Involvement of variety of genes, especially located on Y chromosome, is critical for the regulation of spermatogenesis. In particular, fertility candidate genes such as deleted in azoospermia (DAZ) are believed to have important function in sperm production, since DAZ is frequently deleted in azoospermic and severy oligozoospermic men. The role of the DAZ gene is supported by its exclusive expression in the testis and by its deletion in about 10% of azoospermic and severely oligozoospermic patients. The distribution of DAZ transcripts in seminiferous epithelium of human testis is reported in the present study. The use of Adobe Photoshop and Scion Image softwares allowed for semi-quantitative analysis of in situ RT-PCR (ISRT-PCR) results. The intensity of ISRT-PCR product's fluorescence was different within individual seminiferous tubules. It was clearly shown by using the pseudocolour scale and transforming the intensity of the fluorescence into levels of greyscale images. The more intense fluorescence characterised single spermatogonia and those organized in small groups inside separate tubules. The most intense accumulation of DAZ mRNA was observed in spermatogonia

Andrusiewicz, M

Jankowska, A

Karczewski, M

Szczerba, A

Turkiewicz, W

Warchoł, J B

English

Via Medica Journals

FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 42, No. 2, 2004pp. 119-121Distribution of the DAZ gene transcripts in human testisA. Szczerba1, A. Jankowska1, M. Andrusiewicz1, M. Karczewski2, W. Turkiewicz2 and J.B. Warchoł11Department of Radiobiology and Cell Biology, University of Medical Sciences, Poznan´, Poland and2Department of Transplantology, District Hospital, Poznan´, PolandAbstract: Involvement of variety of genes, especially located on Y chromosome, is critical for the regulation of spermatogen-esis. In particular, fertility candidate genes such as deleted in azoospermia (DAZ) are believed to have important function insperm production, since DAZ is frequently deleted in azoospermic and severy oligozoospermic men. The role of the DAZ geneis supported by its exclusive expression in the testis and by its deletion in about 10% of azoospermic and severelyoligozoospermic patients. The distribution of DAZ transcripts in seminiferous epithelium of human testis is reported in thepresent study. The use of Adobe Photoshop and Scion Image softwares allowed for semi-quantitative analysis of in situ RT-PCR(ISRT-PCR) results. The intensity of ISRT-PCR product’s fluorescence was different within individual seminiferous tubules.It was clearly shown by using the pseudocolour scale and transforming the intensity of the fluorescence into levels of greyscaleimages. The more intense fluorescence characterised single spermatogonia and those organized in small groups inside separatetubules. The most intense accumulation of DAZ mRNA was observed in spermatogonia.Key words: DAZ gene - Transcript localization - Azoospermia - OligozoospermiaIntroductionIt has been presumed that 2-3% of men are consideredto be infertile as a result of various defects in spermproduction [4, 8, 18]. Spermatogenesis is a complexdevelopmental process in which male germ cells pro-gress trough mitotic proliferation, meiotic division anddramatic morphological changes to form mature sperm.Human spermatogenesis is regulated by a network ofgenes located on autosomes and on sex chromosomes,but especially on the Y chromosome. Molecular tech-niques employing Y chromosome-specific markers re-vealed that 5-10% of severely oligozoospermic orazoospermic men have cytogenetically invisible Yqmicrodeletions [9, 12, 13, 14, 16, 20, 22]. These fall intothree classes mapping to discrete subintervals of the Yqregion, termed AZFa, b and c (AZoospermia Factor)[16], and positional cloning strategies have led to theisolation of candidate genes from each subinterval. Mu-tations more frequently involve the DAZ gene in AZFcand could lead to both azoospermia and severe oligo-zoospermia. It is therefore difficult to find a clear rela-tionship between genotype and phenotype. DAZ ispresent in multiple copies in AZFc, and this causes thegene to be difficult to analyze [6]. The critical role forDAZ in human male germ cell development is supportedby the evidence that this gene is testis-specific, withtranscriptions limited to germ cells [5, 7, 11], as well asby its deletion in about 10% of azoospermic and severelyoligozoospermic subjects. The DAZ gene family en-codes a protein with RNA-binding motif near its N-ter-minus [16]. RNA-binding proteins are critical inspermatogenesis, because many of the genes expressedin the sperm are regulated at the level of translation [17].The function of the DAZ gene however, remains unclear,and the postulated RNA binding property of the geneproduct has not yet been demonstrated [4, 5].The aim of our research was to determine distributionand concentration of DAZ transcripts in the seminiferousepithelium of human testis. Materials and methodsMaterial. Human testes were obtained from five fertile organ donors,fixed in 4% formaldehyde in phosphate buffered saline (PBS),dehydrated and embedded according to standard procedure [2, 3].For the procedure approval was obtained from the institutionalcommittee.RT-PCR in situ. RT-PCR in situ was performed on paraffin sectionsof formalin-fixed tissue. Correspondence: A. Szczerba, Dept. Radiobiology and Cell Biol-ogy, University of Medical Sciences, S´wie˛cickiego 6, 60-781Poznan´, Poland; e-mail: abosacka@amp.edu.plIn order to detect the DAZ mRNA, the sections (3 µm) wereprepared according to the procedure described by Bagasra and co-workers [1]. Reverse transcripton in situ was performed using Ex-pand Reverse Transcriptase (Roche Molecular Biochemicals,Mannheim, Germany) with the primer specific for the DAZ gene(reverse primer: 5’-ATGCAAAGCTCAATACTGA-3’, com-plementary to nucleotides 36319-36337, PubMed AC00021). The460 bp fragment of cDNA of the DAZ gene transcript was amplifiedby in situ PCR, using the following primers: sense 5’-TGTTACCA-GAAGGCAAAATC-3’ (nucleotides 1413-1432, PubMedAC00021) and antisense 5’-GCAACTGACATCCAGTGATG-3’(complementary to nucleotides 4613-4632, PubMed AC00021). Flu-orescence-labelled nucleotides CyTM3-dUTP (Amersham Bioscien-ces, Little Chalfont Buckinghamshire, UK) were used in order toidentify the amplified product. DAZ mRNA distribution was exam-ined using Carl Zeiss LSM 510 confocal microscope.Computer generation of pseudocolours. The use of computer-as-sisted image transformation allowed for semi-quantitative analysisof ISRT-PCR results. Images generated in .lsm true colour file formatfrom Zeiss LSM Image Browser Version 3,1,0,99 for Carl ZeissLaser Scanning System LSM 510 (Carl Zeiss GmbH) were exportedto Microsoft Windows® Bitmap File Format and saved. In AdobePhotoshop® 5.5 CEEA (Adobe Systems Inc.1989-1999), the colourmode was changed to greyscale format (8-bit per channel; 256 greylevels) and stored as a Windows® 8 Bitmap File Format. Using ScionImage® (Release beta 4.0.2) for Windows software (Scion Corpora-tion© 2000), the greyscale was changed into 32-colour table modeand stored as a pseudocolours. ResultsThe use of Adobe Photoshop and Scion Image softwaresallowed for semi-quantitative analysis of RT-PCR insitu results. The results of the research based on theISRT-PCR technique demonstrated that active DAZgene products were present in all studied seminiferoustubules of human testis (Fig. 1A). The intensity of ISRT-PCR product fluorescence was different within individ-ual seminiferous tubules, as clearly shown by applyingthe pseudocolour scale and expressing the intensity ofthe fluorescence as levels of greyscale images. The moreintense fluorescence characterised single spermatogoniaand those organized in small groups inside separatetubules. The intensity reached 104th greyscale level(Fig. 1C, D). A weaker fluorescence was detected inprimary spermatocytes, mostly located in the nearestneighbourhood of positively stained spermatogonia(Fig. 1B). The fluorescence intensity of primary sperma-tocytes ranged from 73rd to 84th greyscale levels.A very weak fluorescence (around 50th grey level) wasnoted for the other cells of seminiferous tubules, i.e.secondary spermatocytes and spermatids. This signal,however, was stronger than that found in cells of thebasement membrane, for which the intensity was only10 to 20 grey levels above the background. In case of control reactions in which the reversetranscription step was omitted, the fluorescence productof the PCR reaction was not observed.Discussion The precise biological significance of the DAZ geneis still unknown. Recent studies demonstrated that dele-tion of the gene cause male infertility, but not all themutations of the DAZ gene lead to azoospermia oroligozoospermia. Some authors suggest that the role ofthe DAZ in regulation of spermatogenesis is, if any,rather marginal.The research on the expression and mutations of theDAZ and its homologues in humans and other mammalssuggests that protein products of these genes can mainlyaffect development of germinal cells [15].The aim of the present study was to semi-quantita-tively analyse the expression of the DAZ gene in semi-Fig. 1. Pseudocolour-transformed images ofhuman seminiferous tubules. A. The results ofISRT-PCR. The DAZ gene products visible asgreen to yellow colour are present in all semi-niferous tubules of human testis. B. Cross-sec-tion of single seminiferous tubule. Positivelystained primary spermatocytes are located inthe nearest neighbourhood of yellow sperma-togonia. The cells visible as pale green do notexpress DAZ. C, D. The plots of the fluores-cence intensity of cells measured along thelines shown in A and B, respectively.120 A. Szczerba et al.niferous tubules of the human testis. The results of thestudy clearly showed the strongest fluorescence whichcan correspond to high level of the DAZ gene expressionin spermatogonia and primary spermatocytes. Weakfluorescent signal was also observed in secondary sper-matocytes and spermatids and in interstitial cells. The results of the study did not depend on the thick-ness of the analysed specimens, since confocal micro-scope was used to visualise the fluorescence-labelledproducts of ISRT-PCR. The obtained data partly correspond to the results ofother authors, based on the hybridization techniques andto our previous study which showed the presence ofactive DAZ gene only in spermatogonia and primaryspermatocytes [19, 21]. The observed difference seemsto be due to various fixation methods applied as well asto a choice of new primers. The fluorescence product ofISRT-PCR observed by us also in secondary spermato-cytes and spermatids can be visualized due to highsensitivity of direct detection method based on fluores-cence labeled nucleotides. It made also possible to showthe presence of DAZ mRNA in human spermatozoa [19].Localization of active DAZ gene mainly in spermato-gonia and primary spermatocytes suggests a possible roleof this gene in the first steps of spermatogenesis. DAZ genecan be especially important for regulation of the numberof spermatogonia stem cells and their differentiation.This hypothesis corresponds to the theory of self-renew-ing mechanism of stem cells in germline [10] as well asto the result of Reijo et al. [15] suggesting the role ofDAZ family proteins in male germ cell development.Since the DAZ gene is present in multiple copies inAZFc, it is difficult to analyze and find a clear relation-ship between genotype and phenotype in case of muta-tions causing azoospermia and oligozoospermia.Further research on the role of DAZ can have importantclinical consequences in the diagnostic and therapeutictreatment of the infertile patients, above all when theyare candidates for assisted reproduction techniques.Acknowledgements: The study was supported by National Com-mittee for Scientific Research (KBN), grant no. 4P05E01919.References:[ 1] Bagasra O, Seshamma T, Pomarantz R, Hansen R (1995) Cur-rent Protocols in Molecular Biology; Vol 2, pp 14.8.1-14.8.232[ 2] D’Andrea MR, Nagele RG, Wang HY, Peterson PA, Lee DH(2001) Evidence that neurones accumulating amyloid canundergo lysis to form amyloid plaques in Alzheimer’s disease.Histopathology 38: 120-134[ 3] Duda T, Venkataraman V, Krishnan A, Nagele RG, Sharma RK(2001) Negatively calcium-modulated membrane guanylate cy-clase signaling system in the rat olfactory bulb. 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FoliaHistochem Cytobiol 39: 117-118[22] Wiland E, Kurpisz M (1997) Chromosomal anomalies as apredisposing factor for male infertility. Folia Histochem Cyto-biol 35: 55-62         Accepted January 13, 2004DAZ gene transcripts in human testis 121

Distribution of the DAZ gene transcripts in human testis.

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Distribution of the DAZ gene transcripts in human testis.

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