Accumulated HSV1-TK Proteins Interfere with Spermatogenesis through a Disruption of the Integrity of Sertoli-Germ Cell Junctions

Abstract

Abstract. Transgenic rats show spermatid-specific ectopic expression of the reporter gene, herpes simplex virus type1 thymidine kinase (HSV1-TK), in the testes and have demonstrated male infertility. However, the disruption of spermatogenesis and the underlying molecular mechanisms in these transgenic animals have not been well clarified. In this study, light and electron microscopic observations were performed to characterize the morphological changes in the testes. To explore the molecular mechanisms of male infertility in the HSV1-TK transgenic rat, cDNA microarray and quantitative real-time PCR analyses were performed. The seminiferous tubules of 3-month-old transgenic rats showed morphological alterations including seminiferous epithelial sloughing, vacuolization, and degeneration of spermatogenic cells, suggesting a failure of Sertoli-germ cell interaction. Components of the epididymal lumen from transgenic rats included abnormal spermatozoa, degenerating round spermatids and abnormal elongated spermatids indicating an appearance of direct impairment of spermiogenesis. cDNA microarray and real-time PCRanalyses revealed significant changes (P<0.05) in the gene expression level in six genes, testin, versican, mamdc1, fgf7, ostf1 and cnot7. Among them, testin drew most of our attention, since the testin gene is a sensitive marker for disruption of Sertoli-germ cell adhesion. Thus, our results suggest that the accumulation of HSV1-TK in the spermatids not only directly interferes with spermiogenesis but also disrupts spermatogenesis through a disruption of Sertoligerm cell adhesions. It is important to explore the testicular actions of the HSV1-TK protein in transgenic experimental models and thereby gain clues to find an appropriate treatment for HSV-infected patients exhibiting human male infertility, as has been recently observed. Key words: HSV1-TK, Infertility, Spermatogenesis, Testin, Transgenic rat (J. Reprod. Dev. 58: [544][545][546][547][548][549][550][551] 2012) A suicide gene system, herpes simplex virus type 1 thymidine kinase (HSV1-TK) and its specific substrate, ganciclovir, is widely used for cancer therapy [1,2]. However, previous investigators also reported that HSV1-TK proteins are likely to play a toxic role in the developing mouse male germ cells in the absence of the nucleoside analog, ganciclovir, to induce defects in cell proliferation [3][4][5][6]. The major histological defects of HSV1-TK transgenic mice appeared to be an abnormal nuclear morphology of elongating spermatids and electron microscopy observation disclosed insufficient chromatin condensation and abnormal acrosome structures [4]. Al-Shawi et al. demonstrated that truncated TK polypeptides are translated mainly in the haploid spermatids by a cryptic TATA box-independent promoter and that the severity of male infertility depended on the level of enzymatic activity of HSV1-TK produced [5]. However, precise morphological analyses of the seminiferous tubules of HSV1-TK transgenic mice have not yet been reported.We previously ...

Detection of Human Herpesviruses (HHVs) in Semen of Human Male Infertile Patients

“…More recently, we demonstrated the possibility of Sertoli cell-germ cell disruption possibly by upregulation of Testin [6], the products of which play a crucial role in maintaining the junction between Sertoli cells and germ cells. Though the role of the HHV1-TK protein expressing thymidine kinase activity in the disruption of spermatogenesis is completely unclear at present, our cumulative data [1, 2, 6] certainly indicate a possibility that ectopic expression of infected HHV1 gene causes disruption of male gametogenesis. Elucidation of the molecular mechanism of the ectopic expression of HHV1-TK in round spermatid cells of the TG-rat is an important in vitro approach.…”

“…We previously demonstrated that the transgenic rats show an ectopic expression and accumulation of HHV1-TK proteins in postmeiotic germ cells with disruption of spermatogenesis by the appearance of immature and abnormal sperm and age-dependent loss of germ cells via apoptotic cell death [2]. Continuous investigation by microarray and real-time PCR analyses revealed an increase in gene expression of Testin [6], a sensitive marker for monitoring the integrity of the Sertoli-germ cell junction. These results are suggestive and may provide a clue that may help uncover a relationship between virus infection and male infertility if the viral gene product(s) is expressed in the human male testis and causes male impairment of spermatogenesis.…”

Ectopic Expression of Human Herpesvirus 1 Thymidine Kinase Induces Male Infertility

“…We sorted the genes by their functional categories, such as apoptosis, cell cycle, development, oxidative stress, proteolysis, signal transduction, transcription, translation, transport, metabolism, immune response, and cell adhesion. The highest number of affected genes was linked to metabolism, with 8 genes upregulated and 16 down-regulated by at least 1.5-fold in Tg rat testis ( Table 3) [42].…”

“…Gene ontology classification of genes whose expression changes by at least .5-fold in Tg with respect to normal rats, as revealed by cDNA microarray analysis. Reproduced and modified from [42] with permission from the Society for Reproduction and Development. Notably, the contraceptive adjudin has been reported to induce morphological alterations in the seminiferous tubules similar to the ones we observed here, and target directly testin and actin-myosin [48].…”

“…However, the exact mechanism by which HHV-1-TK disrupts spermatogenesis and adherens junctions will require further work. Reproduced and modified from [42] with permission from the Society for Reproduction and Development. Table 4.…”