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Regular Article |
a Department of Obstetrics & Gynecology, Cedars-Sinai Burns & Allen Research Institute, UCLA School of Medicine, Los Angeles, California 90048
ABSTRACT
The signal initiating ovarian theca cell (TC) differentiation is gonadotropin independent because theca precursor cells do not contain LH receptors. Previously we demonstrated that preantral follicles produce paracrine TC differentiating factors that promote androgen production by an LH-independent mechanism. This study tested the effects of two granulosa cell-produced peptides, insulin-like growth factor-I (IGF-I) and stem cell factor (SCF), on TC differentiation and androgen production. Neutralizing antibodies to either IGF-I or SCF blocked the stimulatory effects of follicle-conditioned medium on TC precursor differentiation more than 90%. The TC isolated from the ovaries of hypophysectomized immature rats by percoll gradient centrifugation were cultured (48 h) with and without SCF (0–100 ng/ml) and IGF-I (0–100 ng/ml) to test their effects on TC differentiation. Androsterone in the medium was measured by RIA. Luteinizing hormone receptor, steroidogenesis acute regulatory protein (StAR), CYP11A, CYP17, and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNAs were measured by specific reverse transcriptase polymerase chain reaction assays. Stem cell factor or IGF-I alone did not stimulate androsterone production but in combination caused a concentration-dependent increase in androsterone levels. Maximum androsterone levels were less than those stimulated by LH (0.1 ng/ml) alone. Although IGF-I synergistically augmented LH stimulation of androsterone production, SCF did not alter LH-stimulated androsterone production in the presence or absence of IGF-I. Stem cell factor alone had no effect on LH receptor, StAR, CYP11A, and 3ß-HSD mRNA expression but decreased CYP17 mRNA levels. Insulin-like growth factor-I alone had no effect on StAR or CYP17 mRNA expression but increased LH receptor, CYP11A, and 3ß-HSD mRNA levels. In combination, SCF plus IGF-I increased the expression of all five mRNAs. These data support the conclusion that IGF-I and SCF are important regulators of TC differentiation.
follicular development, growth factors, theca cells
INTRODUCTION
Mammalian follicular development is the result of a complex progression of cellular interactions culminating with a mature follicle capable of ovulating a fertilizable oocyte. In rats, morphologic studies at the light microscopic level demonstrated a relationship between the size of the oocyte, follicle diameter, the number of layers of granulosa cells (GC), and the formation of the theca interna [1]. In primordial follicles there is a single layer of flattened GC surrounding the oocyte. When the primordial follicle is recruited into the pool of developing follicles, the GC become cuboidal and begin to proliferate. As the second layer of GC develops, stromal cells adjacent to the basal lamina begin to differentiate into a distinct theca interna [2]. At this stage the theca cells (TC) acquire steroidogenic capacity [3], but the GC remain nonsteroidogenic [4]. The characteristics of the thecal precursor cells do not appear to be morphologically distinct from the fibroblast-like cells located in ovarian stroma adjacent to developing follicles [5].
Although LH is the principal hormone regulating mature TC steroidogenesis, theca precursor cells lack LH receptors and do not express the steroidogenic enzyme genes required for androgen biosynthesis, namely cholesterol side-chain cleavage cytochrome P450 (CYP11A), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and 17
-hydroxylase/C17–20 lyase cytochrome P450 (CYP17). Because TC only differentiate adjacent to growing follicles, it was proposed that the signal for TC differentiation originates within the developing follicle [6]. In previous studies we have shown that preantral follicles with two or more layers of GC secrete one or more peptide theca differentiating factors (TDFs) that can stimulate the differentiation of TC independent of LH [6]. The TDFs are secreted in a developmentally specific pattern with activity limited to the preantral stages of development [7]. Importantly the physiological TDFs can stimulate the expression of LH receptor and steroidogenic enzyme mRNAs in theca precursor cells [3].
Insulin-like growth factor-I (IGF-I) is a peptide with properties that make it an attractive candidate as a component of the physiological theca-differentiating signal in preantral follicles. Insulin-like growth factor-I alone stimulates the expression of LH receptor, CYP11A, and 3ß-HSD mRNA expression and augments LH-stimulated androgen production by TC [8–11], but IGF-I has no effect on CYP17 mRNA expression. Therefore IGF-I has some of the properties of the physiological theca-differentiation signal but alone is not sufficient to fully differentiate TC.
The genetic loci Sl (steel) and W (white spotting) are distinct with related functions that were independently identified as coat color mutations in mice. Mutations in the W and Sl loci are known to affect development of the hematopoietic, pigment, and gonadal cell lineages [12, 13].
The W gene encodes the receptor c-kit, a transmembrane receptor with intrinsic tyrosine kinase activity belonging to a family of receptors including colony stimulating factor-1 and platelet-derived growth factor A and B receptors. A variety of mutations in c-kit have been documented leading to phenotypes of differing severity ranging from embryonic lethality to severe macrocytic anemia, depigmentation, and sterility [12]. In the hematopoietic system, c-kit mutations affect stem cells, erythroid precursors, and mast cells, affecting both the migration of cells early in embryogenesis and the survival of mature cells during melanogenesis. Mutations in c-kit alter early germ cell migration and development as well as postnatal oogenesis and spermatogenesis.
The Sl gene encodes a protein ligand of the c-kit receptor known as stem cell factor (SCF), steel factor, mast cell growth factor, or kit ligand [14]. Mutations in SCF result in phenotypes similar to c-kit mutations. Certain mutants do not produce androgens in response to LH [15], and in others follicle development is arrested at the primary stage [16]. Stem cell factor can directly stimulate mature bovine TC growth and androstenedione production [17].
Because mutations of SCF and c-kit can interfere with follicle development and androgen production and SCF can stimulate thecal androgen production, it is reasonable to propose that SCF may combine with IGF-I to stimulate TC differentiation. The purpose of these experiments was to determine if SCF and/or IGF-I are important bioactive components of follicle-conditioned medium, and if the combination of SCF plus IGF-I can mimic the effects of follicle-conditioned medium on TC.
MATERIALS AND METHODS
Reagents
Polymerase chain reaction (PCR) reagents and Taq DNA polymerase were obtained from Perkin Elmer Cetus, Norwalk, CT. Moloney murine leukemia virus reverse transcriptase (RT) was obtained from Gibco-BRL, Gaithersburg, MD. Oligo(dT) was purchased from Pharmacia, Piscataway, NJ. Recombinant human IGF-I and mouse SCF were obtained from R&D Systems, Minneapolis, MN. RNAsin was obtained from Promega, Madison, WI. A cDNA clone for murine steroidogenesis acute regulatory protein (StAR) was generously provided by Dr. Douglas Stocco, Texas Tech University, Lubbock, TX. Neutralizing antisera to human IGF-I (mouse monoclonal IgG1k) and murine SCF were obtained from R&D Systems and Upstate Biotech, Inc. (Lake Placid, NY), respectively.
Cell Culture
In the experiment to determine if SCF or IGF-I are components of follicle-conditioned medium, dispersed ovaries from 4-day-old rat pups were used. Female Sprague-Dawley rat pups were housed with a lactating female until they were killed on the morning of postnatal Day 4 by decapitation as approved by the Institutional Animal Care and Use Committee. The ovaries were collected in ice-cold medium 199 containing 25 mM Hepes and 1 mg/ml BSA and dissected free of nonovarian tissue. The ovaries were dispersed with collagenase and deoxyribonuclease as previously described [18]. Whole ovarian dispersates (1 x 105 viable cells/well) were cultured (2 days) in 96-well plates in the presence and absence of follicle-conditioned medium (100 µl) with and without neutralizing antisera to SCF or IGF-I in a humidified 95% air, 5% CO2 atmosphere at 37°C. Luteinizing hormone (0.3 ng/ml) was used as a control. The medium was collected and frozen (-20°C) until assayed for androsterone by specific RIA as previously described [19]. Follicle-conditioned medium was prepared by incubating preantral follicles with three to five layers of GC in 96-well tissue culture plates containing 200 µl of McCoys 5a medium for 2 days as previously described [7].
For experiments to test the effects of SCF and IGF-I Female Sprague-Dawley rats were hypophysectomized by Harlan Sprague-Dawley (Harlan Industries, Indianapolis, IN) at 21 days of age. Five days later the rats were killed by cervical dislocation after rendering the animals unconscious by CO2 inhalation as approved by the Institutional Animal Care and Use Committee. The ovaries were collected in ice-cold medium 199 containing 1 mg/ml BSA. The rat ovaries were dispersed with collagenase and deoxyribonuclease as previously described [18], and the TC were isolated by discontinuous Percoll gradient centrifugation [20]. The isolated TC (4 x 104 viable cells per well) were cultured in 96-well tissue culture plates containing 200 µl of McCoys 5a medium (Gibco-BRL, Gaithersburg, MD) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 2 mM L-glutamine with and without SCF (0–100 ng/ml), IGF-I (0–100 ng/ml), and LH (0–100 ng/ml). Theca cells were cultured for 48 h in a humidified 95% air, 5% CO2 atmosphere at 37°C. The medium was collected and frozen (-20°C) until assayed for androsterone by specific RIA as previously described [19].
Measurement of mRNA
Extracts were prepared from cultured TC using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol. The RNA from the four replicate wells in each of four individual experiments was pooled and resuspended in 24 µl of RNase-free water and then frozen at -80°C. After reverse transcription in a total volume of 120 µl, aliquots of the cDNA (20 µl for CYP11A, CYP17, 3ß-HSD, LH receptor, StAR, and 4 µl for ß-actin) were measured in each sample (n = 4) by quantitative RT-PCR as previously described [8–11]. Primers for amplification of StAR mRNA were selected to amplify a region of complementarity between the mouse and rat mRNAs. The forward primer was 5'-CTT GGG CAT ACT CAA CAA CC-3' and the reverse primer was 5'-TAA CAC TGG GCC TCA GAG GC-3'. A mutant control template was synthesized from the StAR mRNA by site-directed mutagenesis [21] incorporating a C to A substitution at position 621 of the published sequence (Genebank AB001349). This mutation introduced a unique EcoRI site into the control template. After PCR amplification incorporating 32P-dCTP, the products were precipitated with ethanol and digested with the appropriate restriction enzyme to cut the control products and were then separated on a 2% agarose gel. The DNA in the gel was visualized using ethidium bromide staining. The bands were cut from the gel and counted in a scintillation counter. The cpm from the bands amplified from the cellular mRNA were divided by the cpm from the bands amplified from the mutated control template to control for PCR variation. The data were expressed as a ratio relative to ß-actin to control for procedural variation.
Statistical Analysis
Comparisons of the means were made using one-way analysis of variance with a posthoc Tukeys test. Statistical significance was considered to be P 
0.05.
RESULTS
In previous studies it was shown that conditioned medium from preantral follicles stimulates the differentiation of TC. To determine if SCF or IGF-I were biologically active components of the follicle-conditioned medium, ovarian dispersates from 4-day-old rats containing only theca precursor cells and no differentiated TC were cultured for 2 days in the presence of 50% follicle-conditioned medium from preantral follicles with three to five layers of GC with and without neutralizing antibodies to SCF or IGF-I. Consistent with previous results, cells cultured in medium alone (control) or in the presence of LH (0.3 ng/ml) did not produce detectable amounts of androsterone (Fig. 1). Follicle-conditioned medium stimulated a marked increase in androsterone production. Although neutralizing antibodies to either SCF or IGF-I had no effect on basal androsterone production, both antibodies inhibited the stimulatory effect of follicle-conditioned medium greater than 90%. These data indicate that both SCF and IGF-I are important bioactive components of follicle-conditioned medium.
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To determine the effects of SCF and IGF-I on thecal androgen biosynthesis, isolated TC were cultured with SCF (100 ng/ml) and IGF-I (100 ng/ml) alone and in combination for 2 days to stimulate maximal androgen production (Fig. 2). Neither IGF-I nor SCF alone stimulated TC androgen production above basal levels. In combination IGF-I plus SCF stimulated an approximately 10-fold increase in androsterone production. As shown in Figure 3, the stimulatory effect of SCF on thecal androgen production in the presence of 100 ng/ml of IGF-I was observed at concentrations of 30 ng/ml or higher. Similarly, androgen production was increased by concentrations of IGF-I at or above 30 ng/ml in the presence of 100 ng/ml of SCF (Fig. 4).
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The induction of gene expression for LH receptors, StAR, and steroidogenic enzymes is characteristic of TC differentiation. To determine the effects of SCF plus IGF-I on thecal differentiation, TC were cultured for 2 days in the presence and absence of SCF and IGF-I and the mRNAs for LH receptor, StAR, CYP11A, 3ß-HSD, and CYP17 were measured by quantitative RT-PCR assays. As shown in Figure 5, SCF alone had no effect on LH receptor, StAR, CYP11A, or 3ß-HSD mRNA expression, but there was a significant suppression of CYP17 mRNA expression by SCF (Fig. 5). Insulin-like growth factor-I alone increased LH receptor, CYP11A, and 3ß-HSD mRNAs as expected but did not alter StAR or CYP17 mRNA expression (Fig. 5). The combination of SCF plus IGF-I increased the expression of mRNAs for each of the five genes, LH receptor, StAR, CYP11A, 3ß-HSD, and CYP17. For the LH receptor, CYP11A, and 3ß-HSD mRNAs, addition of SCF did not increase mRNA expression above the levels achieved with IGF-I alone. Thus, the combination of SCF plus IGF-I stimulated the expression of each of the five key mRNAs necessary for TC to become capable of androgen biosynthesis.
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Having demonstrated that IGF-I plus SCF can stimulate TC differentiation and androgen biosynthesis independent of LH, we next tested the effects of IGF-I and SCF on LH-stimulated androgen production. As shown in Figure 6, LH stimulated a concentration-dependent increase in androsterone production. As expected, addition of IGF-I (100 ng/ml) increased LH-stimulated androsterone production approximately threefold at each concentration of LH tested. Stem cell factor (100 ng/ml) did not alter androsterone production stimulated by LH alone or in combination with IGF-I.
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DISCUSSION
Thecal differentiation occurs when the theca precursor cells express LH receptors and the enzymes necessary for androgen biosynthesis. The acquisition of steroidogenic capacity coincides with the morphological differentiation of the theca interna when developing follicles acquire two layers of GC [3]. This corresponds to the time when the preantral follicle secretes small molecular weight peptides that have the capacity to stimulate LH-independent androgen production by TC [7]. The identity of the physiological peptides remains to be proven, but our results indicate that IGF-I and SCF are both important bioactive components of follicle-conditioned medium and that the combination of IGF-I and SCF can mimic the androgen-stimulating activity of follicle-conditioned medium.
Insulin-like growth factor-I has many but not all of the properties of the physiological theca-differentiating signal. Insulin-like growth factor-I is secreted by GC [22] and appears to be limited to the GC of healthy developing follicles [23]. Rat and human TC express IGF-I receptors [24, 25], and IGF-I stimulates androgen production but only in the presence of LH [26]. Previous studies have shown that IGF-I stimulates the expression of LH receptor, CYP11A, and 3ß-HSD mRNAs without a requirement for LH [9–11]. However, IGF-I cannot be the sole theca-differentiating factor because it is incapable of stimulating the expression of CYP17 [8] and StAR mRNAs alone. In support of this conclusion, follicle development is impaired in IGF-I null mice, but differentiation of the theca interna remains intact in those follicles that develop beyond the primary stage [27].
The circulating concentration of IGF-I is approximately 100 ng/ml, but because the majority of IGF-I is complexed to IGF binding proteins [28], the concentration of free IGF-I in the interstitial compartment of the ovary is likely to be much less. It is unknown what the relative importance of circulating IGF-I might be compared to follicle-derived IGF-I with respect to regulation of thecal differentiation. Because theca precursor cells are likely to be exposed continuously to biologically active concentrations of IGF-I regardless of the stage of follicle development, it is probable that IGF-I is not a primary signal for thecal differentiation but amplifies and complements the critical initiating signal.
Stem cell factor is another molecule of the appropriate molecular weight that appears to play a role in developing preantral follicles [29]. Stem cell factor is produced in increasing amounts as follicles develop [30] and the TC contain c-kit, the SCF receptor [31]. Stem cell factor has been shown to promote proliferation of bovine TC and can stimulate thecal androgen production [17]. It is difficult to elucidate the role of SCF in developing follicles in vivo because inactivating mutations of SCF cause the ovaries to form with few or no follicles [32]. It appears that the majority of oocytes undergo apoptosis [33] and few follicles develop [12]. Those follicles that do remain exhibit impaired development [16]. In organ culture SCF promotes rat follicle development and an anti-c-kit antibody (ACK-2) blocks follicle development reminiscent of the effects of inactivating SCF mutations in mice [29]. Thus, it appears that SCF may play an important role in promoting thecal differentiation in small preantral follicles.
Our data support the conclusion that IGF-I and SCF may be important components of the physiological theca-differentiating signal. Both IGF-I and SCF are secreted by healthy developing preantral follicles [22, 30]. This is consistent with our observations that theca-differentiating activity is secreted in a developmental window beginning when follicles acquire two layers of GC and ending when the follicles produce an antrum [3]. Neither molecule alone can mimic the effects of follicle-conditioned medium on TC, but together physiological concentrations of IGF-I plus SCF can stimulate thecal differentiation and androgen production. To our knowledge this is the first combination of nongonadotropin proteins that can stimulate thecal differentiation. Further studies are in progress to determine the role of IGF-I and SCF in stimulating the differentiation of theca precursor cells.
During the developmental window when the thecal differentiating activity is secreted, theca precursor cells develop into morphologically distinct TC and begin to express mRNA for LH receptors. At this time the TC acquire the capacity to respond to LH [3]. The combination of IGF-I plus SCF stimulates the expression of LH receptor mRNA. Interestingly, our data demonstrate that in LH-responsive TC, IGF-I continues to augment TC differentiation and androgen production, but SCF does not modify the LH or IGF-I effects. These data indicate that overexpression of SCF per se is not a plausible mechanism to explain hyperandrogenism in women.
The physiological role of SCF remains to be proven but our data support the hypothesis that SCF plays a limited role in preantral follicles by facilitating initial thecal differentiation. When preantral follicles begin development, the GC begin to secrete IGF-I and SCF. Although IGF-I has been shown to exert a positive influence on TC throughout follicle development, SCF appears to function during a limited time early in follicle development by providing a critical stimulus for theca precursor cells to become LH responsive. Once the TC acquire LH responsiveness, LH becomes the principal regulator of TC function. This concept is supported by the observations that SCF treatment increases the number of TC in bovine follicles [34], that c-kit mRNA expression is decreased by gonadotropins in mouse ovaries [31], and that thecal differentiating activity is not secreted by the GC of antral follicles [7]. Further studies will be required to test this hypothesis.
FOOTNOTES
1 This research was supported by NICHD research grant HD50759. 
2 Correspondence: Denis A. Magoffin, Department of Obstetrics & Gynecology, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Davis 2066, Los Angeles, CA 90048. FAX: 310 423 0302;magoffin{at}cshs.org
3 Current address: Christopher T.F. Huang, Department of Obstetrics and Gynecology, Loma Linda University, Faculty Medical Offices, 11370 Anderson Street, Suite 3950, Post Office Box 1009, Loma Linda, CA 92354.
Accepted: September 11, 2000.
Received: May 16, 2000.
REFERENCES
-hydroxylase messenger ribonucleic acid expression by luteinizing hormone and insulin-like growth factor-I. Endocrinology 1993; 132:1945–1951.[Abstract]-androstane-3,17ß-diol and 5-androstane-3ß,17ß-diol. Steroids 1977; 30:679–689.[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  L. J Spicer, P. Y Aad, D. T Allen, S. Mazerbourg, A. H Payne, and A. J Hsueh Growth Differentiation Factor 9 (GDF9) Stimulates Proliferation and Inhibits Steroidogenesis by Bovine Theca Cells: Influence of Follicle Size on Responses to GDF9 Biol Reprod, February 1, 2008; 78(2): 243 - 253. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Elis, J Dupont, I Couty, L Persani, M Govoroun, E Blesbois, F Batellier, and P Monget Expression and biological effects of bone morphogenetic protein-15 in the hen ovary J. Endocrinol., September 1, 2007; 194(3): 485 - 497. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Honda, M. Hirose, K. Hara, S. Matoba, K. Inoue, H. Miki, H. Hiura, M. Kanatsu-Shinohara, Y. Kanai, T. Kono, et al. Isolation, characterization, and in vitro and in vivo differentiation of putative thecal stem cells PNAS, July 24, 2007; 104(30): 12389 - 12394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Orisaka, K. Tajima, T. Mizutani, K. Miyamoto, B. K. Tsang, S. Fukuda, Y. Yoshida, and F. Kotsuji Granulosa Cells Promote Differentiation of Cortical Stromal Cells into Theca Cells in the Bovine Ovary Biol Reprod, November 1, 2006; 75(5): 734 - 740. [Abstract] [Full Text] [PDF]
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|
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 |
|
 I. Adriaens, R. Cortvrindt, and J. Smitz Differential FSH exposure in preantral follicle culture has marked effects on folliculogenesis and oocyte developmental competence Hum. Reprod., February 1, 2004; 19(2): 398 - 408. [Abstract] [Full Text] [PDF]
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