Histone H4 acetylation and AZFc involvement in germ cells of specimens of impaired spermatogenesis.

OBJECTIVE: To measure histone-H4 acetylation and involvement of the AZFc region in testicular mixed atrophy. DESIGN: Prospective study. SETTING: University-affiliated medical center. PATIENT(S): Azoospermic men (n = 23) who underwent testicular sperm extraction and preparation for intracytoplasmic sperm injection (ICSI) divided into obstructive azoospermia with complete spermatogenesis (group A), testicular mixed atrophy (group B), and testicular mixed atrophy associated with AZFc deletion (group C). INTERVENTION(S): Testicular biopsy evaluation by Western blotting and quantitative immunohistochemistry of histone-H4 hyperacetylation (Hypac-H4) and lysine-12 acetylation (Lys12ac-H4). MAIN OUTCOME MEASURE(S): Percentage of spermatogonia and spermatids stained by Hypac-H4 and Lys12ac-H4 antibodies in retrieved specimens. RESULT(S): The percentage of spermatogonia stained for Hypac-H4 and Lys12ac-H4 in groups B and C was statistically significantly reduced. The percentage of elongated spermatids showing positive staining to Hypac-H4 was statistically significantly lower in group B than group A. The percentage of Lys12ac-H4-labeled spermatids was similar for all groups. Hypac-H4 and Lys12ac-H4 processes were highly correlated in spermatogonia but not in spermatids. CONCLUSION(S): The reduced percentage of spermatogonia with Hypac-H4 and Lys12ac-H4 in groups B and C may contribute to lower sperm production in mixed atrophy. Spermatids Hypac-H4 impairment in mixed atrophy did not deteriorate further by AZFc region deletion.

Detection of calretinin expression in abnormal immature Sertoli cells in non-obstructive azoospermia.

The current study identified for the first time calretinin expression in abnormal Sertoli cells of azoospermic men who underwent testicular biopsy for sperm recovery and application of the retrieved sperm by in vitro fertilization techniques. Testicular biopsies with various spermatogenic impairments were evaluated immunohistochemically for the expression of the calretinin calcium-binding protein and the marker for immaturity of Sertoli cells, cytokeratin-18 (CK-18). Distribution of the markers was assessed in testes demonstrating a histological phenotype of mixed atrophy, Sertoli cell-only, or normal spermatogenesis (obstructive-azoospermia) and in men carrying a deletion in the azoospermia factor region located on the Y chromosome. Calretinin-immunopositive immature Sertoli cells revealed by co-localization of both markers, calretinin and CK-18, were identified in the mixed atrophy group in seminiferous tubules demonstrating spermatogenic failure. Sertoli cells expressing both markers were rarely detected in all other groups. Leydig cells in all the assessed biopsies expressed calretinin and served as a built-in control for immunoreactivity. This pattern of calretinin-selective expression in immature Sertoli cells suggests a functional relationship between calretinin expression and the degree of Sertoli cell differentiation. Disorders of Sertoli cell differentiation as indicated by calretinin and/or CK-18 expression contribute to the multifactorial mechanisms underlying spermatogenic failure.

Sertoli cell inactivation by cytotoxic damage to the human testis after cancer chemotherapy.

OBJECTIVE: To assess Sertoli cell involvement in postchemotherapy azoospermia. DESIGN: Case report. SETTING: Teaching hospital. PATIENT(S): A 31-year-old azoospermic man who underwent cancer cytotoxic chemotherapy for non-Hodgkin's lymphoma at 13 years of age. INTERVENTION(S): Testicular biopsy specimens were obtained for sperm recovery in preparation for intracytoplasmic sperm injection. The biopsy specimens were evaluated by quantitative immunohistochemistry for the immature Sertoli cell markers cytokeratin 18 (CK-18) and D2-40. MAIN OUTCOME MEASURE(S): Extent of immature Sertoli cells. RESULT(S): A fraction of Sertoli cells (13%) in the atrophic tubules of this patient reexpressed the intermediate filament protein CK-18, which is normally absent after puberty, but not the D2-40 antigen, an Mr 40,000 a-linked membrane glycoprotein, whose loss of expression at puberty marks an irreversible step in Sertoli cell maturation. Tubules with normal spermatogenic progression lined by Sertoli cells negative for CK-18 were also observed. CONCLUSION(S): A fraction of Sertoli cells of this patient initially progressed to full maturation at puberty and reverted to a dedifferentiated state marked by reexpression of CK-18 as a consequence of chemotherapy. This inactivation of Sertoli cells caused by the cytotoxicity of the chemotherapeutic drugs may have contributed to the spermatogenic impairment and resulting infertility.

Spermatogonial proliferation patterns in men with azoospermia of different etiologies.

OBJECTIVE: To demonstrate the pattern(s) of spermatogonial proliferation in different spermatogenic disorders. DESIGN: Retrospective case-control study. Teaching hospital. PATIENT(S): Azoospermic men who underwent testicular biopsy for sperm recovery and preparation for intracytoplasmic sperm injection. INTERVENTION(S): Testicular biopsy evaluation by quantitative immunohistochemistry for proliferating cell nuclear antigen (PCNA). MAIN OUTCOME MEASURE(S): The expression of PCNA in spermatogonia as an index of proliferating activity in testes with focal spermatogenesis, spermatocyte maturation arrest, or normal spermatogenesis. RESULT(S): In biopsies with focal spermatogenesis (11 men), there was a statistically significant reduction of PCNA-labeled spermatogonia in seminiferous tubules showing spermatocyte arrest compared with the expression in adjacent tubules with advanced spermatogenic stage or in normal spermatogenesis (obstructive azoospermia, six men). However, PCNA expression in tubules of the group with complete maturation arrest (six men) was significantly higher compared with the same spermatogenic defect-spermatocyte arrest-within focal spermatogenesis biopsies. CONCLUSION(S): Different causes underlie the spermatogenic disorders reported in this study. In focal spermatogenesis, the disorder is associated with the presence of mitotic inactive spermatogonia. The detection of normal active spermatogonia in the spermatocyte arrest group indicates that the spermatogenic defect, which is accompanied by meiosis impairment, is not related to a malfunction of spermatogonial proliferation.

Members of the CDY family have different expression patterns: CDY1 transcripts have the best correlation with complete spermatogenesis.

The CDY family of genes is of special interest because some of them are included in chromosome-Y microdeletions detected among infertile men and are apparently involved in the spermiogenetic process. In this study, we employed the reverse transcriptase/polymerase chain reaction technique to test the RNA expression of the various transcripts of these genes in testicular biopsies of 84 azoospermic men who had been classified by comprehensive histology and cytology analyses. We also evaluated the feasibility of detecting CDY expression in biopsies taken by testicular sperm extraction versus acquisition by aspiration. There was a significant association between the type of testicular impairment and the expression of CDY1 and CDY2 transcripts. CDY2 was expressed whenever germ cells were present, but CDY1 major and especially CDY1 minor and short transcripts were identified almost exclusively when mature spermatids/spermatozoa were detected. The expression of CDY1 minor and short transcripts detected in aspirated specimens was less efficient than that in testicular tissue acquired by extraction. It is suggested that CDY2 is apparently required in the early stages of spermatogenesis, whereas CDY1 transcripts are required later on in the process. The findings of this study imply different functional roles for CDY isoforms during spermatogenesis. However, in consideration of the high levels of identity between CDY1 and CDY2 (98% at the protein level), the delayed up-regulation of CDY1 transcripts could be attributable to temporal changes in dosage requirements.

Immunohistochemistry in the evaluation of spermatogenesis and Sertoli cell's maturation status.

An increasing incidence of male infertility has been noted over the past few decades. This adds urgency to the need to develop immunohistochemical markers for better evaluation of testicular biopsies. We provide evidence that a histopathological evaluation performed according to morphological criteria and assisted by immunohistochemical staining on consecutive sections enhances the sensitivity and accuracy of the diagnosis based on testicular biopsies from infertile men.