Abstract
The seminal plasma is a very complex fluid, which surrounds sperm in semen. It contains numerous proteins including proteases and protease inhibitors that regulate proteolytic processes associated with protein activation and degradation. We previously identified a seminal protein, chicken liver trypsin inhibitor 1 (ClTI‐1) over expressed in semen of roosters with high fertility, suggesting a role in male fertility. In the present study, we showed that ClTI‐1 gene is actually SPINK2. Using normal healthy adult roosters, we showed that SPINK2 amount in seminal plasma was positively correlated with male fertility in chicken lines with highly contrasted genetic backgrounds (broiler and layer lines). Using affinity chromatography combined to mass spectrometry analysis and kinetic assays, we demonstrated for the first time that two chicken acrosin isoforms (acrosin and acrosin‐like proteins) are the physiological serine protease targets of SPINK2 inhibitor. SPINK2 transcript was overexpressed all along the male tract, and the protein was present in the lumen as expected for secreted proteins. Altogether, these data emphasize the role of seminal SPINK2 Kazal‐type inhibitor as an important actor of fertility in birds through its inhibitory action on acrosin isoforms proteins.
Highlights
Understanding the mechanisms involved in animal sperm quality is important to improve fertilization success and to face major challenges including improvement in farming, animal selection, conservation of genetic resources, and biodiversity
We characterized chicken liver trypsin inhibitor 1 (ClTI‐1), one of the seminal plasma proteins identified in our previous study as potential markers of chicken fertility (Labas et al, 2015) and investigated its postgonadic function for the first time
ClTI‐1/SPINK2 is a serine protease inhibitor that was previously identified in chicken liver and found in chicken seminal plasma and Previous in vitro studies showed that Kazal‐type protease inhibitors possess trypsin inhibition properties (Lee et al, 2011; Lin et al, 2008; Slowinska et al, 2014; Zalazar et al, 2012)
Summary
Understanding the mechanisms involved in animal sperm quality is important to improve fertilization success and to face major challenges including improvement in farming, animal selection, conservation of genetic resources, and biodiversity. Using an experimental line of chicken, we recently identified a seminal protein, chicken liver trypsin inhibitor 1 (ClTI‐1) that was present with variable abundance in semen of healthy roosters with contrasted fertility (Labas et al, 2015). This protein was previously purified from chicken liver and seminal plasma using a chromatography based on its trypsin inhibitor property (Kubiak, Jakimowicz, & Polanowski, 2009; Lessley & Brown, 1978) but its function as well as its target proteases remain unknown. We combined several complementary approaches to better decipher the post gonadic biochemical and physiological roles of the seminal ClTI‐1/SPINK2 protein in male fertility of physiologically normal, but with contrasted fertility, breeder males, and identified the physiological proteases that are inhibited by ClTI‐1/SPINK2
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