Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Abstract

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2 .In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3 . Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy.Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosomespreading techniques are used for counting chromosome number 6 . This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs [7][8] . This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error. Video LinkThe video component of this article can be found at https://www.jove.com/video/2851/ Protocol Mouse oocyte collection1. To maximize the number of antral follicles isolated from each mouse, intraperitoneally inject sexually mature female mice (we use 6-week-old CF-1 mice from Harlan) with 5 IU of pregnant mare's serum gonadotropin (PMSG). 2. Prepare the collection medium (MEM/PVP) (3 ml/mouse) by adding milrinone to 2.5 μM and warm it at 37°C. Milrinone is a phosphodiesterase inhibitor that maintains meiotic arrest once oocytes are removed from follicles. Alternatively, 3-isobutyl-1-methylxanthine (IBMX) (0.2 mM) or dibutyryl-cyclic adenosine monophosphate (dbcAMP) (100 μg/ml) can be used. Prepare the culture medium by adding glutamine (1 mM) and mi...

Expanded Satellite Repeats Amplify a Discrete CENP-A Nucleosome Assembly Site on Chromosomes that Drive in Female Meiosis

“…Germinal vesicle (GV)-intact oocytes were collected in bicarbonate-free minimal essential medium with polyvinylpyrrolidone and Hepes (MEM-PVP) [51], denuded from cumulus cells, and cultured in Chatot-Ziomek-Bavister (CZB) [52] medium with 2.5 μM milrinone covered with mineral oil (Sigma) in a humidified atmosphere of 5% CO 2 at 37°C. To induce meiotic entry, oocytes were washed through several drops of CZB media without milrinone.…”

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes

“…Chromosome number and morphology were also assessed within the confines of the cell by treating the oocytes with monastrol. Monastrol binds to and disrupts the function of the kinesin protein KIF11, resulting in monopolar spindles making it easier to distinguish each sister chromatid pair (Stein and Schindler, 2011). Centromere number was counted using an anti-centromere autoantibody (CEN).…”

Knockdown of RBBP7 unveils a requirement of histone deacetylation for CPC function in mouse oocytes

“…46,47 Briefly, eggs were cultured for 1 h in CZB containing 100 μM monastrol (Sigma) to disperse the chromosomes by collapsing the bipolar spindle to a monoploar spindle. Eggs were fixed in freshly prepared 2.5% paraformaldehyde and stained for kinetochores (CREST antibody) and DNA (Sytox Green; Invitrogen, A11012; 1: 5000).…”