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

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).

In mammals, meiosis is initiated at different time points in males and females, but the mechanism underlying this difference is unknown. Female germ cells begin meiosis during embryogenesis. In males, embryonic germ cells undergo G0/G1 mitotic cell cycle arrest, and meiosis begins after birth. In mice, the Stimulated by Retinoic Acid Gene 8 (Stra8) has been found to be required for the transition into meiosis in both female and male germ cells. Stra8 is expressed in embryonic ovaries just before meiotic initiation, whereas its expression in testes is first detected after birth. Here we examine the mechanism underlying the sex-specific timing of Stra8 expression and meiotic initiation in mice. Our work shows that signaling by retinoic acid (RA), an active derivative of vitamin A, is required for Stra8 expression and thereby meiotic initiation in embryonic ovaries. We also discovered that RA is sufficient to induce Stra8 expression in embryonic testes and in vitamin A-deficient adult testes in vivo. Finally, our results show that cytochrome p450 (CYP)-mediated RA metabolism prevents premature Stra8 expression in embryonic testes. Treatment with an inhibitor specific to RA-metabolizing enzymes indicates that a cytochrome p450 from the 26 family (CYP26) is responsible for delaying Stra8 expression in embryonic testes. Sex-specific regulation of RA signaling thus plays an essential role in meiotic initiation in embryonic ovaries and precludes its occurrence in embryonic testes. Because RA signaling regulates Stra8 expression in both embryonic ovaries and adult testes, this portion of the meiotic initiation pathway may be identical in both sexes.

Acetylation within the globular core domain of histone H3 on lysine 56 has recently been shown to play a critical role in packaging DNA into chromatin following DNA replication and repair in budding yeast 1, 2. However, the function or occurrence of this specific histone mark has not been studied in multi-cellular eukaryotes, mainly because the Rtt109 enzyme that is known to mediate acetylation of H3 K56 (H3 K56Ac) is fungal-specific 3 4. Here we demonstrate that in flies and humans the histone acetyl transferases CBP / p300 acetylate H3 K56, while Sir2 / hSirT1 / hSirT2 deacetylate H3 K56Ac. The histone chaperone Asf1 in Drosophila, Asf1a in humans, is required for acetylation of H3 K56 in vivo, while the histone chaperone CAF-1 is required for the incorporation of histones bearing this mark into chromatin. We show that in response to DNA damage, histones bearing acetylated K56 are assembled into chromatin in Drosophila and human cells, forming foci that colocalize with sites of DNA repair. Furthermore, acetylation of H3 K56 is elevated in multiple types of cancer, correlating with elevated levels of Asf1a in these tumors. Our identification of multiple proteins regulating the levels of H3 K56 acetylation in higher eukaryotes will allow future studies of this critical and unique histone modification that couples chromatin assembly to DNA synthesis, cell proliferation and cancer.