Gonadotropin releasing hormone receptor gene and protein expression and immunohistochemical localization in bovine uterus and oviducts.
ABSTRACT Recently GnRH, GnRH-R systems has been demonstrated in various extrahypothalamic and extrapituitary reproductive tissues in different mammalian species, where GnRH acts in an autocrine and or paracrine manner and modulates different biological processes. GnRH-R mRNA has also been demonstrated in bovine ovaries (follicle and corpus luteum) and normal and carcinogenic human endometrium/endometrial cells. This is the first study elucidating presence of GnRH-R mRNA and GnRH-R protein in bovine uterus and oviducts in follicular and luteal phases of the estrous cycle and further localizing the receptors to endometrial and oviductal epithelial cells. To our knowledge this is the first report demonstrating GnRH-R mRNA and protein in mammalian oviducts. We used gene-specific primers and monoclonal GnRH-R antibody to test GnRH-R mRNA and GnRH-R protein through RT-PCR and immunobloting. Immunohistochemistry was employed to localize these receptors to endometrial and oviductal epithelial cells. GnRH-R mRNA and receptor protein were expressed at expected molecular weights of 920bp and 60kD, respectively. Densitometry analysis revealed that expression levels for GnRH-R protein in uterus and oviducts were similar to bovine pituitary. The presence of GnRH receptors in bovine uterus and oviducts is intriguing and it would be imperative to examine the functional role of this system in the regulation of reproductive processes.
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ABSTRACT: The hypothesis of the present study was that a GnRH agonist application at early pregnancy would alter the pattern of the key reproductive hormones LH and FSH, and subsequently that of estradiol (E2) and especially progesterone (P4), and improve the conditions for embryo survival in early pregnant gilts. Therefore, the endocrine effects of a GnRH agonist (GnRHa) application to gilts (n=11 GnRHa treated, n=9 saline Controls) were studied in blood samples from the Vena cava caudalis. GnRHa injected on Day 12 after insemination induced elevated (P<0.01) LH and FSH levels for at least 180 min. However, subsequent LH concentrations were not altered up to Day 21 of pregnancy. LH pulse number, estimated in 6-h period samples on Days 13, 15 and 17, was not influenced by treatment and pregnancy. LH pulse amplitude was decreased (P<0.05) on Days 13 to 17 in pregnant gilts of both groups, but not in nonpregnant animals. In pregnant GnRHa-treated gilts, the basal LH level was elevated compared with the Controls (P<0.01). Additionally, differences (P<0.05) in basal LH were present between the pregnant and nonpregnant animals. The P4 and E2 secretion pattern was not affected by GnRHa. P4 concentrations increased (P<0.01) from Day 10 to Day 14 regardless of the treatment. P4 revealed a pulse-like pattern, but without a definite relation to the LH pulse characteristics. Also, pregnancy rate (73 vs. 67%) and the number of fetuses (12.8 ± 2.3 vs. 11.6 ± 2.3) were unaffected in the treated and Control gilts, respectively. The present study did not confirm the initial hypothesis that a GnRHa-mediated LH effect could alter ovarian steroid secretion and favorably support early embryo development and pregnancy outcome.Journal of Reproduction and Development 12/2010; 57(2):242-8. · 1.76 Impact Factor
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ABSTRACT: The objectives were to compare: (1) preovulatory serum LH concentrations, and (2) synchronization of ovulation, after im or iu administration of the second GnRH treatment of Ovsynch in lactating dairy cows. Lactating cows (N = 23) were presynchronized with two injections of PGF(2α) given 14 days apart (starting at 34 ± 3 days in milk), followed by Ovsynch (GnRH-7 d-PGF(2α)-56 h-GnRH) 12 days later. At the time of the second GnRH of Ovsynch (Hour 0), cows were blocked by parity and randomly assigned to 1 of 3 groups: (1) control group (CON; N = 7) were given 2 mL sterile water im; (2) intramuscular group (IM; N = 8) received 100 μg of GnRH im; and (3) intrauterine group (IU; N = 8) had 100 μg GnRH infused in the uterus (2 mL). Blood samples for serum LH concentrations were collected at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Furthermore, ultrasonography was performed twice daily (12-h intervals) from Hours 0 to 60 to confirm ovulation. The LH concentrations were greater (P < 0.05) in the IM than IU and CON groups at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Although LH concentrations were numerically higher in the IU group, LH concentrations within the IU and CON groups did not change over time. More cows ovulated in the IM (8/8) and IU (7/8) groups within 60 h after the second GnRH administration compared with the CON (2/7) group. In summary, serum LH concentrations were lower in the IU versus IM group, but the proportion of cows that ovulated within 60 h was similar between these two groups. Therefore, iu administration of GnRH may be an alternative route of delivery to synchronize ovulation in beef and dairy cattle.Theriogenology 08/2012; 78(6):1390-7. · 2.08 Impact Factor
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ABSTRACT: Large amounts of protein intake are associated with elevated ammonia and urea concentrations in both plasma and uterine fluid in dairy cows. These increased concentrations affect successful embryo development and subsequent pregnancy establishment. The objective of the present study was to examine the effects of ammonia and urea on the expression of some candidate genes in the endometrium of mid-luteal phase of the estrous cycle of dairy cows. Endometrial explants were cultured and treated with 0, 75, 150, 300, 600μM of ammonium chloride or 0, 4, 8, 12, 16mM of urea. After the RNA extraction and reverse transcription, real time PCR was performed to assess the treatment effects on relative amounts of mRNA of candidate genes. BCL2 mRNA was greater in explants treated with 150μM of ammonium chloride compared to explants treated with 0, 75 and 300μM. Relative amounts of IGFBP1 mRNA were less in explants treated with 600μM of ammonium chloride when compared with other concentrations. Relative FGF2 gene expression was less in explants treated with a greater concentration (600μM) of ammonium chloride or urea (16mM) when compared with lesser concentrations. Expression of HSPA1A, IGFBP3 and SERPINA14 genes was greater in explants exposed to lesser concentrations (150μM) of ammonium chloride or urea (4mM). Relative amounts of IGF1 and BAX mRNA were not affected by any of the ammonium chloride or urea concentrations tested. In conclusion, greater concentrations of ammonia and urea have negative effects on some endometrial gene expression, while moderate concentrations have positive effects.Animal reproduction science 07/2013; · 1.56 Impact Factor