Elevated testicular temperature modulates expression patterns of sperm proteins in Holstein bulls

Abstract

The objective of this study was to determine the effects of elevated testicular temperature on the expression patterns of sperm proteins in bulls. Ejaculates were collected from sexually mature Holstein bulls (n = 6) twice weekly for 10 weeks. Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during Week 2. Triton X-100 extracts of sperm proteins were prepared and subjected to SDS-PAGE. Sperm proteins in the 110 kDa range decreased from pre-thermal insult to Day 28 (start of thermal insult = Day 0), concurrent with decreases in sperm concentration, motility, and morphology, and subsequently increased, approaching pre-thermal insult values by Day 40. Based on mass spectrometry, this band was comprised of angiotensin converting enzyme (ACE), Hexokinase-1, and the alpha-4 subunit of Na(+)/K(+)ATPase. Changes in the expression patterns of these proteins were confirmed by immunoblotting, including the use of a custom antibody against the alpha-4 subunit of Na(+)/K(+)ATPase (testis-specific isoform). Furthermore, a 25 kDa sperm protein (identified as tissue inhibitor of metalloproteinase-2; TIMP-2), had a low expression level in pre-thermal insult samples, increased to Day 28 of post-thermal insult, and subsequently decreased to the pre-thermal insult level by the end of the experimental period. In conclusion, we identified proteins that may serve as molecular markers of impaired sperm function due to elevated testicular temperature, with important implications for fertility predictions. To our knowledge, this is the first report of the identification of a testis-specific isoform of Na(+)/K(+)ATPase in bovine sperm.

Na/K‐ATPase regulates bovine sperm capacitation through raft‐ and non‐raft‐mediated signaling mechanisms

“…The supernatant obtained by high‐speed centrifugation (16,000 × g , 15 min, 4°C) was pre‐cleared with protein‐A beads for 30 min at 4°C on a rocker. Pre‐cleared supernatants from raft and non‐raft fractions of control and capacitated sperm were incubated for 12 hr at 4°C on a rocker with protein‐A beads conjugated with ATP1A4 antibody (3 μg/ml) (Newton, Kastelic, Wong, van der Hoorn, & Thundathil, ) and protein‐G beads conjugated to ATP1A1 antibody (1 μg/ml). After incubation, beads containing the antibody‐antigen and its interacting partners were pelleted by slow‐speed centrifugation (500 × g , 1 min, 4°C).…”

The pumps that fuel a sperm's journey

“…Later, Northern blot analysis with total RNA from different human tissues detected transcripts of the α4 isoform in testis exclusively [4]. The testes-specific sodium pump isoform has now been detected both at mRNA and protein levels in several mammalian species, and, at a cellular level, its expression seems to be confined to gametes [5][6][7]. In both humans and mice, the gene encoding α4 is found on chromosome 1 in close proximity to the gene encoding α2 [8,9], indicating that α4 could originate from a gene duplication of α2, in line with the sequence identity data showing that α4 has slightly higher identity with α2 than with the others (Figure 1).…”

The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

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