127 Chemokine receptor 2 (CCR2) is expressed in growing oocytes, and its deficiency affects follicular activation and long-term female fertility in mice

Abstract

Chemokine receptor 2 (CCR2) has important functions in several biological processes, including activation of PI3K/Akt/mTOR signalling, a key pathway in follicular activation. However, there is no report about the role of CCR2 in ovarian follicular physiology. The objectives of this study were (1) to immunolocalize CCR2 in mice ovaries and (2) to evaluate the effects of CCR2 deficiency on follicular growth during adult life and aging. A total of 74 C57Bl/6 (wild type, WT) and 68 B6.129S4-Ccr2tm1Ifc/J (CCR2^−/− (knockout)) female mice were used. For objective 1, ovaries were collected from WT mice at 1.5 months old (m.o.), fixed in 4% PFA, embedded in paraffin, and used for immunoperoxidase staining with an anti-CCR2 antibody [EPR19698] (ab222496; 1:200) and using anti-rabbit IgG (Ab6721, 1:100) as a secondary antibody. Also, MII oocytes from oviducts of superovulated WT mice were processed with the same primary antibody for immunofluorescence. For objective 2, body and ovarian weight were evaluated. Follicle populations were assessed in WT and CCR2^−/− mice at 1.5, 2.5, 6, 10, and 12 m.o., by serial sectioning; the total follicle population was counted in every third section in the whole ovary. Additionally, ovarian total RNA isolation was performed from WT and CCR2^−/−. Real-time PCR was used to evaluate differential gene expression according to standard protocols, using primers for Bax, Casp3, Bcl2, Fshr, and B-actin (endogenous control). The data were analysed using the GraphPad Prism Software, using t-test, and a P-value of 0.05 was considered as significant. Localization of CCR2 was observed exclusively on the membrane and cytoplasm of growing oocytes in primary, secondary, antral, and atretic follicles, as well as on ovulated MII oocytes membrane and cytoplasm. Body and ovarian weight were similar between WT and CCR2^−/− mice. At 1.5 m.o., CCR2^−/− mice had more primordial follicles and fewer primary and secondary follicles compared with WT (P < 0.05), whereas there was no difference in the antral follicle populations. Follicular activation (primordial to primary transition) and atresia rates were decreased in CCR2^−/− (P < 0.05) at 1.5 m.o. A downregulation of pro-apoptotic genes (Bax and Casp3) was observed on CCR2^−/− (P < 0.05), while anti-apoptotic Bcl2 was upregulated (P < 0.05) compared with WT in 1.5-m.o. animals. A larger ovarian follicular reserve at 1.5, 2.5, and 6 m.o., but not at 10 or 12 m.o., was observed in CCR2^−/− mice. At 6 to 12 m.o., CCR2^−/− ovulated more (P < 0.05) MII oocytes than WT. Altogether, these data may suggest that CCR2 plays an important role in the regulation of ovarian follicular reserve mobilization, potentially affecting reproductive lifespan.

Financial support was provided by Fapemig and CNPq.