Loss of the histone chaperone ASF1B reduces female reproductive capacity in mice.

Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone H3-H4 chaperone involved in the assembly/disassembly of nucleosome and histone modification. Two paralogous genes, Asf1a and Asf1b, exist in the mouse genome. Asf1a is ubiquitously expressed and its loss causes embryonic lethality. Conversely, Asf1b expression is more restricted and has been less studied. To determine the in vivo function of Asf1b, we generated a Asf1b-deficient mouse line (Asf1b(GT(ROSA-ßgeo)437)) in which expression of the lacZ reporter gene is driven by the Asf1b promoter. Analysis of ß-galactosidase activity at early embryonic stages indicated a correlation between Asf1b expression and cell differentiation potential. In the gonads of both male and female, Asf1b expression was specifically detected in the germ cell lineage with a peak expression correlated with meiosis. The viability of Asf1b-null mice suggests that Asf1b is dispensable for mouse development. However, these mice showed reduced reproductive capacity compared with wild-type controls. We present evidence that the timing of meiotic entry and the subsequent gonad development are affected more severely in Asf1b-null female mice than in male mice. In female mice, in addition to subfertility related to altered gamete formation, variable defects compromising the development and/or survival of their offspring were also observed. Altogether, our data indicate the importance of Asf1b expression at the time of meiotic entry, suggesting that chromatin modifications may play a central role in this process.

Apoptosis of Sertoli cells after conditional ablation of murine double minute 2 (Mdm2) gene is p53-dependent and results in male sterility.

Beside its well-documented role in carcinogenesis, the function of p53 family has been more recently revealed in development and female reproduction, but it is still poorly documented in male reproduction. We specifically tested this possibility by ablating Mdm2, an E3 ligase that regulates p53 protein stability and transactivation function, specifically in Sertoli cells (SCs) using the AMH-Cre line and created the new SC-Mdm2(-/-) line. Heterozygous SC-Mdm2(-/+) adult males were fertile, but SC-Mdm2(-/-) males were infertile and exhibited: a shorter ano-genital distance, an extra duct along the vas deferens that presents a uterus-like morphology, degenerated testes with no organized seminiferous tubules and a complete loss of differentiated germ cells. In adults, testosterone levels as well as StAR, P450c17 (Cyp17a1) and P450scc (Cyp11a1) mRNA levels decreased significantly, and both plasma LH and FSH levels increased. A detailed investigation of testicular development indicated that the phenotype arose during fetal life, with SC-Mdm2(-/-) testes being much smaller at birth. Interestingly, Leydig cells remained present until adulthood and fetal germ cells abnormally initiated meiosis. Inactivation of Mdm2 in SCs triggered p53 activation and apoptosis as early as 15.5 days post conception with significant increase in apoptotic SCs. Importantly, testis development occurred normally in SC-Mdm2(-/-) lacking p53 mice (SC-Mdm2(-/-)p53(-/-)) and accordingly, these mice were fertile indicating that the aforementioned phenotypes are entirely p53-dependent. These data not only highlight the importance of keeping p53 in check for proper testicular development and male fertility but also certify the critical role of SCs in the maintenance of meiotic repression.

Rad54 is required for the normal development of male and female germ cells and contributes to the maintainance of their genome integrity after genotoxic stress.

Rad54 is an important factor in the homologous recombination pathway of DNA double-strand break repair. However, Rad54 knockout (KO) mice do not exhibit overt phenotypes at adulthood, even when exposed to radiation. In this study, we show that in Rad54 KO mouse the germline is actually altered. Compared with the wild-type (WT) animals, these mice have less premeiotic germ cells. This germ cell loss is found as early as in E11.5 embryos, suggesting an early failure during mutant primordial germ cells development. Both testicular and ovarian KO germ cells exhibited high radiation sensitivity leading to a long-term gametogenesis defect at adulthood. The KO female germline was particularly affected displaying decreased litter size or sterility. Spermatogenesis recovery after irradiation was slower and incomplete in Rad54 KO mice compared with that of WT mice, suggesting that loss of germ stem cell precursors is not fully compensated along the successive rounds of spermatogenesis. Finally, spermatogenesis recovery after postnatal irradiation is in part regulated by glial-cell-line-derived neurotrophic factor (GDNF) in KO but not in irradiated WT mice, suggesting that Sertoli cell GDNF production is stimulated upon substantial germ cell loss only. Our findings suggest that Rad54 has a key function in maintaining genomic integrity of the developing germ cells.

Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches.

The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10⁻5 M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation and cultured in the presence or not of 10⁻5 M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with ¹4C-MEHP. A 10⁻5 M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10⁻5 M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells.

Exposure of the mouse perinatal testis to radiation leads to hypospermia at sexual maturity.

The first round of mouse spermatogenesis begins from 3 to 4 days after birth through differentiation of gonocytes into spermatogonial-stem cells and type A spermatogonia. Consequently, this step of differentiation may determine generation of the original population of stem cells and the fertility potential of the adult mouse. We aimed to determine the effect of perinatal exposure to ionizing radiation on the testis at the end of the first wave of spermatogenesis and at sexual maturity. Our results show that, radiation sensitivity of the testis substantially decreases from late foetal life to the end of the first week after birth. In addition, partial or full recovery from radiation induced testicular weight loss occurred between the first round of spermatogenesis and sexual maturity, and this was associated with the stimulation of spermatogonial proliferation. Exposure of mice at 17.5 days after conception or at 1 day after birth to gamma-rays decreased the sperm counts at sexual maturity, while exposure of 8 day-old mice had no effect. This suggests that irradiation of late foetal or early neonatal testes has a direct impact on the generation of the neonatal spermatogonial-stem cell pool.

Axotomy-induced motoneuron death is delayed in mice overexpressing PrPc.

The normal function of the cellular prion protein, PrP(c), remains largely unknown. Recently, PrP(c) has been implicated in the regulation of neuronal survival and was shown to confer neuroprotection in the brain. To pursue investigation of the role of PrP(c) in the CNS, we used the facial nerve section, a well-established experimental model of motoneuronal stress. Nerve sections were performed in 2- and 7-day-old newborn mice and in 2 month-old adult mice expressing different levels of PrP(c). We observed no differences in motoneuronal death triggered by facial nerve section between Prnp-/- and wild-type mice, whether in neonatal or adult mice. By contrast, overexpression of PrP(c) in Tga20 newborn mice was correlated with a better survival of motoneurons in the few days following axotomy. The protection was, however transient since motoneuron number in lesioned facial nuclei of Tga20 mice became identical to that of wild-type mice 7 days and 14 days following the lesion when performed in 2- and 7-day-old mice respectively. In Tga20 adult mice, no protection was observed 2 months after the lesion, a time with a significant degree of motoneuron death in adult control mice. These results, while providing further evidence that PrP(c) is endowed with neuroprotective capacity in vivo, also suggest that PrP(c) does not play a physiological role in the regulation of motoneuronal survival.

Characterization of a recombinant encephalomyocarditis virus expressing the enhanced green fluorescent protein.

A recombinant encephalomyocarditis virus (rEMCV2887A-egfp) expressing the enhanced green fluorescent protein (EGFP) was produced. The EGFP gene was inserted in frame within the leader protein coding sequence of a full-length cDNA clone of EMCV. RNA transcripts derived from the recombinant full-length cDNA were synthesized in vitro and transfected into BHK-21 cells. The recombinant transcript RNA remained infectious despite the insertion of EGFP as shown by cytopathic effects on BHK-21 cells and by propagation of the rescued virus. The replication kinetics in BHK-21 cells and the pathogenicity in mice of rEMCV2887A-egfp did not differ significantly from that of the parental virus. The recombinant virus was shown to produce fluorescence in infected cells after at least five passages in BHK-21 cells. However, a decrease of EGFP expression was observed following serial passages, and this was associated with the accumulation of deletion mutations within the EGFP gene. Nevertheless, using EGFP autofluorescence, infected cells were easily detected in the brain of mice infected with the first-passage recombinant virus. These data demonstrate that rEMCV2887A-egfp could be a useful tool to study virus dissemination and pathogenicity when used at low passages.