Defective folliculogenesis in female mice lacking Vaccinia-related kinase 1

Abstract

The Vaccinia-related kinase 1(VRK1), which is generally implicated in modulating cell cycle, plays important roles in mammalian gametogenesis. Female infertility in VRK1-deficient mice was reported to be caused by defective meiotic progression in oocyte at postovulatory stage. VRK1 roles in folliculogenesis, however, remain largely unknown. Here, accurate quantification of folliculogenesis is performed by a direct visualization of 'intact' ovary in 3-dimensions (3-D) using a synchrotron X-ray microtomography. In VRK1-deficient ovaries, the numbers of pre-antral and antral follicles are significantly reduced by 38% and 46%, respectively, comparing to control. The oocytes volumes in antral and Graffian follicles also decrease by 42% and 37% in the mutants, respectively, indicating defects in oocyte quality at preovulatory stage. Genetic analysis shows that gene expressions related to folliculogenesis are down-regulated in VRK1-deficient ovaries, implying defects in folliculogenesis. We suggest that VRK1 is required for both follicle development and oocyte growth in mammalian female reproduction system.

Figure 1. 3-D segmented images of a normal mouse ovary.

(a) Representative segmented image (Supplementary Movie 2) of a whole ovary in 6-week-old normal mouse. navy, pre-antral follicle; green, antral follicle; violet, Graffian follicle; yellow, corpus luteum. (b) Schematic diagram of a whole ovary in mouse. (c) Transparent image of an antral follicle (yellow dashed circle in Fig. 1a; Supplementary Movie 3). (d) Transparent images of a Graffian follicle (white dashed circle in Fig. 1a; Supplementary Movie 4). yellow arrows in (c) and (d), antrum cavity; red arrows in (c) and (d), oocytes. (e) Slice image in a given plane (inset of Fig. 1a).

Figure 2. 3-D segmented images of a VRK1-deficient mouse ovary.

(a) Representative segmented image (Supplementary Movie 5) of a whole ovary in 6-week-old VRK1-deficient mouse. navy, pre-Antral follicle; green, antral follicle; violet, Graffian follicle; yellow, corpus luteum; black; Ruptured follicle. (b) Transparent image of an antral follicle (yellow dashed circle in Fig. 2a; Supplementary Movie 7). (c) Transparent images of a Graffian follicle (white dashed circle in Fig. 2a; Supplementary Movie 8). yellow arrows in (b) and (c), antrum cavity; red arrows in (b) and (c), oocytes. (d) Segmented image of the ruptured follicle, a different view (red arrow head) of the follicle (a white dashed circle) in Fig. 2a (Supplementary Movie 9). (e) Slice image of the ruptured follicle (yellow dashed plane in Fig. 2d). (f) Slice image of the Graffian follicle (asterisk of Fig. 2a). arrows in (e) and (f), granulosa layer with thicknesses as 11 μm and 85 μm, respectively.

Figure 3. Effect of VRK1-deficiency on follicle and CL numbers.

(a) Comparison of average numbers of follicles and CL per ovary in normal and VRK1-deficient mice with 6 weeks of ages (n = 3/genotype). The errors correspond to the s.e.m.. (b) and (c) Comparison of the numbers per ovary for 16-week-old and 43-week-old mice, respectively (n = 3/genotype). preA, pre-Antral follicle; A, anntral follicle; G, Graffian follicle; CL, corpus luteum; P, p-value.

Figure 4. Effect of VRK1-deficiency on oocyte volume.

Comparison of average oocyte volumes in antral (a) and Graffian (b) follicles in normal and VRK1-deficient mice with various ages (6, 16, 43 weeks). The errors correspond to the s.e.m. (n = 6). P, p-value.

Figure 5. Effects of VRK1-deficiency on different pathways involved in folliculogenesis.

(a) Several marker genes, expressed in early folliculogenesis, were detected in primary and pre-antral follicles from whole ovaries of normal or VRK1-deficient mice by qRT-PCR. (b) Several marker genes, expressed in later folliculogenesis, were detected in antral/Graffian follicles from whole ovaries of normal or VRK1-deficient mice by qRT-PCR. Each value of qRT-PCR was normalized to β-actin expression levels and expressed as the fold change relative to the levels detected in normal ovaries, which were set equal to 1. The errors correspond to the s.e.m.. FIGα, Factor in the germline alpha; AMH, anti-müllerian hormone; CX43,Connexin 43; KIT, Kit receptor; KITL, Kit ligand; GDF9, Growth differentiation factor-9; Wnt4, Wingless-related MMTV integration site 4; Foxo3a, Forkhead box O3A; BMP15, Bone morphogenic protein15; FSHR, FSH receptor; SOD1, Superoxide dismutase 1; DDR2, Discoidin domain receptor 2; ERα, Estrogen receptor α; ERβ, Estrogen receptor β; TR4, Testicular orphan nuclear receptor 4; UBE3a, Ubiquitin protein ligase E3A; ADAMTS1, ADAM-metallopeptidase with thrombospondin type 1 motif-1; CCND2, Cyclin D2.