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The thecal layer of the preovulatory follicle of the chicken ovary produces primarily androgens and estrogens. However, the precise cellular sites of androgen and estrogen synthesis in the thecal layer have not been identified. Therefore, our aims were 1) to identify steroidogenic cells in the thecal layer of the preovulatory follicles by localizing specific steroidogenic enzymes in these cells by immunocytochemistry, and 2) to confirm that these cells which contained the specific steroidogenic enzymes secreted the expected steroid in a short term cell incubation. Follicles were collected 2 h after oviposition and prepared for immunocytochemistry and short term cell incubation. Immunocytochemistry for cholesterol side-chain cleavage cytochrome P450 (P450SCC), 17 alpha-hydroxylase cytochrome P450 (P450C17), and aromatase cytochrome P450 (P450AROM) was performed to localize pregnenolone-, androgen-, and estrogen-producing cells, respectively, on frozen sections and paraffin sections of the five largest preovulatory follicles. Interstitial cells showed immunoreactivity for both P450SCC and P450C17, whereas a specific cell population of the theca externa, hereafter termed aromatase cells, showed immunoreactivity for P450AROM. Furthermore, fibroblasts in the theca externa indicated immunoreactivity for P450C17. The immunoreactivity of P450C17 and P450AROM enzymes in specific cells in the theca externa appeared to decrease with follicular maturation. The third largest, fourth largest, and fifth largest follicles were selected for short term cell incubations because the immunoreactivity for P450 enzymes in the thecal layer of these follicles was high. Isolated theca interna cells, theca externa cells, and a combination of interna and externa cells were incubated with/without ovine LH (oLH) for 3 h. The medium was assayed for progesterone, testosterone, and 17 beta-estradiol by RIAs. Incubation of theca interna cells with oLH increased progesterone and testosterone production in a dose-dependent manner. However, we did not observe any production of progesterone and testosterone by theca externa cells. Theca externa cells produced 17 beta-estradiol, and its production was increased significantly when theca interna and externa cells were coincubated in the present of oLH. Based on these data, we propose a multiple cell theory for steroidogenesis in the thecal layer of preovulatory follicles of the chicken ovary which states that interstitial cells in the theca interna produce progestins and androgens, fibroblasts in the theca externa may function as an additional site for the conversion of progestins to androgens, and aromatase cells in the theca externa require androgens as substrate to produce estrogens.

Physiological levels of estrogen enhance humoral immune responses. Several in vitro studies indicate the hormone to have a direct effect on immune cells, and other studies show that estrogen may affect humoral immunity indirectly through the thymus. Therefore, we have conducted experiments to investigate the requirement of the thymus in the enhancement of humoral immune responsiveness by estrogen. In Exp I, adult ovariectomized Lewis rats were thymectomized or sham thymectomized and given estradiol (E2; 0.25 microgram E2 in sesame oil, sc, once every 4 days) or the oil vehicle in a 2 x 2 factorial design, and their antifluorescein responses were followed by enzyme-linked immunosorbant assay across 21 days. Only animals that were thymus intact and given estrogen replacement showed significantly (P less than 0.05) greater serum antifluorescein titers than all other treatments. In Exp II, ovariectomized thymectomized rats were submitted to a 2 x 3 factorial design of oil vehicle or E2 replacement and saline, gelatin, or thymus replacement (thymosin fraction 5; 1 mg/kg in saline, sc). As described above, only the animals receiving both thymosin fraction 5 and E2 replacement displayed antifluorescein titers that were significantly (P less than 0.03) increased over titers of all other treatment groups. These results indicate that the enhancement of in vivo humoral immunity by estrogen requires the thymus, and that a constitutive thymic factor, found in thymosin fraction 5, exerts a permissive influence on the action of E2 outside the thymus to increase a specific humoral immune response.

Progesterone may act locally to modulate follicular maturation and ovulation in the domestic hen. The distribution of progesterone receptors (PR) in the pre- and postovulatory follicles was determined in hens by immunocytochemistry and Western blot analysis. Monoclonal antibodies to chicken PR, PR6, and PR 13, were used. PR were localized in nuclei of theca externa fibroblasts and germinal epithelial cells in stigma and nonstigma regions of the third largest preovulatory follicle (F3). In the largest preovulatory follicle (F1), PR were present in theca externa fibroblasts, germinal epithelial cells, and also in granulosa cells and some of the theca interna fibroblasts in the stigma and nonstigma region. Twenty-four hours after ovulation, PR in the fibroblasts of the theca externa of the postovulatory follicle (POF) were remarkably reduced, but the amount of PR in the granulosa cells was similar to that observed in the F1. A high density of PR was also found in the fibroblasts, arterial wall, and smooth muscle fibers in the loose connective tissue of pre- and postovulatory follicles. Western blot analysis indicated that PR in the granulosa and theca tissue were identical in molecular weight to PR in the shell gland. Western blot analysis also confirmed the changes in the amounts of PR in the pre- and postovulatory follicles as determined by immunocytochemistry. The relative amounts of PR in the granulosa cells as determined by Western blot analysis was F2 less than F1 = POF, and in theca tissue was F2 = F1 greater than POF. The presence of PR in specific ovarian tissues suggests that these tissues are target tissues for progesterone and that progesterone may have a role in regulating follicular maturation and ovulation through receptor-mediated pathways.

We have found that androgens and estradiol-17 beta (E2) produced by theca cells suppress progesterone (P4) secretion by granulosa cells of the domestic hen in a dose-dependent manner. Furthermore, testosterone (T) and E2 inhibited the conversion of cholesterol to pregnenolone (P5) and of P5 to P4, respectively. The aim of this study was to determine if T and E2 suppress P4 biosynthesis by changing activities of the cytochrome P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (Exp I) and the amount of P450scc (Exp II). Granulosa layers of the largest follicle of two to four hens were obtained 22 h before ovulation, pooled, and isolated granulosa cells were prepared. In Exp I, the specific activities of the P450scc and 3 beta-HSD were measured in mitochondrial and microsomal proteins of granulosa cells, respectively, in the presence of T or E2 (0-10 microM). Addition of T to mitochondrial proteins increased the Michaelis-Menten constant (Km) with no change in the maximum velocity (Vmax) of the P450scc, which suggests competitive inhibition (Ki = 30.9 microM), whereas E2 had no effect on Km and Vmax of the P450scc. Likewise, addition of E2 to microsomal proteins increased the Km with no change in the Vmax of the 3 beta-HSD, which suggests competitive inhibition (Ki = 15.1 microM), whereas T had no effect on Km and Vmax of the 3 beta-HSD. In Exp II, granulosa cells (3 x 10(5)/3 ml.tube) were incubated for 0-12 h in triplicate for each combined treatments of 25-OH-cholesterol (8 microM) and cyanoketone (10 microM), T, or E2 (0-10 microM) in the presence or absence of LH (25 ng). Protein content and P5 secretion were measured and the amount of P450scc was determined by Western blot analysis. Incubation of granulosa cells with T decreased the amount of the P450scc in granulosa cells cultured for 12 h and P5 secretion in granulosa cells cultured for 3 h or longer (P less than 0.05), without a change in protein content and cell viability. Our results suggest that P4 production by granulosa cells is suppressed by T and E2 acting as competitive inhibitors of the P450scc and 3 beta-HSD, respectively, and by T decreasing the amount of the P450scc. We conclude that steroidogenesis in the follicle of the chicken is regulated through the interaction of theca and granulosa layers.

Estradiol (E2) enhances humoral immunity, but the quantitative and temporal relationship between serum E2 and antibody titers is unclear. The present study was therefore designed to determine whether the sex of the animal, ovariectomy (OVX), and replacement with various tonic levels of serum E2 influenced the primary and secondary antifluorescein antibody titers of rats. Two experiments were performed. In Exp I, intact male and intact female Lewis inbred rats were given primary and booster (secondary) immunizations with fluorescein-keyhole limpet hemocyanin. Primary and secondary bleedings for antifluorescein antisera were obtained. In Exp II female Lewis inbred rats were randomly distributed among the following treatment groups: sham OVX, sham implanted; OVX, sham implanted; and three groups of OVX and implanted with E2-filled Silastic capsules of 0.5, 1.5, or 5.0 cm lengths. Immunization with fluorescein-keyhole limpet hemocyanin and bleedings for antifluorescein antisera followed the same schedule as in Exp I. In Exp I, females had significantly higher antifluorescein antibody titers than males during the primary and secondary response. In Exp II, OVX significantly depressed the primary and secondary antifluorescein antibody titers relative to sham OVX controls. During the primary antifluorescein antibody response, a significant quadratic relationship was seen between antibody titers and serum E2 when measurements included the pharmacological range of the serum E2 concentrations. A significant linear relationship existed between serum E2 and antifluorescein antibody titers when the same data were examined over a range of 15-130 pg E2/ml. This linear relationship is more significant during the primary response than the secondary response. It is concluded that females generate higher antifluorescein antibody titers than males; OVX significantly depresses the antifluorescein antibody response; E2 is immunoenhancing in the physiological range and immunosuppressive in the pharmacological range; and the presence of E2 during the primary response may be more critical to immunoenhancement.

The aims of this study were to characterize the ontogeny of catecholamines (CA) in the rat ovary, to determine the ability of the immature ovary to synthetize norepinephrine (NE) in vitro, and to correlate between ovarian CA and plasma pituitary hormones. Ovaries, spleen (as a control tissue not subjected to endocrine regulation), and trunk blood were collected at 5-day intervals between days 5 and 40. Ovarian NE concentration increased markedly between days 20 and 35 of life, whereas the major rise in splenic NE concentration occurred between days 10 and 15. Ovarian and splenic tissues from neonatal females were capable of de novo synthesis of NE from tritiated tyrosine without an appreciable accumulation of L-Dopa and dopamine. The rate of NE synthesis by ovarian tissue taken from 20-day-old rats was significantly lower than that from 30- and 40-day-old rats, whereas NE production by splenic tissue from 20-, 30-, and 40-day-old rats were similar. Plasma FSH concentration was significantly elevated between days 10 and 20, whereas the major rise in plasma LH and PRL occurred between days 25 and 40. The following conclusions were reached. The delayed elevation of ovarian NE, compared to splenic NE, is attributable to a decreased production of NE by the ovary on day 20 and may involve a suppression or a delay in development of the activity of a key CA biosynthetic enzyme such as tyrosine hydroxylase. Given the temporal relationship between plasma gonadotropins, in particular FSH, and changes in ovarian NE, it is postulated that ovarian CA during neonatal development are subjected to regulation by circulating pituitary hormones.

The purpose of the present study was to measure norepinephrine (NE) in Graafian follicles and correlate changes in its concentration with circulating gonadotropins secreted endogenously or administered exogenously. Graafian follicles were removed from the ovaries of adult cycling rats. The follicles were pooled in groups of 10-13, and NE was determined by high performance liquid chromatography. Follicular NE(picograms per micrograms protein) did not change between 0900 h (3.61 +/- 0.34) and 1300 h (3.12 +/- 0.25) on proestrus, but was reduced significantly to 1.45 +/- 0.16 at 2100 h, which is 4 h after the peak of the gonadotropin surge. There was a further reduction to 0.83 +/- 0.08 in fresh corpora lutea taken on estrus at 0900 h. The decrease in follicular NE was prevented in estrous rats which were either hypophysectomized 24 h previously or treated with sodium pentobarbital at 1330 h on proestrus. To determine which pituitary hormone was responsible for follicular NE depletion, rats were injected at 0900 h on proestrus with LH (5 micrograms), FSH (20 micrograms), LH plus FSH (5 and 20 micrograms, respectively), or PRL (20 micrograms), and follicular NE was determined 4 h later. FSH reduced follicular NE significantly to 1.86 +/- 0.16 compared to both the control (3.12 +/- 0.25) and the PRL-injected group (2.92 +/- 0.32), whereas LH caused a small but nonsignificant decrease (2.49 +/- 0.2). Both LH and FSH doses used resulted in ovulation, as determined by counting tubal ova 12 h after hormonal treatment. We conclude that 1) NE in Graafian follicles is markedly reduced within 4 h after the preovulatory gonadotropin surge in the normal cycling rat; this reduction is prevented when the surge is abolished; 2) the hormone responsible for follicular NE depletion is FSH rather than LH or PRL; and 3) finally, it is suggested that follicular NE may be involved with the formation and/or functioning of the corpus luteum.

In a previous in vitro study we found that androgens and estradiol (E2) suppress progesterone (P4) production by the granulosa cells isolated from the largest follicle of the domestic hen in a dose-dependent manner. The presence of an aromatase inhibitor did not block the inhibitory action of androgens. The addition of androgen plus E2 to the granulosa cells had an additive effect on suppressing P4 secretion. The aim of this study was to determine the loci in the steroid biosynthetic pathway where androgens and E2 inhibit P4 production by the granulosa cells. Granulosa layers of the largest follicles removed from two or three hens 22 h before ovulation were pooled. Dispersed granulosa cells were incubated for 3 h in triplicate for each treatment, and pregnenolone (P5) and P4 secretion were measured in medium and cells. Experiments were replicated three or four times. Treatment of granulosa cells with cyanoketone (0-100 microM), an inhibitor of 3 beta-hydroxysteroid dehydrogenase, increased P5 production and decreased P4 production in a dose-dependent manner, with maximal production of P5 and suppression of P4 production at 10 microM cyanoketone. The addition of 25-hydroxycholesterol (25OHCh) or P5 (0-16 microM) caused a dose-related increase in basal and LH-stimulated steroid production. The maximal production of P5 or P4 was found at 8 microM 25OHCh or P5. Also, the effect of LH (0-100 ng) on granulosa cell steroidogenesis was examined with or without 8 microM 25OHCh or P5. The half-maximal and maximal doses for P5 or P4 production were 5 and 25 ng LH, respectively. Next, suppression of P5 production by androstenedione, testosterone, dihydrotestosterone, and E2 (each at 0-10 microM) was tested in the presence of 25OHCh plus cyanoketone with or without LH. We found a dose-dependent suppression of P5 production by androgens (1-10 microM), but not by E2. However, when we added the above steroids to granulosa cells in the presence of P5 with or without LH, only E2 (1 and 10 microM) caused a significant suppression of P4 production. Our results suggest that 1) androgens primarily act at the conversion site of cholesterol to P5 to suppress P4 production; and 2) E2 acts at the conversion site of P5 to P4 to suppress P4 production. We conclude that production of androgens and E2 by thecal cells may regulate P4 biosynthesis by granulosa cells in the domestic hen.

The purpose of this study was to correlate changes in catecholamine concentrations in porcine follicular fluid and cyclic events in the ovary. Follicular fluid was aspirated from follicles of ovaries obtained from pigs throughout the 21-day estrous cycle and analyzed for norepinephrine (NE), epinephrine (EPI), and estradiol (E2). Serum was obtained from cycling pigs on days 6-10 and 16-20 of the cycle and assayed for NE, EPI, E2, and progesterone. The concentrations of NE in the follicular fluid were relatively constant during days 1-15 of the luteal phase (1.7 +/- 0.2 ng/ml), but were elevated significantly to 2.9 +/- 0.4 ng/ml during the follicular phase (days 16-20). EPI had a similar profile, but a 6- to 10-fold lower concentration. The follicular fluid E2 concentration increased from 15.6 to 76.5 ng/ml during the luteal phase to 630 ng/ml during the follicular phase. Serum NE and EPI concentrations were similar during midluteal and follicular phases, whereas progesterone and E2 were significantly elevated during the luteal and follicular phases, respectively. These results indicate that catecholamines in follicular fluid are elevated significantly during the follicular phase of the estrous cycle and may have a physiological role in preovulatory events as well as during the luteinization process.

The purpose of the present investigation was to correlate changes in ovarian catecholamines and serum pituitary hormones and sex steroids. As the experimental model we used the prepubertal rat injected with 7.5 IU PMS on day 28 at 0900 h. Within 24 h after the injection, ovarian weight increased, and on day 30 at 2400 h, it was twice that of controls. On day 31, all of the PMS-treated rats but none of the controls had ovulated, and the average number of ova was 9. Serum estradiol in these rats was elevated significantly on the afternoon of day 30 and was followed by a preovulatory surge of LH and FSH. Serum progesterone was also increased on the evening of day 30, and an elevation in serum PRL on the morning of day 30 in the PMS-treated rats was evident, whereas serum GH was unchanged. Ovarian norepinephrine (NE) dropped from 65.5 +/- 6.9 pg/mg ovary (mean +/- SE) before the PMS injection of 14.4 +/- 1.5 pg/mg ovary on the evening of day 30. This depletion proceeded in two apparent phases: an initial drop within 12 h after PMS injection, and a further reduction coincident with the gonadotropin surge. Throughout this period, ovarian NE in the controls increased 50--60%. There was a 2-fold reduction in ovarian dopamine in the PMS-primed rats, but the levels were 10-fold lower than those of NE. The data document that the hormonal profiles and the number of ovulating follicles of the prepubertal rat primed with a low dose of PMS resemble those of the normal cycling rat. A significant depletion of ovarian catecholamines was observed during the preovulatory period, which is temporally related to increases in serum gonadotropins.