Naproxen administration affects murine late folliculogenesis, reduces granulosa cell proliferation and the number of ovulated oocytes.

Naproxen reduces the production of prostaglandins via inhibition of the cyclooxygenase. Studies have shown that its administration in women can be related to failed ovulation. Therefore, preclinical investigations must be performed in order to investigate its effects in experimental models. Thus, the aim of this study was to evaluate the effects of naproxen on murine folliculogenesis, ovulation, and female fertility. Female C57BL/6 mice (n = 128 - 6 weeks old) were divided into Control, low (10 mg/kg), and high naproxen (50 mg/kg) groups, who were treated for 8 days and directed to morphofunctional analyses. Follicular quantification showed a reduced percentage of antral follicles in naproxen-treated animals. These treated animals also showed smaller oocytes included in secondary and antral follicles, and the diameter of secondary and antral follicles was also reduced. A reduction in the percentage of Ki67-positive granulosa cells was observed in treated animals that also showed down-regulation of Igf1r compared to control. After an ovarian stimulation protocol, naproxen-treated animals showed a reduction in the percentage of secondary and antral follicles, a reduced number of ovulated oocytes and, corpora lutea, and an increased number of failed ovulations. Finally, naproxen-treated animals also showed a reduction in mating index and pregnancy rate. Our findings suggested that, in mice, naproxen administration (eight days treatment) negatively affects molecular and morphological aspects related to late folliculogenesis, ovulation, and fertility.

The CC-chemokine receptor 2 is involved in the control of ovarian folliculogenesis and fertility lifespan in mice.

The chemokine receptor 2 (CCR2) was first described as a chemotactic factor involved in immune responses, but it also plays an essential function in several biological processes. The chemokine (C-C motif) ligand 2 (CCL2) binds to CCR2 triggering G protein-coupled receptor (GPCR) signaling in leukocytes, including activation of PI3K/Akt/mTOR, a key pathway that is also related to follicular activation and survival. However, the potential role of CCR2 in ovarian follicular physiology remain unexplored. Thus, we investigated the role of CCR2 on follicular growth during adult life and aging. Ovaries and oocytes were collected from wild type (WT) mice at 1.5 months old (mo), and CCR2 expression was observed predominantly in oocytes included in growing follicles, as well as after ovulation. Follicle populations were assessed in WT and CCR2-/- mice at 1.5 mo, and CCR2-/- mice had more primordial and less primary and secondary follicles, while there were no differences in antral follicle numbers. Pro-apoptotic genes Bax and Casp3 were downregulated, while anti-apoptotic Bcl2 was upregulated in CCR2-/- mice. To further characterize the role of CCR2 in ovarian aging, follicle populations were assessed in WT and CCR2-/- mice at 1.5, 2.5, 6, 10, and 12 mo. A larger ovarian follicular reserve at 1.5-6 mo was observed in CCR2-/- mice. Finally, CCR2-/- aged mice (6-12 mo) ovulated more oocytes than WT mice. Altogether, these data suggest that CCR2 plays an important role in the regulation of murine folliculogenesis, potentially affecting the reproductive lifespan.

Vitrification leads to transcriptomic modifications of mice ovaries that do not affect folliculogenesis progression.

Ovarian tissue cryopreservation is emerging as a promising alternative for fertility preservation of cancer survivors. To date, more than a hundred couples have successfully had babies using this procedure, although it is still considered experimental and demands further investigation. In this work, we evaluated the effects of vitrification, warming and autotransplantation procedures on the morphology and gene expression of murine ovaries. Ovaries were removed from adult female C57BL6 mice (n = 15), vitrified, warmed and autotransplanted (vitrified group), additionally, ovaries were autotransplanted without vitrification (control group, n = 15). After twenty days, grafted ovaries were harvested and used for histological and ultrastructural analysis, germinal vesicle (GV) oocyte collection, RNA sequencing, and Transmission Electron Microscopy (TEM). All classes of follicles and GV were observed in both control and vitrified/warmed transplanted ovaries, and the numbers of primordial, antral and atretic follicles were not different (p > 0.05). Using RNA-seq, we detected 16,602 vs 13,527 expressed genes in vitrified and control ovaries, respectively; and 623 significantly dysregulated genes (fold change >1.5; 332 up-regulated and 291 down-regulated). Cellular membranes, cytoskeletons, and extracellular matrices were found as the main functions of the differentially expressed genes. Moreover, vitrified samples also presented ultrastructural alterations in the cytoskeleton, cell junctions, and endoplasmic reticulum. Taken together, this work showed for the first time that ovarian cells might trigger a compensatory gene regulation mechanism to maintain cellular structure and folliculogenesis progression after vitrification and autotransplantation.