Expression and functional analysis of <i>Tex18</i> and <i>Stra8</i> genes in male germ cells

Abstract

In the present study expression and functional analysis of two testis specific genes, namely Tex18 and Stra8 was performed.RT-PCR, made for the expression analysis of Tex18 gene showed it s expression exclusively in male germ cells. It was observed also in testes of developmental mutants, with exception of W/WV mice (which lack any germ cells), postnatal and prenatal stages. Expression of Tex18 was found also in ES cells. For additional expression analysis transgenic mice, in which EGFP was expressed under the control of 1.6 kb Tex18 promoter, were generated. Histological sections showed that Tex18 is expressed predominantly in the postmeiotic germ cells. FACS analysis performed with testicular cell suspension of transgenic mice confirmed these data. An increased percentage of EGFP positive cells was observed in the case of older animals, in which postmeiotic cells are already present. It was also shown, that FACS positive cells contain an increased ratio of haploid cells, comparing to wild type control.For the functional analysis, knock-out mice were generated on C57 BL/6J x 129/Sv and on 129/Sv backgrounds. Males from the first background were fertile, but 1/3 of the males on 129/Sv background were fully infertile, while others have reduced fertility. Although number of sperm in cauda epididymes was not disturbed (as compared to the wild type controls), both backgrounds showed a reduced number of sperm in uteri and oviducts in VP tests. Reduced motility and progressive movement of sperm on both backgrounds was observed in comparison to wild type controls. An increased number of sperm with abnormal head shape was found in cauda epididymes. Although acrosome shape was changed in spermatozoa with head abnormality, its function was not disturbed since spermatozoa showed normal acrosome reaction. Histological sections of homozygous male testes revealed abnormalities in seminiferous tubules structure. Efficiency of spermatogenesis was disturbed as it was very often arrested at the stage of round spermatid. Apoptotic spermatocytes and spermatids and diploid spermatids were seen regularly in many seminiferous tubules. From these data it can be concluded that Tex18 gene is involved in spermatid differentiation and asthenoteratozoospermia is likely the cause of the infertility of Tex18-deficient males.Similarly like in the case of Tex18, RT- PCR analysis confirmed that expression of Stra8 gene is restricted to germ cells. Expression was observed also in developmental mutants (except W/WV mice), postnatal and prenatal stages. Immunostaining, using Stra8 specific antibody showed that the expression of the gene is restricted to the premeiotic stages of spermatogenesis, as the signal was visible only in cells localised close to the basal lamina of seminiferous tubules. The same pattern was observed on testis sections of transgenic mice (where EGFP was expressed under the control of 1.4 Stra8 promoter). Expression of the human Stra8 gene was found to be restricted to testis, too. It was shown also that the expression of the Stra8 is increased in the RA treated Tera1 human teratocarcinoma cells.For the functional analysis a knock-out strategy was designed, in which exons 2-8 (from 9 existing) were disrupted. Five chimeras were obtained. One female and two male chimeras did not transmit the knock-outed gene to the offspring. Another two males, both showing 5% chimerism, were infertile. Strain specific PCR revealed that in the sperm of one of them the disrupted gene is present. The testis histological section of this male showed serious degeneration of the testis structure and the arrest of spermatogenesis in all seminiferous tubules. These results suggest that Stra8 might be essential for meiotic differentiation.