Expression of Kisspeptin and its receptor in the hypothalamus of cyclic and acyclic buffalo (Bubalus bubalis).

Kisspeptin (Kiss1), neurokinin-B (NKB) and dynorphin (Dyn) neurons regulate the surge and pulsatile centres of gonadotropin releasing hormone (GnRH) in the hypothalamus and are modulated by the ovarian steroids. Accordingly, we studied the temporospatial expression of Kiss1, its receptor and other genes that regulate GnRH in the preoptic area (POA) and arcuate (ARC) regions of hypothalamus at different phases of bubaline estrous cycle. Brain of buffalo (n = 32) was collected immediately after exsanguination and categorized into early luteal (EL), mid luteal (ML), follicular (FL) stages and acyclic (n = 8/group). Total RNA was extracted from the POA and ARC of each stage and real time PCR amplification of Kiss1, Kiss1r, NKB, NKB receptor (NKBR), Dyn, Dyn receptor (OPRK1), GnRH1, ERα, PR, LEPR and GHSR was done using GAPDH as endogenous control and acyclic stage as calibrator group. Further, immunolocalization of Kiss1 and Kiss1r was done on the hypothalamus. In the POA, significant up-regulation of Kiss1 and NKB with a concomitant down-regulation of Dyn transcripts was recorded at FL stage. There was, however, down-regulation of Kiss1 and Kiss1r during the EL perhaps due to the loss of estradiol as a consequence of ovulation. On the other hand, in the ARC, there was a significant up-regulation of Kiss1 and Dyn at FL and ML, while NKB transcript was consistently down-regulated at any stage of estrous cycle. In the POA, expression of ERα was not modulated; however, PR was down-regulated in the EL. In the ARC, the ERα expression was significantly up-regulated in the EL, whereas, PR was moderately expressed irrespective of the stage of estrous cycle. The immunolocalization study revealed the presence of Kiss1 and Kiss1r in the POA and ARC in the cyclic buffalo with relative abundance at FL. The transcriptional profile of the genes suggests that there is estrous cycle stage specific expression of Kiss1, Kiss1r and other GnRH regulating genes in the POA and ARC regions of hypothalamus in the buffalo. Up-regulation of Kiss1r in the POA during ML and ARC during EL indicates the involvement of kisspeptinergic system in the regulation of low LH pulse frequencies during the early and mid luteal phases in the cyclic buffalo.

Quantification and comparison of TLR2 activity in monocyte-derived macrophages of zebu and crossbred cattle.

The present study was conducted to quantify and compare TLR2 (toll-like receptor 2) activity in monocyte-derived macrophages of zebu (Tharparkar) and crossbred (Holstein-Friesian × Jersey × Brown Swiss × Hariana) cattle. The cells were either induced with Pam3CSK4 or kept as control. The TLR2 activity was quantified in terms of IκB-α inhibitory subunit (NFKBIA) messenger RNA (mRNA) copies using real-time, one-step reverse transcription-polymerase chain reaction (RT-PCR). Toll-like receptor 2 activity of induced cells was in the range of 1060421 ± 477937 (n=3) to 3514715 ± 290222 (n=3) copies for Tharparkar cattle (n=7) and in the range of 1365532 ± 47243 (n=3) to 3016510 ± 172340 (n=3) copies for the crossbred cattle (n=7). For uninduced cells, this activity was within the range of 117 ± 51 (n=3) to 293 ± 103 (n=3) copies for the Thraparkar cattle (n=7), and in the range of 182 ± 122 (n=3) to 296 ± 88 (n=3) copies for the crossbred cattle (n=7). The TLR2 activity of induced cells in both groups was found to be significantly higher than that of the respective uninduced cells (P<0.0001). Furthermore, upon comparison, TLR2 activities of induced and uninduced cells of the Tharparkar were not found to be significantly different from those of the crossbred cattle (P=0.8154 and P=0.6670). In the present study, we have quantified and compared, for the first time, TLR2 activity in terms of NFKBIA mRNA copies in monocyte-derived macrophages of Tharparkar and crossbred cattle and found that both have equivalent TLR2 activity.

Expression and localization of ghrelin and its functional receptor in corpus luteum during different stages of estrous cycle and the modulatory role of ghrelin on progesterone production in cultured luteal cells in buffalo.

Evidence obtained during recent years provided has insight into the regulation of corpus luteum (CL) development, function, and regression by locally produced ghrelin. The present study was carried out to evaluate the expression and localization of ghrelin and its receptor (GHS-R1a) in bubaline CL during different stages of the estrous cycle and investigate the role of ghrelin on progesterone (P4) production along with messenger RNA (mRNA) expression of P4 synthesis intermediates. The mRNA and protein expression of ghrelin and GHS-R1a was significantly greater in mid- and late luteal phases. Both factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of ghrelin and GHS-R1a was greater during mid- and late luteal phases. Luteal cells were cultured in vitro and treated with ghrelin each at 1, 10, and 100 ng/mL concentrations for 48 h after obtaining 75% to 80% confluence. At a dose of 1 ng/mL, there was no significant difference in P4 secretion between control and treatment group. At 10 and 100 ng/mL, there was a decrease (P < 0.05) in P4 concentration, cytochrome P45011A1 (CYP11A1), and 3-beta-hydroxysteroid dehydrogenase mRNA expression and localization. There was no difference in mRNA expression of steroidogenic acute regulatory protein between control and treatment group. In summary, the present study provided evidence that ghrelin and its receptor are expressed in bubaline CL and are localized exclusively in the cell cytoplasm and ghrelin has an inhibitory effect on P4 production in buffalo.

Sindbis virus replicon-based DNA vaccine encoding Rabies virus glycoprotein elicits specific humoral and cellular immune response in dogs.

A Sindbis virus (SINV) replicon-based DNA vaccine encoding Rabies virus (RABV) glycoprotein G developed previously (Saxena et al., Vaccine 26, 6592, 2008) was used for immunization of dogs against rabies. The intradermal injection of DNA vaccine into external ear generated protective level of virus neutralizing antibodies. The cellular immune response was specific to RABV, in particular by an increase in CD3+CD4+ and CD3+CD8+ lymphocytes. This study has demonstrated that the SINV replicon-based DNA vaccine encoding RABV G is capable of inducing the protective level of specific immune response in dogs.