Estrogen receptors in ovary and uterus of immature hamster and rat: effects of estrogens

Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1 mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2 mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26. Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52 nM). After ECP treatment, there was a tendency for translocation in all 4 tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.     

In utero exposure of mice to diethylstilbestrol alters neonatal ovarian follicle growth and development

The prenatal exposure of mice to diethylstilbestrol (DES, 10 micrograms/kg on day 15 of gestation) caused both quantitative and structural alterations in ovarian follicles within the neonatal ovary. At birth, control ovaries consisted of small type 1 and 2 ovarian follicles located in the ovarian cortex. By postnatal day 7, ovarian follicle development had advanced to the type 4 stage with larger follicles located within the ovarian medulla. In DES-exposed animals, ovarian follicle maturation was advanced with type 3b and 4 follicles appearing 24 h prior to their appearance in control animals. Also, type 5 ovarian follicles were present on postnatal day 6 in experimental animals but were never seen in control animals. In addition to an alteration in ovarian follicle dynamics, the diameter of individual ovarian follicles was transit time between the various stages of follicular development which results in a greater number of developmentally advanced ovarian follicles being present during neonatal ovarian development. The mechanism by which prenatal exposure to DES alters ovarian follicle dynamics during neonatal development is not known.     

Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.     

Effects of progesterone on the oestrogen-stimulated uterus: a comparative study of the mouse, guinea pig, rabbit and sheep.

To examine more closely the anti-oestrogenic action of progesterone (P), its effect on various parameters in the 17 beta-oestradiol (E2)-primed uterus of the mouse, guinea pig, rabbit and ewe was studied. Changes in uterine wet weight, rate of in vitro protein synthesis, protein : DNA and RNA : DNA ratios, peroxidase activity and the level of cytosol receptors for E2 and P were measured. Considerable between-species differences in the effect of P on these parameters were observed. The anti-uterotrophic action was greater in the mouse than in the guinea pig and was not seen in the rabbit or ewe. P inhibited protein synthesis in the mouse, was without significant effect in the guinea pig and was mildly stimulatory in the rabbit and ewe. Inhibitory effects on protein : DNA and RNA : DNA ratios were substantial in the mouse, minor in the guinea pig and absent in the rabbit and ewe. Peroxidase activity was decrease in the mouse and guinea pig, essentially lacking in the rabbit and not detectable in the ewe. In all species the level of both oestrogen and progesterone cytosol receptors was decreased, although the effect on the E2 receptor was less marked in the ewe. The results indicate that in the species studied an effect of P on the replenishment of the E2 receptor is not necessarily associated with other anti-oestrogenic actions of P and argue against the concept that initial anti-oestrogenic actions of P are mediated via a specific effect on such replenishment.     

Ovarian sympathectomy in the guinea pig

Summary The role of ovarian adrenergic nerves in follicular growth was studied in prepubertal guinea pigs by determining the effect of sympathectomy on 1) follicle populations and 2) follicular development following exogenous gonadotropin administration. Selective unilateral ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian bursa on day 20 postpartum. The contralateral surgically closed ovarian bursa was injected with the vehicle used for 6-hydroxydopamine. On day 25, animals were injected with pregnant mare serum or saline followed by human chorionic gonadotropin or saline 48 h later. All animals were laparotomized on day 28 and blood from utero-ovarian veins was collected bilaterally for androstenedione determination. Ovaries were processed for morphometric analysis of follicles. The sympathectomized ovary in saline-injected animals had a significant decrease in preantral follicles (characterized by 2 layers of granulosa cells without antrum formation), an increase in 310–500 m diameter atretic follicles and an increase in follicles 700 um compared to the contralateral control ovary. There were no differences in androstenedione levels from the two sides, ovarian weights or the total number of follicles per ovary. Neither ovary had corpora lutea. The sympathectomized ovary in animals injected with gonadotropins was not different from the contralateral ovary in any of the parameters measured. Both control and sympathectomized ovaries had newly formed corpora lutea in response to the exogenous gonadotropins. These results suggest that ovarian adrenergic nerves normally participate in follicular development in the prepubertal guinea pig. However, exogenous gonadotropins may override neural influences on the prepubertal ovary.     

Hormonal regulation of LH receptor mRNA and expression in the rat ovary.

Agonist-induced changes in expression and mRNA levels of luteinizing hormone (LH) receptors were compared during stimulation of ovarian follicular maturation and luteinization by gonadotropic hormones. Three major species of LH receptor mRNA, 5.8, 2.6 and 2.3 kb, were present throughout differentiation and changed similarly, the 5.8 kb species being consistently more abundant than the smaller forms. The increased expression of plasma-membrane LH receptors in preovulatory follicles and luteinized ovaries and their homologous down-regulation during follicular and luteal desensitization were closely correlated with the steady-state receptor mRNA levels. The reappearance of LH receptors following desensitization during the luteal stage was preceded by an increase in mRNA levels. These studies have demonstrated that the expression of LH receptors during follicular maturation, ovulation and desensitization is related to the prevailing levels of receptor mRNA in the ovary.     

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.     

Expression of GDF-9, BMP-15 and their receptors in mammalian ovary follicles

The synergetic process of folliculogenesis is mainly regulated by GDF-9 and BMP-15 as well as their receptors, such as BMPR2, TβR1 and BMPR1B. Expressions of these factors and the receptors are significant different among species. This study was designed to detect expression of GDF-9, BMP-15 and their receptors in mouse, porcine and human healthy follicles by immunohistochemistry. Three ages of human ovary were studied according to ovarian developmental schedule, i.e. gestational week (GW) 16, puberty (14 year-old) and adult (40 year-old). The results showed that both GDF-9 and BMP-15 were detectable in oocytes from primary follicles onward, besides, BMP-15 also presented in granulosa cells (GCs) and follicular follicle of mature follicles in mouse. However, they were maintained in oocytes and GCs from primordial to mature follicles in porcine except that GDF-9 was undetectable in GCs of mature follicles. For human ovary, GDF-9 presented in oocytes of primordial follicles in all samples, whereas BMP-15 was only observed in primordial follicle of adult ovary. Receptors, BMPR2, TβR1 and BMPR1B were found in oocytes and GCs of all follicles in mouse and porcine. In human, they were stained in oocytes from primordial follices but BMPR1B was not expressed in pubertal primordial follicles. Furthermore, we found that GDF-9, BMP-15 and three receptors distributed in adult corpus lutea. Collectively, our studies suggested that GDF-9, BMP-15 and their receptors might correlate with primordial follicular recruitment in pig and human. Positive expression of the receptors (BMPR2, TβR1 and BMPR1B)in primordial follicles of mouse ovaries indicated that these receptors might interact with others ligands besides GDF-9 and BMP-15 to regulate primordial follicular activity in mouse. Moreover, presence of GDF-9 in oocytes and BMP-15 in oocytes and GCs of mature follicles from mice and porcine elucidated coordinated roles of GDF-9 and BMP-15 in cumulus oophorus expansion. Additionally, expression of these factors in adult human corpus lutea suggested they play roles in corpus luteum activity.     

Vascular endothelial growth factor stimulates preantral follicle growth in the rat ovary

The regulation of preantral follicle growth in mammals is poorly understood. The availability of an adequate vascular supply to provide endocrine and paracrine signals may be important during the early states of follicle growth as well as the later states of follicle selection and dominance. The objective of the present study was to investigate whether vascular endothelial growth factor (VEGF) plays a role in preantral follicular development in the rat ovary. Immature (age, 21 days) Sprague-Dawley rats were injected with 500 ng of VEGF in saline or 50 microg of diethylstilbestrol (DES) in oil under the bursa of one ovary. The contralateral ovary was injected with a corresponding volume of vehicle. Rats were killed 48 h later, and the ovaries were removed and analyzed histologically. Intrabursal administration of VEGF significantly increased the number of primary and small secondary, but not of large secondary, preantral follicles in the ovary, similar to the effect of DES (P < 0.05). The VEGF stimulated preantral follicle growth in a time- and dose-dependent manner. Subcutaneous DES administration increased the number of primary and secondary follicles, and both s.c. and intrabursal estrogen administration stimulated VEGF protein expression in the rat ovary. These data indicate that VEGF stimulates preantral follicular development in the rat ovary, is regulated by estrogen, and may be one of the factors that participate in the regulation of early follicle growth in the rat.     

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.     

The effects of age and sex steroids on the macrophage population in the ovary of the chicken, Gallus domesticus

The role of macrophages in the function of the hen ovary has not yet been described, although these cells may be an important regulator of ovarian function in mammals. The aim of this study was to determine the changes in the frequency of macrophages during ageing and follicular atresia, and the effects of sex steroids on the macrophage population in the hen ovary. Cryostat sections of ovarian tissues of immature, young laying and old laying hens and those of immature hens treated with or without diethylstilboestrol (DES) or progesterone were immunostained for macrophage cells using mouse anti-chicken macrophage monoclonal antibody. Macrophages were observed under a light microscope and counted using a computer assisted image analyser. The frequency of macrophages in both the stroma and theca of primary follicles was significantly greater in young laying hens than in immature and old laying hens and these cells were more frequent in old laying hens than in immature hens (P < 0.01). Macrophages were more frequent in atretic follicles than in normal follicles (P < 0.01). The number of macrophages in both the stroma and theca of primary follicles of DES-treated birds was significantly greater than in those of progesterone-treated and control birds (P < 0.01). Progesterone had no significant effect on the population of macrophages. These results suggest that macrophages in the ovary increase in association with sexual maturation of birds and atresia of follicles and decrease during ageing. Oestrogen may be one of the factors that affect the population of macrophages in the hen ovary.     

Ovarian sympathectomy in the guinea pig

The influence of ovarian adrenergic nerves on follicular growth during the estrous cycle in the adult guinea pig was ascertained by comparing follicular development in control and chemically sympathectomized ovaries from the same animal. Selective ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian membranous sac (bursa) on day 2 of the cycle (day 1 = day of estrus). The contralateral surgically closed ovarian bursa was injected with solvent used for 6-hydroxydopamine. Animals were laparotomized on days 5, 10 and 14 of the cycle. Blood from the utero-ovarian vein was collected bilaterally for measurement of progesterone and androstenedione. The ovaries were processed for histologic examination, and the number of follicles in each ovary was analyzed morphometrically. Sympathectomy on day 2 caused a decrease in healthy preovulatory follicles (greater than 700 micron diameter) on day 10 of the cycle. There were no differences in ovarian weights or the total number of follicles per ovary at this time. On days 5 and 14 of the cycle, there were no differences in ovarian weights, total number of follicles per ovary or follicles in any size classification. Sympathectomy did not alter progesterone levels in the utero- ovarian vein as compared to contralateral control levels. From control ovaries, there was a significant increase in progesterone in the blood of the utero-ovarian vein on day 10 but venous levels of progesterone from sympathectomized ovaries were not significantly different at any day of the cycle. In the venous effluent from sympathectomized ovaries, androstenedione was elevated at day 5 compared to days 10 and 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of buffalo follicular fluid treatment on follicle population and ovulation rate in guinea pigs

A number of workers have studied the effect of follicular fluid (FF) on the secretion of follicular stimulating hormone (FSH) but little is known about its potential as a regulator of ovarian activity, including ovulation rate. This paper describes the effect of charcoal treated-buffalo follicular fluid (buFF) treatment on follicular growth and ovulation rate in guinea pigs. Eighteen guinea pigs in three groups of 6 each were given 0.2 ml buFF at 12 hr interval for 3 days at different stages of estrous cycle viz., early-luteal, mid-luteal or follicular phase. One control group received equal volume of saline. Estrus was monitored every morning and evening by inspection of the opening of vaginal membrane and its cytology. All animals were sacrificed at 24 hr after the onset of estrus. Both the ovaries were dissected out, weighed and number of ovulation points recorded. One ovary from each animal was processed for histological examination to determine the population of healthy and atretic follicles. In early-luteal and follicular phase-treated animals the onset of estrus was delayed (P < 0.01) and ovulation rate was not affected. However, estrus occurred at normal when the treatment was initiated at midluteal stage and 50% animals failed to ovulate in this group. The total follicle population at metestrus increased significantly in all treated animals because of increase in number of follicles of size class II (400 to < 600 microns diam.). Atresia was also declined due to treatment. These results demonstrated that the buFF contained some inhibitory substances that delayed the onset of estrus in guinea pigs.     

Effect of tamoxifen on the Notch signaling pathway in ovarian follicles of mice

ABSTRACT

We investigated the effect of tamoxifen (TAM) treatment on the Notch signaling pathway in mouse ovary. Mice were randomly divided into four groups. Control group A animals were untreated. Control group B animals were treated with the vehicle only. Animals of the 0.5 TAM group received 0.5 mg/day TAM. Animals of the 1.5 TAM group received 1.5 mg/day of TAM. TAM was injected subcutaneously for 5 days. Body weights were measured at the start and end of the experiment. Sections were stained using Crossman’s modified trichrome to examine general ovarian structure. Other sections were immunostained to demonstrate Jagged 1, Ki 67 and Notch 2. The TUNEL method was used to detect apoptosis. No significant differences in body weight or ovarian weight were found among the experimental groups. The number of primordial follicles was greater in the treatment groups than in the control groups, while the number of antral follicles and corpora lutea were reduced in the treatment groups. Cell proliferation rates were decreased by TAM treatment and cystic follicles were formed in the ovarian stroma. Notch 2 expression in the granulosa cells was increased following TAM administration, but no change was found in Jagged 1 expression. TAM administration suppressed follicular development and exhibited a negative effect on ovarian morphology. Our findings suggest that the Notch pathway participates in the action of TAM. We suggest that it may be useful to use Notch pathway regulators to adjust the effects of TAM on the ovary.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Presence of immunoreactive atrial natriuretic peptide in follicular fluid, ovary and ovarian perfusates

Immunoreactive atrial natriuretic peptide (ir-ANP) was measured in the follicular fluid of pig ovarian follicle, and rabbit ovarian homogenates and perfusates using a specific radioimmunoassay (RIA). Serial dilution curves made with the extracts of follicular fluid, ovarian homogenates and perfusates using SepPak C18 cartridges were parallel with the RIA standard curve. On gel filtration chromatography and reverse phase HPLC, all extracted materials showed high and low molecular weight forms of ir-ANP. The amount of ir-ANP in rabbit ovary was 40.70 +/- 0.39 pg/mg and that in follicular fluid of pig ovarian follicle was 18.88 +/- 2.49 pg/ml.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

Diethylstilbestrol facilitates masculine and feminine copulatory behavior in female rats

Diethylstilbestrol (DES), which binds to estrogen receptor without crossreacting to androgen receptor, was tested for its ability to facilitate male-typical mounting behavior. The administration of either 30 or 90 μg of DES per day to adult ovariectomized female rats activated mounting behavior in 94% of the animals (last three tests). The response to the two dosages did not differ. The mount frequency response to 30 μg of DES of 6.8 (mean of last three tests) was only about half as high as the response to 100 μg of testosterone propionate. All animals receiving DES also responded with lordosis behavior when mounted. The lower dosage of DES depleted 91% of hypothalamic cytosol estrogen receptors, and the higher dosage 96%. Androgen receptors were not depleted. Thus the synthetic estrogen DES can facilitate masculine sex behavior by interacting with estrogen receptors without binding to or translocating androgen receptors.     

Effects of pregnant mare serum gonadotropin (eCG) on follicle development and granulosa-cell apoptosis in the pig

The effect of eCG on follicular development and granulosa-cell apoptosis in sexually mature and immature gilts and on granulosa-cell apoptosis in vitro were studied. The sexually mature gilts were treated with eCG on Day 11 of the estrous cycle, and effects were analyzed at different times after treatment with untreated animals at corresponding stages of the cycle as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), hematoxylin and eosin staining, and DNA ladder. The proportion of apoptotic cells in atretic follicles (39%) was significantly higher (P<0.01) than that in healthy follicles (9%). At 24h after eCG treatment in mature gilts, the total number of follicles visible on the ovarian surface (57 per ovary), the number of small (<3mm) follicles (31.5 per ovary) and the number of medium-sized (3-5mm) follicles (23 per ovary) were significantly higher (P<0.05) than those of control animals (28, 20 and 6.5 per ovary, respectively), and declined gradually thereafter to below the level of control animals. The number of large (>or=5mm) follicles began to show a marked increase at 72h after eCG (8.5 versus 2.5, P<0.05). At 24h after eCG treatment, the proportions of apoptotic cells in small (7.2%) and medium-sized follicles (7.4%) were markedly lower (P<0.01) than those in controls (21.5 and 21%, respectively) and increased gradually thereafter to approach the level in controls. The percentage of apoptotic cells in large follicles (10% at 24h post-eCG) did not change significantly. Before eCG treatment, there were markedly fewer follicles of all types on ovaries of immature gilts than of mature gilts (9 versus 25 per ovary) and the proportion of apoptotic cells in small and medium follicles was high (25 and 34%, respectively). After eCG treatment, the changes in follicle number and proportion of apoptotic cells in the immature gilts followed a similar pattern to that of the mature gilts. Equine chorion gonadotropin inhibited apoptosis of granulosa cells cultured either in vitro or in intact follicles in a dose-dependent manner. Thus, follicular atresia in the pig, as in other animals, was characterized by apoptosis of large numbers of granulosa cells, and eCG promoted follicular development by inhibition of granulosa-cell apoptosis.