (P < 0.01) to normal in baboons treated with CGS 20267 plus estradiol. Activin beta(A) was detected in oocytes and pregranulosa cells at midgestation and in oocytes and granulosa cells of primordial follicles at late gestation. Activin beta(B) was also expressed in pregranulosa cells and granulosa cells at mid and late gestation, respectively, but was not detected in oocytes. Neither the pattern nor the apparent level of expression of activin beta(A) or beta(B) were altered in fetal ovaries of baboons treated with CGS 20267 or CGS 20267 and estrogen. Activin receptors IA, IB, IIA, and IIB were detected by Western blot analysis in fetal ovaries at mid and late gestation, and expression was not altered by treatment with CGS 20267 or CGS 20267 and estrogen. Activin receptors IB and IIA were localized to oocytes and pregranulosa cells at midgestation and to granulosa cells and oocytes of primordial follicles at late gestation. Thus, the decrease in the number of follicles in the primate fetal ovary of baboons deprived of estrogen in utero was associated with increased expression of alpha-inhibin. Therefore, we propose that estrogen regulates fetal ovarian follicular development by controlling alpha-inhibin expression and, thus, the intraovarian inhibin:activin ratio.

Expression of estrogen receptors alpha and beta in the fetal baboon testis and epididymis.

Although studies in transgenic mice suggest that estrogen is important for development of the testis, very little is known about the potential role of estrogen in maturation of the primate fetal testis. Therefore, as a first step to determine whether estrogen regulates maturation of the fetal primate testis, we used immunocytochemistry to determine estrogen receptor (ER) alpha and beta expression in the fetal baboon testis. Second, we established methods to quantify ERbeta mRNA levels by competitive reverse transcription-polymerase chain reaction in Sertoli cells isolated by laser capture microdissection (LCM) from the fetal baboon testis. ERbeta protein expression was abundant in the nuclei of Sertoli, peritubular, and interstitial cells in baboon fetuses at mid (Day 100) and late (Day 165) gestation (term is 184 days). ERbeta mRNA level was 0.03 attomole/femtomole 18S rRNA in Sertoli cell nuclei and associated cytoplasm isolated by LCM. ERalpha was expressed in low level in seminiferous tubules and in moderate level in peritubular cells on Day 165. Germ cells expressed very little ERalpha or ERbeta protein, whereas the baboon fetal epididymis exhibited extensive ERalpha and ERbeta immunostaining at mid- and late gestation. In contrast to the robust expression of ERbeta, androgen receptor protein was not demonstrable within the cells of the seminiferous cords but was abundantly expressed in epididymal epithelial cells of the fetal baboon. In summary, the results of this study show that the fetal baboon testis and epididymis expressed the ERalpha and ERbeta, and we suggest that our nonhuman primate baboon model can be used to study the potential role of estrogen on maturation of the fetal testis.

Localization and developmental expression of the activin signal transduction proteins Smads 2, 3, and 4 in the baboon fetal ovary.

We recently demonstrated that the reduction in the number of primordial follicles in ovaries of near-term baboon fetuses deprived of estrogen in utero was associated with increased expression of alpha-inhibin, but not activin betaA and betaB or the activin receptors. Therefore, we proposed that estrogen regulates fetal ovarian follicular development by controlling the intraovarian inhibin:activin ratio. As a prelude to conducting experiments to test this hypothesis, in the current study we determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation, and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB, which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen deprivation in utero. These findings, coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and upregulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.