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

BIOLOGY OF REPRODUCTION 59, 1187–1194 (1998) In Vitro Culture of Hamster Ovarian Primary Interstitial Cells: Effect of Serum1 James R. Schwartz3 and Shyamal K. Roy2,3,4 Department of Obstetrics and Gynecology,3 Leland J. and Dorothy H. Olson Center for Women’s Health, Division of Reproductive Endocrinology, and Department of Physiology and Biophysics,4 University of Nebraska Medical Center, Omaha, Nebraska 68198–4515 ABSTRACT INTRODUCTION 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 (P450scc) and 17a-hydroxylase/C17,20-lyase (CYP17a; [9]). Johnson and Crane [10] have shown that rat interstitial cells express high levels of 17a-hydroxylase activity and the enzyme activity increases significantly after a single Accepted July 6, 1998. Received April 2, 1998. 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. MATERIALS AND METHODS 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 378C for 30 min in a shaking water bath. An efficient collagenase digestion was achieved by repeated 1187 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]thymidine incorporation, labeling index, and DNA content of the culture. All cells exhibited active 3b-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. injection of hCG. Likewise, increased CYP17a mRNA expression in rat theca-interstitial cells due to LH and insulinlike growth factor I exposure has also been reported [11]. However, a dramatic decrease in ovarian androstenedione production and CYP17a activity following the preovulatory LH surge has been reported in the hamster [12, 13]. In contrast to that in the rat ovary, CYP17a 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, Cyp17a and P450scc 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. 1188 SCHWARTZ AND ROY Culture of Interstitial Cells and Analysis of Cell Growth Cells (1 3 104) were cultured in 24-well culture plates (Costar, Cambridge, MA) in DMEM containing 1% ITS1 (insulin, transferrin, and selenium; Collaborative Research, Waltham, MA) [18] and antibiotics for 72 h at 378C 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. (...truncated)