In Vitro Culture of Hamster Ovarian Primary Interstitial Cells: Effect of Serum¹

^a Department of Obstetrics and Gynecology, Leland J. and Dorothy H. Olson Center for Women's Health, Division of Reproductive Endocrinology, and ^b Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198–4515

	ABSTRACT

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The function of ovarian interstitial cells has been largely addressed using rat theca-interstitial cell culture. However, this preparation is primarily enriched with theca and secondary interstitial cells, which make it difficult to address selectively the function of the primary interstitial cells. We have developed an in vitro culture of hamster ovarian primary interstitial cells. Cells were isolated from postnatal hamster ovaries by collagenase digestion and purified over a Percoll gradient. The preparation contained 90% viable, pure interstitial cells, which anchored to the plastic and glass culture surface in the presence of fetal bovine serum. Cell proliferation was noted in the presence of serum dosages higher than 0.2%; however, reduction of serum concentration to 0.1% or complete serum starvation did not affect cell viability but almost completely abolished cell proliferation as determined by [³H&; incorporation, labeling index, and DNA content of the culture. All cells exhibited active 3ß-hydroxysteroid dehydrogenase and P450 side chain cleavage immunoreactivity, which corresponded to basal progesterone and androstenedione accumulation. Replacement of serum to starving cells resulted in the induction of the "S" phase and "M" phase specific cyclins, and resumption of cell proliferation. Our results indicate that hamster primary interstitial cells can be cultured in vitro as a monolayer, and the anchorage and proliferation of these cells depend on serum supplement; however, a viable monolayer can be maintained for several days without serum. This model will be useful for addressing the mechanisms of differentiation of ovarian interstitial cells.

	INTRODUCTION

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Formation of thecal cells is essential for successful follicular development. Although it is logical to postulate that thecal cells arise from the surrounding interstitium, the mechanisms and spatio-temporal events leading to interstitial cell differentiation are unclear. Numerous studies have been done on the function of interstitial cells and their response to hormone and growth factors in vitro [1–8]; however, the cell preparation always contains substantial thecal and secondary interstitial cell contamination, which obscures the true function of primary interstitial cells and precludes the determination of interstitial cell response to various growth stimuli.

Among the major steroidogenic enzymes, ovarian interstitial cells in the adult express both P450 side chain cleavage (P450_scc) and 17-hydroxylase/C17,20-lyase (CYP17; [9]). Johnson and Crane [10] have shown that rat interstitial cells express high levels of 17-hydroxylase activity and the enzyme activity increases significantly after a single injection of hCG. Likewise, increased CYP17 mRNA expression in rat theca-interstitial cells due to LH and insulin-like growth factor I exposure has also been reported [11]. However, a dramatic decrease in ovarian androstenedione production and CYP17 activity following the preovulatory LH surge has been reported in the hamster [12, 13]. In contrast to that in the rat ovary, CYP17 expression is mainly localized in the theca cells of adult hamsters [13–15] and has been shown to decrease significantly in the afternoon of proestrous, i.e., following the preovulatory gonadotropin surge [12, 15]. We have observed that in the postnatal hamster ovary, Cyp17 and P450_scc immunoreactivities are not visible until postnatal Day 13 (unpublished observations), when follicles with 5–6 layers of granulosa cells are present and serum levels of LH are detectable [16, 17].

Because the theca-interstitial cell preparation from immature rats contains cells that already exhibit theca-specific changes, the usefulness of this model to address the onset of interstitial cell differentiation is limited. Therefore, a cell preparation consisting of undifferentiated primary interstitial cells will be useful to understand the mechanisms of interstitial cell differentiation. Ten-day-old postnatal hamster ovaries provide such an option, since these ovaries contain primordial and preantral follicles with 1–3 layers of granulosa cells, and interstitial cells are undifferentiated. The objectives of the present studies were to establish an ovarian primary interstitial cell culture from 10-day-old postnatal hamsters and to monitor the growth characteristics of these cells in vitro.

	MATERIALS AND METHODS

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Animals

Golden Syrian hamsters were obtained from Sasco (Kingston, NY) and maintained in a climate-controlled animal facility according to the NIH guidelines for the Care and Handling of Experimental Animals. Females with at least 3 consecutive estrous cycles were placed with males (2 males/female per cage) in the afternoon of proestrous. The presence of sperm in the vaginal smear on the next morning was considered Day 1 of pregnancy, and pregnant females were housed separately. Ten-day-old female pups were anesthetized with 0.1 ml of Nembutal (sodium pentothal; Sigma Chemical Company, St. Louis, MO; 8 mg/100 g BW). The ovaries were removed, placed in Kreb's Ringer bicarbonate media, pH 7.4, containing 0.1% glucose (KRBG), and used within 30 min.

Isolation of Interstitial Cells

Ovaries were dissected free of oviduct and bursa, and minced with a razor blade, and ovarian cells were dissociated with 1208 U/ml collagenase (type XI; Sigma) in 2 ml of KRBG and 123 U/ml of deoxyribonuclease (type I; Sigma) at 37°C for 30 min in a shaking water bath. An efficient collagenase digestion was achieved by repeated pipetting at 15-min intervals. The digest (2 ml) was filtered through a 60-µm nylon filter (Tetko, Elmsford, NY). Undigested debris and large cell clusters were pelleted at 41.3 x g for 5 min at 4°C. The supernatant containing the single cell suspension was placed into another 15-ml polypropylene conical tube and centrifuged at 372 x g for 5 min, to pellet cells. After centrifugation, the cell pellet was suspended in 1 ml of single-strength Dulbecco's Modified Eagle medium (DMEM) containing 100 U/ml of penicillin G, 100 µg/ml streptomycin sulfate, 0.25 µg/ml amphotericin B, and 5 mg/ml BSA [18], and layered over a discontinuous Percoll (Sigma) density gradient. An iso-osmotic Percoll working stock (specific gravity 1.123 g/ml) was made by diluting 9 volumes of stock Percoll (specific gravity, 1.13 g/ml) with 1 part of 10-strength DMEM without BSA. All subsequent concentrations were obtained by diluting the Percoll working stock with single-strength DMEM. A discontinuous Percoll density gradient was prepared by layering sequentially 1 ml of 44% Percoll (specific gravity, 1.06 g/ml), 2 ml of 35% Percoll (specific gravity, 1.049 g/ml), and 1.5 ml of 22% Percoll (specific gravity, 1.033 g/ml) in a 10-ml Falcon polypropylene tube (Falcon Plastics, Los Angeles, CA). One milliliter of cell suspension in DMEM was layered on top of the gradient and centrifuged for 20 min at 372 x g at 4°C. The interstitial cells were isolated from the 35% and 44% Percoll layer interphase and washed twice with DMEM. Cell viability and number were determined by using a fluorescent Live/Dead assay kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions, and the Trypan blue dye exclusion methods, respectively.

Culture of Interstitial Cells and Analysis of Cell Growth

Cells (1 x 10⁴) were cultured in 24-well culture plates (Costar, Cambridge, MA) in DMEM containing 1% ITS⁺ (insulin, transferrin, and selenium; Collaborative Research, Waltham, MA) [18] and antibiotics for 72 h at 37°C under 5% CO₂ in air.

Experiment 1 Because interstitial cells from the adolescent hamster ovary can grow as a monolayer in the absence of serum (unpublished results), Percoll-purified ovarian cells from the postnatal hamsters were cultured in the absence of fetal bovine serum (FBS) to determine their serum dependency.

Experiment 2 On the basis of the results of experiment 1, cells were cultured for 72 h in the presence of 0.1%, 0.2%, 1%, 2%, and 5% heat-inactivated FBS (Gibco-BRL, Grand Island, NY), and medium was replaced every 48 h. To determine the growth potential of neonatal interstitial cells, 1 µCi/ml of [³H&; (specific activity 40 Ci/mmol; Amersham Corp., Arlington Heights, IL) was added 48 h after culture, and the culture was continued for additional 24 h. The plates were placed on ice, medium was removed, and cells were rinsed with 0.5 ml ice-cold PBS. After adding 0.5 ml distilled water to each culture well, cells were scraped with a rubber cell scraper, and the suspension was transferred to a 1.5-ml microfuge tube and stored at -20°C until used for DNA assay.

Experiment 3 To determine the long-term effect of serum and the effect of its reduction on interstitial cell growth in vitro, cells were cultured as mentioned above for up to 96 h either in the presence of 5% FBS throughout the culture period or in the presence of 5% FBS for 24 h followed by 0.1% FBS up to 96 h. The objective was to assess whether cells would remain viable under a low-serum environment, which will be desirable for subsequent use of this model. Medium was changed every 48 h, and 1 µCi/ml [³H&; was added to the culture 24 h before termination. Cultures were terminated every 24 h, and cells were processed for quantitation of DNA and [³H&; incorporation.

Experiment 4 To gain insight into the rate of cell proliferation, cells were cultured in 2-chambered glass slides (Nunc, Naperville, IL) following the design of experiment 3. Cultures were terminated at 24-h intervals, and cells were rinsed with ice-cold PBS, fixed overnight in Bouin's fixative at room temperature under 100% humidity, dehydrated in grades of ethanol, and air-dried before coating with Kodak NTB2 autoradiographic emulsion (Eastman Kodak, Rochester, NY).

Experiment 5 To determine whether serum replacement after a withdrawal would engage cells into the proliferative phase or whether cells would differentiate permanently once the initial serum stimulation was withdrawn, expression of the "S" and "G2/M" phase-specific antigens, such as proliferating cell nuclear antigen (PCNA), cyclins D2, E, and B1, were assessed in neonatal interstitial cells. Cells were seeded for 24 h in the presence of 5% FBS, and then the medium was replaced with fresh medium without FBS. After cells were cultured for an additional 96 h without serum, serum concentration was raised to 5% and the culture was continued for 24 h. In a parallel experiment, 1 µCi/ml [³H&; was added to the culture to assess the resumption of DNA synthesis. Cells were rinsed with ice-cold PBS and either fixed in 4% formaldehyde in PBS for immunofluorescent detection of cell cycle antigens or fixed in Bouin's fixative for [³H&; autoradiography and labeling index (LI).

DNA Quantitation, Analysis of [³H]Thymidine Incorporation, and Assessment of the LI

DNA was quantified essentially as described by Roy and Greenwald [19]. Briefly, the cell suspension was sonicated for 3 sec at 50 watts, and DNA was coprecipitated with BSA with 10% ice-cold trichloroacetic acid. The precipitate was dissolved in solubilization buffer and heated for 10 min at 37°C, and 10 µl of the sample was added to 2 ml of assay buffer containing bisbenzimide (Hoechst dye). The fluorescence was recorded in a Perkin-Elmer spectrofluorometer, and the amount of DNA in the samples was determined from a standard curve run simultaneously with the samples. The results were expressed as nanograms of DNA per culture well.

For the LI, cells grown in culture slides were fixed overnight in Bouin's fixative to remove any free [³H&; After ethanol dehydration, slides were coated with Kodak NBT2 liquid photographic emulsion, air-dried, exposed in the dark for 7 days, developed in Dektol (Kodak), and stained with hematoxylin and eosin. Cell nuclei with or without silver grains were counted using a Leica DMR research microscope (Leica Corp. GmbH, Wetzlar, Germany), and the percentage of labeled cells was expressed as the LI.

Immunofluorescent detection of cyclins E, B1, and D2 was done using antigen-specific rabbit polyclonal antibodies (Santa Cruz Biotech, Santa Cruz, CA) and streptavidin-fluorescein isothiocyanate (FITC; Vector Laboratories, Burlingame, CA). PCNA was detected by a monoclonal anti-PCNA antibody (Santa Cruz Biotech). Briefly, cells were fixed with 4% formaldehyde in PBS, blocked with 10% goat serum for 1 h at room temperature, and exposed overnight to 1 µg/ml of either of the antibodies at 4°C. After a 10-min exposure to biotinylated goat anti-rabbit IgG at room temperature, cells were exposed to streptavidin-FITC in PBS/0.05% Tween-20 for 30 min at room temperature. The signal was detected using a Leica DMR epifluorescence microscope and recorded using an Optronics video camera and Image Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD). To determine the specificity of the antibodies and to rule out any autofluorescence, an immunofluorescence study was done using antibody host-specific nonimmune serum.

To determine whether cells growing in vitro represented interstitial cells, cells cultured for 96 h in vitro were subjected to 3ß-hydroxysteroid dehydrogenase/^5–4-isomerase (3ß-HSD) histochemistry using nitroblue tetrazolium as electron acceptor [20]. Formazan-labeled cells were considered steroidogenically competent interstitial cells. In addition, 3ß-HSD and P450_scc immunoreactivities were assessed by immunocytochemistry using enzyme-specific rabbit polyclonal antipeptide antibodies and a peroxidase-based aminoethylcarbazole protocol [17]. Moreover, the steroidogenic property of hamster interstitial cells grown in vitro was assessed by measuring the production of progesterone and androstenedione using specific RIAs [21]. The results were expressed as picograms per milliliter of medium.

Statistical Analysis

Data were analyzed by one-way ANOVA with Scheffe's F test. The level of significance was at 5%. All experiments were repeated 3 times, and each value represents a mean of 4 replicates.

	RESULTS

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Enzymatic digestion dissociated interstitial cells from 10-day-old hamster ovaries, but early primary follicles remained intact and were easily removed by membrane filtration. Because of the relatively larger size, interstitial cells from postnatal hamsters migrated at the junction between 44% and 37% Percoll while smaller granulosa-like cells precipitated at the bottom through 44% Percoll. No oocyte-like structure was present in the large cell population as assessed by phase contrast microscopy. Assessment of the cell viability by fluorescence vital staining indicated that 90% of the interstitial cells were viable (Fig. 1). The principle of the live/dead detection system is based on ubiquitous intracellular esterase activity, which is active only in live cells. The vital dye mixture contained a nonfluorescent cell-permeant calcein AM, and a red fluorescent ethidium homodimer (EthD-1); EthD-1 cannot pass through intact cell membrane—a characteristic of live cells. Nonfluorescent calcein enters cells, and upon esterase activation is converted into practically cell-nonpermeant, intensely green fluorescent calcein. On the other hand, EthD-1 can enter cells once the membrane is damaged (a hallmark of dead cells) and emits bright red fluorescence upon binding to nucleic acids. Therefore, only dead cells will be red (Fig. 1B) while all live cells will be green (Fig. 1A). The usefulness of the vital fluorescent dyes was validated using granulosa cells of preovulatory hamster follicles and compared with the conventional Trypan blue dye exclusion method. The majority of large cells retrieved from 35% and 44% Percoll interphase were green while all small cells were red. In addition, the percentage of red cells was appreciably low in the green cell population, resulting in a consistent 90–95% pure large cell preparation. The results of Trypan blue dye exclusion compared well with the fluorescent dye method; however, the latter was much more specific and convenient. Moreover, the apparent size difference between granulosa and interstitial cells was also clearly discernible (Fig. 1).

View larger version (7K): [in this window] [in a new window]   FIG. 1. Determination of live versus dead cells in hamster interstitial cell preparation using fluorescent dyes. A) Live cells recovered from the Percoll interphase and cultured as interstitial cells show green fluorescence. B) Dead cells present in the interstitial cell preparation show red fluorescence. Note the size difference between live and dead cells. The size of the dead cells corresponded to granulosa-like cells. Whereas the number of live small cells was scanty, large dead cells were not visible. Magnification x100.

The attachment of the interstitial cells to the tissue culture surface was very sluggish in the absence of serum, and attached cells grew extremely slowly (data not shown); therefore, serum-based culture was adopted. In the presence of 5% FBS, large cells adhered to the plastic surface within few hours and formed a monolayer by 24 h. Cell growth became prominent by 48 h (Fig. 2A) and continued through 96 h (Fig. 2B); however, none of the small cells apparently adhered to the culture plate, and they were washed away with the change in the medium, resulting in a pure primary interstitial cell culture. That cells growing in the monolayer were indeed interstitial was evident from the presence of active 3ß-HSD activity (data not shown) and immunoreactivity (Fig. 3A), and side chain cleavage enzyme immunoreactivity in all cells of the monolayer after 96-h culture (Fig. 3B). That steroidogenic enzymes were functional was evident from the production of progesterone and androstenedione in vitro (Fig. 4).

View larger version (83K): [in this window] [in a new window]   FIG. 2. Phase contrast micrographs showing hamster primary interstitial cell culture 48 and 96 h after plating. Cells anchored well and plated by 24 h, and formed distinct monolayer by 48 h in the presence of 5% FBS (A) and showed further growth with the extension of the culture period to 96 h (B). Magnification x200 (reproduced at 75%).

View larger version (101K): [in this window] [in a new window]   FIG. 3. Immunocytochemical localization of P450 scc and 3ß-HSD immunoreactivities in hamster primary interstitial cells grown in culture for 96 h in the presence of 5% FBS. A) The presence of P450scc-positive mitochondrial structures is visible (arrowhead). B) A uniform 3ß-HSD staining was visible in all cells; however, some cells expressed relatively more enzyme immunoreactivities (arrowhead). No staining was observed when the antibodies were withdrawn (data not shown). Magnification x200 (reproduced at 87%).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Accumulation of progesterone and androstenedione in the culture medium of hamster primary interstitial cells. Cells were cultured for 96 h in the presence of 5% FBS, and the amount of steroids accumulated during 24-h culture was determined by RIAs. A relatively higher production of androstenedione reflects differentiated interstitial cell property. *Value was significantly (p < 0.05) different from progesterone. The bars represent mean ± SEM.

Serum dose dependently supported interstitial cell growth and stimulated DNA synthesis. An almost linear increase in [³H&; incorporation was noted when serum concentration was raised from 0.2% (Fig. 5), and DNA synthesis maximized at 5% serum concentration. Although cells incorporated [³H&; with 0.1% serum, no significant increase in DNA synthesis was noted when the serum concentration was raised to 0.2%, indicating that 0.1–0.2% serum basically maintained a viable monolayer without much stimulation to cell proliferation.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Serum dose response to [3H&; incorporation by hamster interstitial cells in vitro. Cells were cultured for 72 h and [3H&; was added 24 h before culture termination. Note an almost linear increase in cpm incorporated when serum concentration was raised from 0.2%. Significant (p < 0.05) increases in the rate of DNA synthesis were noted at serum concentrations 1% (*), 2% (**), and 5% (***) compared to 0.1 and 0.2%. The bars represent mean ± SEM.

Interstitial cells proliferated in the presence of 5% serum throughout the culture period, as evidenced by the increase in DNA content of the monolayer, and the growthwas asynchronous (Fig. 6). Cellular incorporation of [³H&; increased significantly (p < 0.05) 48 h after the beginning of culture (Fig. 7); this increase was followed by a moderate decrease by 72 and 96 h; however, there was a corresponding increase in the DNA content (Fig. 6). Reducing the serum concentration to 0.1% after 24-h exposure to 5% serum resulted in a significant reduction in [³H&; incorporation by 48 h, which fell further by 96 h (Fig. 7), with a consequent cessation of DNA synthesis throughout the culture period (Fig. 6). LI data revealed that cells entered the "S" phase at different times, resulting in a steady-state LI up to 96 h; however, reduction of serum concentration dramatically reduced the number of labeled cells after 24 h, and only 2–3% of cells incorporated [³H&; by 72 h (Fig. 8). A complete serum starvation after 24 h also resulted in a reduction of LI similar to that observed with 0.1% serum; however, administration of 5% serum after 96-h serum starvation resulted in 70% resumption in the LI (Fig. 9), suggesting that cells, once they formed the monolayer, remained viable without serum for several days and could resume the cell cycle after serum replacement. The resumption of the cell cycle was further evident from the significant induction of PCNA, cyclin D2, cyclin E, and cyclin B1 in the interstitial cells after 24-h culture with 5% serum (Fig. 10) following 96 h of serum starvation.

View larger version (20K): [in this window] [in a new window]   FIG. 6. DNA content of hamster interstitial cells cultured up to 96 h in the presence of 5% or 0.1% FBS. For the low-serum dose, cells were plated in the presence of 5% serum for 24 h; then medium was removed, the cultures were rinsed with serum-free medium, and, finally, cells were placed in fresh medium containing 0.1% FBS. Note a stationary DNA amount reflecting a cessation of cell proliferation in the low-serum concentration. * Values were significantly (p < 0.05) different from those of the corresponding low-serum groups. The bars represent mean ± SEM.

View larger version (23K): [in this window] [in a new window]   FIG. 7. Incorporation of [3H&; by hamster primary interstitial cells cultured up to 96 h in vitro in the presence of 5% or 0.1% FBS. Cultures were plated for 24 h with 5% FBS and then either continued under the same serum concentration or under 0.1% FBS from 48–96 h. The incorporation of [3H&; in cells was detected 24 h after the administration of radioactive nucleotide. A sharp decline in [3H&; incorporation was noted after the reduction in serum concentration. * Values were significantly (p < 0.05) different from those of the corresponding low-serum groups. The bars represent mean ± SEM.

View larger version (20K): [in this window] [in a new window]   FIG. 8. LI of hamster primary interstitial cells cultured up to 96 h in vitro in the presence of 5% or 0.1% FBS. Cultures were plated for 24 h with 5% FBS and then either continued under the same serum concentration or under 0.1% FBS from 48–96 h. [3H]Thymidine was added 24 h before the termination of cultures. Reduction in serum concentration to 0.1% resulted in a sharp decrease in the LI. * Values were significantly (p < 0.05) different from those of the corresponding low-serum groups. The bars represent mean ± SEM.

View larger version (12K): [in this window] [in a new window]   FIG. 9. Effect of serum replacement on the LI of hamster primary interstitial cells cultured in vitro. Cells were initially seeded for 24 h with 5% FBS and then cultured up to 96 h without any serum. Cells were then cultured for additional 24 h in the presence or absence of 5% FBS, and with 1 µCi/ml [3H&; Serum replacement (*) resulted in a significant (p < 0.05) resumption in the LI. The bars represent mean ± SEM.

View larger version (22K): [in this window] [in a new window]   FIG. 10. Immunofluorescence detection of cell cycle-specific antigens in hamster primary interstitial cells cultured in vitro for up to 120 h. Cells were seeded with 5% serum for 24 h, then cultured in serum-free medium for 96 h. Cells were then cultured for additional 24 h in the absence (A, C, E, G) or presence (B, D, F, H) of 5% FBS, and used for the detection of PCNA (A, B), cyclin E (C, D), cyclin D2 (E, F), and cyclin B1 (G, H). Note distinct expression of cell cycle antigens in the interstitial cells after the serum replacement. No expression was visible for cells cultured without serum.

	DISCUSSION

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The results of this study document that hamster primary interstitial cells can be obtained from neonatal ovaries and cultured in vitro. This culture is free of granulosa, theca, and secondary interstitial cells&; latter two cell types present major limitations that are prevalent in existing interstitial cell culture models [1–2, 5]. Because 10-day-old neonatal hamster ovaries contain only primordial and primary follicles and the primary follicles can be separated intact using collagenase, this source of interstitial cells is certainly superior to that using cells of adolescent animals. Moreover, because cells were isolated from ovaries long before the formation of definitive thecal layers, the presence of secondary interstitial cells is unlikely. Therefore, these primary interstitial cells provide an opportunity to study directly their differentiation process in vitro. The large size of the interstitial cells also makes cell purification on Percoll gradient easy. A similar protocol has also been used successfully to obtain rat theca-interstitial cells [2].

The serum requirement for the anchorage and growth of hamster interstitial cells indicates that these cells are undifferentiated and that some serum factors may maintain their growth in vivo, at least during the early stage of ovarian development. Culture of rat theca-interstitial cells (TIC) without serum has been documented [1, 2]; however, to our knowledge, the proliferation of primary interstitial cells in vitro has not been documented. Similar to rat TIC, interstitial cells from adolescent hamster ovaries (Days 20–24 postnatal) can anchor and form a monolayer without serum supplement (data not shown), suggesting that primary interstitial cells lose their serum dependency once the onset of follicular development is complete. Alternatively, it is likely that peripubertal rodent ovaries contain primarily secondary interstitial cells that originated from atretic follicles, and these cells make factor(s) that are adequate for their anchorage on the culture surface. Our results suggest that interstitial cells of early postnatal ovaries require serum support for monolayer formation but can remain viable for several days when the serum is withdrawn. The resumption of the cell cycle after serum replacement suggests that hamster interstitial cells follow the same general trends as observed for other nonovarian proliferating cells. The presence of 3ß-HSD activity and immunoreactivity, and side chain cleavage enzyme immunoreactivity in 100% of the plated cells, and the production of progesterone and androstenedione in vitro, suggest strongly that these cells are steroidogenic in nature (for references see [4]). Granulosa cells of primordial, primary, and early secondary follicles from the adult hamster do not express discernible 3ß-HSD activity even after 96-h culture (unpublished). In the neonatal hamster ovary, 3ß-HSD and side chain cleavage enzyme immunoreactivities do not appear in vivo until postnatal Day 14, when serum LH levels start rising [16, 17]. Moreover, 15-day-old hamster ovary produces a significant amount of progesterone in response to LH or cyclic AMP [22]. Gelety and Magoffin [6] have demonstrated that thecal cells appear in the rat ovary by postnatal Day 5, when hCG responsiveness to steroidogenesis can be observed in whole ovarian dispersates, and the response increases progressively to postnatal Day 10. Therefore, it appears that interstitial cells in the rat ovary receive their differentiation-promoting signal before postnatal Day 5. In the hamster, however, definitive thecal cells do not appear until follicles have 5–6 layers of granulosa cells, which do not develop before postnatal Day 13 [17], thus ruling out any theca cell contamination in the present interstitial cell preparation.

That interstitial cells undergo proliferation in response to serum is evident from the increases in the LI, [³H&; incorporation, and DNA content. The pattern of [³H&; incorporation with respect to DNA content suggests that different populations of cells enter the cell cycle at different times. The increase in [³H&; incorporation by 48 h and DNA content by 72 h in the presence of serum indicates DNA duplication rather than repair. The gradual fall in [³H&; incorporation and a corresponding increase in DNA content also suggest that cells, after completion of one round of division, may not enter the cell cycle before 24 h. The exact cell cycle time for these cells, however, is not yet known. The increase in DNA synthesis correlates well with the induction of the "S" phase cyclins, such as cyclins D2 and E [23], and PCNA [24]. Cyclin D2 expression has been demonstrated in proliferative murine granulosa cells, and the expression is FSH- and cyclic AMP-dependent [25]. Moreover, cyclin D2 expression has been shown to be growth factor-dependent in many other cell types [26, 27]. Because intraovarian growth factors affect interstitial cell growth (for references see [28]), a serum-induced growth factor cascade controlling the cell cycle progression in hamster interstitial cells may exist. Alternatively, serum may directly induce G1/S phase cyclins via activation of a cyclic AMP signal pathway. Induction of cAMP by serum has been reported for many cell types [29, 30] and a cAMP-inducing property has been attributed to lysophosphatidic acid, a serum component that stimulates cell proliferation by activating a pertussis toxin-sensitive G-protein mechanism [31, 32]. The induction of cyclin B1 in the presence of serum suggests that interstitial cells do complete their cell cycle in vitro since the entry into mitosis is signaled by the activation of the primary mitotic kinase, the cyclin B-cdc2 complexes (for references see [33]).

In summary, a hamster primary interstitial cell culture model has been developed to address the mechanism of ovarian nonfollicular cell differentiation. This model utilizes undifferentiated primary interstitial cells from the postnatal hamster ovary, which is completely devoid of thecal and secondary interstitial cells. Moreover, these primary interstitial cells proliferate in vitro in the presence of serum and express steroidogenic enzymes characteristic of ovarian interstitial cells.

	FOOTNOTES

 
¹ This work was supported by grants HD28165 from the National Institute of Child Health and Human development, NIH, and the Olson Foundation to S.K.R.

² Correspondence: Shyamal K. Roy, Bennett Hall 5005, Departments of OB/GYN and Physiology/Biophysics, 984515 University of Nebraska, Medical Center, Omaha, NE 68198–4515. FAX: 402 559 6164.

Accepted: July 6, 1998.

Received: April 2, 1998.

	REFERENCES

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