Francesca Gioia Klinger

Inhibition of the c-Abl–TAp63 pathway protects mouse oocytes from chemotherapy-induced death

Germ cells are sensitive to genotoxins, and ovarian failure and infertility are major side effects of chemotherapy in young patients with cancer. Here we describe the c-Abl–TAp63 pathway activated by chemotherapeutic DNA-damaging drugs in model human cell lines and in mouse oocytes and its role in cell death. In cell lines, upon cisplatin treatment, c-Abl phosphorylates TAp63 on specific tyrosine residues. Such modifications affect p63 stability and induce a p63-dependent activation of proapoptotic promoters. Similarly, in oocytes, cisplatin rapidly promotes TAp63 accumulation and eventually cell death. Treatment with the c-Abl kinase inhibitor imatinib counteracts these cisplatin-induced effects. Taken together, these data support a model in which signals initiated by DNA double-strand breaks are detected by c-Abl, which, through its kinase activity, modulates the p63 transcriptional output. Moreover, they suggest a new use for imatinib, aimed at preserving oocytes of the follicle reserve during chemotherapeutic treatments.

Derivation in culture of primordial germ cells from cells of the mouse epiblast: phenotypic induction and growth control by Bmp4 signalling.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes of the adult. PGCs derive from cells of the most proximal part of the cup-shaped epiblast corresponding to the presumptive region of the extraembryonic mesoderm. At 7.2 days post coitum (dpc) a small group of PGCs located at the base of the allantois can be recognised due to a strong alkaline phosphatase activity. Thus far, scant information was available on the mechanism(s) controlling the lineage of PGCs in the mouse embryo. However, results obtained in mice defective for bone morphogenetic protein-4 (Bmp4) secreted molecule revealed that this growth factor has important functions for the derivation of PGCs from extraembryonic mesoderm cells. In this paper, we have studied the effects in culture of Bmp4 on epiblast cells obtained from egg-cylinder stage mouse embryos (5.5-6.0 dpc) and PGCs from 11.5 dpc embryos. We found that Bmp4 treatment enables recruitment of pluripotent cells to a PGC phenotype by a multi-step process involving an initial pre-commitment of epiblast cells and a following stage of PGC phenotypic determination. We further provide evidences that Bmp4 may promote the growth of gonadal PGCs through a Smad1/4 signalling.

Establishment of oocyte population in the fetal ovary: primordial germ cell proliferation and oocyte programmed cell death

Strict control of cell proliferation and cell loss is essential for the coordinated functions of different cell populations in complex multicellular organisms. Oogenesis is characterized by a first phase occurring during embryo-fetal life and in common with spermatogenesis, during which mitotic proliferation of the germline stem cells, the primordial germ cells (PGC), prevails over germ cell death. The result is the formation of a relatively high number of germ cells depending on the species, ready to enter sex specific differentiation. In the female, PGC enter into meiosis and become oocytes, thereby ending their stem cell potential. After entering into meiosis in the fetal ovary, oocytes pass through leptotene, zygotene and pachytene stages before arresting in the last stage of meiotic prophase I, the diplotene or dictyate stage at about the time of birth. The most part of oocytes die during the fetal period or shortly after birth. It is widely accepted that in mammals a female is born with a fixed number of oocytes within the ovaries, which over the years progressively decreases without possibility for renewal. Once the oocyte reserve has been exhausted, ovarian senescence, driving what is referred to as the menopause in women, rapidly ensues. The fertile lifespan of a female depends by the size of the oocyte pool at birth and the rapidity of the oocyte pool depletion. Which mechanisms control PGC proliferation? Why do most of the oocytes die during fetal life and what are the mechanisms of such massive degeneration? Is it possible to prolong the lifespan of a female by reducing oocyte lost during the fetal life? This review reports some of the most recent results obtained in an attempt to answer these questions.

Poly(ADP-ribosyl)ation Acts in the DNA Demethylation of Mouse Primordial Germ Cells Also with DNA Damage-Independent Roles

Poly(ADP-ribosyl)ation regulates chromatin structure and transcription driving epigenetic events. In particular, Parp1 is able to directly influence DNA methylation patterns controlling transcription and activity of Dnmt1. Here, we show that ADP-ribose polymer levels and Parp1 expression are noticeably high in mouse primordial germ cells (PGCs) when the bulk of DNA demethylation occurs during germline epigenetic reprogramming in the embryo. Notably, Parp1 activity is stimulated in PGCs even before its participation in the DNA damage response associated with active DNA demethylation. We demonstrate that PARP inhibition impairs both genome-wide and locus-specific DNA methylation erasure in PGCs. Moreover, we evidence that impairment of PARP activity causes a significant reduction of expression of the gene coding for Tet1 hydroxylases involved in active DNA demethylation. Taken together these results demonstrate new and adjuvant roles of poly(ADP-ribosyl)ation during germline DNA demethylation and suggest its possible more general involvement in genome reprogramming.

Identification of multipotent cytotrophoblast cells from human first trimester chorionic villi.

In this article we used immunohistochemistry and FACS analyses to show that cells expressing markers typical of human stem cells such as SSEA4, OCT-4, ALP, and CD117 are present within the cytotrophoblastic tissue of human fetal chorionic villus samples (CVSs). After immunoselection of CV cells for SSEA4, FACS analyses showed an increased number of cells positive for OCT-4 and ALP and a small percentage (around 4%) of side population (SP) cells. In the same cell population, RT-PCR indicated the presence of OCT-4, NANOG, and SOX2 transcripts, also typical of stem cells. Depending on the in vitro conditions, a subset of SSEA4+ cells formed colonies resembling hESCs, with limited self renewal ability. At the same time, these cells were able to differentiate in vitro into derivatives of all three germ layers. When inoculated into immunocompromised mice, SSEA4+ cells did not form teratomas but were able to populate depleted hematopoietic tissues. Moreover, after injection into mouse blastocysts, they were incorporated into the inner cell mass and could be traced into several tissues of the adult chimeric mice. Finally, we show that SSEA4+ cells isolated from fetuses affected by Spinal Muscular Atrophy (SMA) can be genetically corrected with high efficiency in culture by Small Fragment Homologous Recombination (SFHR), a gene targeting approach. Taken together, our results indicate that SSEA4+ cells obtained from human CVSs contain a subpopulation of multipotent cells that we propose to name Human Cytotrophoblastic-derived Multipotent Cells (hCTMCs). These cells may be a safe and convenient source of cells for cell-based therapy, as well as an ideal target for in utero fetal gene therapy.

Minimal Concentrations of Retinoic Acid Induce Stimulation by Retinoic Acid 8 and Promote Entry into Meiosis in Isolated Pregonadal and Gonadal Mouse Primordial Germ Cells

ABSTRACT In the present study, we demonstrate that minimal concentrations (≤1 nM) of retinoic acid (RA), equivalent to the quantity contaminating serum-containing culture medium, are sufficient to promote meiotic entry and progression through meiotic prophase I (MPI) stages in isolated 12.5-days postcoitum (dpc) XX and XY mouse primordial germ cells (PGCs) in culture. Similarly, we found that the same low RA concentration up-regulated or induced stimulation by retinoic acid 8 (Stra8) in such cells, both at mRNA and protein level. In preleptotene/leptotene germ cells, STRA8 was localized in nuclear dots that disappeared at later MPI stages. In addition to Stra8, other meiotic genes such as Dmc1 and Rec8 appeared stimulated by RA directly in PGCs with similar concentration-dependent trends. Finally, we found that RA induced Stra8, Sycp3, Dmc1, and Rec8 transcripts, promoting meiotic entry in culture also in pregonadal 10.5-dpc PGCs of both sexes. When cultured isolated from somatic cells, such PGCs, however, were unable to progress through MPI stages, while after entering meiosis, they progressed through MPI when cultured within aorta/gonad/mesonephros tissues. We conclude that besides RA, germ cell intrinsic factors and other exogenous signals from the surrounding somatic cells are probably necessary for meiotic entry and progression in mouse PGCs.

Hematopoietic activity in putative mouse primordial germ cell populations

In the present paper, starting from the observation of heterogeneous expression of the GOF-18ΔPE-GFP Pou5f1 (Oct3/4) transgene in putative mouse PGC populations settled in the aorta-gonad-mesonephros (AGM) region, we identified various OCT3/4 positive populations showing distinct expression of PGC markers (BLIMP-1, AP, TG-1, STELLA) and co-expressing several proteins (CD-34, CD-41, FLK-1) and genes (Brachyury, Hox-B4, Scl/Tal-1 and Gata-2) of hematopoietic precursors. Moreover, we found that Oct3/4-GFP(weak) CD-34(weak/high) cells possess robust hematopoietic colony forming activity (CFU) in vitro. These data indicate that the cell population usually considered PGCs moving toward the gonadal ridges encompasses a subset of cells co-expressing several germ cell and hematopoietic markers and possessing hematopoietic activity. These results are discussed within of the current model of germline segregation.

c-Flip expression and function in fetal mouse gonocytes

Apoptosis is a key mechanism in spermatogenesis, and in testis, most gonocytes degenerate at fetal and postnatal ages to select a cell subset committed to become germ stem cells. The aim of the present study is to investigate mechanisms controlling the massive apoptosis of fetal gonocytes. We evaluated the expression and function of c‐Flip, an apoptosis inhibitor known to interfere with the proapoptotic Fas‐signaling pathway in a variety of cell types, but never investigated in fetal testis. Expression of c‐Flip long isoform (c‐FlipL) within fetal testis was localized in gonocytes at 16.5 and 18.5 days post coitum (dpc), both at the mRNA and protein level, while it was weakly expressed or undetectable at earlier stages. Moreover, Fas protein was found in fetal testes at 13.5, 16.5, and 18.5 dpc. Testes at 18.5 dpc, expressing high levels of c‐FlipL, were resistant to Fas‐induced apoptosis while they became highly sensitive when c‐FlipL was inhibited by antisense c‐Flip oligos. In addition, there was an inverse relation between gonocyte spontaneous apoptosis sensitivity and c‐FlipL levels. Furthermore, caspase‐10 activity was inversely related with c‐FlipL expression, suggesting that caspase‐10 might be a target of c‐FlipL. These data represent the first evidence demonstrating c‐Flip expression in fetal testes and its role in protecting gonocytes from Fas‐dependent apoptosis.

Stage-variations of anandamide hydrolase activity in the mouse uterus during the natural oestrus cycle

Recent studies have demonstrated that the endogenous cannabinoids are important modulators of fertility in mammals. In particular, a role of the endocannabinoid system in early stages of embryo development, oviductal transport of embryos, pregnancy maintenance and labour has been demonstrated in rodents and/or in humans. In the present paper, we report the analysis of FAAH activity and protein content in the mouse uterus as a function of the natural oestrus cycle stages. Variations of FAAH activity are discussed in relationship to changes in sex steroid levels and to the possible action of AEA on remodelling of uterine tissues.

Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63

The survival rate of cancer patients is steadily increasing, owing to more efficient therapies. Understanding the molecular mechanisms of chemotherapy-induced premature ovarian insufficiency (POI) could identify targets for prevention of POI. Loss of the primordial follicle reserve is the most important cause of POI, with the p53 family member p63 being responsible for DNA-damage-induced apoptosis of resting oocytes. Here, we provide the first detailed mechanistic insight into the activation of p63, a process that requires phosphorylation by both the priming kinase CHK2 and the executioner kinase CK1 in mouse primordial follicles. We further describe the structural changes induced by phosphorylation that enable p63 to adopt its active tetrameric conformation and demonstrate that previously discussed phosphorylation by c-Abl is not involved in this process. Inhibition of CK1 rescues primary oocytes from doxorubicin and cisplatin-induced apoptosis, thus uncovering a new target for the development of fertoprotective therapies.p63 activation in response to DNA damage leads to oocyte death and loss of fertility in women receiving chemotherapy. Activation requires sequential phosphorylation by CHK2 and CK1 kinases, and inhibition of these kinases rescues oocytes from apoptosis induced by chemotherapy.