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.

[Antibacterial antibodies in human immunoglobulins and sera: past and present].

AIM: To measure levels of several types of antibacterial antibodies in preparations of normal human immunoglobulin as well as in samples of donor sera obtained in 1965 and 2009. MATERIALS AND METHODS: Five batches of human normal immunoglobulin manufactured in 1965 and five batches manufactured in 2009 as well as 77 and 28 blood serum samples respectively were tested by agglutination assay for the presence of antibodies to enterobacteria, Brucella species, tularemia agent, Rickettsia burnetii, Rickettsia prowazekii, and several species of opportunistic bacteria. RESULTS: Higher antibody titers to Salmonella typhi, Salmonella paratyphi A and B, Salmonella enteritidis, Salmonella typhimurium, Shigella flexneri and Shigella sonnei were revealed in immunoglobulin preparations and donor sera obtained in 1965 compared to that obtained in 2009. There was no difference in antibody titers to Shigella boydii, Salmonella choleraesuis, Escherichia coli O-55, Pseudomonas aeruginosa, Proteus vulgaris, Serratia marcescens and E. coli. Antibodies to Brucella species, tularemia agent, R. burnetii, R. prowazekii were not detected in normal human immunoglobulin. CONCLUSION: Decrease of antibody levels to several pathogenic enterobacteria in human immunoglobulin preparations as well as in sera of donors for 40 years could be linked with decrease of number of immunized persons, changes in circulation of pathogenic bacteria, decrease of rate of asymptomatic infections. Stability of antibody titers to opportunistic bacteria is a rationale to use them for assessment of humoral immunity function.

[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.

[Response of the spermatogenic system to chemical mutagen dipin in SAMP1 senesce-accelerated mice].

Specific features of spermatogenesis were studied in senesce-accelerated mice of the line SAMP-1 after one-time injection of the chemical mutagen dipin. Quantitative and histomorphological changes in the spermatogenic epithelium proved to develop gradually. Cell loss and disorganization of spermatogenesis reached the peak as late as on days 28 and 35 after the injection. Differentiating spermatogonia manifested increased sensitivity to dipin. In prophase I of meiosis, developing spermatocytes proved to be less sensitive to the cytotoxic action of dipin at the pachytene than at the preleptotene-leptotene stages. Spermatogenesis in most seminiferous tubules was restored by day 56 after dipin treatment. At the end of the experiment (day 100), both quantitative parameters and morphological pattern of spermatogenesis did not differ significantly from those in the control. Thus, the cytotoxic action of dipin does not lead to irreversible structural disorganization of the spermatogenic epithelium in SAMP1 mice. Radioautography revealed a large proportion of highly differentiated Sertoli cells with 3H-thymidine-labeled nuclei in experimental animals. In some cases, structures resembling embryonic seminiferous tubules were revealed in the vicinity of rete testis in testis sections of experimental mice. These structures contained the cells morphologically similar to gonocytes and young Sertoli cells.

[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.

[Analysis of spermatogenesis in senescence-accelerated mice].

A comparative analysis of age-related dynamics of spermatogenesis has been performed in mutant mouse lines predisposed or resistant to accelerated senescence (SAMP1 and SAMR1 respectively). The results show that quantitative and morphohistological trends in the development of sperm cells and Sertoli cells in both lines are similar in both lines. Their comparison with data obtained in our previous studies (Zakhidov et al., 2001; Gordeeva et al., 2001) shows that sharp quantitative and qualitative changes in the structure of the spermatogenic system have occurred in senescence-accelerated mice of new generations, which confirms the fact of dynamic instability of the germinal lineage. The role of stem spermatogonial cells in restoration of spermatogenesis in animals reaching the critical age is discussed.

Carnosine-induced changes in the development of spermatogenic epithelial cells in senescence accelerated SAM mice.

Carnosine significantly increased the number of spermatogonia and Sertoli cells in mice prone (SAMP1) and resistant (SAMR1) to accelerated aging and appreciably reduced cell yield in meiosis and spermiogenesis in SAMP1 mice. In experimental SAMP1 mice catastrophic changes in the number of gametes were paralleled by intensive degradation of the spermatogenic epithelium. In SAMR1 mice treated with carnosine highly ordered spermatogenic structure was preserved.

Carnosine modifies the incidence of genetically abnormal sex cells in the testes of senescence-accelerated mice.

The quantitative micronucleus test showed that the natural dipeptide carnosine increases the count of aberrant spermatogonia and round spermatids in the testes in SAMR1 mice resistant to accelerated aging by 64 and 85%, respectively, compared to the control. However, this agent did not modify the incidence of chromosome mutations in spermatogenic cells in SAMP1 mice predisposed to accelerated aging.