Role of poly(ADP-ribosyl)ation during human spermatogenesis.

OBJECTIVE: Genomic stability of cells is known to be linked to their poly(ADP-ribosyl)ation capacity. We aimed to demonstrate, for the first time, the patterns of poly(ADP-ribosyl)ation during human spermatogenesis. DESIGN: Retrospective case-control study. SETTING: Teaching hospital. PATIENT(S): Azoospermic men who underwent testicular biopsy for sperm recovery. INTERVENTION(S): Testicular biopsy evaluation by immunohistochemistry for the expression of poly(ADP-ribose) polymerase-1 (PARP-1) enzyme and of poly(ADP-ribose) (PAR) (an indicator for PARP activity.) MAIN OUTCOME MEASURE(S): The subcellular localization of both markers in testes with full spermatogenesis (obstructive azoospermia), spermatocyte maturation arrest, or Sertoli cell-only syndrome. RESULT(S): Expression of both markers was localized in germ cell nuclei in full spermatogenesis: PAR expression, indicating PARP activity, was exhibited in round and elongating spermatids and in a subpopulation of primary spermatocytes. Strong immunoreactivity for PAR was identified in all of the spermatocytes in maturation arrest at the spermatocyte level. Sertoli cells lacked immunoreactivity for both markers, whereas other somatic testicular cells were rarely immunostained. CONCLUSION(S): The detection of PAR expression in germ-line cells and its subcellular localization in meiotic and postmeiotic prophases demonstrates chromatin modifications occurring during spermatogenesis and establishes a key role for poly(ADP-ribosyl)ation in germ cell differentiation, presumably to safeguard DNA integrity.

Reduced human germ cell-less (HGCL) expression in azoospermic men with severe germinal cell impairment.

Germ cell-less (GCL) protein is a nuclear envelope protein highly conserved between the mammalian and Drosophila orthologues. In Drosophila, maternal GCL protein is required to establish the germ lineage during embryonic development. In mammals, it is suggested that the GCL function is mainly in spermatogenesis and that it might be related to the ability of mouse GCL to repress transcription. Using reverse transcriptase-polymerase chain reaction analyses, we investigated the role of human GCL (HGCL) in spermatogenesis by studying its expression in the testicular tissue of 67 azoospermic men with normal karyotype and no Y-chromosome microdeletion. Their testicular biopsy specimens underwent meticulous histological and cytological analysis as well as molecular analysis with various markers of spermatogenesis (RBM1, DAZ, and CDY1). The rate of X-Y and 18 chromosome bivalent formation during meiosis was additionally assessed in 22 of these biopsy specimens and correlated to HGCL expression. Expression of HGCL was affected in parallel with the severity of testicular impairment found. Defective sperm motility was associated with the absence of HGCL. Nevertheless, the absence of HGCL expression did not influence the normal process of chromosome bivalent formation in meiosis. Our results suggest that HGCL is not essential for the chromosomal events of meiosis but might be involved in later aspects of spermatogenesis.

Double immunolabeling by the RBM and the PLAP markers for identifying intratubular (in situ) germ cell neoplasia of the testis.

Identification of intratubular germ cell neoplasia (carcinoma in situ, CIS) of the testis is a diagnostic challenge, and markers are sorely needed to assist in accurately identifying the lesion. RNA-binding motif (RBM) protein, encoded by the Y chromosome, is expressed exclusively and consistently in differentiated male germ cells, while it is absent in neoplastic germ cells. Another immunohistochemical marker, placental alkaline phosphatase (PLAP), is commonly used for the detection of undifferentiated germ cells. The current study demonstrates that simultaneous use of the immunohistochemical markers, RBM and PLAP, by double immunolabeling enhances the accuracy of diagnosing CIS, a preinvasive testicular neoplasm.

Lack of RBM expression as a marker for carcinoma in situ of prepubertal dysgenetic testis.

Individuals with various intersex states who carry Y-chromosome material bear a high risk of developing testicular neoplasia. In order to gain more insight into the pathogenesis of this neoplasia, the current study evaluates the differentiation of the seminiferous epithelium in 46,XY dysgenetic male pseudohermaphroditism. Immunohistochemical evaluation was performed using the germ cell-specific RNA-binding motif (RBM) protein (encoded by the Y-chromosome) to identify normal germ cells, whereas placental alkaline phosphatase (PLAP) was used to detect neoplastic germ cells. Differentiation of somatic Sertoli cells was assessed using cytokeratin-18 (CK-18) and anti-Müllerian hormone (AMH) as markers for immature Sertoli cells. Specimens were taken from surgically removed dysgenetic gonads of five children (46XY karyotype). Intratubular germ cell neoplasia (carcinoma in situ [CIS] of the testis) was detected in all of them. Normal germ cells revealed immunoreactivity for RBM, whereas the PLAP-positive neoplastic germ cells were negative for RBM expression. Sertoli cells revealed an immature phenotype indicated by AMH expression in their cytoplasm. The design of the current study is unique in its assessment of the state of germ cell differentiation in dysgenetic gonads by the use of the RBM protein, which was expressed only in normal germ cells but not in those of CIS. Testicular dysgenesis interrupted the normal differentiation of the germ line and had no effect on the immature phenotype of the prepubertal Sertoli cells. This points toward the germinal component of CIS as the precursor for the promotion of testis cancer.

Localization of the germ cell-specific protein, hnRNP G-T, in testicular biopsies of azoospermic men.

The increasing interest in the application of in vitro fertilization techniques in human reproduction has led to a wide use of testicular biopsies to identify the presence of spermatogenic foci in testes of azoospermic men. Histopathologic evaluation of these testicular biopsies is required to determine the spermatogenic state with respect to fertility potential and to rule out preinvasive testicular lesions. Heterogeneous nuclear ribonucleoprotein G-T (hnRNP G-T) is a germ cell-specific protein expressed most prominently during meiosis. We studied the usefulness of hnRNP G-T antibody in the evaluation of these biopsies and reasoned that its germ cell-restricted expression pattern might provide a marker to improve accuracy of diagnosis. Testicular biopsies with various spermatogenic impairments were evaluated immunohistochemically for hnRNP G-T expression. In biopsies exhibiting normal spermatogenesis (obstructive azoospermia), hnRNP G-T was localized in meiotic pachytene spermatocytes and round spermatids. Immunostaining was barely detected when maturation was arrested at the spermatocyte level and not at all in cases of Sertoli cell-only syndrome. Biopsies with a mixed histologic phenotype and minute concentrations of spermatogenesis demonstrated strong immunostaining only in tubules with full spermatogenesis. This distribution pattern of hnRNP G-T enabled instant identification of spermatogenic foci. Thus, exploitation of the hnRNP G-T marker, which is expressed preferentially as meiosis proceeds, enhances sensitivity and accuracy of diagnosis in the histologic evaluation of testicular biopsies.

Sertoli cell maturation in men with azoospermia of different etiologies.

OBJECTIVE: To evaluate the involvement of Sertoli cell in different spermatogenic disorders. DESIGN: Retrospective case-control study. SETTING: Teaching hospital. PATIENT(S): Azoospermic men who underwent testicular biopsy for sperm recovery in preparation for intracytoplasmic sperm injection. INTERVENTION(S): Testicular biopsy evaluation by quantitative immunohistochemistry for the immature Sertoli cell markers anti-Müllerian hormone and cytokeratin 18 (CK-18). MAIN OUTCOME MEASURE(S): Relative area of immature Sertoli cells in testes with focal spermatogenesis, spermatocyte maturation arrest, or normal spermatogenesis. RESULT(S): The relative area occupied by immature Sertoli cells, as revealed by anti-Müllerian hormone and CK-18 expression, was highest in the 11 men with focal spermatogenesis. In the group representing normal spermatogenesis (obstructive azoospermia, 6 men) and in the group characterized by spermatocyte maturation arrest (6 men), the areas occupied by anti-Müllerian hormone- and CK-18-positive cells were minimal. CONCLUSION(S): Different etiologies underlie the spermatogenic disorders reported in this study. In focal spermatogenesis with high anti-Müllerian hormone and CK-18 expression, the spermatogenic impairment is associated with the presence of immature Sertoli cells. The detection of normal mature Sertoli cells in the spermatocyte maturation arrest group indicates that the spermatogenic defect that is accompanied by an impairment of meiosis is intrinsic to the germ line without affecting Sertoli cell differentiation.

Localization of a specific germ cell marker, DAZL1, in testicular germ cell neoplasias.

DAZ-like 1( DAZL1) is a germ cell-specific protein expressed in both male and female gonads. The DAZL1 gene, which maps to chromosome 3 in humans, is an autosomal homologue to the Deleted in AZoospermia ( DAZ) gene(s) located on the Y chromosome. We studied the expression of DAZL1 by means of immunohistochemistry in order to determine its distribution among testicular germ cell neoplasias and among the intratubular lesions in their vicinity. Our results demonstrated that expression of DAZL1 protein was consistently observed in scattered cells in all intratubular germ cell neoplasias (IGCN) of the unclassified type, as well as in some of the intratubular seminomas. Foci of DAZL1 immunopositive cells were detected in pure seminomas, while single immunopositive cells were dispersed in the seminomatous component of mixed germ cell neoplasias. All the nonseminomatous components were negative for DAZL1 expression. These findings demonstrate an antigenic heterogeneity of seminoma cells. The localization of a specific germ cell protein, DAZL1, in the putative ontogenic progenitor, IGCN, and in their putative derivative, seminoma, provides further support for the hypothesis that IGCN is the precursor of germ cell neoplasias.