|Female Germ Cell Aneuploidy and Embryo Death in Mice Lacking the Meiosis-Specific Protein SCP3|
Li Yuan, Jian-Guo Liu, Mary-Rose Hoja, Johannes Wilbertz, Katarina Nordqvist, and Christer Höög
Materials and Methods
Generation of the SCP3-deficient mice has been described previously (1). C57BL/6NCrlBR wild-type male mice (Charles River, Uppsala) were mated with 12- to 24-week-old SCP3-null females or control siblings (C57BL/6NCrlBR 129/Ola). Two females were caged with one male after 16.00 and examined daily thereafter for vaginal plugs between 08.00 and 09.00. The day when the plug was found was recorded as day 0.5 of pregnancy, referred to as "0.5 dpc". Cages were monitored daily, and number and size of litters were recorded. For embryo loss assessment, mice were killed at 11.5 dpc and the resorbing and nonresorbing embryos counted. Percentage of embryo loss was calculated using the formula: 100 Resorbing embryos/(Viable + Resorbing embryos). Animal husbandry practices followed guidelines established by the transgenic mouse core facility at Karolinska Institutet.
Cytogenetic analysis of MI oocytes
Virgin females at 13-14 weeks were primed with 5 IU pregnant mare serum and killed 42-44 h later by cervical dislocation. The ovaries were removed and placed in a pre-equilibrated culture medium, -MEM (GibcoBRL), containing 0.3 mg/ml bovine serum albumin, 100 IU/ml penicillin, and 100 IU/ml streptomycin. Under a dissecting microscope, the antral follicles were punctured with a fine needle to release the oocyte-cumulus cell complexes. Only oocytes with easily removable cumulus cells and a germinal vesicle were selected, washed twice in fresh medium, and cultured in microdrops of medium under oil at 37°C in 5% CO2. After 2 h in culture, oocytes were scored for germinal vesicle breakdown, indicating resumption of meiosis I, and any oocytes remaining at the germinal vesicle stage were excluded from the experiment. Selected oocytes were incubated for an additional 3 h, and air-dried chromosome spreads were then prepared from the MI oocytes for cytogenetic analysis (2, 3). The preparations were stained with 2% (w/v) buffered Giemsa and only complete oocytes were scored. Univalents were classified as described previously (4).
Cytogenetic analysis of one-cell zygotes
Virgin females at 12-16 weeks of age were superovulated with an intraperitioneal (i.p.) injection of 5 IU pregnant mare serum followed 48 h later by an i.p. injection of 5 IU human chorionic gonadotropin. Females were paired (2:1) with C57BL/6NCrlBR wild-type males, checked for the presence of a vaginal plug the morning following mating. Mated females were i.p. injected with 2 g/g body weight of 0.2 ml colchicine dissolved in sterile water (250 g/ml, Sigma) 26 h after injection of human chorionic gonadotropin to arrest the cells at first-cleavage metaphase and then killed 4 h later by cervical dislocation (5). Cytogenetic preparation of the zygotes was based on described procedures (6). Zygotes were flushed from the oviducts with a needle using M2 medium. Residual cumulus cells were removed by brief treatment with hyaluronidase. After two rinses in M2 medium, zygotes were transferred to a hypotonic solution of 0.3% sodium citrate for 30 min at room temperature, and exposed to a freshly prepared fixative mixture of 3:1 methanol:acetic acid. Chromosomes were then air-dried and stained with 2% (w/v) buffered Giemsa (Sigma).
Cytogenetic analysis of diakinesis oocytes
SCP3-null females and control siblings were primed with 5 IU pregnant mare serum (Intervet) and killed 42-44 h later by cervical dislocation. The ovaries were removed and placed in M2 medium. Antral follicles were punctured with fine needles to obtain immature oocytes at the germinal vesicle stage. Such oocytes were cultured in M16 culture medium at 37°C for 2 h, allowing the oocytes to enter diakinesis. The oocytes were stained with 2% (w/v) buffered Giemsa (Sigma) and karyotyped (2, 3).
Crossing over frequency
C57BL/6NCrlBR SCP3+/- mice were derived by six repeated backcrosses of SCP3+/- mice with a mixed genetic background (C57BL/6NCrlBRx129/Ola) onto the C57BL/6NCrlBR background (7). C57BL/6NCrlBR SCP3+/- females/males were mated to C57BL/6NCrlBRx129/Ola SCP3+/- males/females to generate F1 hybrid mice. Those F1 hybrid female mice nullizygous or wild-type for SCP3 were mated with C57BL/6NCrlBR wild-type males to generate F2 offspring. Tail DNA from F2 offspring and their parents was isolated with DNAeasy Tissue kit (QIAGEN) and typed with four pairs of polymorphic microsatellite markers on four separate chromosomes (chromosome 12: D12Mit46 and D12Mit14, chromosome 15: D15Mit26 and D15Mit29, chromosome 17: D17Mit93 and D17Mit123 and chromosome 18: D18Mit64 and D18Mit74, Invitrogen). PCR using microsatellite makers was performed under standard conditions, the products were analyzed on 4% (3:1) NuSieve agarose gels and the recombinants counted (8).
Crossing-over frequency was evaluated by chi2 testing derived from the 2 2 contingency test. Two values were compared in each test class: recombinant and non-recombinant. Litter size and live embryos from two different genotypes at the same age were evaluated by Student's t-test.
Histological, immunocytological and SC analysis
Ovaries were dissected from 12-week-old female mice for histology, fixed in Bouin's solution, dehydrated and embedded in paraffin wax. Sections (4 m) were prepared from the embedded ovaries and stained with hematoxylin and eosin. SCP3+/- females were mated with SCP3+/- males and the copulation plug date noted (vaginal plug = 0.5 dpc). Fetal oocytes at the zygotene to pachytene stages of meiosis were obtained by sampling fetuses at 16.5-18.5 dpc. The ovaries were dissected from female embryos and spread using a "dry-down" technique (9). Briefly, the isolated cells were fixed using 1% paraformaldehyde and 0.15% Triton X-100, and then promptly immunostained as described previously (1). The different sera were diluted as follows: MLH1, 1:300 (Pharmingen); CREST, 1:3000 (1); SCP1, 1:20 (1), DMC1, 1:500 (10). Secondary antibodies were TRITC-conjugated sheep -mouse (1:100, Boehringer Mannheim), FITC-conjugated swine -rabbit (1:100, DAKO) and Cy5-conjugated goat -human (1:2000, Amersham). Analysis was performed at a magnification of 630 using a Leica DMRXA fluorescence microscope and imaged with a Hamamatsu C4742-95 digital camera and Openlab 3.0 software. Final images were prepared using Adobe Photoshop 5.0.
Supplemental Figure 1. SC formation is compromised in SCP3-deficient oocytes. (A) Surface-spread oocyte preparations were made from 16.5- to 18.5-day-old fetal ovaries by a modified method of Speed (11). Primary oocytes were spread on the surface of slides in 0.5% NaCl, fixed in 4% paraformaldehyde and stained with 50% silver nitrate. The preparations were examined using a Zeiss Axioscope. The SC is clearly visible in nuclei of wild-type pachytene oocytes (indicated by arrow) but no such structures are seen in the nuclei of SCP3-deficient oocytes. (B) The cellular distribution of SCP2 is affected by the absence of SCP3. Pachtene ocytes were isolated from wild-type or SCP3-deficient females, fixed and analyzed by immunofluorescence microscopy (8). The distribution of SCP2, CREST and the merged images are shown. The sera were diluted as follows: CREST, 1:3000 (1); SCP2, 1:600 (12). Secondary antibodies were FITC-conjugated swine -rabbit (1:100, DAKO) and Cy5-conjugated goat -human (1:2000, Amersham).
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