Mitochondrial Function in Modulating Human Granulosa Cell Steroidogenesis and Female Fertility

Abstract

Ovarian follicle steroidogenesis associated with embryo quality results in a successful pregnancy. Each follicle consists of an oocyte surrounded by granulosa cells, which secrete several steroid and peptide hormones. Follicles harvested from women who conceived after assisted reproductive therapy (ART) had significantly higher estradiol levels in follicular fluids than the follicles from women who failed to conceive after ART. The higher follicular estradiol levels correlate well with successful fertilization following ART. Mitochondria are the central sites for steroid hormone biosynthesis. The first and rate-limiting step in the biosynthesis of steroid hormones occurs in the mitochondria of granulosa cells. In the present study, we hypothesized that the mitochondria in granulosa cells are critical for maintaining oocyte quality and fertility capacity. This study aims to clarify the relationship between mitochondrial function and granulosa cell steroidogenesis, and the relationship between hormone levels and fertility capacity. Sera, follicular fluids and granulosa cells were obtained from individuals undergoing IVF-ET treatment. The oocyte numbers, oocyte quality, fertilization rate, and pregnancy rate were also recorded. The patients who provided the granulosa cells were further classified into four groups: endometriosis, ovarian endometrioma, endometriosis without ovarian endometrioma, and polycystic ovary syndrome (PCOS); patients with other female factor infertility and male factor infertility were used as controls. We measured the levels of estradiol (E2) by radioimmunoassay. Concurrently, we analyzed the mitochondrial mass and membrane potential, and apoptosis by flow cytometry using nonyl acridine orange, TMRE, Annexin V-FITC and PI. Mitochondrial morphology was visualized by transfection with pLV-mitoDsRed. In addition, we assessed the protein levels of steroidogenic enzymes, steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) by Western blot. The results showed significantly decreased serum E2 and follicular E2 levels, and decreased IVF outcomes, in the patients with endometriosis. Reduced mitochondrial mass and decreased mitochondrial membrane potential were correlated with lower E2. Furthermore, a significant decrease in StAR and 3β-HSD was found in patients with ovarian endometrioma. The enzyme levels of StAR and 3β-HSD were highly correlated with E2 levels. Finally, elevated cumulus cell apoptosis was found in the patient group with ovarian endometrioma and PCOS. In conclusion, mitochondrial dysfunction of human granulosa cells may contribute to the decline of steroidogenesis, decreased fertilization rate, oocyte maturation rate, and oocyte quality, and it can ultimately jeopardize fertility.

Keywords: 3β-HSD.; StAR; estradiol; fertilization rate; granulosa cells; mitochondrial mass; progesterone; steroidogenesis.

Figure 1

Comparison of the pregnancy and fertilization in the different groups as an indication of infertility. The (A) fertilization rate and (B) pregnancy rate in each group were counted. The infertile individuals were characterized into six groups: other female factors (O, female factor without endometriosis and PCOS), endometriosis (E), ovarian endometrioma (EC), endometriosis without ovarian endometrioma (ENC), male factor (M), and polycystic ovary syndrome (P). The fertilization rate and pregnancy outcome values were significantly lower in the EC group than they were in the other groups. The p-value for each group versus the group with other female factors was determined by an unpaired t-test.

Figure 2

Comparison of oocyte quality and quantity in the different groups as an indication of infertility. The (A) follicle number, (B) oocyte number, and (C) number of mature oocytes (metaphase II) from all infertility patient groups. The infertile individuals were grouped into six groups: male factor (as control group, M), endometriosis (E), ovarian endometrioma (EC), endometriosis without ovarian endometrioma (ENC), other female factors (O), and polycystic ovary syndrome (P). The p-value for each group versus the male factor group was determined by an unpaired t-test.

Figure 3

The relationship between serum estradiol (E2) and cycle follicle outcomes in the various infertile groups. (A) Serum from each infertile group was collected, and serum E2 (pg/mL) was analyzed by radioimmunoassay (RIA). (B) Serum E2 per follicle in each group was compared. The E2 content in the serum before removing the eggs was divided by the number of follicles. The p-value for each group versus the male factor group was determined by an unpaired t-test.

Figure 4

The relationship between serum P4 and cycle follicle outcomes in the various infertile groups. (A) Serum from each infertile group was collected, and serum P4 was analyzed by radioimmunoassay (RIA). (B) The serum P4 per follicle was compared in the various infertile groups of each patient group. The p-value for each group versus the male factor group was determined by an unpaired t-test.

Figure 5

Comparison of the levels of E2 and P4 in the follicular fluid of the various infertile groups. (A) E2 content in the follicular fluid. (B) P4 content in the follicular fluid. The follicular fluid of the patient was collected, and the contents of E2 (pg/mL) and P4 (ng/mL) in the follicular fluid were analyzed by radioimmunoassay (RIA). The p-value for each group versus the male factor group was determined by an unpaired t-test.

Figure 6

Comparison of mitochondrial membrane potential in the various infertile groups. The mitochondrial membrane potential was measured by (A) N, N, N’, N’-tetramethyl-ethylenediamine (TMRE) and (B) MitoTracker Red staining and measured by flow cytometry. (C) The relationships between the mitochondrial membrane potential (TMRE or MitoTracker Red) and the serum E2 content were assessed by linear regression.

Figure 7

Comparison of mitochondrial mass in the various infertile groups. (A) The mitochondrial mass was measured by nonyl acridine orange (NAO) staining and flow cytometry. Reduced mitochondrial mass in cumulus cells was found in patients with endometriosis, other female factors, and PCOS. (B) The relationship between the mitochondrial potential and mitochondrial mass in the various groups was analyzed.

Figure 8

Representative immunofluorescent images for mitochondrial morphology in male factor (M), endometriosis (E), other female factors (O), and polycystic ovary syndrome (P). Images of cumulus cells from in vitro fertilization (IVF) patients were analyzed by confocal microscopy to visualize the mitochondrial network and cumulus cell expansion. Representative images show the fragmentation of the mitochondria in the endometriosis group and PCOS group. Scale bar = 25 µm.

Figure 9

Alteration of key steroidogenic enzymes in various infertile groups. Steroidogenic acute regulatory protein (StAR) and 3 beta-hydroxysteroid dehydrogenase (3β-HSD) are key steroidogenic enzymes. (A) Representative immunoblots reveal StAR (30 kDa) and 3β-HSD (43 kDa) levels in granulosa cells, as determined by Western blot; α-tubulin (50 kDa) was used as an internal control. (B) Data quantification of StAR and 3β-HSD. (C). Linear regression analysis was performed on serum E2 content with StAR or serum E2 content with 3β-HSD in granulosa cells.

Figure 10

Alteration of granulosa cell apoptosis in various groups. Apoptosis was measured by staining the cell membrane with annexin V- fluorescein-5-isothiocyanate FITC, staining the nucleus with propidium iodide (PI), and measuring the proportion of Annexin V-positive or PI-positive cells by flow cytometry. The top panel shows a scatter plot of each group [male factor (M), endometriosis (E), other female factors (O), and polycystic ovary syndrome (P)] in a flow cytometer. The bottom panel shows quantitative flow cytometry data. The p-value for each group versus the male factor group was determined by an unpaired t-test.

Figure 11

Schematic diagram summarizing the mitochondrial function in modulating human granulosa cell steroidogenesis and female fertility. Mitochondrial dysfunction of human granulosa cells may contribute to the decline of steroidogenesis, decreased fertilization rate, oocyte maturation rate, and oocyte quality, and it can ultimately jeopardize fertility.

Figure 12

Mitochondrial function in modulating human granulosa cell steroidogenesis and female fertility. Cholesterol transferred from granulosa cells, by low-density lipoprotein (LDL) receptor-mediated endocytosis, into theca cells, where it is used as a substrate for steroidogenesis. The conversion of cholesterol to pregnenolone is initiated by the binding of luteinizing hormone (LH) to the LH receptor (LHR), and subsequent conversion of androgens to E2 is initiated by the binding of follicle-stimulating hormone (FSH) to the follicle-stimulating hormone receptor (FSHR). Mitochondria are the central sites for steroid hormone biosynthesis. The first step in the biosynthesis of steroid hormones is the transfer of cholesterol to the mitochondrial outer membrane, which is facilitated by StAR. Then, cytochrome P450scc (CYP11A1) initiates steroidogenesis by converting cholesterol to pregnenolone at the mitochondrial inner membrane, and the enzyme 3β-HSD binds with P450scc to form a complex inserted into the mitochondrial inner membrane of the mitochondria to synthesize progesterone. The mitochondrial intermembrane proton gradient is essential for the 3β-HSD activity. Mitochondrial dysfunction of the human granulosa cells may contribute to the decline of steroidogenesis.