Erythropoietin-producing hepatocellular A7 triggering ovulation indicates a potential beneficial role for polycystic ovary syndrome

Abstract

Background: The ovulatory dysfunction mechanisms underlying polycystic ovary syndrome (PCOS) are not completely understood. And the roles of EPHA7 and EPHA7-regulated pathway factors in the pathogenesis of anovulation remain to be elucidated.

Methods: We used human granulosa cells (hGCs) of PCOS and non-PCOS patients to measure EPHA7 and other target gene expressions. We performed in vitro experiments in KGN cells to verify the molecular mechanisms. Additionally, we conducted in vivo loss- and gain-of-function studies using EPHA7 shRNA lentivirus and recombinant EPHA7-Fc protein injection to identify the ovulation effects of EPHA7.

Findings: EPHA7 functions as a critically positive upstream factor for the expression of ERK1/2-mediated C/EBPβ. This protein, in turn, induced the expression of KLF4 and then ADAMTS1. Moreover, decreased abundance of EPHA7 was positively correlated with that of its downstream factors in hGCs of PCOS patients. Additionally, a 1-week functional EPHA7 shRNA lentivirus in rat ovaries contributed to decreased numbers of retrieved oocytes, and a 3-week functional lentivirus led to menstrual disorders and morphological polycystic changes in rat ovaries. More importantly, we found that EPHA7 triggered ovulation in rats, and it improved polycystic ovarian changes induced by DHEA in PCOS rats.

Interpretation: Our findings demonstrate a new role of EPHA7 in PCOS, suggesting that EPHA7 is an effective target for the development of innovative medicines to induce ovulation. FUND: National Key Research and Development Program of China, National Natural Science Foundation, Shanghai Municipal Education Commission--Gaofeng Clinical Medicine, and Shanghai Commission of Science and Technology.

Keywords: EPHA7; Female fertility; Ovulation; PCOS.

Fig. 1

Decreased mRNA expression of EPHA7, C/EBPβ, KLF4, and ADAMTS1 in granulosa cells of PCOS patients. Relative mRNA abundances of EPHA7 (A), C/EBPβ (B), KLF4 (C) and ADAMTS1 (D) in ovarian granulosa cells from 32 non-PCOS patients and 40 PCOS patients calculated by qRT-PCR analysis. β-actin values were used for normalization. * P < .05, *** P < .001.

Fig. 2

EPAH7 promotion of KLF4 expression via C/EBPβ, leading to induction of ADAMTS1 expression in KGN cells. (A) mRNA and protein abundances of KLF4 and ADAMTS1 after KLF4 knockdown in KGN cells detected by western blot analysis and qPCR analysis. The panel (left-to-right) shows representative images of western blot assays, we quantified protein abundances by measuring the densitometry of the immunoreactive bands. (B) mRNA and protein abundances of KLF4 and ADAMTS1 after KLF4 overexpression in KGN cells. (C) mRNA and protein abundances of C/EBPβ, KLF4 and ADAMTS1 after C/EBPβ knockdown in KGN cells. Left, a representative western blot is shown. Right, the immunoreactive bands were densitometrically quantified (above); and mRNA abundance is presented (below). (D) mRNA and protein abundances of C/EBPβ, KLF4 and ADAMTS1 after LAP overexpression in KGN cells. (E) mRNA and protein abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 after EPHA7 knockdown in KGN cells. (F) mRNA and protein abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 after EPHA7 overexpression and further incubation with PD98059 (ERK1/2 inhibitor) in KGN cells. Above, a representative western blot is shown (left), and the immunoreactive bands for ERK1/2 phosphorylation were quantified densitometrically (right). Middle, the immunoreactive bands for other proteins were also quantified densitometrically. Below, mRNA abundance is presented. (G) Above, we used a ChIP assay to detect the enrichment of C/EBPβ at the KLF4 promoter in KGN cells in response to EPHA7 overexpression. IgG served as the negative control. Below, sequence of the KLF4 promoter spanning–879 to–865 base pairs (bp). Boxed letters indicate putative transcription factor binding sites. TSS, transcription start site. β-actin or GAPDH were used as loading controls for western blot and for qPCR analyses. Blots are representative and data are presented as means ± SEM from 3 to 5 experiments. * P < .05, ** P < .01, *** P < .001 against si-NC cells or against Control-vector cells; # P < .05, ## P < .01 against EPHA7-vector cells.

Fig. 3

Ovulatory dysfunction in rats due to low expression levels of EPHA7 and its downstream factors. (A) Immunohistochemistry demonstrates the distribution of EPHA7 in granulosa cells (triangle), theca cells (arrow) and corpus luteum (asterisk) of the rat ovaries. (B) Body weight (left) and ovarian weight (right) of rats. (C) Representative images of oocytes retrieved per rat. (D) Number of oocytes retrieved per rat. (E) mRNA and protein whole-ovary abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 in rats, detected by western blot and qPCR analyses. The panel (left-to-right) shows representative images of western blot assays, immunoreactive bands were quantified by densitometry; and relative mRNA abundances by qPCR. β-actin or GAPDH were used as loading controls. N = 6 per group. Data are presented as means ± SEM. * P < .05, ** P < .01, *** P < .001.

Fig. 4

Long-term effects of EPHA7 shRNA lentivirus contributing to irregular estrus cycles and polycystic ovaries in rats. (A) Estrous cycles were detected in rats injected with control shRNA (above) or EPHA7 shRNA lentivirus (below). D, diestrus; P, proestrus; E, estrus; M, metestrus. (B) Body weight changes (left), ovarian weight (middle), and representative image of rats' ovaries (right). (C) Rats' serum LH (left) and FSH (middle) levels, and LH/FSH ratio (right) detected by ELISA analysis. (D) Histology of microscopic ovarian structures of rats injected with control shRNA (above) or EPHA7 shRNA lentivirus (below). Asterisk stands for corpus luteum. N = 4 per group. Data are presented as means ± SEM.

Fig. 5

EPAH7 triggers ovulation after induction in rats. (A) Number of oocytes retrieved per rat. (B) mRNA abundances of EPHA7, C/EBPβ, KLF4, and ADAMTS1 in the rats' ovaries were quantified by qPCR analyses. (C) Serum EPHA7 levels in rats before and every 4 h after PBS, hCG, or EPHA7 injection detected by ELISA. (D) Serum LH levels in rats before and every 4 h after PBS, hCG, or EPHA7 injection detected by ELISA. (E) Serum E₂ levels in rats before and every 4 h after PBS, hCG, or EPHA7 injection detected by ELISA. β-actin was used as a loading control. N = 5 per group. Data are presented as means ± SEM. * P < .05.

Fig. 6

Improvement of the 16-h function of EPAH7 in PCOS rats. (A) Estrous cycles were detected during the eight last days after the establishment of the PCOS rat model. The panel (top-to-bottom) shows the CONTROL, CONTROL+EPHA7, DHEA, and DHEA+EPHA7 groups. D, diestrus; P, proestrus; E, estrus; M, metestrus. (B) Body weight (left) and ovarian weight (right) changes in rats. (C) Glucose tolerance test and AUC values were obtained after each daily injection (s.c.) with DHEA for 20 consecutive days. (D) Rats' serum EPHA7 levels detected by ELISA. (E) Rats' serum LH (left) and FSH (middle) levels, and LH/FSH ratio (right) detected by ELISA. (F) Rats' histological microscopic ovarian structures. The panel (left-to-right) shows the CONTROL, CONTROL+EPHA7, DHEA, and DHEA+EPHA7 groups. (G) mRNA and protein abundances of StAR and CYP11A1 in rats' ovaries detected by western blot and qRT-PCR analyses. The panel (left-to -right) shows representative images of western blot assays (the immunoreactive bands were densitometrically quantified), and relative mRNA abundances. (H) Rats' serum progesterone levels detected by ELISA. (I) mRNA and protein abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 in the rats' ovaries. β-actin or GAPDH were used as loading controls. N = 6 per group. Blots are representative, and data are presented as means ± SEM. * P < .05, ** P < .01, *** P < .001 against the group marked in the figure or against control rats; ## P < .01 against DHEA rats.