Genome Analysis Laboratory, Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar 243122, India.
Domest Anim Endocrinol. 2013 Aug;45(2):55-63. doi: 10.1016/j.domaniend.2013.05.001. Epub 2013 Jun 4.
Ghrelin, a novel motilin-related endogenous ligand for growth hormone secretagouge receptor, is implicated in various biological functions, including regulation of female reproduction. But the presence of ghrelin and its role in reproductive functions in buffalo, a species with poor reproductive efficiency, is not known. In the present study full-length ghrelin cDNA was isolated from bubaline abomasum, which encodes the entire prepropeptide of 116 amino acids. The deduced amino acid sequence of ghrelin of buffalo showed >95% and 31% identity with that of ruminants (cattle, sheep, and goat) and humans, respectively. Analysis of synonymous and nonsynonymous nucleotide substitutions in the coding region of ghrelin indicated that these sequences of different species have been under purifying selection. The 3995-bp amplicon of ghrelin gene consisting of 4 exons and 3 introns was cloned with genomic DNA from buffalo. Further, ghrelin expression was determined by quantitative real-time PCR, in situ hybridization, and immunohistochemistry in bubaline endometrial tissues at different stages of the estrous cycle and early pregnancy. Our results indicated the persistent expression of ghrelin mRNA and protein in the endometrium during stage I (day 3-5), stage II (day 6-15), and stage III (day 16-21) of the estrous cycle and also during early (~day 30-40) pregnancy. Immunohistochemistry and quantitative real-time PCR experiments indicated the relatively higher expression of ghrelin in the endometrium during stage II (day 6-15) of the estrous cycle and early pregnancy than during stage I (day 3-5) and stage III (day 16-21) of the estrous cycle, but no statistically significant difference in ghrelin expression was observed among stages. To conclude, the results of the present study indicate the persistent expression of ghrelin in the uterine endometrium throughout the estrous cycle and in early pregnancy which might be helpful in determining its role in buffalo reproduction.
胃饥饿素是一种新型的促生长激素释放肽受体同源内源性配体，与多种生物学功能有关，包括对雌性生殖的调节。但是，在繁殖效率低下的水牛中，胃饥饿素的存在及其在生殖功能中的作用尚不清楚。本研究从水牛皱胃中分离出全长胃饥饿素 cDNA，该 cDNA 编码 116 个氨基酸的完整前肽。水牛胃饥饿素的推导氨基酸序列与反刍动物（牛、绵羊和山羊）和人类的分别具有>95%和 31%的同一性。胃饥饿素编码区同义与非同义核苷酸取代的分析表明，这些不同物种的序列一直受到纯化选择的影响。从水牛基因组 DNA 克隆了包含 4 个外显子和 3 个内含子的 3995bp 胃饥饿素基因扩增子。进一步通过定量实时 PCR、原位杂交和免疫组织化学法检测了发情周期和早孕不同阶段水牛子宫内膜组织中胃饥饿素的表达。我们的结果表明，在发情周期的 I 期（第 3-5 天）、II 期（第 6-15 天）和 III 期（第 16-21 天）以及早孕（约第 30-40 天）期间，胃饥饿素 mRNA 和蛋白在子宫内膜中持续表达。免疫组织化学和定量实时 PCR 实验表明，在发情周期的 II 期（第 6-15 天）和早孕期间，胃饥饿素在子宫内膜中的表达相对较高，而在发情周期的 I 期（第 3-5 天）和 III 期（第 16-21 天）中表达较低，但在各期之间，胃饥饿素的表达没有统计学上的显著差异。总之，本研究结果表明，胃饥饿素在发情周期和早孕期间持续表达于子宫子宫内膜，这可能有助于确定其在水牛繁殖中的作用。
