Marcia G. Burch

Estrogen Regulates 11β-Hydroxysteroid Dehydrogenase-1 and -2 Localization in Placental Syncytiotrophoblast in the Second Half of Primate Pregnancy

We recently demonstrated that the 11β-hydroxysteroid dehydrogenase enzymes catalyzing cortisol-cortisone reduction (11β-hydroxysteroid dehydrogenase-1) and oxidation (11β-hydroxysteroid dehydrogenase-2) are located in different regions of the baboon and human placental syncytiotrophoblast. Moreover, there was a 2-fold increase in the ratio of 11β-hydroxysteroid dehydrogenase-2 to 11β-hydroxysteroid dehydrogenase-1 in syncytiotrophoblast membranes contiguous with the basal membrane (BMm) between mid and late baboon gestation. Our laboratories have also shown that estrogen regulates syncytiotrophoblast functional differentiation. Therefore, the current study determined whether the change in the ratio of 11β-hydroxysteroid dehydrogenase-2 to 11β-hydroxysteroid dehydrogenase-1 in the BMm was regulated by estrogen. Placentas were obtained on d 165 of gestation (term = d 184) from baboons that were untreated or were treated daily beginning on d 100 with the aromatase inhibitor CGS 20267, which reduced uterine a...

Up-Regulation of α-Inhibin Expression in the Fetal Ovary of Estrogen-Suppressed Baboons Is Associated with Impaired Fetal Ovarian Folliculogenesis

Abstract 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 α and β, 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 α-inhibin, activin βA, activin βB, 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 α-inhibin was minimal to nondetectable in fetal ovaries from untreated baboons. In contrast, in baboons depleted of estrogen, α-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 α-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 βA was detected in oocytes and pregranulosa cells at midgestation and in oocytes and granulosa cells of primordial follicles at late gestation. Activin βB 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 βA or βB 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 α-inhibin. Therefore, we propose that estrogen regulates fetal ovarian follicular development by controlling α-inhibin expression and, thus, the intraovarian inhibin:activin ratio.

Expression of the mRNAs and Proteins for the Na+/H+ Exchangers and Their Regulatory Factors in Baboon and Human Placental Syncytiotrophoblast

In polarized epithelial cells of several organ systems, e.g. the kidney, a family of Na(+)/H(+) exchangers (e.g. Na(+)/H(+) exchanger-1 and -3) and their regulatory proteins, Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein play a major role in regulating Na(+)/H(+) exchange integral to cellular homeostasis. Because the primate placenta regulates exchange of Na(+) and H(+) between the mother and fetus critical to fetal-placental homeostasis, the current study determined whether Na(+)/H(+) exchanger-1 and -3 were compartmentalized and associated with expression of Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein in baboon and human syncytiotrophoblast. Using RT-PCR, single 413-bp Na(+)/H(+) exchanger-1 and 190-bp Na(+)/H(+) exchanger-3 products were expressed by baboon and human syncytiotrophoblasts. The 104-kDa Na(+)/H(+) exchanger-1 protein was detected by Western blot in microvillus membranes and to a much lesser extent in the basal membranes of the baboon and human syncytiotrophoblasts. In contrast, the 85-kDa Na(+)/H(+) exchanger-3 protein was detected primarily in membranes contiguous with the basal membranes of the syncytiotrophoblast of both species. Differential localization of Na(+)/H(+) exchanger-1 and -3 was confirmed by immunocytochemistry. The Na(+)/H(+) exchanger-3 regulatory protein, Na(+)/H(+) exchanger-3 kinase A regulatory protein, resided almost exclusively in the basal membranes, whereas Na(+)/H(+) exchanger regulatory factor was localized primarily to the microvillus membranes in the baboon and human syncytiotrophoblast. Collectively, these results are the first to show that the baboon and human term placental syncytiotrophoblast expressed the mRNAs and proteins for Na(+)/H(+) exchanger-1 and -3 and their regulatory factors and that Na(+)/H(+) exchanger-1 and Na(+)/H(+) exchanger regulatory factor resided primarily in the microvillus membranes, whereas Na(+)/H(+) exchanger-3 and Na(+)/H(+) exchanger-3 kinase A regulatory protein were localized to membranes contiguous with the basal membranes and to the basal membranes, respectively. We conclude that a complete Na(+)/H(+) exchange system is present in the baboon and human term placental syncytiotrophoblast and suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na(+)/H(+) exchange between the placenta and the maternal and fetal circulations.

Interconversion of cortisol and cortisone in the baboon placenta at midgestation: Expression of 11β-hydroxysteroid dehydrogenase type 1 messenger RNA

At midgestation in the baboon, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalysed interconversion of cortisol (F) and cortisone (E) during transuterine passage favors the formation of F. Because the site(s) of oxidation/reduction of F and E are not clear, the present study compared F-E interconversion in placenta, decidua and chorion in vitro. In addition, because the reduction of E to F is catalysed only by the 11beta-HSD-1, we also determined whether the mRNA for this enzyme was expressed in baboon placenta, including placental syncytiotrophoblast cells; the site of fetal-maternal exchange. Placentas were obtained on day 100 of gestation (term = day 184) and villous tissue, decidua and chorion isolated, minced in HBSS and incubated (300-400 mg) in duplicate for 0.1-24 h in Medium 199 containing 10% fetal bovine serum and [3H]F and [14C]E. Radiolabelled F and E were purified from incubates and the percentage conversion of F to E and E to F was calculated. In decidua, mean (+/- SE; n = 3) conversion of E to F (69 +/- 2%) was greater than oxidation of F to E (26 +/- 2%). Conversion of E to F in placenta (50 +/- 1%) and chorion (39 +/- 9%) was also extensive and greater than or equal to that for the oxidation of F to E (39 +/- 4% and 32 +/- 4%, respectively). The apparent ratio of 11beta-HSD reductive/oxidative activity was maintained when respective tissues from four baboons were incubated for 18 h with or without 4.4 microM excess radioinert substrate. Expression of 11beta-HSD-1 mRNA was determined by Northern blot by hybridization of poly (A) + -enriched RNA from tissues obtained at midgestation with [32P]labelled human 11beta-HSD-1 cDNA. This cDNA hybridized to a single mRNA species of 1.6 kb in decidua, whole placental villous tissue and syncytiotrophoblast cells, but not to RNA isolated from fetal baboon kidney. The results of the present study demonstrate that at midgestation in the baboon, 11beta-HSD activity in intact placenta and decidua in vitro favored the formation of F from E, presumably catalysed by the 11beta-HSD-1 enzyme protein, the mRNA for which is present in placental syncytiotrophoblasts. Moreover, based on overall mass and extensive vascularity, we suggest that the net formation of F from E during transuterine passage in vivo at this stage of gestation results from the 11beta-HSD-1 in the syncytiotrophoblast cells of the baboon placenta.

Localization and Developmental Expression of the Activin Signal Transduction Proteins Smads 2, 3, and 4 in the Baboon Fetal Ovary

Abstract 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 α-inhibin, but not activin βA and βB 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.

Regulation of Expression of Microvillus Membrane Proteins by Estrogen in Baboon Fetal Ovarian Oocytes

Abstract 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.