Genetic Influences on Ovulation of Primary Oocytes in LT/Sv Strain Mice

REPRODUCTIONRESEARCH Genetic influences on ovulation of primary oocytes in LT/Sv strain mice Clare A Everett1,2, Catherine A Auchincloss1, Matthew H Kaufman3, Catherine M Abbott1 and John D West2 1Medical Genetics, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK, 2Division of Reproductive and Developmental Sciences, Genes and Development Group, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK and 3Division of Biomedical Sciences, Genes and Development Group, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK Correspondence should be addressed to John West; Email: [email protected] Abstract A high proportion of LT/Sv strain oocytes arrest in meiotic metaphase I (MI) and are ovulated as diploid primary oocytes rather than haploid secondary oocytes. (Mus musculus castaneus 3 LT/SvKau)F1 3 LT/SvKau backcross females were analysed for the proportion of oocytes that arrested in MI and typed by PCR for a panel of microsatellite DNA sequences (simple sequence repeat polymorphisms) that differed between strain LT/SvKau and M. m. castaneus. This provided a whole genome scan of 86 genetic markers distributed over all 19 autosomes and the X chromosome, and revealed genetic linkage of the MI arrest phenotype to markers on chromosomes 1 and 9. Identification of these two chromosomal regions should facilitate the identification of genes involved in mammalian oocyte maturation and the control of meiosis. Reproduction (2004) 128 565–571 Introduction The control of meiosis is a complex process involving many genes, some of which have been identified (Sagata The inbred mouse strain LT/Sv is unusual in ovulating a 1997, Taieb et al. 1997, Schafer 1998, Stojkovic et al. high proportion of its oocytes prematurely as primary 1999), but the primary genetic defect in LT/Sv strain mice oocytes at meiotic metaphase I (MI), rather than secondary remains unknown. M-phase promoting factor, MPF (pre- oocytes at meiotic metaphase II (MII) (Kaufman & Howlett 1986, O’Neill & Kaufman 1987, Spiers & Kaufman 1990, viously known as maturation promoting factor), plays an Eppig & Wigglesworth 1994, Maleszewski & Yanagimachi important role in both meiotic and mitotic cell cycles, 1995), and producing a high frequency of ovarian terato- whereas cytostatic factor (CSF) is specifically associated mas (Stevens & Varnum 1974, Eppig et al. 1977). These with meiosis and is responsible for the normal arrest of primary oocytes may be activated parthenogenetically and oocytes at meiotic metaphase II. MPF is a protein kinase, begin development (Stevens & Varnum 1974, Anderson et consisting of a heterodimer of Cdc2 kinase and cyclin B al. 1984, Kaufman & Howlett 1986, Maleszewski & (regulatory subunit), and it phosphorylates and regulates Yanagimachi 1995, Eppig et al. 1996) or they may be proteins involved in cell division. MPF activity varies fertilised to produce dygynic triploid embryos (Kaufman & according to the stage of the cell cycle. It increases at the Speirs 1987, O’Neill & Kaufman 1987, Maleszewski & end of each interphase and drives the cell into metaphase, Yanagimachi 1995). The MI arrest occurs in LT/Sv oocytes but cyclin B is destroyed at anaphase, so MPF levels fall denuded of cumulus cells (Ciemerych & Kubiak 1998), as the cell exits M-phase and enters the next interphase. and experiments with chimeric reconstituted ovaries CSF involves the action of Mos (Moloney sarcoma onco- showed that MI arrest in the related strain LTXBO was a gene protein), a serine/threonine protein kinase, and evi- 2 2 consequence of the genotype of the oocyte rather than the dence from Mos / knockouts showed that Mos activates surrounding follicle cells (Eppig et al. 2000). MI arrest MAPK (mitogen-activated protein kinase), a key com- occurred frequently in LTXBO oocytes surrounded by ponent of CSF (Araki et al. 1996, Verlhac et al. 1996). wild-type follicle cells, but not in the reciprocal Activation of MAPK by Mos involves an activation cas- combination. cade: Mos activates MAPK kinase kinase, which activates q 2004 Society for Reproduction and Fertility DOI: 10.1530/rep.00325 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/29/2021 06:54:56AM via free access 566 C A Everett and others MAPK kinase, which, in turn, activates MAPK. CSF (Mos- (CAST £ LT/SvKau)F1 £ LT/SvKau backcross females and MAPK) is produced only during meiosis and is involved in (C57BL/Ws £ LT/SvKau)F1 £ LT/SvKau backcross females stabilising MPF. MAPK, induced by Mos, may act to inhi- were produced for analysis. (Female mice are shown first bit cyclin B degradation and so sustain high levels of MPF for all genetic crosses.) and cause MII arrest (Hirao & Eppig 1997). At fertilisation, CSF is degraded, so MSF levels fall and allow the oocyte Cytogenetic analysis of oocytes to complete the second meiotic division. The involvement of Mos in the normal MII arrest Oocytes from (C57BL/Ws £ LT/SvKau)F1 £ LT/SvKau back- suggested that Mos might also be involved in the abnor- cross females were analysed after maturation in vivo, mal MI arrest of LT/Sv oocytes. In LT/Sv oocytes arrested in essentially as described previously (West et al. 1993). MI, cyclin B1 and MPF fail to decline (Hampl & Eppig Oocytes in the main experiment using (CAST £ 1995), which is consistent with the possibility that the MI LT/SvKau)F1 £ LT/SvKau backcross females were analysed arrest occurs because CSF stabilises MPF prematurely in cytogenetically after in vitro culture as described by Ever- MI LT/Sv oocytes (West et al. 1993). However, experimen- ett and Searle (1995). Female mice were injected with tal evidence suggests Mos is involved in maintaining this 5 IU pregnant mares’ serum gonadotrophin (PMS) at abnormal MI arrest, but not in initiating it (Hirao & Eppig 1200 h. Approximately 45 h later, ovaries were dissected 1997), and that the MI arrest of LT/Sv is caused by delayed into M2 handling medium in a solid watch glass, trimmed transition from metaphase-I to anaphase-I rather than of fat and pricked with 25G needles to burst the follicles. direct involvement of CSF (Ciemerych & Kubiak 1998). The oocytes were freed from the follicles and cleaned of A more recent study suggests that the delayed entry into most of the adhering cumulus cells. Germinal vesicle anaphase-I (MI arrest) seen in LT/Sv oocytes involves stage oocytes were collected with a micropipette, trans- abnormalities in regulation of protein kinase C (PKC) ferred to a drop of M2 handling medium and then to sev- (Viveiros et al. 2001). eral wash drops of M16 culture medium (Whittingham A genetic approach may help uncover the primary 1971), pre-equilibrated in 5% CO2 in air at 37 8C, and defect(s) responsible for the meiotic abnormality in LT/Sv finally transferred to a culture drop of M16 culture med- oocytes. Genetic crosses between LT/SvKau and ium under liquid paraffin oil in a Petri dish. After over- C57BL/Ws strain mice were compatible with segregation night culture for approximately 17 h at 37 8Cin5%CO2 of a co-dominant autosomal gene (with incomplete pene- in air (during which time the oocytes underwent germinal trance and variable expressivity) that has a major influ- vesicle breakdown), chromosome preparations were ence on the incidence of ovulation of primary oocytes made. The oocytes were transferred to M2 handling (West et al. 1993). This putative gene was given the provi- medium, ensuring that no paraffin oil remained on the sur- sional name primary oocyte ovulation (provisional gene face of the drop, and then a few at a time were transferred symbol Poo). However, analysis of recombinant inbred to a drop of freshly made 1% sodium citrate. Within strains, derived from the progenitor strains of LT/Sv mice 10 min, the oocytes appeared swollen and were trans- (C58/J and BALB/cJ), implied that both MI arrest and ferred to a clean glass microscope slide in the smallest parthenogenetic activation were controlled by more than possible volume of sodium citrate. A drop of freshly pre- one gene (Eppig et al. 1996). As Eppig et al. (1996) point pared ethanol:acetic acid (3:1) fixative was dropped onto out, these conflicting results can be reconciled if LT/SvKau the drop of sodium citrate containing the oocytes and air and C57BL/Ws strains differ for alleles of Poo but both dried. The slides were post-fixed in 3:1 fixative for 30 min, carry the same alleles of one or more genes that are rinsed in tap water, hydrolysed in 0.5 M HCl for 30 min, necessary but insufficient to induce MI arrest. rinsed in tap water and stained with Giemsa. MI oocytes The present study was undertaken to try to map the were identified cytogenetically as those with chiasmata. gene or genes responsible for the MI arrest that leads to ovulation of primary oocytes in LT/Sv mice, to help Genetic analysis uncover the primary molecular and cellular defect(s) responsible for this meiotic abnormality. This genetic trait is difficult to analyse because it shows incomplete penetrance and variable expressivity (West et al. 1993). We considered that it would be unreliable to Materials and Methods estimate a quantitative value from the proportion of MI oocytes ovulated by each female because the number of Mice analysable oocytes was small and varied among females. Inbred LT/SvKau and C57BL/Ws mice were bred under Instead, we followed the approach used previously with conventional conditions in the Centre for Reproductive this trait (West et al. 1993) and classified each female Biology, University of Edinburgh. Mus musculus castaneus according to whether the proportion of MI oocytes ovu- mice of strain CAST/Ei were obtained from the MRC lated was above the normal range.

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