Gerald J. Pepe

Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues. In situlocalization of DNA strand breaks in fixed human and baboon ovarian tissue sections indicated that apoptosis was essentially restricted to granulosa cells of atretic antral follicles. Biochemical analysis of DNA oligonucleosomes in individual follicles isolated from baboon ovaries during the ovulatory phase revealed the presence of apoptotic DNA fragments in subordinate but not dominant follicles, thus substantiating the in situ labeling studies. Messenger RNA transcripts encoded by the bax death susceptibility gene, the bcl-xlong survival gene, the bcl-xshort pro-apoptosis gene, the p53 tumor suppressor gene, and two members of the CASP gene family (CASP-2/Ich-1, CASP-3/CPP32), were detected by Northern blot analysis of total RNA prepared either from human ovaries or from Percoll-purified granulosa-lutein cells obtained from patients undergoing assisted reproductive technologies. Lastly, immunohistochemical localization of the BAX death-susceptibility protein in the human ovary revealed abundant expression in granulosa cells of early atretic follicles, whereas BAX protein was extremely low or non-detectable in healthy or grossly-atretic follicles. We conclude that apoptosis occurs during, and is probably responsible for, folicular atresia in the human and baboon ovary. Moreover, apoptosis in the human ovary is likely controlled by altered expression of the same cohort of cell death regulatory factors recently implicated as primary determinants of apoptosis induction or suppression in the rodent ovary.

Developmental Regulation of Baboon Fetal Ovarian Maturation by Estrogen

Abstract 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 α and β 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 mm2) 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 mm2 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.

Estrogen regulation of placental angiogenesis and fetal ovarian development during primate pregnancy.

During human and nonhuman primate pregnancy, an extensive blood vessel network is established within the villous placenta to support fetal growth and follicles develop within the fetal ovary to provide a pool of oocytes for reproductive function in adulthood. These two important developmental events occur in association with a progressive increase in placental estrogen production and levels. This review will describe the developmental processes required for placental vascularization and fetal follicular maturation and recent studies which show that estrogen has an important role in regulating these events.

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.

Effect of Estrogen on Vascular Endothelial Growth/Permeability Factor Expression by Glandular Epithelial and Stromal Cells in the Baboon Endometrium

Abstract The ovarian steroid hormones, estrogen and progesterone, have important roles in establishing the new vascular bed within the endometrium during each menstrual cycle; however, little is known about the mechanisms underlying this process. We recently showed that mRNA and protein levels for the angiogenic factor vascular endothelial growth/permeability factor (VEG/PF) in endometrial glandular epithelial and stromal cells of baboons were decreased to very low levels by ovariectomy, and we proposed that the levels of estrogen and progesterone exhibited during the menstrual cycle regulate endometrial VEG/PF expression in the primate. To test this hypothesis, VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in glandular epithelial and stromal cells isolated by laser-capture microdissection from, and VEG/PF protein was determined by immunocytochemistry in the endometrium of baboons after ovariectomy and chronic administration of estradiol and progesterone in levels designed to replicate the hormonal profiles that are characteristic of the proliferative and secretory phases of the menstrual cycle. Administration of estradiol to ovariectomized baboons in levels that replicated the late-proliferative phase of the menstrual cycle (209 ± 40 pg/ml serum) increased/restored VEG/PF mRNA to levels in the glands (5.57 ± 1.53 amol/fmol 18S rRNA, P < 0.01) and stroma (2.61 ± 1.57 amol/fmol 18S rRNA, P < 0.02) that were approximately 10-fold greater than those observed after ovariectomy alone (0.52 ± 0.21 and 0.22 ± 0.11 amol/fmol 18S rRNA, respectively) and were similar to those previously shown in intact baboons. Concomitant administration of estradiol and progesterone to ovariectomized baboons in levels that replicated the midsecretory phase of the menstrual cycle (44 ± 15 pg/ml serum and 9.8 ± 2.2 ng/ml serum, respectively) resulted in glandular epithelial (3.65 ± 1.42 amol/fmol 18S rRNA) and stromal (1.25 ± 0.77 amol/fmol 18S rRNA) VEG/PF mRNA levels that were not significantly different from those exhibited after ovariectomy or ovariectomy and estradiol treatment. Comparable results were obtained for VEG/PF mRNA expression in whole-endometrial tissue, although the relative 2-fold increase (P < 0.03) in VEG/PF mRNA levels induced by estrogen in mixed endometrial cells of ovariectomized baboons appeared to be less marked than that in isolated glandular epithelial and stromal cells. After ovariectomy, endometrial width (0.98 ± 0.09 mm) was approximately one-third of that in intact baboons (3.58 ± 0.32 mm), and endometrial VEG/PF protein expression was low. Estradiol restored endometrial width (3.00 ± 0.12 mm, P < 0.01) and VEG/PF protein expression to normal. In summary, estrogen has a significant role in regulating and maintaining VEG/PF expression by glandular epithelial and stromal cells of the endometrium during the menstrual cycle.

Expression of estrogen receptors α and β in the baboon fetal ovary

Abstract 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 ERα and/or ERβ 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, ERα protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERβ 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, ERα expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ERα expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERβ 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 ERβ, ERβ 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 ERα and ERβ were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ERα and ERβ 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.

Effect of estrogen on dehydroepiandrosterone formation by baboon fetal adrenal cells in vitro

The present study determined if estrogen modulates the responsivity of the adrenal gland of the baboon fetus to tropic hormones such as adrenocorticotropic hormone and prolactin. Adrenal glands were obtained from seven baboon fetuses at midgestation (days 100 to 105). Adrenal cells were dispersed in medium 199 with 0.2% collagenase for 10 minutes at 37 degrees C. Approximately 10(5) cells/4.0 ml of medium 199 were incubated for 24 hours at 37 degrees C with 10 nmol of adrenocorticotropic hormone or 10 nmol of ovine prolactin in the presence or absence of 10(-5) or 10(-6) mol/L of estradiol. The major steroid formed and secreted into the medium was dehydroepiandrosterone. Mean +/- standard error basal formation of dehydroepiandrosterone was 176 +/- 64 ng/10(5) cells/24 hours. Dehydroepiandrosterone formation was increased (p less than 0.05) 3.5-fold and five-fold by adrenocorticotropic hormone and prolactin, respectively. Estradiol at 10(-5) mol/L prevented the response in dehydroepiandrosterone obtained with adrenocorticotropic hormone alone. Estradiol alone had no effect on dehydroepiandrosterone. The results suggest that estrogen modulates the regulatory effects of adrenocorticotropic hormone on dehydroepiandrosterone formation by the adrenal gland of the baboon fetus.

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...

d-cycloserine improves sociability and spontaneous stereotypic behaviors in 4-week old mice

Balb/c mice are a model of impaired sociability and social motivation relevant to autism spectrum disorders (ASDs). Impaired sociability of 8-week old Balb/c mice is attenuated by agonists of the glycine(B) site on the NMDA receptor, such as d-cycloserine. Although ASDs are often recognized in toddlerhood, there is interest in earlier identification (e.g., before 6 months) and disease-modifying interventions to improve functional outcomes. Thus, we wondered if d-cycloserine could improve sociability in 4-week old Balb/c mice, similar to its effects in 8-week old mice. d-Cycloserine improved measures of impaired sociability in 4-week old (i.e., one-week post-weanling) Balb/c mice. Moreover, because stereotypies can compete with the salience of social stimuli, we compared Balb/c and Swiss Webster mice on several spontaneous stereotypic behaviors emerging during social interaction with a social stimulus mouse. Interestingly, similar to 8-week old mice, spontaneous stereotypic behaviors during social interaction were more intense in the 4-week old Swiss Webster mice; furthermore, d-cycloserine reduced their intensity. Thus, d-cycloserine improves both sociability and stereotypic behaviors, but these effects may lack strain-selectivity. A prosocial effect of d-cycloserine was observed at a dose as low as 32.0mg/kg in Balb/c mice. d-cycloserine has the therapeutic properties of a desired medication for ASDs; specifically, a medication should not improve stereotypic behaviors at the expense of worsening sociability and vice versa. The data suggest that targeting the NMDA receptor can have promising therapeutic effects on two prominent domains of psychopathology in ASDs: impaired sociability and spontaneous stereotypic behaviors.

Immunocytochemical identification of the oestrogen receptor in the nuclei of cultured human placental syncytiotrophoblasts

We have shown that oestrogen has a central integrative role in regulating key components of the progesterone biosynthetic and corticosteroid metabolic pathways within syncytiotrophoblasts that govern placental function and maturation of the fetal pituitary-adrenocortical axis. Studies utilizing classic binding procedures and RNAse protection have demonstrated that human placental villous tissue exhibits specific high affinity oestrogen binding and expresses the mRNA for the oestrogen receptor. However, it is not known whether the oestrogen receptor is expressed specifically in syncytiotrophoblasts. Therefore, the present study determined whether the oestrogen receptor protein was detectable by immunocytochemistry in cultured human syncytiotrophoblast maintained in a low oestrogen/progestin environment. Cytotrophoblasts were isolated from human term placentae by trypsin dispersion and Percoll gradient centrifugation and cultured for 5, 7 or 10 days. Incubation of syncytiotrophoblast with 5-10 micrograms/ml of the anti-oestrogen receptor rat monoclonal antibody D-75, which is specific for the primate oestrogen receptor, resulted in identification of the oestrogen receptor in the nuclei of these cells. In contrast, there was no reactivity of the trophoblasts to either rat IgG or an irrelevant rat monoclonal antibody IgG2a against mouse common leukocyte antigen T200. Collectively, these findings indicate that oestrogen receptor is expressed in the nuclei of human placental syncytiotrophoblasts and support the suggestion that the syncytiotrophoblast is an oestrogen-responsive tissue.