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Identification of protein candidates in spermatozoa of water buffalo (Bubalus bubalis) bulls helps in predicting their fertility status

ABSTRACT: Abstract The water buffalo (Bubalus bubalis) is an indispensable part of the Indian dairy sector and in several instances, the farmers incur economic losses due to failed pregnancy after artificial insemination (AI). One of the key factors for the failure of conception is the use of semen from the bulls of low fertilizing potential and hence, it becomes important to predict the fertility status before performing AI. In this study, the global proteomic profile of high fertile (HF) and low fertile (LF) buffalo bull spermatozoa was established using a high-throughput LC-MS/MS technique. A total of 1385 proteins (≥ 1 high-quality PSM/s, ≥ 1 unique peptides, P < 0.05, FDR < 0.01) were identified out of which, 1002 were common between both the HF and LF groups while 288 and 95 proteins were unique to HF and LF groups respectively. We observed 211 and 342 significantly upregulated (log Fc ≥2) and downregulated in HF (log Fc ≤0.5) spermatozoa (p <0.05). Gene ontology analysis revealed that the fertility associated upregulated proteins were involved in spermatogenesis, sperm motility, acrosome integrity, zona pellucida binding and other associated sperm functions. Besides this, the downregulated proteins were involved in glycolysis, fatty acid degradation and inflammation. Furthermore, fertility related differentially abundant proteins (DAPs) on sperm viz., AKAP3, Sp17 and DLD were validated through Western blotting and immunocytochemistry which was in coherence with the LC-MS/MS data. The DAPs identified in this study may be used as potential protein candidates for predicting fertility in buffaloes. Our findings provide an opportunity in mitigating the economic losses that farmers incur due to male infertility.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Seema Karanwal

LAB HEAD: Dr Rakesh Kumar

PROVIDER: PXD039680 | Pride | 2023-05-10

REPOSITORIES: Pride

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Identification of protein candidates in spermatozoa of water buffalo (<i>Bubalus bubalis</i>) bulls helps in predicting their fertility status.

Karanwal Seema S Pal Ankit A Chera Jatinder Singh JS Batra Vipul V Kumaresan Arumugam A Datta Tirtha K TK Kumar Rakesh R

Frontiers in cell and developmental biology 20230220

The water buffalo (<i>Bubalus bubalis</i>) is an indispensable part of the Indian dairy sector and in several instances, the farmers incur economic losses due to failed pregnancy after artificial insemination (AI). One of the key factors for the failure of conception is the use of semen from the bulls of low fertilizing potential and hence, it becomes important to predict the fertility status before performing AI. In this study, the global proteomic profile of high fertile (HF) and low fertile ( ...[more]

PMID: 36891514

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Project description:Abstract Buffalo bulls are the backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of buffalo dairy farms. Seminal plasma (SP) of bulls harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility associated proteins which contribute to the fertilizing potential of spermatozoa. In this study, we explored various parameters of EVs such as size, concentration and the complete proteome profiles of SP EVs from two distinct fertility groups in order to unravel the differentially abundant proteins that potentially affect the overall fertility of bulls. Through Dynamic Light Scattering (DLS) it was found that the purified EVs were present in 7 to 14 size exclusion chromatographic (SEC) fractions with sizes ranging from 145 to 256 nm in the high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively across all the fractions. No significant difference was observed in the size and concentration of seminal EVs between the HF and LF groups. We identified a total of 1862 and 1807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1754 proteins were common in both the groups and about 87 proteins were highly abundant in HF group while 1292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that the highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. In addition, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins of HF EVs, namely protein disulfide-isomerase A4 (PDIA4) and gelsolin (GSN), were successfully immunolocalized on spermatozoa, indicating that these proteins might be transferred to spermatozoa through EVs. The proteins; PDIA4 and GSN are intricately associated with sperm-oocyte fusion and acrosome reaction, respectively, thus they are vital for regulating the fertilizing capacity of sperm. Our evidences clearly support that the protein repertoires in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, the current investigation clearly indicates that SPEVs possess crucial protein repertoires which are essential for enhancing the sperm fertilizing capacity.

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