Transmeiotic differentiation of male germ cells in culture

Inaba

The Journal of Veterinary Medical Science

1998

ABSTRACT. In male hypogonadic mutant rat (hgn/hgn), gonocytes degenerate and peritubular cells form multiple layers around seminiferous tubules during early postnatal testicular development. Alkaline phosphatase (AP) activity has been used as not only a tracer for the primordial germ cells (PGCs) but also a histochemical marker for the peritubular myoid cells. In the present study, we examined the localization of AP activity during the postnatal testicular development in the hgn/hgn and phenotypically normal (+/+ or +/hgn) rat. In the normal testis, high AP activity was located in the surface of the PGCs on 3 days of age. As the PGCs differentiated into spermatogonia, the AP activity drastically decreased in intensity on 7 days and was completely lost by 12 days. In the hgn/hgn, the PGCs showing high AP activity occupied the inside of dilated seminiferous tubules on 3 and 7 days of age. The luminal AP activity declined gradually by 18 days and disappeared on 21 days, when the germ cells completely degenerated in the hgn/hgn testis. In the normal, high AP activity emerged in a layer of the peritubular cells surrounding the tubules on 7 days and afterwards, indicating that the peritubular cells were differentiating into myoid cells. In the hgn/hgn, the peritubular cells formed multiple layers around the tubules and showed weak AP activity on 3 to 18 days of age. On 21 days of age, high AP activity emerged in a single layer of the peritubular cells directly attached to the basement membrane in the hgn/hgn. These results indicate that the PGCs showing AP activity kept remained at later stage in the hgn/ hgn and that the single layer of mature myoid cells showing high AP activity appeared much later in the hgn/hgn testis than in normal. -KEY WORDS: alkaline phosphatase, hypogonadism, peritubular myoid cell, primordial germ cell, testicular development.J. Vet. Med. Sci. 60(6): 671-679, 1998 cells, the abnormal cell division of the gonocytes, the degeneration of the gonocytes, and the formation of multiple layers of the peritubular cells around the seminiferous tubules [32]. Alkaline phosphatases (APs) are membranelinked enzymes that hydrolyze orthophosphate monoesters at alkaline pH [4]. In normal murine testis, AP activity is predominantly localized in the gonocytes and the myoid cells [2,11,16,22]. In this study, we examine the temporal and spatial distribution of AP activity in the postnatal hgn/ hgn and normal (+/+ or hgn/hgn) testis to investigate the distribution and development of the hgn/hgn gonocytes and peritubular myoid cells. MATERIALS AND METHODS Animals:Male normal and hgn/hgn rats used in this study were obtained from matings between proven +/hgn males and females derived from the 21st generation of hypogonadic rat (HGN) line maintained in our department [29]. The rats were kept in a conventional animal room, and were fed a mixed grain diet and water ad libitum [31].Preparation of cryosectioned testis samples: The rats were sacrificed on 3, 7, 12, 18, 21, and 40 days after birth by overdose ether. Th...

Section: Resultsmentioning

confidence: 99%

Temporal and Spatial Distribution of Alkaline Phosphatase Activity in Male Hypogonadic Rat (hgn/hgn) Testis during Postnatal Development.

Inaba

The Journal of Veterinary Medical Science

1998

“…Transplanted GC-1 cells were capable of differentiating into spermatids in an in vivo microenvironment (Wang et al 2009). The 15P1 cell line exhibits some characteristics of Sertoli cells, including their transcription of Wilms' tumor (Wt1) and Kitl (Steel) genes (Rassoulzadegan et al 1993), and their phagocytic activity (Grandjean et al 1997). The cells also express KITL (KL) protein, a ligand of the KIT receptor, which is also expressed in Sertoli cells (Vincent et al 1998, Vidal et al 2001.…”

Section: Methodsmentioning

confidence: 99%

SUMO proteins are involved in the stress response during spermatogenesis and are localized to DNA double-strand breaks in germ cells

Shrivastava¹,

Pekar²,

Grosser³

et al. 2010

Reproduction

Small ubiquitin-like modifiers (SUMO) proteins have been implicated in cellular stress response in different tissues, but whether sumoylation has a similar role during spermatogenesis is currently unknown. In this study, changes in the levels of both free SUMO isoforms and high-molecular weight (HMW) SUMO conjugates were monitored before and after the induction of different types of cellular stresses. Using cell lines and primary cells freshly isolated from mouse testes, significant changes were detected in the levels of SUMO1 and SUMO2/3 conjugates following short exposure of the cells to heat stress and oxidative stress. While high concentrations of H 2 O 2 caused an increase in protein sumoylation, low concentrations of H 2 O 2 mostly caused protein desumoylation. Immunofluorescence studies localized SUMO to the sites of DNA double-strand breaks in stressed germ cells and during meiotic recombination. To study the effect of oxidative stress in vivo, animals exposed to tobacco smoke for 12 weeks were used. Changes in sumoylation of HMW proteins were consistent with their oxidative damage in the tobacco-exposed mice. Our results are consistent with the important roles of different SUMO isoforms in stress responses in germ cells. Furthermore, this study identified topoisomerase 2 a as one of the targets of sumoylation during normal spermatogenesis and under stress.

“…Several experiments have identified physical associations and even cellular junctional attachments between germ cells and Sertoli cells under culture conditions (Smith et al, 1992). Other studies have demonstrated maturation or brief differentiation sequences in vitro (Hofmann et al, 1992(Hofmann et al, , 1994Rassoulzadegan et al, 1993). Often cells remaining in culture retain some morphological characteristics of spermatogonia, such as large nuclear to cytoplasmic ratio, and it seems likely these primitive cells can be maintained in culture for several days or perhaps a few weeks (Kierszenbaum, 1994).…”

Section: Introductionmentioning

confidence: 99%

Culture of mouse spermatogonial stem cells

et al. 1998

Spermatogenesis occurs within the seminiferous tubules of mammals by a complex process that is highly organized, extremely efficient and very productive. At the foundation of this process is the spermatogonial stem cell that is capable of both self-renewal and production of progeny cells, which undergo differentiation over a period of weeks to months in order to generate mature spermatozoa. It had been thought that germ cells survive only a brief period in culture, generally less than a few weeks. However, an accurate assessment of the presence of spermatogonial stem cells in any cell population has only recently become possible with development of the spermatogonial transplantation technique. Using this technique, we have demonstrated that mouse spermatogonial stem cells can be maintained in culture for approximately 4 months and will generate spermatogenesis following transplantation to the seminiferous tubules of an appropriate recipient. Extensive areas of cultured donor cell-derived spermatogenesis are generated in the host, and production of mature spermatozoa occurs. Cultivation of the testis cells on STO feeders is beneficial to stem cell survival. These results provide the first step in establishing a system that will permit spermatogonial stem cells to be cultivated and their number increased in vitro to allow for genetic modification before transplantation to a recipient testis.