LH Replacement Research Articles

Expression of Estrogen Receptor α 36 (ESR36) in the Hamster Ovary throughout the Estrous Cycle: Effects of Gonadotropins

Estradiol-17β (E) plays an important role in ovarian follicular development. Evidence indicates that some of the effect of E is mediated by the transmembrane estrogen receptor. In this study, we examined the spatio-temporal expression of recently discovered ERα36 (ESR36), a splice variant of Esr1 and a receptor for non-genomic E signaling, in the hamster ovary during the estrous cycle and the role of gonadotropins and ovarian steroid hormones in ESR36 expression. ESR36 expression was high on estrus (D1∶0900 h) and declined precipitously by proestrus (D4∶0900 h) and remained low up to D4∶1600 h. Immunofluorescence findings corroborated immunoblot findings and revealed that ESR36 was expressed only in the cell membrane of both follicular and non-follicular cells, except the oocytes. Ovarian ESR36 was capable of binding to the E-affinity matrix, and have different molecular weight than that of the ESR1 or GPER. Hypophysectomy (Hx) resulted in a marked decline in ESR36 protein levels. FSH and LH, alone or combined, markedly upregulated ESR36 protein in Hx hamsters to the levels observed in D1 hamsters, but neither E nor P had any effect. Inhibition of the gonadotropin surge by phenobarbital treatment on D4∶1100 h attenuated ESR36 expression in D1∶0900 h ovaries, but the decline was restored by either FSH or LH replacement on D4 afternoon. This is the first report to show that ESR36, which is distinct from ESR1 or GPER is expressed in the plasma membrane of ovarian follicular and non-follicular cells, binds to E and its expression is regulated directly by the gonadotropins. In light of our previous findings, the results suggest that ovarian cells contain at least two distinct membrane estrogen receptors, such as GPER and ESR36, and strongly suggest for a non-genomic action of E regulating ovarian follicular functions.

The effect of progesterone replacement on gene expression in the corpus luteum during induced regression and late luteal phase in the bonnet monkey (Macaca radiata)

BackgroundIn higher primates, although LH/CG play a critical role in the control of corpus luteum (CL) function, the direct effects of progesterone (P4) in the maintenance of CL structure and function are unclear. Several experiments were conducted in the bonnet monkey to examine direct effects of P4 on gene expression changes in the CL, during induced luteolysis and the late luteal phase of natural cycles.MethodsTo identify differentially expressed genes encoding PR, PR binding factors, cofactors and PR downstream signaling target genes, the genome-wide analysis data generated in CL of monkeys after LH/P4 depletion and LH replacement were mined and validated by real-time RT-PCR analysis. Initially, expression of these P4 related genes were determined in CL during different stages of luteal phase. The recently reported model system of induced luteolysis, yet capable of responsive to tropic support, afforded an ideal situation to examine direct effects of P4 on structure and function of CL. For this purpose, P4 was infused via ALZET pumps into monkeys 24 h after LH/P4 depletion to maintain mid luteal phase circulating P4 concentration (P4 replacement). In another experiment, exogenous P4 was supplemented during late luteal phase to mimic early pregnancy.ResultsBased on the published microarray data, 45 genes were identified to be commonly regulated by LH and P4. From these 19 genes belonging to PR signaling were selected to determine their expression in LH/P4 depletion and P4 replacement experiments. These 19 genes when analyzed revealed 8 genes to be directly responsive to P4, whereas the other genes to be regulated by both LH and P4. Progesterone supplementation for 24 h during the late luteal phase also showed changes in expression of 17 out of 19 genes examined.ConclusionThese results taken together suggest that P4 regulates, directly or indirectly, expression of a number of genes involved in the CL structure and function.

Transforming Growth Factor-β1 Null Mutation Causes Infertility in Male Mice Associated with Testosterone Deficiency and Sexual Dysfunction

TGFbeta1 is a multifunctional cytokine implicated in gonad and secondary sex organ development, steroidogenesis, and spermatogenesis. To determine the physiological requirement for TGFbeta1 in male reproduction, Tgfb1 null mutant mice on a Prkdc(scid) immunodeficient background were studied. TGFbeta1-deficient males did not deposit sperm or induce pseudopregnancy in females, despite an intact reproductive tract with morphologically normal penis, seminal vesicles, and testes. Serum and intratesticular testosterone and serum androstenedione were severely diminished in TGFbeta1-deficient males. Testosterone deficiency was secondary to disrupted pituitary gonadotropin secretion because serum LH and to a lesser extent serum FSH were reduced, and exogenous LH replacement with human chorionic gonadotropin (hCG) induced serum testosterone to control levels. In the majority of TGFbeta1-deficient males, spermatogenesis was normal and sperm were developmentally competent as assessed by in vitro fertilization. Analysis of sexual behavior revealed that although TGFbeta1 null males showed avid interest in females and engaged in mounting activity, intromission was infrequent and brief, and ejaculation was not attained. Administration of testosterone to adult males, even after neonatal androgenization, was ineffective in restoring sexual function; however, erectile reflexes and ejaculation could be induced by electrical stimulation. These studies demonstrate the profound effect of genetic deficiency in TGFbeta1 on male fertility, implicating this cytokine in essential roles in the hypothalamic-pituitary-gonadal axis and in testosterone-independent regulation of mating competence.

Gonadotropin and steroid regulation of matrix metalloproteinases and their endogenous tissue inhibitors in the developed corpus luteum of the rhesus monkey during the menstrual cycle.

The factors regulating the dynamic expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the primate corpus luteum (CL) during the menstrual cycle are unknown. We hypothesized that LH or progesterone (P) regulate interstitial-collagenase (MMP-1), the gelatinases (MMP-2 and -9), TIMP-1, and TIMP-2 in the CL. Hormone ablation/replacement was performed in rhesus monkeys on Days 9-11 of the luteal phase in five treatment groups (n = 4/group): control (no treatment), antide (GnRH antagonist), antide + LH; antide + LH + trilostane (TRL; 3beta-hydroxysteroid dehydrogenase inhibitor), and antide + LH + TRL + R5020 (nonmetabolizable progestin). On Day 12, the CL was removed and the RNA and protein isolated for real-time polymerase chain reaction and immunoassays, respectively. The MMP-1 mRNA increased 20-fold with antide, whereas LH replacement maintained MMP-1 mRNA at control levels. Likewise, TRL increased MMP-1 mRNA 54-fold, and R5020 prevented this effect. Immunodetectable MMP-1 protein also increased with antide or TRL; these increases were abated with LH or R5020. Gelatinase mRNA and/or protein levels increased with antide (e.g., 3-fold, MMP-2 mRNA), and LH replacement reduced protein levels (e.g., 11-fold, MMP-2). The TRL increased MMP-9, but not MMP-2, expression; however, R5020 replacement had no effect on mRNA or protein levels. The LH treatment increased TIMP-1 and -2 mRNA and TIMP-1 protein expression compared to controls and antide groups, whereas R5020 enhanced only immunodetectable TIMP-1. These data strongly suggest that LH suppresses MMP-1 in the primate CL via P and that it also suppresses gelatinases, either at the mRNA (MMP-2) or protein (MMP-2 and -9) levels, perhaps in part via steroids, including P. In contrast, LH promotes TIMP expression, perhaps via steroids, including P.

Titrating luteinizing hormone replacement to sustain the structure and function of the corpus luteum after gonadotropin-releasing hormone antagonist treatment in rhesus monkeys.

These studies were designed to identify 1) a regimen of a third generation GnRH antagonist that abolishes primate luteal function, and 2) the amount of LH replacement required to maintain the structure and functional life span of the corpus luteum of the menstrual cycle after GnRH antagonist treatment. A single injection of antide at 3 or 5 mg/kg BW on day 6 of the luteal phase suppressed serum progesterone levels within 1 day of treatment, but levels recovered within 4 days. Administration of antide (3 mg/kg) for 3 days (luteal days 6-8) reduced (P < 0.05) serum progesterone below 1 ng/mL and maintained these low levels for the entire sampling period; in subsequent experiments, all monkeys received this antide regimen. Fixed doses (5, 10, or 20 IU) of recombinant human LH administered at 8-h intervals during and after antide treatment stimulated progesterone production in a dose-dependent manner; these monkeys menstruated earlier than controls regardless of treatment group. Replacement with an escalating dose regimen (5-20 IU) of LH resulted in typical serum progesterone and relaxin levels throughout a luteal phase of normal length. Corpora lutea removed on day 10 from monkeys treated with antide alone had decreased wet weight (P < 0.05) and few large luteal cells; coadministration of the escalating dose regimen of LH maintained luteal structure similar to that seen in time-matched controls. Antide-only treatment increased progesterone receptor (PR) messenger ribonucleic acid, but decreased PR immunostaining in luteal tissue; the escalating dose regimen of LH maintained PR messenger ribonucleic acid and immunostaining similar to those in controls. This study indicates that during GnRH antagonist administration, an escalating dose regimen of LH replacement is optimal for maintenance of the structure and functional life span of the primate corpus luteum.

Leydig cell peroxisomes and sterol carrier protein-2 in luteinizing hormone-deprived rats.

We investigated the effects of 8 days of LH withdrawal on rat Leydig cell peroxisomal volume, total and intraperoxisomal catalase and sterol carrier protein-2 (SCP2) contents, and LH-stimulated testosterone secretion in vitro. Three groups of adult male Sprague-Dawley rats, i.e. control, TE-implanted (testosterone-17 beta-estradiol-filled Silastic implants to suppress LH), and TELH-implanted (TE-implanted and LH replacement via Alzet mini osmotic pumps), were used. After 8 days, Leydig cell organelle volumes (stereology), intraperoxisomal catalase and SCP2 contents (immunocytochemistry), LH-stimulated testosterone secretion by isolated Leydig cells in vitro (determined by RIA), and total catalase and SCP2 contents in equal numbers of Leydig cells (immunoblot analyses) were determined. Results showed that the TELH-implanted rats were identical to controls in every parameter tested. Testis volume and Leydig cell number per testis in control and TE-implanted rats were not significantly different; however, reductions (P < 0.05) were observed in the average volume of a Leydig cell (one third of controls) and the volume of Leydig cells per testis. All Leydig cell organelle volumes tested were significantly lower in TE-implanted rats than in the controls; however, the volumes of smooth endoplasmic reticulum (SER) and peroxisomes were the most reduced (lowered to one sixth of control values). LH-stimulated testosterone secretion per Leydig cell in vitro correlated well with these changes in the volumes of Leydig cell SER and peroxisomes. Intraperoxisomal catalase in Leydig cells was unchanged in TE-implanted rats, although immunoblotting demonstrated a loss of total catalase content (which reflected the reduction in the volume of peroxisomes). SCP2 in Leydig cells of TE-implanted rats was undetectable with immunoblot analysis (explained by the reductions in Leydig cell peroxisome volume and intraperoxisomal SCP2). These results demonstrate that the organelles SER and peroxisomes and the protein SCP2 in Leydig cells are more LH dependent than the other organelles (e.g. mitochondria, lysosomes) and protein catalase, respectively. Moreover, the findings of this study are consistent with the hypothesis that Leydig cell peroxisomes play a significant role in testosterone production.

The Role of Luteinizing Hormone in Regulation of Testicular Inhibin α and β-B Subunit Messenger RNAs in Immature and Adult Animals*

In vivo and in vitro evidence indicates that FSH is a primary regulator of testicular inhibin production. However, recent reports suggest that LH may also promote inhibin secretion in vivo. To investigate whether LH regulates inhibin subunit messenger RNA (mRNA) expression as well, we examined the effects of hypophysectomy and LH replacement on the testicular content of inhibin alpha and beta-B subunit mRNAs in sexually immature and adult rats. Twenty- and 60-day-old intact and hypophysectomized rats received saline or 0.25, 2.5, 25.0, or 250 micrograms ovine LH/100 g body wt sc, twice daily for 7 days beginning on the day after hypophysectomy (n = 5/group in three separate experiments). The inhibin subunit mRNA content of each testis was measured for statistical analysis by dot-blot hybridization, and experimental results were confirmed by northern analysis of poly(A) RNA from each sample. In immature animals, the testicular content of both inhibin subunit mRNAs was decreased after hypophysectomy; inhibin alpha and beta-B mRNA levels were decreased to 6.9 +/- 0.9% and 31.7 +/- 2.7% of intact control values, respectively. In adult animals, hypophysectomy resulted in a more modest decrease in inhibin alpha subunit mRNA per testis (44.9 +/- 3.1% of controls) but had no effect on beta-B subunit mRNA. Replacement of LH to immature hypophysectomized animals did not alter levels of mRNA for either inhibin subunit. However, in adults LH restored testicular inhibin alpha subunit mRNA content in testes of hypophysectomized animals to levels seen in intact, saline-treated control animals. LH replacement also slightly but consistently decreased testicular beta-B subunit mRNA content compared to levels seen in hypophysectomized, saline-treated rats. These results indicate that although the response of inhibin subunit mRNAs to pituitary input decreases as a function of sexual maturation, LH may play an important role in regulation of inhibin subunit mRNA expression in adult but not immature testes. The mechanism(s) of LH action on testicular inhibin subunit gene expression is currently unknown, but may involve either direct actions of LH on Leydig cell inhibin subunit mRNAs or indirect actions of interstitial cell factors on Sertoli cell inhibin subunit gene expression.

Effects of luteinizing hormone on luteal cell populations in hypophysectomized ewes

To examine the effect of purified LH on development and function of luteal cells, 27 ewes were assigned to: (1) hypophysectomy plus 2 micrograms ovine LH given i.v. at 4-h intervals from Days 5 to 12 of the oestrous cycle (oestrus = Day 0; Group H + LH; N = 7); (2) hypophysectomy with no LH replacement (Group N-LH; N = 6); (3) control (no hypophysectomy) plus LH replacement as in Group H + LH (Group S + LH; N = 7); (4) control with no LH treatment (Group S-LH; N = 7). Blood samples were collected at 4-h intervals throughout the experiment to monitor circulating concentrations of LH, cortisol and progesterone. On Day 12 of the oestrous cycle corpora lutea were collected and luteal progesterone concentrations, unoccupied receptors for LH and number and sizes of steroidogenic and non-steroidogenic luteal cell types were determined. Corpora lutea from ewes in Group H-LH were significantly smaller (P less than 0.05), had lower concentrations of progesterone, fewer LH receptors, fewer small luteal cells and fewer non-steroidogenic cells than did corpora lutea from ewes in Group S-LH. The number of large luteal cells was unaffected by hypophysectomy, but the sizes of large luteal cells, small luteal cells and fibroblasts were reduced. LH replacement in hypophysectomized ewes maintained luteal weight and the numbers of small steroidogenic and non-steroidogenic luteal cells at levels intermediate between those observed in ewes in Groups L-LH and S-LH. In Group H + LH ewes, luteal and serum concentrations of progesterone, numbers of luteal receptors for LH, and the sizes of all types of luteal cells were maintained. Numbers of small steroidogenic and non-steroidogenic cells were also increased by LH in hypophysectomized ewes. In Exp. II, 14 ewes were assigned to: (1) sham hypophysectomy with no LH replacement therapy (Group S-LH; N = 5); (2) sham hypophysectomy with 40 micrograms ovine LH given i.v. at 4-h intervals from Day 5 to Day 12 of the oestrous cycle (Group S + LH; N = 5); and (3) hypophysectomy plus LH replacement therapy (Group H + LH; N = 4). Experimental procedures were similar to those described for Exp. I. Treatment of hypophysectomized ewes with a larger dose of LH maintained luteal weight, serum and luteal progesterone concentrations and the numbers of steroidogenic and non-steroidogenic luteal cells at control levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible Role of Luteinizing Hormone and Follicle-Stimulating Hormone in Modulating Inhibin Secretion and Expression during the Estrous Cycle of the Rat*

In the female rat, plasma immunoreactive inhibin alpha (irl alpha) levels show marked changes during proestrus and estrus. We investigated the modulating effect of LH and FSH on these changes by injecting the GnRH antagonist DNal-DCpa-DPal-Dpr-(Ac)Dal-Leu-Arg-Pro-Asn-NH2, with or without exogenous LH replacement. Administration of the antagonist at noon on proestrus abolished the primary (proestrus) LH and FSH surge and markedly reduced the secondary (estrus) FSH surge. This treatment also reduced the release of irl alpha normally measured during proestrus afternoon, and partially prevented the decrease in irI alpha secretion on proestrus evening. Exogenous LH injected at 1545 h on proestrus had no measurable effect on irI alpha or FSH levels in control rats; however, in antagonist-treated animals, it restored the secondary FSH surge to control values while augmenting the late proestrus fall in irI alpha. This suggests that the decrease in inhibin secretion measured after exogenous LH treatment represents the mechanism through which LH induced the secondary FSH surge in antagonist-blocked rats. We also used in situ hybridization techniques to examine the changes in the expression of inhibin subunits in the ovary at 0200 h on estrus. The antagonist reduced expression of the alpha-, beta A-, and beta B-subunits in all follicle and tissue types, with the exception of the granulosa cells of large tertiary (possibly preovulatory) follicles where the signal appeared greatly enhanced. These changes were reversed by LH. The alteration in inhibin subunit messages caused by blockade of the primary gonadotropin surge suggests the presence of a cross-regulation between LH and inhibin/activin secretion, so that a decline in circulating LH levels might stimulate inhibin/activin secretion in the granulosa cells of preovulatory follicles, while reducing the production of these proteins in less mature follicles and in other ovarian cell types.

Anterior pituitary hormonal regulation of testicular gonadotropin-releasing hormone receptors.

Testicular GnRH receptors are increased 2-fold 1 day after hypophysectomy and remain elevated for up to 6 days. Consequently, the present study was undertaken to determine which pituitary hormone(s) regulated testicular GnRH receptors. Adult male rats were hypophysectomized and injected sc every 8 h for 2 days with LH, FSH, GH, PRL, estradiol, testosterone, 5 alpha-dihydrotestosterone, and vehicle (controls), beginning within 5 h of surgery. The animals were decapitated on the third day after hypophysectomy, the testes were excised, and interstitial tissue was teased from seminiferous tubules before storage at -70 C until assayed. The analog D-Ala6-des-Gly10-GnRH ethylamide was used to assess GnRH receptors on 10,800 X g-membrane fractions of interstitial tissue. The administration of GH, PRL, and FSH at concentrations which maintained LH receptors in adult or immature hypophysectomized rats did not prevent the increase in GnRH receptors, whereas LH replacement prevented the rise in GnRH analog binding in a dose related manner. LH also reduced preexisting posthypophysectomy increases in GnRH receptor concentrations. Injections of estradiol (5 micrograms/day) partially inhibited the posthypophysectomy increase in GnRH receptors, whereas the androgens 5 alpha-dihydrotestosterone and testosterone were ineffective. These results indicate that LH can regulate testicular GnRH receptors. Since GnRH directly inhibits testosterone secretion, inhibition of testicular GnRH receptors by LH may be one of the mechanisms by which LH replacement enhances testosterone production after hypophysectomy.

Luteinizing hormone increases the number of ova shed in the cyclic hamster and guinea-pig.

Osmotic minipumps containing 400 micrograms ovine LH installed subcutaneously on day 1 (oestrus) of the cycle in the hamster induced superovulation of 30.0 +/- 2.1 ova (n = 5) at the next expected oestrus. Controls ovulated 12.0 +/- 0.8 ova (n = 6). Bovine LH, human LH, porcine LH, human chorionic gonadotrophin and pregnant mare serum gonadotrophin were effective in approximately doubling the number of ova spontaneously shed in the hamster. Ovine FSH (200 micrograms/pump) was most effective in increasing the number of ova spontaneously shed (55 +/- 6, n = 5) in the hamster. Infusion of ovine LH on days 1-4 prevented the reduction of the number of antral follicles that occurs normally between days 3 and 4 of the 4-day cycle. Since this reduction in follicular numbers in control cyclic hamsters is due to atresia, the exogenous LH might prevent atresia of the developing follicles. In the hamster, exogenous ovine LH significantly increased the serum concentrations of androstenedione, oestradiol and LH but not of FSH. Hamsters were hypophysectomized on the day of oestrus, given immediate LH (400 micrograms) or FSH (200 micrograms) replacement therapy and autopsied on day 4. Ovarian histology revealed that immediate LH treatment after hypophysectomy sustained development of histologically normal preovulatory follicles but had no effect on the number of smaller sizes of follicles. Immediate FSH treatment after hypophysectomy increased only the number of smaller sized follicles. Since LH did not increase the smaller sized follicles, no 'FSH-like' effect on follicular development was observed. In the hamster, the ability of various preparations of LH to induce superovulation did not correlate with their ability to displace 125I-labelled ovine FSH from its ovarian binding sites. The superovulatory action of LH required the presence of the pituitary gland, indicating that LH might synergize with FSH and/or prolactin (or hamster LH) for spontaneous superovulation and it appears that exogenous LH might induce superovulation by prevention of atresia. Infusion of LH into the guinea-pig beginning on day 12 of the cycle (day 1 is the day of ovulation) doubled the ovulation rate whereas in the cyclic rat and mouse LH treatment throughout the cycle was ineffective in increasing the number of ova shed.

Inhibition of thecal androstenedione production by exogenous progesterone in the cyclic hamster.

Implants of progesterone on the day of dioestrus II in the hamster induced on the following day an increase in circulating levels of progesterone (6.0 +/- 0.7 ng/ml, N = 8; sesame oil controls, less than 0.5 ng/ml, N = 6) and a decline in serum levels of LH (5.3 +/- 0.4 ng/ml; controls 12 +/- 2 ng/ml) and oestradiol (10 +/- 2 pg/ml; controls 69 +/- 5 pg/ml). The production of androstenedione and oestradiol by antral follicles in vitro was reduced in progesterone-treated hamsters when compared with controls, but progesterone production was not affected. Aromatizing activities of antral follicles were the same in progesterone-treated and sesame oil-treated hamsters. Androstenedione production by theca was significantly less in progesterone-treated hamsters than in controls. On dioestrus II, LH replacement therapy (200 micrograms ovine LH by osmotic minipump inserted s.c.) prevented the decline in follicular androstenedione and oestradiol production induced by progesterone alone, and also prevented the decline in thecal androstenedione production in vitro. The results indicate that exogenous progesterone on dioestrus II lowers circulating levels of LH by the following day, inhibits thecal androstenedione production and thus reduces follicular oestradiol production without alteration in aromatizing ability.