PMS Gonadotropin Research Articles

Transient periovulatory expression of pituitary adenylate cyclase activating peptide in rat ovarian cells.

Pituitary adenylate cyclase activating polypeptide (PACAP), a potent stimulator of cAMP formation, has a widespread distribution in neuronal elements of the central nervous system and a number of peripheral organs. Recently, PACAP has also been shown to be stage specifically expressed in the spermatogenic cells from the rat testis. This prompted us to examine the cell-specific expression of PACAP during the estrous cycle in the rat ovary by in situ hybridization with a digoxiginin UTP-labeled RNA probe and by immunohistochemistry using a specific monoclonal antibody. Ovaries from PMS gonadotropin (PMSG)/human CG (hCG) treated immature animals were examined 0, 12, 24, 48, 54, 57, 58.5, 60, 61.5, 63, 66, 72, and 96 h after PMSG treatment. Ovaries from adult cyclic rats were examined at 1000 h on each day of the estrous cycle and at 0400 h on estrus morning. PACAP positive cells were observed in the preovulatory and ovulatory period, i.e. 6-18 h following hCG injection in the PMSG induced cycle and at 0400 h and 1000 h on estrus morning in adult cyclic animals. Both PACAP messenger RNA (mRNA) and PACAP immunoreactivity were observed in the majority of granulosa and cumulus cells from large preovulatory follicles, in the majority of the cells comprising the interstitial glandular tissue and in solitary theca cells of growing and mature follicles. In PMSG/hCG treated animals, positive signals were also observed in solitary theca and interstitial cells 12 h after PMSG injection. PACAP immunoreactive nerve fibers were observed in all ovaries examined, located in the interstitial glandular tissue or in the loose connective tissue. Northern blotting of ovarian tissue confirmed the transient preovulatory and ovulatory expression of PACAP. Three transcripts were observed. The most predominant band had a size of approximately 1.2 kilobase pairs, corresponding to the transcript observed in the testis. Two bands of 2.4 and 3.0 kilobase pairs, corresponding to the transcripts from neural tissue, were also observed. The complex distribution of PACAP positive cells and nerve fibers suggests that the peptide could be a local regulator of a number of the events that take place in the periovulatory period as well as during early folliculogenesis in the ovary.

Stimulation of progesterone and prostaglandin E accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells.

The present investigation was performed to characterize the accumulation of progesterone and prostaglandin E (PGE) by rat granulosa cells exposed to LHRH and LHRH analogs in vitro. Granulosa cells were obtained from immature rats 48 h after the injection of 20 IU PMS gonadotropin and incubated for up to 5 h, followed by determination of the total progesterone and PGE accumulated in cells and medium. LHRH and several LHRH agonists, at concentrations of 10-2000 ng/ml, caused 2- to 3-fold increases in progesterone accumulation. This stimulation was consistently much less than the 15- to 30-fold increase produced by LH or FSH. A LHRH agonist also stimulated PGE synthesis by rat granulosa cells, but in this case the stimulation was similar in magnitude and time course to the effect of LH. In addition, the injection of a LHRH agonist into hypophysectomized, PMS gonadotropin-treated immature rats resulted in elevated ovarian PGE content 5 h later. The effect of LHRH on progesterone and PGE accumulation by rat granulosa cells was blocked by a LHRH antagonist. Lh stimulation of progesterone and PGE accumulation, on the other hand, was not affected by this antagonist. Incubation of granulosa cells with LHRH agonist did not reduce the progesterone or PGE response to LH or FSH, even at suboptimum concentrations of gonadotropin. The presence of 10(-7) M testosterone did not modify the effect of a LHRH agonist on basal or LH-stimulated progesterone and PGE accumulation. In acute incubations of granulosa cells from mature follicles, therefore, LHRH specifically stimulates progesterone and PGE accumulation when present alone, and does not modify the stimulation of progesterone accumulation by LH or FSH.

Prolactin modifies follicle-stimulating hormone-induced prostaglandin synthesis by the rat granulosa cell.

Female rats that had been hypophysectomized and implanted with a diethylstilbestrol capsule at 21 days of age were sacrificed 7 days after treatment with various combinations of ovine FSH, PMS gonadotropin, hCG, and ovine PRL (oPRL). Suspensions of granulosa cells obtained from the different groups were incubated at 37 C for 2 h with or without 5 × 10-4 M indomethacin. Prostaglandins (PG) E and F2α extracted from the suspensions and partially purified by chromatography on silicic acid columns were quantitated by specific RIA. The amounts of PG synthesized by the diethylstilbestrol-treated controls ranged from 10–2000 pg/106 cells and were not significantly altered by treatments with a single 5-IU hCG injection and/or 100 μg oPRL every 4 h. Three injections of 100 μg FSH markedly increased the rates of synthesis of PGE and PGF2α up to 2400 and 5600 pg/106 cells, respectively. These rates were increased still further by the injection of a FSH and hCG mixture. Additional injections of 100 μg oPRL every 4 h decreased the synthesis of PGE by 79– 89% and that of PGF2α by 35–56% in the FSH or FSH plus hCG groups, respectively. However, hourly injections of graded low doses of oPRL into PMS gonadotropin- plus hCG-treated animals stimulated both PGE and PGF2α synthesis; maximal stimulation was a 3-fold increase over the control level and occurred at a dose of 1–2.5 μg oPRL‐h for PGE and at 1 μg oPRL‐h for PGF2α. PRL treatment altered progesterone levels in a dosedependent fashion, gradually increasing from 0 to 1124 pg/ml in the 0‐50 μg oPRL/h treatment groups, respectively. This indicates that PRL may alter both ovarian steroidogenesis and the ovulatory process by modifying the FSH-related stimulation of PG synthesis.

Biosynthesis of proteoglycans by rat granuloma cells cultured in vitro: modulation by gonadotropins, steroid hormones, prostaglandins, and a cyclic nucleotide.

Rat ovarian granulosa cells were isolated from immature female rats 48 h after stimulation with 5 IU PMS gonadotropin and then maintained in culture. The effects of ovine (o)FSH, oLH, hCG, testosterone, 17 beta-estradiol, progesterone, prostaglandins E1, E2, F1 alpha, F2 alpha, N,O'-dibutyryl cAMP, and theophylline on proteoglycan synthesis by the granulosa cells in vitro were examined using [35S]sulfate as a precursor. oFSH, oLH, hCG, prostaglandins E1 and E2, N,O'-dibutyryl cAMP, theophylline and testosterone stimulated the production of 35S-labeled proteoglycans compared to control cultured. 17 beta-Estradiol, progesterone, and the prostaglandins F1 alpha, and F2 alpha, showed no stimulation. Two major species of proteoglycan are synthesized and secreted into the medium: 1) a population with large hydrodynamic size (Kav = 0.30 on Sepharose CL-2B); and 2) a population with relatively small size (Kav = 0.60 on Sepharose CL-2B). The stimulatory effect of the hormones was accounted for entirely by a net increase in synthesis of the smaller proteoglycan population. The hormones exert their stimulatory effects on proteoglycan synthesis at concentrations similar to those required for other in vitro biological responses of granulosa cells.

Gonadocrinins

A newly discovered small peptide purified from rat follicular fluid stimulates the pituitary to release FSH and LH in vitro as well as in vivo. Dialysates of crude acid extracts of ovarian follicular tissue and fluid from rats pretreated with PMS gonadotropin stimulate the secretion of both LH and FSH, but not PRL, GH, or TSH, in a pituitary monolayer culture system. This stimulating factor, named gonadocrinin for operational facility, is smaller than 3500 daltons; its biological activity disappears after treatment with trypsin. Gonadocrinin is not recognized by two antisera binding the decapeptide LRF even though D-Phe2,D-Trp6-LRF, an LRF analog antagonist, competitively inhibits the activity of ovarian gonadocrinin. Cultured rat granulosa cells also secrete substances with gonadocrinin activity in vitro, indicating that the granulosa cells probably are in vivo the source of gonadocrinin. A crude preparation of gonadocrinin given iv to rats on the second aday of diestrus induced secretion of LH comparabl...

Effect of luteinizing hormone and follicle-stimulating hormone on progesterone synthesis by cultured rat cumulus cells.

This study examined the responsiveness of the rat cumulus oophorus to gonadotropin in terms of progesterone (P) secretion. Immature female rats pretreated with a single injection of PMS gonadotropin were used. Oocyte-cumulus complexes were isolated aseptically from large preovulatory follicles or from the oviduct shortly after ovulation and cultured in modified Eagle's medium. Ten cumuli from each rat were cultured for 24 or 48 h, with change of medium after 12 and 24 h. P accumulation in the culture medium was determined by RIA, and cell morphology was examined. Cumuli isolated from rats killed before the endogenous LH/FSH surge and cultured in hormone-free medium secreted low amounts of P (<10 pg/cumulus/ day) throughout the culture period. By contrast, cumuli isolated 3–5 h after the surge secreted high amounts of P (300–400 pg/day). This increment in P secretion was abolished by pentobarbital treatment of the rats during the critical period. Postovulatory cumuli isolated from the oviduct 5–9 h after o...

Gonadocrinins: peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins.

A newly discovered small peptide purified from rat follicular fluid stimulates the pituitary to release FSH and LH in vitro as well as in vivo. Dialysates of crude acid extracts of ovarian follicular tissue and fluid from rats pretreated with PMS gonadotropin stimulate the secretion of both LH and FSH, but not PRL, GH, or TSH, in a pituitary monolayer culture system. This stimulating factor, named gonadocrinin for operational facility, is smaller than 3500 daltons; its biological activity disappears after treatment with trypsin. Gonadocrinin is not recognized by two-antisera binding the decapeptide LRF even though D-Phe2,D-Trp6-LR, an LRF analog antagonist, competitively inhibits the activity of ovarian gonadocrinin. Cultured rat granulosa cells also secret substances with gonadocrinin activity in vitro, indicating that the granulosa cells probably are in vivo the source of gonadocrinin. A crude preparation of gonadocrinin given iv to rats on the second day of diestrus induced secretion of LH comparable to that produced by a 250-ng LRF injection. Gonadocrinin has chemical characteristics different from those of LRF. When purified gonadocrinin or LRF was applied to an identical isocratic high pressure liquid chromatography system, LRF was eluted at a position different from that of gonadocrinin, indicating that, chemically, gonadocrinin is not identical to the hypothalamic decapeptide, LRF.

Gonadotropin-releasing hormone surge: possible modulation through postsynaptic alpha-adrenoreceptors and two pharmacologically distinct dopamine receptors.

We have studied modulation of the release of gonadotropin-releasing hormone (GnRH) by central monoaminergic neurons. The test system was the GnRH surge that occurs at 1700 h on day 32 in female rats injected on day 30 with PMS gonadotropin (PMSG). The release of GnRH, measured by RIA of pituitary stalk blood, was correlated with the release of LH, measured by RIA of external jugular venous blood. All blood samples were collected under Althesin (alphaxalone plus alphadolone acetate) anesthesia. The heights of the PMSG-induced surges of GnRH and LH were reduced in animals which had been injected with either αmethyl-p-tyrosine (an inhibitor of tyrosine hydroxylase), or diethyldithiocarbamate or fusaric acid (inhibitors of dopamine-β-hydroxylase). The action of all three inhibitors was reversed by administering dihydroxyphenylserine, and the inhibitory action of diethyldithiocarbamate was potentiated by L-dopa, The height of the PMSG-induced LH surge was reduced by phenoxybenzamine, but was not affected by ph...

Progesterone and prostaglandin secretion by ovulated rat cumulus cell-oocyte complexes.

Hormone production (progesterone and prostaglandin) by oocyte-cumulus cell complexes was studied in the postovulatory period. Cumulus-oocyte complexes were collected from oviducts of prepuberal rats after the synchronization of follicle development and ovulation with PMS gonadotropin and hCG injections. Cumulus masses from individual animals were cultured in roller vessels containing Eagles' Minimal Essential Media with or without added heat-inactivated rat sera (5% CO2-air; 37 C). Culture media were collected after 4.5 h of incubation and analyzed by RIA for prostaglandin E2 (PGE), PGF, and progesterone. Extensive progesterone secretion occurred during the culture period and was markedly stimulated by the addition of rat serum; however, cumulus masses did secrete some progesterone in the absence of rat serum and without exogenous gonadotropins. The addition of aminoglutethimide, a steroidogenesis inhibitor, suppressed progesterone secretion but did not alter the secretion of PGF or PGE. Both PGF and PGE were detected in the culture medium, and in all cases, PGE was the predominant PG detected. Indomethacin, an inhibitor of PG synthesis, suppressed PGE and PGF secretion but had no effect on progesterone synthesis. Small amounts of PGs were also detected in nonincubated cumulus-oocyte masses. Hormone secretion in culture was measured after cumulus mass dissociation by enzymatic digestion. Somatic cumulus cells, rather than germ cells, were the primary cellular source of progesterone and PGs. The results demonstrate that cumulus-oocyte complexes can synthesize and secrete steroid and PG hormones subsequent to ovulation, and thus, these masses should be considered functional endocrine tissues. The physiological significance of the endocrine activities of the cumulus-oocyte complex are discussed in terms of oviductal and gametic processes. (Endocrinology 108: 457, 1981)

Ovarian gonadotropin-releasing hormone receptors. II. Regulation and effects on ovarian development.

The direct antagonistic actions of gonadotropinreleasing hormone (GnRH) and its agonist analogs on hormonal (hCG and epinephrine) activation of progesterone and cAMP production in isolated luteal cells were described in the preceding paper. These effects are mediated by specific, high affinity GnRH receptors demonstrated in both ovarian membranes and isolated luteal cells. The present studies show that the administration of GnRH agonist analogs in vivo markedly inhibits PMS gonadotropin (PMSG)-induced follicular development and the increased formation of LH receptors after hCG-induced luteinization. Three GnRH agonist analogs with structural modifications at positions 6 and 10 given by osmotic minipump infusion, caused complete inhibition of the trophic actions of both gonadotropins. An antagonist analog which binds to the ovarian GnRH receptor with high affinity did not inhibit the gonadotropin-induced changes. Using the PMSG/hCG-primed immature rat, the effects of a single injection of GnRH were studied and compared to the action of hCG. Whereas the latter caused a marked loss of GnRH and LH receptors, GnRH caused a loss of ovarian LH receptors but a consistent increase in ovarian GnRH receptors; PRL receptors also showed a transient decline, and there was a fall in serum progesterone. In hypophysectomized rats, GnRH and hCG similarly caused a decrease in ovarian LH receptors and a transient change in the PRL receptor concentration. The GnRHinduced decrease in LH and PRL receptors in hypophysectomized animals indicates that GnRH analogs can act directly on the ovary and that their effects are not only due to the secondary release of gonadotropins from the pituitary gland. The direct inhibitory actions of this class of GnRH peptides upon gonadal function constitutes an important component of the antifertility effects of these compounds.

Regulation of uterine angiotensin II receptors by estrogen and progesterone.

The characteristics of uterine receptors for angiotensin II and their regulation during the estrous cycle and after gonadal steroid treatment were investigated in the rat and rabbit. Binding of [125I]iodoangiotensin II to rat uterine particles was temperature dependent and saturable, and Scatchard analysis showed a single population of binding sites with Ka of about 109m−1. The concentration of uterine angiotensin II receptors changed markedly during the estrous cycle of the rat and after treatment with gonadal steroids. During the ovarian cycle, the concentration of angiotensin II receptors was about 4 times higher at proestrus than at diestrus II. Similar changes were observed when estrous cycles were induced in immature rats by a single injection of PMS gonadotropin. Ovariectomy was followed by a progressive decrease in uterine angiotensin II receptors, reaching 50% of the control values 8 days after castration. Conversely, injection of 17β-estradiol caused a dose-dependent increase in angiotensin II receptors measured 24 h after steroid treatment. Similarly, short term infusion of 17β-estradiol caused a progressive increase in angiotensin II receptors, which reached a maximum of 4- to 6-fold above control values at 36 h. However, after long term infusion of estradiol for 5–8 days, angiotensin receptors returned to or below the control values. Progesterone infusion for 7 days caused uterine angiotensin II receptors decrease by 93%. When immature rabbits were primed with estrogen, a 4-fold increase in the concentration of uterine angiotensin II receptors was measured. In progesterone-primed uteri, angiotensin II receptor concentration was no longer augmented by estrogen. No effects of steroids upon the affinity of the receptors for angiotensin II were detected, all changes in binding being due to alterations in the tissue content of receptor sites. These studies have revealed marked changes in the concentration of angiotensin receptors during the estrous cycle and after steroid treatment. Thus, receptors for angiotensin II are regulated by estrogen in manner similar to the control of receptors for other oxytocic hormones. These findings may explain the increased contractile sensitivity of estrogen-primed uteri to angiotensin II and suggest a role for angiotensin II in the physiological control of myometrial activity. (Endocrinology106: 5, 1980)

An Explanation for Anovulation in Immature Alloxan-Diabetic Rats Treated with Pregnant Mare's Serum Gonadotropin: Reduced Pituitary Response to Gonadotropin-Releasing Hormone*

We have recently reported that immature alloxan- diabetic rats injected with PMS gonadotropin do not ovulate because the preovulatory LH surge is absent. In the present studies, the hypothesis was tested that alloxan-induced diabetes might alter hypothalamic gonadotropin-releasing hormone (GnRH) depletion and⁄or pituitary response to GnRH. Immature rats were injected with PMSG at 30 days of age. In one experiment, the rats were killed either before or during the expected proest-rous LH surge (day 32), and the hypothalami were removed and assayed for GnRH content. In other experiments, the effects of GnRH on ovulation and serum and pituitary LH were tested by injecting GnRH early on presumed proestrus (day 32). A significant and similar degree of depletion of hypothalamic GnRH was found in diabetic and control rats after the time of the expected LH surge in nondiabetic rats. Although the degree of pituitary exposure to endogenous GnRH is unknown, these data suggest that the hypothalamic release of GnRH is ...

Chronic Effects of [D-Trp6,Pro9-NEt]Luteinizing Hormone-Releasing Factor on Reproductive Processes in the Male Rat*

Chronic administration of a potent LRF agonist, [D-Trp6,Pro9-NEt]LFR (A), to intact adult male rats induced a dose-related decrease in testicular and accessory organ weights, diminished production of testosterone (T), and disrupted tubular morphology, indicative of suppression of spermatogenesis. Transient elevations of plasma progesterone levels and a marked reduction of PRL secretion were also observed in A-treated rats. The effects of A on testicular weights, T levels, and seminiferous tubule morphology were already apparent 3 days after the first injection. Administration of A was equally effective when administered daily or at 1- to 4-day intervals in lowering steroid levels and testicular weights. After a 1-week daily exposure to A, seminal vesicles and prostate weights as well as T levels returned to control values within 1--2 weeks. Testicular weights, however, were still depressed 4 weeks after cessation of treatment, and germinal epithelium was disrupted in 30--40% of the tubules. Measurement of T and LH plasma levels after each daily injection of A indicated a blunting of succeeding A-induced elevations of LH and T, which lasted up to 72 h after a single administration of A. The observation that cultured pituitary cells repeatedly exposed to A showed a similar blunting of LRF-induced LH elevation suggests that the decrease in pituitary responsiveness was not exclusively due to steroid feedback. Administration of T propionate to A-treated intact rats was unable to restore normal testicular weights and seminiferous tubule morphology, whereas in hypophysectomized rats, T propionate maintained qualitatively normal spermatogenesis even in the presence of A. These results coupled with the observation that high levels of hCG and PMS gonadotropin had effects similar to those of A on the tubular morphology of intact rats suggest the importance of pituitary secretion in mediating the deleterious effects of A on reproductive functions in the male rat.

Androgen and estrogen metabolism in male rhesus monkeys.

Using constant infusions of 3H-labeled androgens and 14C-labeled estrogens, the dynamics of androgen and estrogen metabolism have been studied in male rhesus monkeys. The mean (+/-) MCR, as measured in whole blood, for testosterone (T; 140 +/- 10 liters/day) was significantly smaller than the MCRs for androstenedione (A), estrone (E1), and estradiol (E2; 650 +/- 8, 1010 +/- 160, and 500 +/- 20 liters/day, respectively). These findings are compatible with the specific globulin binding of T in this species. The mean blood productions were 0.42 +/- 0.11 and 0.55 +/- 0.04 mg/day for A and T and 40 +/- 16 and 13 +/- micrograms/day for E1 and E2, respectively. The interconversions of the androgens [fractional conversion rates of A to T ([rho]A,TBB) and T to A ([rho]T,ABB)] were not different but were lower than the interconversions of the estrogens ([rho]E1,E2BB and [rho]E2,E1BB). The mean [rho]T,E2BB values were 0.015 +/- 0.001 and 0.0024 +/- 0.0003, which are remarkably similiar to those values in men. Administrations of PMS gonadotropin did not alter any measured values of androgen or estrogen metabolism, hCG resulted in a decrease in the conversion ratio of A to T, T administration increased the MCR of T, and estrogen administration resulted in no changes. The aromatization rate remained unaffected by all treatments.

Ovarian Responses of Pregnant Mare Serum Gonadotropin- and Human Chorionic Gondotropin-Primed Rats: Desensitizing, Luteolytic, and Ovulatory Effects of a Single Dose of Human Chorionic Gonadotropin*

We conducted a study to determine the morphological appearance and functional responsiveness of ovarian tissues after administration of hCG to 28-day-old rats primed 65 h earlier with PMS gonadotropin (PMSG) and after administration of a second dose of hCG 5 days later, i.e. to 33-day-old rats containing heavily luteinized ovaries. Sixty-five hours after the administration of 50 IU PMSG sc to 25-day-old rats, ovaries already contained an abundance of luteinized follicles and an adenylyl cyclase (AC) system that was responsive to LH, epinephrine, and NaF. The administration of 50 IU hCG sc at this time initially resulted in a loss of LH-responsive ovarian AC. Within 4 days of the hCG injection, the ovaries of the now 32-day-old rats were heavily luteinized, and ovarian AC was highly responsive to LH, epinephrine, and NaF. The administration of a single sc dose of 200 IU hCG to 33-day-old PMSC- and hCG-primed rats with luteinized ovaries resulted in a rapid desensitization of the ovarian AC to LH and a drop in serum progesterone levels, During the subsequent 7 days, serum progesterone levels continued to decline, while total ovarian AC reacquired responsiveness to LH by days 4--5 after the densensitizing dose of hCG. Dissection of ovarian components revealed, however, that the AC system of the corpora lutea originally present at the time of the second hCG injection remained permanently refractory to LH and that the AC in corpora lutea newly formed from freshly ovulated follicles exhibited a significant responsiveness to LH, epinephrine, and NaF. However, these new corpora lutea were not fully active, since serum progesterone never rose. Subcutaneous administration of 50 IU hCG to 33-day-old PMSG- and hCG-primed rats also promoted a rapid loss of AC responsiveness to LH. This lower concentration of hCG was not sufficient to promote follicular development or ovulation, and the ovarian AC remained refractory to LH for at least 7 days. Intravenous administration of 75 IU hCG to 33-day-old PMSG- and hCG-primed rats similarly promoted a rapid and permanent loss of luteal AC responsiveness to LH; again, follicles did not mature to a preovulatory state and, in fact, appeared to undergo atresia rather than ovulation. These results indicate that in heavily luteinized ovaries 1) hCG promotes desensitization of rat luteal AC to LH, 2) Desensitization of AC to LH stimulation in corpora lutea is permanent and irreversible, and 3) only under conditions where follicles mature and ovulate and new corpora lutea are formed does total ovarian AC reacqure responsiveness during the subsequent week.

Enzymological Studies of Rat Luteinized Ovaries in Relation to Acute Reduction of Aromatizable Androgen Formation and Stimulated Production of Progestins*

Female rats received an injection of 10 IU PMS gonadotropin (PMSG) sc at 21 days of age. One group of the animals was then injected with 30 IU hCG sc at 23 days of age (PMSG-hCG treatment), and the other was not given hCG (PMSG treatment). All the animals were sacrificed at 24 days of age. The ovarian weight was increased by the PMSG treatment and further increased by additional hCG, whereas the uterine weight was increased by PMSG but decreased by hCG. The supernatant fluid obtained by centrifugation at 10,000 × g of homogenates of the ovaries was incubated with 14C-labeled steroids in the presence of the required cofactor to measure steroid-metabolizing enzyme activities. After PMSG-hCG treatment, activities of 17α-hydroxylase and C-17-C-20 lyase per mg protein or per pair of ovaries decreased markedly compared with those of the PMSG-treated group, whereas the aromatase and 20α-hydroxysteroid dehydrogenase activities were enhanced significantly. The activity of Δ5-3β-hydroxysteroid dehydrogenase coupled...

Chronic Effects of [D-Trp6-Pro9-NEt]Luteinizing Hormone-Releasing Factor on Reproductive Processes in the Female Rat*

LRF and one of its highly potent analogs, [D-Trp6-Pro9-NEt]LRF (A), were tested for their effects on pregnancy. Daily doses of A prevented implantation and/or caused resorption of implanted blastocysts in mated female rats. This effect was dose related and was most evident when given in regimens including days 5-7 or days 9-10 or pregnancy. LRF also has antigonadal effects, but at much higher doses than A. The use of corn oil vs. 0.1% gelatin-saline improved the effectiveness of low doses of A. Termination of pregnancy in A- or LRF-treated rats is most probably due to the markedly diminished progesterone secretion, which could result from the secretion of biologically inactive gonadotropins, reduced pituitary responsiveness to endogenous LRF, or down-regulation of gonadal receptors for LH.

Prolactin, Growth Hormone, Luteinizing Hormone Receptors, and Seasonal Changes in Testicular Activity in the Golden Hamster*

In adult male hamsters, 2 months of exposure to a short photoperiod (5 h of light:19 h of darkness) caused testicular regression and a precipitous decline in plasma PRL, in agreement with earlier reports from other laboratories. Depressed release of PRL cannot be explained by a reduction in testicular steroidogenesis, because castration of males kept in a long photoperiod did not reduce PRL levels and administration of testosterone to males kept in a short photoperiod failed to reverse the decline in plasma PRL concentration. Treatment of such "regressed" animals with PRL, GH, or ectopic pituitary transplants stimulated growth of the testes and the accessory reproductive glands, increased the concentration of LH receptors in the testes, and elevated plasma testosterone levels. A single injection of 250 microgram PRL was sufficient to increase testicular LH binding, and chronic treatment with pituitary grafts completely reversed testicular regression. The effectiveness of exogenous PRL in stimulating testicular growth and LH receptors was significantly influenced by the timing of the injection. In some experiments, gonadotropin levels appeared elevated in animals injected with PRL, but these differences were not statistically significant. In hamsters with gonadal regression induced by exposure to a short photoperiod, daily administration of 20 microgram H and/or 150 microgram FSH had no apparent effect on testicular function. However, treatment with large doses of hCG and/or PMS gonadotropin resulted in significant stimulation of testicular growth and steroidogenesis. Chronic treatment of males maintained in a long photoperiod (14 h of light:10 h of darkness) with an inhibitor of PRL release, 2-Br-alpha-ergocryptine, resulted in a decreased weight of the testes and seminal vesicles. Administration of this inhibitor for a longer period (2 months) produced a significant increase in body weight but had little effect on testicular function. These results indicate that changes in the release of PRL (and possibly also GH) may plan an important role in mediating the effects of the photoperiod on testicular function in the golden hamster.

Changes in steroidogenesis in preovulatory rat ovaries induced to ovulate with pregnant mare serum gonadotropin.

The pattern of steroids produced by the immature rat ovary is different from that observed in the adult cycling rat. Changes in steroid production were studied several hours before the first ovulation, as it was anticipated that during this transitional period both the immature and mature pattern of steroid formation may coexist in the ovary. Ovarian homogenates (1000 ×g supernatant) obtained from 26-day-old rats treated 52 h previously with 25 IU PMS gonadotropin were incubated in the presence of NADPH with either [14C]progesterone, 20α- [3H]hydroxy -4 - pregnen - 3 - one (20α-OHP), or [14C]progesterone and 3α-[3H]hydroxy- 5α-pregnan-20-one (5α -3α -ol-20-one). The dose of PMS gonadotropin used induces an endogenous LH surge in the afternoon of day 26, which is followed by ovulation. After incubation, radioactive metabolites were isolated, identified, and measured by thin layer and gas-liquid chromatography, with derivative formation and recrystallization to constant specific activity. 5α -Pregnane-3α,20...

Etiology of anovulation in the immature alloxan-diabetic rat treated with pregnant mare's serum gonadotropin: absence of the preovulatory luteinizing hormone surge.

The effects of alloxan-induced diabetes on ovulation and other ovarian responses were investigated in immature rats injected with PMS gonadotropin (PMSG, 15 IU/100 g) on day 30 of age. Rats were killed on day 32 (presumed proestrus) or on day 33, at which time the oviducts were examined for ova. Ovarian weight gain was similar in control and diabetic rats and Graafian follicles were present in both groups on day 32. None of the diabetic rats ovulated while 96% of the control rats ovulated. Anovulation in diabetic rats could not be attributed to a drug side-effect of alloxan or to a lack of ovarian responsiveness, as 90% of the animals ovulated after treatment with insulin or with hCG (5 IU). Measurements of serum estradiol and LH on the morning of presumed proestrus revealed that concentrations of these hormones were not different in control and diabetic rats. However, measurements of LH in blood samples taken in the afternoon from control rats showed an LH surge, whereas no LH surge was found in diabetic rats. Thus, anovulation in immature diabetic rats treated with PMSG is not caused by an attenuation of ovarian responsiveness or by decreased secretion of estradiol, but rather is due to the loss of the LH surge.