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Biology of Reproduction 60, 1078-1086 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Expression of Granulosa Cell-Specific Genes and Induction of Apoptosis in Conditionally Immortalized Granulosa Cell Lines Established from H-2Kb-tsA58 Transgenic Mice

Norbert Walther1,a, Almuth Einspanierb, and Martina Jansena

a Institute for Hormone and Fertility Research, University of Hamburg, D-22529 Hamburg, Germany b German Primate Center, D-37077 Göttingen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Granulosa cell lines have been established from H-2Kb-tsA58 transgenic mice. Using immunocytochemistry, significant amounts of insulin-like growth factor-I (IGF-I) were found in all cell lines investigated, whereas estrogen and progesterone receptor expression could be detected in only some of the lines. All cell lines showed low basal production of the gonadal steroids estradiol and progesterone. The genes for the ovarian paracrine regulators IGF-I and basic fibroblast growth factor were expressed, as well as the genes for anti-Müllerian hormone and for the P450 side-chain cleavage enzyme (P450scc). Expression of P450scc could be shown to be up-regulated in the cell lines under conditions mimicking the hormonal environment of the luteinizing granulosa cells in vivo. Inactivation of the temperature-sensitive SV40 T antigen by a shift of the cell lines to the nonpermissive temperature of 39.5°C led to massive induction of apoptosis in several cell lines. These cell lines will allow a detailed study of the mechanisms regulating the expression of granulosa cell-specific functions, as well as the induction of granulosa cell apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of a preovulatory ovarian follicle during each cycle in the female is the prerequisite for the development of the oocyte and hence for female fertility. The granulosa cells that form the inner cell layer of the ovarian follicle are recruited by the oocyte early in follicle development and differentiate to fulfill their specialized functions. They nurse the developing oocyte and constitute the major site of steroidogenesis in the female. The granulosa cells of the dominant follicle after ovulation take part in the formation of the corpus luteum [1–3]. The function of the nonselected follicles is terminated by the process of atresia, in which the apoptotic death of the granulosa cells plays an important role [4].

A number of hormones and paracrine factors have been implicated in the regulation of granulosa cell proliferation and differentiation [5]. The differentiation of granulosa cells during follicular growth is promoted by FSH released from the pituitary. Paracrine factors have been shown to be necessary for granulosa cell proliferation, e.g., basic fibroblast growth factor (bFGF) in bovine [6] and insulin-like growth factor-I (IGF-I) in rat granulosa cells [7]. Moreover, IGF-I acts synergistically with FSH to induce the expression of granulosa cell differentiation markers [8–10]. In contrast to the differentiation of the granulosa cells of the dominant follicle to luteal cells, the granulosa cells of the nonselected follicles follow an alternate pathway ending in atresia of these follicles. This process involves induction of granulosa cell apoptosis, thereby terminating the function of the atretic follicle. The same factors regulating proliferation of granulosa cells and the expression of cell-specific genes have also been found to be implicated in the suppression of granulosa cell apoptosis [4]. In order to study the regulatory mechanisms in granulosa cells in greater detail, model systems have to be devised that allow application of molecular biology methods.

One major problem in studying the complex regulation of granulosa cell proliferation, luteal differentiation, and granulosa cell apoptosis is the fact that although granulosa cells can be easily isolated and cultured, they undergo spontaneous luteinization in tissue culture. Main indicators used are an increase in progesterone production and the down-regulation of estradiol synthesis. In order to develop experimental systems better suited for the study of granulosa cell functions, numerous granulosa cell lines have been established from several species, mostly by immortalization of primary granulosa cells by viral oncogenes or stimulation of granulosa cell growth by mitogens [11–25]. The majority of these cell lines represent luteinizing granulosa cells, demonstrating that the process of immortalization does not prevent the spontaneous luteinization of granulosa cells in culture. One cell line conserving FSH receptor expression and the stimulation of estradiol production by FSH has been described [14]; two other lines show induction of P450 aromatase (P450arom) expression by cAMP [19] or constitutive P450arom expression [23]. Recently, one cell line has been described that responds to FSH by an irreversible stop of proliferation followed by cell differentiation [25]. This cell line apparently represents early granulosa cells, but the cells do not express P450arom after FSH induction. In summary, all the granulosa cell lines reported so far express only subsets of the specific granulosa cell functions expressed in vivo. The establishment of granulosa cell lines seems to impair the coordinated expression of specific granulosa cell functions to some extent, but immortalized cell lines still provide useful model systems for the detailed investigation of the regulatory mechanisms involved in the control of granulosa cell functions. Cell lines that allow manipulation of the expression of granulosa cell-specific functions experimentally in tissue culture will be extremely helpful in these studies. In the study presented here, we describe the establishment of conditionally immortalized granulosa cell lines from H-2Kb-tsA58 transgenic mice. The temperature-sensitive SV40 T antigen these mice carry in their germ line allows these cell lines to grow permanently at the permissive temperature, whereas after transfer to the nonpermissive temperature, growth is stopped. This treatment results in specific changes in expression of granulosa cell-specific genes and induction of apoptosis. Therefore these cell lines open up the possibility of studying important events in regulation of granulosa cell proliferation, gene expression, and apoptosis in tissue culture.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Establishment of Granulosa Cell Lines from H-2Kb-tsA58 Transgenic Mice

Primary cultures of granulosa cells from H-2Kb-tsA58 transgenic mice [26] were set up according to an established protocol [27]. The ovaries of approximately 10-wk-old female H-2Kb-tsA58 transgenic mice were removed and transferred to a Petri dish containing tissue culture medium M199. Large follicles were carefully dissected from the ovaries and washed with the same medium. The follicles were transferred to another Petri dish containing the same medium, and granulosa cells were released by puncturing the follicle wall with an injection needle and squeezing the punctured follicle. After washing by centrifugation, the pooled granulosa cells were suspended in tissue culture medium M199 supplemented with 0.1% BSA (Sigma-Aldrich, Deisenhofen, Germany), 1% fetal calf serum (Gibco-BRL, Eggenstein, Germany), and 20 U/ml recombinant mouse interferon-{gamma} (Genzyme Diagnostics, Cambridge, MA); they were then seeded into a T25 tissue culture flask coated with fibronectin (Sigma-Aldrich) and incubated at 33°C in an atmosphere containing 5% CO2. For passaging, the cells were detached from the flask by incubation at 37°C with 200 µg/ml EDTA in Ca- and Mg-free PBS. Where the cells did not detach readily after 10 min, EGTA was added to a final concentration of 200 µg/ml and the incubation continued for another 5 min. After centrifugation, the cells were suspended in a 1:1 mixture of fresh culture medium with conditioned medium from the confluent flask and transferred to two new fibronectin-coated T25 flasks. After several passages, cell clones were isolated from the primary cultures by the method of limiting dilution essentially as described previously [28]. Fourteen clones could be grown into immortalized cell lines at the permissive temperature of 33°C in the presence of interferon-{gamma}. For serial passaging of the cell lines obtained, fibronectin coating of the flasks was omitted; after a number of passages the cells were cultivated in the culture medium described above but without the addition of conditioned medium. For investigation of granulosa cell-specific gene expression under different growth conditions, growing cells were cultivated at 33°C as described above, and parallel cultures for induced cells were washed and re-fed with medium without interferon-{gamma} but supplemented with 1 µg/ml insulin (Boehringer-Mannheim, Mannheim, Germany) and 10 µM forskolin (Calbiochem, La Jolla, CA). These cultures were incubated for 48 h at the nonpermissive temperature of 39.5°C. Total RNA was prepared from all cultures using the RNA-Clean system (AGS, Heidelberg, Germany).

Immunocytochemistry

Cells (104) of selected granulosa cell lines were seeded in 300 µl of culture medium into each chamber of 8-chamber Lab-Tek chamber slides (Nunc, Wiesbaden, Germany) and cultured for several days at 33°C. After cell morphology was microscopically checked, the medium was removed and the cells snap-frozen and stored at -80°C until further processing for immunocytochemistry. Cultured cells were thawed at room temperature for 2 h, postfixed in 4% paraformaldehyde, and then rinsed in PBS (Instamed Dulbecco [Biochrom, Berlin, Germany] without Ca2+ and Mg2+, prepared to a concentration of 9.55 g/L, pH 7.5). Immunocytochemistry was carried out as described previously [27]. Briefly, the sections were incubated for 30 min in diluted normal rabbit or mouse serum. After a wash step in PBS, sections were incubated with primary antibodies directed against progesterone receptor (mouse monoclonal antibody, AT 4.14; Dianova, Hamburg, Germany; 1:50), estradiol receptor (mouse monoclonal antibody, B 10; Euromedex, Strasbourg, France; 1:1000), or IGF-I (rabbit polyclonal antibody; 1:200; [29]). Immunoreactivity was visualized using the alkaline phosphatase technique (APAAP-complex; Dianova) and the conjugated avidin-biotin method (LSAB-Kit; Dakopatts, Glostrup, Denmark) for each antibody. The specificity of staining reactions was monitored in control sections where the primary antibody had been omitted or had been replaced by normal mouse or rabbit IgG. All sections were mounted in Mowiol (Hoechst, Frankfurt/Main, Germany) and examined using a Zeiss (Carl Zeiss, Oberkochen, Germany) microscope.

Measurement of Steroid Hormone Production

Cells (105/well) of selected granulosa cell lines were seeded in 1 ml of culture medium into 24-well tissue culture plates (Nunc). After culture for 48 h at 33°C, supernatants were collected for determination of progesterone production. After addition of fresh medium containing 1 µM testosterone, the cells were incubated for another 3 h at 33°C and supernatants were collected for measurement of conversion of testosterone to estradiol. Culture supernatants were stored at -20°C until assayed, and secreted hormones were analyzed using a validated enzyme immunoassay. Progesterone in media was determined by a direct, nonextraction enzyme immunoassay using an antiserum raised in sheep against progesterone-11-{alpha}-hemisuccinate-BSA and alkaline phosphatase linked to progesterone-11-glucuronide as enzyme conjugate. The assay has previously been described in detail [30]. Estradiol immunoreactivity was detected using a polyclonal rabbit antiserum against estradiol-6-carboxymethyloxime-BSA. Estradiol-3-carboxymethyloxime coupled with peroxidase was used as label. Further details are described elsewhere [31].

Detection of Granulosa Cell Transcripts by Reverse Transcription-Polymerase Chain Reaction

Total RNA (4 µg) from growing and induced cells was used in a random oligonucleotide-primed reverse transcription (RT) reaction with Superscript II RNase H- reverse transcriptase (Gibco-BRL) according to the manufacturer's instructions. Aliquots of the reverse-transcribed cDNAs were used directly for amplification of mRNA sequences by the polymerase chain reaction (PCR). Primers were synthesized using published sequences (mouse P450 side-chain cleavage enzyme [P450scc]: 5'-AGGACTTTCCCTGCGCT-3' and 5'-GCATCTCGGTAATGTTGG-3' [32], PCR product size 951 base pairs [bp]; mouse IGF-I: 5'-GGACCAGAGACCCTTTGCGGGG-3' and 5'-GGCTGCTTTTGTAGGCTTCAGTGG-3' [33], PCR product size 210 bp; rat bFGF: 5'-AAGCGGCTCTACTGCAAG-3' and 5'-AGCCAGACATTGGAAGAAACA-3' [34], PCR product size 372 bp). Primers for human anti-Müllerian hormone (AMH [35]) were a kind gift of H. Ungefroren (Institute for Hormone and Fertility Research, Hamburg, Germany): 5'-CACCCAGCGCAGACCCCTTC-3' and 5'-CACTCGGTGGCCACCATGTTG-3', PCR product size 820 bp. The primers for FSH receptor and LH receptor [28] were a kind gift of D. Jähner (Institute for Hormone and Fertility Research)—FSH receptor: 5'-ACTGGCTGTGTCATTGCTCT-3' and 5'-CTGAGAGATCTCTATTTTCTC-3', PCR product size 179 bp; LH receptor: 5'-AATCCCATCACAAGCTTTCAG-3' and 5'-TGCCTGTGTTACAGATGC-3', PCR product size 215 bp. PCR amplification and identification of the specific reaction products by agarose gel electrophoresis, Southern blotting, and hybridization to internal 32P-labeled probes were performed as described previously [28].

Induction of Apoptosis and Detection of Apoptotic Cell Death

For induction of apoptosis, cultures of the conditionally immortalized granulosa cell lines growing at 33°C in the presence of interferon-{gamma} were washed and re-fed with culture medium without interferon-{gamma} in order to reduce the level of SV40 T antigen expression. After transfer to the nonpermissive temperature of 39.5°C that leads to inactivation of the residual thermolabile SV40 T antigen, cellular growth and cell death in the cultures was followed by regular microscopic inspection. After 2 and 4 days of culture at 39.5°C, the dead cells were collected from the supernatant by centrifugation, and genomic DNA was isolated. For detection of apoptotic cell death, genomic DNA (5 µg) was separated by electrophoresis on a 2% agarose gel, and the endonucleolytic cleavage pattern was visualized by ethidium bromide staining of the DNA. In order to follow cellular growth and apoptosis in the granulosa cell lines cultured at 39.5°C, time-lapse videomicroscopy [36] was performed.

For retroviral infection of GK9 cells, a cell line producing the helper-free recombinant retrovirus pBabePuro P175, overexpressing a p53 protein containing an amino acid exchange (Arg to His) at position 175 [37, 38], was grown to confluency. The culture supernatant was filtered through a 0.2-µm membrane filter and added to a growing culture of GK9 cells. A parallel culture was infected with the supernatant of a cell line producing the empty pBabePuro retroviral vector as a control. After 24 h, the medium on the GK9 cultures was removed, and the cultures were re-fed with their normal culture medium supplemented with 2 µg/ml puromycin for selection of infected cells. After death of the noninfected cells, the puromycin-resistant GK9 cells were cultured continually in the presence of 2 µg/ml puromycin. In order to estimate the influence of mutant p53 overexpression on cell death induction, cultures of retrovirus-infected GK9 cells (75 000 cells per 35-mm Petri dish) were treated with insulin and/or forskolin as described above and incubated at 33°C or 39.5°C. After 2 days the dead cells were collected from the supernatant and counted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of Conditionally Immortalized Granulosa Cell Lines

Granulosa cells were prepared from large ovarian follicles of approximately 10-wk-old female H-2Kb-tsA58 transgenic mice. The primary cell cultures were cultivated at the permissive temperature of 33°C in the presence of interferon-{gamma} to induce SV40 T antigen expression, thereby stimulating permanent growth. After limiting dilution of the primary cultures, 14 cell clones were isolated. These clones could be grown into immortalized cell lines at the permissive temperature of 33°C in the presence of interferon-{gamma} and analyzed for the expression of granulosa cell-specific genes. After establishment of the cell lines, the induction of high levels of SV40 T antigen expression apparently is no longer absolutely essential for permanent growth, as selected cell lines could be propagated at 33°C in the absence of interferon-{gamma} without any detectable changes in growth rate and morphology. A number of cell lines were selected for investigation of expression of granulosa cell-specific genes by immunocytochemistry and RT-PCR studies.

Detection of Steroid Hormone Receptor and Growth Factor Expression by Immunocytochemistry

Immunocytochemistry was performed using specific antibodies against the nuclear receptors for the gonadal steroids estrogen and progesterone and against the growth factor IGF-I, a paracrine regulator known to play an important role in the regulation of granulosa cell proliferation and differentiation. The results are shown for selected cell lines in Figure 1 and summarized for all cell lines that were investigated in Table 1. The intraovarian factor IGF-I could be detected immunologically in the cytoplasm of the immortalized granulosa cells, with a more intensive perinuclear staining. In some cases a weak intranuclear staining was also detected. All cell lines investigated expressed relatively high amounts of IGF-I, with the expression of this paracrine factor in cell line GK18 being significantly higher than in the other lines. In contrast, the cell lines differed in their pattern of steroid hormone receptor expression. Some granulosa cell lines showed positive nuclear expression of steroid hormone receptors, such as GK2 and GK5 for the progesterone receptor and GK3 for the estradiol receptor. Moreover, where one receptor was expressed the other appeared to be absent or only very weakly expressed. Whereas estrogen receptor was expressed only in GK3 cells, progesterone receptor expression could be detected in cell lines GK2 and GK5. The lines GK9 and GK18 appeared to lack expression of both receptors.



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  		FIG. 1. Immunocytochemical detection of estrogen receptor, progesterone receptor, and IGF-I in immortalized granulosa cell lines. Selected granulosa cell lines were cultured and processed for immunocytochemistry as described in Materials and Methods. The results for cell line GK3 are shown in A–C and the results for cell line GK2 in D–F. Primary antibodies directed against estrogen receptor (A and D), progesterone receptor (B and E), or IGF-I (C and F) were used. x175 (A, D, and E) or x350 (B, C, and F). Bar = 50 µm.


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  		TABLE 1. Expression of estrogen receptor (ER), progesterone receptor (PR), and IGF-I in conditionally immortalized granulosa cell lines.a

Production of Gonadal Steroids

The production of the gonadal steroids estrogen and progesterone from selected immortalized granulosa cell lines was determined as a measure of expression of the steroidogenic enzymes in these cells. Only low levels of basal estrogen and progesterone production could be detected (Fig. 2). The production of estradiol appeared to differ in the cell lines investigated, with line GK9 producing more and line GK18 less estradiol than the average, whereas the levels of progesterone production in the cell lines were more uniform with a slightly lower production in lines GK9 and GK18. These results show that the immortalized granulosa cell lines retain the ability to produce gonadal steroids, the low levels presumably resulting from down-regulation of the expression of several genes coding for steroidogenic enzymes, namely P450arom and 3ß-hydroxysteroid dehydrogenase (3ßHSD) (see below).



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  		FIG. 2. Production of gonadal steroids from immortalized granulosa cell lines. Granulosa cell lines GK2, GK3, GK5, GK9, GK12, and GK18 were cultured, and the steroids produced were measured in the culture supernatants as described in Materials and Methods. The levels of estradiol and progesterone produced are given as mean and SD.

Expression of Granulosa Cell-Specific Genes

For further characterization of the expression of granulosa cell functions, RT-PCR was used to screen the immortalized cell lines for a number of granulosa cell-specific transcripts. A number of lines were selected; the cultures were split for simultaneous cultivation at 33°C in the presence of interferon-{gamma}, inducing expression of the SV40 T antigen, or at 39.5°C in the absence of interferon-{gamma} but in the presence of insulin and the adenylate cyclase activator forskolin, conditions that have been shown to induce oxytocin expression and progesterone production in bovine granulosa cells [10]. Forty-eight hours after the temperature shift, the two parallel cultures were lysed. Total RNA was prepared, reverse-transcribed into cDNA, and used for PCR amplification of partial sequences of granulosa cell-specific transcripts using sequence-specific primers (Fig. 3). AMH, known to be expressed in fetal Sertoli cells, where it plays an essential role in inducing Müllerian duct regression during male fetal development [39], has also been shown to be expressed in ovarian follicles [40]. Therefore AMH was chosen as an example of a gene expressed during granulosa cell differentiation and down-regulated before luteinization [41]. The steroidogenic enzyme P450scc, on the other hand, is expressed late in granulosa cell development, being responsible for the considerable increase in progesterone production during luteinization [5]. AMH and P450scc were both expressed in all granulosa cell lines investigated, showing that the immortalized cells simultaneously express markers for early and later stages of granulosa cell differentiation; this unexpected finding presumably results from the conflict between permanent proliferation of the immortalized cell lines and the spontaneous luteinization of the cells in culture. Although RT-PCR experiments of the type employed here cannot be interpreted as quantitative measurements of transcript levels, it appears that the expression of P450scc is induced in all cell lines in the presence of insulin and forskolin under culture conditions suppressing cellular growth, suggesting that the mechanisms of transcriptional regulation of this gene are conserved. However, quantitative measurements of transcript levels under different culture conditions will be necessary to clarify this point. The growth factors IGF-I and bFGF have been shown to be important factors maintaining viability and controlling proliferation of granulosa cells [6, 7]. All granulosa cell lines investigated could be shown to express both of these factors. IGF-I and bFGF may be important for survival and growth of the cell lines in culture, as RT-PCR studies suggested that the receptors for these growth factors, IGF-I receptor, and FGF receptors 1 and 2 are expressed in all cell lines (data not shown). Unexpectedly, expression of the genes coding for the steroidogenic enzymes P450arom and 3ßHSD could not be detected, not even by RT-PCR. Taken together, the results of the RT-PCR experiments, as well as the immunocytochemical studies described above, show that the conditionally immortalized granulosa cell lines derived from H-2Kb-tsA58 transgenic mice have conserved the expression of some important granulosa cell-specific functions, whereas other granulosa cell-specific genes appear to have been shut off during establishment of the cell lines.



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  		FIG. 3. Expression of granulosa cell-specific genes in immortalized cell lines. Granulosa cell lines GK2, GK3, GK5, GK9, GK12, and GK18 were tested for expression of AMH, P450scc, IGF-I, and bFGF by RT-PCR using the specific primers described in Materials and Methods. Cells were grown at 33°C (lanes 1, 3, 5, 7, 9, 11, labeled "g") or induced by insulin and forskolin at 39.5°C (lanes 2, 4, 6, 8, 10, 12, labeled "i"). Water was used as a negative (lane 13) and mouse testis as a positive (lane 14) control for PCR.

The expression of gonadotropin receptor genes was investigated in another set of RT-PCR experiments. The expression of gonadotropin receptors on granulosa cells changes during follicular development. FSH receptor is induced early during follicle development, whereas LH receptor expression is induced later, before this receptor becomes essential for transducing the signal of the preovulatory LH surge that induces ovulation and luteinization of the granulosa cells [5]. Therefore gonadotropin receptor expression was investigated as a marker for the differentiation status of the immortalized granulosa cell lines. Complementary DNAs from parallel cultures of the cell lines were used for PCR amplification of partial sequences of gonadotropin receptor transcripts as described above (Fig. 4). Relatively weak expression of both gonadotropin receptor transcripts could be shown for most of the cell lines investigated, with the exception of cell line GK5, which apparently does not express the LH receptor gene. However, the levels of expression were very low compared with those of other granulosa cell-specific genes investigated. In a parallel study with conditionally immortalized Sertoli cells [28], comparable levels of gonadotropin receptor expression found by RT-PCR could not be detected by other methods, not even by a highly sensitive RNase protection assay. The very small amounts of gonadotropin receptor transcripts in the granulosa cell lines are too low to expect functional receptor expression. This was confirmed by the finding that it was not possible to induce significantly the production of gonadal steroids in cultures of the immortalized cell lines by incubation with either hormone (data not shown). These results show that the majority of the conditionally immortalized granulosa cell lines described in this study represent luteinizing granulosa cells, as indicated by expression of the LH receptor gene. However, the cell lines did not show the high levels of progesterone production characteristic for luteinizing granulosa cells, apparently due to down-regulation of steroidogenic enzyme gene expression.



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  		FIG. 4. Expression of gonadotropin receptor genes in granulosa cell lines. FSH and LH receptor gene expression was investigated in granulosa cell lines GK2, GK3, GK5, GK9, GK12, and GK18 by RT-PCR as described in Materials and Methods. Cells were grown at 33°C (lanes 1, 3, 5, 7, 9, 11, labeled "g") or induced by insulin and forskolin at 39.5°C (lanes 2, 4, 6, 8, 10, 12, labeled "i"). Water was used as a negative (lane 13) and mouse testis as a positive (lane 14) control for PCR. As controls for the tissue specificity of expression, the Sertoli cell line TM4 (lane 15) and the pituitary gonadotroph cell line {alpha}T3–1 (lane 16) were included.

Induction of Apoptosis in the Conditionally Immortalized Granulosa Cell Lines

Whereas cells derived from the H-2Kb-tsA58 transgenic mouse strain at the permissive temperature of 33°C readily enter the cell cycle and grow due to the immortalizing action of the SV40 T antigen, at the nonpermissive temperature (39.5°C) the thermolabile T antigen protein is inactivated, and cell divisions come to a stop [26]. In an earlier study in which Sertoli cell lines were established from the same mouse strain, slowing down of cellular growth was accompanied by dramatic morphological changes [28]. In order to investigate the effects of SV40 T antigen inactivation on the immortalized granulosa cell lines, growing cultures of selected cell lines were transferred to the nonpermissive temperature of 39.5°C in the absence of interferon-{gamma} and observed microscopically. Massive cell death was observed in all cultures after 2–4 days as indicated by the appearance of many small rounded phase-bright cells detached from the culture flask. Addition of insulin and/or the adenylate cyclase activator forskolin did not significantly influence the induction of cell death in these cultures.

In order to assess whether the massive cellular death in the conditionally immortalized cell lines might be a sign for induction of apoptosis (the pathway of granulosa cell death known to be involved in the process of atresia in vivo), cell line GK9, showing strongest induction of cell death, was chosen for an experiment using overexpression of a mutant p53 protein in order to suppress intracellular regulatory pathways leading to apoptosis. GK9 cells were infected with a retroviral vector expressing a mutated p53 protein that by complexing with the cellular p53 leads to inactivation of the p53 complex and thereby to suppression of established apoptotic pathways [42]. The cells were cultivated at 33°C and 39.5°C in the presence or absence of insulin and the adenylate cyclase activator forskolin, agents known to have major influences on granulosa growth and differentiation. As the mutation of the retrovirus-transduced p53 does not exhibit a temperature-sensitive phenotype, p53 action in this experiment is not influenced by the temperature differences between the cultures. After 2 days of culture under the conditions described, the cells that had died during culture were counted. The relation of the number of dead GK9 cells in the cultures at 39.5°C to the numbers in the control cultures (33°C) was calculated; the results are shown in Table 2. Although stimulation of GK9 cells by insulin and/or forskolin appears to influence cell survival to a certain extent, the effect of mutant p53 expression on the incidence of cell death was far more prominent. The numbers of dead cells were reduced in GK9 cultures overexpressing the mutant p53 by a factor of approximately 7, demonstrating clearly that suppression of apoptotic pathways interferes with the induction of cell death in GK9 cells.


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  		TABLE 2. Fold-increase in numbers of dead cells in GK9 cultures at 39.5°C compared to 33°C with or without overexpression of a mutant p53 protein.

As large numbers of dead cells could easily be collected from the tissue culture medium by centrifugation, electrophoretic analysis of the genomic DNA from these cells was chosen as a reliable marker for death by either apoptosis or necrosis. For three of the cell lines (GK3, GK9, and GK18), the appearance of an endonucleolytic cleavage pattern typical of apoptotic cell death could be demonstrated (Fig. 5). GK3 and GK9 genomic DNAs exhibited prominent bands at multiples of approximately 170 bp, representing the internucleosomal cleavage of the chromatin by an endonuclease activated exclusively during apoptosis [43]; in contrast, a smear resulting from random endonucleolytic cleavage was superimposed upon the cleavage pattern detectable in genomic DNA from GK18 cells. This finding correlates well with the observation that GK18 cells do not die in large numbers after inactivation of the temperature-sensitive T antigen (see below), but grow to confluence and eventually detach from the flask. The results of this experiment show that apoptosis is induced in a number of the conditionally immortalized granulosa cell lines after inactivation of the SV40 T antigen. The efficiency of induction of apoptosis, however, appears to differ between individual cell lines.



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  		FIG. 5. Fragmentation of genomic DNA from apoptotic granulosa cells. Dead cells were collected from supernatants of cultures of conditionally immortalized cell lines GK3, GK9, and GK18 after several days of culture at the nonpermissive temperature (39.5°C). The nucleosomal ladder originating from endonucleolytic cleavage during apoptosis was resolved by electrophoresis of 5 µg of genomic DNA on a 2% agarose gel. M, DNA size marker (100 bp ladder).

In order to follow the process of induction of apoptosis in the granulosa cell lines, cultures of cell lines GK3, GK9, and GK18 were observed by time-lapse videomicroscopy for 60 h after the transfer to 39.5°C. A field of 30–150 cells was observed, and the exact times of the individual apoptotic and mitotic events were registered by analysis of the videotapes. The data presented in Figure 6 clearly show that the induction of apoptosis after the shift to the nonpermissive temperature of 39.5°C followed very similar kinetics in cell lines GK3 and GK9. After a lag period of 20–30 h, cells began to die by apoptosis. The number of apoptotic events observed per hour remained approximately constant during the rest of the observation time, indicating a constant probability for each single cell to enter this pathway. Mitotic cell divisions in these two cell lines came to a stop completely after 30–40 h, confirming the temperature-sensitive phenotype of the immortalizing T antigen. In contrast, mitoses continued to occur in cell line GK18 over the entire duration of the experiment, whereas the incidence of apoptotic events in this cell line was very low compared with that for GK3 and GK9. Permanent growth of GK18 cells appears to be independent of the expression of functional T antigen, thereby reducing the probability for the cells to enter the apoptotic pathway. Figure 7 shows the video frames for the cultures described above, from the beginning of the experiment and after 60 h of incubation at 39.5°C. In contrast to observations in the Sertoli cell lines described in a previous study [28], no dramatic morphological changes in the granulosa cell lines were observed during the course of the experiment. Only cell line GK3 acquired a somewhat more extended shape after prolonged culture at the nonpermissive temperature. When all the findings are taken together, the investigation of apoptotic cell death in the conditionally immortalized granulosa cell lines shows that under conditions in which cellular growth is halted, simultaneous induction of expression of granulosa cell-specific genes and induction of cellular death occur. Elucidation of the regulatory mechanisms involved will require further studies.



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  		FIG. 6. Time course of cellular growth and apoptotic cell death in cultures of conditionally immortalized granulosa cell lines at the nonpermissive temperature. Cultures of cell lines GK3, GK9, and GK18 were continually observed by time-lapse videomicroscopy for 60 h after the shift from the permissive (33°C) to the nonpermissive temperature (39.5°C). The exact times of the individual apoptotic (triangles) and mitotic events (circles) were registered by analysis of the videotapes.



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  		FIG. 7. Apoptotic cell death in cultures of conditionally immortalized granulosa cell lines cultured at the nonpermissive temperature. Cultures of cell lines GK3, GK9, and GK18 were continually observed by time-lapse videomicroscopy after transfer from the permissive (33°C) to the nonpermissive temperature (39.5°C). The video frames from the beginning of the experiment and after 60 h of incubation at 39.5°C are shown. The accumulating apoptotic cells appear as small phase-bright spheres.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The conditionally immortalized mouse granulosa cell lines described in the study presented here, like the other granulosa cell lines reported in the literature, express a subset of the genes that are known to be specifically up-regulated during granulosa cell development and differentiation in vivo. A special feature of our cell lines is expression of the genes coding for the growth factors IGF-I and bFGF and their receptors, which play important roles in constituting paracrine and autocrine regulatory systems controlling granulosa cell growth and differentiation. Expression of the genes coding for the receptors for gonadal steroids, however, could be detected only in some of the lines, with one line (GK3) showing expression of estrogen receptor and two lines (GK2 and GK5) of progesterone receptor. Cultures of primary granulosa cells luteinize spontaneously [44–47] and express progesterone receptors during culture [48–50]. The down-regulation of the receptors for gonadal steroids in some of our cell lines presumably is caused by regulatory events during the establishment of the individual cell lines. Permanent growth of the immortalized cell lines possibly may interfere with the expression of some granulosa cell- or luteinization-specific genes. The finding that some of these genes are selectively shut off in the immortalized cell lines is confirmed by the RT-PCR studies. Expression of the genes coding for the steroidogenic enzymes P450arom and 3ßHSD was found to be down-regulated to levels below the detection level of this method, explaining the low steroidogenic activity of the cells. Most of the granulosa cell lines described in the literature express only some of the functions known to be expressed in granulosa cells in vivo. In only three cases, the cell lines conserve the expression of aromatase [14, 19, 23]. The GK cell lines described here express granulosa cell-specific genes like AMH and the steroidogenic enzyme P450scc, with AMH representing a gene specifically expressed in growing follicles [41] and P450scc being up-regulated around the time of ovulation with high levels of expression in luteinized granulosa cells [5]. It could be shown that P450scc is also up-regulated in the GK cell lines under conditions designed to simulate the culture conditions that promote luteinization of granulosa cells in vitro. The simultaneous expression of marker genes expressed sequentially in vivo hints at a certain loss of regulation presumably effected by the immortalization process. Additionally, the genes for the membrane receptors for the gonadotropins FSH and LH are strongly down-regulated in the cell lines, so that gonadotropins are not able to exert effects on the GK cell lines. This finding was not unexpected, as there are only two reports in the literature indicating that FSH receptor expression was conserved in established granulosa cell lines [14, 25]. Apparently, immortalized granulosa cell lines like those described by us tend to down-regulate FSH receptor expression, thus removing an important external signal that could interfere with permanent growth and induce differentiation.

Although differentiation cannot be induced by the stimuli acting on granulosa cells in vivo, the conditionally immortalized granulosa cell lines described in the present study offer the opportunity to stop growth of the cells simply by a shift to the nonpermissive temperature of the immortalizing SV40 T antigen. Under these conditions the P450scc gene, which is expressed to high levels in luteinizing granulosa cells in vivo, can be significantly up-regulated. Therefore these cell lines provide an easily accessible model system for analysis of the mechanisms involved in the regulation of granulosa cell functions. In the majority of the cell lines, up-regulation of granulosa cell-specific gene expression occurs simultaneously with induction of apoptotic cell death. Only one of the cell lines investigated, GK18, does not show massive induction of apoptosis at the nonpermissive temperature, irrespective of the absence of the immortalizing SV40 T antigen. The possible reason for this behavior may be the relatively high expression of IGF-I in this cell line, presumably leading to suppression of apoptosis and induction of cellular growth by action of IGF-I in an autocrine fashion via its specific membrane receptor.

In summary, we have established a set of conditionally immortalized mouse granulosa cell lines that express important granulosa cell-specific genes. The possibility of regulating expression of these genes by changing the growth conditions of the cell lines will allow us to analyze in greater detail the mechanisms involved in gene regulation during granulosa cell differentiation. Investigations of the chromatin structure of granulosa cell-specific genes and the in vivo binding of specific transcription factors to regulatory DNA elements can be performed by comparing a number of our cell lines showing specific differences in the regulation of these genes. These experiments—which can be performed neither with primary granulosa cells nor with heterologous cell lines—will broaden our view of the mechanisms controlling granulosa cell-specific gene expression. In the majority of the cell lines described, apoptosis can be induced by inactivation of the immortalizing temperature-sensitive SV40 T antigen, opening up the possibility of studying the control of apoptosis in these lines. The initial experiments using cell line GK9 that are shown in this study demonstrate the usefulness of this approach. Further experiments with the two cell lines, GK9 and GK18, that show dramatic differences in their apoptotic responses will allow a more detailed characterization of the process of apoptosis in these granulosa cell lines. For example, supplementing cell line GK9 with factors expressed to significantly higher levels in line GK18 might help to characterize survival factors involved in specific processes suppressing apoptosis of granulosa cells. Our cell lines probably are much more suited for the elucidation of granulosa cell apoptosis than other existing cell lines or primary granulosa cells, as they exhibit a number of different patterns of responses to regulatory stimuli. It may be possible to use these cell lines not only to study apoptosis but also to investigate processes involved in granulosa cell development and luteinization. After further characterization, our granulosa cell lines will be well suited to complement existing granulosa cell culture systems. They will allow an approach to specific problems of intracellular regulation and thus will prove useful as tools in the investigation of granulosa cell functions and development.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the cooperation of Drs. M. Noble and P. Jat (Ludwig Institute for Cancer Research, London, UK), who gave us the opportunity to use the H-2Kb-tsA58 mice for this study, and of Dr. A. Groves (Ludwig Institute for Cancer Research, London, UK), who helped us in setting up the primary cultures. We are especially indebted to Drs. G. Evan and C. Gilbert (Imperial Cancer Research Fund Laboratories, London, UK) for suggesting the detailed analysis of apoptosis and for the performance of time-lapse videomicroscopy. We thank Drs. H. Land and F. Obermüller (Imperial Cancer Research Fund Laboratories, London, UK) for the gift of the retroviral vectors used in this study, Drs. D. Jähner and H. Ungefroren for the gifts of specific PCR primers, and G. Tillmann and A. Jurdzinski for expert technical assistance. Above all, we are grateful to Professor F. Leidenberger and Professor R. Ivell for their generous support of the project and their constant encouragement.


   FOOTNOTES
 
1 Correspondence: Norbert Walther, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, D-22529 Hamburg, Germany. FAX: 49 40 56190864; walther{at}ihf.de Back

Accepted: December 1, 1998.

Received: June 8, 1998.


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