Background Low conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins. Results The raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments. Conclusions The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

The present study was conceived with the aim of modulating the cumulus expansion characteristics of poor quality (BCB-) buffalo oocyte complexes (COCs) in order to improve their fertilization outcomes. BCB- COCs were subjected to in vitro maturation (IVM) in presence of Interleukin-1 beta (IL-1β) along with BCB- (control) and good quality (BCB+) COCs. Results were assessed morphologically, by scanning electron microscopy (SEM) and by expression analysis of cumulus expansion related genes. Also, numbers of zona pellucida bound spermatozoa were counted and development rates of oocytes were monitored under different groups. Expression of versican isoforms and ADAMTS-1 was observed to be significantly different between cumulus cells of BCB+ and BCB- COCs. Upon IL-1β supplementation, ADAMTS-1 expression increased in BCB- COCs along with corresponding cumulus expansion rates. SEM analysis also revealed improved cumulus expansion in IL-1β supplemented BCB- COCs. HAS2 and TNFAIP-6 were significantly up-regulated after IL-1β supplementation while PTGS2 expression remained unaffected. Significantly more numbers of sperms crossed the cumulus barrier, especially in 100 ng/mL IL-1β supplemented COCs. Besides, cleavage and blastocyst development rates were also improved upon IL-1β addition. We concluded that IL-1β supplementation in IVM medium can improve cumulus expansion and development ability of poor quality buffalo oocytes.

SummaryMaturing oocytes have diverse developmental potential and good quality oocytes exhibit a better ability to attain physiological milestones in a time-dependent manner. This situation necessitates the confirmation of oocyte developmental status more precisely under an in vitro embryo production (IVEP) regime. The aim of this study was to explain timely events in germinal vesicle breakdown (GVBD), an important milestone of oocyte nuclear maturation, to delineate the developmental capacity of Bubalus bubalis oocytes. In addition, the expression profile of genes responsible for GVBD was assessed in order to understand the molecular context responsible for GVBD. The chronology of GVBD events at different time intervals during in vitro maturation (IVM) suggests that the rate at which oocytes undergo GVBD was strikingly different in the brilliant cresyl blue (BCB)+ and BCB− groups. The expression of AKT and CDC25B genes for BCB+ oocytes was maximum at 8 h of IVM, and CCNB (cyclin B) peaked at around 10 h, which suggested that GVBD was finished after 10 h in BCB+ oocytes, whereas the expression of AKT and CDC25B was found to peak at around 12–14 h of IVM. This difference consequently delays the GVBD event by 2–4 h in BCB− oocytes. Poor abundance of gene transcripts was mainly implicated in delay and lower rate of GVBD in BCB− oocytes which in turn strongly affected the translational ability of oocytes to blastocysts. The findings of this study support the idea that there is a propensity in sub-optimal grade oocytes for delayed GVBD that compromises the developmental ability of low grade buffalo oocytes. The study highlights the very small, but importantly vital and separate, time window of the GVBD event during which the competence levels of buffalo oocytes are altered along with their translational ability to develop into the prospective embryos.

Precise early pregnancy diagnosis in dairy animals is of utmost importance for an efficient dairy production system. Not detecting a dairy animal pregnant sufficiently early after the breeding results to extending the unproductive time of their milk production cycle and causes substantial economic loss for a dairy producer. At present, the most conventional and authentic pregnancy confirmation practice in cows and buffaloes is rectal palpation of the reproductive organs at Days 35–40 after insemination, which sometime leads to considering an animal as false pregnant. Other alternative methods available for early pregnancy diagnosis lack either accuracy or reproducibility or require elaborate instrumentation and laboratory setup not feasible to practice at farmers’ doorstep. The present study was aimed at establishment of the microRNA (miRNA) repertoire of the placentome in buffaloes, which could capture the event of the cross talk between a growing embryo and a dam, through fetal cotyledons and maternal caruncles, and thus could hint at the early pregnancy establishment event in ruminants. Total RNA was isolated from buffalo placentome tissues during early stages of pregnancy (at Day < 25 and Days 30–35), and global small RNA analysis was performed by using Illumina single-end read chemistry and Bubalus bubalis genome. A total of 2,199 miRNAs comprising 1,620 conserved and 579 non-conserved miRNAs were identified. Stringent functional miRNA selection criteria could predict 20 miRNAs worth evaluating for their abundance in the plasma of pregnant, non-pregnant, cyclic non-bred, and non-cyclic prepubertal animals. Eight of them (viz., miR-195-5p, miR-708-3p, miR-379-5p, miR-XX1, miR-XX2, miR-130a-3p, miR-200a-3p, and miR-27) displayed typical abundance patterns in the plasma samples of the animals on Day 19 as well as Day 25 post-insemination, thus making them ambiguous candidates for early pregnancy detection. Similarly, higher abundance of miR-200a-3p and miR130a-3p in non-pregnant animals was indicative of their utility for detecting the animals as not pregnant. Most interestingly, miR-XX1 and miR-XX2 were very characteristically abundant only in pregnant animals. In silico target prediction analysis confirmed that these two miRNAs are important regulators of cyclooxygenase-2 (COX-2) and cell adhesion molecule-2 (CADM-2), both of which play a significant role in the implantation process during feto-maternal cross talk. We interpret that circulatory miR-XX1 and miR-XX2 in blood plasma could be the potential biomarkers for early pregnancy detection in buffaloes.

Germinal vesicle breakdown (GVBD) is the first milestone that an oocyte needs to achieve toward completing the maturation and gaining potential to fertilize. Significantly lower in vitro embryo production rate in buffaloes can be attributed to heterogeneity of GVBD occurrence among oocytes obtained from abattoir derived ovaries. Evidence from our earlier work had suggested that different qualities of buffalo oocytes differ significantly in their timing of GVBD. Besides, these oocytes also differ in terms of volume of Akt phosphorylation, which initiates the process of GVBD. With objective of synchronizing the oocytes for GVBD, immature buffalo oocytes were subjected to a two-step culture protocol, initially in the presence of GVBD inhibitors and subsequently, in vitro maturation (IVM) with added SC79 (activates Akt). Expression of developmentally important genes was assessed along with embryo development rate and blastocyst health to interpret the consequences. Oocytes subjected to a short GVBD inhibition period of 6 h followed by IVM with SC79 resulted in improved cleavage and blastocyst rates. Resultant blastocysts also possessed higher ICM: TE ratio. Further, GVBD inhibited oocytes displayed a sustained cytoplasmic maturation status in terms of reorganization of cortical granules (CGs), mitochondrial membrane potential, and glutathione levels during the period of inhibition. We conclude that a temporary GVBD arrest of buffalo oocytes and modulation of Akt improves the in vitro embryo development rate as well as quality of resultant embryos. Besides, our meiotic arrest protocol does not affect the cytoplasmic maturation.

In breast cancer therapy, Gemcitabine (Gem) is an antineoplastic antimetabolite with greater anticancer efficacy and tolerability. However, effectiveness of Gem is limited by its off-target effects. The synergistic potential of MUC1 (mucin 1) inhibitors and Gem-loaded polymeric nanoparticles (NPs) was discussed in this work in order to reduce dose-related toxicities and enhance the therapeutic efficacy. The double emulsion solvent evaporation method was used to prepare poly(ethylene glycol) methyl ether-block-poly-caprolactone (PEG-PCL)-loaded Gem and MUC 1 inhibitor NPs. The average size of Gem and MUC 1 inhibitor-loaded NPs was 128 nm, with a spherical shape. Twin-loaded NPs containing Gem and the MUC1 inhibitor decreased IC 50 and behaved synergistically. Furthermore, in vitro mechanistic studies, that is, loss of MMP, clonogenic assay, Annexin V FITC assay, and Western blotting to confirm apoptosis with simultaneous induction of autophagy using acridine orange (AO) staining were performed in this study. Furthermore, the investigated NPs upon combination exhibited greater loss of MMP and decreased clonogenic potential with simultaneous induction of autophagy in MCF-7 cells. Annexin V FITC clearly showed the percentage of apoptosis while Western blotting protein expression analysis revealed an increase in caspase-3 activity with simultaneous decrease in Bcl-2 protein expression, a hallmark of apoptosis. The effectiveness of the Ehrlich ascites solid (EAT) mice treated with Gem-MUC1 inhibitor NPs was higher than that of the animals treated alone. Overall, the combined administration of Gem and MUC1 inhibitorloaded NPs was found to be more efficacious than Gem and MUC1 inhibitor delivered separately.

Female germ cell and its intricate milieu regulate key processes of folliculogenesis and early embryonic development. However, the composition and dynamics of the oocyte transcriptome defines its future fertilizing ability which in turn depends on a number of oocyte specific genes whose identities are still unknown. In this context, the construction of buffalo oocyte specific subtracted cDNA library has raised fresh challenges of defining the importance of a battery of oocyte expressed transcripts in oocyte maturation. The present study tried to characterize these hitherto unknown transcripts and further to assess their expression dynamics in buffalo oocytes of different quality. For this purpose, three ESTs were selected from the library and subjected to 5' and 3' RACE for generating their full length sequences. These constructed full length sequences were validated by amplifying them in oocytes. Further these sequences were extensively analyzed for their coding potential and possible role using coding potential calculator and miRNA database. Besides, their expression was monitored during in vitro maturation in good (BCB+) and poor quality (BCB-) oocytes which was interestingly found to be differing significantly. All the three sequences under study were interpreted as long intergenic non-coding RNAs with the possibility of two of them acting as a miRNA precursors. Also, their differential expression trends in competitively diverse oocytes hints at their possible involvement in oocyte maturation and future embryonic development which needs to be explored further. J. Cell. Biochem. 118: 1712-1721, 2017. © 2016 Wiley Periodicals, Inc.

Background The microRNAs (miRs) secreted by the trophectoderm (TE) cells have recently been implicated in the conceptus‐endometrial cross talk during implantation and placentation. These miRs modulate various cellular processes during conception and throughout the pregnancy by regulating the gene expression in the foetal and maternal tissues. Objectives This study was undertaken to elucidate the function of TE secreted miRNAs in the maternal‐foetal cross‐talk during implantation/placentation in buffalo. Methods The in vitro produced blastocysts were cultured on a cumulus feeder layer for 21 days. The relative expression profiles of a selected panel of miRs was generated using the spent media collected on Days 0, 7, 12, 16, and 21. A custom‐designed mirVana™ miRNA mimic was used to transfect the endometrial epithelial cells (EECs) in order to determine the role of miRNA exhibiting highest expression on Days 21 and 21. Results The expression of miR‐1246 ( p < 0.001) and let‐7b ( p < 0.01) was found to be significantly higher on Day 21 of TE culture in comparison to the control (Day 0). This elevated expression indicated the involvement of these miRs in the maternal‐foetal cross‐talk. Interestingly, after the transfection of EECs with miRNA mimic for miR‐1246 (a novel molecule vis‐à‐vis implantation), the expression of beta‐catenin and mucin1 in these cells was found to be significantly ( p < 0.05) downregulated vis‐à‐vis the control, that is, the IFN‐τ primed EECs (before transfection). Conclusions The TE secreted miR‐1246 appeared to lower the expression of the endometrial receptivity genes (mucin1 and beta‐catenin) which apparently assists the endometrium in preparing for placentation.