ERβ regulated ovarian kisspeptin plays an important role in oocyte maturation

Abstract

Kisspeptin (KISS1) signaling in the hypothalamic-pituitary (H-P) axis plays an essential role in regulating gonadotropin secretion. KISS1 and KISS1 receptor (KISS1R) are also expressed in the ovary; however, the role of intraovarian KISS1 signaling remains unclear. Granulosa cell (GC)-specific expression of KISS1, and oocyte-specific expression of KISS1R indicate that GC-derived KISS1 may act on oocytes. Expression of KISS1 in GCs is induced by gonadotropins but it is absent in estrogen receptor β knockout (Erβ^null) rat ovaries. We also observed that gonadotropin stimulation failed to induce maturation of Erβ^null oocytes. Interestingly, KISS1 treatment of cumulus oocyte complexes (COCs) isolated from antral follicles promotes in vitro maturation of oocytes. Treatment of oocytes with KISS1 induced intracellular Ca²⁺ release, and increased activation of MAP kinase ERK1/2. KISS1 treatment also induced the expression of oocyte genes that are crucial for differentiation of GCs, and maturation of oocytes. Our findings suggest that ovarian KISS1-signaling plays an important role in gonadotropin induced follicle development and oocyte maturation.

Keywords: And oocyte maturation; Estrogen receptor β; Gonadotropin; Granulosa cells; Kisspeptin; Kisspeptin receptor.

Fig. 1.. Abnormal oocyte maturation in Erβ^null rats.

8–12 wk-old Erβ^null adult rats are infertile and they failed to ovulate either during natural estrus cycle (A) or induced by exogenous gonadotropins to 4 wk-old rats (C). Oocytes isolated from 4-wk-old wildtype (WT) and Erβ^null rat ovaries at different stages of gonadotropin treatment were examined after removal of cumulus cells. Although unstimulated (Basal D and E) oocytes of both genotypes appeared similar, abnormal division and fragmentation was observed in Erβ^null oocytes after PMSG 48h (F and G), 4h after hCG (H and I), and 24h after hCG (J and K) treatment. n = 6. *P ≤ 0.05.

Fig. 2.. Kiss1 and Kiss1r expression in rat ovary.

Oocytes and granulosa cells (GCs) were isolated from the ovaries collected 4h after hCG administration to PMSG treated 4-wk-old rats. RT-qPCR analyses showed that expression of Kiss1 was remarkably higher in (GCs) (A), whereas Kiss1r in the oocytes (B). RNA sequencing analyses showed a significantly higher expression of Kiss1 in wildtype (WT) GCs compared to Erβ^null GCs (C), but a similar level of Kiss1r expression in both WT and Erβ^null oocytes (E). RT-qPCR data from GCs and oocytes further confirmed the results of RNA-sequencing (D and F). RNA-sequencing data are presented in triplicates. RT-qPCR data are represented as mean ± SE. n = 6. *P ≤ 0.05.

Fig. 3.. Role of KISS1 in oocyte maturation.

48h after PMSG treatment, cumulus oocyte complex (COCs) were isolated from 4-wk-old wildtype and Erβ^null rats and cultured in BO-IVM in presence or absence of 1nM of rat kisspeptin 10 (KP-10) for 20h. Approximately 55% of wildtype (A and C) and only 35% of Erβ^null oocytes achieved meiotic maturation (MII) in BO-IVM culture (control) (D and F). Remarkably, addition of KP-10 to BO-IVM increased the in vitro maturation to 78% of wildtype (B and C) and 56% of Erβ^null oocytes (E and F). Each treatment group contained at least 25 oocytes at each time point and repeated three times. Cont, Control.

Fig. 4.. Role of KISS1 in inducing calcium efflux and ERK signaling.

The expression of KISS1R was significantly higher in oocytes compared to granulosa cells (GCs) as shown by western blot analysis (A, B). Oocytes from PMSG treated rats were examined for calcium efflux using a Fluo-3 assay kit. Calcium efflux increased after the treatment with kisspeptin 10 (KP-10) (F, I) compared to control (D). 1nM of KP-10 also markedly elevated ERK1/2 phosphorylation in oocytes (G, J) but not AKT (H, K). Ph, Phase contrast; Fluo, Fluorescence microscopy; Cont, Control. Calcium efflux from 15 oocytes and Western blot data from three different samples were quantified by ImageJ analyses. Data represent the mean ± SE. *P < 0.05, n ≥ 3.

Fig. 5.. KP-10 treatment regulated genes involved in inducing oocyte maturation.

48h after PMSG treatment, cumulus oocyte complex (COCs) were isolated from 4-wk-old wildtype female rats and treated with 1nM of rat kisspeptin 10 (KP-10) for 30 min. KP-10 treatment upregulated the mRNA levels of Gdf9, Bmp15, but downregulated Bmp7 (A-C). It also upregulated the mRNA levels of protein kinases cMos, Kit, and Aurka (D-F), as well as Wee2 and Cdc25b (G and H) important for meiotic maturation. Moreover, KP-10 treatment upregulated the mRNA levels of RNA binding proteins Dazl, Btg4, and Papbc1l (J-L) required for selective mRNA stability during oocyte maturation. RT-qPCR data represent the mean ± SE. *P < 0.05, n = 6.

Fig. 6.. Intraovarian role of ovarian derived KISS1.

Based on our findings, we conclude that the preovulatory LH surge induces the expression of KISS1 (KP) by granulosa cells (GCs), which is ERβ-dependent. GC-derived KP acts on the KISS1R (KR) expressed in oocytes to increase Ca2+ release, ERK phosphorylation, and modulation of genes, which are important for inducing oocyte maturation.