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In the present study, we describe the proteome of porcine cauda epididymis fluid and spermatozoa by means of Multidimensional Protein Identification Technology (MudPIT). Ten sexually mature healthy boars were surgically castrated and epididymides were dissected to obtain the cauda epididymal content. Polled protein extracts of cauda epididymal fluid (CEF) and spermatozoa (CESperm) were loaded in an Agilent 1100 quaternary HPLC and peptides eluted from the microcapillary column were electro-sprayed directly into a LTQ Orbitrap XL mass spectrometer. Using bioinformatics, identified proteins were classified by their molecular functions, involvement in biological processes and participation in relevant metabolic pathways associated with spermatozoa physiology, fertility potential and protection. A total of 645 proteins were identified in the CEF, with epididymal-specific lipocalin-5, beta-hexosaminidase subunit beta precursor and phosphatidylethanolamine-binding protein 4 being the most abundant proteins found. A total of 2886 proteins were identified in the CESperm proteome with 81 proteins being considered more abundant (spectral counts > 100). CEF and CESperm data were compared and 345 proteins were present in both proteomes. Phosphatidylethanolamine-binding protein 4 precursor was the only protein found most abundant in both CEF and CESperm proteomes. Based on Gene Ontology analysis, we identified CEF and CESperm proteins associated with sperm protection against ROS and immune mediated response, glycosaminoglycan degradation, ubiquitin-proteasome system, metabolic process and maturation, modulation of acrosome reaction and ZP binding and oocyte penetration. These results provide a better comprehension about the molecular process and biological pathways involved in sperm epididymis maturation and establishment of the cauda epididymis sperm reservoir.

Secondary hypogonadism is a consequence of congenital or acquired diseases that affect the hypothalamus and/or pituitary gland, impairing secretion of gonadotrophin-releasing hormone (GnRH). Androgen deficiency resulting from reduced GnRH secretion is likely to have disrupting effects in epididymal epithelial cells, impairing the sperm maturation process. The aim of this study was to describe changes in the proteome of epididymal spermatozoa in a porcine model of secondary hypogonadism. Cauda epididymal spermatozoa were obtained from 10 boars previously immunised against GnRH (Vivax; Pfizer) and from 10 healthy boars. Protein extracts were analysed by multidimensional protein identification technology. In all, 1322 unique proteins were identified in the protein extracts of cauda epididymal spermatozoa, with significant changes in the abundance of key proteins involved in sperm metabolism (enolase, pyruvate dehydrogenase), acrosome reaction and capacitation (oxoprolinase, acrosomal protein SP-10, dihydrolipoyl dehydrogenase) and sperm–oocyte interactions (zona pellucida-binding protein, zonadhesin, sperm adhesion molecule 1). In addition, the abundance of mitochondrial proteins was severely affected, with significant changes in proteins of Complex I and II, as well as ATPase of the oxidative phosphorylation chain. The proteins identified in this study are potential sperm biomarkers of testicular and epididymal dysfunction related to disruption of the hypothalamus–pituitary–testis axis. Secondary hypogonadism is an important endocrine disorder that may decrease libido, impair erectile function, induce muscle weakness, increase adiposity, depress mood, decrease vitality and cause infertility. This study investigated the effects of secondary hypogonadism on the protein profile of epididymal spermatozoa in a swine model. Secondary hypogonadism induced significant changes in the proteome profile of epididymal spermatozoa, altering the abundance of key proteins involved in sperm metabolism, protection and fertilisation. [ABSTRACT FROM AUTHOR]