Gonadotropin-releasing Hormone Treatment Research Articles

Modulation of periovulatory endocrine profiles in beef cows: consequences for endometrial glucose transporters and uterine fluid glucose levels

In beef cattle, proestrus estradiol and subsequent progesterone (P4) concentrations can regulate the endometrial characteristics and thereby determine maternal receptivity toward the embryo. However, the underlying mechanisms linking periovulatory endocrine profiles to receptivity, which is crucial to obtain pregnancy, need to be elucidated. We hypothesized that the size of the preovulatory follicle (POF) and subsequent circulating P4 concentrations, during early diestrus, modulate endometrial levels of glucose transporter transcripts and proteins, and subsequently affect the luminal glucose availability in the uterus. Therefore, follicle growth of Nelore cows was manipulated, and cows were assigned to 2 experimental groups: (1) large follicle and large corpus luteum (LF-LCL) group with a large POF and corpus luteum (CL); and (2) small follicle and small corpus luteum (SF-SCL) group with a small POF and CL. At day 7 post gonadotropin-releasing hormone induced ovulation (gonadotropin-releasing hormone treatment = day 0), animals were slaughtered (n = 18 per group), and uterine tissues and washings were collected for characterization of glucose transporters and glucose levels, respectively. The diameter of POF was larger (P < 0.05) in the LF-LCL cows compared with their SF-SCL counterparts (12.8 ± 0.4 vs 11.1 ± 0.4 mm). Furthermore, CL size (17.49 ± 0.88 vs 14.48 ± 0.52 mm) and circulating P4 concentrations at day 7 (4.5 ± 1.0 vs 3.3 ± 1.1 ng/mL, P < 0.05) were significantly higher in the LF-LCL cows compared with the SF-SCL cows. No differences (P > 0.05) were detected in gene expression patterns of SLC2A1, SLC2A3, SLC2A4, SLC2A5, SLC5A1, ATP1A2, ATP1B2, and SLC37A4. However, the protein abundance of endometrial SLC2A1was increased in the LF-LCL group compared with the SF-SCL group (P < 0.05). SLC2A1 and SLC2A4 protein products were mainly identified at the endometrial luminal and glandular epithelium membranes as well as in the endometrial stroma. Glucose concentrations in uterine washings were similar between groups. In conclusion, we provided information on the potential link between endocrine profiles and glucose transport pathways in the bovine endometrium. More specifically, our data reveal that the size of the POF, and subsequent P4 concentrations, do not functionally affect the main endometrial glucose transporter pathways or uterine fluid glucose concentrations during diestrus.

Trichostatin A specifically stimulates gonadotropin FSHβ gene expression in gonadotroph LβT2 cells

Trichostatin A (TSA) is a selective inhibitor of mammalian histone deacetylase. In the present study, TSA was found to selectively increase gene expression of the pituitary gonadotropin β-subunit of follicle-stimulating hormone (FSH). Stimulation of mouse pituitary gonadotroph cell lines, LβT2, with TSA for 24 h resulted in no change in mRNA expression of the α- and LHβ-subunit. On the other hand, FSHβ-subunit mRNA expression was significantly increased in a dose-dependent fashion. Similarly, specific induction of the FSHβ-subunit gene with TSA stimulation was observed in primary cultures of rat pituitary cells. Histone acetylation in whole cell lysates of LβT2 cells was significantly increased after TSA treatment, but not gonadotropin-releasing hormone (GnRH) treatment. The effect of TSA on FSHβ mRNA expression was prominent compared to that of GnRH; however, TSA-stimulated FSHβ mRNA expression was significantly reduced with combined TSA and GnRH treatment. TSA caused a slight increase in extracellular signal-regulated kinase (ERK) phosphorylation, while GnRH-increased ERK phosphorylation was potentiated in the presence of TSA. In addition, TSA, but not GnRH, significantly stimulated gene expression of retinaldehyde dehydrogenase 1 (RALDH1), a retinoic acid (RA) synthesizing enzyme involved in cell differentiation. These findings demonstrate that TSA specifically increases FSHβ subunit gene expression with a concomitant increase in whole cell histone acetylation. Moreover, although GnRH is a stimulator of FSHβ gene expression, it interfered with the stimulatory effect of TSA on FSHβ mRNA expression, without modification of TSA-increased whole cell histone acetylation. This suggests that the mechanisms of TSA and GnRH-induced gonadotropin subunit gene expression are entirely distinct.

Interaction of Gonadal Steroids and Gonadotropin-Releasing Hormone on Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP Receptor Expression in Cultured Rat Anterior Pituitary Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are expressed in the hypothalamus, the gonadotrope cells of the anterior pituitary gland, and the gonads, forming an autocrine-paracrine system in these tissues. Within the pituitary, PACAP functions either alone or synergistically with gonadotropin-releasing hormone (GnRH) to stimulate gonadotropin gene expression and secretion. Our goal was to define the hormonal regulation of pituitary PACAP and PACAP receptor (PAC1) gene expression by dihydrotestosterone (DHT), estradiol, and progesterone alone or in conjunction with GnRH. Treatment of adult male rat pituitary cell cultures with DHT or progesterone augmented GnRH-mediated increase in PACAP messenger RNA (mRNA) levels, but neither had an effect when present alone. Conversely, estradiol treatment blunted PACAP gene expression but did not alter GnRH effects on PACAP expression. Expression of PACAP receptor mRNA was decreased by GnRH treatment, minimally increased by DHT treatment, but not altered by the addition of estradiol or progesterone. DHT and GnRH together blunted PACAP receptor gene expression. Taken together, these results suggest that the activity of the intrapituitary PACAP-PAC1 system is regulated via the complex interaction of gonadal steroids and hypothalamic GnRH.

Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH

SummaryAging is a result of gradual and overall functional deteriorations across the body; however, it is unknown if an individual tissue works to primarily mediate aging progress and lifespan control. Here we found that the hypothalamus is important for the development of whole-body aging in mice, and the underlying basis involves hypothalamic immunity mediated by IKKβ/NF-κB and related microglia-neuron immune crosstalk. Several interventional models were developed showing that aging retardation and lifespan extension are achieved in mice through preventing against aging-related hypothalamic or brain IKKβ/NF-κB activation. Mechanistic studies further revealed that IKKβ/NF-κB inhibits GnRH to mediate aging-related hypothalamic GnRH decline, and GnRH treatment amends aging-impaired neurogenesis and decelerates aging. In conclusion, the hypothalamus has a programmatic role in aging development via immune-neuroendocrine integration, and immune inhibition or GnRH restoration in the hypothalamus/brain represent two potential strategies for optimizing lifespan and combating aging-related health problems.

Inflammation links ageing to the brain

Inflammation-activated signalling pathways in the brain's hypothalamus control the production of ageing-related hormones. This finding provides a link between inflammation, stress responses and systemic ageing. See Article p.211 Ageing is closely associated with both nutrition and inflammation, and certain neurons can mediate environmental influences on ageing in Caenorhabditis elegans and Drosophila. This study focuses on the hypothalamus, a brain region that is critical for the neuroendocrine interaction between the central nervous system and the periphery. Guo Zhang et al. show that activation of IKK-β and NF-κB in the hypothalamus of mice accelerates the ageing process and shortens lifespan. Inhibition of IKK-β and NF-κB, mediators of metabolic inflammation, delays ageing and extends lifespan. NF-κB activation results in a decline of gonadotropin-releasing hormone (GnRH) levels and suppresses neurogenesis. GnRH treatment amends ageing-impaired neurogenesis and decelerates ageing. These results suggest that NF-κB-mediated inhibition of GnRH may shut down reproduction and ensure the quality of the species, but it initiates systemic ageing at the same time.

Reducing the duration between gonadotropin-releasing hormone (GnRH) and prostaglandin F2α treatment in the Ovsynch protocol to 6 days improved ovulation to second GnRH treatment, but inclined to reduce fertility

The aim of this study was to test Ovsynch (OVS) versus modified OVS (decreasing the interval between first GnRH and PGF2α to 6d) protocols on pregnancy per artificial insemination (AI) and OVS outcomes in cyclic dairy cows. Cyclic cows (n=920) were assigned to 1 of 2 groups: the OVS7 group (n=459) received the OVS protocol [GnRH treatment, PGF2α treatment 7d later, a second GnRH (GnRH2) treatment 56h later, and timed AI (TAI) 16 to 18h after the GnRH2 treatment], and the OVS6 group (n=461) received a modified OVS protocol, in which the interval between the first GnRH and PGF2α was decreased to 6d (GnRH treatment, PGF2α treatment 6d later, GnRH2 treatment 56h later, and TAI 16 to 18h after the GnRH2 treatment). The response to the first GnRH of OVS was similar between OVS7 (54.5%, 250/459) and OVS6 (54.2%, 250/461) groups. The ovulatory response to GnRH2 of OVS was higher in OVS6 (91.3%, 421/461) than OVS7 (84.5%, 388/459). The follicle size (mean ± standard error of the mean) at the time of TAI was smaller in OVS6 (15.23±0.11mm) than OVS7 (16.04±0.11mm). When all cows were evaluated, the pregnancy per AI at 31d tended to be lower in OVS6 (38.0%, 175/461) than in OVS7 (43.8%, 201/459). Moreover, the pregnancy per AI at 31d was lower in OVS6 (40.9%, 172/421) compared with OVS7 (50.3%, 195/388) in synchronized cows. In conclusion, although the modified OVS protocol decreased the follicle size at the time of AI and increased the ovulatory response to GnRH2 of OVS, it unexpectedly reduced fertility in cyclic lactating dairy cows.

702 CLINICAL FEATURES AND TESTICULAR MORPHOLOGY IN INFANTILE PATIENTS WITH KALLMANN SYNDROME

You have accessJournal of UrologyPenis/Testis/Urethra: Benign & Malignant Disaese (I)1 Apr 2013702 CLINICAL FEATURES AND TESTICULAR MORPHOLOGY IN INFANTILE PATIENTS WITH KALLMANN SYNDROME Hidenori Nishio, Kentaro Mizuno, Satoshi Kurokawa, Hideyuki Kamisawa, Makoto Imura, Yoshinobu Moritoki, Yasuhiro Shibata, Akihiro Nakane, Toshiki Kato, Tetsuji Maruyama, Yoshiyuki Kojima, Yutaro Hayashi, and Kenjiro Kohri Hidenori NishioHidenori Nishio Nagoya, Japan More articles by this author , Kentaro MizunoKentaro Mizuno Nagoya, Japan More articles by this author , Satoshi KurokawaSatoshi Kurokawa Nagoya, Japan More articles by this author , Hideyuki KamisawaHideyuki Kamisawa Nagoya, Japan More articles by this author , Makoto ImuraMakoto Imura Nagoya, Japan More articles by this author , Yoshinobu MoritokiYoshinobu Moritoki Nagoya, Japan More articles by this author , Yasuhiro ShibataYasuhiro Shibata Nagoya, Japan More articles by this author , Akihiro NakaneAkihiro Nakane Nagoya, Japan More articles by this author , Toshiki KatoToshiki Kato Nagoya, Japan More articles by this author , Tetsuji MaruyamaTetsuji Maruyama Nagoya, Japan More articles by this author , Yoshiyuki KojimaYoshiyuki Kojima Nagoya, Japan More articles by this author , Yutaro HayashiYutaro Hayashi Nagoya, Japan More articles by this author , and Kenjiro KohriKenjiro Kohri Nagoya, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.260AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Kallmann syndrome (KS) is a genetic disorder characterized by the simultaneous occurrence of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia. Although there have been some reports about genes responsible for KS, their impact on the testicular tissue of KS during infantile periods remains unclear. We retrospectively reviewed the patients clinically diagnosed KS, and evaluated the testicular morphology of KS these patients by performing testicular biopsies during orchiopexy. METHODS A retrospective assessment was conducted of 4 cryptorchid patients diagnosed with KS. The assessment included a determination of the chief complaints of the patients upon presentation, the age at which the orchiopexy was performed, and the results of hormone tests, including tests for luteinizing hormone-releasing hormone and human chorionic gonadotropin (hCG). In addition, testicular morphology was assessed during testicular biopsy, and histrogical examinations were performed. Especially the presence of Leydig cells was examined by immunohistochemistry using antibodies against Ad4BP/SF1. RESULTS Laboratory testing showed low values of LH, FSH, and testosterone, confirming hypogonadotropic hypogonadism. LH and FSH did not increase in response to intravenous gonadotropin-releasing hormone treatment; hCG tests also failed to show changes in testosterone levels. Head magnetic resonance imaging scans failed to show evidence of pituitary tumors but showed deficiencies in the olfactory bulbs and grooves. Histologic examinations of the testes showed immature seminiferous tubules with mild interstitial fibrosis and edema; spermatogonia were not detected. Leydig cells were detectable in the testis of the interstitium and Sertoli cells in the seminiferous tubules by immunohistochemistry using antibodies against Ad4BP/SF1 in the testes of one patient. CONCLUSIONS Although the diagnosis of KS before puberty is difficult, LH-RH and hCG tests were useful for the differential diagnosis. The present study showed the presence of Leydig cells in the testis of one patient with IHH using immunohistochemistry for Ad4BP/SF1, even though Leydig cells were reportedly absent. Testicular morphology in the patients with KS is more varied than expected, and further investigations are required to elucidate the hormonal effects on normal testicular development. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e289 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hidenori Nishio Nagoya, Japan More articles by this author Kentaro Mizuno Nagoya, Japan More articles by this author Satoshi Kurokawa Nagoya, Japan More articles by this author Hideyuki Kamisawa Nagoya, Japan More articles by this author Makoto Imura Nagoya, Japan More articles by this author Yoshinobu Moritoki Nagoya, Japan More articles by this author Yasuhiro Shibata Nagoya, Japan More articles by this author Akihiro Nakane Nagoya, Japan More articles by this author Toshiki Kato Nagoya, Japan More articles by this author Tetsuji Maruyama Nagoya, Japan More articles by this author Yoshiyuki Kojima Nagoya, Japan More articles by this author Yutaro Hayashi Nagoya, Japan More articles by this author Kenjiro Kohri Nagoya, Japan More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Differentially Expressed miRNAs after GnRH Treatment and Their Potential Roles in FSH Regulation in Porcine Anterior Pituitary Cell

Hypothalamic gonadotropin-releasing hormone (GnRH) is a major regulator of follicle-stimulating hormone (FSH) secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs) are small RNA molecules that control gene expression by imperfect binding to the 3′-untranslated region (3′-UTR) of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA) at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHβ mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR). Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHβ mRNA 3′-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially expressed miRNAs resolved in this study provide the first guide for post-transcriptional regulation of pituitary gonadotrope FSH secretion in pig, as well as in other mammals.

Utilization of a 5- or 14-day progestin-based estrous synchronization protocol with sorted semen in beef heifers

Estrous synchronization protocols that utilize progestin via a controlled internal drug-release insert (CIDR) in conjunction with gonadotropin releasing hormone (GnRH) and prostaglandin (PG) treatments have been shown to be effective in beef heifers. Sorted semen is more costly than unsorted semen; thus, AI pregnancy rates need to be maximized when sorted semen is used. The objective of this study was to compare the effectiveness of a 5-day versus a 14-day CIDR protocol when sorted semen is utilized for AI in beef heifers. A secondary objective was to determine the effect of timing of insemination on pregnancy rates when using sorted semen.

Gonadotropin-releasing hormone treatment improves locomotor activity, urinary function and neurofilament protein expression after spinal cord injury in ovariectomized rats

It was reported that the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH) produces neurotrophic effects and that the spinal cord possesses GnRH receptors. The aim of the present study was to determine whether administration of GnRH improves locomotor activity, urinary function and neurofilament (NFs) protein expression after spinal cord injury (SCI) in ovariectomized rats. SCI was induced by balloon inflation model resulting in paraplegia. Locomotion was evaluated according to the Basso, Beattie, and Bresnahan Scale. Rats were subjected to bladder compression, twice daily until bladder reflex was established. NFs of 68, 160 and 200kDa from spinal cords were analyzed by electrophoresis. GnRH (60μg/kg) or physiologic NaCl solution was administered at 1 day after SCI and then daily for 15 days and the functional evaluation was realized for 5 weeks. Our results indicate that locomotor activity, restoration of urinary dysfunction and NFs expression of 160 and 200kDa were improved in SCI animals given GnRH compared to those without treatment. These findings suggest that GnRH acts as a neurotrophic factor and may be used as a potential therapeutic agent for treatment of SCI.

GnRH dose reduction decreases pituitary LH release and ovulatory response but does not affect corpus luteum (CL) development and function in llamas

Gonadotrophin releasing hormone (GnRH) is commonly used in llamas to induce ovulation; however, the consequence of reduced doses of GnRH on luteinizing hormone (LH) release, ovulatory response, and subsequent corpus luteum (CL) development and function have apparently not been investigated. Hence, we examined the effect of gradual reduction of gonadorelin acetate (GnRH) dosage on pituitary LH release, ovulatory response, CL development, and plasma progesterone concentrations in llamas. Non-pregnant, non-lactating adult llamas were examined once daily by transrectal ultrasonography, and those with a follicle ≥8 mm in diameter that had grown for three consecutive days were randomly assigned to receive 50 (GnRH50, n = 23), 25 (GnRH25, n = 29), 12.5 (GnRH12.5, n = 29), or 6.25 μg (GnRH6.25, n = 29) of GnRH, or 0.5 mL of PBS (Control group, n = 16) im. In a subset (7 or 8 animals/group), intense blood sampling was done to measure LH concentrations. All females were examined by ultrasonography every 12 h from treatment (Day 0) to Day 2 to determinate ovulation, and thereafter on alternate days until Day 16 to evaluate CL development (9–13 animals/group). Also, blood samples for progesterone determination were taken (9 or 10 animals/group) on alternate days from Days 0–16. Ovulatory response (%) was highest (P < 0.05) in the GnRH50 (82.6), intermediate in the GnRH25 (72.3) and GnRH12.5 (75.9) groups, and lowest in the GnRH6.25 group (48.3). No ovulations were detected in the Control group. Mean peak LH concentrations (ng/mL) were highest (P < 0.05) for GnRH50 (6.2), intermediate for GnRH25 (4.4) and GnRH12.5 (2.9), and lowest for GnRH6.25 (2.2) groups. In addition, based on regression analysis, llamas with an LH peak <4 ng/mL were less likely to ovulate. Llamas given 50 μg of GnRH released more (P < 0.05) pituitary LH and had an LH surge of longer duration than those given 25, 12.5, or 6.25 μg. However, in those that ovulated, neither GnRH treatment nor treatment by time interaction affected (P > 0.05) CL diameter or plasma progesterone concentrations. In summary, reducing the dose of GnRH gradually decreased the magnitude of the preovulatory LH surge and ovulatory response; however, subsequent CL development and plasma progesterone concentrations were not affected.

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.

Selective cleavage of ErbB4 by G‐protein‐coupled Gonadotropin‐Releasing Hormone Receptor in Cultured Hypothalamic Neurons

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). GnRH neurons have a GnRH receptor belonging to the G-protein-coupled receptors. The stimulation of this receptor activates extracellular signal-regulated kinase (ERK). In the present study, we found that epidermal growth factor receptor (EGFR) and ErbB4 were expressed in immortalized GnRH neurons (GT1-7 cells). AG1478, a relatively specific inhibitor of the ErbB family, and small interfering RNA (siRNA) for ErbB4 inhibited the GnRH-induced activation of ERK in GT1-7 cells, suggesting that EGFR and ErbB4 were necessary for the activation. In addition, GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment. After treatment of the cells with 50 nM GnRH for 5 min, about 80% of ErbB4 was cleaved. Biotinylation of cell surface proteins revealed that more than 70% of the cell surface ErbB4 was cleaved by GnRH treatment. A higher concentration and longer treatment were necessary for GnRH to induce ErbB4 cleavage than ERK activation. TAPI-2, an inhibitor of tumor necrosis factor-α-converting enzyme (TACE), and siRNA for TACE inhibited the cleavage of ErbB4, suggesting that TACE was involved. After ErbB4 cleavage, the activation of ERK by neuregulin 1 was almost completely inhibited. These results suggest that the down-regulation of ErbB4 expression is induced by G-protein-coupled receptor stimulation.

Characterization of Seasonal Variations in Responsiveness of Pituitary Gland to Different Doses of Gonadotropin Releasing Hormone in Buffalo Cows

In tropical countries such as India, it has been observed that a number of buffalo cows experience seasonal anestrous during summer months. This might be due to seasonal changes in responsiveness of pituitary gland to gonadotropin releasing hormone (GnRH) and/or decreased hypothalamic GnRH release. Attempts were made to characterize the responsiveness of pituitary gland to a range of doses (0.1, 1, 3, 10 and 33 µg) of GnRH in terms of LH and progesterone (P4) secretions during summer (April-May) and rainy (September-November) months. As a part of these studies, a radioimmunoassay method for estimation of circulating LH in buffalo cows was standardized. During summer months, it was observed that in the presence of low circulating P4 levels the minimum dose of GnRH required for eliciting a significant increase in circulating LH levels was 10 μg/animal that corresponded to a dose of ~28 ng/kg BW. However, during rainy months, administration of the same dose of GnRH failed to elicit a response suggesting that the pituitary gland is not responsive to low doses of exogenous GnRH. On the other hand, buffalo cows receiving a dose of 100 µg of GnRH during rainy months elicited a surge-like increase in circulating LH that peaked at 2 h and the increase in LH concentrations lasted for nearly 6 h post GnRH treatment. The results appear to suggest that during summer months the pituitary gland function is not affected, but there may be lowered hypothalamic GnRH input to the pituitary gland.

Progesterone concentrations and pregnancy rates of repeat breeder cows following postinsemination PRID and GnRH treatments*

The objective of this study was to determine the effects of progesterone (PRID) application and gonadotropin-releasing hormone (GnRH) injection after artificial insemination on serum progesterone concentration and pregnancy rates in repeat breeder cows. In group 1, repeat breeder cows received an injection of GnRH on day 12 after artificial insemination. In group 2, repeat breeder cows received PRID from day 4 to day 11; group 3 received PRID from day 11 to day 18. Blood samples were obtained from all treated cows to assay serum progesterone concentrations on application days, 7 days after application, and 21 days after artificial insemination. In the control group repeat breeder cows did not receive any treatment. Blood samples were obtained from all control repeat breeder cows periodically after artificial insemination. Pregnancy rates were 20% in group 1, 26.6% in group 2, 40% in group 3, and 20% in the control group. Serum progesterone concentrations were significantly higher in group 1 and group 3 (P < 0.05) than the control group, 7 days after application. Application of PRID and GnRH after artificial insemination did not significantly improve pregnancy rates, despite the fact that serum progesterone concentrations were higher in treatment groups.

Impact of circulating concentrations of progesterone and antral age of the ovulatory follicle on fertility of high-producing lactating dairy cows

Dairy cow infertility negatively affects profit of dairy production enterprises around the world, and enhancing conception rates of dairy cows is a critical management issue to resolve. It appears that conception rates of dairy cows are attenuated due to reduced progesterone concentrations in circulation during growth of the ovulatory follicle. It is not clear how reduced progesterone influences fertility, but data presented in this brief review suggest that it can be somewhat reversed through increasing concentrations of progesterone during the growth of the ovulatory follicle before luteolysis. Ovsynch protocols may be utilised to enhance progesterone concentrations through the induction of an accessory corpus luteum (CL) following the initial gonadotropin-releasing hormone (GnRH) treatment. Cows at Day 13 of the oestrous cycle with a 7-day-old accessory CL had ~50% more progesterone at the time of prostaglandin injection of Ovsynch compared with cows with only a Day 13 CL. Ovsynch can consistently induce an accessory CL following the initial GnRH treatment if cows are on Days 6 or 7 of the oestrous cycle at the time of treatment. Pre-synchrony strategies are critical to enhance the probability that cows will be on Days 6 or 7 at first GnRH treatement of Ovsynch.

Effect of gonadotropin-releasing hormone (GnRH) treatment on multiple births in Afshari ewes

The purpose of this study was to investigate the effects of gonadotropin-releasing hormone (GnRH) administration on the induction of multiple births in synchronized Afshari ewes. 16 cycling, multiparous fat-tailed Iranian Afshari ewes, weighing 66.5 ± 2.5 kg, were used in the trail. Estrus was synchronized using controlled internal drug release CIDR’s; inserted for 14 days. Pregnant mare serum gonadotrophin (PMSG) (300 IU) was injected to ewes a day before CIDR removal. The ewes was randomly allocated to two groups (n = 8). Synchronization treatment was initiated with a week interval in two groups (weeks 1 and 2). 24 h following CIDR removal (day 1), GnRH (50 µg) was administered to half of each group ewes (GnRH and control groups), and ewes mated with the proven rams. Pregnancy was diagnosed with the aid of transabdominal ultrasonograpy 30 days after mating. Ovarian follicular activity was monitored with the aid of transrectal ultrasonograpy on the days of CIDR removal and at estrus (approximately day 1). Following PMSG treatment, the mean number of large follicles on total ovaries increased from CIDR removal to estrus 1.48 ± 0.71 vs. 2.85 ± 0.82 (P < 0.05). Lambing rate in GnRH group (162.5%) was higher (P < 0.05) compared to the control group (125%). The mean weights of lambs born in the GnRH and control groups were 5.5 ± 0.1 and 5.2 ± 0.1, respectively.

Molecular characterization and regulation of the angiotensin-converting enzyme type 2/Angiotensin-(1-7)/MAS receptor axis during the ovulation process in cattle

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE(2), NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE(2), NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased (p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE(2), NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE(2), NEP and PEP were differentially expressed after GnRH treatment (p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE(2), NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

Expression of genes for oestrogen and progesterone receptors in the cervix of anoestrous ewes treated with gonadotrophin releasing hormone with or without progesterone priming

The aim was to determine the oestrogens receptor alpha (ERα) mRNA and the binding capacity of oestrogens (ER) and progesterone receptor (PR) in the cervix of anoestrous ewes treated with gonadotrophin-releasing hormone (GnRH) with or without progesterone (P) priming, at the expected time of induced ovulation and early luteal phase. In Experiment 1, ewes were treated with P for 10 days ( n = 4), with nine micro-doses of GnRH followed by a GnRH bolus injection ( n = 4), or with P plus GnRH treatments ( n = 3), and tissues were harvested either without treatment ( n = 4), when P was removed, or 24 h after the GnRH bolus injection. In Experiment 2, ewes were treated with the same GnRH or P plus GnRH treatments and tissues were harvested on Day 1 ( n = 12) or Day 5 ( n = 10) after the GnRH bolus injection. In the cranial cervix, the P treatment decreased and the GnRH treatment (after P treatment) increased the ERα mRNA, ER and PR concentrations ( P < 0.002). The ERα mRNA and ER concentrations were greater on Day 1, than on Day 5 in P plus GnRH treated ewes ( P < 0.0005). In the caudal cervix, lesser ERα mRNA, ER and PR concentrations than cranial cervix were found ( P < 0.0001). In conclusion, the ERα transcriptional activity and ER and PR binding capacity were strongly influenced by P and/or GnRH treatments in the cranial cervix, while the steroid receptors binding capacity remained unchanged in the caudal cervix of anoestrous ewes at the expected time of induced ovulation and early luteal phase.

P-cadherin cooperates with insulin-like growth factor-1 receptor to promote metastatic signaling of gonadotropin-releasing hormone in ovarian cancer via p120 catenin

Gonadotropin-releasing hormone (GnRH) is a potent prometastatic factor in ovarian cancer, but the intracellular signaling events are not well understood. The classical Gα(q)-phospholipase C signal transduction pathway known to operate in the pituitary is not involved in GnRH actions at non-pituitary targets. Here we showed that GnRH treatment of ovarian cancer cells led to a rapid and remarkable tyrosine phosphorylation of p120 catenin (p120(ctn)), which was mediated by P-cadherin. The use of P-cadherin small interfering RNA or neutralizing antibodies to inhibit P-cadherin expression and function resulted in diminished p120(ctn) activation, confirming that the effect was P-cadherin specific. On exploring how P-cadherin, which lacks intrinsic kinase activity, might regulate the activation of p120(ctn), we found that P-cadherin could induce the ligand-independent activation of insulin-like growth factor-1 receptor (IGF-1R). Inhibition of IGF-1R expression or its activity significantly inhibited GnRH-induced p120(ctn) activation, and the subsequent cell migration and invasion. In addition, we showed that IGF-1R regulation by P-cadherin was associated with complex formation between IGF-1R and P-cadherin, and this regulation was also observed to be in vivo correlated with metastasis. Furthermore, using a mouse model of ovarian cancer metastasis, GnRH receptor knockdown was shown to diminish peritoneal dissemination of tumors and ascites formation. These findings suggest for the first time that GnRH can initiate an outside-in p120(ctn) signal transduction through the cross-talk between P-cadherin and IGF-1R, thus providing a novel molecular mechanism by which GnRH may control the high level of aggressiveness and invasion and metastasis potential that are characteristic of ovarian cancer.