PAR6, a potential marker for the germ cells selected to form primordial follicles in mouse ovary

Abstract

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage.

Figure 1. Immunohistochemical localization of PAR6 in the mouse ovary.

The germ cells with the cyst structure in the 13.5 dpc (A) and 15.5 dpc (B) fetal ovaries did not show positive staining for PAR6 in contrast to the strong staining in somatic cells. At 17.5 dpc, the germ cells moved to the cortex (long arrow), and their cytoplasm began to express PAR6 (C). After 19.5 dpc, PAR6 staining was strong in the nuclei of some germ cells (D, 19.5 dpc; E, 1 dpp) and almost all oocytes in primordial follicles, while the oocytes nuclei of growing follicles showed weak expression of PAR6 (F, 3 dpp; G, 8 weeks). No reaction was observed in control sections (3 dpp) after replacement of the anti-PAR6 antibody with nonimmune serum (H). Black arrow, small or shrunk germ cell; red arrow, growing follicle; ring, primordial follicle. A–E, Bar = 20 µm; F–H, Bar = 40 µm. The numbers of germ cells, PAR6 positive germ cells and follicles in the largest cross-section of the ovary with different stages (I). Each independent data came from six ovaries. Different superscripts denote differences (P<0.05) in the total germ cells or follicles, respectively.

Figure 2. The relationship between PAR6 expression and apoptosis of germ cells.

Two adjacent serial sections of 2 dpp mouse ovaries were stained by anti-PAR6 antibody (A) and TUNEL (B), respectively. PAR6 positive germ cells with negative TUNEL (black ring), while TUNEL positive germ cells with negative PAR6 (red ring). Bar = 20 µm.

Figure 3. The effect of anti-PAR6 antibody on the follicle formation.

15.5 dpc ovaries were treated with anti-PAR6 antibody or goat IgG (control ) for 7 days, and then the shape (A, B), the numbers (C) of follicles and naked germ cells, and the mRNA expression of the germ cell-specific transcript factor Figα (D) were examined. The shape and number of follicles in anti-PAR6 antibody treatment were abnormal compared with that of control (arrows noted the defective follicles). Bar = 20 µm. Each follicular data came from five ovaries, and RT-PCR was repeated at least three times with similar results. Different letters were considered significant (P<0.05) respectively.

Figure 4. Loss of PAR6 disrupted ovarian histogenesis and reduced follicular formation.

15.5 dpc ovaries were treated with control shRNA plasmid or shRNA for 12 hours, cultured for 6.5 days, then the shape of ovaries and the localization of PAR6 (A and C, control; B and D, shRNA ),the number of follicles and naked germ cells (E), the expression of mRNA and protein (F) were examined. Arrows noted the naked germ cells and red rounds noted the cysts with PAR6 negative germ cells. Each data came from five ovaries, different superscripts denote differences (P<0.05) respectively. RT-PCR and Western blot were repeated at least three times with similar results. A and B, Bar = 40 µm; C and D, Bar = 20 µm.

Figure 5. The effect of CBX on the PAR6 expression and follicles formation.

15.5 dpc ovaries were treated with or without CBX for 4 or 8 days, and then the follicular formation (A, B control; C, D, E CBX treatment) and the expression of Figα gene (F) were examined. A large number of primordial follicles were observed with strong staining of PAR6 after ovaries were cultured for 4 days (A) or 8 days (B). The germ cells stayed in cyst stage with slightly staining of PAR6 in CBX treated ovaries for 4 days (C, CBX), and no follicle was observed for 8 days (D, CBX+). Some primordial follicles were observed with strong staining of PAR6 when the ovaries were treated with CBX for 4 days and then without CBX for further 4 days (E, CBX−). Bar = 20 µm. CBX obviously decreased the mRNA level of Figα (F). The experiment was repeated at least three times with similar results. CBX, carbenoxolone.