MARF1 regulates essential oogenic processes in mice

Abstract

Development of fertilization-competent oocytes depends on integrated processes controlling meiosis, cytoplasmic development, and maintenance of genomic integrity. We show that meiosis arrest female 1 (MARF1) is required for these processes in mammalian oocytes. Mutations of Marf1 cause female infertility characterized by up-regulation of a cohort of transcripts, increased retrotransposon expression, defective cytoplasmic maturation, and meiotic arrest. Up-regulation of protein phosphatase 2 catalytic subunit (PPP2CB) is key to the meiotic arrest phenotype. Moreover, Iap and Line1 retrotransposon messenger RNAs are also up-regulated, and, concomitantly, DNA double-strand breaks are elevated in mutant oocytes. Therefore MARF1, by suppressing levels of specific transcripts, is an essential regulator of important oogenic processes leading to female fertility and the development of healthy offspring.

Fig. 1. Meiotic arrest and loss of Marf1 expression in Marf1 mutant oocytes

(A) Number of oocytes (left) and histology of cumulus-oocyte complexes (right) ovulated in oviducts by WT, HET, and mutant Marf1^ENU females 14 h after hCG injection. GV: germinal vesicle; GVB: GV breakdown; Pb1: first polar body. Scale bars, 50 µm. Arrowheads indicate MII spindle and Pb1. Arrow indicates GV. (B) qRT-PCR of Marf1 exon 16 (left) and 5’- (right) mRNA expression in WT, HET, and mutant Marf1^ENU FGOs. In this and subsequent figures, graphs show mean ± SEM. *P<0.05, compared to WTs. Bars connected with different letters are significantly different, P<0.05. (C) Western blot of MARF1 and ACTB protein in WT, HET and mutant Marf1^ENU FGOs (left), WT, HET (GT/+) and mutant Marf1^GT (GT/GT) (middle), Marf1^GT/+ (GT/+), Marf1^ENU/+ (ENU/+) and Marf1^GT/ENU (GT/ENU) (right panel).

Fig. 2. Expression of Marf1 by mouse oocytes

(A) In situ hybridization of Marf1 mRNA in ovaries of 22-d old B6SJLF1 mice 46 h after eCG injection. Bright and dark field images are in the top and bottom panels, respectively. Arrowheads indicate oocytes, arrow indicates cumulus, and asterisk indicates mural granulosa cells. (B) qRT-PCR (left) and Western blot (right) analyses of Marf1 mRNA and protein levels in oocytes, cumulus cells (CC), and mural granulosa cells (MGC) of eCG primed F1 mice. (C) X-gal staining of Marf1^GT/GT ovaries from eCG (46 h) primed 22-d old mice. Scale bars, 50 µm.

Fig. 3. Meiotic reversal by CDC25B and alteration of transcriptomes in Marf1^ENU/ENU oocytes

(A) Effect of microinjection of Cdc25b mRNA (Cdc25b) on meiotic progression (GVB and Pb1 emission) in Marf1^ENU/ENUoocytes. (B) Marf1^ENU/ENUoocytes matured after Cdc25b-injection. Left: DIC image. Scale bar, 50 µm. Right: chromosomes (blue) and spindles (green) staining. Scale bar, 10 µm. (C) MPF and MAPK activity in Marf1^ENU/ENUoocytes with or without Cdc25b mRNA microinjection (Cdc and Ctl group, respectively), or treated with or without 2.5 µM okadaic acid (Ctl and OA group, respectively). (D) Distribution of significantly changed transcripts (represented by Affymetrix probe sets) at various magnitudes of difference in expression levels between Marf1^ENU/ENU (Mutant) and WT FGOs detected by microarray analysis. The number and the percentage of each group of transcripts in total changed transcripts are indicated above each bar. (E) Levels of heterogeneous nuclear RNA (hnRNA) and mRNAs measured by qRT-PCR in WT and Marf1^ENU/ENU (Mutant) growing oocytes.

Fig. 4. Up-regulation of Ppp2cb, defective developmental competency, and dysregulation of retrotransposons and DNA double-strand breaks in Marf1^ENU/EMU oocytes

(A) Rpl19 and Ppp2cb mRNA expressed by WT and Marf1^ENU/ENU (Mutant) FGOs. (B) Effect of okadaic acid on GVB in Marf1^ENU/ENU FGOs. (C–D) Effect of microinjecting Marf1^ENU/ENU FGOs with siRNAs (C) and morpholinos (MO) (D) on GVB. (E) Mature (induced by Cdc25b-injection) Marf1^ENU/ENUoocytes 24 h after IVF. Scale bar, 50 µm. (F) Levels of retrotransposon mRNAs measured by qRT-PCR in WT and Marf1^ENU/ENU (Mutant) FGOs. (G) Confocal microscopy of FGOs stained with anti-γH2AX (red) and DAPI (blue). Scale bars, 5 µm. Bar graph shows the number of oocytes with various numbers of foci in the nucleus.