Ontogeny of Steroidogenic Enzyme Gene Expression in Ovarian Theca-Interstitial Cells in the Rat: Regulation by a Paracrine Theca-Differentiating Factor Prior to Achieving Luteinizing Hormone Responsiveness

BIOLOGY OF REPRODUCTION 56, 938-945 (1997) Ontogeny of Steroidogenic Enzyme Gene Expression in Ovarian Theca-Interstitial Cells in the Rat: Regulation by a Paracrine Theca-Differentiating Factor Prior to Achieving Luteinizing Hormone Responsiveness' Timothy J. Gelety 3 and Denis A. Magoffin2 Department of Obstetrics and Gynecology, CSMC Burns and Allen Research Institute, UCLA School of Medicine, Los Angeles, California 90048 ABSTRACT INTRODUCTION In the female ovary, a mature preovulatory follicle represents the culmination of a long process of cellular proliferation and differentiation involving the intraovarian processes of follicular recruitment, selection, and dominance [1]. These processes are regulated by the tightly coordinated interactions of the hypothalamic-pituitary-ovarian axis and are critical for successful gamete maturation, meiosis, and release of a fertilizable ovum at ovulation. Ovarian Accepted November 26, 1996. Received October 3, 1996. 'This research was supported by NICHD grant HD28154. A preliminary report of portions of this data was presented at the 41st Annual meeting of the Society for Gynecologic Investigation, March 24, 1994. 2Correspondence: Denis A. Magoffin, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Davis 2066, Los Angeles, CA 90048. FAX: (310) 652-8010; e-mail: 3 Current address: Department of Obstetrics and Gynecology, University of Arizona School of Medicine, 1501 N. Campbell Ave., Tucson, AZ 85724. 938 The theca cells (TC) first become identifiable in preantral follicles after the granulosa cells (GC) begin to divide. It remains unknown when the TC first respond to LH and acquire the capacity to produce androgens. The signal initiating TC differentiation is also unknown since pre-theca cells do not contain LH receptors. Since the first wave of follicle development in the rat occurs postnatally, we correlated the function of dispersed ovarian cells from 4-, 5-, 6-, 7-, and 10-day-old rats with the morphological differentiation of TC. The largest follicles in ovaries from 4-day-old rats were primary follicles without associated TC. These cells were unable to produce cAMP or steroids in vitro in response to hCG. At 5 days, the first theca were associated with follicles containing 2-3 layers of GC. These cells were responsive to hCG, producing cAMP, progesterone, androstenedione, and androsterone. Responses to hCG increased progressively through 10 days of age. Cholesterol side-chain cleavage (P450cc), 3p1-hydroxysteroid dehydrogenase (3p1-HSD), and 17o-hydroxylase/C,7_20 lyase (P4501 7 ) enzymes were localized exclusively to the theca interna. Messenger RNAs for LH receptor, P450cc,, 3-HSD, and P450 7 , were expressed prior to the time the TC become responsive to LH or morphologically differentiated. To determine the source of the signal regulating TC differentiation, dispersed cells from 4-day-old rat ovaries that were unresponsive to LH were treated with preantral follicleconditioned medium containing thecal differentiating factor (TDF) activity. The TDF activity stimulated androgen production and expression of LH receptor, P450,,, 31-HSD, and P450,,7 mRNAs. These data demonstrate that a paracrine signal from the preantral follicle can initiate TC differentiation prior to expression of LH receptors. TC become responsive to LH and capable of producing androgens coincident with morphological differentiation. follicle development begins with primordial follicle recruitment into the population of growing follicles. The oocyte grows to full size and the granulosa cells (GC) proliferate into a multilayered band of endocrine cells surrounding a fluid-filled antrum and contained within a basal lamina. Immediately adjacent to the basal lamina, several layers of stromal cells differentiate into steroidogenic cells to form the theca interna (TI). Primordial follicles, however, appear to contain only the immature oocyte and GC but lack a morphologically distinct TI. The primary role of the TI in providing aromatizable androgen substrate for GC estradiol production is a basic principle of the two-cell, two-gonadotropin concept of follicular estrogen biosynthesis [2]. Therefore, the timing and control of theca cell (TC) differentiation is of central importance to an understanding of ovarian function. However, little is known regarding the timing and regulation of differentiation of pre-theca cells into functional endocrine cells. Morphological studies at the light microscopic level in the rat have demonstrated a relationship between the size of the oocyte, follicle diameter, the number of layers of GC, and the appearance of the TI. Whereas primordial follicles do not contain discernible TC, differentiation of morphologically distinct TC begins only after follicle development has begun and the GC have become cuboidal and begun to proliferate [3]. The origin of the steroidogenically active TC is unclear; however, the uptake of tritiated thymidine by stromal cells adjacent to primary follicles suggests that undifferentiated pre-thecal cells exist in the interfollicular stroma [4]. Since the TI is associated only with developing follicles, it seems reasonable to propose that a signal originates from preantral follicles that stimulates TC differentiation. We have recently shown that the GC in preantral rat follicles beginning with 2 layers of GC secrete a lowmolecular-weight peptide that stimulates the differentiation of isolated TC [5, 6]. These data demonstrate that preantral follicles produce a paracrine signal that can stimulate the formation of a steroidogenically active TI. There is a large body of evidence demonstrating that LH is the principal hormone regulating TC differentiation and steroidogenesis in TC that contain LH receptors [7, 8]; however, the pre-theca cells do not contain LH receptors. Two goals of these studies were to determine when the TC attain steroidogenic capacity in relation to the timing of morphological differentiation and to determine whether the theca cell differentiation factor (TDF) secreted by the GC of preantral follicles is capable of stimulating TC differentiation prior to the acquisition of LH responsiveness. Evidence from studies unrelated to the pattern of initial TC differentiation has led to the concept that the TC differentiate sequentially into progesterone-producing cells and later become androgen-producing cells [7]. The final goal of these experiments was to determine whether the expression ONTOGENY OF THECAL GENE EXPRESSION MATERIALS AND METHODS Reagents Ovine LH (AFP-5551B; 2.3 IU/mg), recombinant human FSH (rFSH; R1), and hCG (CR-127; 14 900 IU/mg) were a gift of the National Hormone and Pituitary Program (Rockville, MD). McCoy's 5a medium, Medium 199, penicillin-streptomycin solution, L-glutamine solution, BSA, and trypan blue stain were obtained from Gibco (Santa Clara, CA). Collagenase (CLS; 144 U/mg) was obtained from Worthington (Freehold, (...truncated)