In vitro spermatogenesis: In search of fully defined conditions.

A complete reconstruction of spermatogenesis in vitro under fully defined conditions still has not been achieved. However, many techniques have been proposed to get closer to that aim. Here we review the current progress in the field. At first, we describe the most successful technique, the organ culture method, which allows to produce functional haploid cells. However, this method is based on the culturing of intact testis tissue with unknown factors acting inside it. Then we discuss different types of 3D-cultures where specific testicular cell populations may be aggregated and the impact of each cell population may be examined. Unfortunately, germ cell development does not proceed further than the pachytene stage of meiosis there, with rare exceptions. Finally, we describe recent studies that focus on germ cells in a conventional adherent cell culture. Such studies thoroughly examine issues with in vitro meiosis and provide insight into the mechanisms of meiotic initiation.

Creation of a Model of Co-Culturing of Sertoli-Like Mouse Cells with Spermatogonial Cells.

Sertoli-like cells is a cell population in the testes of adult mice capable of growth in culture and expressing many genes typical of Sertoli cells and supporting the development of germ cells in the gonad. A technique of co-culturing of Sertoli-like cells with spermatogonial cells was proposed that allows maintaining the growth and viability of germ cells and inducing their differentiation. This technique can provide the basis for obtaining fully differentiated germ cells in culture through using Sertoli-like cells as the supporting somatic cells.

[Interaction of herpesviruses with mature human spermatozoa in the model system in vitro].

The DNA of human herpesviruses (HHV), including the herpes simplex virus (HSV) and cytomegalovirus (CMV), is often identified in ejaculates of patients with urogenital diseases and infertility. At least a part of viral DNA is associated with cell fraction of ejaculate. However, it remains unclear how the semen is infected by the virus. It can be located in gametes or be capable of infecting mature germ cells, including motile sperm cells. In order to resolve this issue, interactions of the CMV and HSV with human sperm cells were studied using an original optimized model of the herpesviral infection of male gametes in vitro. The analysis of the immunofluorescent staining of gametes for viral antigens has shown that CMV infected 2% gametes, while HSV infected 17.26 ± 2.58% gametes. The fraction of progressively motile sperm cells contained 13.99 ± 4.64% infected cells. Localization of HSV was studied by the confocal microscopy. Sometimes, viral gB protein was found on sperm cell membrane. In addition, optical scanning of other cells has shown the intracellular localization of the viral proteins. In the majority of spermatozoa, the viral proteins were observed in the head and neck. In some cells, they were located in the middle piece or, rarely, in the equatorial segment. In general, after in vitro infection HSV antigens were located in the same areas of the sperm cells as in ejaculates from infected patients. According to DNA-DNA hybridization in situ, gametes containing HSV DNA accounted for 16.94 ± 5.28%, which is consistent with the results obtained in the immunofluorescence assay. It can be concluded that mature male gametes are infected by HHV in the genital tract, where the virus binds to the sperm cell membrane and enters the cell. Interaction of HHV with progressively motile sperm cells implies a vertical viral transmission upon fertilization and points to the necessity of testing ejaculate for herpesviruses infections.

[Spermatogenesis recovery following allogeneic transplantation of undifferentiated Sertoli cells in experimental model of bilateral abdominal cryptorchidism].

This study explores the effect of transplantation of undifferentiated Sertoli cells in the testicular tissue of an experimental animal model of bilateral abdominal cryptorchidism. Effectiveness of undifferentiated Sertoli cell transplantation was assessed after 1 and 3 months after injection. Partial restoration of seminiferous tubules was found to occur after transplantation. In a third of them germ cells of all stages of the differentiation were discovered while they were not found in the control group.

Detection and quantification of human herpes viruses types 4-6 in sperm samples of patients with fertility disorders and chronic inflammatory urogenital tract diseases.

Acute and chronic infections of the seminal tract are among the most common causes of male infertility. As at least half of male infertility cases are classified as idiopathic, some of these cases might be attributed to asymptomatic infection. The detection and quantification of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpes virus type 6 (HHV-6) DNA in semen samples were performed. A total of 232 patients were divided into five groups: (i) infertile men with varicocoele; (ii) men with idiopathic infertility; (iii) infertile men with chronic inflammatory urogenital tract diseases (IUTD); (iv) fertile men with IUTD and (v) men whose partners had a history of pregnancy loss. In the study population, the prevalence of viral DNA was 17.7, 3.4% for EBV, 5.2% for CMV, 6.5% for HHV-6, 0.43% for EBV + CMV, 0.87% for EBV + HHV-6 and 1.3% for CMV + HHV-6. The median viral loads for EBV, CMV and HHV-6 were 500, 2250 and 250 copies/mL respectively. Of the sperm cell fractions, derived from infected samples 87.5% contained viral DNA. No association between EBV and fertility disorders or IUTD was found. CMV detection was much higher in the group of patients with infertility and concomitant IUTD compared with the other groups combined (18.5% vs. 5.4%, p = 0.03) and associated with reduced sperm cell count (39.5 × 10(6) /mL vs. 72.5 × 10(6) /mL, p = 0.036). Immunostaining of spermatozoa from infected samples and in vitro-infected cells detected CMV in sperm heads, tails and connecting pieces and revealed attachment to sperm membrane and intracellular localization. HHV-6 was the more common in fertile men with chronic IUTD than in the other groups combined (19% vs. 6.3%, p = 0.018) and had no effect on sperm parameters. The results suggest that both CMV and HHV-6 may contribute to the aetiology of IUTD and, moreover, CMV-associated IUTD can lead to male sterility.

[Destructive changes in the mice testes in retrograde infection with herpes simplex virus].

Herpes simplex virus (HSV) causes inflammatory diseases of the genitourinary system of males, infects male sex cells, and its presence in the ejaculate is associated with infertility. However, information on the pathways of HSV in the testicles, the extent of damage of spermatogenic tissue and the effect on spermatogenesis are insufficient. This work was aimed to the evaluation of effect of HSV on mice spermatogenesis in retrograde infection with the virus. Molecular (RT-PCR), virologic, morphological and immunohistochemical methods were used. Analysis showed that after virus inoculation directly into seminiferous tubules the viral protein is found in all layers of seminiferous epithelium. On the third day of infection the proportion of tubules containing HSV protein was 4.9%, reached a maximum on day 6 - 23,5 and 18% for the high and low doses of HSV, respectively, and then decreased; viral protein was not detected on 21th and 45th day. HSV DNA was detected in the testes at all stages of infection. Since the 14th day after infection, testes weight was significantly reduced compared to the control: 7,9-fold decrease at 45th day with a high dose of HSV, and 4,9-fold decrease with low dose. The infection with HSV led to the development of orchitis and considerable destructive changes in the spermatogenic tissue. The proportion of morphologically normal tubules was reduced to 6 and 15% at day 14 and remained at a low level up to 45th day. Approximately half of the seminiferous tubules (46.5%) at the 14th and 21th day had no somatic Sertoli cells needed for the restoration of spermatogenic tissue. These data suggests that retrograde infection of male gonads with HSV leads to the structure damage of testis and death of germ and somatic cells, indicating the irreversibility of degenerative changes in infected testes.

[Characterization of cultured Sertoli cells under high-temperature and hypoxic conditions].

Sertoli cells (SCs) isolated from adult C57Bl/6 mice were characterized under four different cell culture conditions: standard conditions (34 degrees C, 21% O2 - 34_21), high-temperature conditions (37 degrees C, 21% O2 - 37_21), hypoxic conditions (34 degrees C, 5% O2 - 34_5), and combination of these conditions (37 degrees C, 5% O2 - 37_5). Proliferation and viability were promoted when SCs were grown under hypoxia: 28.5 and 24.6% of SCs were BrdU-positive at the peak of proliferation, 92.7 and 92.7% of SCs were viable after 15 days in culture at 34_5 and 37_5, respectively, versus 20.2 and 88.9% at 34_21, respectively. In SCs grown under high-temperature conditions proliferation was slightly increased, but viability was decreased: 23.1% of SCs were BrdU-positive, and only 74.9% of SCs were viable at 37_21. At the same time cultivation of SCs at 37 degrees C promoted their dedifferentiation: after 15 days in culture 98.8 and 98.6% of cells at 37_5 and 37_21, respectively, expressed a marker of immature SCs--cytokeratin-18, compared to 26.5% at 34_5 and 6.6% at 34_21. Expression of Wt1, a transcription factor controlling cell-cell junction formation and germ cell development, disappeared in most cells after 3 days in culture under all culture conditions. However, SCs forming colonies restored Wt1 expression at day 15 in culture under high-temperature conditions: 59.1 and 29.5% of SCs were Wt1-positive at 37_21 and 37_5, respectively, versus 11.1 and 3.6% at 34_21 and 34_5, respectively. Cultured SCs expressed other SC markers (vimentin, clusterin, Gata-4) under all culture conditions. Our results show that cultured SCs may be useful for reproductive biology and regenerative medicine.

Regeneration potential of transplanted adult mouse sertoli cells.

The regeneration potential of differentiated Sertoli cells subjected to thermal treatment was studied by the method of cell transplantation. Cells from mice with artificial cryptorchism (1.5 months after fixation of the testes in the body) and after culturing (10 days, 37°C) were transplanted. Transplantation of Sertoli cells from 2-3-month-old and 2-day-old mice served as controls. The cells were transplanted into the testes of recipient mice, from which sex cells and Sertoli cells were removed by busulfan and cadmium salt treatment. Adult mouse Sertoli cells exposed to thermal treatment exhibited much higher regeneration potential than intact cells. Two months after transplantation, mature Sertoli cells subjected to thermal treatment populated the recipient testicular tubules, formed new tubules, and in some cases supported the development of sex cells similarly as immature cells from newborn mice.

[Ageing of the spermatogenesis system].

This review summarizes the data characterizing the effect of ageing on the development of male germ cells and their hereditary structures. We have studied causes of spermatogenesis reduction at late stages of ontogenesis. We have focused on age-specific changes of the structural-functional integrity of stem spermatogonial cells and their microenvironment (niche). We also examined several unique and specific features of the spermatogenic system in senescence-accelerated mutant mice (SAM), with accelerated ageing.

[Estimation of the frequencies of induced mutations in spermatogenic cells of senescence-accelerated prone mice of the SAMP1 strain].

The results obtained in this work demonstrate the dynamics of cytogenetic changes of spermatogenic cells in senescence-accelerated prone mice, strain SAMP1, after a single exposure to a chemical mutagen, dipin, at a genetically active dose of 30 mg/kg. In the time interval between days 3 and 28 the frequency of induced spermatogonial micronuclei does not significantly exceed the level of spontaneous mutagenesis. The lack of an experimental effect of micronuclei in this time interval is probably a consequence of mitotic delay and (or) of the death of a considerable part of genetically defective cells in the spermatogonial compartment. Different stages of meiosis exhibit different chemical sensibilities: the yield of round spermatids with micronuclei is maximum after treatment of early primary spermatocytes (preleptotene-leptotene stage) with dipin. The high sensibility of preleptotene and leptotene spermatocytes is confirmed by the sperm head shape abnormality assay. Chromosome damage caused by dipin in spermatogonial stem cells is irreversible, as evidenced by a sharp increase in the frequencies of spermatogonial and meiotic micronuclear aberrations within long periods after treatment. Increased genetic instability in the stem compartment does not lead to irreversible degradation of the system of development of male sex cells in senescence-accelerated SAMP1 mice.