Differential proteomic profile of X- and Y- sorted Sahiwal bull semen

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Highlights

  • Highly upregulated protein viz. Armadillo repeat containing 12 protein, NDC1 transmembrane nucleoporin, Beta-nerve growth factor, C-type natriuretic peptide, Nucleobindin 2, Phosphoglycerate mutase 2, Calmodulin along with one uncharacterised protein with accession number F1MN9 were identified.

  • These proteins may have the potential to be used as the biomarker for separating bovine X- and Y- sperm.

  • Ropporin-1, sperm acrosome membrane associated protein 3, seminal plasma protein PDC 109, calmodulin, glyceraldehyde 3-phosphate dehydrogenase testis specific proteins related to sperm motility and capacitation were also upregulated in Xsperm which may also have potential for X- and Y- sperm differentiation.

  • In future, it may further be analysed on large number by more sensitive and specific protein characterization methods to validate the present findings.

Abstract

The identification of differential proteins between X- and Y-sperm may be useful for immunological sexing of sperm. Hence, the present study was aimed to compare the protein profile of X- and Y-sorted Sahiwal bull semen using SDS-PAGE and Liquid Chromatography coupled with Mass Spectrometry (Nano LC-MS). Semen sample (n = 6) were categorized into three groups i.e., group I (X-sorted), group II (Y-sorted) and control group (both X- and Y- sperms). SDS PAGE revealed specific proteins of molecular weight between 18 and 24 kDa and between 30 and 37 kDa were present in X-sorted sperms. Also, band corresponding to 25 kDa was specific to Y-sorted sperms. Data obtained from Nano LC/MS is analysed by search engine database i.e., MASCOT and SEQUEST HT. Total, 241 proteins were identified, out of which 113 were differentially expressed between X- and Y-sorted sperms, in which 54 proteins showed at least two unique peptides. Out of 54 proteins, 27 were upregulated in X-sorted sample, 3 were upregulated in Y-sorted sample and 24 were differentially downregulated. Highly upregulated protein in X-sperm viz. Armadillo repeat containing 12 protein, NDC1 transmembrane nucleoporin, β-nerve growth factor, C-type natriuretic peptide, Nucleobindin-2, Phosphoglycerate mutase 2, Calmodulin along with one uncharacterised protein having accession number F1MN9 may have potential to be used as biomarker for separating X and Y sperm.

Introduction

Animal Husbandry plays a significant role in Indian economy and development of country. India stood first in world cattle population i.e. 192.49 million (https://www.nddb.coop/information/stats/pop). One of the possible ways to expand dairy industry and to increase female calves in the herd is control of foetal sex. For dairy industry development, farmers consider male calf vain and are often slaughtered. Thus, around half of the germplasm get wasted. Managemental cost is also enhanced due to rearing of undesired male calves. To avoid all these consequences, there is need to enhance female calf birth in a herd.

To increase number of female foetuses, sexed semen or embryo sexing technique can be used. Technique of embryo sexing is quite complicated, expansive, time consuming and reduces embryo viability (Sharma et al., 2017). Sperm sexing has substantial effect on cattle production (Sharma and Sharma, 2016). Various technologies used for sperm sexing till date are not up to mark because of lower quality and poor conception of sex sorted semen as compared to unsorted semen especially when sorted by Flow Cytometry (Beyhan et al., 1999; Fetrow et al., 2007). Basically, difference in DNA content, size, motility, charge and surface antigen were considered for developing various sperm sexing techniques (Garner and Seidel, 2008; Boro et al., 2016), but none of the method was efficient with 100% accuracy in sorting of semen. Flow cytometry is only viable technique that is used for semen sexing with maximum (∼90%) efficiency (Seidel Jr., 2012). It is completely based on difference in DNA content among X- and Y- chromosome i.e. In bovine X chromosome has 3.8% more DNA as compared to Y chromosome. Use of fluorescence activated cell sorting resulted in lower conception rates, as it damages the sperm cell membrane. Furthermore, frequency of sperm sorting per ejaculate was very low (Mallory et al., 2013; Seidel Jr., 2014). Furthermore, this cell sorting method may cause damage to sperm membrane leading to lower conception rate (Spinaci et al., 2006; Bermejo-Alvarez et al., 2008). Therefore, there is an urgent need of new semen sexing technique which would overcome the limitations of flow cytometry semen sexing and does not affect the viability and longevity of spermatozoa after sexing.

In spite of all, the use of FACS gives opportunity to identify variation in proteins among X- and Y sorted sperm population, which can be exploited further to introduce new immunological technique of sperm sexing.

Immunological method of sexing could be used to overcome the limitations of flowcytometry. Sharing of gene product among X and Y spermatozoa through intracellular bridge is not complete, so there is a possibility of sex specific protein expression (Hendriksen, 1999; Blecher et al., 1999). Direct comparison of protein levels in various cells can identify the markers responsible for differences between the cells (Park et al., 2013; Kwon et al., 2014). The presence of a specific protein in either the spermatozoa with an X-bearing chromosome or in the spermatozoa with a Y-bearing chromosome would be an essential requirement for the development of the immunological sexing of sperm (Galeraud-Denis et al., 2007; Hu and Namekawa, 2015).

Manifold attempts to search for differences between X- and Y-sperm have been reported using two-dimensional gel electrophoresis (2-DE). However, this technique was insignificant in providing any information about sex specific protein (Hendriksen et al., 1996). However, some indirect evidences suggested that variation may exist between X- and Y-sperm (Grant et al., 2008).

Sex sorted semen could be used in search of sex-specific antigens and these could further be used as preliminary molecular markers to differentiate X and Y sperm. Therefore, it is obvious to search an attractive alternative that can be used to detect the low level of specific X and Y protein of sperm. Various trials have been done to search the sex specific protein of exotic bull sperm but for indigenous bull specifically Sahiwal bull it is lacking. Unquestionably, the identification of protein difference between X- and Y- chromosome bearing spermatozoa will give an opportunity for developing an efficient and promising immune-sexing technique. For development of such technology detection, identification and characterization of differential protein between X- and Y- sperm is needed. Keeping in view the above facts, the present study was proposed with the objective of evaluating and comparing the total protein of X- and Y-sorted Sahiwal bull semen and to study the protein profile of X- and Y-sorted Sahiwal bull semen.

Section snippets

Reagents

Reagents used in this study were high purity research grade and were procured from Sigma-Aldrich chemicals, GeNei Pvt. Ltd. and Hi-media laboratory Pvt. Ltd.

Samples and animals

X- and Y-sorted as well as unsorted Sahiwal bull semen was used to conduct the study. The samples were categorized into three groups, on the basis of type of sorted semen, Group I- X-sorted semen, Group II- Y-sorted semen and Group III- unsorted or control group. Further, in each group number of samples were kept six i.e. n = 6. Semen

Total protein

The total protein concentration was 2.098 ± 0.033 mg/ml, 1.79 ± 0.042 mg/ml and 2.189 ± 0.035 mg/ml in group I (X-sorted semen), group II (Y-sorted semen) and group III (Unsorted semen) respectively. Significantly (P < 0.05) higher protein concentration was observed in unsorted semen compared to Y-sorted semen. Similarly, significantly (P < 0.05) higher in X-sorted semen compared to Y-sorted Sahiwal bull semen (Fig. 1).

One-dimensional SDS-PAGE analysis

SDS-PAGE analysis showed a specific weaker labelling of bands at 18–20 kDa

Discussion

In our study significantly (P < 0.05) higher protein concentration was observed in unsorted semen compared to Y-sorted semen. Similarly, significantly (P < 0.05) higher in X-sorted semen compared to Y-sorted Sahiwal bull semen. More number of proteins in unsorted semen as compared to X-sorted or Y-sorted semen is may be due to protein loss during sorting process.

Contrary to our findings, Hendriksen et al. (1996) and Howes et al. (1997) reported a non-significant difference in protein

Conclusion

The total protein concentration (mg/ml) reveals, significantly (P < 0.05) higher protein concentration was observed in unsorted semen compared to Y-sorted semen. Similarly, significantly (P < 0.05) higher in X-sorted semen compared to Y- sorted Sahiwal bull semen. Protein between 18–24 kDa and 30–37 kDa might be specific to X-sperm sorted Sahiwal bull semen. But further studies are required at large level to validate the results. Highly upregulated protein viz. Armadillo repeat containing 12

Acknowledgements

Authors are highly thankful to Vice Chancellor, GBPUAT, Director, DES and Dean, CVASc for their valuable official and financial help to conduct the research work.

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