Our understanding of the post-transcriptional mechanisms involved in follicular atresia is limited; however, an important development has been made in understanding the biological regulatory networks responsible for mediating follicular atresia. MicroRNAs have come to be seen as a key regulatory actor in determining cell fate in a wide range of tissues in normal and pathological processes. Profiling studies of miRNAs during follicular atresia and development have identified several putative miRNAs enriched in apoptosis signaling pathways. Subsequent in vitro and/or in vivo studies of granulosa cells have elucidated the functional role of some miRNAs along with their molecular pathways. In particular, the regulatory roles of some miRNAs have been consistently observed during studies of follicular cellular apoptosis. Continued work should gradually lead to better understanding of the role of miRNAs in this field. Ultimately, we expect this understanding will have substantial benefits for fertility management at both the in vivo or/and in vitro levels. The stable nature of miRNA holds remarkable promise in clinical use as a diagnostic tool and in reproductive medicine to solve the ever-increasing fertility problem. In this review, we summarize current knowledge of the involvement of miRNAs in follicular atresia, discuss the challenges for further work and pinpoint areas for future research.

Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility.

Mastitis is one of the major diseases causing economic loss to the dairy industry by reducing the quantity and quality of milk. Thus, the objective of this scientific study was to find new biomarkers based on genes for the early prediction before its severity. In the present study, advanced bioinformatics including hierarchical clustering, enrichment analysis, active site prediction, epigenetic analysis, functional domain identification, and protein docking were used to analyze the important genes that could be utilized as biomarkers and therapeutic targets for mastitis. Four differentially expressed genes (DEGs) were identified in different regions of the mammary gland (teat cistern, gland cistern, lobuloalveolar, and Furstenberg’s rosette) that resulted in 453, 597, 577, and 636 DEG, respectively. Also, 101 overlapped genes were found by comparing 27 different expressed genes. These genes were associated with eight immune response pathways including NOD-like receptor signaling pathway (IL8, IL18, IL1B, PYDC1) and chemokine signaling pathway (PTK2, IL8, NCF1, CCR1, HCK). Meanwhile, 241 protein-protein interaction networks were developed among overlapped genes. Fifty-seven regulatory events were found between miRNAs, expressed genes, and the transcription factors (TFs) through micro-RNA and transcription factors (miRNA-DEG-TF) regulatory network. The 3D structure docking model of the expressed genes proteins identified their active sites and the binding ligands that could help in choosing the appropriate feed or treatment for affected animals. The novelty of the distinguished DEG and their pathways in this study is that they can precisely improve the detection biomarkers and treatments techniques of cows’ Escherichia coli mastitis disease due to their high affinity with the target site of the mammary gland before appearing the symptoms.

Serine/threonine kinase 3 (AKT3) is a protein-coding gene that is associated with several cattle immune diseases including different tumors and cancers. The objective of this study was to investigate the differences in structures and functions of AKT3 of cow and buffalo cattle. The sequence differences of gene-coding sequence (CDS) and core promoter region of AKT3 in cow and buffalo were analyzed by using bioinformatics tools and PCR sequencing. Also, the functional analysis of promoter regulating gene expression by RT-PCR was performed using 500 Holstein cows and buffalos. And, evaluation of AKT3 inflammatory response to the lipopolysaccharide (LPS)-induced mastitis was performed between both species. The results revealed the variation in 6 exons out of 13 exons of the two species of CDS. Also, 4 different regions in 3-kb promoters of the AKT3 gene were significantly different between cow and buffalo species, in which cow’s AKT3 promoter sequence region was started from − 371 to − 1247, while in buffalo, the sequence was started from − 371 to − 969 of the promoter crucial region. Thus, the promoter was overexpressed in cows compared to buffaloes. As a result, significant differences (P < 0.05) between the two species in the AKT3 gene expression level related to the LPS stimulation in their mammary epithelial cell line. This study emphasized the great importance of the structural differences of AKT3 between the animal species on their different responses against immune diseases like mastitis.

Inhibin is a molecule that belongs to peptide hormones and is excreted through pituitary gonadotropins stimulation action on the granulosa cells of the ovaries. However, the differential regulation of inhibin and follicle-stimulating hormone (FSH) on granulosa cell tumor growth in mice inhibin-deficient females is not yet well understood. The objective of this study was to evaluate the role of inhibin and FSH on the granulosa cells of ovarian follicles at the premature antral stage. This study stimulated immature wild-type (WT) and Inhibin-α knockout (Inha−/−) female mice with human chorionic gonadotropin (hCG) and examined hCG-induced gene expression changes in granulosa cells. Also, screening of differentially expressed genes (DEGs) was performed in the two groups under study. In addition, related modules to external traits and key gene drivers were determined through Weighted Gene Co-Expression Network Analysis (WGCNA) algorithm. The results identified a number of 1074 and 931 DEGs and 343 overlapping DEGs (ODEGs) were shared in the two groups. Some 341 ODEGs had high relevance and consistent expression direction, with a significant correlation coefficient (r² = 0.9145). Additionally, the gene co-expression network of selected 153 genes showed 122 nodes enriched to 21 GO biological processes (BP) and reproduction and 3 genes related to genomic pathways. By using principal component analysis (PCA), the 14 genes in the regulatory network were fixed and the cumulative proportion of fitted top three principal components was 94.64%. In conclusion, this study revealed the novelty of using ODEGs for investigating the inhibin and FSH hormone pathways that might open the way toward gene therapy for granulosa cell tumors. Also, these genes could be used as biomarkers for tracking the changes in inhibin and FSH hormone from the changes in the nutrition pattern.

Brucellosis is a contagious and zoonotic disease, affecting human and animals equivocally. It is a seriously neglected disease in underdeveloped countries, causing heavy financial losses in the livestock sector in terms of abortion, and a decrease in milk production. To better cope with socioeconomic impact of brucellosis, this study was conducted in district Swabi with the aim to detect brucellosis in high-risk human population through serological Rose Bengal plate test, Serum plate agglutination test, Enzyme-Linked Immunosorbent Assay, and molecular Polymerase chain rection techniques with respect to different risk factors and clinical history. A total of 250 blood samples (n=250 each from Human) were collected through predesigned questionnaire and were processed for detection of brucellosis through different techniques in Veterinary Research Institute, Peshawar. Detection of brucellosis frequency was performed through SPAT, RBPT, Indirect ELISA (IgM, IgG) and PCR as 12.4, 10.4, 11.6, 12 and 9.6% respectively. A numerical difference was observed through detection of different tools, but the difference was statistically non-significant (p>0.05). The present research contributes to the existing prevalence data concerning brucella infection in humans and emphasizes the benefits and effectiveness of the molecular technique of PCR compared to serological tests.