S.K. Bhure

Expression of leptin and its receptor in corpus luteum during estrous cycle in buffalo (Bubalus bubalis)

Leptin is supposed to play a crucial role in ovarian luteal dynamics. The present study was aimed to investigate the importance of leptin and its receptors in buffalo corpus luteum (CL) obtained from different stages of the estrous cycle. Real-time RT-PCR (qPCR), western blot and immunohistochemistry techniques were applied to investigate mRNA expression, protein expression and localization of examined factors. Additionally to assess the contribution of leptin in progesterone production the expression profiles of StAR, P450scc and HSD were also investigated. In general, we demonstrated presence of leptin and its receptors in buffalo CL during the estrous cycle. The mRNA levels of leptin and its receptors were significantly up regulated in (P<0.05) in all the stages and highest levels were observed in mid and late luteal stages consistent with in vivo luteinization of buffalo CL and declined coincidental to luteal regression. The expression of StAR, P450scc and HSD factors maintained low in early luteal phase, after that level of expression increased steadily to show a significant rise (P<0.05) in mid luteal phase followed by gradual decline in late luteal phase and regressed CL and this correlates well with the Ob and ObR receptor activity, verifying their key role in progesterone and other steroids production in functional CL. As revealed by immunohistochemistry, leptin protein was localized predominantly in large luteal cells however leptin receptor (Ob-R) was localized in large luteal cells as well as in endothelial cells. It can be concluded from our study that leptin via its autocrine/paracrine effects play a significant role in promoting angiogenesis, steroidogenesis and also acts as key survival factor in bubaline CL.

Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo Spermatozoa

Advancements in reproductive technologies have shown seminal plasma (SP) as a nutritive-protective medium for spermatozoa metabolism, function and transport. At the same time quality variables and thus freezability of spermatozoa are influenced by SP proteins originating from male reproductive tract. One such protein, viz. PDC-109 is reported to influence freezability of spermatozoa in cattle. Thus the present investigation was designed to evaluate effect of seminal PDC-109 protein concentration on post-thaw cholesterol content and semen quality variables (SQP) as an indicator of membrane integrity and freezability, respectively of buffalo spermatozoa. Ejaculates (n=42) selected on the basis of mass activity and individual motility were divided into three parts, first part for SP proteins isolation, second for cholesterol estimation and third part was cryo-preserved to evaluate freezability based on post-thaw SQP, viz. individual progressive motility, viability and acrosome integrity of spermatozoa. A total of 28 (66.7%) and 14 (33.3%) ejaculates from four bulls were found as freezable or non-freezable, respectively. Though total seminal plasma protein (TSPP) concentration was found similar in freezable and non-freezable ejaculates, the heparin binding proteins (HBP) content in non-freezable semen was greater (P<0.01) than freezable ejaculates. There was a similar trend for the PDC-109 protein content in respective ejaculates. Cholesterol content of spermatozoa and SQP were greater (P<0.05 and 0.01, respectively) in freezable as compared to non-freezable ejaculates of each bull at post-thaw stage. This study showed that concentrations of HBP and PDC-109 in non-freezable semen might be responsible for greater cryo-damage reflecting in poor freezability of buffalo spermatozoa.

Localization of IGF proteins in various stages of ovarian follicular development and modulatory role of IGF-I on granulosa cell steroid production in water buffalo (Bubalus bubalis)

The present study aimed to determine the expression of insulin like growth factor (IGF) genes in the bubaline ovarian follicles and modulatory role of IGF-I on progesterone production from granulosa cells (GC) of pre-ovulatory follicle in vitro. According to size, follicles were classified into four groups: GI (small), GII (medium), GIII (large) and GIV (preovulatory). All IGF genes were expressed in both GC and theca interna (TI) cells. The relative expression of IGF-I and IGF receptor I (IGFR-I) genes increased with follicle size and was greatest in the pre-ovulatory follicle (P<0.05). Expression of IGF-II and IGFR-II genes was minimal in GC but was readily detected in TI cells. In TI cells, the gene expression was greater in medium and large as compared to small and pre-ovulatory follicles. The expression of all binding protein (IGFBP) genes was detected in both GC and TI cells. Expression of IGFBP-3 gene increased with follicle size and was greatest in pre-ovulatory follicles (P<0.05). The expression of IGFBP-2 and IGFBP-4 was less in pre-ovulatory follicles but expression of IGFBP-5 and IGFBP-6 genes were greater at this stage. The GC culture was conducted for three time durations and with three doses of IGF-I. Expression of steroidogenic genes (StAR, CYP11A1, HSD3B) and progesterone concentration were increased in a dose and time dependent fashion. The present study, therefore, provided evidence of an autocrine/paracrine role of IGFs in follicular development and a stimulatory role of IGF1 in steroid production in GC of preovulatory follicles in the bubaline species.

Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei.

Blackleg, an economically important and highly fatal disease of ruminants, is caused by anaerobic bacillus, Clostridium chauvoei. Identification and differentiation of the causative agent is crucial for implementation of therapeutic and control measures in real time. Most of the diagnostic tests available for blackleg are PCR based, and only a couple of serological tests have been reported. In this study, we targeted flagellin, an important immunogenic protein of C. chauvoei, to develop a sandwich ELISA for detection of C. chauvoei. Sequence analysis of flagellin gene of related Clostridium species showed that central region of flagellin gene is unique to C. chauvoei. Hence, we cloned and expressed central region of flagellin in a prokaryotic expression system. Antiserum against recombinant flagellin was generated in rabbits and chickens. A sandwich ELISA was developed, in which rabbit anti-flagellin antibodies were used as capture antibodies and chicken anti-flagellin antibodies as detecting antibodies. The test was specific and sensitive in detection of up to 10(4) CFU/ml of C. chauvoei. This study shows that assay developed can be used for detection of C. chauvoei in suspected samples.

Regucalcin is widely distributed in the male reproductive tract, and exerts a suppressive effect on in vitro sperm capacitation in the water buffalo (Bubalus bubalis)

Regucalcin is a multi‐functional, calcium‐binding protein with roles in calcium homeostasis, apoptosis, cell proliferation, and free radical neutralization. Regucalcin is broadly expressed in the male reproductive organs of rat and bovine; here, we report its expression in the reproductive tract of male buffalo—especially in testis, epididymis, seminal vesicle, prostate, and bulbourethral gland of buffalo—as analyzed by real‐time PCR, Western blot, and immunolocalization. Regucalcin degradation in seminal plasma, despite its high abundance in vesicular fluid, was demonstrated using recombinant regucalcin co‐incubated with buffalo seminal plasma. This depletion of regucalcin appears to be related to its suppressive effect on in vitro sperm capacitation, observed using the chlortetracycline assay after treating buffalo spermatozoa with recombinant protein. Indeed, addition of recombinant regucalcin to capacitating media significantly reduced (P < 0.05) the percentage of capacitated spermatozoa to 6.1 ± 0.6 from 36.4 ± 1.8 in the untreated group. Taken together, the wide distribution of regucalcin in male buffaloes, versus its degradation in the seminal plasma and suppressive effects on in vitro capacitation of spermatozoa, indicate its possible anti‐capacitation role in the reproductive tract. Mol. Reprod. Dev. 84: 212–221, 2017.

Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis)

The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.

Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis)

Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein‐30 (SMP‐30), is a calcium‐binding protein with multiple roles that include calcium homeostasis, anti‐oxidative, anti‐apoptosis, and anti‐proliferation. In Drosophila, RGN is reportedly a putative cold‐tolerance gene and a cytoprotective role for RGN against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN has anticapacitatory effect and abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 μg/ml), as a supplement for Tris‐egg yolk‐based semen extender. Post‐thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 μg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post‐thaw progressive motility of spermatozoa (50.6 ± 3.5% vs 40.6 ± 2.6%; p < 0.01), acrosome integrity (53.3 ± 7.4 vs 75.6 ± 6.8; p < 0.05), and zona pellucida binding (31.6 ± 14.0 vs 191.9 ± 12.3 bound spermatozoa; p < 0.01) compared to control conditions without RGN. Thus, ∼1 μM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender.

Transcriptional and translational abundance of Bone morphogenetic protein (BMP) 2, 4, 6, 7 and their receptors BMPR1A, 1B and BMPR2 in buffalo ovarian follicle and the role of BMP4 and BMP7 on estrogen production and survival of cultured granulosa cells

BMPs and their receptors modulate the granulosa cell (GC) function in the follicle of domestic animals. Since little is known on BMPs in the buffalo, the present study was aimed to investigate the expression of BMP2, 4, 6, 7 and their receptors BMPR1A, BMPR1B, BMPR2 in the GC and theca cells (TC) of ovarian follicles and the role of BMP4 and BMP7 on buffalo GC. Follicles were classified into four groups based on size and E2 level in the follicular fluid as follows: (i) Group1(4-6 mm; <0.5 ng/mL) (ii) Group 2 (7-9 mm; 0.5-5 ng/mL) (iii) Group 3 (10-13 mm; 5-40 ng/mL) and (iv) Group 4 (dominant follicle) (>13 mm; >180 ng/mL). The results revealed that except BMP6, BMP2, 4 7 and receptors BMPR1A, BMPR1B and BMPR2 showed a minimum of 1.5-2 fold increase in mRNA expression in the GC of dominant follicle as compared to other follicle classes. In the dominant follicle, a two-fold increase in BMP4 and BMP7 expression was observed in the TC. At 100 ng/mL, the BMP4 and BMP7 either alone or in combination maximally down-regulated CASPASE3 and stimulated the transcripts of PCNA, FSHR and CYP19A1 that was supported by E2 secretion in the granulosa cell culture suggesting their role in cell survival and E2 production. In conclusion, GC and TC of dominant follicles express BMP 2, 4, 6, 7 and their receptors BMPR1A, BMPR1B and BMPR2. BMP4 and BMP7 stimulate E2 production and promote GC survival.

Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium-regulating multifunctional protein

Regucalcin (RGN) is a calcium-regulating, anti-apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real-time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34-kDa, 28-kDa and 24-kDa isoforms, wherein the 34-kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium-related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.

Altered Expression Pattern of Major Seminal Plasma Protein Genes in Seminal Vesicles of Water Buffalo (Bubalus bubalis)

The binder of sperm proteins (BSP) are secretary products of seminal vesicles. Secretions from the seminal vesicles contribute more to seminal plasma which determine the quality of the semen. The concentration of BSPs in seminal plasma has been negatively correlated with cryopreservation. An analysis of expression profile of seminal plasma proteins could be critical for better freezability of semen. Present study was carried out to assess the expression levels of major seminal plasma protein genes viz., BSP1, BSP3 and BSP5 from seminal vesicles of water buffalo using Real Time PCR. The study consisted of identification of a suitable reference gene in seminal vesicles followed by the expression analysis of major bovine seminal plasma protein (BSP) genes. The most stable reference gene among the three (glyceraldehyde 3-phosphate dehydrogenase, ribosomal protein S15a (RPS15a) and beta-actin), was found to be RPS15a and therefore, was used for normalization of target gene expression. Data analysis of major BSPs showed highest relative expression of BSP3 followed by BSP1 and BSP5. The ratio of relative expression of BSP1:BSP3:BSP5 was 10:18:1. The results from the study also showed an altered expression pattern of major BSP genes in buffalo seminal vesicles. Thus, it can be concluded that the altered expression of these genes at mRNA level would also be reflected at protein level in the seminal plasma. This could be one of the probable reasons for poor freezability of buffalo semen as compared to cattle and the finding helps in designing better cryopreservation strategies for buffalo semen.