Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes

Abstract

We produced XX<-->XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX* germ cells developed as "eggs" within the seminiferous tubules of XX*<-->XY chimeric testes. These cells were indistinguishable from XX* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes. The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.

Fig. 1.

XX*↔XY chimeras containing XX* cells in their testes. (A) Strategy of producing XX*↔XY chimera. Males containing the EGFP transgene on the X chromosome were bred with wild-type females. The male and female embryos were separated, and XX*↔XY chimera embryos were made by aggregation. These embryos were transferred to pseudopregnant females. (B) A testicular section from a newborn XX*↔XY chimera (no. 50). (Upper Left) EGFP-positive XX cells. (Scale bar: 50 μm.) (Upper Center) Immunolabeling (red) for TRA98, a germ cell-specific antigen. (Lower) Higher magnification showing XX* cells (arrows) and XY germ cells (arrowheads) in seminiferous tubules. (Scale bar: 50 μm.) (C) Testicular section from a 5-week-old sexually mature XX*↔XY chimera (no. 50). XX* Sertoli cells are present (Upper); however, XX* spermatogenic cells are absent. (D) Flow cytometric analysis of newborn testicular cells from the no. 139 mouse line. The forward-scatter and side-scatter dot-plot gated fraction was shown to divide into three peaks (negative, medium, and bright) in which the brightest peak consisted of >98% germ cells, proven by TRA98 staining (see Results).

Fig. 2.

Characterization of germ cells in testes of newborn XX*↔XY chimera (no. 139). DMR methylation of paternal methylated genes Dlk1-Gtl2/Meg3 and Igf2-H19 in newborn germ cells, analyzed by bisulfite genomic sequencing. Filled ovals indicate methylated CpGs, and open ovals indicate unmethylated CpGs. As expected, DMRs of spermatogonia (MG) showed hypermethylation, and oocytes (FG) showed hypomethylation. XX spermatogonia (CG) showed hypermethylation.

Fig. 3.

Testicular eggs in seminiferous tubules. (A)(Left) Large cells in testis indicated as XX* by EGFP fluorescence (no. 139). (Center and Right) Higher magnification of three EGFP-expressing cells in a separated seminiferous tubule. (B) A testicular section from a 7-dpp XX*↔XY chimera. Granulosa-like cells, which surround testicular eggs, are indicated by arrowheads. (C) A testicular section of XX*↔XY chimera at 17 dpp (no. 139). GFP-positive testicular eggs are indicated by asterisks, and GFP-negative, SCP3-positive spermatocytes are marked by arrowheads. (Scale bars: A Left, 500 μm; A Center, B, and C, 50 μm.)

Fig. 4.

Characterization of testicular eggs in chimeric testes. (A) Immunofluorescence staining of ZP3 in zona pellucida of testicular eggs in 1-week-old chimeric testes and oocytes in “normal” ovaries. (B) Sperm were added to eggs preloaded with Hoechst 33342. Fused sperm (arrowheads) are stained with Hoechst because of dye transfer from the eggs. (C) BrdUrd staining to detect onset of meiosis in testicular eggs. In two Left panels, BrdUrd was injected at 12.5 dpc, and the testicular eggs were recovered at 1 week after birth. In two Right panels, BrdUrd was injected every day from 0 to 5 dpp, and the testicular eggs were recovered at 6 dpp. (D) SCP3-positive cells in 17.5-dpc chimeric testis. EGFP indicates the XX* cells. TRA98 staining (with 7-amino-4-methylcoumarin-3-acetic acid) shows germ cells. SCP3 staining (with tetramethylrhodamine isothiocyanate) shows synaptonemal complexes. Note some XX germ cells (EGFP and TRA98 double positive) also contained SCP3 (arrowheads), whereas others did not (arrows). SCP3 staining in 17.5-dpc ovarian eggs is shown for comparison. (Scale bars: 50 μm.) (E) DMR methylation in maternally methylated (Igf2r and Snrpn) and paternally methylated (Dlk1-Gtl2/Meg3 and Igf2-H19) genes in 3-week-old testicular eggs. Methylation of Igf2r-DMR, but not Snrpn-DMR, was similar in testicular eggs and ovarian eggs. The paternal methylated gene was almost completely nonmethylated in testicular eggs, similar to ovarian eggs.

Fig. 5.

XX germ cells are reported to develop as spermatogonia when deposited in a testicular environment (6, 10). However, proof of male type differentiation in molecular bases was not available. As shown in the present study, once XX germ cells were inhibited from entering meiosis, they were demonstrated to acquire paternal imprinting, which indicates the development of XX prospermatogonia. In 1-dpp testes, we found approximately as many spermatogonia surviving as in the 0-dpp testes. However, the XX* spermatogonia were seldom seen at 2 dpp (data not shown). The reason for this disappearance is not known. Occasionally, some XX germ cells initiated meiosis in seminiferous tubules in their embryonic stage and were arrested in 4n stage. Despite continuous exposure to male factors inside seminiferous tubules during the embryonic stage, these cells did not acquire paternal imprinting as XX prospermatogonia. Instead, they resumed meiosis after birth and obtained a maternal imprinting pattern in the testicular environment. Because the maternal imprinting starts after birth in the growing oocytes, the imprinting in testicular eggs may also start after birth, together with their growth in size. Taking these facts together, we postulate that the pattern of genomic imprinting is designated when the germ cells choose the sex to develop at ≈12.5 dpc and that it is not influenced by environmental factors when methylation takes place.