Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival.

Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gßγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

Correction: LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse.

Following publication of their article, the authors reported that the name of the fifth author had been formatted incorrectly in PubMed. Instead of "Rella FD" it should be "Di Rella F".

Programmed cell death in the human ovary.

During a woman's reproductive life, only about 400 primordial follicles will develop into a preovulatory follicle and undergo ovulation, releasing an oocyte available for fertilization. The process of formation and selection of these follicles is complex and involves a multistep process characterized by a balance between survival and death of the oocytes and the surrounding follicular cells. Although the mechanisms underlying such process are not completely clarified yet, it is common idea that they can occur through various types of programmed cellular death (PCD). Since atresia is the principal destiny of the ovarian follicles, it is relevant to understand how this process takes place and how it is regulated. In this review, after a summary description of folliculogenesis in humans, the main mechanisms of atresia reported to occur during folliculogenesis from birth to adult age, in the human ovary and in other mammals when appropriate, are described.

LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse.

Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy.

Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability.

Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.

Programmed cell death in mouse primordial germ cells.

In a number of mammalian species, the main events of development of the primordial germ cells (PGCs), the embryonic precursors of the oocytes and spermatozoa, were described during the early twentieth century. Actually, the concept of the origin of germ cells in extragonadal sites before the formation of the gonadal anlagen, was put forward for the human embryo around the first decade of the 1900s (for a review, see De Felici, 2013). PGC development is characterized by two major cellular processes, a movement from the wall of the yolk sac, where the germline is determined, to the gonadal anlagen and an increase in number due to active proliferation. As far as we know, the notion that programmed cell death (PCD) might physiologically occur in mammalian PGCs was for the first time put forward by us in 1993 in the case of the mouse. How we arrived to such a concept and the progress made up to now in the characterization of this process in our and other laboratories mainly in the mouse are the topics of the present review.

Multifaceted programmed cell death in the mammalian fetal ovary.

From previous and more recent works reviewed in the present paper, it appears that mammalian fetal oocytes face several challenges to survive throughout the stages of meiotic prophase I up to the block at the diplotene/dictyate stage and the primordial follicle assembly. Depending on the period of development and experimental conditions, these oocytes can undergo different forms of programmed cell death (PCD) and cross-talking pathways. We hypothesize that they require the continuous support of growth factors to accomplish the activities required to overcome PCD during prophase I. An extraordinary level of DNA double strand break (DSB) tolerance characterizes oocytes during the first stages of meiotic prophase I. However, the activation of a p63/p53-and PCNA-dependent DNA damage checkpoint, plays a major role in eliminating defective oocytes when they reach the diplotene stage. Before oocytes are enclosed into a primordial follicle, the shortness of nutrients/growth factors might activate protective autophagy but this can turn into their death if starvation is prolonged. Actually, clarifying the relationships among growth factor signalling (mainly AKT cascade), apoptotic and autophagic proteins that seem to coexist in fetal oocytes, could be the key to understanding PCD in these cells.

Effect of Culture in Simulated Microgravity on the Development of Mouse Embryonic Testes.

BACKGROUND: All known organisms develop and evolve in the presence of gravitational force, and it is evident that gravity has a significant influence on organism physiology and development. Microgravity is known to affect gene expression, enzyme activity, cytoskeleton organization, mitotic proliferation and intracellular signaling. OBJECTIVES: The aim of the present study was to study some aspects of the development in vitro of mouse embryonic testes in simulated microgravity. MATERIAL AND METHODS: Testes from mouse embryos (12.5-16.5 days post coitum, d.p.c.) were cultured in simulated microgravity and standard static culture conditions. The microgravity condition was provided by a Rotary Cell Culture System (RWV) bioreactor, an apparatus designated for 3D tissue and small organ cultures. After 48 h of the culture in the RWV, testis morphology and size was evaluated. RESULTS: The first observation was that the culture in the RWV bioreactor had a beneficial effect on the testis growth and on the survival of germ cells in comparison to static 2D culture methods. Moreover, we found, that RWV culture caused disorganization the gonadal tissues, namely of the testis cords. CONCLUSIONS: The results suggest that the maintenance of testis cord could be sensitive to microgravity. We hypothesize that while the effect on testis growth is due to a better nutrient and oxygen supply, the testis cord's disorganization might depend on the microgravity conditions simulated by the bioreactor. Considering the complexity of the processes involved in the formation of the testis cords and their dynamic changes during the embryo fetal period, further studies are needed to identify the causes of such effect.

Embryotoxicity assays for leached components from dental restorative materials.

BACKGROUND: Currently, there are no suitable assays available to evaluate the embryotoxicity of leached components from restorative dental materials. METHODS: The effect of the medium conditioned by composites and amalgam on mouse blastocysts in vitro was tested. The materials were also subcutaneously implanted, and the effect of the medium supplemented with serum from the host blood was evaluated in the embryotoxicity assay. The embryo implantation rate in the material-transplanted mothers was also evaluated. RESULTS: The results show that while the culture in media conditioned by amalgams did not affect blastocyst development, the medium conditioned by composites caused blastocyst degeneration and apoptosis. The development of blastocysts in a medium containing serum obtained from animals after transplantation was, however, without effect. Finally, inconsistent reduction in the implantation rate in transplanted mothers was observed. CONCLUSIONS: In this study, we provide examples of in vitro and in vivo tests that may be used to evaluate embryotoxicity for dental materials. Our results show that leached components from our composite-material induced embryotoxicity in vitro, however, no toxicity was observed when subcutaneously implanted in vivo. This highlights the necessity of integrated in vitro and in vivo tests for valuable predictive estimation of embryotoxicity for complex materials.

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.

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.

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.

Kit regulatory elements required for expression in developing hematopoietic and germ cell lineages.

The Kit (White) gene encodes the transmembrane receptor of stem cell factor/Kit ligand (KL) and is essential for the normal development/maintenance of pluripotent primordial germ cells (PGCs), hematopoietic stem cells (HSCs), melanoblasts, and some of their descendants. The molecular basis for the transcriptional regulation of Kit during development of these important cell types is unknown. We investigated Kit regulation in hematopoietic cells and PGCs. We identified 6 DNase I hypersensitive sites (HS1-HS6) within the promoter and first intron of the mouse Kit gene and developed mouse lines expressing transgenic green fluorescent protein (GFP) under the control of these regulatory elements. A construct driven by the Kit promoter and including all 6 HS sites is highly expressed during mouse development in Kit+ cells including PGCs and hematopoietic progenitors (erythroid blast-forming units and mixed colony-forming units). In contrast, the Kit promoter alone (comprising HS1) is sufficient to drive low-level GFP expression in PGCs, but unable to function in hematopoietic cells. Hematopoietic expression further requires the addition of the intronproximal HS2 fragment; HS2 also greatly potentiates the activity in PGCs. Thus, HS2 acts as an enhancer integrating transcriptional signals common to 2 developmentally unrelated stem cell/progenitor lineages. Optimal hematopoietic expression further requires HS3-HS6.