Differential abundance of microRNAs in seminal plasma extracellular vesicles (EVs) in Sahiwal cattle bull related to male fertility.

Sahiwal cattle, known for their high milk yield, are propagated through artificial insemination (AI) using male germplasm, largely contingent on semen quality. Spermatozoa, produced in the testes, carry genetic information and molecular signals essential for successful fertilization. Seminal plasma, in addition to sperm, contains nano-sized lipid-bound extracellular vesicles (SP-EVs) that carry key biomolecules, including fertility-related miRNAs, which are essential for bull fertility. The current study focused on miRNA profiling of SP-EVs from high-fertile (HF) and low-fertile (LF) Sahiwal bulls. SP-EVs were isolated using size exclusion chromatography (SEC) and characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Western blotting detected the EV-specific protein markers TSG101 and CD63. The DLS analysis showed SP-EV sizes of 170-180 nm in HF and 130-140 nm in LF samples. The NTA revealed particle concentrations of 5.76 × 1010 to 5.86 × 1011 particles/mL in HF and 5.31 × 1010 to 2.70 × 1011 particles/mL in LF groups, with no significant differences in size and concentration between HF and LF. High-throughput miRNA sequencing identified 310 miRNAs in SP-EVs from both groups, with 61 upregulated and 119 downregulated in HF bull. Further analysis identified 41 miRNAs with significant fold changes and p-values, including bta-miR-1246, bta-miR-195, bta-miR-339b, and bta-miR-199b, which were analyzed for target gene prediction. Gene Ontology (GO) and KEGG pathway analyses indicated that these miRNAs target genes involved in transcription regulation, ubiquitin-dependent endoplasmic reticulum-associated degradation (ERAD) pathways, and signalling pathways. Functional exploration revealed that these genes play roles in spermatogenesis, motility, acrosome reactions, and inflammatory responses. qPCR analysis showed that bta-miR-195 had 80% higher expression in HF spermatozoa compared to LF, suggesting its association with fertility status (p < 0.05). In conclusion, this study elucidates the miRNA cargoes in SP-EVs as indicators of Sahiwal bull fertility, highlighting bta-miR-195 as a potential fertility factor among the various miRNAs identified.

MAPKs signaling is obligatory for male reproductive function in a development-specific manner.

Mitogen-activated protein kinases (MAPKs) represent widely expressed and evolutionarily conserved proteins crucial for governing signaling pathways and playing essential roles in mammalian male reproductive processes. These proteins facilitate the transmission of signals through phosphorylation cascades, regulating diverse intracellular functions encompassing germ cell development in testis, physiological maturation of spermatozoa within the epididymis, and motility regulation at ejaculation in the female reproductive tract. The conservation of these mechanisms appears prevalent across species, including humans, mice, and, to a limited extent, livestock species such as bovines. In Sertoli cells (SCs), MAPK signaling not only regulates the proliferation of immature SCs but also determines the appropriate number of SCs in the testes at puberty, thereby maintaining male fertility by ensuring the capacity for sperm cell production. In germ cells, MAPKs play a crucial role in dynamically regulating testicular cell-cell junctions, supporting germ cell proliferation and differentiation. Throughout spermatogenesis, MAPK signaling ensures the appropriate Sertoli-to-germ cell ratio by regulating apoptosis, controlling the metabolism of developing germ cells, and facilitating the maturation of spermatozoa within the cauda epididymis. During ejaculation in the female reproductive tract, MAPKs regulate two pivotal events-capacitation and the acrosome reaction essential for maintaining the fertility potential of sperm cells. Any disruptions in MAPK pathway signaling possibly may disturb the testicular microenvironment homeostasis, sperm physiology in the male body before ejaculation and in the female reproductive tract during fertilization, ultimately compromising male fertility. Despite decades of research, the physiological function of MAPK pathways in male reproductive health remains inadequately understood. The current review attempts to combine recent findings to elucidate the impact of MAPK signaling on male fertility and proposes future directions to enhance our understanding of male reproductive functions.

The Female Reproductive Tract Microbiota: Friends and Foe.

We do not seem to be the only owner of our body; it houses a large population of microorganisms. Through countless years of coevolution, microbes and hosts have developed complex relationships. In the past few years, the impact of microbial communities on their host has received significant attention. Advanced molecular sequencing techniques have revealed a remarkable diversity of the organ-specific microbiota populations, including in the reproductive tract. Currently, the goal of researchers has shifted to generate and perceive the molecular data of those hidden travelers of our body and harness them for the betterment of human health. Recently, microbial communities of the lower and upper reproductive tract and their correlation with the implication in reproductive health and disease have been extensively studied. Many intrinsic and extrinsic factors influences the female reproductive tract microbiota (FRTM) that directly affects the reproductive health. It is now believed that FRTM dominated by Lactobacilli may play an essential role in obstetric health beyond the woman's intimate comfort and well-being. Women with altered microbiota may face numerous health-related issues. Altered microbiota can be manipulated and restored to their original shape to re-establish normal reproductive health. The aim of the present review is to summarize the FRTM functional aspects that influence reproductive health.

Buffalo sperm membrane glycan-binding proteins reveal precise and preferential binding signatures with specific glycans targets on oviduct epithelium and zona pellucida-an implication in fertilization.

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3'-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 µg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3'-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(ß1-4)GlcNAc further supports the abundance of 3'-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3'-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.

Biodegradable PEG-PCL Nanoparticles for Co-delivery of MUC1 Inhibitor and Doxorubicin for the Confinement of Triple-Negative Breast Cancer.

Combating triple-negative breast cancer (TNBC) is still a problem, despite the development of numerous drug delivery approaches. Mucin1 (MUC1), a glycoprotein linked to chemo-resistance and progressive malignancy, is unregulated in TNBC. GO-201, a MUC1 peptide inhibitor that impairs MUC1 activity, promotes necrotic cell death by binding to the MUC1-C unit. The current study deals with the synthesis and development of a novel nano-formulation (DM-PEG-PCL NPs) comprising of polyethylene glycol-polycaprolactone (PEG-PCL) polymer loaded with MUC1 inhibitor and an effective anticancer drug, doxorubicin (DOX). The DOX and MUC1 loaded nanoparticles were fully characterized, and their different physicochemical properties, viz. size, shape, surface charge, entrapment efficiencies, release behavior, etc., were determined. With IC50 values of 5.8 and 2.4 nm on breast cancer cell lines, accordingly, and a combination index (CI) of < 1.0, DM-PEG-PCL NPs displayed enhanced toxicity towards breast cancer cells (MCF-7 and MDA-MB-231) than DOX-PEG-PCL and MUC1i-PEG-PCL nanoparticles. Fluorescence microscopy analysis revealed DOX localization in the nucleus and MUC1 inhibitor in the mitochondria. Further, DM-PEG-PCL NPs treated breast cancer cells showed increased mitochondrial damage with enhancement in caspase-3 expression and reduction in Bcl-2 expression.In vivo evaluation using Ehrlich Ascites Carcinoma bearing mice explicitly stated that DM-PEG-PCL NPs therapy minimized tumor growth relative to control treatment. Further, acute toxicity studies did not reveal any adverse effects on organs and their functions, as no mortalities were observed. The current research reports for the first time the synergistic approach of combination entrapment of a clinical chemotherapeutic (DOX) and an anticancer peptide (MUC1 inhibitor) encased in a diblock PEG-PCL copolymer. Incorporating both DOX and MUC1 inhibitors in PEG-PCL NPs in the designed nanoformulation has provided chances and insights for treating triple-negative breast tumors. Our controlled delivery technology is biodegradable, non-toxic, and anti-multidrug-resistant. In addition, this tailored smart nanoformulation has been particularly effective in the therapy of triple-negative breast cancer. Supplementary Information: The online version contains supplementary material available at 10.1007/s10924-022-02654-4.

Beta-defensins as marker for male fertility: a comprehensive review†.

Bovine male fertility in animals has a direct impact on the productivity of dairy herds. The epididymal sperm maturations involve extensive sperm surface modifications to gain the fertilizing ability, especially by absorptions of the plethora of biomolecules, including glycoprotein beta-defensins (BDs), enzymes, organic ions, protein, and phospholipids. Defensins are broad-range nonspecific antimicrobial peptides that exhibit strong relations with innate and adaptive immunity, but their roles in male fertility are relatively recently identified. In the course of evolution, BD genes give rise to different clusters with specific functions, especially reproductive functions, by undergoing duplications and nonsynonymous mutations. BD polymorphisms have been reported with milk compositions, disease resistance, and antimicrobial activities. However, in recent decades, the link of BD polymorphisms with fertility has emerged as an appealing improvement of reproductive performance such as sperm motility, membrane integrity, cervical mucus penetration, evading of uterus immunosurveillance, oviduct cell attachment, and egg recognition. The reproductive-specific glycosylated BD class-A BDs (CA-BDs) have shown age- and sex-specific expressions in male reproductive organs, signifying their physiological pleiotropism, especially in the sperm maturation and sperm transport in the female reproductive tract. By considering adult male reproductive organ-specific BD expressions, importance in sperm functionalities, and bioinformatic analysis, we have selected two bovine BBD126 and BBD129 genes as novel potential biomarkers of bovine male fertility. Despite the importance of BDs, however, genomic characterization of most BD genes across most livestock and nonmodel organisms remains predictive/incomplete. The current review discusses our understanding of BD pleiotropic functions, polymorphism, and genomic structural attributes concerning the fertilizability of the male gamete in dairy animals.

Analysis of amplification and association polymorphisms in the bovine beta-defensin 129 (BBD129) gene revealed its function in bull fertility.

ß-defensins are adsorbable on the sperm surface in the male reproductive tract (MRT) and enhance sperm functional characteristics. The beta-defensin 129 (DEFB129) antimicrobial peptide is involved in sperm maturation, motility, and fertilization. However, its role in bovine fertility has not been well investigated. This study examines the relationship between the bovine BBD129 gene and Bos indicus x Bos taurus bull fertility. The complete coding sequence of BBD129 mRNA was identified by RNA Ligase Mediated-Rapid Amplification of cDNA End (RLM-RACE) and Sanger sequencing methodologies. It consisted of 582 nucleotides (nts) including 5' untranslated region (UTR) (46nts) and 3'UTR (23nts). It conserves all beta-defensin-like features. The expression level of BBD129 was checked by RT-qPCR and maximal expression was detected in the corpus-epididymis region compared to other parts of MRT. Polymorphism in BBD129 was also confirmed by Sanger sequencing of 254 clones from 5 high fertile (HF) and 6 low fertile (LF) bulls at two positions, 169 T > G and 329A > G, which change the S57A and N110S in the protein sequence respectively. These two mutations give rise to four types of BBD129 haplotypes. The non-mutated TA-BBD129 (169 T/329A) haplotype was substantially more prevalent among high-fertile bulls (P < 0.005), while the double-site mutated GG-BBD129 (169 T > G/329A > G) haplotype was significantly more prevalent among low-fertile bulls (P < 0.005). The in silico analysis confirmed that the polymorphism in BBD129 results in changes in mRNA secondary structure, protein conformations, protein stability, extracellular-surface availability, post-translational modifications (O-glycosylation and phosphorylation), and affects antibacterial and immunomodulatory capabilities. In conclusion, the mRNA expression of BBD129 in the MRT indicates its region-specific dynamics in sperm maturation. BBD129 polymorphisms were identified as the deciding elements accountable for the changed proteins with impaired functionality, contributing to cross-bred bulls' poor fertility.

Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction.

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.

Promising role of defensins peptides as therapeutics to combat against viral infection.

Antimicrobial peptides (AMPs) are ubiquitously present small peptides, which play a critical function in the innate immune system. The defensin class of AMPs represented an evolutionarily ancient family containing cationic cysteine residue and frequently expressed in epithelial or neutrophils cells. It plays myriad functions in host innate immune responses against various infection. Defensin has a broad spectrum of antimicrobial activities, including anti-bacteria, anti-viruses (AVPs), anti-fungi, anti-cancers, and also overcoming bacterial drug resistance. In this review, we compiled the progress on defensin, particularly incorporating the mechanism of action, their application as an antiviral agent, prospects in different areas, and limitations to be solved as an antiviral peptide. Defensins were explored, in particular, their capacity to stimulate innate and adaptive immunity by trigging as anti-coronavirus (COVID-19) peptides. The present review summarised its immunomodulatory and immunoenhancing properties and predominantly focused on its promising therapeutic adjuvant choices for combat against viral infection.