The seminal acrosin‐inhibitor ClTI1/SPINK2 is a fertility‐associated marker in the chicken

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.

Seminal Plasma Proteome as an Indicator of Sperm Dysfunction and Low Sperm Motility in Chickens

“…Its more-abundance in the seminal plasma of LSM roosters make us speculate that failure of SPINK2 to bind to the membrane and acrosome of the damaged and disfunction spermatozoa. In a previous study, SPINK2 was reported to be more-abundant in the seminal plasma of roosters with high fertility (6,29). This inconstancy might be due to the different causal factors between studies.…”

“…Seminal SPINK2, also named chicken liver trypsin inhibitor 1 (ClTI-1) (29), originates from testis, epididymis, and genital tract. It functions as an Chicken Sperm Motility Related Seminal Plasma Proteins 27 acrosin/trypsin inhibitor protecting the spermatozoa against damage by binding to the sperm membrane (29). Its more-abundance in the seminal plasma of LSM roosters make us speculate that failure of SPINK2 to bind to the membrane and acrosome of the damaged and disfunction spermatozoa.…”

“…It is involved in the digestion of the innermost glycoprotein layer of the oocyte during the gamete fusion of both mammals and birds (56,57). ACR is typically stored in the acrosome of mature spermatozoa, and its abundance in the LSM seminal Chicken Sperm Motility Related Seminal Plasma Proteins 29 plasma probably caused by the degradation of the acrosome cap of sperm in the ejaculate (6). Previous studies have demonstrated that altered abundance of seminal plasma proteins, including ACR, Pyruvate kinase, and Superoxide Dismutase 1 (SOD1), may be related to acrosome dysfunction in humans (17,58), as suggested by our findings.…”

“…Cytochrome c (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and interleukin 6 signal transducer (IL6ST), were performed following the method of Thélie et al, where total protein served as a loading control(29). The seminal plasma proteins were separated on 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Merck-Millipore, Darmstadt, Germany) at 200 mA for 90 min.…”

Cross comparison of seminal plasma proteins from cattle and buffalo (Bubalus bubalis)

“…Acrosin inhibitor is produced by accessory sexual glands and secreted in the seminal plasma of ruminants (Cechová & Fritz, 1; Cechová, Jonáková, Sedláková, & Mach, ; Codognoto et al, ; Davidová, Jonáková, & Manásková‐Postlerová, ; Martins et al, ; Torska & Strzezek, ; Tschesche, Kupfer, Klauser, Fink, & Fritz, ), and it functions to prevent early sperm proteolysis induced by acrosin, an acrosomal enzyme responsible for zona pellucida hydrolysis during acrosomal reaction and fertilization (Adham, Nayernia, & Enzel, ; Crosby, Jones, Barros, & Carvalho, ; Gurupryaia et al, ; Martins et al, ). Acrosin inhibitor and acrosin are proteins related to in vivo and in vitro fertility in humans (Goodpasture, Zavos, Cohen, & Zaneveld, ; Kennedy, Reid, Kaminski, Jeyendran, & Zaneveld, ; Mohsenian, Syner, & Moghissi, ) and chickens (Thélie et al, ). In fact, the upregulation of acrosin inhibitor in cattle can contribute to cell protection during cryopreservation, since cold shock is able to induce acrosomal morphological alterations (Quinn & White, 1), and enzyme loss can damage the spermatozoa and fertility in boar (Johnson & Pursel, ) and bovine (Church & Graves, ).…”