Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland.During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes.In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.     

Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.     

Ovarian responses of seasonally anestrous ewes administered progesterone,PMS, HCG and(or) GnRH

One hundred and sixty ewes were assigned to sixteen groups in a 2 × 2 × 4 factoral design and were treated during the anestrous season. The main effects were progesterone pretreatment (non-implanted and implanted for 14 days), PMS pretreatment (no pretreatment and pretreatment with 500 IU at the time of progesterone implant removal) and treatments (none, GnRH in saline, GnRH in gelatin capsules and HCG). GnRH in saline (250 μg) and HCG (500 IU) were administered intramuscularly and GnRH in gelatin capsules (250 μg) was administered subcutaneously 24 hours after the time of progesterone implant removal.Ewes were classified into one of four progesterone response categories: cyclic, transient, prolonged and no response. An injection of GnRH in saline induced a prolonged progesterone response in only one ewe (13%) which was similar to the response in the untreated ewes (0%). More ewes administered GnRH in gelatin capsules (56%) and more ewes administered HCG (89%) had a prolonged progesterone response than GnRH (in saline) treated or untreated ewes. A higher percentage of ewes that were pretreated with PMS and treated with GnRH in saline (78%) had a prolonged progesterone response than ewes treated with either PMS (22%) alone or with GnRH (in saline; 13%) alone.     

The effect of method of administration on ovarian responses of seasonally anestrous ewes administered gonadotrophin releasing hormone

Mature ewes were treated during the anestrous season with saline (I) or GnRH either intramuscularly in saline (II), subcutaneously in carboxymethylcellulose (CMC) (III) or subcutaneously in gelatin capsules (IV). Fifty μg of GnRH or 1 ml of saline were administered to 22 ewes in experiment 1. In experiments 2 and 3, forty-seven and 10 ewes received 250 μg GnRH or 1 ml of saline. Ewes were bled for progesterone determination prior to treatment and up to 12 or 13 days after treatment. In experiment 3, ovaries were observed via mid-ventral laparotomy 4 days after treatment and ovarian structures recorded. Ewes were classified into one of four progesterone response categories: cyclic, transient, prolonged or no response. The only treatment that changed the progesterone response from the saline-treated controls was GnRH in gelatin capsules. More ewes in this group were classified with a prolonged progesterone response (40%) than in the saline control group (0%). GnRH (in gelatin capsules)-treated ewes in the prolonged progesterone response category had higher concentrations of plasma progesterone than GnRH (in saline or CMC)-treated ewes with a prolonged progesterone response. For the GnRH (in gelatin capsule)-treated ewes, the prolonged progesterone response was similar to progesterone in ewes during the estrous cycle and all ewes in the prolonged progesterone category had corpora lutea (experiment 3). In summary, implanting the GnRH in gelatin capsules subcutaneously in seasonally anestrous ewes increased the ovulation response and enhanced corpus luteum function over ewes administered GnRH in saline intramuscularly.     

Short photoperiod-induced gonadal regression: effects on the gonadotropin-releasing hormone (GnRH) neuronal system of the white-footed mouse, Peromyscus leucopus

The peroxidase-antiperoxidase method was used to determine quantitatively the effect of short photoperiod-induced gonadal regression on the immunoreactive gonadotropin-releasing hormone (GnRH) neuronal system of female Peromyscus leucopus. In mice exposed to either long (16L:8D) or short (8L:16D) photoperiod, immunoreactive cell bodies were loosely organized into six groups: olfactory peduncle, diagonal band of Broca, septum, preoptic area (POA), anterior hypothalamus (AH), and basal hypothalamus. The POA and AH contain the largest number of cell bodies, which supply the major GnRH innervation to the median eminence (ME) and several extrahypothalamic brain sites. Exposure to short photoperiod increased the number of immunoreactive cell bodies within the anterior hypothalamus and preoptic area (AHPOA) and also increased the optical density for staining of immunoreactive cell bodies in the AHPOA and olfactory peduncle. The ME of mice exposed to short photoperiod had a higher density of GnRH fibers relative to that of mice exposed to long photoperiod, and the content of GnRH fibers in the rostral ME was correlated with the optical content for immunostaining of cell bodies in the AHPOA. These results are evidence that gonadal regression induced by short photoperiod (mediated by the pineal gland) involves alterations of GnRH neuronal activity. Notably, data from this study are consistent with the hypothesis that suppressed release of GnRH from neurovascular terminals in the ME, rather than lack of availability of the decapeptide, promotes gonadal regression.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor: structure, distribution and regulation of expression

Reproduction in mammals is controlled by interactions between the hypothalamus, anterior pituitary and gonads. Interaction of GnRH with its cognate receptor is essential to regulating reproduction. Characterization of the structure, distribution and expression of GnRH receptors (GnRH-R) has furthered our understanding of the physiological consequences of GnRH stimulation of pituitary gonadotropes. Based on the putative topology of the amino acid sequence of the GnRH-R and point mutation studies, key elements of the GnRH-R have been identified to play a role in ligand recognition and binding, G-protein activation and internalization. Normally, reproductive function is mediated by GnRH-R expressed only on the membranes of pituitary gonadotropes. The density of GnRH-R on gonadotropes determines their ability to respond to GnRH. This density is highest just prior to ovulation and likely is important for complete expression of the pre-ovulatory surge of LH. Therefore, knowledge regarding what regulates the density of GnRH-R is essential to understanding changes in pituitary sensitivity to GnRH and ultimately, to expression of the LH surge. Regulation of GnRH-R gene expression is influenced by a multitude of factors including gonadal steroid hormones, inhibin, activin and perhaps most importantly GnRH itself.     

Hypothalamic sites of catecholamine inhibition of luteinizing hormone in the anestrous ewe.

Norepinephrine (NE) and dopamine (DA) actively inhibit the release of LH in anestrous ewes. This can be detected as an increase in LH pulse frequency following i.v. injection of NE and DA antagonists. The objective of this study was to determine the sites of these inhibitory actions in the ovine hypothalamus by using local administrations of the NE antagonist, phenoxybenzamine (PBZ), or the DA antagonist, pimozide (PIM), into specific hypothalamic areas. Each neurotransmitter antagonist was administered via a chronically implanted steel guide tube into either the preoptic area (POA), retrochiasmatic area (RCh), or the median eminence region (ME). Blood samples were taken every 15 min for 2 h before and 4 h during implantation of these drugs and were analyzed for LH and prolactin by RIA. Control (no treatment) samples were obtained similarly from the same animals on another day. Placement of PBZ into the POA significantly increased LH pulse frequency and mean LH concentrations over control values whereas PIM did not. In contrast, PIM significantly increased LH pulse frequency and mean LH concentrations when placed in the ME or in the RCh, but PBZ did not. No effects of PIM on prolactin concentrations were detected. These results suggest that an NE neural system operates in the POA and that a DA system acts in the medial basal hypothalamus (RCh or ME) to suppress GnRH pulse frequency in the ovary-intact anestrous ewe.     

Sex differences and the development of the rabbit brain: effects of vinclozolin

The preoptic/anterior hypothalamic area (POA/AH) is one of the most sexually dimorphic areas of the vertebrate brain and plays a pivotal role in regulating male sexual behavior. Vinclozolin is a fungicide thought to be an environmental antiandrogen, which disrupts masculine sexual behavior when administered to rabbits during development. In this study, we examined several characteristics of the rabbit POA/AH for sexual dimorphism and endocrine disruption by vinclozolin. Pregnant rabbits were dosed orally with vinclozolin (10 mg/kg body weight) or carrot paste vehicle once daily for 6 wk beginning at midgestation and continuing through nursing until Postpartum Week 4. At 6 wk, offspring were perfused with 4% paraformaldehyde and brains processed for immunocytochemical localization of tyrosine hydroxylase, calbindin, gonadotropin-releasing hormone (GnRH), or Nissl stain. There were significant sex differences in the distribution of calbindin in the POA/AH and the size of cells in the dorsal POA/AH (values greater in females than in males), but not in the number or distribution of tyrosine hydroxylase or GnRH neurons. In both sexes, exposure to vinclozolin significantly increased calbindin expression in the ventral POA/AH and significantly decreased number of GnRH neurons selectively in the region of the organum vasculosum of the lamina terminalis (OVLT) but not more caudally in the POA/AH. This is the first documentation of a sexually dimorphic region in the rabbit brain, and further supports the use of this species as a model for studying the influence of vinclozolin on reproductive development with potential application to human systems.     

Progesterone exposure of the preovulatory follicle in the seasonally anestrous ewe alters the expression of angiogenic growth factors in the early corpus luteum

Gonadotrophin releasing hormone (GnRH)-induced ovulation in seasonally anestrous ewes is associated with a high incidence of defective corpora lutea (CL), which can be completely eliminated by priming ewes with progesterone before GnRH treatment, but the physiological basis of this has remained elusive. This study tested the hypothesis that progesterone priming eliminates defective luteal function by altering the expression of Vascular Endothelial Growth Factor (VEGF), its receptor VEGFR-2, and angiopoietin (ANG)-1, ANG-2 and their receptorTIE-2 in the early CL. Fifteen seasonally anestrous ewes were treated by i.m. injection with 20 mg of progesterone 3 days before the start of GnRH treatment, while another 15 animals served as controls. Intravenous injections of 500 ng GnRH were given to all the ewes every 2 h for 28 h, followed by a 300 μg GnRH bolus injection to synchronize the preovulatory luteinizing hormone (LH) surge. Corpora lutea were collected 1, 2 and 4 days after ovulation and analyzed for protein and mRNA expression of VEGF, VEGFR-2, ANG-1, ANG-2 and Tie-2 using Western Immunoblotting and in situ hybridization. VEGF, VEGFR-2 and ANG-1 expression was significantly higher (P ≤ 0.05) in the CL of progesterone-primed animals compared to non-primed ones. However, no differences were observed in the ANG-2 or Tie-2 expression levels between the two treatment groups. These data suggest that progesterone priming of the preovulatory follicle alters the expression of some angiogenic growth factors in the early CL, leading to greater vascular stability and thereby normal luteal function.     

No evidence for pituitary priming to gonadotropin-releasing hormone in relation to luteinizing hormone (LH) secretion prior to the preovulatory LH surge in ewes

The purpose of this study was to determine the occurrence of and the regulatory mechanisms involved in priming of the pituitary to GnRH before the preovulatory LH surge in sheep. Experiment 1: Forty-two ewes had progestagen devices removed after 14 days and were assigned to luteal (Lut) or follicular (Foll) groups. Fifteen days later, blood sampling was initiated either immediately or 36 h after induced luteolysis in groups Lut and Foll, respectively. After 4 h, ewes were administered either saline (n = 5) or 250 ng (n = 8) or 10 microg (n = 8) of GnRH. Five ewes per treatment group were killed 1 h later, while remaining animals were blood sampled for a further 7 h. Experiment 2: Eighteen ewes were allocated to Lut and Foll groups (described above). Blood samples were collected from 2 h before GnRH (10 microg) treatment until 7 h after. Despite up-regulated GnRH-R mRNA levels in Foll ewes, pituitary content and plasma levels of LH and LHbeta mRNA levels were similar between groups. Mean FSHbeta mRNA and plasma FSH levels were elevated in Lut ewes but declined after GnRH treatment. Inversely, plasma estradiol and inhibin-A concentrations were higher in Foll ewes and declined after GnRH treatment. Fewer LH(+ve)/secretogranin II(-ve) (SgII(-ve)) granules were present in gonadotropes of Foll ewes, coincident with increased basal LH levels. Fewer smaller sized granules were present after GnRH treatment. In conclusion, there was no evidence of self-priming before onset of the preovulatory LH surge. Constitutive release of LH(+ve)/SgII(-ve) granules may maintain basal LH levels while smaller sized, presumably mature granules may be preferentially released after GnRH stimulation.     

The role of GABA(A) and GABA(B) receptors in the control of GnRH release in anestrous ewes

The paper reviews data concerning the involvement of GABA(A) and GABA(B) receptors in the control of GnRH secretion in anestrous ewes. Generally, GABA influences the GnRH release through GABA(A) and GABA(B) receptors located on perikaria of the GnRH neurons in the preoptic area (MPOA) or through the influence on beta-endorphinergic and catecholaminergic systems activity in MPOA and in ventromedial-infundibular region of the hypothalamus (VEN/NI). Stimulation of GABA(A) receptors in VEN/NI and MPOA attenuates GnRH release, while activation of GABA(B) receptors in MPOA decreases GnRH secretion, and in VEN/NI increases concentration of GnRH. The different neural mechanisms could be involved in this process: direct ligand action on the GABA(A) and GABA(B) receptors located on GnRH cells and axon terminals or indirect effect through the changes in the beta-endorphinergic and catecholaminergic systems activity in these structures of the brain.     

Effects of time of PMSG and fixed-time GnRH injections on estrus incidence and fertility in physiologically different ewes pre-treated with progestogen-impregnated vaginal sponge during the nonbreeding season

A 2 × 2 factorial study for hormonal treatment was designed in 85 seasonally anestrous ewes with physiologically different status (nonparous, dry, and postpartum). All ewes were pre-treated with 60 mg of 6-methyl-17-acetoxy-progesterone (MAP) vaginal sponge for nine days and divided into four groups: Group I (22 ewes) — an i. m. injection of 600 i.u. pregnant mare's serum gonadotropin (PMSG) at the sponge removal (Day 0) and a single i.m. injection of 100 ug synthetic gonadotropin releasing hormone (GnRH) at 36 h after the sponge removal; Group II (21 ewes) — a PMSG injection at Day 0 and a saline injection at 36 h after the sponge removal; Group III (21 ewes) — a PMSG injection two days before the sponge removal and the GnRH injection at 24 h after the sponge removal; and Group IV (21 ewes) — a PMSG injection at Day -2 and a saline injection at 24 h after the sponge removal. The treated ewes were allowed to mate once with rams for five days after treatment. Estrus incidence and lambing rates were low (69.4% and 27.1%, respectively), probably due to the mating system and poor body condition of ewes used in the study. No effect of PMSG injection two days before with-drawal of MAP sponge and the fixed-time GnRH injection were observed in estrus incidence, fertility, and prolificacy. The present study indicates that the physiological status of ewes combined with management systems including feeding and mating would be important for out-of-season breeding with hormonal treatment.     

Neurosecretory neurons of the nucleus preopticus (NPO) express salmon GnRH mRNA and show reproduction phase-related variation in the female Indian major carp, Cirrhinus cirrhosus

We studied the expression of sGnRH mRNA in the neurons of the nucleus preopticus (NPO) of the Indian major carp, Cirrhinus cirrhosus, and their correlation with the reproductive status of the fish. Non-radioisotopic in situ hybridization histochemistry protocol employing biotinylated-oligonucleotide probes complementary to salmon GnRH, cichlid GnRH I, catfish GnRH, chicken GnRH II (from cichlid and catfish), and mammalian GnRH, were applied to the sections through the POA of the female Indian major carp Cirrhinus cirrhosus. Incubation with the probe complimentary to salmon GnRH (sGnRH) mRNA from salmon, produced distinct hybridization signal in the cytosol of several neurosecretory neurons of the magnocellular and parvocellular subdivisions of the NPO of the fish collected during February-April (preparatory phase) and May-June (prespawning phase). However, no signal was detected in the NPO of fish collected during July-August (spawning phase). Application of other antisense probes, or sense probe for salmon GnRH mRNA, produced no signal. We suggest that NPO neurons in C. cirrhosus may express sGnRH mRNA, produce GnRH peptide, and play a role in regulation of pituitary-ovary axis.     

Endocrine and ultrasound evaluation of the response to PGF 2alpha and GnRH given at different stages of the luteal phase in cyclic ewes

To investigate the effects of prostaglandin (PGF 2alpha) plus GnRH at different stages of the luteal phase 13 ewes received PGF 2alpha on Day 9 of the synchronized cycle, followed 36 h later by GnRH. This control regimen resulted in ovulation and normal corpus luteum (CL) function. In the next cycle, the ewes were treated simultaneously with PGF 2alpha and GnRH either on Day 4 (early, n = 7) or Day 9 (late, n = 6). Ovarian activity was monitored daily by ultrasonography, and blood samples were obtained to monitor hormonal patterns. Size of the largest follicle present when GnRH was administered was similar in all groups, but the preceding growth rate was greatest for the early group. In the 36 h after injection of PGF 2alpha, serum progesterone (P4) had declined to basal levels in the control cycles when GnRH was administered, but P4 concentrations were higher in the early group and were highest in the late group when the GnRH was administered with PGF 2alpha. The LH surges induced by GnRH were highest in the control cycles, and were lower in the 2 treated groups. In the early group, 6 of 7 ewes demonstrated ovulation within 48 h of GnRH, resulting in the formation of normal CL. In the late group, ovulation was delayed for about 5 d in 4 of 6 ewes, and subsequent luteal function was normal; no ovulation was detected in the other 2 ewes of this group, but the follicles became luteinized, resulting in a normal P4 profile in one and subnormal in the other. These results suggest that follicles present during the early luteal phase are capable of ovulating and forming fully functional CL in response to exogenous GnRH. In contrast, follicles present during the late luteal phase fail to ovulate in response to GnRH while P4 levels are high, even though the LH stimulus is adequate; however, these follicles persist and subsequently ovulate after P4 levels have decreased. Therefore, the endocrine milieu to which a follicle was exposed may be more important than its size in determining its ability to undergo ovulation and development into a normal CL.     

Estrogen and progesterone receptor content in the pituitary gland and uterus of progesterone-primed and gonadotropin releasing hormone-treated anestrous ewes

The objective of this work was to investigate the effect of progesterone (P) and gonadotropin-releasing hormone (GnRH) treatment on estrogen receptor (ER) and P receptor (PR) concentrations in the pituitary gland and uterus of anestrous ewes. Ewes were either not treated (group C, n = 4); were treated with 0.33 g P-controlled internal drug release (P-CIDR) for 10 days (group P, n = 4), with GnRH, 6.7 ng i.v. injections every 2 h for 18 h followed by a 4 microg bolus administration of Receptal at 20 h (group GnRH, n = 4), or with a combination of the P and GnRH treatment (group P + GnRH, n = 3). Ewes were humanely killed either at the beginning of the experiment (group C), when the CIDR was removed (group P), or 24 h after the GnRH bolus treatment (groups GnRH and P + GnRH). Progesterone treatment increased serum P concentrations, indicating that the treatment was effective. All GnRH treated ewes had similar luteinizing hormone (LH) surges, which lasted 8 h. At slaughter, estradiol (E2) concentrations in the GnRH group were higher than in groups C, P, and P + GnRH. Treatment with GnRH increased more than 10-fold the content of ER and PR in the pituitary gland without altering steroid receptor concentrations in the uterus. When GnRH was combined with P the uterine receptor contents were higher than with P treatment alone. The treatment with P decreased ER and PR content in the uterus, but had no effect on the pituitary gland. The results show that regulation by P and GnRH of ER and PR content in anestrous ewes is tissue-specific.     

Inter-breed variation in the postpartum ewe response to continuous low dose infusion of GnRH

During the nonbreeding season the pituitary and ovarian responses to a subcutaneous GnRH infusion were investigated in acyclic, lactating Mule ewes which exhibit a deep seasonal anestrus and in Finn x Dorset ewes in which seasonal anestrus is ill-defined. Each breed received 10 d of progestagen priming before being subdivided into 3 groups. In Group L + G, 5 lactating ewes received GnRH (250 ng/h sc) for 96 h; in Group D + G, 5 dry ewes received GnRH (250 ng/h sc) for 96 h; in Group L, 5 lactating ewes received saline vehicle for 96 h. The infusions began when lactating and dry ewes were approximately 28 d and 120 d post partum, respectively. Blood samples were collected for LH, progesterone and estradiol analysis. Estrous behavior was monitored between Day -4 and Day +7. On Day +7 the reproductive tract was also examined. In the Mule ewes the mean plasma LH concentration increased (P < 0.05) following minipump insertion in each treatment group, although mean LH levels were greater (P < 0.05) in Group D + G, than in either Group L + G or Group L. Following the GnRH infusion, mean plasma estradiol levels increased (P < 0.05) in Group D + G but not in Group L + G. A preovulatory LH surge and subsequent ovulation occurred in 5 5 , 2 5 and 0 5 ewes from Group D + G, L + G and L, respectively, and estrus was recorded in 5 5 , 1 5 and 0 5 of these ewes, respectively. The LH surges began earlier (P < 0.05) (43.2 +/- 6.8 h vs 77.0 +/- 1.0 h) and the ovulation rate was greater (2.2 +/- 0.37 vs 1.00 +/- 0.00) in Group D + G than Group L + G. In the Finn x Dorset ewes mean LH concentrations increased (P < 0.05), to a similar level following minipump insertion in Groups D + G and L + G, but not Group L. The elevated LH levels were accompanied by increased (P < 0.05) plasma estradiol levels in Group D + G, but not in Group L + G. The GnRH infusion culminated in an LH surge and estrous behavior in 5 5 , 1 5 and 0 5 ewes from Groups D + G, L + D and L, respectively. The interval to the LH surge was similar between Group D + G (48.4 +/- 6.6 h) and Group L + G (46.0 h). Ovulation was evident in those ewes which exhibited an LH surge plus one additional ewe from Group L + G. The mean ovulation rate was greater in Group D + G (4.00 +/- 1.05) than in Group L + G (1.5 +/- 0.50). These data show that continuous GnRH infusion can consistently induce out of season breeding in the nonlactating Mule and Finn x Dorset ewe but can not break combined seasonal and lactational anestrous in these breeds. Further, between-breed differences are evident in the site along the hypothalamic-pituitary-ovarian axis at which reproduction is compromised in ewes at the same chronological stage post partum.