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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 4  |  Page : 195-203

Luteotropic roles of glucocorticoids in rat granulosa cells


Department of Pharmacy, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing 210004, Jiangsu, China

Date of Submission02-Sep-2020
Date of Decision15-Oct-2020
Date of Acceptance29-Oct-2020
Date of Web Publication31-Dec-2020

Correspondence Address:
Xin-Yan Huang
Women's Hospital of Nanjing Medical University, No. 123, Tianfei Lane, Nanjing 210004, Jiangsu
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.305933

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  Abstract 


Objective: The study objective was to investigate whether endogenous glucocorticoids directly impact the functions and proliferation/apoptosis of ovarian granulosa cells.
Methods: Primary rat ovarian granulosa cells were cultured and treated with graded concentrations of corticosterone either alone or in the presence of the indicated drugs. After 48 h of treatment, the cells and growth media were collected to measure intracellular and extracellular progesterone/estradiol concentrations, and steroid secretion ratios were obtained by parameter calculation. The number of granulosa cells was determined by Cell Counting Kit-8. To determine the impact on cell numbers, granulosa cell proliferation was detected using the BrdU incorporation method and cell apoptosis was detected by flow cytometry.
Results: First, high corticosterone concentrations significantly stimulated progesterone synthesis/secretion and inhibited estradiol synthesis in cultured granulosa cells. Second, accompanied by follicle-stimulating hormone, high corticosterone concentrations promoted progesterone synthesis/release and estradiol release. Last, high corticosterone concentrations increased the cell number and suppressed apoptosis but did not induce cell proliferation.
Conclusions: These indicate that high glucocorticoid concentrations may play luteotropic roles in the functions and number of corpora lutea.

Keywords: Apoptosis; Glucocorticoids; Granulosa Cells; Ovary; Steroid


How to cite this article:
Huang XY, Xu YY, Xie J, Wang L. Luteotropic roles of glucocorticoids in rat granulosa cells. Reprod Dev Med 2020;4:195-203

How to cite this URL:
Huang XY, Xu YY, Xie J, Wang L. Luteotropic roles of glucocorticoids in rat granulosa cells. Reprod Dev Med [serial online] 2020 [cited 2021 Sep 17];4:195-203. Available from: https://www.repdevmed.org/text.asp?2020/4/4/195/305933




  Introduction Top


Glucocorticoids are the most important regulator of stress response in the body, and endogenous glucocorticoid levels significantly increase in patients with depression and Cushing syndrome. A growing body of evidence indicates that high glucocorticoids have important effects on reproductive processes, such as follicular maturation, ovulation, and pregnancy.[1],[2],[3] Glucocorticoids are able to indirectly modulate ovarian function by acting on the hypothalamus and pituitary axes to alter the levels of circulating gonadotropins.[3] Glucocorticoids can also directly modulate ovarian function through its receptors in ovarian cells. There are some contradictions in the current research, where some researchers believe that glucocorticoids exert agonistic effects, whereas others persist in the idea that glucocorticoids exert antagonistic effects. These contradictions may be explained through differences in glucocorticoid levels and different stages of follicular development.

These findings have revealed a substantial gap in our knowledge concerning the influence glucocorticoids exert on the corpus luteum. Glucocorticoid receptors are present on ovarian surface epithelium cells, ovarian follicles, and the corpus luteum of rats and humans.[4] The presence of glucocorticoid-metabolizing enzymes has previously been verified in the corpus luteum and in luteinized granulosa cell cultures.[5],[6],[7],[8] These findings raise the possibility of a physiological role for glucocorticoids in the corpus luteum, whereby glucocorticoids are directly involved in luteal cell processes independent of the hypothalamic–pituitary–ovary axis. However, whether glucocorticoids play a positive role (such as a luteotropic role) or a negative role (such as a luteolytic role) in the corpus luteum is unknown.

To determine the direct regulatory effects of endogenous glucocorticoids on the corpus luteum, we used cultured rat ovarian granulosa cells as the cellular model and examined the effects of endogenous glucocorticoids on corpus luteum functions (progesterone/estradiol synthesis and secretion) and structure (cell number, proliferation, and apoptosis).


  Methods Top


Granulosa cell cultures

Animal maintenance and use procedures were in strict accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the Ethics Committees on Research of Nanjing Maternal and Child Health Hospital. The permit number was 201361.

Female rats (6–8 weeks, Wistar strain) were primed with pregnant mare serum gonadotropin (10 U, injected intraperitoneally) to stimulate the development of follicles, and the rats were sacrificed by decapitation 48 h later. Ovarian granulosa cells were collected and resuspended in DMEM/F12 medium containing 10% fetal bovine serum, 0.1 μmol/L androstenedione, and 2 mmol/L L-glutamine and then, seeded into culture dishes. The medium was changed on the first day after plating and replaced every 2 days thereafter. Cultures were maintained in an incubator at 37°C for 5 days or 10 days. The culture medium was then replaced with serum-free medium to begin the drug treatments. Depending on the experiment, cells were treated with either graded concentrations of corticosterone (0 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, and 10-5 mol/L) (Sigma-Aldrich, St. Louis, MO, USA) either alone or in the presence of the indicated drugs and follicle-stimulating hormone (FSH) (0.01 μmol/L).[9] After 48 h of treatment, the cells and medium were collected for study.

Steroid assay

Granulosa cells were treated with the indicated drugs for 48 h and the cells and culture medium were then collected to measure intracellular and extracellular progesterone/estradiol concentrations, which were determined by direct measurements with ELISA kits according to the manufacturer's instructions (Elabscience, Wuhan, Hubei, China).

Cell number, proliferation, and apoptosis analysis

The total number of granulosa cells was determined by Cell Counting Kit-8 (7 seabiotech, Shanghai, China). The granulosa cells were cultured in 96-well plates and incubated with the indicated drugs for 48 h. The cells were then treated according to the kit manufacturer's instructions and the absorbance at 450 nm was measured with a microplate reader. Finally, the number of granulosa cells was obtained indirectly.

Granulosa cell proliferation was measured with a bromodeoxyuridine (BrdU, Sigma, USA) incorporation assay. The granulosa cells were cultured in 12-well plates and treated with drugs for 48 h. During the last 24 h of culture growth, 10 μmol/L BrdU was added to the dividing cells. The granulosa cells were fixed and blocked, and then incubated with anti-BrdU antibodies (1:100; Abcam, Cambridge, UK). Next, the cells were exposed to fluorescein isothiocyanate (FITC)-conjugated secondary antibodies at a dilution of 1:300 for 60 min. Finally, the cells were mounted in antifade mounting medium with 4',6-diamido-2-phenylindole hydrochloride and analyzed under a fluorescence microscope (Axio Imager, Oberkochen, Baden-Wurttemberg, Germany).

Granulosa cell apoptosis was examined with the Annexin V-FITC/PI Apoptosis Detection Kit (Keygentech, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Cells were subjected to flow cytometry to measure cell apoptosis.

Western blot analysis

Expression of the P450 aromatase (P450arom) protein was analyzed by Western blot. After 48 h of drug treatments, the granulosa cells were harvested and lysed. The protein samples (30 μg total protein) were loaded onto an SDS-polyacrylamide gel, electrophoresed, and transferred to a PVDF membrane. The membranes were blocked and incubated with P450arom (1:1,000, Abcam, UK) and β-actin (1:2,000, Abcam, UK) antibodies diluted in Tris Buffered Saline Tween (TBST). Then, the membranes were incubated for 2 h at room temperature with IgG and horseradish peroxidase-conjugated secondary antibodies in TBST. Finally, immunoreactivity was visualized with the ECL Detection Kit according to the manufacturer's instructions.

Statistical analysis

Data were presented as the means ± standard error of the mean from three experiments. Comparisons between multiple groups were analyzed with one-way ANOVA (one factor), followed by the Bonferroni test. Comparisons between two groups were analyzed with a two-tailed Student's t-test. Differences were considered statistically significant when P < 0.05.


  Results Top


Glucocorticoids regulate progesterone and estradiol synthesis and release from granulosa cells cultured for 5 days

Granulosa cells in the corpus luteum are responsible for the synthesis and secretion of progesterone/estradiol during the estrous cycle and early pregnancy. To confirm the effects of glucocorticoids on steroid synthesis and release, we cultured rat ovarian granulosa cells for 5 days, treated those cells with corticosterone (glucocorticoids in rodents) at graded concentrations of 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, and 10-5 mol/L for 48 h, and then collected the cells and culture medium to measure progesterone/estradiol concentrations. Intracellular progesterone levels decreased signi?cantly from 1.88 ± 0.19 ng/106 cells/48 h (control) to 0.89 ± 0.04 ng/106 cells/48 h (at 10-5 mol/L corticosterone) [Figure 1]Aa. The extracellular progesterone levels increased from 0.75 ± 0.01 ng/mL (control) to 15.02 ± 0.31 ng/mL (at 10 -5 mol/L corticosterone) [Figure 1]Ab. Total progesterone production markedly increased to 15.91 ± 0.33 ng/106 cells/48 h after treatment with 10-5 mol/L corticosterone but not after treatment with corticosterone at any other concentration [Figure 1]Ac. The progesterone secretion ratio gradually increased and reached a maximum level of 94.38% ± 0.18% at 10-5 M corticosterone [Figure 1]Ad. From these results, we conclude that high glucocorticoid concentrations promote progesterone synthesis by and release from granulosa cells when those cells are cultured for 5 days.
Figure 1: Effect of glucocorticoids on progesterone/estradiol synthesis and release from granulosa cells. (A) Graphs showing the intracellular progesterone levels (a), extracellular progesterone levels (b), total progesterone levels (c), and progesterone secretion ratios (d) of granulosa cells that were cultured for 5 days and then treated. (B) Graphs showing the intracellular estradiol levels (a), extracellular estradiol levels (b), total estradiol levels (c), and estradiol secretion ratios (d) of granulosa cells that were cultured for 5 days and then treated. (C) Graphs showing the intracellular progesterone levels (a), extracellular progesterone levels (b), total progesterone levels (c), and progesterone secretion ratios (d) of granulosa cells that were cultured for 10 days and then treated. (D) Graphs showing the intracellular estradiol levels (a), extracellular estradiol levels (b), total estradiol levels (c), and estradiol secretion ratios (d) of granulosa cells that were cultured for 10 days and then treated. Granulosa cells were cultured for the indicated days and then treated with corticosterone (0 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, and 10-5 mol/L) for 48 h. Granulosa cells treated with vehicle alone (0 mol/L corticosterone) were the control group. Data are presented as the mean ± SEM of three separate experiments. *P < 0.05,P < 0.01 versus control. One-way ANOVA. SEM: Standard error of the mean.

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With the graded concentrations of corticosterone, the intracellular estradiol level and total estradiol production markedly decreased to low levels [Figure 1]Ba and [Figure 1]Bc. Total estradiol production reduced from 9.43 ± 1.18 ng/106 cells/48 h (control) to 3.09 ± 0.07 ng/106 cells/48 h (at 10-5 mol/L corticosterone). Conversely, at 10-5 mol/L corticosterone, the extracellular estradiol levels increased from 0.31 ± 0.07 ng/106 cells/48 h to 0.79 ± 0.02 ng/106 cells/48 h and the estradiol secretion ratio increased from 3.25% ± 0.35% to 25.73% ± 1.14% [Figure 1]Bb and [Figure 1]Bd. We conclude that high corticosterone concentrations promote estradiol release but suppress estradiol synthesis by granulosa cells when those cells are cultured for 5 days.

Glucocorticoids regulate progesterone and estradiol synthesis and release from granulosa cells cultured for 10 days

It is well known that luteinized granulosa cell functions change with the physiological luteal cycle and pregnancy, so we speculated that the effects of glucocorticoids on granulosa cell functions may change as the culture time is prolonged. We cultured granulosa cells for 10 days, then treated them with corticosterone for 48 h, and determined the progesterone/estradiol concentrations in the cells and medium.

Total progesterone production increased significantly from 2.50 ± 0.17 ng/106 cells/48 h (control) to 18.68 ± 2.88 ng/106 cells/48 h (at 10-5 mol/L corticosterone) [Figure 1]Cc. After stimulation with 10-5 mol/L corticosterone, the extracellular progesterone level increased from 1.12 ± 0.09 ng/106 cells/48 h to 16.93 ± 2.76 ng/106 cells/48 h and the progesterone secretion ratio increased from 44.86% ± 2.03% (control) to 90.38% ± 0.94% [Figure 1]Cb and [Figure 1]Cd. Therefore, high corticosterone concentrations also promote progesterone synthesis in, and release from, granulosa cells when cells are cultured for 10 days.

Conversely, under the same conditions, extracellular estradiol levels, total estradiol production, and the estradiol secretion ratio all showed downward trends that reached statistical significance at multiple corticosterone concentrations [Figure 1]Db [Figure 1]Dd. In summary, when cell culture is extended to 10 days, the stimulatory effects of high glucocorticoid concentrations on progesterone synthesis and secretion remain unchanged. With time, the inhibitory effects of high glucocorticoid concentrations on total estradiol production remain the same, but the stimulatory effects of those concentrations on estradiol secretion become inhibitory effects.

Glucocorticoids regulate progesterone and estradiol synthesis and release from follicle-stimulating hormone-stimulated granulosa cells

FSH is the most important physiological hormonal regulator of ovarian follicle growth, and it is widely used in the field of assisted reproductive techniques. We were interested in the corticosterone responses of FSH-stimulated granulosa cells. Therefore, we examined steroidogenesis in, and steroid release from, granulosa cells cultured for 5 days and treated with FSH (0.01 μmol/L) in combination with corticosterone (0 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, and 10-5 mol/L) for 48 h.

As observed with unstimulated granulosa cells, progesterone synthesis in, and release from, FSH-stimulated granulosa cells increased when the cells were treated with corticosterone. The steroid assay showed that at 10-5 mol/L corticosterone, these FSH-stimulated granulosa cells had signi?cant increases in both total progesterone production, from 3.14 ± 0.03 ng/106 cells/48 h to 8.40 ± 1.35 ng/106 cells/48 h, and extracellular progesterone levels, from 2.39 ± 0.16 ng/106 cells/48 h to 7.65 ± 1.41 ng/106 cells/48 h [Figure 2]Ab and [Figure 2]Ac. The progesterone secretion ratio also showed a clear upward trend. Thus, these data suggest that high corticosterone concentrations also promote progesterone synthesis by, and release from, FSH-stimulated granulosa cells.
Figure 2: Effect of glucocorticoids on progesterone/estradiol synthesis and release from FSH-stimulated granulosa cells. (A) Graphs showing the intracellular progesterone levels (a), extracellular progesterone levels (b), total progesterone levels (c), and progesterone secretion ratios (d) of granulosa cells cultured for 5 days and then treated. (B) Graphs showing the intracellular estradiol levels (a), extracellular estradiol levels (b), total estradiol levels (c), and estradiol secretion ratios (d) of granulosa cells cultured for 5 days and then treated. Granulosa cells were treated with FSH (0.01 μmol/L) in combination with corticosterone (0 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, and 10-5 mol/L) for 48 h. Granulosa cells treated with vehicle alone (0 mol/L corticosterone) were the control group. Data are presented as the mean ± SEM of three separate experiments. *P < 0.05 versus control. One-way ANOVA. FSH: Follicle-stimulating hormone; SEM: Standard error of the mean.

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Corticosterone treatment had no effect on total estradiol production by FSH-stimulated granulosa cells at any concentration. However, at 10 - 5 mol/L corticosterone, those FSH-stimulated granulosa cells showed increased extracellular estradiol levels, from 0.06 ± 0.02 ng/106 cells/48 h to 0.30 ± 0.05 ng/106 cells/48 h, and an increased estradiol secretion ratio, from 5.56% ± 1.96% to 28.78% ± 3.91% [Figure 2]Bb and [Figure 2]Bd. Thus, high corticosterone concentrations stimulated estradiol secretion from, but not estradiol synthesis by, FSH-stimulated granulosa cells. Thus, it is clear that when granulosa cells are stimulated with FSH, high corticosterone concentrations still have some positive effects on progesterone and estradiol synthesis and release.

Glucocorticoids do not affect P450arom protein expression

Although it is not known precisely how glucocorticoids affect steroidogenesis and secretion, they presumably function through the mediation of enzymes in these processes. P450arom is considered to be a representative enzyme in the steroidogenic pathway. However, when granulosa cells were stimulated with 10 -5 mol/L corticosterone, the expression level of the P450arom protein (as quantified by Western blot) remained unchanged [Figure 3]a and [Figure 3]b. We hypothesized that changes in enzymatic activity influenced the process of steroid synthesis and secretion, and determined that protein chip experiments were necessary to identify target enzymes.
Figure 3: Glucocorticoids do not affect P450arom protein expression. (a) Immunoblots showing the P450arom protein in granulosa cells cultured for 5 days and then treated with 0 mol/L or 10-5 mol/L corticosterone for 48 h. (b) Graph showing the ratio of P450arom to β-actin in granulosa cells cultured for 5 days and then treated with 0 mol/L or 10-5 mol/L corticosterone for 48 h. Granulosa cells treated with vehicle alone (0 mol/L corticosterone) were the control group. Data are presented as the mean ± SEM of three separate experiments. Student's t-test. (c-h) Graphs showing the concentrations of all the sex steroids (c and f), the progesterone proportion (d and g), and the estradiol proportion (e and h) in granulosa cells cultured for 5 or 10 days and then treated with corticosterone (0 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, and 10-5 mol/L) for 48 h. Granulosa cells treated with vehicle alone (0 mol/L corticosterone) were the control group. Data are presented as the mean ± SEM of three separate experiments. *P < 0.05,P < 0.01 versus control. One-way ANOVA. SEM: Standard error of the mean.

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Before performing further experiments, we examined several details of the steroidogenesis process and noticed some interesting secretion-related enzyme activities. First, the total sex steroid levels showed a spoon-shaped change pattern. At 10 -5 mol/L corticosterone, the levels increased to 6.19 ± 0.08 × 10 -2 nmol/106 cells/48 h and 7.31 ± 0.85 × 10 -2 nmol/106 cells/48 h when cells were cultured for 5 days and 10 days, respectively [Figure 3]c and [Figure 3]f. These results indicated that high glucocorticoid concentrations may promote intracellular transport of cholesterol and induce cellular cholesterol uptake. Second, we measured the levels of progesterone synthesis and found that as the corticosterone concentration increased, progesterone proportion increased [Figure 3]d and [Figure 3]g, while estradiol proportion decreased [Figure 3]e and [Figure 3]h. This suggests that corticosterone significantly stimulates the steroidogenic enzymes of progesterone, while only weakly stimulating, or possibly inhibiting, estradiol synthase.

Glucocorticoids suppress apoptosis of granulosa cells but do not affect cell proliferation

The corpus luteum is an active, transient, and dynamic endocrine gland, and its structure undergoes extensive tissue remodeling via processes that include cell proliferation and apoptosis. We examined the proliferation and apoptosis of granulosa cells that were cultured for 5 days and then treated with 10-5 mol/L corticosterone to evaluate the structural remodeling of the corpus luteum. First, treatment with 10-5 mol/L corticosterone increased the total number of cells quantified with the Cell Counting Kit-8 by 28.9% ± 2.6% compared with that of the control [Figure 4]a. Second, BrdU incorporation experiments showed that corticosterone treatments had no significant effect on the ratio of BrdU-positive cells in granulosa cells [Figure 4]b and [Figure 4]c. Finally, treatment with 10-5 mol/L corticosterone decreased the apoptosis rate by 22.0% ± 3.7% (quantified with the Annexin V-FITC/PI staining cell apoptosis assay) compared with that of the control [Figure 4]d and [Figure 4]e. These data indicate that high corticosterone concentrations increased the total granulosa cell number, which could be due to suppressed cell apoptosis because high corticosterone concentrations did not induce cell proliferation.
Figure 4: Glucocorticoids suppress granulosa cell apoptosis but do not induce cell proliferation. (a, b and e) Graphs showing the total cell number (a), the ratio of BrdU-positive cells (b), and the apoptosis rate (e) of granulosa cells cultured for 5 days and then treated with 0 mol/L or 10-5 mol/L corticosterone for 48 h. (c and d) Representative analysis of BrdU-labeled cells (blue) (c) and the apoptosis rate (d) of granulosa cells cultured for 5 days and then treated with 0 mol/L or 10-5 mol/L corticosterone for 48 h. Granulosa cells treated with vehicle alone (0 mol/L corticosterone) were the control group. Data are presented as the mean ± SEM of three separate experiments. *P < 0.05 versus control. Student's t-test. Scale bars = 100 μm. DAPI: 4',6-diamido-2-phenylindole hydrochloride; BrdU: Bromodeoxyuridine; SEM: Standard error of the mean.

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  Discussion Top


It is well accepted that glucocorticoids have positive and negative effects in the body in general and in the ovary in particular.[3],[10],[11] These differential effects may primarily be due to differences in the exposure duration and dosage. Time-limited exposure to low doses of glucocorticoids may produce beneficial changes, but prolonged or continuous exposure to high dosages may result in profound suppression outcomes. Due to different animal categories and discrepancies between in vitro and in vivo experimental models,[12] it is difficult to determine which concentration and duration are beneficial and which are detrimental to cultured granulosa cells. Based on previous studies,[13],[14],[15] we used a wide range, with graded concentrations of corticosterone, to clarify the effects of glucocorticoids on granulosa cells. When granulosa cells were cultured for 5 days, corticosterone discernibly upregulated progesterone synthesis/release and estradiol release, and there was a significant difference at the high corticosterone concentration of 10 - 5 mol/L. When we extended the culture time to 10 days, corticosterone treatments still promoted progesterone synthesis/release but suppressed estradiol synthesis/release. It is clear that treatments with high corticosterone concentrations induced progesterone synthesis and secretion regardless of how long cell culture was extended. When glucocorticoids were used in combination with FSH, both progesterone synthesis/release and estradiol release were stimulated significantly.

For patients with luteal dysfunction, we can use glucocorticoid treatment either alone or in combination with other stimulatory regimens, such as FSH, to stimulate progesterone production to support the luteal cycle and early pregnancy. These treatment strategies may improve both corpora luteal functions and pregnancy rates, ultimately promoting the development of assisted reproduction technology in future. In fact, some researchers have explored the use of glucocorticoids in assisted reproduction to improve folliculogenesis and pregnancy rates.[16] If the safety and effectiveness of glucocorticoid administration to support the luteal cycle and early pregnancy are verified, many infertile women will benefit from these treatment strategies.

Because the glucocorticoid concentration in the follicular fluid of preovulatory follicles, where it fluctuates dramatically, is significantly different from that in circulation,[17],[18] we still do not know how to determine the proper dose for use in vivo from our studies in vitro. Additional studies are needed to explain the relationship between the local glucocorticoid environment in the ovarian corpus luteum and in body circulation before clinical applications can be evaluated.

The corpus luteum synthesizes and secretes progesterone/estradiol as normal physical functions. We use four parameters to demonstrate these functions: intracellular progesterone/estradiol levels, extracellular progesterone/estradiol levels, total progesterone/estradiol production, and the progesterone/estradiol secretion ratio. Total progesterone/estradiol production represents the ability of granulosa cells to synthesize these hormones, and the progesterone/estradiol secretion ratio represents the ability of these cells to secrete the hormones into the extracellular space. Extracellular and intracellular progesterone/estradiol levels are related to both the cellular synthesis and secretion of these hormones, and these levels represent, in part, the ability of granulosa cells to synthesize and secrete. In our experiments, these four parameters thoroughly reflect the regulatory effects of glucocorticoids on steroid synthesis and secretion. This evaluation method, comprised of the four parameters in our research, is better than a method utilizing a single parameter, such as steroid levels in the medium, to clarify the regulatory effects of glucocorticoids on cell functions.

Granulosa cell proliferation and apoptosis play important roles in corpus luteum structural remodeling, and glucocorticoids may have a mediatory role in these biological processes. As expected, our experiments confirmed that glucocorticoid treatments indeed suppressed the apoptosis of granulosa cells. Glucocorticoids have long been suspected of acting in cell apoptosis, and glucocorticoids are recognized survival factors capable of inhibiting apoptosis during folliculogenesis.[19] As reviewed by Quirk et al., suppression of granulosa cell apoptosis can induce cell proliferation.[20] However, our data showed that glucocorticoids did not promote cell proliferation. We suspect that this may be due to the periodicity of follicular development. Before the granulosa cells were collected, the rats received injections of pregnant mare serum gonadotropin, which led to the rapid growth of follicles and the rapid proliferation of granulosa cells. When the cells were cultured in vitro, they had gone through a period of rapid proliferation and entered into the luteal phase. Therefore, in our experiments, glucocorticoids suppressed granulosa cell apoptosis, which may be beneficial for maintaining corpus luteum structure.

Granulosa cells in particular have been demonstrated to play a major role in deciding the fate of the corpus luteum.[21],[22] Granulosa cells are steroidogenic cells that provide proteins and steroids for a rich microenvironment.[23] By studying the effects of glucocorticoids on granulosa cells, we could provide important insights into the relationship between glucocorticoids and the corpus luteum. With our cultured granulosa cell model, we reveal that high glucocorticoid concentrations induce remarkably levels of progesterone synthesis and secretion. The luteotropic regulatory role of glucocorticoids in granulosa cells will have important implications in the field of assisted reproduction. We speculate that an appropriate in vivo dose of glucocorticoids may be good for inducing granulosa cells to secrete progesterone and estradiol to support the luteal cycle and early pregnancy after ovulation. Additional studies are required to develop a better understanding of these findings and apply them, ultimately, to the development of a new clinical strategy.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (Nos. 81401170, 81501271, and 81503087), Natural Science Foundation of Jiangsu province (BK20130073), Nanjing Medical Science and Technique Development Foundation (YKK15149), and Foundation of Nanjing Medical University (2014NJMUZD047).

Conflicts of interest

There are no conflicts of interest.



 
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