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The presence and role of the c-kit proto-oncogene protein was investigated in the mature sperm of the human. A polyclonal antibody against the c-kit peptide was used to perform immunohistochemical (IHC) staining, electron microscopy (EM) studies, and Western blot analysis. The acrosomal region of fresh sperm specifically stained with the antibody. No acrosomal staining or staining limited to the equatorial region was noted in the acrosome-reacted (AR) sperm. EM studies demonstrated immunogold label on the plasma membrane (PM) of the acrosome, and confirmed the lack of binding following the acrosome reaction. A 150 kDa band was detected by Western blot analysis. This protein was released from the sperm surface during sperm capacitation and the acrosome reaction. Antibody against the c-kit receptor significantly inhibited the acrosome reaction and increased sperm agglutination, but did not significantly inhibit sperm motility. These results suggest that the c-kit receptor protein is present in mature human sperm and is released during capacitation and/or the acrosome reaction. The assessment of the c-kit receptor may also be a useful assay for sperm function in male infertility., ((c) 2004 Wiley-Liss, Inc.)

Purpose: The c-kit receptor is a proto-oncogene important in germ cell migration and maturation and has also been demonstrated on the acrosomal region of mature sperm. The purpose of the present study was to examine the ultrastructural location of the c-kit receptor in mouse testis and sperm., Materials and Methods: Testis and sperm from mature male mice were examined for the c-kit receptor utilizing electron microscopy and Western blot analysis techniques. Thin sections of mouse testis and sperm were stained with immunogold-labeled anti-c-kit antibodies. The protein from these testes and sperm was also utilized for Western blot analysis., Results: The c-kit protein was localized within the mouse testes to the type A spermatogonia, the round spermatids, and the mature testicular spermatozoa. The c-kit receptor was noted to migrate from the lumen of the acrosomal vesicles in the early spermatids to the plasma membrane of the late spermatids. It was also noted in the acrosomal region of the testicular spermatozoa, as well as the sperm from the epididymis. Sperm undergoing the acrosome reaction demonstrated association of the c-kit receptor with the plasma membrane of the acrosome, but not on the acrosomal membrane itself. Western blot analysis demonstrated protein bands of 150 kDa in testis and intact sperm., Conclusions: The present study confirms the presence of the c-kit receptor in mouse testis and sperm. It also demonstrates that this receptor is localized to the region of the developing acrosome.

Objective: To examine the expression of the c-kit receptor and its ligand, stem cell factor, and their possible relation with apoptosis in infertile men., Design: Prospective laboratory study., Setting: Urology laboratory in a university hospital., Patient(s): Men undergoing testicular biopsy during an investigation of subfertility., Intervention(s): None., Main Outcome Measure(s): Expression of the c-kit receptor protein, stem cell factor, and apoptosis in the testes., Result(s): The c-kit receptor was strongly present in Leydig cells and type A spermatogonia of normal testes, with decreased staining in Leydig cells and type A spermatogonia of testes with maturational arrest, and staining in only Leydig cells of Sertoli cell-only specimens. Stem cell factor was demonstrated in Leydig cells and Sertoli cells in all specimens. Western blotting demonstrated the 150-kd c-kit protein in the normal testes and the testes with maturational arrest, but not in the testes with the Sertoli cell-only pattern. Stem cell factor was expressed in all specimens, with a protein size of 45 kd. Increased apoptosis was demonstrated in type A spermatogonia and spermatocytes of tissue with maturational arrest compared with normal testicular tissue., Conclusion(s): C-kit receptor expression is decreased in subfertile testicular tissue compared with normal testicular tissue. Stem cell factor expression is present in Leydig cells and Sertoli cells. Increased apoptosis is seen in tissue with maturational arrest compared with normal tissue.

The presence and role of the c-kit protein was investigated in the mature sperm of the mouse. The c-kit monoclonal antibody (mAb) ACK2 reacted specifically with the acrosomal region and the principal piece of fixed noncapacitated sperm but did not react with the acrosome region in acrosome-reacted sperm. ACK2 significantly inhibited the acrosome reaction; this inhibition was relieved by the calcium ionophore A23187. The kit ligand stem cell factor (SCF) significantly increased the percentage of sperm undergoing acrosome reaction. This increase was partially inhibited by the calcium channel inhibitor (verapamil), the PI3k inhibitor (wortmannin), and the PLC inhibitor (U-73122). ACK2 predominantly recognized c-kit proteins of 33, 48, and 150 kDa by Western blotting of mouse sperm extracts. The 48- and 150-kDa protein bands were released into the media and tyrosine autophosphorylated at low basal levels during acrosome reaction. On stimulation with SCF, the level of c-kit phosphorylation increased significantly. These findings suggest that c-kit is present in mature sperm, and its binding to SCF may result in the activation of PLC gamma 1 and PI3K, leading to receptor autophosphorylation, and ultimately may play a role in capacitation and/or the acrosome reaction.

The presence and role of the c-kit protein were examined in mature sperm of the mouse. Monoclonal antibodies (mAbs) against the c-kit protein were used to perform immunohistochemical staining, electron microscopy studies, and Western blot analysis. The acrosomal region of both fixed and unfixed noncapacitated sperm stained with the antibodies. No acrosomal staining was noted in acrosome-reacted (AR) sperm. Electron microscopy studies demonstrated immunogold label on the plasma membrane of the acrosome and confirmed the lack of binding following the acrosome reaction. Proteins corresponding to 33 kDa, 48 kDa, and 150 kDa were detected by the antibodies utilizing Western blot analysis. The 48-kDa and 150-kDa proteins were released into the media during sperm capacitation, and release from the acrosome was dependent upon the acrosome reaction. The mAbs significantly inhibited the acrosome reaction and increased sperm agglutination. Monoclonal antibody ACK1 significantly inhibited the motility of the sperm, whereas mAbs ACK2 and NCL-ckit did not. These results suggest that c-kit-related proteins are present in mature sperm and may play a role in capacitation and/or the acrosome reaction.

Objectives: To examine the localization and expression of the c-kit receptor protein in the testes of the mouse, rat, and human, and then compare these among the three species., Methods: Testis tissue from all three species was obtained through biopsy or orchiectomy. Immunohistochemistry was used for the localization, using a monoclonal antibody to the c-kit receptor. The expression of the c-kit receptor protein was examined in the testes and sperm with Western blot analysis., Results: Localization was noted in the early spermatogenic cells, most likely type A spermatogonia, as well as in the acrosomal region of more mature germ cells, such as the round spermatids. The c-kit receptor was localized in analogous sites in all three species. The Western blot data revealed testicular expression of the c-kit receptor protein in all three species as well. Similar bands were recognized on the Western blots of all three species in testes at approximately 75 kDa and approximately 90 kDa, and sperm at approximately 90 kDa only., Conclusions: The c-kit receptor protein is expressed in the early spermatogenic cells, as well as the later stages of spermatogenesis, specifically, the acrosomal granules of the round spermatids, and the acrosomal region of testicular spermatozoa, in the mouse, rat, and human. All three species exhibit similar expression of the c-kit receptor protein in both testis and sperm, although to a varying degree. We believe that these observations allow direct valid comparisons concerning the expression of the c-kit receptor to be made cautiously to the human condition from experimental data obtained from rodents.

The c-KIT proto-oncogene encodes for a transmembrane receptor and is associated with maturation of several cell types, including germ cells. The ligand of the receptor has been identified as stem cell factor (SCF). Loss or alteration of the expression of either of these factors leads to anemia, albinism, and/or sterility in mice. We examined the expression of c-KIT and SCF by immunohistochemistry in specimens from normal and infertile human testis. All specimens were obtained in the evaluation of male subfertility. We were able to demonstrate staining for c-KIT in Leydig cells in all specimens. Normal testis stained for c-KIT in the cytoplasm of early spermatogenic cells, as well as the acrosomal granules of the round spermatids and the acrosome of testicular spermatozoa. However, staining in testis demonstrating maturation arrest failed to demonstrate acrosomal staining, and Sertoli-only specimens demonstrated staining for c-KIT in Leydig cells only. The results for SCF demonstrated an overall uniform staining of Leydig cells in all specimens. The intensity of staining of Sertoli cells increased from normal to maturation arrest to Sertoli-only specimens. Germ cell staining was consistently negative. We hypothesize that these staining patterns for SCF are due to either lack of staining of the receptor-ligand complex or overexpression of the kit ligand in tissue that does not express the kit receptor. It appears that the c-kit receptor is expressed in the acrosome of developing germ cells, as well as in Leydig cells and early spermatogenic cells, suggesting a role in the acrosome reaction, as well as germ cell maturation and differentiation.