The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins

Abstract

Background and Objectives: Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. Materials and Methods: The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, AEBSF. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native PAGE, co-immunoprecipitation, and mass spectrometry, with interrogation of protein-protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin-containing complex in sperm function. Results: Testisin surface expression increased significantly in capacitated spermatozoa (p < 0.001), increased further following acrosome reaction (p < 0.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes; co-immunoprecipitation revealed interactions of testisin with a multitude of zona pellucidabinding proteins, including ZPBP, ZAN, acrosin, several heat-shock proteins, and components of the TCP1 complex.Conclusion: Testisin appears to form part of the zona pellucida-binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.

Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa

“…ARSA -a protein vital for zona pellucida adhesion and binding (Carmona et al 2002, Redgrove et al 2012a,b, Bromfield et al 2015a) -has been identified resolving at 68 kDa in capacitated boar sperm (Jiménez et al 2006), and shares 90% sequence identity with equine ARSA. Testisin was recently characterized in stallion spermatozoa, and holds a functional relationship with HSPA2 (Swegen et al 2019). Localized to both the sperm tail and the inner acrosomal membrane, testisin has been implicated as an integral member of the zona pellucida-binding complex (Netzel-Arnett et al 2009, Swegen et al 2019.…”

“…Testisin was recently characterized in stallion spermatozoa, and holds a functional relationship with HSPA2 (Swegen et al 2019). Localized to both the sperm tail and the inner acrosomal membrane, testisin has been implicated as an integral member of the zona pellucida-binding complex (Netzel-Arnett et al 2009, Swegen et al 2019. The significant degradation of bands resolving at 64-70 kDa in the present study confirms previous findings that, even in low concentrations, cytotoxic aldehydes can have a significant effect on overall mammalian cell function (Bromfield et al 2015a,b, Hall et al 2017.…”

Genome‐wide association study for age at puberty in young Nelore bulls

“…The PRSS21 (serine protease 21) gene, as well as TEX14 , also participates in the male gamete generation (GO:0048232) biological process, which encodes a cell‐surface anchored serine protease known as testisin, which is abundantly expressed by premeiotic testicular germ cells and sperm (Scarman et al, ). Deficient PRSS21 ‐mouse displays sperm with several defects that occur during epididymal transit, such as heterogeneity in sperm form and angulated flagella, reduced numbers of motile sperm, and abnormal sperm volume, suggesting that testisin is a proteolytic factor that is responsible for epididymal sperm cell maturation, spermatozoa‐fertilizing ability (Netzel‐Arnett et al, ) and contributes to the zona pellucida‐binding complex in stallion spermatozoa, which could be involved in the proteolytic cascade that arranges the sperm surface for interaction with the oocyte (Swegen et al, ).…”

Research Progress on the Microregulatory Mechanisms of Fertilization: A Review

“…The AR is an important indicator for evaluating sperm function during fertilization. In addition to intracytoplasmic sperm injection (ICSI), in vivo fertilization and in vitro fertilization (IVF) require the sperm AR to support contact, fusion, and fertilization between the sperm and egg (2). Completion of the AR is marked by complete fusion of the outer acrosomal membrane with the sperm cell membrane.…”