The Corticosterone-Glucocorticoid Receptor-AP1/CREB Axis Inhibits the Luteinizing Hormone Receptor Expression in Mouse Granulosa Cells.

Under stress conditions, luteinizing hormone (LH)-mediated ovulation is inhibited, resulting in insufficient oocyte production and excretion during follicular development. When the body is stressed, a large amount of corticosterone (CORT) is generated, which will lead to a disorder of the body's endocrine system and damage to the body. Our previous work showed that CORT can block follicular development in mice. Since LH acts through binding with the luteinizing hormone receptor (Lhcgr), the present study aimed to investigate whether and how corticosterone (CORT) influences Lhcgr expression in mouse ovarian granulosa cells (GCs). For this purpose, three-week-old ICR female mice were injected intraperitoneally with pregnant mare serum gonadotropin (PMSG). In addition, the treatment group was injected with CORT (1 mg/mouse) at intervals of 8 h and the control group was injected with the same volume of methyl sulfoxide (DMSO). GCs were collected at 24 h, 48 h, and 55 h after PMSG injection. For in vitro experiments, the mouse GCs obtained from healthy follicles were treated with CORT alone, or together with inhibitors against the glucocorticoid receptor (Nr3c1). The results showed that the CORT caused a downregulation of Lhcgr expression in GCs, which was accompanied by impaired cell viability. Moreover, the effect of the CORT was mediated by binding to its receptor (Nr3c1) in GCs. Further investigation revealed that Nr3c1 might regulate the transcription of Lhcgr through inhibiting the expression of Lhcgr transcription factors, including AP1 and Creb. Taken together, our findings suggested a possible mechanism of CORT-induced anovulation involving the inhibition of Lhcgr expression in GCs by the CORT-Nr3c1-AP1/Creb axis.

Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG).

BACKGROUND: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). RESULTS: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24 h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by > 2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses. CONCLUSIONS: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

In vitro maturation of immature rat oocytes under simple culture conditions and subsequent developmental ability.

Rat oocytes can be produced artificially by superovulation. Because some strains show low sensitivity to superovulation treatment, in vitro maturation is an alternative method to produce numerous matured oocytes. Furthermore, establishment of an in vitro maturation system with simple culture conditions is cost effective and leads to easy handling of oocytes. This study examined developmental ability of rat germinal vesicle (GV) oocytes maturing in vitro under simple culture conditions. Significantly different numbers of ovulated oocytes reached the second metaphase of meiosis (MII) among Jcl:Wistar (17.0), F344/Stm (31.0), and BN/SsNSlc (2.2) rats in whom superovulation was induced by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. However, similar numbers of GV oocytes were obtained from ovaries of PMSG-injected Wistar (27.7), F344 (34.7), and BN (24.7) rats. These GV oocytes were cultured in vitro in HTF, αMEM, and a 1:1 HTF + αMEM or TYH + αMEM mixture. High proportions of Wistar and F344 oocytes that matured to MII in αMEM were parthenogenetically activated by strontium chloride treatment (78% and 74%, respectively). Additionally, 10% of matured oocytes of both strains developed into offspring after intracytoplasmic sperm injection and embryo transfer to foster mothers. Although BN oocytes cultured in αMEM could be parthenogenetically activated and developed into offspring, the success rate was lower than that for Wistar and F344 oocytes. This study demonstrated that numerous GV oocytes were produced in rat ovaries by PMSG injection. This simple in vitro maturation system of immature oocytes could be further developed to maintain valuable rat strains experiencing reproductive difficulties.

The influence of nutrition on the insulin-like growth factor system and the concentrations of growth hormone, glucose, insulin, gonadotropins and progesterone in ovarian follicular fluid and plasma from adult female horses (Equus caballus).

BACKGROUND: Feed intake affects the GH-IGF system and may be a key factor in determining the ovarian follicular growth rate. In fat mares, the plasma IGF-1 concentration is high with low GH and a quick follicular growth rate, in contrast to values observed in thin mares. Nothing is known regarding the long-term effects of differential feed intake on the IGF system. The objective of this experiment was to quantify IGFs, IGFBPs, GH, glucose, insulin, gonadotropin and progesterone (P4) in blood and in preovulatory follicular fluid (FF) in relation to feeding levels in mares. METHODS: Three years prior to the experiment, Welsh Pony mares were assigned to a restricted diet group (R, n = 10) or a well-fed group (WF, n = 9). All mares were in good health and exhibited differences in body weight and subcutaneous fat thickness. Follicular development was scanned daily and plasma was also collected daily. Preovulatory FF was collected by ultrasound-guided follicular aspiration. Hormone levels were assayed in FF and plasma with a validated RIA. RESULTS: According to scans, the total number of follicles in group R was 53% lower than group WF. Insulin and IGF-1 concentrations were higher in WF than in R mares. GH and IGF-2 concentrations were lower in plasma from WF mares than from R mares, but the difference was not significant in FF. The IGFBP-2/IGFBP-3 ratio in FF was not affected by feeding but was dramatically increased in R mare plasma. No difference in gonadotropin concentration was found with the exception of FSH, which was higher in the plasma of R mares. On the day of puncture, P4 concentrations were not affected by feeding but were higher in preovulatory FF than in plasma. CONCLUSIONS: The bioavailability of IGF-1 or IGF-2, represented by the IGFBP2/IGFBP3 ratio, is modified by feed intake in plasma but not in FF. These differences partially explain the variability in follicular growth observed between well-fed mares and mares on restricted diets.

Distribution and Y397 phosphorylation of focal adhesion kinase on follicular development in the mouse ovary.

Several protein tyrosine kinases (PTKs) are identified as follicle survival factors that suppress apoptosis in granulosa cells. Focal adhesion kinase (FAK/PTK2) interacts with numerous signaling partners and is important for cell adhesion, survival and other vital processes in which FAK autophosphorylation at Y397 (pY397 FAK) is critical for activating signaling pathways. Despite its important roles in apoptosis, the expression and function of FAK in the ovaries remain unknown. Here, we describe FAK expression, including pY397 FAK, in normal healthy mouse ovaries and its association with follicular development and/or atresia. Normal healthy mouse ovaries were used for western blot (n > 60) and immunohistochemical (n > 180) analyses. Western blot results in immature and mature mice revealed that total FAK and pY397 FAK were highly expressed in the ovary and immunohistochemistry results in 3-week-old mice showed they were localized to granulosa cells of ovarian follicles, especially preantral follicles. In 3-week-old mice treated with 5 IU pregnant mare serum gonadotropin (for obtaining homogenous populations of growing or atretic follicles), western blotting revealed that follicular atresia progression involved decreased phosphorylation of Y397 at 72 and 96 h after treatment, particularly in granulosa cells of atretic follicles, as shown by immunohistochemistry results at 72 h after treatment. Moreover, immunostaining patterns of FAK and cleaved caspase-3 were negatively correlated in serial sections of 3-week-old mouse ovaries. These results suggest that FAK is most active in ovarian follicle granulosa cells and that its phosphorylation at Y397 is histologically meaningful in follicular development in normal healthy ovaries.

Morphological changes in mouse ovary due to hormonal hypersecretion and matrix metalloproteinase -2 activity.

We analyzed whether aberrant gonadotropin secretion affects the morphological remodeling of murine ovarian tissues facilitated by activated matrix metalloproteinase (MMP) enzymes. Six mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG) or human chorionic gonadotropin (HCG) every two days after estrus synchronization. Morphology and expression of various MMPs were assessed following the successful induction of hormonal secretion in these tissues. HCG treatment, but not PMSG treatment, resulted in the expanded production of granulose second follicular cells. In addition, the number of developing follicular cells in the HCG group increased compared with that in the PMSG group. Ovarian diameters were also very small in the PMSG group. Immunohistochemistry revealed decreased MMP-2 protein activity in the HCG group and increased MMP-2 activity in the PMSG group. Activity was particularly high in theca and granulose cells of the PMSG group, but only partial activity was observed in the theca cells of the HCG group. Vascular endothelial growth factor activity was increased in both the external and internal theca cell walls in the PMSG group while the HCG group showed high overall expression of this protein in the internal theca cells. These data indicate that follicular cell activity and remodeling of the ovaries differ based on the type of secretory hormone signals they receive. Inappropriate gonadotropin secretion may induce functional changes in the ovaries, and follicular remodeling may be facilitated by the activity of various MMPs.

In vivo and in vitro effects of interleukin-1beta on equine oocyte maturation and on steroidogenesis and prostaglandin synthesis in granulosa and cumulus cells.

We analysed the effect of interleukin-1 on oocyte maturation and on steroid and prostaglandin production by equine granulosa and cumulus cells. In Experiment 1, interleukin-1beta (IL-1beta) was injected into the growing dominant follicle, which was punctured 38 h later. Follicular fluid was assayed for steroids and prostaglandin-F2alpha (PGF2alpha). Granulosa cells were analysed for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), progesterone receptor (PR), cyclooxygenase 1 and 2 (Cox 1 and Cox 2) and steroidogenic acute regulatory protein (StAR) mRNAs. In Experiment 2, cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries and cultured in different media: control group (TCM199 + BSA); Group 2 (+ IL-1beta); Group 3 (+ EGF); Group 4 (+ EGF + IL-1beta); and Group 5 (+ EGF + IL-1beta + IL-1RA). Cumulus cells were analysed for 3beta-HSD, PR, Cox 1, Cox 2 and StAR mRNAs. After injections of crude equine gonadotropin (CEG; LH effect) or IL-1beta, progesterone and PGF2alpha levels increased, whereas 17beta-oestradiol decreased. EGF induced an increase in the rate of in vitro maturation (P < 0.05), whereas IL-1beta had a limited effect. IL-1beta significantly decreased the rate of EGF-induced oocyte maturation (P < 0.05). Cox 2 mRNA level increases in granulosa cells after CEG injection (P = 0.07). In cumulus cells, StAR and PR mRNAs were lower in Group 2 and 3beta-HSD mRNA was higher in Groups 4 and 5. These data confirm that IL-1 is involved in equine oocyte in vitro maturation. We demonstrated in vivo that IL-1beta has an effect on steroids and PGF2alpha secretion in the preovulatory follicle.

Gonadotrophin subunit and GnRH receptor gene expression in the pars distalis of the equine pituitary.

In the horse, pronounced changes in fertility occur annually in response to photoperiod. However, the mechanisms regulating gonadotrophin synthesis and release in this species remain unclear. Here, we investigated the expression of gonadotrophin subunits and GnRH receptor (GnRH-R) mRNA in the pituitary glands of Thoroughbred horses during the breeding (BS) and non-breeding (NBS) season. Seasonal effects on the prevalence of gonadotrophs in the pars distalis were also examined. GnRH-R and common alpha-, LHbeta- and FSHbeta-subunit mRNA contents were determined by Northern analysis and the prevalence of LH-gonadotrophs assessed by immunohistochemistry in pituitaries from sexually active females (mares) in the BS, and sexually inactive mares in the NBS. These variables were then measured in castrated male horses (geldings). In mares, pituitary content of FSHbeta mRNA was significantly higher in the NBS (P<0.01). Conversely, the content of common alpha-subunit mRNA was significantly higher during the BS (P<0.05). In contrast, GnRH-R and LHbeta mRNA abundance were unaffected by season. Interestingly, whereas no seasonal effects were apparent on the number of LH-gonadotrophs/field, the proportion of LH cells (in relation to all other cells) was higher in BS than NBS animals (P<0.05); this resulted from an increased number of non-gonadotroph cells during the NBS (P<0.05). In geldings, no significant seasonal effects were detected for any of the variables investigated (P>0.05). These results reveal robust seasonal effects on common alpha-subunit and FSHbeta gene expression in the pituitary of the mare, in the absence of detectable changes in the content of LHbeta or GnRH-R mRNA.

Equine chorionic gonadotropin.

Cells from the chorionic girdle of the equine trophoblast invade the maternal endometrium at day 36 of gestation and become established as secretory elements known as the endometrial cups. These structures, which persist for 40-60 days, produce a gonadotropin which can be found in circulation until about day 130 of gestation. This glycoprotein has been identified in the horse and the donkey, with the former having received much better characterization. It consists of 2 noncovalently linked peptide chains; an alpha-subunit of 96 amino acids, which is common to that found in other horse glycoprotein hormones. The beta-subunit of 149 amino acids is identical to horse LH beta. Horse CG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. The alpha-subunit has two and the beta-subunit one N-linked glycosylation site, and the beta-chain has in excess of four O-linked glycosylation sites. The N-linked glycans have some oligosaccharides that are not found on other glycoprotein hormones. The sialic component of glycosylation confers an exceptionally long half-life on CG compared to other glycoprotein hormones. Horse CG has LH-like activity in horse receptor and in vitro bioassays. In spite of the amino acid homology, it has lower LH activity than does horse LH. Its most intriguing, and as yet unexplained, characteristic is its pronounced FSH and LH activity in species other than the horse. Horse CG binds to FSH receptors of virtually all mammalian species, other than the horse, in which it has been tested and will produce biological effects peculiar to FSH. It has similar and potent interaction with LH receptors. The structural basis of this duality is not known but may be related to the region 90-110 of the beta-chain. Horse CG is believed to be constitutively expressed by the trophoblastic cells until the endometrial cups degenerate. The role of CG in equine gestation is not completely understood. It is believed to act as an LH-like hormone to induce supplementary ovulation and/or luteinization of follicles in the mare. It has not been established whether CG or the accessory corpora lutea are necessary for successful horse pregnancy. They may serve as a redundant system to assure that there is sufficient secretion of the primary corpus luteum to maintain pregnancy until the placenta assumes its role as the principal steroidogenic organ of gestation.

ADAMTS-1 is involved in normal follicular development, ovulatory process and organization of the medullary vascular network in the ovary.

To clarify the role of disintegrin-like and metalloproteinase with thrombospondin type I motifs-1 (ADAMTS-1) in ovarian function, we examined abnormalities in ovulatory processes, folliculogenesis and the vascular system of ADAMTS-1 null ovaries. First, when immature female mice were treated with pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG), the number of ovulated oocytes was markedly decreased in ADAMTS-1 null mice in comparison to ADAMTS-1 (+/-) controls. The proportion of anovulated follicles to total mature follicles was significantly higher in ADAMTS-1 null females when compared with controls. The numbers of growing follicles at each stage were counted. The number of follicles at type 5b (late preantral) and later stages was markedly reduced in ADAMTS-1 null mice, irrespective of gonadotropin treatment (no gonadotropins, PMSG alone or PMSG/hCG). These data demonstrate that impairment of ovarian function to ovulate oocytes in ADAMTS-1 null mice occurs at two different levels: in the development of growing follicles and ovulatory processes. Furthermore, ADAMTS-1 null ovaries included a number of unusual atretic follicles that showed no sign of oocyte degeneration but lost the surrounding granulosa cell layers and were considered to be derived from type 4 or 5a follicles. These results suggest that ADAMTS-1 is important for follicular development beyond the type 4 and/or 5a and for maintaining normal granulosa cell layers in follicles. Finally, the number of large blood vessels in the medullar zone was significantly decreased in ADAMTS-1 null mice ovaries, suggesting that ADAMTS-1 is also involved in the organization of the medullary vascular network.

Phosphodiesterase regulation is critical for the differentiation and pattern of gene expression in granulosa cells of the ovarian follicle.

Feedback regulations are integral components of the cAMP signaling required for most cellular processes, including gene expression and cell differentiation. Here, we provide evidence that one of these feedback regulations involving the cyclic nucleotide phosphodiesterase PDE4D plays a critical role in cAMP signaling during the differentiation of granulosa cells of the ovarian follicle. Gonadotropins induce PDE4D mRNA and increase the cAMP hydrolyzing activity in granulosa cells, demonstrating that a feedback regulation of cAMP is operating in granulosa cells in vivo. Inactivation of the PDE4D by homologous recombination is associated with an altered pattern of cAMP accumulation induced by the gonadotropin LH/human chorionic gonadotropin (hCG), impaired female fertility, and a markedly decreased ovulation rate. In spite of a disruption of the cAMP response, LH/hCG induced P450 side chain cleavage expression and steroidogenesis in a manner similar to wild-type controls. Morphological examination of the ovary of PDE4D-/- mice indicated luteinization of antral follicles with entrapped oocytes. Consistent with the morphological finding of unruptured follicles, LH/hCG induction of genes involved in ovulation, including cyclooxygenase-2, progesterone receptor, and the downstream genes, is markedly decreased in the PDE4D-/- ovaries. These data demonstrate that PDE4D regulation plays a critical role in gonadotropin mechanism of action and suggest that the intensity and duration of the cAMP signal defines the pattern of gene expression during the differentiation of granulosa cells.

Purification and characterization of the gonadotropin secreted by cultured horse trophoblast cells.

Equine chorionic gonadotropin (eCG) secreted by horse trophoblast cells cultivated in vitro was isolated and its chemical, immunochemical, and biological characteristics were compared to those of the gonadotropin secreted in vivo and isolated from PMS. It was also compared with eCG isolated from the tissue of origin, the endometrial cups. The gonadotropin secreted in vitro had a smaller molecular size, contained appreciably less carbohydrate, and showed different amino-terminal residues from that secreted in vivo. In addition, there were significant differences in amino acid composition between eCG secreted in vivo and that secreted in vitro. The reactivity of eCG (isolated from the medium of cultured trophoblast cells) in homologous eCG (isolated from PMS) and equine LH and FSH RIAs suggested close antigenic similarities to eCG isolated from PMS. However, compared to serum-derived eCG, eCG secreted in vitro showed reduced activity in both LH (13%) and FSH (9-24%) bioassays and in LH and FSH radioreceptor assays (40-50%). The differences in bioassay potencies may be accounted for in part by the lower sialic acid content of the gonadotropin secreted in vitro. With respect to both bioactivity and chemical composition, the eCG secreted in vitro more closely resembled the eCG isolated from endometrial cups than that isolated from serum.

Immunochemical studies of equine chorionic gonadotropin (eCG), eCG alpha, and eCG beta.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

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.

Differences in the behavior of luteinizing hormones of various species at the rat gonadal cell receptor site.

The ability of different LH-like hormones, such as hCG, PMSG/equine (e) CG, ovine (o) LH, eLH, and rat (r) LH, to bind to and stimulate steroidogenesis in two types of rat gonadal cells was studied under the same experimental conditions. In both Leydig and granulosa cells, the maximal steroidogenic responses elicited by optimal doses of different LHs present during a 2-h incubation were comparable. However, if the cells were exposed to the different LHs for a brief period and then subjected to interference with hormone action by removing the unbound hormone from the medium by washing or adding specific antisera, differences were observed in the amount of steroid produced during subsequent incubation in hormone-free medium. Thus, in the case of hCG, either of these procedures carried out at 15 or 30 min of incubation had little inhibitory effect on the amount of steroid produced at 2 h, the latter being similar to that produced by cells incubated in the continued presence of hCG for 2 h. With eCG and rLH, the effect was dramatic, in that there was a total inhibition of subsequent steroidogenic response. In cells exposed to eLH and oLH, inhibition of subsequent steroidogenesis due to either removal of the free-hormone or addition of specific antisera at 15 or 30 min was only partial. Although all of the antisera used were equally effective in inhibiting the steroidogenic response to respective gonadotropins when added along with hormones at the beginning of incubation, differences were observed in the degree of inhibition of this response when the same antisera were added at later times of incubation. Thus, when antisera were added 60 min after the hormone, the inhibition of steroidogenesis was total (100%) for eCG, partial (10-40%) for eLH and oLH, and totally lacking in cells treated with hCG. From this, it appears that hCG bound to the receptor probably becomes unavailable for binding to its antibody with time, while in the case of eCG and other LHs used, the antibody can still inhibit the biological activity of the hormone. Studies with 125I-labeled hormones further supported the conclusion that hCG differs from all other LHs in being most tightly bound and, hence, least dissociable, while eCG and rLH dissociate most readily; oLH and eLH can be placed in between these hormones in the extent of their dissociability.

Adenylyl cyclase activities in ovarian tissues. III. Regulation of responsiveness to LH, FSH, and PGE1 in the prepubertal, cycling, pregnant, and pseudopregnant rat.

In the ovaries of prepubertal rats, responsiveness of adenylyl cyclase (AC) to LH, FSH, and PGE1 is acquired at day 10, coinciding with the appearance of the ability to produce steroids in response to gonadotrophins. The activity and responsiveness of AC on day 11 were similar to those at puberty on day 40 and relatively stable in between. In the follicles of the cycle, the responsiveness of AC to LH and FSH was poor (ca 2-fold stimulation) on metestrus and diestrus, and became high (ca 10-fold stimulation) between 1000 h of diestrus and 1000 h of proestrus. Thereafter, the system slowly became desensitized to LH and FSH, being unresponsive by the morning of estrus. Nembutal injected at 1230 h and again at 1500 h on proestrus blocked ovulation and prevented the decline in LH- and FSH-stimulated AC activity. In the CL of the cycle, the AC was unresponsive to LH on day 1 (estrus), became responsive by the morning of day 2 (metestrus), and maintained responsiveness throughout that day. Thereafter, the responsiveness and basal AC activity declined rapidly. In the CL of pregnancy, LH-stimulated AC was indistinguishable from that of the CL of the cycle during days 1 and 2, then increased steadily until day 9, showing a transient decrease on days 10 and 11, followed by a sharp rise to maximal activity on days 15 and 16. Thereafter, activity declined as parturition approached. In the CL of pseudopregnancy (PSP), LH-stimulated AC was very similar to that of the CL of pregnancy during the first 11 days. Thereafter, it decreased coincident with the termination of PSP. Injections of PRL (100 mug SC twice daily, from metestrus through estrus, and from proestrus through proestrus) or estradiol-17 beta (20 mug, SC at 1230 h on metestrus) resulted in "rescue" of the CL-AC system, which remained at metestrus levels when measured on the days of expected proestrus or estrus. Injections of pregnant mare serum gonadotrophin into prepubertal rats at day 26 (3 IU, iv), induced by day 28 a highly responsive AC system in follicles, with activities equivalent to those found in Graafian follicles on proestrus. By day 29, synchronous ovulation had occurred with a concomitant loss of LH-stimulated AC such as seen in the 1-day-old CL of mature rats. Our results suggest that the LH-sensitive AC may be indicative of the final development of ovulability of the follicles, and that it may correlate with the functional capacity of CL during various reproductive stages of the rat.

Purification and characterization of donkey chorionic gonadotrophin.

Serum of the pregnant donkey, like that of the mare, contains a gonadotrophin of chorionic origin. The chorionic gonaditrophin of the donkey (dCG) has been isolated in purified form from the serum of pregnant donkeys using methodology previously employed for the purification of pregnant mare chorionic gonadotrophin (eCG). Unlike eCG, dCG is predominatly an LH in biological tests. In the in-vitro rat Leydig cell assay, dCG was as active as eCG, but in the in-vitro rat seminiferous tubule assay for FSH and in the augmentation assay, dCG was considerably less potent than eCG (1-10%). Specific rat testis radioreceptor assays for LH and FSH also showed dCG to be at least nine times more potent in LH than in FSH activity. Chemically, dCG was found to be similar to eCG in fractionation behaviour and glycoprotein nature. However, dCG had significantly less carbohydrate (31%) than had eCG (45%) and several differences were noted in a comparison of amino-acid compositions. A single amino-terminal residue, phenylalanine, was detected in dCG. Immunologically, dCG cross-reacted in homologous radio-immunoassays for eCG, equine LH and equine FSH, but its inhibition curves were all nonparallel with those of the respective equine gonadotrophin standards.

Biological and binding activities of equine pituitary gonadotrophins and pregnant mare serum gonadotrophin.

The biological and binding activities of pregnant mare serum gonadotrophin (PMSG) were compared with those of highly purified FSH and LH from the pituitary gland of the same species. Pregnant mare serum gonadotrophin showed activity in bioassays considered to be specific for both FSH (e.g. the Steelman-Pohley ovarian augmentation test and cyclic AMP production by rat seminiferous tubules) and LH(androgen production by rat Leydig cells), as well as activity in a variety of radioreceptor assay systems previously considered to be specific for one of the two types of gonadotrophin. The potency of PMSG was high compared with that of purified ovine FSH or LH standards in all assays but PMSG was considerably less active than equine FSH and LH in vitro. In radioreceptor assays employing rat, pig and horse tissues, the activity of PMSG was equivalent to only 1--5% of equine FSH in competing for FSH-binding sites and only 3--35% of equine LH in competing for LH-binding sites. Pregnant mare serum gonadotrophin was least active in homologous binding assays with horse testis and equine LH as radioligand. In the rat Leydig cell bioassay, the activity of PMSG was only 2.0% that of equine LH. Furthermore, in some assays equine LH was found to resemble PMSG in exhibiting a high degree of FSH-like activity that could not be accounted for by cross-contamination. The FSH immunoactivity of equine LH was less than 0.5% that of equine FSH, but equine LH was up to 63% as potent as equine FSH in competition for FSH-binding sites and it was 20% as active in the Steelman-Pohley ovarian augmentation bioassay. Equine LH did not, however, show the expected activity in the cyclic AMP production bioassay. Thus, the FSH-binding sites and physiological receptors may not be identical. Overall, comparison of PMSG with pituitary gonadotrophins from homologous species shows that the apparent dual activity of PMSG may not be a unique feature of this pregnancy hormone since equine LH also exhibits some FSH activities. The chemical resemblance between PMSG and equine LH is noteworthy in this regard.

Pregnant mare serum gonadotrophin: ratio of follicle-stimulating hormone and luteinizing hormone activities measured by radioreceptor assay.

Specific radioreceptor assays for FSH and LH, which employ tissue receptors from rat testis and highly purified human FSH (LER 1575-C) and LH (Hartree IRC-2, 24/6/69) as standards, have been developed to determine the FSH-like and LH-like activities in pregnant mare serum gonadotropin (PMSG). Measurements of FSH and LH concentrations in the serum of six pregnant Pony mares showed that the ratio of these two activities did not vary significantly between mares and remained constant between days 40 and 80 of gestation with a value of 1-45 +/- 0-04 (S.E.M.). The FSH:LH ratio of PMSG produced by cultured equine trophoblast cells was found to be 0-72 +/- 0-03 (S.E.M.) and that of partially purified serum extracts of PMSG 1-08 (range 0-87-1-30).

Expression of horse and donkey LH in COS-7 cells: evidence for low FSH activity in donkey LH compared with horse LH.

Horse (Equus caballus) luteinizing hormone (eLH) and chorionic gonadotrophin (eCG), which have the same amino acid sequence, are unusual in that, although they express only LH activity in equids, they express dual LH and FSH activities in all other species tested. Donkey (Equus asinus) LH (dkLH) and CG (dkCG), which also share an identical peptide backbone, have been less well characterized and conflicting results concerning their FSH activity in heterologous species have appeared in the literature. In order to assess and compare the intrinsic LH and FSH activities of the horse and donkey LHs in heterologous species, recombinant eLH (r.eLH/CG) and recombinant dkLH (r.dkLH/CG) were expressed, for the first time, in COS-7 cells. Their LH activities were assessed in a rat Leydig cell bioassay, and their FSH activities were estimated in a bioassay using Y1 cells stably expressing the human FSH receptor. Human CG (hCG) was expressed (r.hCG) and analysed in the same system. The results showed that, whereas r.dkLH/CG was about twice as active as r.eLH/CG in the LH bioassay, it was five times less active than r.eLH/CG in the FSH bioassay; r.hCG was about three times less active than r.eLH/CG in the LH bioassay but was completely inactive in the FSH bioassay. These results confirm that dkLH/CG possesses significant FSH activity in heterologous species that is not attributable to contamination with FSH.