Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells

Susanna Dolci ) 2 3 Lauretta Levati 1 2 Manuela Pellegrini 2 3 Isabella Faraoni 0 2 Grazia Graziani 0 2 Anna Di Carlo 2 3 Raffaele Geremia 2 3 0 Dipartimento di Neuroscienze, Universita' di Roma Tor Vergata , Rome , Italy 1 Istituto Dermopatico dell'Immacolata (IDI, IRCCS) , Rome , Italy 2 Key words: Kitl , Kit, Telomerase, Germ cells, Meiosis, Proliferation, PI3K 3 Dipartimento di Sanita' Pubblica e Biologia Cellulare , Sezione di Anatomia - The discovery of sterility in the descendants of telomerasenull mutant mice, owing to the lack of spermatogonia proliferation, has drawn attention to the role of telomerase activity in mouse spermatogenesis. Since spermatogonia proliferation is under Kitl control, we explored its possible role in the regulation of telomerase activity. We show that Kitl induces telomerase activity in mitotic spermatogonia and increases the mRNA levels of both the catalytic subunit form and the telomerase RNA template. The increase of telomerase activity by Kitl is blocked by the presence of the PI3K inhibitor LY294002. Kit-positive proliferating male Telomerase is a ribonucleoprotein that adds hexameric repeats to the mammalian telomeres to compensate for the loss of basepairs that occurs after subsequent rounds of DNA replication. This enzyme consists of an RNA-dependent DNA polymerase (TERT), which synthesizes the hexameric repeats on the 3 end of the telomeres using a RNA molecule (TR) as a template (Blackburn et al., 1991; Greider et al., 1996). Progressive shortening of the telomeric ends, which occurs in the absence of telomerase, has been suggested to act as a mitotic checkpoint that contributes to cell senescence and mortality (Harley, 1991). Telomerase activity has been shown to be present in human and mouse tumors, immortalized cell lines, stem cells of self-renewing tissues and germ cells (Kim et al., 1994; Wright et al., 1996). Human somatic cells do not express telomerase activity, whereas most mouse somatic cells express detectable amounts of telomerase (Kipling, 1997). However, homologous recombination of the TR gene does not alter the normal phenotype of the knock-out mice, and primary cells obtained from these animals can be oncogenically transformed after tumorigenic viral infection (Blasco et al., 1998), which may suggest that telomerase activity may not be limiting for tumorigenesis in the murine system. Interestingly, after several generations of breeding, the telomerase-null mice develop progressively worse effects on the reproductive and hematopoietic organs (Lee et al., 1998). In particular, homozygous male mice show a reduced testis weight compared with control animals, absence of germ cells within the tubules, impairment of long term renewal of hematopoietic stem cells and increased apoptosis of activated primordial germ cells (PGCs) show low levels of telomerase activity, but they increase telomerase activity upon Kitl stimulation. Diplotene-arrested growing oocytes that reexpress Kit do not increase telomerase activity upon Kitl stimulation. Our data suggest that the induction of telomerase by Kitl may contribute to the self-renewing potential of male germ cells and of PGCs. splenocytes, suggesting that telomerase is involved in the control of normal cell growth and survival. In the mouse testis, telomerase activity has been reported mainly in proliferating spermatogonia (type A spermatogonia), and it is downregulated in the differentiating spermatocytes and spermatids and is no longer present in spermatozoa (Ravindranath et al., 1997; Eisenhauer et al., 1997). As a result of telomerase activity, sperm cells have long telomeres, of about 10-20 kb in humans and 50 kb in mice, that apparently do not shorten with the aging of the organism (Allsopp et al., 1992). Type A spermatogonia are pluripotent stem cells in the testis, which undergo many rounds of duplications giving rise to type B spermatogonia and to other type A spermatogonia. The mechanisms of germ cell mitogenesis are poorly understood, but we have recently shown that stem cell factor (Kit ligand, Kitl) can induce 3H-thymidine incorporation in type A, Kit expressing spermatogonia in vitro (Rossi et al., 1993). In light of the recent reports that spermatogonia lacking telomerase undergo arrest of mitosis and apoptosis, we investigated whether Kitl is able to regulate testicular stem cell telomerase activity. Since Kitl is a survival/proliferation factor for proliferating primordial germ cells (PGCs) but not for Kitexpressing growing oocytes, we investigated its role in regulating telomerase activity in these cell types. Materials and methods Male PGCs were obtained from 12.5, 14.5 and 15.5 day post-coitum (dpc) CD-1 embryos according to De Felici and McLaren (De Felici and McLaren, 1982). Briefly, gonads were collected in PBS and incubated for 15 minutes in PBS-EDTA. Germ cells were released in PBS+BSA (1 mg/ml) by puncturing the gonads with fine needles under a stereomicroscope. Cells were then collected by a mouthoperated micropipette and cultured in suspension for 24 hours in 10% FCS with D-MEM added, in the presence or absence of 100 ng/ml Kitl. An equal number of surviving cells, which were trypan-blue negative, at each experimental point were immediately frozen. Contaminating somatic cells were less than 10% of the total, as judged by both alkaline phosphatase staining and nuclear morphology of Giemsa-stained cells. Growing oocytes were obtained from 10 day post-natum (dpn) mice by puncturing ovaries with fine needles under a stereomicroscope. At the beginning of the culture (T0) and after 24 hours of culture in the presence or absence of Kitl, groups of 15 viable oocytes were collected in 5 m l of PBS+BSA and immediately frozen. The occurrence of germinal vesicle breakdown (GVBD) was evaluated in three different experiments by direct observation of cells under a stereomicroscope. Spermatogonia were obtained from 7-8 days old Swiss CD-1 mice, as previously reported by Rossi et al. (Rossi et al., 1993). Briefly, germ cell suspensions were obtained by sequential collagenasehyaluronidase-trypsin digestions of freshly withdrawn testes. A 3 hour period of culture in 10% FCS with E-MEM added was performed to facilitate adhesion of contaminating somatic cells to the plastic dishes. At the end of this pre-plating treatment, which was considered to be T0, enriched germ cell suspensions were rinsed from FCS, and spermatogonia were then cultured in E-MEM supplemented with 2 mM Na-pyruvate and 1 mM Na-lactate in the presence or absence of Kitl (100 ng/ml, Genzyme). To obtain mitotic indexes, nuclear morphologies from at least 103 cells per treatment were assessed by microscope observations of the cell cultures previously fixed in 3:1 methanol-acetic acid and stained with Giemsa, according to Meistrich et al. (Meistrich et al., 1973). The purity of the spermatogonia was about 80% after the pre-plating treatment. During the 24 hours of culture, the contaminating soma (...truncated)