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Hum. Reprod. Advance Access originally published online on November 10, 2006
Human Reproduction 2007 22(2):401-406; doi:10.1093/humrep/del408
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Gonadotrophin stimulation of non-luteinized granulosa cells increases steroid production and the expression of enzymes involved in estrogen and progesterone synthesis

M. Lindeberg1,3, K. Carlström1, O. Ritvos2 and O. Hovatta1

1 Department of Clinical Science Investigation and Technology, Division of Obstetrics and Gynecology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 2 Programme for Developmental and Reproductive Biology, Biomedicum Helsinki and Department of Bacteriology and Immunology, Haartman Institute, 00014 University of Helsinki, Helsinki, Finland

3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, K57, Karolinska Institute, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. E-mail: maria.lindeberg{at}ki.se


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In regular IVF treatment, mature oocytes are collected with their luteinized granulosa cells (GCs). When in vitro maturation (IVM) of the oocytes is performed, non-luteinized GCs can be collected. We have investigated how these cells respond to gonadotrophin stimulation in culture. METHODS: GCs were collected from patients undergoing IVM treatment and compared with GCs from IVF patients. The cells were stimulated with FSH and/or hCG. After 48 h, culture media were collected for hormone analysis, and RNA was isolated for gene expression analysis. RESULTS: In IVM GCs, hCG and FSH alone and in combination induced significantly increased progesterone production, and FSH alone and in combination with hCG increased estrogen production. We also studied the gene expression of P-450aromatase and P-450scc and the receptors for FSH and LH. In non-luteinized GCs, the expression levels of P-450aromatase increased with all treatments, and P-450scc expression increased with the combined FSH and hCG treatment. LHR expression increased with FSH treatment, but the FSH receptor expression did not change with different treatments. CONCLUSIONS: Non-luteinized GCs behaved differently from luteinized GCs in culture. The data help understand the final stages of maturation of human oocytes and follicles.

Key words: in vitro maturation of human oocytes/immature granulosa cells/gonadotrophin receptors/aromatase/culture


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ovarian follicular development is a complex process that involves endocrine, paracrine and autocrine actions. Follicles develop through primordial, primary, secondary and pre-antral stages before they finally undergo atresia, or through cyclic gonadotrophin stimulation, they reach the pre-ovulatory antral stage and ovulate (Gougeon, 1996Go). During this process, interactions between the different ovarian cell types are essential. The main triggers of folliculogenesis are the gonadotrophins, FSH and LH. Their effect on the ovarian steroid production is well documented. LH stimulates the theca interna cells to produce aromatizable androgens (Hillier et al., 1994Go; Karnitis et al., 1994Go). These are then converted to estrogens in the GCs by cytochrome P-450aromatase (CYP19) that is induced by FSH (Hillier et al., 1994Go; Smyth et al., 1995Go). In differentiated GCs, FSH also induces cytochrome P-450scc (CYP11A) that catalyses the rate-limiting conversion of cholesterol to pregnenolone, promoting the production of increasing amounts of progesterone (Richards et al., 1998Go). Progesterone receptors (PRs) appear on the GCs at the gonadotrophin surge (Natraj and Richards, 1993Go; Park-Sarge and Mayo, 1994Go; Park-Sarge and Sarge, 1995Go). More knowledge about the interactions between the different ovarian cell types at different time points of follicular development will increase the understanding of ovarian physiology, which will help to improve protocols for infertility treatment.

In conventional infertility treatment, the ovaries are hyperstimulated to induce simultaneous follicular growth. This is obtained by FSH injections after down-regulation by GnRH agonists or antagonists (Hughes et al., 1992Go; Liu et al., 1992Go; Minaretzis et al., 1995Go). Oocyte maturation is then induced by hCG administration before transvaginal oocyte retrieval. The hormone treatment can be difficult for the patient, and some women develop a severe life-threatening side effect, ovarian hyperstimulation syndrome (OHSS). This syndrome is characterized by massive enlargement of the ovaries, ascites, pleural effusion, oligures, haemoconcentration and thromboembolic phenomena. Women with polycystic ovary syndrome (PCOS) are more likely to develop OHSS (Aboulghar and Mansour, 2003Go).

In vitro maturation (IVM) of oocytes is used as an alternative to conventional infertility treatment. IVM treatment can be carried out completely without hormone stimulation, or using low doses of FSH to initiate follicular development. The low dose of FSH is helpful in timing of the oocyte retrieval in PCOS patients (Mikkelsen and Lindenberg, 2001Go). The oocytes are then aspirated when at least one follicle has reached a diameter of 10–14 mm. The oocytes are then cultured for 28–36 h in maturation media enriched with recombinant FSH, recombinant LH and serum collected from the patient (Mikkelsen et al., 1999Go). IVM treatment is particularly suitable for patients who are at risk of developing OHSS, such as women with PCOS.

When oocytes are retrieved from IVM patients, a lot of GCs are also aspirated from the follicle. These GCs have not yet entered the luteinized phase. It is only possible to retrieve such non-luteinized GCs in IVM cycles, and hence, they have been previously very little studied. Learning more about these cells will improve our knowledge of the last steps of follicular development and oocyte maturation. We compared the gonadotrophin response in non-luteinized GCs from patients undergoing IVM treatments with luteinized GCs from IVF patients. We measured estrogen and progesterone production in the cell culture media and also studied the gene expression of cytochrome P-450aromatase and cytochrome P-450scc enzymes involved in the synthesis of these hormones, respectively. We also measured the expression of the FSH receptor (FSHR) and the LH receptor (LHR) in non-luteinized GCs.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
GC culture
Mural GCs were collected from follicular aspirates after oocyte retrieval. Cells were centrifuged out of follicular fluid and then treated with hyaluronidase for 30 min in 37°C. To separate the GCs from red blood cells, the cell suspension was layered on Ficoll (Amersham, Uppsala, Sweden) and centrifuged at 300 x g for 30 min. The GC fraction was then washed with phosphate-buffered saline (PBS), and the cells were seeded in five different wells, 50 000 cells/well in a 24-well plate in 500 µl of basic media, TCM-199 (Invitrogen-Gibco, Paisley, UK), 2.5% fetal calf serum (FCS), 1% penicillin/streptavidin (Invitrogen-Gibco) overnight to attach to the plastic and to be separated from white blood cells (Mäkinen et al., 2004Go). The following day, one of five different stimulations was given to the cells: (i) control basic media; (ii) basic media and 1 µM androstenedione (Sigma); (iii) basic media, 1 µM androstenedione and 30 ng/ml of recombinant hCG (Ovitrelle®; Serono Nordic, Stockholm, Sweden); (iv) basic media, 1 µM androstenedione (Sigma) and 30 ng/ml of recombinant FSH (Gonal F®; Serono Nordic); or (v) basic media, 1 µM androstenedione (Sigma), 30 ng/ml of recombinant hCG and 30 ng/ml of recombinant FSH. After 48 h, culture media were collected for hormone measurements, and the cells were collected and homogenized in lysis buffer RLT with 10 µl/ml of beta-mercaptoethanol (beta-ME) for mRNA preparation and RT–PCR analysis.

Cell cultures from two groups of patients were studied: (i) non-luteinized GCs from IVM patients (n = 12; mean age 31.7 ± 3.7) and (ii) luteinized GCs from IVF patients as a control group (n = 6; mean age 33 ± 5.0). As a second control, we used luteinized GCs from IVF patients (n = 6; mean age 34 ± 2.3) and cultured them for 72 h before stimulation. This was done to see whether the IVF GCs can regain a more native reaction pattern after a 72-h pre-culture period before stimulation.

In the IVM group, 6 of 12 patients had PCO, and all except two of the patients were given low doses of FSH to time the follicles for oocyte retrieval.

The Ethics Committee of the Karolinska Institute approved the study. Informed consent was obtained from all participants.

Hormone measurements
Concentrations of estradiol-17beta (E2) and progesterone in the culture fluid were determined by clinical routine immunoassays intended for the analysis of human serum. The culture fluid was found to possess the same matrix properties as serum in both assays. Culture fluid concentrations of E2 and progesterone were determined by competitive chemiluminescence immunoassay using commercial kits obtained from Roche Diagnostica GmbH (Mannheim, Germany; E2: Modular E170) and from Diagnostic Products (Los Angeles, CA; progesterone: Immulite 2000). Practical detection limits and within- and between-assay coefficients of variation were 100 pmol/l, 4% and 5%, respectively, for E2 and 0.6 nmol/l, 8% and 10%, respectively, for progesterone.

RNA extraction, RT reaction and real-time PCR
RNA was prepared according to Rneasy Mini-kit (Qiagen, Hilden, Germany) protocol for animal cells. The synthesis of cDNA was performed using the SuperscriptIII First Strand Synthesis System for RT–PCR (Gibco BRL, Grand Island, NY, USA). Fourty nanograms of mRNA, 1 µl of random hexamers (50 ng), 1 µl of dNTP-mix (10 mmol/l) and diethyl pyrocarbonate (DEPC)-treated water up to 10 µl were mixed and incubated at 65°C for 5 min and then immediately placed on ice. Next, a mixture of 2 µl of RT buffer (20 mM Tris–HCl, 50 mM KCl), 4 µl of MgCl (5 mM), 2 µl of dithiothreitol (DTT, 10 mM) and 40 IU of RNaseOUT Recombinant Ribonuclease Inhibitor was added, and 50 IU of Superscript II Reverse Transcriptase was also added. cDNA synthesis was carried out at room temperature for 10 min and at 42°C for 50 min. Next, the reaction was stopped by incubating at 85°C for 5 min. The samples were then stored at –20°C until real-time PCR was performed.

Primer sequences and the size of the expected PCR products for cytochrome P-450aromatase, cytochrome P-450scc, FSHR and LHR as well as the endogenous control gene GAPDH are presented in Table I. To detect amplification, we used Sybrgreen.


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  		Table I. Primer sequences and the size of the expected PCR products for cytochrome P-450aromatase, cytochrome P-450scc, FSH receptor (FSHR) and LH receptor (LHR) as well as the endogenous control gene GAPDH

 
Real-time PCRs were performed using an ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA, USA). The reaction mixture consisted of 200 nM of each primer for cytochrome P-450aromatase and cytochrome P-450scc, and 500 nM for the FSHR and the LHR primers, 1x Sybrgreen PCR mastermix and 4 µl of the RT reaction mixture, corresponding 0.5 ng mRNA in a total reaction volume of 25 µl. The cDNA was then heated to 50°C for 2 min and denatured at 95°C for 10 min. The template was then amplified over 50 cycles of 15 s of melting at 95°C and 1 min at 62°C for annealing and extension. During the PCR, an argon ion laser excites fluorescent dyes in each reaction. Fluorescence data were acquired by measurements taken approximately every 7 s and presented as a plot of fluorescence intensity versus cycle number.

Standard curves were constructed using duplicates of serial dilutions of cDNA prepared from total placenta RNA (for cytochrome P-450aromatase and cytochrome P-450scc) and ovarian RNA (for FSHR and LHR) of known concentration. One standard curve was generated and run in parallel with the unknown samples in each experiment. Fluorescence was measured, and the threshold cycle (Ct) values at each point in the standard curve were plotted against the log (ng) of the initial concentration (Higuchi et al., 1993Go). The relative concentration of unknown samples was determined from the standard curve and normalized to the endogenous control gene GAPDH. The different treatments were then calibrated against the controls.

Differences in gene expression were analysed using two-tailed t-test, and significance is reported at the 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Hormone measurements
In all hormone measurements, there was a large variability between patients. Therefore, we choose to look at differences between control and treatment samples and compare them. However, the absolute values are also presented to show the different levels of hormone production in the different cell stages (Figure 1A and B).


Figure 1
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  		Figure 1. Hormone synthesis in culture media from granulosa cells (GCs) obtained by in vitro maturation (IVM) of oocytes, IVF GCs and IVF GCs +72 h culture. (A) Estrogen synthesis in absolute values (pmol/l). (B) Progesterone synthesis in absolute values (nmol/l). (C) Estrogen synthesis proportions. (D) Progesterone synthesis proportions. In (C) and (D), values are calibrated to the control to illustrate differences. Mean values (±SEM) are presented. Asterisk (*) indicates significant differences at P = 0.05 compared with control using Student’s t-test.

 
Estrogen production in GCs from patients going through IVM treatment was low in culture media from untreated cells and increased when the cells were stimulated with hCG, FSH or both hCG and FSH. When only androstenedione was added to the culture media, an increase in estrogen synthesis was also observed. When the different treatments were compared with the control with androstenedione, no difference was seen with hCG treatment, but in cells treated with FSH and FSH in combination with hCG, estrogen production increased significantly. (Figure 1C).

Progesterone synthesis in IVM GCs was low in culture media from untreated cells. It increased significantly with all three different treatments with the highest production in the media from cells treated with both FSH and hCG (Figure 1D).

In control IVF GCs and in IVF GCs that were cultured for 72 h before stimulation, a different pattern was observed. Estrogen concentrations increased in the culture media with all treatments, including the one with only androstenedione, when compared with untreated cells. The production was equal between the different treatment groups (Figure 1B). The concentrations of progesterone were, as expected, generally much higher in the IVF GCs and in the IVF GCs that were cultured for 72 h before stimulation. Different treatments did not influence the hormone levels significantly in IVF GCs, but in IVF GCs that were cultured for 72 h before stimulation, the progesterone synthesis increased significantly with hCG treatment and with the combined treatment (Figure 1D).

Gene expression
The expression of cytochrome P-450aromatase in IVM GCs was significantly higher in cells treated with hCG, FSH or both hCG and FSH compared with untreated cells. In cells treated with only androstenedione, the expression was similar to that in the untreated cells (Figure 2A). Cytochrome P-450scc was expressed at a relatively low level in untreated IVM GCs. When the cells were treated with FSH or hCG, the expression changed but varied between patients. Therefore, no significant difference was observed. However, with the combined hCG and FSH treatment, the expression level significantly increased (Figure 2A).


Figure 2
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  		Figure 2. Gene expression of cytochrome P-450aromatase and cytochrome P-450scc in granulosa cells (GCs) obtained by in vitro maturation (IVM) of oocytes (A) and IVF GCs (B). Values are normalized to the endogenous control gene GAPDH, and gonadotrophin-treated cells are calibrated against the control cells. Mean values of the differences (±SEM) are presented. Asterisk (*) indicates significant differences at P = 0.05 compared with control and {dagger} significant difference at P = 0.05 when combined treatment is compared with single treatments using Student’s t-test.

 
In control IVF GCs, the cytochrome P-450aromatase expression level was significantly lower in cells treated with hCG alone or in combination with FSH. In cells only treated with FSH, the expression level did not change significantly. The androstenedione did not influence the expression level of cytochrome P-450aromatase (Figure 2B).

The general expression level of cytochrome P-450scc was, as expected, much higher in luteinized IVF GCs. With hCG or FSH treatment, the expression decreased. However, with the combined hCG and FSH treatment, a significant decrease could not be shown. Androstenedione did not influence the cytochrome P-450scc expression (Figure 2B).

The expression of the FSHR in non-luteinized GCs did not change significantly with the different treatments. However, LHR expression increased significantly in cells stimulated with FSH or FSH in combination with hCG. Only hCG did not influence the expression of LHR (Figure 3).


Figure 3
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  		Figure 3. Gene expression of FSH receptor (FSHR) and LH receptor (LHR) in granulosa cells (GCs) obtained by in vitro maturation (IVM) of oocytes. Values are normalized to the endogenous control gene GAPDH, and gonadotrophin-treated cells are calibrated against the control cells. Mean values of the differences (±SEM) are presented. Asterisk (*) indicates significant differences at P = 0.05 compared with control.

 
We did not observe any differences between the PCOS and the non-PCOS patients in any of the analyses we performed.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
We describe here, for the first time, how non-luteinized human GCs responded to gonadotrophin stimulation by increased production of both estrogen and progesterone. The expression of the enzymes involved in the synthesis of these steroids, cytochrome P-450aromatase and cytochrome P-450scc, increased in the GCs at the same time. There is a clear difference in the response in luteinized GCs.

FSH and LH and their receptors are two of the main actors in follicular development. Both the FSHR and LHR use the G-protein/adenyl cyclase/cAMP signalling pathway (McFarland et al., 1989Go; Sprengel et al., 1990Go). Despite the high degree of homology between LH and FSH, the expression of the receptors and the effects of the gonadotrophins vary throughout the different stages of GC differentiation. The FSHR is expressed in both immature and mature GCs, but only the mature GCs express the LHR. The effects also differ between stages of differentiation; in immature cells, FSH promotes cell differentiation and induces the expression of aromatase and LHR. In contrast, mature GCs respond to the LH surge by cell cycle arrest and suppression of aromatase and LHR expression, increased progesterone synthesis and luteinization (Pierce and Parsons, 1981Go; Pierce, 1988Go).

These divergent effects caused by two structurally similar receptors that signal through cAMP could be explained in several different ways. There may be other signalling pathways coupled to the FSHR and LHR and a branching of the signalling at the membrane accounts for different outcomes. Furthermore, the properties and distribution of the cAMP may be the reasons for the different outcomes. Finally, the GCs of different stages may react differently, and the cAMP signal will be differently distributed. Consequently, different kinase cascades and transcription factors will be induced and will activate the expression of different genes (Conti, 2002Go).

In this study, we have investigated the hormone production and gene expression of hormones regulating enzymes in different stages of GC development. It would have been of interest to investigate this with a dose–response assay, but because of the rarity of the material, we were only able to test a single concentration of each gonadotrophin and a combination of the two. We found that the patterns differ a lot between different stages. In the non-luteinized GCs, as we expected, the estrogen production increased in cells treated with FSH and in cells treated with a combination of FSH and hCG. The expression level of cytochrome P-450aromatase paralleled the pattern of estrogen secretion. Our measurements showed increased expression in all treatments with the largest increase in the combined FSH and hCG treatment. Our results confirm this well-documented process of estrogen synthesis induction in pre-ovulatory GCs (Hillier et al., 1994Go; Smyth et al., 1995Go).

In the culture media from luteinized GCs, estrogen synthesis was already high in control cells with androstenedione, and not much difference was observed in the treated cells, indicating that they already reached their peak production. The expression level of cytochrome P-450aromatase in the luteinized cells was decreased in the cells treated with hCG and in cells treated with hCG and FSH in combination. This decline was expected, as the estrogen production is about to drop during the luteinization induced by the LH surge. It has also been shown in the rat that the expression of cytochrome P-450aromatase declines after hCG-induced ovulation (Tetsuka et al., 1998Go).

Progesterone synthesis in the control non-luteinized GCs was very low, and it was increased with all treatments. Similar to estrogen synthesis, the effect of FSH was larger than that of hCG, and the highest synthesis of progesterone was observed in the combined FSH and hCG treatment. The expression of cytochrome P-450scc varied a lot between different patients, and some patients showed an increase in expression and others a decrease. In cells treated with hCG or FSH alone, we could therefore not show any significant change, but in the combined treatment, the expression of cytochrome P-450scc significantly increased. The large variation between patients probably indicated that the cells have reached different degrees of maturation. The cells that did not respond with increased cytochrome P-450scc expression may not yet have up-regulated the LHR and were not receptive to LH stimulation yet. When we looked in individual patients, we could see a correlation with high up-regulation of the LHR and increased cytochrome P-450scc expression in the non-luteinized GCs that we studied (data not shown).

In luteinized GCs, the control cells already produced very high concentrations of progesterone, and the different treatments did not influence the production significantly, indicating that they probably had reached their maximum level of synthesis.

However, when the cells had been cultured for 74 h before stimulation, they regained some of their responsiveness and increased progesterone production in response to hCG or hCG in combination with FSH, although the baseline in the non-treated controls was much higher compared with that in the non-luteinized cells.

The expression of cytochrome P-450scc in luteinized GCs was generally very high in the non-treated cells, ~10 times higher than that in the non-luteinized FSH- and hCG-treated cells. The expression decreased slightly in hCG- and FSH-treated cells but was still much higher than in all cells from IVM patients. In the cells treated with the combination, no difference in cytochrome P-450scc expression was observed. In vivo, the maximal stimulation of progesterone production takes place in the early luteal phase (Fisch et al., 1989Go). Our results indicated that the expression of cytochrome P-450scc had already reached its peak and that further gonadotrophin stimulation did not augment it any more. It has been shown earlier that after the LH surge the cytochrome P-450scc expression is maintained at a constant level and will not be increased by gonadotrophin stimulation in humans (Voutilainen et al., 1986Go) and in rats (Oonk et al., 1989Go). The FSH-stimulated induction of the LHR is a well-known phenomenon from other species (Richards et al., 1976Go; LaBarbera and Ryan, 1981Go), but because of the lack of available human material, this has not been shown previously in human GCs.

Although luteinized GCs provide a nice model for many studies, there is the problem that they have received superphysiological doses of gonadotrophins before they are put to culture. The fact that they have entered the luteinized stage is also a problem, as they have passed the very interesting phase where they participate in the maturation process of the follicle and the oocytes.

Our new culture model, although it offers fairly small amounts of cells to work with, can together with modern techniques provide good expression data. Altogether, this can improve our knowledge regarding the basic physiology of follicular development.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Sirpa Mäkinen for her expertise and advice concerning GC culture. We are also very grateful to the laboratory staff at the IVF-unit Huddinge University Hospital, for their enthusiastic collaboration in collecting GCs. The Swedish Research Council supported this work.


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 Discussion
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 References
 
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Submitted on July 5, 2006; resubmitted on September 4, 2006; accepted on September 25, 2006.


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