Molecular characterization, putative structure and function, and expression profile of OAS1 gene in the endometrium of goats (Capra hircus).

An interferon-inducible gene, 2'-5'-oligoadenylate synthetase-1 (OAS1), plays an essential role in uterine receptivity and conceptus development by controlling cell growth and differentiation in addition to anti-viral activities. As OAS1 gene has not yet been studied in caprine (cp), so present study was designed with the aim to amplify, sequence, characterize and in-silico analyze the coding sequence of the cpOAS1. Further, expression profile of cpOAS1 was performed by quantitative real-time PCR and western blot in the endometrium of pregnant and cyclic does. An 890 bp fragment of the cpOAS1 was amplified and sequenced. Nucleotide and deduced amino acid sequences revealed 99.6-72.3% identities with that of ruminants and non-ruminants. A constructed phylogenetic tree revealed that Ovis aries and Capra hircus differ from large ungulates. Various post-translational modifications (PTMs), 21 phosphorylation, two sumoylation, eight cysteines and 14 immunogenic sites were found in the cpOAS1. The domain, OAS1_C, is found in the cpOAS1 which carries anti-viral enzymatic activity, cell growth, and differentiation. Among the interacted proteins with cpOAS1, Mx1 and ISG17 well-known proteins are found that have anti-viral activity and play an important role during early pregnancy in ruminants. CpOAS1 protein (42/46 kDa and/or 69/71 kDa) was detected in the endometrium of pregnant and cyclic does. Both cpOAS1 mRNA and protein were expressed maximally (P<0.05) in the endometrium during pregnancy as compared to cyclic does. In conclusion, the cpOAS1 sequence is almost similar in structure and probably in function also to other species along with its higher expression during early pregnancy.

Kiss1 and its receptor: molecular characterization and immunolocalization in the hypothalamus and corpus luteum of the buffalo.

ABSTARCT The neuropeptide kisspeptin (Kp) through its receptor Kiss1r regulates the HPG axis by controlling GnRH release. Since buffalo is a seasonal breeder with problems of delayed puberty and postpartum anestrus, we characterized the Kiss1 and Kiss1r and investigated the immunolocalization in the hypothalamus and corpus luteum (CL). Kiss1 and Kiss1r genes were amplified from gDNA covering the coding region, cloned and sequenced with accession numbers MF168937 and MG820539, respectively. The Kiss1 DNA sequence had two exonic segment contained coding sequence (cds); 408 bp encoding a predicted protein of 136 aa with conservation of Kp-10 and shared 94.5-98.3% identity with ruminants. Kiss1r DNA sequence consisted of five exons with a cds of 1134 bp encoding a protein of 378 aa. Phylogenetic analysis of Kiss1 and Kiss1r revealed that it formed a monophyletic clade with cattle, which branched from sheep and goat. Immunofluorescence study revealed the presence of Kiss1 and Kiss1r in the neuronal soma and perinuclear area of preoptic and arcuate regions of the hypothalamus and luteal cells of the CL. This is the first report on molecular characterization of bubaline Kiss1 and Kiss1r genes that confirmed the presence of conserved Kp-10 like other ruminants and kisspeptinergic system is present in the hypothalamus and CL.

Endometrial transcripts of proinflammatory cytokine and enzymes in prostaglandin synthesis are upregulated in the bitches with atrophic pyometra.

Inflammatory markers of endometrial origin are valuable in order to differentiate the pyometra from cystic endometrial hyperplasia in the bitch. In the present study, we hypothesized that histological categorization would distinguish the differential regulation of the proinflammatory genes in the endometrium of bitches with pyometra. Ovariohysterectomy was done on bitches with confirmatory diagnosis of pyometra (n = 18). Using endometrium to myometrium ratio of 0.79 as threshold, the uteri (n = 8/group) were categorized into hyperplastic pyometra (HP) and atrophic pyometra (AP). Two samples were excluded as the diagnosis was inconclusive. In parallel, endometrial tissue was collected for total RNA extraction to study the differential expression of TLR4, IL-6, IL-8, COX-2 and PGFS through real time PCR. Diestrus uterus of non-pyometra bitches (n = 6) served as control. The mean fold change (2-ΔΔCt) for the target genes was determined using ß-actin as endogenous control and non-pyometra uterus as calibrator group. Except TLR4, other inflammatory genes were upregulated significantly by 1.82 to 3.74 times in the AP as compared to HP with maximum upregulation of COX-2 and PGFS. Further, correlation matrix with Spearman's rho revealed that IL-8 had strong positive correlation with COX-2 and PGFS in the AP group (P < 0.05). It is concluded that histological grading of pyometra into HP and AP revealed differential regulation of inflammatory cytokines and enzymes in the PG synthetic pathway in the canine endometrium that has diagnostic potential under clinical settings.