Genetically-biased fertilization in APOBEC1 complementation factor (A1cf) mutant mice

Abstract

Meiosis, recombination, and gametogenesis normally ensure that gametes combine randomly. But in exceptional cases, fertilization depends on the genetics of gametes from both females and males. A key question is whether their non-random union results from factors intrinsic to oocytes and sperm, or from their interactions with conditions in the reproductive tracts. To address this question, we used in vitro fertilization (IVF) with a mutant and wild-type allele of the A1cf (APOBEC1 complementation factor) gene in mice that are otherwise genetically identical. We observed strong distortion in favor of mutant heterozygotes showing that bias depends on the genetics of oocyte and sperm, and that any environmental input is modest. To search for the potential mechanism of the 'biased fertilization', we analyzed the existing transcriptome data and demonstrated that localization of A1cf transcripts and its candidate mRNA targets is restricted to the spermatids in which they originate, and that these transcripts are enriched for functions related to meiosis, fertilization, RNA stability, translation, and mitochondria. We propose that failure to sequester mRNA targets in A1cf mutant heterozygotes leads to functional differences among spermatids, thereby providing an opportunity for selection among haploid gametes. The study adds to the understanding of the gamete interaction at fertilization. Discovery that bias is evident with IVF provides a new venue for future explorations of preference among genetically distinct gametes at fertilization for A1cf and other genes that display significant departure of Mendelian inheritance.

Figure 1

Mendelian segregation and deviations from expectations. (A) Mendelian segregation. ‘m’ and ‘+’ designate the mutant and wild-type allele, respectively. Intercrosses between inbred strains make F1 hybrids. Hybrids are then crossed to either parental strain for backcrosses or inter se for intercrosses. Genotype ratios are based on Mendelian expectations. (B) Distinct segregation ratios, litter size, and genotype loss depending on the basis for non-Mendelian segregation. With embryonic lethality, mutant alleles that compromise viability of mm homozygotes can also result in loss of some m+ heterozygotes (partial dominance), which can further reduce litter size. Intercrosses can show an excess (< 1: > 2) or a deficiency (1:1) of m+ relative to ++.

Figure 2

A1cf segregation in in vivo test and control crosses. A1cf genotype segregation is shown for two study locations: the Maine Medical Center Research Institute (MMCRI, panel A) and the University of Hawaii (UH, panel B). ++ refers to the wild-type genotype and m+ to the heterozygous genotype. Homozygotes were not found in either intercross. c² goodness-of-fit tests are shown with point-wise p-values; results remain significant after correcting for testing multiple hypotheses. ‘Comment’ refers to test results for the fit between total observed genotype numbers with Mendelian expectations for wild-type and heterozygous mice. Sex ratios did not differ significantly from 1:1 expectation (not shown). SEM, standard error of the mean; n, total number of litters.

Figure 3

Reproductive features of A1cf mice. Female (A) and male (B) A1cf m+ and ++ mice were examined in regard to their reproductive features. The n shown under each graph represents a number of mice, unless stated otherwise. The number of oocytes per oviduct was assessed during IVF so each value was derived by dividing the total number of oocytes by the number of oviducts from which they were released in each IVF. The females used for IVF were subjected to hormonal stimulation. All mice were 8–10 weeks old. CL, corpora lutea; CL White, old CL that regressed to a fibrous scar; CL Red, fresh CL that was still hemorrhagic. Statistical analysis (t-test) revealed no significant differences between A1cf m+ and ++ mice for any measured parameter except sperm number.

Figure 4

RNA-binding proteins and genoinformativity markers. (A) RNA-binding proteins (RBPs) and genoinformativity markers (GIMs). Source of information: Chromosome (informatics.jax.org), RBPs, and GIMs, non-GIMs and sweeps. The approximate distance (kb) of “bias” genes from the center of the sweep is provided in parentheses. The Ppp2cb transcript was detected but did not reach the threshold to determine whether it is a GIM or non-GIM. The ninth ‘bias’ gene, apolipoprotein B transcript (Apob), which is not known to be an RBP, was not detected. (B) Genoinformativity scores. Left: genoinformativity scores and 95% confidence intervals are displayed. Four are confident GIMs (red), two confident non-GIMs (black), and two “remaining genes” (gray). Right: the kernel density showing that most mouse genes had low genoinformativity scores.

Figure 5

Network of interactions and functional dependencies. Genes were included if they show no more than two steps (nodes) from a bias gene. The Eif and Ppp gene families are shown in both red and black font because some family members are GIMs, others non-GIMs, and still others are ‘remaining genes’ or were not detected. Red asterisks denote non-Mendelian segregation. See Table S5 and Fig. S3 for additional information.

Figure 6

Two models for Biased Fertilization. Two models for putative effects of sperm and oocyte genotype on oocyte selection and meiosis are shown. According to the Oocyte Selection Model, sperm from m+ males preferentially or perhaps exclusively join with genetically heterologous ++ oocytes (or ++ sperm with m+ oocytes). By contrast, with the Reversed Meiosis Model, the genetics of fertilizing sperm determines which chromatid remains in the oocyte and which goes to the second polar body. In A1cf mice, the preference is to retain the chromatid with the opposite allele to that sperm brings so that heterozygous embryos predominate. Gametes with recombinant chromosomes are not shown.