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. 2009 Oct 19;4(10):e7501.
doi: 10.1371/journal.pone.0007501.

Cyp26b1 expression in murine Sertoli cells is required to maintain male germ cells in an undifferentiated state during embryogenesis

Hui Li  1 , Glenn MacLean, Don Cameron, Margaret Clagett-Dame, Martin Petkovich
Affiliations

Cyp26b1 expression in murine Sertoli cells is required to maintain male germ cells in an undifferentiated state during embryogenesis

Hui Li et al. PLoS One. .

Abstract

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of Sertoli cell-specific Cyp26b1 knockout mice (Cyp26b1SC−/SC−).
(A) Floxed Cyp26b1 locus showing position of primers (P1, P2, P3) used for genotyping. LoxP sites are indicated by triangles, and the exons of Cyp26b1 are numbered. In Sertoli cells, exons 3–6 will be excised (Cyp26b1SC−/SC−), thus allowing for PCR amplification of a 364 bp product using P1 and P3. (B) PCR genotyping using P1, P2 and P3 showing detection of an excised allele (364 bp) only in the testes of a Cyp26b1fl/fl mouse also expressing Cre (mouse #1).
Figure 2
Figure 2. Stra8 expression is elevated in the absence of CYP26B1.
Reverse transcription- PCR was performed with RNA collected from E15.5 and E16.5 Cyp26b1SC+/SC− and Cyp26b1SC−/SC− testes. Stra8 is only detected in RNA from E16.5 Cyp26b1SC−/SC− testes. Mvh expression was analyzed as a positive control for RNA integrity.
Figure 3
Figure 3. Loss of germ cells in adult Cyp26b1SC−/SC− animals.
3-month-old Cyp26b1SC+/SC+ and Cyp26b1SC−/SC− testes stained with hematoxylin and eosin (A and B) or antibodies to TRA98 (a germ cell marker, red in C and D), GATA-1 (a Sertoli cell marker, red in E and F) and 3βHSD (a Leydig cell marker, green in E and F). Note the decrease in the seminiferous tubule size in testes from Cyp26b1SC−/SC− mice (F) compared to Cyp26b1SC+/SC+ mice (E). Seminiferous tubules devoid of germ cells are indicated by asterisks. Bars, 20 µm.
Figure 4
Figure 4. Neonatal loss of germ cells in the absence of CYP26B1.
Postnatal day 0 (P0) testes stained for TRA98 show a loss of germ cells in Cyp26b1SC−/SC− mice. Bar, 20 µm.
Figure 5
Figure 5. Cyp26b1SC−/SC− male germ cells enter meiosis prematurely.
Sections of testes from Cyp26b1SC+/SC+ (A) and Cyp26b1SC−/SC− (B) littermates at E16.5 stained for the meiotic marker SCP3 (green). Sections were counterstained with DAPI. Bar, 20 µm.
Figure 6
Figure 6. Re-entry into mitotic cell cycle in embryonic Cyp26b1SC−/SC− male germ cells.
Sections of testes from Cyp26b1SC+/SC+ (A, B, C, G, H, I) and Cyp26b1SC−/SC− (D, E, F, J, K, L) littermates at E15.5 (A–F) and E16.5 (G–L) stained for the mitotic marker Ki67 (B, E, H, K, red) and the germ cell marker MVH (A, D, G, J, green). Overlays of images show Ki67 expressing germ cells are observed only in Cyp26b1SC−/SC− fetuses (F, arrowheads). Bar, 20 µm.
Figure 7
Figure 7. Proposed model for the role of CYP26B1 in maintaining male germ cells in an undifferentiated state during embryogenesis.
In wild-type gonads, germ cells exhibit sex-specific divergence during embryogenesis as male germ cells enter mitotic arrest, while female germ cells enter mitosis followed by meiosis. However, in Cyp26b1SC−/SC− fetuses, Cyp26b1 activity is inactivated after E15.5, thus elevating levels of retinoic acid within the testes. As a result, male germ cells exit from G0 to re-enter the cell cycle and initiate meiotic prophase, which subsequently culminates in loss of male germ cells.
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