While it is known that Retinoic Acid (RA) induces meiosis in mouse female fetal gonads, the mechanisms which regulate this process during spermatogenesis are poorly understood. We show that the All trans RA derivative (ATRA) and Kit Ligand (KL) increase meiotic entry of postnatal mouse spermatogonia in vitro without synergism. Competence to enter meiosis is reached by spermatogonia only at the stage in which they undergo Kit-dependent divisions. Besides increasing Kit expression in spermatogonia, ATRA also upregulates KL expression in Sertoli cells. Both ATRA and KL increase the expression of Stimulated by Retinoic Acid Gene 8 and Dmc1, an early meiotic marker. A specific Kit tyrosine kinase inhibitor prevents the increase in the number of meiotic cells induced by both the two factors, suggesting that they converge on common Kit-dependent signalling pathways. Meiotic entry induced by ATRA and KL is independent from their ability to affect germ cell viability, and is mediated by the activation of PI3K and MAPK pathways through Kit autophosphorylation. ATRA-induced phosphorylation of the two downstream kinases is mediated by a non-genomic mechanism.These data suggest that RA may control the timing of meiosis by influencing both the somatic and the germ cell compartment of the postnatal testis through the activation of the KL/Kit system.

SummaryIn the mouse, three genes that are homologous to the Drosophila Nanos (Nos) gene have been identified. Deletion of one of these genes, Nanos2, results in male sterility, owing to loss of germ cells during fetal life. Before apoptosis, Nanos2-null gonocytes enter meiosis, suggesting that Nanos2 functions as a meiotic repressor. Here, we show that Nanos2 is continuously expressed in male germ cells from fetal gonocytes to postnatal spermatogonial stem cells. We observed that the promeiotic factor AtRA, an analog of retinoic acid (RA), downregulates NANOS2 levels, in both fetal and postnatal gonocytes, while promoting meiosis. Interestingly, FGF9, a growth factor crucial for sex differentiation and survival of fetal gonocytes, upregulates levels of NANOS2 in both male and female primordial germ cells (PGCs) and in premeiotic spermatogonia. This effect was paralleled by an impairment of meiotic entry, suggesting that FGF9 acts as an inhibitor of meiosis through the upregulation of Nanos2. We found that NANOS2 interacts with PUM2, and that these two proteins colocalize in the ribonucleoparticle and polysomal fractions on sucrose gradients, supporting the notion that they bind RNA. Finally, we found that recombinant NANOS2 binds to two spermatogonial mRNAs, Gata2 and Taf7l, which are involved in germ-cell differentiation.

The development of an anterior–posterior (AP) polarity is a crucial process that in the mouse has been very difficult to analyse, because it takes place as the embryo implants within the mother. To overcome this obstacle, we have established an in-vitro culture system that allows us to follow the step-wise development of anterior visceral endoderm (AVE), critical for establishing AP polarity. Here we use this system to show that the AVE originates in the implanting blastocyst, but that additional cells subsequently acquire AVE characteristics. These 'older' and 'younger' AVE domains coalesce as the egg cylinder emerges from the blastocyst structure. Importantly, we show that AVE migration is led by cells expressing the highest levels of AVE marker, highlighting that asymmetry within the AVE domain dictates the direction of its migration. Ablation of such leading cells prevents AVE migration, suggesting that these cells are important for correct establishment of the AP axis.

SummaryHow Kit expression is regulated in the germline remains unknown. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, are involved in spermatogonia and oocyte differentiation. In the male, deletion of each factor causes loss of Kitexpressing spermatogonia in the prepuberal testis. In the female, SOHLH1 and SOHLH2 ablations cause oocyte loss in the neonatal ovary. To investigate whether Kit expression is regulated by these two factors in male germ cells, we examined SOHLH1 and SOHLH2 expression during fetal and postnatal mouse development. We found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal at Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with all-trans retinoic acid. We found that E-box sequences within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing Sohlh1 or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box-containing probe. In vivo coimmunoprecipitations indicated that the two proteins interact and overexpression of both factors increases endogenous Kit expression in embryonic stem cells. SOHLH1 was found by ChIP analysis to occupy an E-box-containing region within the Kit promoter in spermatogonia chromatin. Our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signaling involved in spermatogonia differentiation and spermatogenetic progression.

These data identify a new effect of PDE5 inhibitors on the modulation of cardiomyocyte response to adrenergic stimulation via PDE5-PDE2-mediated cross-talk.

SummaryHow Kit expression is regulated in the germline remains unknown. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, are involved in spermatogonia and oocyte differentiation. In the male, deletion of each factor causes loss of Kitexpressing spermatogonia in the prepuberal testis. In the female, SOHLH1 and SOHLH2 ablations cause oocyte loss in the neonatal ovary. To investigate whether Kit expression is regulated by these two factors in male germ cells, we examined SOHLH1 and SOHLH2 expression during fetal and postnatal mouse development. We found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal at Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with all-trans retinoic acid. We found that E-box sequences within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing Sohlh1 or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box-containing probe. In vivo coimmunoprecipitations indicated that the two proteins interact and overexpression of both factors increases endogenous Kit expression in embryonic stem cells. SOHLH1 was found by ChIP analysis to occupy an E-box-containing region within the Kit promoter in spermatogonia chromatin. Our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signaling involved in spermatogonia differentiation and spermatogenetic progression.

We demonstrated for the first time a direct relationship between Pde2A impairment and the onset of mouse congenital heart defects, highlighting a novel role for cAMP in cardiac development regulation.

SummaryMeiosis is a crucial process for the production of functional gametes. However, the biological significance of many genes expressed during the meiotic phase remains poorly understood, mainly because of the lethal phenotypes of the knockout mice. Functional analysis of such genes using the conditional knockout approach is hindered by the lack of suitable Cre transgenic lines. We describe here the generation of transgenic mice expressing Cre recombinase under the control of the meiotic Spo11 gene. Using LacZ-R26 loxP and EYFP-R26 loxP reporter mice, we show the specific expression and activity of Cre during meiosis in males and females. Spo11 Cre mice were then crossed with floxed Nbs1 and JAM-C mice to produce conditional knockouts. A strong reduction of Nbs1 and JAM-C protein levels was found in the testis. Although Nbs1-deleted mice developed minor gonadal abnormalities, JAM-C-knockout mice showed a spermiogenetic arrest, as previously described for the null mice. These results provide strong evidence that Spo11Cre transgenic mice represent a powerful tool for deleting genes of interest specifically in meiotic and/or in postmeiotic germ cells.