Detection of DAZ mRNA distribution in human testis using reverse transcription in situ PCR technique (RT-ISPCR). Warchoł J B,Augustyniak S,Stecewicz D,Jankowska A Folia histochemica et cytobiologica Normal course of spermatogenesis reflects appropriate expression of specific genes in spermatogenic cells. In the present study, the localization of DAZ mRNA in human testis obtained from two fertile organ donors has been determined. It was established that DAZ mRNA is located in the germinal epithelium of seminiferous tubules. The most intensive accumulation of DAZ transcript was observed in primary spermatocytes. The results indicate that expression of DAZ gene begins in spermatogonia and that distribution of DAZ mRNA most likely correlates with the stage of the human seminiferous cycle.

Expression of DAZ (deleted in azoospermia), DAZL1 (DAZ-like) and protamine-2 in testis and its application for diagnosis of spermatogenesis in non-obstructive azoospermia. Lee J H,Lee D R,Yoon S J,Chai Y G,Roh S I,Yoon H S Molecular human reproduction Spermatogenesis is regulated by hormones, local regulatory factors in the testes and specific gene expression of spermatogenic cells in humans. In this study, we have detected the expression of the deleted in azoospermia (DAZ), the DAZ-like autosome (DAZL1), and the protamine-2 genes in spermatogenic cells. Spermatogenesis in 38 male infertility patients was evaluated by the semen analysis and histological examination. Patients were diagnosed as Sertoli cell-only syndrome (n = 20), maturation arrest (n = 6), hypospermatogenesis (n = 6), and obstructive azoospermic patients with normal spermatogenesis (n = 6). After microscopic observation of the wet preparation of the testis tissues, seminiferous tubule contents were used for reverse transcription-polymerase chain reaction (RT-PCR) analysis of DAZ, DAZL1 and protamine-2. In cases with Sertoli-cell only syndrome, we found spermatogenic cells in 30% of patients (6/20) by the wet preparation method. There was no difference between the histology and the wet preparation results in maturation arrest and obstructive azoospermia; however, in one case of hypospermatogenesis, spermatozoa were not detectable by the wet preparation method. Using in-situ hybridization with DAZ and protamine-2 ribonuclear probes, we confirmed spermatogenic cell-specific expression of DAZ (spermatogonia/early spermatocyte) and protamine-2 (spermatid/spermatozoon). DAZ and protamine-2 expression can therefore be considered spermatogenic cell markers and could be useful in molecular diagnosis of spermatogenesis. In 13 patients with spermatozoa under the wet preparation, the expression of DAZ, DAZL1 and protamine-2 was detected in all the preparations. In one wet preparation showing only spermatogonia/spermatocyte, only DAZ and DAZL1 RNA were detected. In 14 wet preparations showing no spermatogenic cells, DAZ, DAZL1 and protamine-2 were not detected except in one preparation where DAZL1 expression was detected. In 10 wet preparations representing spermatogonia/spermatocyte to spermatids, but showing no spermaozoa, DAZ and DAZL1 were detected in eight and nine preparations respectively, and protamine-2 was detected in six preparations. These results of gene expression were similar to the wet preparation results. RT-PCR for DAZ, DAZL1 and protamine-2 was informative for the existence of germ cells, germ cell physiology and differentiation. From these results, we suggest that the analysis of DAZ, DAZL1 and protamine-2 expression by RT-PCR and wet preparation might offer a better method for finding the spermatogenic cells compared to the histological method. 10.1093/molehr/4.9.827

Restricted expression of the human DAZ protein in premeiotic germ cells. Huang William J,Lin Yi-Wen,Hsiao Kuang-Nan,Eilber Karyn S,Salido Eduardo C,Yen Pauline H Human reproduction (Oxford, England) BACKGROUND:The role of the Y chromosome-encoded Deleted in Azoospermia (DAZ) gene family in spermatogenesis remains unclear. The ability of men without the DAZ gene to produce sperm, as well as the lack of selective pressure on DAZ exon sequences during evolution, casts doubts on its functional significance. Most men have four DAZ genes encoding protein isoforms that differ significantly in size. However, published western blots showed only a single "DAZ" band, raising the possibility that not all four DAZ genes are expressed. METHODS:RT-PCR, western blotting and immunostaining were used to study the expression of the four DAZ genes and the autosomal DAZL gene in human testes and in tissue culture cells. RESULTS:RNA transcripts of all four DAZ genes were found in the testis, but at much lower levels than that of the DAZL transcripts. Expression in cultured somatic cells showed that DAZ transcripts encoding multiple DAZ repeats were translated inefficiently. No DAZ proteins could be unambiguously identified on western blots when the testicular samples from three patients without the DAZ genes were used as negative controls. Nonetheless, low levels of DAZ were detected in the cytoplasm of spermatogonia by immunostaining. CONCLUSIONS:The expression of DAZ proteins in adult human testes is restricted to the spermatogonia and suggests a premeiotic role. 10.1093/humrep/den099

Vascular endothelial growth factor A is a putative paracrine regulator in seasonally controlled spermatogenesis: insights from a ruminant model, the roe deer. Schön Jennifer,Blottner Steffen,Gabler Christoph,Fickel Jörns Growth factors (Chur, Switzerland) Vascular endothelial growth factor A (VEGFA) influences spermatogenesis, but its impact on seasonally regulated sperm production is still not fully understood. Thus, we investigated both expression levels and localisation of VEGFA and its receptors VEGFR1 and 2 in roe buck testis via real-time reverse transcription polymerase chain reaction and immunohistochemistry in relation to seasonal changes in the cellular composition of the testis. VEGFA was expressed by interstitial cells while its receptors were found on endothelial and perivascular cells. Inside the tubules, VEGFA was located in spermatogonia and spermatocytes, VEGFR1 was present on elongating spermatids and VEGFR2 on Sertoli cells. VEGFR1 mRNA was expressed tenfold lower than VEGFR2 and VEGF mRNAs. Relative VEGF and VEGFR2 expression (divided by the number of VEGFA and VEGFR2 expressing cells) showed an increase towards the rut (July/August) and a decrease thereafter. The results suggest involvement of VEGFA in the adjustment of vascular permeability as well as in spermiogenesis and the proliferation of spermatogonia. 10.3109/08977191003587668

Screening for biomarkers of spermatogonia within the human testis: a whole genome approach. von Kopylow Kathrein,Kirchhoff Christiane,Jezek Davor,Schulze Wolfgang,Feig Caroline,Primig Michael,Steinkraus Volker,Spiess Andrej-Nikolai Human reproduction (Oxford, England) BACKGROUND:A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type. METHODS:The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets. RESULTS:Highly significant differences in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast growth factor receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-1 (CTAG1A/B), undifferentiated embryonic cell transcription factor 1 (UTF1) and synaptosomal-associated protein, 91 kDa homolog (SNAP91) were shown to represent specific biomarkers of human spermatogonia. CONCLUSIONS:These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation. 10.1093/humrep/deq053