Intact Immature Female Rats Research Articles

DT56a (Femarelle): A natural selective estrogen receptor modulator (SERM)

A selective estrogen receptor modulator (SERM) is defined as a substance with dissimilar effects on different tissues: agonist in some and antagonists in others. The natural compound DT56a (Femarelle) was shown to activate estrogen receptors in human cultured female derived osteoblasts. It was also shown to relieve menopausal symptoms and to increase bone mineral density with no effect on sex steroid hormone levels and on the endometrial thickness. DT56a, similarly to estradiol-17β (E 2), stimulated the specific activity of creatine kinase (CK) in skeletal and vascular tissues of female rats, as a marker of estrogen receptor (ER) activation. However, in the uterus, CK was activated only by E 2 but not by DT56a. In order to prove that DT56a is a SERM, we examined the mutual interaction between DT56a and E 2, at supra physiological doses, in different tissues in both intact and ovariectomized female rats, as well as in human cultured vascular and bone cells. Administration of DT56a or E 2 stimulated CK in all tissues tested, but when given simultaneously, in intact immature female rats, DT56a completely abolished E 2 stimulation of CK in all organs except in the diaphyseal bone where the inhibition was partial. In ovariectomized female rats, DT56a abolished E 2's stimulation of CK in diaphyseal bone, thymus, uterus and pituitary but caused a partial inhibition in aorta, left ventricle and epiphyseal cartilage. In human bone cells E 2 stimulation of CK, of alkaline phosphatase (AP) activity and of DNA synthesis was completely abolished by DT56a in post-menopausal cells and partially inhibited in pre-menopausal cells. In human vascular cells, inhibition of DNA synthesis by E 2 was completely abolished by DT56a and E 2-induced CK was partially inhibited by DT56a. The results support the finding that DT56a is a SERM; it stimulated different parameters similar to E 2, but when given simultaneously, at supra physiological doses, inhibited these E 2's effects. Further investigations regarding intra and extra cellular mechanism of action of DT56a are currently performed.

Evidence that progesterone modulates anterior pituitary neuropeptide Y levels during the progesterone-induced gonadotropin surge in the estrogen-primed intact immature female rat.

In a previous study we reported that in vivo estrogen-priming alone, without subsequent progesterone-treatment, was sufficient to maximize NPY potentiation of gonadotropin hormone-releasing hormone responsiveness exhibited in vitro by the rat anterior pituitary. This observation suggests that the necessity, as reported by others, for both estrogen-priming and progesterone-treatment to maximize NPY potentiation of GnRH responsiveness in vivo may be due to progesterone acting primarily at the hypothalamus. Consequently, the current study was performed to determine whether progesterone facilitates gonadotropin secretion in vivo by acting to stimulate hypothalamic synthesis of NPY and the subsequent elevation of anterior pituitary tissue levels of NPY. Intact immature female rats were injected with estradiol at 1700 h on days 27 and 28. On day 29 at 0900 h, the animals received an injection of progesterone (2 mg/kg) or vehicle and were subsequently sacrificed at 1200, 1330 and 1500 h. Rats which received only estradiol injections were used as controls. Surge levels of serum LH and FSH were observed at 1330 and 1500 h. Hypothalamic levels of NPY mRNA at 1200 h on day 29 were higher (P < 0.01) in estradiol-primed rats which received progesterone; there was no accompanying statistically significant change in hypothalamic NPY content. NPY content in the anterior pituitary was significantly increased (P < 0.01) at 1200 h on day 29 in estradiol-primed rats which received progesterone; there was no accompanying significant change in anterior pituitary NPY mRNA levels. Hypothalamic GnRH mRNA content was significantly increased (P < 0.01) at 1330 h on day 29 concomitant with the peak of the gonadotropin surge in the estradiol-primed, progesterone-treated rat. The data indicate that progesterone modulates hypothalamic NPY mRNA and anterior pituitary NPY levels as well as GnRH mRNA levels and that modulation of NPY levels in the hypothalamic-pituitary axis occurs prior to modulation of GnRH gene expression. These studies support the hypothesis that in the estrogen-primed rat, progesterone facilitates the induction of the gonadotropin surge by maintaining hypothalamic synthesis of NPY as well as by modulating anterior pituitary NPY tissue levels.

In vivo action of activin-A on pituitary-gonadal system.

Activin, a dimer of the beta-subunits of inhibin, has been found to stimulate FSH secretion from the cultured pituitary cells. However, in vivo action of activin is poorly elucidated. Daily sc injections of 40 micrograms activin-A over a period of 1-3 days to intact immature female rats caused a significant increase in serum FSH, inhibin, estradiol, uterine weight, and ovarian FSH receptors. Daily sc injections of 5 micrograms or 20 micrograms activin-A for 6 days caused a marked increase in ovarian weight and the development of large ovarian follicles. However, daily sc injections of 20 micrograms activin-A to hypophysectomized immature female rats for 3 days induced no significant changes in ovarian and uterine weight, serum inhibin, estradiol, and progesterone levels. Simultaneous injections of both activin-A and 5 IU PMSG induced a significant increase in ovarian and uterine weight, serum inhibin, and estradiol levels, compared to simultaneous injections of both vehicle and PMSG in the hypophysectomized immature female rats. These results demonstrate that activin-A induces not only an increase of FSH secretion from the pituitary but also a direct autocrine or paracrine ovarian stimulation resulting in an increase of the number of ovarian FSH receptors and ovarian and uterine weight, as well as an increase in the level of inhibin and estradiol secretion from the ovary.

Inhibition by diethylstilboestrol of proliferative potential of follicles of different sizes in immature rat ovaries

Intact immature female rats were treated with 1, 2, 3 or 4 subcutaneous injections of 2 mg diethylstilboestrol (DES)/rat at intervals of 24 h and then killed. Ovaries were collected, cleaned, enzymically digested and serially filtered through Teflon sieves to yield follicles of diameter less than 200 microns (small), 200-400 microns (medium) and greater than 400 microns (large). Follicular supernatant was collected and granulosa cells were extracted from these isolated follicles. There was a general increase in [3H]thymidine incorporation in all sizes of follicles after 1 or 2 DES injections, the increase in the medium and large follicles being significant after 2 doses. With 3 and 4 injections of DES, there was a sudden decrease in the rates of [3H]thymidine incorporation, particularly in the medium-sized follicles, which also had higher concentrations of follicular supernatant protein. Protein contents in small and large follicles did not change significantly. The follicular supernatant protein had a specific and dose-dependent inhibitory effect on [3H]thymidine incorporation when added to cultures of rapidly dividing granulosa cells. Addition of the same amounts of bovine serum albumin (BSA) to the cultures had no effect. Heat-denaturing did not abolish the inhibition by the protein. Removal of the protein from the cultures after the first 48 h resulted in a rebound increase in [3H]thymidine incorporation during the following 48 h, showing that the inhibitory effects were reversible. Though aromatase activity after 1 or 2 DES injections abruptly decreased after 3 and 4 injections, follicular supernatant protein had no effect on steroidogenesis in cultured granulosa cells. Taken together, these findings suggest that oestrogen can inhibit follicular development, depending on the duration of exposure. We propose that the inhibitory effects of DES on cell proliferation are mediated via the synthesis of a specific peptide factor which is produced in high amounts in the medium-sized follicles only, on prolonged exposure to the oestrogen. This factor may be autocrine or paracrine, serving as an in-built autoregulatory control mechanism for follicle development, particularly at pro-oestrus, when oestrogen concentrations are highest.

Effect of dihydrotestosterone on the growth and function of ovarian follicles in intact immature female rats primed with PMSG

Intact, immature female rats were primed with PMSG and treated with 4 injections of DHT. DHT given at 0, 12, 24 and 36 h caused a significant decrease in the ovulation rate 72 h after the PMSG treatment. Concurrent treatment with oestrogen reversed the inhibitory effects of the androgen. The androgen effect was apparently exerted directly on the ovary since DHT did not alter the surge of LH and FSH which occurred at 58 h after PMSG treatment. The DHT inhibition of ovulation was observed in the treatment cycle as well as in subsequent cycles which followed a second PMSG injection. This finding suggests that intermediate size follicles were also adversely affected by the androgen. To confirm that androgen affects follicles of all size ranges, follicles less than 200 microns, 200-400 microns and greater than 400 microns in diameter were isolated from the ovaries of rats treated with PMSG and DHT or the vehicle. The follicles were isolated by density gradient separation of follicles followed by filtration with pre-calibrated Teflon sieves. In some experiments, granulosa cells were also harvested from isolated follicles. DHT treatment did not affect the numbers of follicles of any size but did reduce the oestrogen content of follicles of all sizes. Follicles from DHT-treated animals contained fewer granulosa cells and the cells from treated animals had lower aromatase activity than did cells from control rats. Taken together, these findings suggest that DHT reduces the ovulation rate by decreasing the number of granulosa cells/follicle and by altering the oestrogen synthetic abilities of the cells. All follicles, regardless of size, were sensitive to androgen treatment.

Tissue-Type Plasminogen Activator in Rat Oocytes: Expression during the Periovulatory Period, after Fertilization, and during Follicular Atresia*

The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.

Inhibin-mediated feedback control of follicle-stimulating hormone secretion in the female rat.

The secretion of follicle-stimulating hormone (FSH) by the anterior pituitary gland is regulated by the interaction of hypothalamic and gonadal hormones. Recently, proteins termed inhibins that selectively suppress FSH secretion have been purified and characterized from the gonadal fluids of several species. Antibodies to a synthetic peptide encompassing the amino terminal 25 residues of the recently characterized porcine inhibin were used to develop a specific radioimmunoassay (RIA) for inhibin and to neutralize endogenous inhibin during the estrous cycle of the rat. The administration of 20 international units of pregnant mare serum gonadotropin (PMSG) stimulated the secretion of inhibin in intact immature female rats, whereas ovariectomy caused an abrupt decrease in plasma inhibin concentrations that were not prevented by the injection of PMSG. The infusion of a polyclonal antiserum to inhibin, from 12 noon on proestrus to 1 a.m. on the morning of estrus, as well as its acute intravenous injection during diestrus I or II, caused an increase in plasma FSH (but not luteinizing hormone) concentrations. These results support the hypothesis of a feedback loop between the release of ovarian inhibin and FSH in the female rat.

Prolactin inhibits pituitary luteinizing hormone-releasing hormone receptors in the rat.

Pituitary LHRH receptors were studied in intact immature and mature female rats as well as in adult castrated animals of both sexes under experimental conditions leading to marked alterations of plasma PRL levels. Implantation of three anterior pituitary glands under the kidney capsule on the morning of estrus prevented the increase in pituitary LHRH receptor content measured 3 days later on the morning of expected proestrus. Pituitary implants slightly decreased pituitary LHRH receptor levels in ovariectomized adult rats while simultaneous treatment with 17β-estradiol (E2; 1 μg daily) markedly potentiated the inhibitory effect of pituitary implants and led to much higher plasma PRL concentrations. Although pituitary implants alone had no effect on plasma gonadotropin levels, they potentiated the inhibitory effect of E2 on both plasma LH and FSH on both plasma LH and FSH on both plasma LH and FSH levels. Treatment with the dopaminergic agonist 2α-bromoergocryptine (CB-154) completely reversed the inhibito...

Diethylstilbestrol regulates the number of α- and β-adrenergic binding sites in incubated hypothalamus and amygdala

Previous work has identified an effect of circulating estrogens on the number of central adrenergic binding sites. We have further characterized this effect by performing the experiments in vitro and have taken advantage of a well-described hypothalamic preparation in which diethylstilbestrol (DES), is known to elevate cAMP levels through a pathway which involves adrenoreceptors. We show that DES induces a reciprocal change in the numbers of α- and β-adrenergic binding sites in incubated hypothalami obtained from intact immature female rats as well as from ovariectomized adult rats. The α-adrenergic binding sites are reduced by 25–30% whereas the β-adrenergic sites are increased by 60–100%. The effect is maximal at 3 h in vitro (20 μM DES) and largely reversible following a 2 h wash in the absence of DES. Using the change in β-adrenergic binding sites as a probe, we were further able to show that estradiol (100 μM) and 2-hydroxyestradiol (50 μM) had no effect. Further, the effect of DES was not blocked by the anti-estrogens clomiphene or tamoxifen. Since DES is able to elevate β-adrenergic binding sites in hypothalamus and amygdala (brain areas known to contain high levels of estrogen receptors) but has no effect in cerebellum, we conclude that we have observed an effect of DES not shared by estradiol but which may be confined to estrogen target areas of the brain.

Effect of androstanediol sulfates on luteinizing hormone release in ovariectomized rats.

Androstanediols are the major products of the immature rat ovary and are present in peripheral circulation mainly as sulfate conjugates. In this paper we identified 5 alpha-androstane-3 beta, 17 beta-diol-3-monosulfate (3 beta-A-MS) as one of the forms found in blood and subsequently synthesized and administered it to ovariectomized rats at a dose of 100 microgram/100 g BW . day from 21-45 days of age. This dose effectively inhibits postcastrational LH elevation. Other androstanediols examined, like 5 alpha-A-3 alpha, 17 beta-diol-disulfate, 5 alpha-A-3 beta, 17 beta-diol-disulfate, and the free 5 alpha-A-3 beta, 17 beta-diol do not exert such an effect on LH release. The MCR of 3 beta-A-MS was 441 +/- 64 ml/h, independent of the infusion rate between 0.15-15.0 microgram/h, and its production rate was calculated to be 37 microgram/day at the age of 30 days. The quantitative relations of the steroid level in serum to its capacity to inhibit LH release was studied using Silastic capsules. A steady concentration of 1.1 ng 3 beta-A-MS/ml serum inhibits postcastrational LH release in the immature female rat. Since a similar or higher concentration of the steroid is present in the intact rat, it is assumed that 3 beta-A-MS controls pituitary LH release in the intact immature female rat. (Endocrinology 108: 500, 1981)

Evidence for a role of endogenous estrogen on follicular growth in immature female rats.

The effect of gonadotropin and endogenous estrogen on the growing follicles (especially in relation to the presence or absence of theca cell layers) was studied morphometrically in intact immature female rats, using a chemical antiestrogen.When pregnant mare serum gonadotropin (PMSG) was given in combination with clomiphene, the uterine weight decreased significantly as compared with that in the animals treated with PMSG alone (p<0.001). The administration of clomiphene and PMSG did not have a significant effect on the total number of follicles (growing plus atretic). The number of atretic follicles in those treated with PMSG alone tended to decrease as compared with sesame oil-treated animals. However, when PMSG was given in combination with clomiphene, the number of atretic follicles increased significantly as compared with the animals treated with PMSG alone (p<0.05). The number of growing follicles in the animals treated with PMSG alone tended to increase as compared with the animals treated with neither PMSG nor clomiphene. In contrast, when PMSG was given in combination with 5mg/day of clomiphene, the number of growing follicles tended to decrease. The administration of PMSG alone and PMSG combined with 1 and 5mg/day of clomiphene caused a significant decrease in the number of growing follicles without theca cell layers, as compared with sesame oil-treated animals (p<0.05, p<0.01 and p<0.001, respectively). On the other hand, the number of growing follicles with theca cell layers was increased markedly by the administration of PMSG alone (p<0.05).It is suggested that the early stage of follicular growth in the absence of theca cell layers may be promoted by endogenous estrogen. In the presence of theca cell layers, in addition to being mediated by estrogen, gonadotropin-induced follicular growth may be promoted by the maturative effect of gonadotropin itself.

Androgen receptor in rat skeletal muscle: characterization and physiological variations.

Androgen binding has been studied in the quadriceps femoris of recently castrated adult and intact immature male and female rats using a variety of techniques for separating and measuring hormone-receptor complexes. [3H]Testosterone, [3H]androstanolone (or 5 alpha-dihydrotestosterone). [3H]methyltrienolone (a potent synthetic androgen), and [3H]estradiol bind to the androgen receptor. Affinities are identical for the first two hormones (Kd = approximately 70 pM) and lower for estradiol (Kd = approximately 0.2 nM), as determined by Scatchard plots of binding data. Competition experiments indicate that in addition to the nonradioactive steroids corresponding to the above-cited tritiated compounds, progesterone, cyproterone acetate (an antiandrogen), and spironolactone compete for [3H]androgen binding by the receptor, but diethylstilbestrol, moxestrol (a potent synthetic steroidal estrogen), and cortisol do not. 3 alpha- and 3 beta-androstanediols slightly inhibit testosterone binding. Therefore, striated muscle androgen receptor specificity is identical to that of all androgen receptors of target tissues which have been previously studied. Binding is abolished by pronase and heat treatment, and displays an approximate 7S sedimentation coefficient in low salt ultracentrifugation gradient analysis. Preliminary observations suggest hormone-induced receptor translocation into the nucleus. No evidence has been found for an independent estrogen receptor. In the course of the binding experiments, extensive metabolism of androstanoloe and testosterone was observed in muscle cytosol at 0-4 C, during the 2-h incubation period used for most binding studies. Metabolite formation can jeopardize the binding data, specifically altering the significance of competition experiments with relatively high concentrations of steroids approaching the Km of metabolizing enzymes. Therefore, most quantitative studies were performed in enzyme-free, receptor-containing cytosol preparations. In adult male rats castrated for 2 days, the concentration of receptor in the cytosol was of the order of 1 fmol/mg protein and corresponded to 72 fmol/mg tissue DNA (that is, 100 and 20 times less than that in corresponding prostatic cytosol, respectively). In the adult female rat 2 days after castration, the concentration of receptor in the cytosol was 0.34 fmol/mg protein. Treatment with testosterone pellets (20 mg for 15 days) increased androgen receptor concentration significantly. In spite of the relatively low concentration of androgen-binding sites, the typical binding specificity of the androgen receptor and the regulatory effects of androgens on their own receptor support the possibility that some effect(s) of androgens upon skeletal muscles may be initiated directly at the cellular level through this receptor, a concept which is also in agreement with recently demonstrated in vitro effects of androgens on cultured myoblasts.

A Role for the Ovaries in Maturational Processes of Hypothalamic Neurons Containing Luteinizing Hormone- Releasing Hormone*

The role of the ovaries in the maturation of the hypothalamic LHRH neuron has been investigated. Female rats were castrated at 4 weeks of age, and the subcellular distribution as weLl as the content of LHRH in the hypothalamic homogenates was analyzed 2.5, 12, and 20 weeks after ovariectomy. Hypothalami were homogenized in an isoosmotic sucrose solution, and a 900 X g supernatant fluid was prepared. Free granules and synaptosomes containing LHRH were separated by means of continuous sucrose density gradient centrifugation. In hypothalamic homogenates of intact immature female rats, 35% of the LHRH was associated with free granules and 65% was associated with synaptosomes; in homogenates of adult rats, 57% of the LHRH was associated with free granules and 43% was associated with synaptosomes. Ovariectomy did not alter the postnatal changes in the fractional distribution of LHRH between these two subcellular compartments The LHRH content of the hypothalamus as well as the amount of LHRH sequestered in free ...

Estrogen Treatment of Immature Rats Inhibits Ovarian Androgen Production in Vitro*

The effects of estrogen treatment of intact vs. hypophysectomized immature female rats on the subsequent in vitro steroidogenic responses of the ovary to LH and dibutyryl cAMP were studied. Treatment of intact rats with 17β-estradiol (E2; 1 mg) for 3 days inhibited the ability of the ovaries to respond to LH with the production of testosterone but not progesterone, whereas both LH-induced testosterone and progesterone levels were attenuated by E2 in hypophysectomized animals. Addition of dibutyryl cAMP to the incubation media of ovaries from the E2-treated rats (both intact and hypophysectomized) markedly stimulated progesterone production but failed to elicit any significant production of testosterone. Ovaries from E2-treated hypophysectomized rats were deficient in converting exogenous progesterone to testosterone. In the E2- treated intact rats, the thecal cells of the largest follicles accumulated significantly greater quantities of progesterone in response to LH when compared to those from similarly ...

Stimulatory Action of Androgen Administration in Vivo on Ovarian Responsiveness to Gonadotropins*

In intact immature female rats, ovarian progesterone levels were significantly elevated 1 h after iv injection of either LH (NIH-LH-B8, 10 μg) or FSH (NIH-FSH-S11; 100 μg). Testosterone treatment (5 mg daily, sc) for 3 consecutive days enhanced this ovarian progesterone response to LH or to FSH. 17β-Estradiol (1 mg daily for 3 days) had no effect on stimulation of ovarian progesterone production by LH. Although 17β-estradiol slightly enhanced ovarian progesterone levels in response to FSH stimulation, its effect was significantly less than that of testosterone. By contrast, 17β-OH-5α-androstan-3-one (DHT), when administered systemically (10 mg, sc), had no effect on the ovarian responsiveness to either LH or FSH. However, when DHT was administered by means of Silastic minicapsules implanted locally, ovarian progesterone response to FSH, but not LH, was significantly enhanced when compared to the response in controls. A stimulatory action of androgens on the ovary could be explained, at least in part, by t...

Acute Effects of Estradiol and Follicle-Stimulating Hormone on Specific Binding of Human [125I]Iodo-Follicle-Stimulating Hormone to Rat Ovarian Granulosa Cellsin vivoandin vitro*

Twenty-four-day-old hypophysectomized and intact immature female rats were used to determine the time-dependent effects of estradiol on FSH stimulation of FSH receptor in granulosa cells. In hypophysectomized rats, a single 2 μg injection of human (h) FSH (LER-1577) given alone, concomitantly with, or 6 h after 1.5 mg estradiol caused a 50% increase in the available FSH receptor content in granulosa cells by 24 h. The same dose of hFSH given 12 or 24 h after estradiol caused a 300% increase in available FSH receptor by 24 h. To establish the accuracy of these in vitro FSH-binding assays, it was imperative to determine if the hFSH administered in vivo occupied binding sites and thereby masked increases in total available FSH receptor occurring within the first 24 h. Therefore, 20 × 106 cpm of 125I-labeled hFSH, equivalent to 250 ng hFSH LER-1577, was administered sc to estradiolpiimed hypophysectomized rats 1 h after an injection of saline or 2 μg hFSH. The amount of labeled hormone bound in vivo to 30,000...

The in vivo behavior of human chorionic gonadotropin after dissociation into subunits.

The plasma half-life (T½) and target organ uptake of the subunits of hCG (hCG-α and hCG-β) were investigated. For these studies, tritiated (3H) hCG, hCG-α, and hCG-β were injected iv into intact immature female rats. At timed intervals plasma concentration was determined by specific homologous radioimmunoassays and liquid scintillation counting for 3H. T½ was also determined by radioimmunoassay for unlabeled hCG, hCG-α, hCG-β and recombined subunits, hCG-αβ. Tissue distribution (renal, hepatic, splenic, ovarian) versus time was determined by 3H counting. All substances had exponential disappearance curves with fast and slow components. The T½ (rapid) was 141, 6.2 and 11.1 min for hCG, hCG-α and hCG-β, respectively (p < 0.01) and T½ (slow) of 725, 58 and 81 min, respectively. Slopes of the tritiated and homologous unlabeled compounds were indistinguishable as were those of the native hCG and recombined subunits. 3H-hCG, 3H-hCG-α and 3H-hCG-β were rapidly concentrated in the kidney with lesser concentration...

Physiological studies on the identity of the gonadotropic and progonadotropic substance(s) in sera of ewes immunized against HCG.

The sera of two ewes immunized against hCG were studied intensively over a period of 3 yr for anti-, pro- and gonadotropic activities in an attempt to determine the factors responsible for these activities. Data from an additional ewe (9–437) were used for limited studies. Gonadotropic and progonadotropic responses were studied in intact immature female rats and in hypophysectomized male and female rats. In a few instances the LH contents of the sera were determined by radioimmunoassay. Gonadotropic responses were obtained in intact immature females and in hypophysectomized males but not in hypophysectomized females. On the other hand, administration of immunized ewe serum concurrently with crude or purified FSH resulted in an enhancement of the FSH response (progonadotropic activity) in both intact and hypophysectomized females. The concurrent injection of the serum with ovine LH into hypophysectomized males augmented the ventral prostate response to ovine LH. Thus, it was shown for the first time that a...

Gonadotropic and antigonadotropic activity of ewe serum following chronic treatment with human chorionic gonadotropin (HCG).

SummaryThree ewes were immunized with HCG. All ewes had high anti-HCG titers (>200 rat units/ml) and elevated serum LH levels as determined by ventral prostate weight assays and radioimmunoassays following the third series of immunizing injections. A significant increase in uterine weight was produced by the antisera in intact immature female rats. The antisera failed to neutralize the biological activity of ovine LH in the ventral prostate weight assay, but the antisera were able to block the occurrence of estrus to varying degrees in all ewes and blocked ovulation in two of the ewes as judged by the absence of corpora lutea at the time of laparotomy. One of the ewes showed an enlarged vulva and mammary development 6 weeks following cessation of the third series of injections. It is postulated that the elevated serum LH levels and blockage of estrus and ovulation are due to the ability of the anti-HCG to block the action of endogenous LH on its end organ, thus altering the secretion of the gonadal hormones.

Further studies on mare serum gonadotrophins secreted in response to immunization with HCG or crude ovine FSH.

Summary. The gonadotrophic activity of sera from two mares treated either with human chorionic gonadotrophin or with crude ovine pituitary fsh as antigens has been studied in intact and hypophysectomized immature female and male rats. The studies were projected over a 4-year period during which time the mares were subjected to three and four sequences of immunization, respectively. One-third millilitre of serum from the mare treated with ovine fsh increases ovarian and uterine weights in intact immature female rats, while 5 ml of serum tends to depress these weights because of the antifsh present in the serum. Thirty millilitres of serum approximately doubles the seminal vesicle and prostate weights in intact males, whereas the testis weights are either unaffected or depressed. Based on the ventral prostate responses in Sprague-Dawley hypophysectomized males, the serum is estimated to contain about 25-\g=m\gequivalents of nih-lh-s8/ml. Both the serum of this mare and that of an untreated non-pregnant mare depressed the ovarian ascorbic acid level significantly, but the slopes of the responses to graded doses were flat and thus lh values could not be estimated by this procedure. The biological activity of the serum from the mare treated with hcg as an antigen has been previously described. In earlier studies, Long\p=m-\Evans hypophysectomized males, operated upon at 23 to 26 days, were used with treatment starting 15 days later. The testes were enlarged but the ventral prostate weights were unaffected by 16 ml of serum. In more recent findings, it has been found that if the animals are operated upon at 21 days and injections begun 4 days later significant ventral prostate weight responses can be obtained with 8 ml of serum. Attempts have been made to separate the gonadotrophic and anti-gonadotrophic activities in this serum by chemical fractionation. From the localization of activities in the \g=g\-globulinfractions ofthe ammonium sulphate\p=m-\ethanol procedure, and the \g=a\-and \g=b\-globulinfractions of the deae-cellulose procedure we conclude that the gonadotrophic and anti-gonadotrophic activities are not following any of the classical serum proteins for which