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.     

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.     

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.     

Developmental maturation of primate placental trophoblast: placental cytosolic and secretory phospholipase A2 expression after estrogen suppression of baboons

We have demonstrated that the baboon placenta expressed the mRNAs and proteins for secretory and cytosolic phospholipase A2 (PLA2) enzymes and that cPLA2 expression increased with advancing gestation in association with the increase in placental estrogen production. To determine whether estrogen regulates placental PLA2 expression, as it does other aspects of syncytiotrophoblast functional differentiation, we compared sPLA2 and cPLA2 mRNA levels in placentas obtained on day 165 of gestation (term = day 184) from baboons that were untreated or treated during the second half of gestation with the aromatase inhibitor CGS 20267 or CGS 20267 and estradiol. Maternal saphenous and uterine vein estradiol levels were reduced (P < 0.05) by approximately 95% by treatment with CGS 20267 and restored by concomitant administration of CGS 20267 and estrogen. However, sPLA2 and cPLA2 mRNA levels expressed as a ratio of beta-actin were similar in whole villous placenta from baboons that were untreated or treated with CGS 20267 or CGS 20267 plus estrogen. PLA2 expression in an enriched fraction of nontrophoblast cells of the baboon placenta was also not altered by CGS 20267 treatment. Collectively these findings indicate that placental cPLA2 and sPLA2 expression is not estrogen-dependent. Because estrogen has been shown to regulate other aspects of placental steroidogenesis, we suggest that the regulatory role of estrogen on syncytiotrophoblast functional maturation is specific.     

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.     

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.     

Effect of estrogen on the metabolism of cortisol and cortisone in the baboon fetus at midgestation

To determine whether the metabolism of cortisol (F) and cortisone (E) in the baboon fetus is regulated by estrogen, fetal interconversion of F/E was measured at midgestation after an experimental increase in placental estradiol (E2) production. Six baboons (Papio anubis) received increasing numbers of androstenedione implants (50 mg) inserted s.c. at 8-day intervals between Days 70 and 100 of gestation (term = Day 184) to elevate the production of estrogen; controls (N = 8) received no treatment. On Day 100 of gestation, each animal was anesthetized with ketamine:halothane/nitrous oxide, the fetus was exteriorized and [3H] F/[14C] E was infused via a fetal femoral vein for 70 min. Blood samples were then obtained from the contralateral fetal femoral vein, the umbilical vein/artery, and a maternal saphenous vein. After purification of F and E, the metabolic clearance rate (MCR), peripheral interconversion, and placental extraction of F and E were calculated. Maternal serum E2 concentrations (ng/ml; mean +/- SE) between Days 80 and 100 of gestation were greater (p less than 0.01) in androstenedione-treated baboons (2.2 +/- 0.2) than in untreated controls (1.2 +/- 0.1). Although the MCR of F was similar in control (5.2 +/- 0.3 1/day) and treated (7.7 +/- 1.0 1/day) animals, the MCR of E (13.5 +/- 2.0 1/day) was increased (25.8 +/- 2.5 1/day; p less than 0.05) by androstenedione treatment. Placental extraction of F (59 +/- 9%) was lower (p less than 0.01) than that of E (82 +/- 5%) in untreated baboons and was not affected by androstenedione treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.     

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

In the mammalian ovary, FGF10 is expressed in oocytes and theca cells and is a candidate for paracrine signaling to the developing granulosa cells. To gain insight into the participation of FGF10 in the regulation of fetal folliculogenesis, we assessed mRNA expression patterns of FGF10 and its receptors, FGFR1B and FGFR2B, in relation to fetal follicle dynamics and localized FGF10 protein in bovine fetal ovaries at different ages. Primordial, primary, secondary, and antral follicles were first observed on Days 75, 90, 150, and 210 of gestation, respectively. The levels of GDF9 and BMP15 mRNA, markers for primordial and primary follicles, respectively, increased during fetal ovary development in a consistent manner with fetal follicle dynamics. CYP17A1 mRNA abundance increased from Day 60 to Day 75 and then from Day 120 to Day 150, coinciding with the appearance of secondary follicles. FGF10 mRNA abundance increased from Day 90, and this increase was temporally associated with increases in FGFR1B mRNA abundance and in the population of primary follicles. In contrast, FGFR2B mRNA expression was highest on Day 60 and decreased thereafter. FGF10 protein was localized to oogonia and oocytes and surrounding granulosa cells at all fetal ages. The present data suggest a role for FGF10 in the control of fetal folliculogenesis in cattle.     

Activation of bovine and baboon primordial follicles in vitro

Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles.     

Differential response to gonadotropins and prostaglandin E2 in ovarian tissue during prenatal and postnatal development in cattle.

In cattle, growing follicles are present in fetal ovaries during the last part of gestation. This study examines the extent of changes in basal and hormone-stimulated adenylyl cyclase (AC) activity in ovaries of the bovine fetus when the first follicles begin to grow. The first growing follicles appeared in fetal ovaries around Day 180 and consisted mainly of primary and secondary follicles; few antral follicles were present before Day 220 of gestation. Basal AC activity in ovarian membranes increased simultaneously with the beginning of follicle growth in the fetus (5.8 +/- 0.9 vs. 9.3 +/- 1.3 pmol cAMP/mg protein/min at 130-180 and 180-210 days of gestation, respectively p less than 0.05). During the same time period, there was a significant increase in both the absolute (16.1 +/- 1.2 to 39.9 +/- 1.4 pmol cAMP/mg protein/min) and the relative (2.8 +/- 0.1 to 4.3 +/- 0.3 times the basal level, p less than 0.05) effects of guanosine triphosphate (GTP). After birth, basal and GTP-stimulated AC activities (pmol cAMP/mg protein/min) increased markedly in ovarian membranes of 1-wk-old calves and then decreased with age; the lowest levels were measured in mature cyclic cows. However, the relative effect of GTP (times the basal level) did not show this age-related variation. Prostaglandin E2 (PGE2) stimulation of AC in ovarian membranes from fetuses was high even on Day 120 (2.1 +/- 0.3 times the control level).(ABSTRACT TRUNCATED AT 250 WORDS)     

Source and regulation of 17 alpha-hydroxyprogesterone during baboon pregnancy

The present study determined the source and regulation of 17 alpha-hydroxyprogesterone (17-OHP4) during mid-late baboon pregnancy. Serum 17-OHP4 (ng/ml) in 5 untreated baboons increased from low values at mid-late gestation to a mean (+/- SEM) of 0.49 +/- 0.02 during the final 20 days of gestation. Fetectomy of 5 baboons resulted in serum 17-OHP4 concentrations which declined to and remained at baseline. Serum 17-OHP4 concentrations were 5- to 10-fold greater (P less than 0.001) in the uterine, utero-ovarian, and umbilical veins than peripherally. Apparently the fetal adrenal provides precursors for placental 17-OHP4 formation because the fetal adrenal gland develops delta 5-3 beta-hydroxysteroid dehydrogenase only late in gestation, and because the fetal adrenal and not the placenta has the capacity for 17-hydroxylation. Thus, at mid-late gestation the placenta appears to supply a major, and at term the corpus luteum a minor portion of the total 17-OHP4. Administration of the estrogen antagonist ethamoxytriphetol (MER-25, 15 mg/kg BW) to 4 baboons did not affect 17-OHP4 during mid-late gestation, when the placenta was the only source of 17-OHP4. However, MER-25 resulted in serum 17-OHP4 concentrations (ng/ml) at term which were greater (1.08 +/- 0.10, P less than 0.001) than in untreated baboons (0.49 +/- 0.02). Prior removal of the corpus luteum of pregnancy in 4 animals subsequently given MER-25 prevented this rise in 17-OHP4. This suggests that the marked elevation in 17-OHP4 observed near term after MER-25 administration was of luteal origin and that antiestrogen enhanced 17-OHP4 secretion by the corpus luteum.     

Immunolocalization of GnRHRI,gonadotropin receptors,PGR, and PGRMCI during follicular development in the rabbit ovary

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.     

Regulation of placental low-density lipoprotein uptake in baboons by estrogen: dose-dependent effects of the anti-estrogen ethamoxytriphetol (MER-25)

In the present study, increasing amounts of the anti-estrogen 1-(p-2-diethylaminoethoxyphenyl)-1-phenyl-2-p-methoxyphenoletha nol (MER-25) were administered to pregnant baboons (Papio anubis) to block the action of endogenous estrogen and to determine effect on placental low-density lipoprotein (LDL) uptake. Pregnant baboons were untreated (n = 8) or received MER-25 orally at a dosage of 25 (n = 10), 50 (n = 8), or 75 (n = 4) mg/kg BW daily on Days 140-170 of gestation (term = 184 days). Placentas were removed on Day 170 of gestation and villous tissue was dispersed with 0.1% collagenase. Placental cells (10(6] were incubated in Medium 199 for 12 h at 37 degrees C with increasing amounts of 125I-LDL, with or without a 100-fold excess of unlabeled baboon LDL. Mean (+/- SEM) placental uptake (ng/micrograms cell protein) of 125I-LDL was 55% (6.4 +/- 1.0), 75% (3.6 +/- 0.7), and 81% (2.7 +/- 0.2) lower (p less than 0.001) in baboons that received MER-25 in doses of 25, 50, and 75 mg/kg BW, respectively, than in untreated baboons (14.2 +/- 1.3 ng/micrograms cell protein). Maximal effect occurred with 50 mg MER-25, because LDL uptake was not further decreased with greater levels of MER-25. Dissociation constants for placental LDL uptake, as determined by Scatchard analysis, were unaltered by anti-estrogen treatment. The amount of 125I-LDL degradation by placental cells of untreated and MER-25-treated baboons was proportional to LDL uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological development and characterization of aromatase and estrogen receptors alpha and beta in fetal ovaries of cattle from days 110 to 250

To better understand the role of estradiol-17β in fetal ovarian development, presence and localization of cytochrome P450 aromatase (P450arom) and estrogen receptors alpha (ERα) and beta (ERβ) proteins were characterized in fetal ovaries of cattle using immunohistochemistry. Fetal cattle ovaries were collected from an abattoir and sorted into fetal age groups (days 110, 130, 150, 170, 190, 210, 230, 250+) based on crown-rump length. In addition to immunohistochemistry, morphological analysis of ovarian and follicular formation was made. Ovaries appeared lobular at day 110, but by the end of gestation (day 250+) ovaries were oval-shaped similar to those found in adult animals. Ovarian structures within different lobes appeared to be at different developmental stages. At day 110, oocytes and pre-granulosa cells were observed in ovigerous cords that were still open to the surface epithelium. Most ovigerous cords appeared to be closed to the surface epithelium on day 130, all closed by day 150 and were no longer present at day 210. Ovarian follicles were classified as follows: Type 1(primordial): single layer of flattened granulosa cells, Type 1a (transitory): single layer of mixed flattened and cuboidal granulosa cells, Type 2 (primary): at least one but less than two layers of cuboidal granulosa cells, Type 3 (small preantral): two to three layers of granulosa cells, Type 4 (large preantral): four to six layers of granulosa, and the theca layer is forming around the follicle, Type 5 (antral): contain greater than six layers of granulosa cells, several layers of theca cells and the antrum has formed. Type 1 follicles were observed in day 110 ovaries. Follicle Types 1a and 2 were first observed on day 130. Type 3 follicles were first observed on day 150 and Types 4 and 5 were first observed on day 170. P450arom protein was localized in granulosa cells of follicle Types 2–5 and cells of rete tubules throughout the experimental period. There was punctate expression within stroma and rete masses. There was ERα protein localization in pre-granulosa cells and germ cells of ovigerous cords and all surface epithelial cells. There was also localization in granulosa cells and oocytes of all follicle types and cells of rete tubules. There was punctate ERα protein expression in stroma and rete masses. ERβ protein was localized in pre-granulosa cells and germ cells of ovigerous cords. Expression was also localized to granulosa cells of all follicle types and cells of rete tubules. ERβ protein was punctate in oocytes of follicles, surface epithelial cells, stroma and rete masses. Thus, the fetal ovary of cattle has the steroidogenic enzyme (P450arom) to convert androgens to estradiol-17β, and estrogen receptors α and β to facilitate an estrogen response within the fetal ovary.     

Ontogeny of hematological cell and biochemical profiles in maternal and fetal baboons (Papio species)

The normal ranges of hematological cell profiles and biochemistry are documented in adult non-pregnant, pregnant, juvenile, and neonatal baboons. Despite the extensive use of the baboon as a model for the study of various aspects of pregnancy, there is no data from paired mothers and their fetuses at different stages of gestation. Hematologic and biochemical profile data were obtained from eight non-pregnant female baboons, 37 mothers and 38 fetal baboons at 30 +/- 2, 90 +/- 2, 125 +/- 2, and 175 +/- 2 days of gestation (mean +/- range; dGA; term, 180 dGA). Changes observed in fetal and maternal blood during normal baboon pregnancy were similar to those reported in human pregnancy. The level of alkaline phosphatase was two times higher in fetal blood circulation than that reported in human pregnancy.     

The ontogeny of components of the renin–angiotensin system in the porcine fetal ovary

There is an autonomous renin–angiotensin system (RAS) in the adult ovary. Renin is present in the primitive kidney, and the fetal ovary develops from the nephrogenic ridge. We hypothesised that components of the ovarian RAS would be present from early gestation, with potential roles in ovarian development. We studied fetal pig ovaries from approximately day 45 (～0.39 gestation) to term and measured mRNA (RT-PCR) for prorenin, angiotensinogen and the angiotensin II (AngII) Type 1 and 2 receptors (AT₁ and AT₂), and protein expression (Western blot) and localization (immunohistochemistry) of the AT₁ and AT₂ receptors. mRNA for prorenin was present in relatively low abundance from at least day 45 and rose to ～day 75 of gestation, whilst mRNA for angiotensinogen rose steadily. mRNA for the AT₁ receptor was present from approximately day 45 and did not alter significantly with increasing gestation but AT₂ receptor mRNA was initially high, falling sharply through pregnancy. The AT₁ receptor protein abundance fell steadily to term, whereas the AT₂ receptor protein did not change during gestation. Both receptors were localised in the surface epithelium and egg nests, the granulosa cells of primordial, primary and secondary follicles, and the oocytes of all except the secondary follicles. Collectively, our results support the hypothesis that there is a functional RAS in the fetal ovary from at least approximately day 45 of gestation until term and that it may have a paracrine role in ovarian growth and development.