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. 2017 Feb;53(2):167-178.
doi: 10.1007/s11626-016-0096-z. Epub 2016 Oct 3.

Zscan4 Is Expressed Specifically During Late Meiotic Prophase in Both Spermatogenesis and Oogenesis

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Free PMC article

Zscan4 Is Expressed Specifically During Late Meiotic Prophase in Both Spermatogenesis and Oogenesis

Kei-Ichiro Ishiguro et al. In Vitro Cell Dev Biol Anim. .
Free PMC article

Abstract

Mouse zinc finger and SCAN domain containing 4 (Zscan4) proteins, which are encoded by multiple copies of Zscan4 genes, are expressed specifically in preimplantation embryos in vivo and embryonic stem (ES) cells in vitro. However, the expression patterns of mouse Zscan4 in vivo have been largely elusive. Here, we show that Zscan4 proteins are expressed in adult ovaries and testes. In ovaries, Zscan4 proteins were detected in germinal vesicle (GV) stage oocytes in antral follicles, indicating that Zscan4 genes are activated during the diplotene/dictyate stage in meiotic prophase I. Remarkably, Zscan4 showed different spatial localization patterns between two distinct GV oocytes, which can be distinguished by global chromatin organization-surrounded nucleolus (SN) and non-surrounded nucleolus (NSN). These spatiotemporal differences in Zscan4 localizations correlated with the transition of RNA polymerase II-mediated transcriptional status during GV oocyte maturation. In testes, Zscan4 proteins were detected in spermatocytes at late pachytene/diplotene stages and in Sertoli cells. These results suggest that Zscan4 may play critical roles during late meiotic prophase in both males and females.

Keywords: ES cell; GV oocyte; Ovary; Preimplantation embryo; SN and NSN oocyte; Testis.

Conflict of interest statement

Author contribution

K. I. and M.S.H.K. designed the study, conducted experiments, and wrote the manuscript. K. I. and M.M. performed experiments. T.A., H.K., N.C., M.S., S.S., and S.B.H.K. contributed to data acquisition.

Competing interests

M.S.H.K. is a co-founder of Elixirgen, LLC, which promotes clinical application of human ZSCAN4.

Figures

Figure 1.
Figure 1.
Zscan4 mRNA expression in the testis and ovary. (A) Tissue specificity of mRNA expression for the indicated genes was analyzed by RT-PCR. E16 fetal ovary at embryonic day 16; 4w 4-wk ovary; Em− and Em+ Emerald GFP negative and positive populations of mouse MC1 ES cells enriched by FACS, respectively; Syce1 and Sycp3: meiosis specific markers. MuERV-L retrotransposon supposed to be highly expressed in Em+ ES cell. (B) Expression of endogenous Zscan4 and exogenous Emerald GFP, which is knocked-in at the Zscan4c locus, was analyzed by RT-PCR. RNA was extracted from the isolated oocytes and testis in Emerald GFP knock-in (Z4EmKI) and wild-type mice and from Z4EmKI ES cells. Schematic Z4EmKI locus is shown (upper). Em− and Em+ Emerald negative and positive enriched populations of Z4EmKI ES cells, respectively. Meikin meiosis I specific marker (Kim et al. 2015). RT− control PCR without reverse transcription.
Figure 2.
Figure 2.
Zscan4 protein is expressed in oocytes of antral follicles. Ovary sections were immunostained as indicated, showing Z-stack sections of Zscan4 immunofluorescence of primordial, primary, secondary, preantral, and antral follicles. Scale bars, 20 μm.
Figure 3.
Figure 3.
Zscan4 protein is expressed in GV oocytes and preimplantation embryos. (A) GV oocytes were immunostained as indicated. Two types of immunostaining patterns in GV oocytes are shown. The upper example shows the spotty immunostaining pattern of Zscan4, some of which surround a nucleolus. The lower example shows the faint diffusive immunostaining pattern of Zscan4. Enlarged images of the nuclei are shown on the right. Arrow indicates cumulus granulosa cells associated with zona pellucida of GV oocyte. Asterisk indicates nucleolus. (B) Preimplantation embryos at different developmental stages were immunostained as indicated. Enlarged deconvolution images of partial Z projection for two cells are shown on the right, emphasizing intense Zscan4 foci. Arrowhead indicates polar body in two cells. Scale bars, 20 μm.
Figure 4.
Figure 4.
Zscan4 localization in SN- and NSN-type GV oocytes. (A) GV oocytes, classified as SN type and NSN type by heterochromatin morphology relative to nucleolus (shown by asterisk), were immunostained as indicated. The spatial localization pattern of Zscan4 was classified as spotty (limited number of foci) and diffusive (dispersed in nuclei). The immunostaining pattern of Zscan4 is quantified in the right graph. The number of analyzed GV oocytes (pooled from four independent experiments) is indicated. **P < 0.001 (Pearson’s chi-square test). (B) Intensity profile of Zscan4 immunostaining signal obtained with the FluoView Software from representative GV oocytes with a SN spotty (top, n = 21), SN diffuse (middle, n = 11), and NSN (bottom, n = 23). The value of pixel intensity on Z-axis is equal to 200 for the spotty signal (SN spotty) and to 180 (SN diffuse, NSN) for the oocytes with diffuse signal as inferable from the picture. Corrected total cell fluorescence (CTCF, arbitrary units) is shown in the bottom graph with SEM. C Zscan4 and DAPI signals were scanned across a nucleolus in SN-type GV oocyte (dashed line). Signal intensity of DAPI (green) and Zscan4 (gray bar) is shown on the bottom. D SN-type and NSN-type GV oocytes were immunostained for KAP1: a heterochromatic protein which is potentially associated with Zscan4. E SN-type GV oocyte was immunostained for anti-centromere antigen (ACA). Scale bars, 10 μm.
Figure 5.
Figure 5.
Localization patterns of Zscan4 correlate with Pol II-mediated transcription activity in GV oocytes. (A) SN- and NSN-type GV oocytes were immunostained for Pol2(S2P): RNA polymerase II CTD repeat (phospholylated Ser2), a marker for active transcriptional elongation. Weak Pol2(S2P) in Zscan4-spotty SN-type oocytes(top, n = 27/27), intense Pol2(S2P) in Zscan4-diffusive SN-type oocytes (middle, n = 2/2), intense Pol2(S2P) in Zscan4-diffusive NSN-type oocytes (bottom, n = 3/3). (B) SN- and NSN-type GV oocytes were immunostained for Pol2(S5P): RNA polymerase II CTD repeat (phospholylated Ser5), a marker for active transcriptional initiation.
Figure 6.
Figure 6.
Zscan4 protein is expressed in late prophase spermatocytes and Sertoli cell. (A) Seminiferous tubule sections were immunostained as indicated. Stage X-XI tubule is shown (upper). Higher magnitude image is shown (lower). Lep leptotene, late P. late pachytene, Dip diplotene, e St. elongated spermatid, Ser. Sertoli cell. (B) Zscan4 immunostaining of spermatocytes at different stages of meiotic prophase and Sertoli cells are shown. Scale bars, 5 μm.

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