Identification and Characterization of Cystatin-Related Epididymal Spermatogenic Protein in Human Spermatozoa: Localization in the Equatorial Segment1

Wassler, Michael; Syntin, Patrick; Sutton-Walsh, H.G.; Hsia, Nelson; Hardy, Daniel M.; Cornwall, Gail A.

doi:10.1095/biolreprod.102.003970

Cited by 30 publications

(25 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6E and H). Other sperm-associated proteins that exhibit this band-like pattern are cystatin-related epididymal spermatogenic and equatorial sperm proteins (Wassler et al 2002, Wolkowicz et al 2003. In the case of these two proteins the characteristic band-like pattern is located between the principal and the equatorial segments of the sperm head.…”

Section: Discussionmentioning

confidence: 99%

The PATE gene is expressed in the accessory tissues of the human male genital tract and encodes a secreted sperm-associated protein

Soler-García

Maitra²,

Kumar³

et al. 2005

Reproduction

View full text Add to dashboard Cite

ThePATEgene is expressed in prostate and testis. To determine if PATE is expressed in other accessory tissues of the male genital tract, RT-PCR of the epididymis and seminal vesicle was performed. PATE mRNA was highly expressed in the epididymis and seminal vesicle.In situhybridization of the testis showed PATE mRNA is strongly expressed in the spermatogonia. ThePATEgene encodes a 14-kDa protein with a predicted signal sequence and a cleavage site between residues G21 and S22. To determine if PATE is a secreted protein, 293T cells were transfected with a pcDNA-PATE-myc-His plasmid and protein immunoprecipitated with anti-myc monoclonal antibody. Western blot analysis showed the presence of PATE-myc-His protein was in the medium and the cell lysate. Confocal microscopy demonstrated that PATE-myc-His protein is found in the endoplasmic reticulum. The polyclonal antibody SOL-1 was generated by immunization of rabbits with recombinant PATE protein expressed and purified fromEscherichia coli.Western blots were performed on extracts of prostate, testis, seminal vesicle and ejaculated spermatozoa, but PATE protein was only detected in the spermatozoa. Immunostaining of sperm smears revealed that PATE is located in a band-like pattern in the sperm head. Our data indicate that PATE is made by various sexual accessory tissues and secreted into the semen where it becomes associated with sperm, suggesting that PATE is a novel sperm-associated protein with a possible role in mammalian sperm maturation.

show abstract

Section: Discussionmentioning

confidence: 99%

The PATE gene is expressed in the accessory tissues of the human male genital tract and encodes a secreted sperm-associated protein

Soler-García

Maitra²,

Kumar³

et al. 2005

Reproduction

View full text Add to dashboard Cite

show abstract

“…7A and 7B). CRES is present in round spermatids of humans where it also localizes to the equatorial segment (Wassler et al, 2002). The 1.6 kb of the CRES promoter contains DNA elements necessary for germ cell expression but not epididymal expression (Hsia et al, 2005).…”

Section: Microscopy Research and Techniquementioning

confidence: 99%

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: Developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane

Hermo¹,

Pelletier²,

Cyr³

et al. 2009

Microscopy Res & Technique

View full text Add to dashboard Cite

Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review.

show abstract

“…Similar to CRES, CRES2, CRES3 and cystatin E2 are expressed by the principal cells in the mouse initial segment epididymal epithelium Li et al, 2003Li et al, , 2005. The human homologs of all CRES subgroup genes have been identified and the expression of four genes established, including CRES and CRES2 in the human testis and/or epididymis suggesting conserved functions in human reproduction (Wassler et al, 2002;Frygelius et al, 2010). However, except for studies showing that CRES2 and CRES3 are associated with human and mouse spermatozoa in the epididymis, respectively, little is known about the remaining CRES subgroup family members (Hamil et al, 2002;Li et al, 2005).…”

Section: Introductionmentioning

confidence: 97%

Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen

et al. 2016

Self Cite

View full text Add to dashboard Cite

STUDY QUESTION: Do the CRES (cystatin-related epididymal spermatogenic) subgroup members, including CRES2, CRES3 and cystatin E2, contribute to the formation of a nonpathological, functional amyloid matrix in the mouse epididymal lumen?SUMMARY ANSWER: CRES2, CRES3 and cystatin E2 self-assemble with different aggregation properties into amyloids in vitro, are part of a common amyloid matrix in the mouse epididymal lumen and are present in extracellular vesicles.WHAT IS KNOWN ALREADY: Although previously thought only to be pathological, accumulating evidence has established that amyloids, which are highly ordered protein aggregates, can also carry out functional roles in the absence of pathology. We previously demonstrated that nonpathological amyloids are present in the epididymis; specifically, that the reproductive cystatin CRES forms amyloid and is present in the mouse epididymal lumen in a film-like amyloid matrix that is intimately associated with spermatozoa. Because the related proteins CRES2, CRES3 and cystatin E2 are also expressed in the epididymis, the present studies were carried out to determine if these proteins are also amyloidogenic in vitro and in vivo and thus may coordinately function with CRES as an amyloid structure. STUDY DESIGN, SAMPLES/MATERIALS, METHODS:The epididymides from CD1 and Cst8 (CRES)129SvEv/B6 gene knockout (KO) and wild-type mice and antibodies that specifically recognize each CRES subgroup member were used for immunohistochemical and biochemical analyzes of CRES subgroup proteins. Methods classically used to identify amyloid, including the conformation-dependent dyes thioflavin S (ThS) and thioflavin T (ThT), conformation-dependent antibodies, protein aggregation disease ligand (which binds any amyloid independent of sequence) and negative stain electron microscopy (EM) were carried out to examine the amyloidogenic properties of CRES subgroup members. Immunofluorescence analysis and confocal microscopy were used for colocalization studies. MAIN RESULTS AND THE ROLE OF CHANCE:Immunoblot and immunofluorescence analyzes showed that CRES2, CRES3 and cystatin E2 were primarily found in the initial segment and intermediate zone of the epididymis and were profoundly downregulated in epididymides from CRES KO mice, suggesting integrated functions. Except for CRES3, which was only detected in a particulate form, proteins were present in the epididymal lumen in both soluble and particulate forms including in a film-like matrix and in extracellular vesicles. The use of amyloid-specific reagents determined that all CRES subgroup members were present as amyloids and colocalized to a common amyloid matrix present in the epididymal lumen. Negative stain EM, dot blot analysis and ThT plate assays showed that recombinant CRES2, CRES3 and cystatin E2 formed amyloid in vitro, albeit with different aggregation properties. Together, our studies demonstrate that a unique amyloid matrix composed of the CRES family of reproductive-specific cystatins and cystatin C is a normal component of the mouse ...

show abstract

Identification and Characterization of Cystatin-Related Epididymal Spermatogenic Protein in Human Spermatozoa: Localization in the Equatorial Segment1

Cited by 30 publications

References 32 publications

The PATE gene is expressed in the accessory tissues of the human male genital tract and encodes a secreted sperm-associated protein

The PATE gene is expressed in the accessory tissues of the human male genital tract and encodes a secreted sperm-associated protein

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: Developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane

Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen

Contact Info

Product

Resources

About