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.

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.

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.

The dynamics of connexin expression, degradation and localisation are regulated by gonadotropins during the early stages of in vitro maturation of swine oocytes.

Gap junctional communication (GJC) plays a primordial role in oocyte maturation and meiotic resumption in mammals by directing the transfer of numerous molecules between cumulus cells and the oocyte. Gap junctions are made of connexins (Cx), proteins that regulate GJC in numerous ways. Understanding the dynamic regulation of connexin arrangements during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and consequently in vitro development of fully competent oocytes. However, physiological events happening during the early hours of IVM may still be elucidated. The present study reports the dynamic regulation of connexin expression, degradation and localization during this stage. Cx43, Cx45 and Cx60 were identified as the main connexins expressed in swine COC. Cx43 and Cx45 transcripts were judged too static to be a regulator of GJC, while Cx43 protein expression was highly responsive to gonadotropins, suggesting that it might be the principal regulator of GJC. In addition, the degradation of Cx43 expressed after 4.5 h of IVM in response to equine chorionic gonadotropin appeared to involve the proteasomal complex. Cx43 localisation appeared to be associated with GJC. Taken together, these results show for the first time that gonadotropins regulate Cx43 protein expression, degradation and localisation in porcine COC during the first several hours of IVM. Regulation of Cx43 may in turn, via GJC, participate in the development of fully competent oocytes.

Bioassay for follicle stimulating activity of equine gonadotropic hormone in mare serum using frozen/thawed transiently transfected reporter cells.

The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive ß-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this bioassay were highly correlated with estimates obtained with the Steelman-Pohley hCG augmentation assay. The colorimetric end point permitted the use of this assay as a rapid screen for FSH bioactivity without the need for animal use or complex cell culture facilities.

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.

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.

Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants.

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus-Sf9 insect cell system either as a single-chain with the C-terminus of the beta-subunit fused to the N-terminus of the alpha-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of alpha-subunit and the other at the C-terminus of the beta-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 degrees C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T(1/2), 74-77 degrees C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

Regulation of the expression of prostate apoptosis response protein 4 (Par-4) in rat granulosa cells.

The par-4 gene, directs the expression of a protein in the rat ventral prostate after apoptotic stimuli but not growth stimulatory, growth arresting or necrotic signals. Since Par-4 expression appears to be ubiquitous we investigated the possibility of Par-4 having a role in the rat ovary granulosa cells apoptotic death. Par-4 mRNA was detected by RT-PCR with oligonucleotides designed to prime Par-4 leucine zipper in the ovaries of 12 day old rats and reached the higher levels in 24 days old rats. In situ hybridization analysis revealed that Par-4 expression is restricted to granulosa cells. PMSG priming of 24 day old rats for 2 days greatly reduced Par-4 expression in granulosa cells as determined by in situ hybridization, RT-PCR of mRNA and protein immunodetection with Western blot. Granulosa cells placed in serum-fee culture, exhibited increased levels of Par-4 mRNA and protein, in good correlation with the degree of apoptosis. The culture-induced increases in Par-4 are significantly prevented by FSH. Transient transfection of granulosa cells with Par-4 leucine zipper domain that functions as a dominant-negative regulator of Par-4 activity resulted in lower rates of apoptosis while overexpression of the full length Par-4 counteracted FSH effects on apoptosis. Par-4 association with PKCzeta which is supposed to inhibit this kinase mediated antiapoptotic way is also prevented by FSH and, FSH antiapoptotic effects are counteracted by a PKCzeta specific inhibitor. These findings indicate that FSH by suppressing Par-4 expression in the ovary activates PKCzeta-dependent antiapoptotic pathway and suggest that Par-4 is part of the mechanism underlying granulosa cells apoptotic demise.

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.

Involvement of equine chorionic gonadotropin (eCG) carbohydrate side chains in its bioactivity; lessons from recombinant hormone expressed in insect cells.

Natural eCG consists of as much as 45% carbohydrate side chains. The present paper deals with the analysis of the roles of the N- and O-linked saccharides of this hormone in the different steps of its activity and its possible replacement by recombinant eCG expressed in baculovirus-insect cell systems.

Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic insect cells.

Recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) was expressed in Mimic insect cells, that are commercial stably transformed Spodoptera frugiperda (Sf9) cells expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex-type monosialylated N-glycans. We previously showed that it exhibited no in vivo bioactivity although expressing full in vitro bioactivity, and it was suspected that this was because of insufficient sialylation of eLH/CG N-glycans. Lectin binding analyses were performed with recombinant dimeric eLH/CG or its alpha subunit, secreted in the serum-containing supernatant of infected Sf9 and Mimic cells. Two types of specific lectin affinity assays (blot analyses and enzyme-linked immunosorbent assay) were used to compare the ability or inability of natural and recombinant gonadotropins to bind to various lectins. In natural equine chorionic gonadotropin (eCG), complex-type N-glycans terminating with both Siaalpha2,3Gal (based on Maackia amurensis agglutinin [MAA] binding) and Siaalpha2,6Gal (based on Sambucus nigra agglutinin [SNA] binding) were found, but in the alpha subunit dissociated from natural eCG, we only detected Siaalpha2-6Gal. In eLH/CG and its alpha subunit produced by Sf9 cells, N-glycans were found to be terminated by mannosyl residues (based on Galanthus nivalis agglutinin [GNA] binding), whereas those produced in Mimic cells were terminated by galactoses (based on binding to Ricinus communis agglutinin I [RCA I] , but not to SNA or MAA). This is in agreement with the fact that the nucleotide donor substrate of sialic acid is not naturally synthesized in insect cells. On the basis of binding to Arachis Hypogaea agglutinin [PNA], O-glycans exhibited the Galbeta1-3GalNAc structure in recombinant-free alpha and eLH/CG from both Sf9 and Mimic cell lines. Both N- and O-linked carbohydrate side chains synthesized in Mimic cells should thus be amenable to further acellular sialylation.

Ovarian activity and oocyte development during follicular development in pigs at different reproductive phases estimated by the repeated endoscopic method.

The aim of the present study was to assess follicular and oocyte development in the same gilts during three phases of their reproductive life [prepuberal gilts (PP; 6.0 months of age), puberal gilts (P; 9.5 months of age) and primiparous sows (S)]. Follicular development was stimulated by the injection of 1,000 IU of equine chorionic gonadotropin (eCG) followed by 500 IU of human chorionic gonadotropin (hCG) 72 h later. Cumulus-oocyte-complexes (COCs) were recovered by endoscopic ovum pick up/aspiration from preovulatory follicles of the left ovary, and the follicular fluid (FF) from the right ovary was collected 34 h after the hCG treatment by endoscopy. Altogether, 19 pigs were used in the PP and P trials and 12 in the S trial. From the left ovaries, 168, 190 and 82 follicles were aspirated and 106, 125 and 42 COCs, respectively, were recovered (recovery rate 61 +/- 27, 63 +/- 21 and 53 +/- 22%, respectively). The mean number of follicles was greater in the P phase than in the PP phase (19.7 +/- 6.8 vs. 15.7 +/- 6.8; p=0.06) and S phases (14.2 +/- 4.0; p<0.05). More uniform oocytes with an expanded cumulus were aspirated in the P and PP phases than in the S phase (90 and 78 vs. 46%; p<0.05). Furthermore, the meiotic configuration in oocytes (T I/M II stage) differed between the three phases (56 and 62 vs. 0%; p<0.05). Progesterone (P4) levels in FF decreased from 590.0 +/- 333.6 (PP) to 249.1 +/- 72.6 (P) and 161.4 +/- 75.2 ng/ml (S) (p<0.05). Estradiol-17beta (E2) levels differed between PP and P gilts and S sows (9.3 +/- 2.9, 21.9 +/- 10.6 and 94.0 +/- 15.9 pg/ml, respectively; p<0.05), and the P4/E2 ratio was 72, 15 and 5, respectively. These results indicate differences in follicular and oocyte development between the reproductive phases investigated. Puberal gilts should preferably be used in IVF and breeding programs. The lower reproductive potential of primiparous sows must be taken into consideration in breeding. Any prediction of lifetime performance based on individual ovarian reactions of prepuberal gilts is unreliable.

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.

Expression of ovarian prolactin receptor in relation to hormonal changes during induction of ovulation in the rat.

We examined the relationships between the expression of the short and long forms of the prolactin (PRL) receptor (PRLR) mRNA in the ovary and changes in the levels of serum hormones such as sex steroid hormones and PRL during induction of ovulation in the rat. The expression of both forms of PRLR mRNA in the ovary was examined by Northern blot analysis in immature female rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ovarian tissues and blood samples were obtained before treatment, 24 and 48 h after PMSG injection and 4, 6, 8, 12, 24 and 48 h after hCG treatment. Serum levels of 17beta-estradiol, progesterone and PRL were determined by radioimmunoassay. Serum levels of 17beta-estradiol rapidly increased to a maximal level 48 h after PMSG injection and then rapidly declined until 4 h after hCG injection. Serum levels of progesterone gradually increased after PMSG treatment, markedly increased to 114.2 nmol/l 8 h after hCG treatment and remained high until 48 h after hCG treatment. The serum level of PRL peaked at 66.2 microg/l (p < 0.01) 48 h after PMSG injection, and a temporary decrease after hCG treatment was followed by a continuously high level from 8 to 48 h. The expression of the long form of PRLR mRNA increased significantly (p < 0.01) to 688% of the control level after PMSG treatment, while that of the short form increased to only 184% of the control level. The expression of the long form of PRLR-mRNA rapidly declined until 6 h and then gradually increased until 48 h after hCG treatment. On the other hand, the expression of the short form of PRLR mRNA decreased to a nadir 12 h after hCG injection and then increased significantly (p < 0.01) to 142% of the control level. Our results showed that the changes in the short and long forms of PRLR mRNA differed in a time-specific manner and that these two forms are involved in different functions in the rat ovary during induction of ovulation. It is thought that the long form of PRLR mRNA is involved in folliculogenesis, while the short form of PRLR mRNA may play an important role in the formation and maintenance of the corpus luteum in the rat ovulatory cycle.

Luteal deficiency and embryo mortality in the mare.

Four separate components combine to produce the progesterone and biologically active 5 alpha-reduced pregnanes needed to maintain pregnancy in the mare. The primary corpus luteum (CL) is prolonged beyond its cyclical lifespan by the down-regulation of endometrial oxytocin receptors to prevent activation of the luteolytic pathway and its waning progesterone production is supplemented from day 40 of gestation by the formation of a series of accessory CL which develop in the maternal ovaries as a result of the gonadotrophic actions of pituitary FSH and the equine chorionic gonadotrophin (eCG). From around day 100 the allantochorion secretes progesterone and progestagens directly to the endometrium and underlying myometrium and, in the last month of gestation, the enlarging foetal adrenal gland secretes appreciable quantities of pregnenelone which is also utilized by the placenta to synthesize progestagens. Between 10 and 15% of mares undergo foetal death and abortion at some time in gestation and the majority of these losses occur during the first 40 days of gestation when the primary CL is the sole source of progesterone. Yet, all the available evidence suggests that untoward luteolysis is not common in this period and the losses that do occur have other underlying causes. Beyond day 40 the secondary CL receive powerful luteotrophic support from eCG and from day 80-100 until term the supply organ (placenta) and target tissues (endometrium and myometrium) are in direct contact with each other over their entire surface. In the face of this interlocking and failsafe system for progestagen production throughout pregnancy, and despite a paucity of evidence that a deficiency of progesterone production is a cause of pregnancy loss in the mare, it is surprising, and worrying, that annually many thousands of pregnant mares throughout the world are given exogenous progestagen therapy during part or all of their gestation as a form of preventative insurance against the possibility of pregnancy failure. Basic investigative research is required urgently to validate or debunk the practice.

Expression of an in vitro biologically active equine LH/CG without C-terminal peptide (CTP) and/or beta26-110 disulphide bridge.

The C-terminal region of the beta subunit of the human chorionic gonadotrophin (hCG) is implied in heterodimer stability (beta26-110 disulphide bridge), in vitro LH bioactivity (region beta102-110) and in in vivo LH bioactivity (beta CTP). Like the hCG beta, the equine eLH and eCG beta subunits, also possess a C-terminal extension (CTP). But, in contrast to hCG, eLH and eCG bind to both LH and FSH receptors in species other than the horse. This allows investigation of the roles of the beta subunit C-terminal region of a eLH/CG recombinant molecule on both LH and FSH activities. To do so, the CTP was deleted and/or the beta26-110 disulphide bond was mutated and the resulting mutated beta subunits were transiently co-expressed with common alpha subunit in COS7 cells. These regions were also deleted in a betaalphaeLH/CG single chain also expressed in COS7 cells. The hormones produced were characterized by different ELISAs and in vitro LH and FSH bioassays. Mutation of the 26-110 disulphide bond and deletion of the betaCTP led to a decrease in eLH/CG heterodimer production. Double mutation promoted an additive effect on production of the heterodimer and of the corresponding tethered eLH/CG. The elimination of the beta26-110 disulphide bond in the betaalpha single chain had no effect on its production. However, neither the 26-110 disulphide bond nor the CTP mutations affected dimer stability and bioactivities of the secreted heterodimers and/or single chain molecules. Therefore, in contrast to hCG, the 26-110 S-S bond of the recombinant eLH/CG beta subunit does not seem to be essential for eLH/CG dimer stability upon secretion and expressing LH and FSH bioactivities.