DEAD-box protein-103 (DP103, Ddx20) is essential for early embryonic development and modulates ovarian morphology and function

Abstract

The DEAD-box helicase DP103 (Ddx20, Gemin3) is a multifunctional protein that interacts with Epstein-Barr virus nuclear proteins (EBNA2/EBNA3) and is a part of the spliceosomal small nuclear ribonucleoproteins complex. DP103 also aggregates with the micro-RNA machinery complex. We have previously shown that DP103 interacts with the nuclear receptor steroidogenic factor-1 (SF-1, NR5A1), a key regulator of reproductive development, and represses its transcriptional activity. To further explore the physiological function of DP103, we disrupted the corresponding gene in mice. Homozygous Dp103-null mice die early in embryonic development before a four-cell stage. Although heterozygous mice are healthy and fertile, analysis of steroidogenic tissues revealed minor abnormalities in mutant females, including larger ovaries, altered estrous cycle, and reduced basal secretion of ACTH. Our data point to diverse functions of murine DP103, with an obligatory role during early embryonic development and also in modulation of steroidogenesis.

Figure 1

Expression and localization of DP103 in steroid-producing organs. Immunofluorescence was performed for DP103 (green) and TOPRO-3 iodide for nuclear counterstaining (blue) as described in Materials and Methods. A, Mouse ovarian sections stained with preimmune rabbit serum, serving as a negative control. B, Mouse ovarian sections. Arrow indicates primary follicle; arrowhead indicates antral follicle. CL, Corpus luteum. C, Mouse testicular sections. D, Female adult mouse adrenal sections showing adrenal medulla (M) cortex (C), and x-zone (X). E, Expression of DP103 in follicular stages, including (from left) negative control, secondary follicle, antral follicle, and postrupture follicle, detected using immunohistochemistry as described in Materials and Methods. Data shown are from one experiment, which represents at least three repeats, each with qualitatively similar results. Scale bar, 50 μm.

Figure 2

Targeted disruption of mouse Dp103 (Ddx20/Gemin3) gene. A, Schematic maps of the targeting vector, wild-type allele, and targeted allele. The filled boxes and solid lines between them represent exons and introns, respectively. The middle drawing represents the 5′ end of mouse Dp103 gene with the boxes labeled E1 and E2 representing exons 1 and 2, respectively. The targeting vector was linearized by PmeI. Restriction sites were ClaI (C), PstI (P), and SpeI (S). Neo, Neomycin resistance cassette. B, A representative Southern blot analysis (reproduced more than 10 times) of DNA extracted from wild-type and heterozygote mice for Dp103. Genomic DNA was digested with SpeI. The wild-type and targeted alleles yield 6.8- and 9.2-kb fragments, respectively (internal probe indicated; an external probe was used for ES cell screening). C, Detection of DP103 (red) and GFP (green) proteins in two-cell mouse embryos. The zygotes in the three panels were stained for both DP103 and GFP, demonstrating (from left) Dp103 wild-type, null, and heterozygous zygotes. Images shown are from one experiment, which represents four repeats, each with similar results.

Figure 3

The weight of steroid-producing tissues and ovarian histology. A, Weights of the ovary, testis, and adrenal in male and female mice obtained from wild-type (WT) animals (n = 20) and Dp103 heterozygous (Het) animals (n = 24–26). Values are mean ± sd. *, P < 0.01. B, DP103 expression in ovarian follicles from Dp103 heterozygotes. Sections of adult ovaries were immunostained for DP103 protein, and the presence or absence of DP103 immunofluorescence in follicles was ascertained. C, The frequency of follicular phenotypes as shown in B, in wild-type mice (n = 15–17, one section per animal) and Dp103 heterozygous mice (n = 9–13, one section per animal). Data are represented as mean ± sd. *, P < 0.05 (t test).

Figure 4

Assessment of the estrous cycle in wild-type and Dp103 heterozygous mice. Determination was performed as described in Materials and Methods by investigators blinded to the mouse genotype. A, Daily assessment of estrus (E), diestrus (D), or metestrus/proestrus (M/P); B, fraction of time in estrus for mice in either genotypic group (n = 4 for each genotype). Data were analyzed by combining the total number of days spent in estrus for each of the two groups (based on the data presented in A and compared using χ² test, P < 0.03).

Figure 5

Increased expression of Cyp17a1 in the ovary of Dp103^+/− mice. Expression of key steroidogenic enzymes was assessed by quantitative RT-PCR using total RNA from ovary (A), testis (B), and female adrenal (C). Results are expressed as fold expression (mean ± sd) in Dp103 heterozygote vs. wild-type mice (with levels in wild-type defined as one) and represent three to five experiments, each performed in duplicate. *, P < 0.05 (t test).

Figure 6

ACTH and corticosterone levels in wild-type and Dp103^+/− mice. Basal plasma hormone levels were measured 2 h after lights on (AM) and 1 h before lights off (PM) or after 30 min of restraint stress, all performed at the same time every morning as described in Materials and Methods by investigators blinded to the mouse genotype. A, ACTH. The differences between stress vs. morning or evening were significant for males and for females (n = 4; P < 0.05, ANOVA with post hoc Bonferroni test). The difference for females at evening or stress were significant (P < 0.05) between the two Dp103 genotypes. The other differences between the two Dp103 genotypes within each paradigm were insignificant. B, Corticosterone. The differences between stress vs. morning or evening were significant for males, and the differences between morning, evening, and stress were significant for females. The differences between the two Dp103 genotypes within each paradigm were insignificant.