Elsevier

Journal of Proteomics

Volume 75, Issue 18, 22 October 2012, Pages 5861-5871
Journal of Proteomics

Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC–MSE) reveals altered proteomic signatures in asthenozoospermia

https://doi.org/10.1016/j.jprot.2012.07.003Get rights and content

Abstract

Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC–MSE, the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.

Highlights

► Sixty-six phosphoproteins differentially expressed in severe asthenozoospermia ► Deregulated phosphoproteins reveal pathways important for sperm motility. ► Deregulated proteins correlate with ultra-structure defects in these spermatozoa. ► Signature molecules could be used for diagnosis of asthenozoospermia and improve treatment efficacy.

Introduction

Sperm motility is one of the most easily observable phenomena but not very well understood. Poor sperm motility on routine semen analysis is only a clinical indicator of male infertility. However, the underlying cause for this manifestation is elusive and therefore treatment non-specific. In order to improve the treatment efficacy, it is important to identify the precise proteins and the pathways involved in sperm movement. Our knowledge on the proteins important for sperm motility has been mainly through studies on single gene defects in humans, knock out mouse models [1], [2], [3], [4], [5], [6], [7], [8], and differential proteomics [9], [10], [11], [12], [13]. Although these approaches have yielded valuable information, the missing links between the molecules still remain to be deciphered. Post-translational modifications especially phosphorylation play an important role in sperm motility. Tash and Means were the first to propose the role of phosphorylation in initiation of motility [14], [15]. They demonstrated cAMP/PKA signaling and calcium signaling as the key pathways central to normal sperm motility and that cAMP dependent phosphorylation of flagella proteins is responsible for initiation and maintenance of activated flagellar motility. Subsequently a few proteins such as valosin-containing protein and two members of the A kinase-anchoring protein (AKAP) family [9], fibrous sheath protein 95 [1] and calcium binding tyrosine phosphorylation regulated protein [3] have been identified to be phosphorylated during capacitation.

Although human sperm proteome has been studied, and literature is available on proteins phosphorylated during capacitation (hyperactivated motility), very few phosphoproteins related exclusively to activated motility have been identified. This is probably due to the fact that only 1–2% of the proteome pool is phosphorylated at any given point of time [16] and that owing to their low stoichiometry and low abundance, phosphoproteins are poorly represented on 2D gels. Hence, in the present study, immobilized metal affinity chromatography (IMAC) was employed to selectively enrich phosphoproteins and the expression of phosphoproteins in asthenozoosperm vis-a-vis that in normal spermatozoa was discerned by Nano UPLC–MSE. The phosphoproteins differentially expressed in asthenozoosperm were then analyzed by Ingenuity Pathways Analysis to decipher the key pathways involved in sperm motility and disruption of which could affect activated sperm movement. We also attempted to correlate the differential expression of the protein to the sperm deficiencies observed at ultra-structural level and validated the expression of a few deregulated proteins by Western blot analysis.

Section snippets

Sample collection

Sixteen semen samples from eight individuals (four with normal spermatozoa having sperm concentration > 20 million cells/ml, progressive motility > 50% and normal morphology, and four with severe asthenozoospermia having sperm concentration > 20 million cells/ml, progressive motility < 10% and normal morphology) were collected by masturbation following 3–4 days of abstinence. For the Western blot and immunoprecipitation experiments, asthenozoospermic samples were selected as per WHO definition (progressive

Results and discussion

Asthenozoospermia is one of the clinical manifestations of male infertility and could arise due to several reasons very well reviewed by Turner [21]. Two pathways known to be central for activated sperm movement are cAMP/protein kinase A pathway and calcium signaling. But are these the only pathways operative in the spermatozoa and can the pathogenesis of poor sperm motility be explained solely on the basis of our current understanding of sperm motility? Information is available; however it is

Conclusion

Treatment for idiopathic asthenozoospermia is at present non-specific and based on trial and error (patients are prescribed anti oxidants/cell detoxifiers/cellular energizers. If these are not effective, then the patient is advised ICSI/ART). This being so, much of the precious time is lost (age of the patient advances) and this adds to the financial burden and increases the psychological pressure on the individual. The present study has identified the phosphoproteins and the pathways relevant

Authors' roles

P.P.P. was instrumental in the study conceptualization and design, interpretation and analysis of the data and preparation of the manuscript. P.R. has done all the proteomic work with assistance from P.P.P. and was involved in manuscript writing. S.R. and V.L. have done the validation experiments. S.J.D. has done the electron microscopic studies. R.G. and V.K. screened and recruited normozoospermic and asthenozoospermic individuals for the study.

Funding

This project was carried out with the financial support from the Indian Council of Medical Research and Department of Biotechnology, India and their support is gratefully acknowledged. The Department of Biotechnology, India, and the Lady Tata Trust provided the JRF and SRF, respectively to Parimala Rao.

Acknowledgments

The authors express their sincere gratitude to Dr Ian Edwards and Dr Joanne Connolly, Waters Corporation, Manchester, UK, with the protein identifications using Nano UPLC–MSE, and Dr Jyoti Paniyadi (Ingenuity® Systems) for her assistance with the pathway analysis. Also acknowledged is the support of Dr Vrinda Khole and the team of the Proteomics Core facility at the Institute (Dr Amol Suryawanshi, Dr Kaushiki Kadam, Dr Shalmali Dharma, and Mayura Nakade) during the pilot proteomic experiments

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