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First published online October 12, 2007
doi: 10.1242/10.1242/dev.007930

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Broad, ectopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice

Naoko Yoshida^1,*, Manami Amanai^1,*, Tomoyuki Fukui¹, Eriko Kajikawa¹, Manjula Brahmajosyula¹, Akiko Iwahori¹, Yoshikazu Nakano¹, Shisako Shoji¹, Joachim Diebold², Harald Hessel³, Ralf Huss³ and Anthony C. F. Perry^1,

¹ Laboratory of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
² Institute of Pathology, University of Munich, Thalkirchner Str. 36, 80337 Munich, Germany.
³ Roche Diagnostics GmbH, Nonnenwald 2, D-82372 Penzberg, Germany.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Plcz1 expression in wild-type and transgenic mice. (A) Plcz1 RT-PCR on testes (t) and brains (br) from duplicate wild-type males at the ages shown (upper) and from wild-type (wt) and F2 transgenic females on brain (br), heart (h), lung (lu), liver (li), spleen (sl), kidney (k), skeletal muscle (sm) and ovaries (o) (beneath). (B) Configurations of Plcz1 promoter-mapping constructs with Cre and rPLCZ1 reporters, showing cAMP-responsive element binding protein (CREBP) cognate sequences (green), the putative transcriptional start site (red, boxed), a region of high Z-DNA potential (bold, italicised) and the putative translational start codon (red, underlined). Sequences unique to each construct are indicated in blue, with Cre uppermost. Beneath are shown the structures of downstream open reading frames. (C) Structures (not to scale) of CV and CS tg constructs. Arrows mark the start and direction of translation. (D) Immunoblotting to show rPLCZ1 and PLCZ1 expression in heart (h), skeletal muscle (sm), testes (t) and ovaries (o) of CS and CV hemizygotes (tg) and aged-matched, non-transgenic littermates (wt).

View larger version (45K): [in this window] [in a new window]   Fig. 2. Parthenogenesis follows normal meiotic progression in rPLCZ1-expressing hemizygous female mice. (A) Immunofluorescence microscopy of oocytes matured in vitro at <2 (GV) or 8-10 (mI) hours after meiotic resumption, or 13 hours post-hCG (mII). Oocytes were from hemizygotes (CS) and age-matched non-transgenic littermates (wt). TUBA2 labelling is green and genomic DNA red. Arrows and arrowheads respectively mark the first polar body (Pb1) and mII plate. (B) Proportion of age-matched oocytes upon collection at mII 13 (white) and 24 (grey) hours post-hCG. (C) Hofmann image of F4 oocytes 13.5-14 hours post-hCG, showing metaphase or anaphase-telophase distortions (arrowheads) and Pb1 (arrows). (D) Immunofluorescence microscopy of different oocytes from C, 14 hours post-hCG, at early (upper) and late (beneath) stages of spindle rotation, represented diagrammatically to the right. Staining and key to arrow and arrowheads are as for A. (E) Ratiometric Fura 2-AM [Ca2+]i imaging of representative oocytes at different stages, performed as for A but with mII oocytes 14.5 hours post-hCG. Oocytes were from non-transgenic control (wt, upper) and age-matched transgenic (CS, beneath) females. (F,G) Hofmann images (F) and bar chart (G) showing development in vitro of CS16 F3 (CS) and SrCl2-induced wild-type (wt) haploid parthenogenotes at the times shown after oocyte collection or activation. Pronuclei and Pb2 in F are respectively indicated with arrowheads and arrows. (H) Preimplantation development following nuclear transfer (nt) from CS16 F4 cumulus cell nuclei into enucleated wild-type oocytes without exogenous activation, shown at the times indicated (hours) post-nt. Pronuclei are indicated with arrowheads. (I) Developmental rates in vitro following nt of CS16 F3 and F4, or age-matched non-transgenic or pCAGmtVenus transgenic (Shoji et al., 2006) cumulus cells (control) into enucleated wild-type oocytes. m/b (in G,I), morula/blastocyst. Scale bars: 20 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Timing and early in vivo consequences of rPLCZ1 expression. (A) Epifluorescence microscopy of mouse ovarian follicle section showing Venus expression (left) within a maturing primary oocyte (arrowhead), stained for DNA (right). (B) Asynchronous parthenogenotes recovered from hemizygotes 16 hours post-hCG. (C) Hematoxylin and Eosin staining of an 11-week-old hemizygous ovary which appeared macroscopically normal, at low and higher (inset) magnification, showing a nascent follicular choriocarcinoma or yolk sac tumour. Scale bars: 50 µm in A,C; 20 µm in B.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Ovarian tumours in PLCZ1-expressing female mice. (A) Hemizygous F2 CV3 and CS16 females at 6 months, with age-matched control (wt) showing abdominal distensions (arrowheads) and the tumours that caused them. (B) Percentages of transgenic females with tumours at the ages shown. (C) Reproductive tissue from a hemizygote at 6 months with a small tumour (arrowhead) and three regressing implantation fossa (arrows). (D) Dosages of tgs (rPlcz1) determined for 12 ovarian tumours relative to levels of the native genes Actb and H2afz. All tumours were from hemizygous females. Somatic tissue from homozygotes (CS1006h and CS1008h) gave expected tg dosages of 2. Error bars (±s.e.m.) were produced from results obtained for different (n) samples from the same tumour. Discrete portions from the tumour CS832-1 (bracketed) possessed disparate relative tg dosages. Scale bars: 1 cm.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Plcz1 expression in tumours and in the tumourigenic strain LT/Sv, and expression of its human orthologue in tumours. (A) Relative mRNA levels determined by qPCR for tumours from CV3 (stippled) and CS16 (grey) relative to wild-type ovary, or combined and expressed relative to skeletal muscle (white). Error bars, ±s.e.m.; *, not detected. Corresponding P-values are shown for unpaired Student's t-tests. (B) Tumour transcript levels of maternally- (white) and paternally- (grey) expressed imprinted genes determined by qPCR and represented relative to respective levels in wild-type ovary (1.0). Error bars, ±s.e.m. P-values (above), unpaired Student's t-tests. (C) RT-PCR analysis of two CV3 (CV) and four CS16 (CS) tumours, wild-type placenta (pl) and ovaries (o), for placental marker and metastasis-associated gene expression. (D) RT-PCR (35 cycles) of selected tissues from an 8-week-old LT/Sv female. Key as per Fig. 1. (E) RT-PCR for PLCZ1 and control ACTB mRNAs in ten independent human ovarian germline tumour (hOGT) biopsy samples as indicated. t, mouse testis control.

View larger version (117K): [in this window] [in a new window]   Fig. 6. Histopathology of rPLCZ1-induced ovarian tumours. (A) Mature cystic teratoma (epidermoid cyst) with keratinisation. (B) Intestinal epithelium with goblet cells. (C) Neurectodermal rosettes with tubules and glia-like stroma. (D) Mixed germ cell tumour showing yolk sac tumour (right), choriocarcinoma (top) and dysgerminoma (left). (E) Dysgerminoma (right) with yolk sac tumour. (F) Nests of dysgerminoma cells with follicle. (G) Mucinous cystadenoma with borderline malignancy of columnar epithelium. (H) Mixed germ cell tumour showing yolk sac tumour (right) and choriocarcinoma (top). Sections stained with Hematoxylin and Eosin are from three CV3 (A-E) and two CS16 (F-H) F2 female mice. Scale bars: 100 µm.