miR-1246 is implicated as a possible candidate for endometrium remodelling facilitating implantation in buffalo (Bubalus bubalis).

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.

Codelivery of Gemcitabine and MUC1 Inhibitor Using PEG-PCL Nanoparticles for Breast Cancer Therapy.

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 IC50 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 inhibitor-loaded NPs was found to be more efficacious than Gem and MUC1 inhibitor delivered separately.

Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls.

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic bulls.

Absolute copy number differences of Y chromosomal genes between crossbred (Bos taurus × Bos indicus) and Indicine bulls.

BACKGROUND: The Y chromosome in mammal is paternally inherited and harbors genes related to male fertility and spermatogenesis. The unique intra-chromosomal recombination pattern of Y chromosome and morphological difference of this chromosome between Bos taurus and Bos indicus make it an ideal model for studying structural variation, especially in crossbred (Bos taurus × Bos indicus) bulls. Copy Number Variation (CNV) is a type of genomic structural variation that gives information complementary to SNP data. The purpose of this study was to find out copy number differences of four Y chromosomal spermatogenesis-related candidate genes in genomic DNA of crossbred and purebred Indicine bulls. RESULT: Four Y chromosomal candidate genes of spermatogenesis namely, sex determining gene on Y chromosome (SRY), DEAD box polypeptide 3-Y chromosome (DDX3Y), Ubiquitin specific peptidase 9, Y-linked (USP9Y), testis-specific protein on Y chromosome (TSPY) were evaluated. Absolute copy numbers of Y chromosomal genes were determined by standard curve-based quantitative real time PCR. Copy numbers of SRY and TSPY genes per unit amount of genomic DNA are higher in crossbred than Indicine bulls. However, no difference was observed in DDX3Y and USP9Y gene copy numbers between two groups. CONCLUSION: The present study demonstrates that the structural organization of Y chromosomes differs between crossbred and Indicine bulls which are reproductively healthy as observed from analysis of semen attributes. The absolute copy numbers of SRY and TSPY genes in unit mass of genomic DNA of crossbred bulls are significantly higher than Indicine bulls. No alteration in absolute copies of DDX3Y and USP9Y gene was found between the genome of crossbred and Indicine bulls. This study suggests that the DDX3Y and USP9Y are likely to be single copy genes in the genome of crossbred and Indicine bulls and variation in Y chromosome length between crossbred and Indicine bulls may be due to the copy number variation of SRY gene and TSPY array.

Involvement of transcription factor GATA-4 in regulation of CYP19 gene during folliculogenesis and luteinization in buffalo ovary.

CYP19 gene encode aromatase, the key enzyme of estrogen biosynthesis, is regulated in species- and tissue-specific manner by alternate use of different promoters. Previously, we have reported the cloning and characterization of tissue-specific promoter and transcripts in buffalo ovary and placenta. In human and rat ovary, FSH induces the phosphorylation of transcription factor CREB (cAMP response element binding protein) through PKA pathway which binds to cAMP response element like sequence (CLS) in CYP19 gene ovarian promoter. However, in buffalo as well as in bovine, in silico analysis of ovary specific promoter sequence identified a single base pair deletion in CLS site and is designated as CLS-like sequence. To understand if CLS with a point mutation is still a functional cis-element and is involved in FSH stimulated transactivation of CYP19 gene in buffalo ovary, the present study was thus aimed to functionally characterize the role of buffalo CLS in CYP19 gene transactivation. We also studied the involvement of GATA-4, having consensus binding sites in CYP19 gene ovarian promoter in the vicinity of CLS during different stages of the buffalo estrus cycle. Reporter construct analyses and EMSA results showed that CLS is playing no significant role in CYP19 gene regulation in buffalo ovary. Real time absolute quantification of GATA-4 showed the differential expression of GATA-4 mRNA during folliculogenesis and luteinization with significantly higher transcript abundance in large follicle in comparison to other tissues. Western blot analysis of granulosa cells nuclear protein isolated from different stage of follicular development (small and large follicles) and differentiation (corpus luteum) showed that abundance of phosphorylated GATA-4 (Ser261) was significantly higher in granulosa cell nuclear protein of large follicles as compared to small follicles and corpora lutea. Interestingly, binding studies using ChIP showed significantly enhanced binding to the CYP19 gene promoter in large follicle which was seen to be declined in the luteal tissue. Similar results were obtained in the in vitro experiments as well. Finally, RNAi experiments were performed to validate the involvement of GATA-4 in CYP19 gene regulation. Results of RNAi showed that knockdown of GATA-4 mRNA significantly declined CYP19 gene mRNA as well as 17ß-estradiol contents. In conclusion, result of the present study indicated that that in the absence of consensus CRE (cAMP response element); GATA-4 could be a downstream effector of cAMP/PKA pathway in regulation of CYP19 gene during folliculogenesis and luteinization.

Characterization of the luteinizing hormone beta (LH-beta) subunit gene in the Indian river buffalo (Bubalus bubalis).

The cDNA sequence of leuteinizing hormone (LH) beta subunit was determined to understand the molecular basis for silent oestrus behavior and poor response to superovulation in buffalo. The LH-beta cDNA contains an open reading frame 426 bp long. The deduced sequence of the LH-beta is 141 amino acids in length. The amino acid sequences of the Indian river buffalo LH-beta subunit showed overall similarity to those of other mammals. The nucleotide sequence variability of LH-beta was studied in more than approximately 350 Indian buffaloes covering five different breeds. The results of the sequence analysis showed that the buffalo LH-beta gene is not highly conserved and non-synonymous mutations are not rare, at least in the samples collected randomly from five different breeds and buffalo populations. A total of seven different variants were obtained. In spite of its crucial role in reproduction, variation of the LH-beta gene was found present in this species. The polymorphisms found were unique in the Indian river buffalo population.