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Regular Article |
-Hydroxylase Messenger Ribonucleic Acid and Protein in the Neonatal Hamster Ovary1
a Department of Obstetrics and Gynecology, Leland J. and Dorothy H. Olson Center for Women's Health, and
b Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
ABSTRACT
The temporal and spatial expression of cytochrome P450 side-chain cleavage (CYP11A1) and cytochrome P450 17
-hydroxylase (CYP17) mRNA and protein during thecal cell differentiation in developing hamster ovaries were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence histochemistry, respectively. Ovaries were collected from 15-day fetal through 20-day-old postnatal hamsters and used either for immunofluorescence detection of enzyme protein or RT-PCR evaluation of enzyme mRNA. Immunoreactivity of CYP11A1 first appeared in the interstitial cells on Day 10 postnatal (PN), and the intensity increased significantly with further ovarian development beyond 11 days of age. In contrast, CYP17 immunostaining was first detected in a few interstitial cells closer to large preantral follicles by Day 12 PN, and their number increased appreciably by Day 14 PN. By age 18–20 days, CYP17-positive cells were localized primarily in the thecal layer of large preantral follicles. A low level of CYP17 and CYP11A1 mRNA was present in fetal ovaries. The CYP17 mRNA levels increased sharply by Day 1 PN but decreased to a low baseline level by Day 2 PN and remained low up to Day 9 PN. Both CYP11A1 and CYP17 mRNA levels increased significantly by Day 10 PN compared to Day 9 PN; however, the increase for CYP11A1 was greater than CYP17. The CYP11A1 mRNA levels decreased noticeably on Day 11 PN and remained relatively stable until Day 14 PN; however, mRNA levels started increasing by Day 15 PN and increased sharply by Day 17 PN onward, corresponding to the increase in CYP11A1 protein in the ovarian interstitium and thecal compartments. On the other hand, CYP17 mRNA expression increased progressively through Day 12 PN. A sharp increase in CYP17 mRNA was noted on Day 13 PN in conjunction with the morphological development of thecal cells; mRNA levels remained steady afterward. The correlation of the increase in enzyme mRNA and protein, especially of CYP17, with the morphological development of thecal layers suggests that the differentiation of interstitial cells into theca may be modulated by multilayered preantral follicles, and the expression of enzyme protein occurs prior to an increase in serum LH.
developmental biology, follicle, FSH, ovary, theca cells
INTRODUCTION
Maturation of ovarian follicles coincides with their ability to synthesize estradiol from androgen precursor [1], that originates from thecal cells surrounding multilayered preantral and antral follicles [2]. Despite a wealth of information about follicular dynamics in the adult ovary, information about the spatiotemporal development of steroidogenic activity in ovarian interstitial cells during neonatal development is meager. The presence of cytochrome P450 side-chain cleavage (CYP11A1) and cytochrome P450 17-hydroxylase (CYP17) mRNA, and CYP17 activity in 10-day-old mice has been demonstrated [3]. Although it is axiomatic that LH controls thecal androgen biosynthesis, its role on the induction of ovarian steroidogenic activity during neonatal development remains unclear.
The newborn hamster ovary contains mitotic germ cells interspersed within cords of somatic cells [4, 5]. By Day 13 of postnatal (PN) age, secondary follicles with five layers of granulosa cells begin to appear [4]. The concentration of FSH in the serum begins to increase between Days 10 to 12, and peaks around Days 22–24, while the secretion of LH begins to increase on Day 15 and peaks from Days 19–21 [5, 6]. Large antral follicles do not develop until around Days 25–26 PN, and ovulation occurs at about Day 30 [7]. The first morphologically distinct thecal layer appears on Day 14 PN when secondary follicles with six layers of granulosa cells develop [5, 8]. These lines of evidence suggest that the onset of thecal cell differentiation in the hamster ovary may occur prior to 14 days PN and prior to the increase in serum LH. The production of steroids, particularly androstenedione, denotes thecal cell differentiation. The expression of CYP17 enzyme in the adult hamster ovary occurs primarily in thecal cells, suggesting that the onset of CYP17 expression may herald the initiation of thecal cell differentiation. CYP11A1 is a mitochondrial enzyme that converts cholesterol to pregnenolone in all steroidogenic cells, while CYP17 catalyzes the transformation of C21 steroids to C19 steroids [8, 9].
The objectives of the present studies were to determine the temporal expression of CYP11A1 and CYP17 mRNA and spatiotemporal expression of enzyme protein in the developing hamster ovary from 24 h before birth to Day 20 PN development with special reference to thecal cell differentiation.
MATERIALS AND METHODS
Animals
Golden Syrian hamsters were obtained from Sasco (Kingston, NY) and housed in a climate-controlled facility in accordance with the National Institutes of Health and Institutional Animal Care and Use Committee (IACUC) guidelines for the care and handling of experimental animals. The use of animals for the experiment was approved by the IACUC. Females were mated on the afternoon of proestrus, and the presence of sperm in the vagina the next morning was considered Day 1 of pregnancy. Ovaries were removed from 15-day-old fetal and 1- through 20-day-old neonatal hamsters. Fetal ovaries were removed after anesthetizing the mother with nembutal (pentobarbital sodium, 8 mg/100 g body weight), while neonatal ovaries were removed following anesthetizing the pups. One ovary from each pup was snap frozen in liquid nitrogen and sectioned at 6 µm in a Leica cryostat microtome (North Central Instruments, Minneapolis, MN) for immunofluorescence localization of CYP11A1 and CYP17 protein. The other ovary was used for reverse transcription-polymerase chain reaction (RT-PCR) evaluation of enzyme mRNA.
Fluorescent Immunocytochemistry of CYP11A1 and CYP17
Frozen sections were fixed in fresh 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature and blocked with 10% goat serum for 1 h at room temperature. Next, the sections were incubated overnight with polyclonal anti-CYP11A1 (1:1200) or anti-CYP17 (1:1000) antiserum (kindly provided by Drs. M.J. Soares and D.C. Johnson, University of Kansas Medical Center, Kansas City, KS) at 4°C in a humidified chamber. The sections were washed twice in PBS for 5 min each and then exposed for 30 min to a mixture of Hoechst dye (bisbenzamide, 2 µg/ml) and goat anti-rabbit IgG-Alexa 488 (Molecular Probes, Eugene, OR) and IgG-Alexa 596 for CYP11A1 and CYP17, respectively. The sections were then washed in PBS two times, 5 min each, and mounted with Prolong Antifade (Molecular Probes). The immunofluorescence signal was detected with a Leica DMR epifluorescence microscope equipped with an Optronics video camera and Image Pro Plus image analysis software (Media Cybernetics, Baltimore, MD).
Semiquantitative Analysis of CYP11A1 and CYP17 mRNA Expression in the Developing Hamster Ovary
Ovarian RNA from three or more hamsters on each day of development was isolated using TRI reagent (Molecular Research Inc., Cincinnati, OH). Extreme care was taken to avoid any DNA contamination that was further verified by running PCR without reverse transcription. The RT-PCR procedure was similar to that reported previously [10, 11] with appropriate modifications. First, to clone hamster CYP11A1 and CYP17 cDNA partially, ovarian poly (A+) RNA was reverse transcribed using gene-specific reverse primer. The cDNA was amplified for 30 cycles after 4 min of heating at 94°C. The reverse and forward primers for CYP11A1 were 5'-TACAACACTGGT-GATGGACT and 5'-CTCGATCCTTCAATGAGATC, respectively, and the reverse and forward primers for CYP17 were 5'-CAACCACGGGAATATGTCCACCAG and 5'-ACTCT-AGGCCTCTTGTCGGACCAA, respectively. The primers for CYP11A1 and CYP17 were designed from rat [12] and mouse [13, 14] cDNA, respectively. The PCR conditions for CYP17 were: 1 min at 94°C, 2 min at 55°C, and 2 min at 72°C. The PCR amplification was repeated for 30 cycles. For PCR of CYP11A1 cDNA, the annealing temperature was 56°C, and the conditions were repeated for 35 cycles. The authenticity of the PCR products was verified by restriction digestion and sequencing (Applied Biosystem automated sequencer, Eppley DNA sequencing core laboratory, University of Nebraska Medical Center) following directional insertion into a PGEM-T easy vector (Promega, Madison, WI). The sequences were compared with available sequences in the GeneBank to identify similarities and differences between hamster CYP11A1 and CYP17 cDNA and other published sequences. Subsequently, total ovarian RNA from developing hamsters was subjected to semiquantitative RT-PCR evaluation of CYP11A1 and CYP17 using a protocol [11] to minimize error in RNA sampling and to ensure reproducibility. In brief, mRNA was reverse transcribed in the presence of random hexamers (Amersham-Pharmacia, Piscataway, NJ). Aliquots of each RT product mixture were amplified for CYP11A1, CYP17, and S4 gene transcripts using a gene-specific primer pair. Therefore, the relative levels of three gene transcripts in each RT tube were evaluated simultaneously. For CYP11A1, a nested primer pair designed from hamster CYP11A1 cDNA was used. The forward and reverse primers for hamster CYP11A1 were 5'-CTTCCCCCGTGACAATGGTTGGCTAAAC-3' and 5'-GGAACAGGTCATCGCTGATGTCCCCTG-3', respectively. To determine the specificity of steroidogenic enzyme gene expression, CYP11A1 and CYP17 mRNA expression were normalized against S4 mRNA that encodes a hamster ribosomal protein. The S4 mRNA was amplified using a reverse 5'-CCCAAGTTAGCACCTCC-3' and forward 5'-GGCGATGAAGTCAAGAAG primer pair derived from the hamster S4 cDNA sequence [15]. The S4 cDNA was amplified for 30 cycles after a 4-min incubation at 94°C. For PCR, 1.5 µl of the RT reaction product was amplified in a final volume of 25 µl. The PCR incubation conditions for S4 were as follows: 1 min at 94°C, 2 min at 56°C, and 2 min at 72°C.
The PCR products were resolved in a 1% agarose gel, transferred to a Zeta Probe nylon membrane (Bio-Rad, Hercules, CA), and hybridized with respective 32P-labeled antisense oligonucleotide probe that annealed to the sequence in the midstream of a hamster-specific S4, CYP11A1, or CYP17 cDNA sequence. Membranes for a specific gene transcript were hybridized simultaneously to avoid interassay variation. The hybridization signal was quantified using a Cyclone phosphorimager (Packard Instruments; Meridan, CT), and the results were expressed as CYP11A1 or CYP17 digital light units (DLU) relative to S4 DLU.
Statistical Analysis
All quantitative data were analyzed by one-way ANOVA, and significant (P < 0.05) differences between days were determined by Scheffe's test.
RESULTS
Immunolocalization of CYP11A1 and CYP17 in the Developing Ovary
The expression of CYP11A1 protein was first noted in the ovary on Day 10 PN (Fig. 1A). The CYP11A1-immunopositive interstitial cells appeared on the periphery of developing follicles; however, their location was random. Moreover, CYP11A1 immunoreactivity was localized in punctate vesicle-like structures reminiscent of mitochondria. The expression of CYP11A1 protein decreased somewhat by Day 11 (Fig. 1B) but increased again by Days 12–13 PN (Figs. 1C and 2A). By Days 14–17 PN, CYP11A1 immunoreactivity was localized primarily in the interstitium surrounding secondary follicles (Fig. 2, B and C). Subsequently, the majority of the interstitial cells became CYP11A1 immunoreactive by Days 18–20 (Fig. 3, A and B). No CYP11A1 immunoreactivity was present in the granulosa cells of any follicle, thus validating the specificity of the immunolocalization. In contrast to that of CYP11A1, CYP17 immunoreactivity first appeared in a few randomly scattered cells on Day 12 PN (Fig. 1F). However, cells expressing strong CYP17 immunoreactivity did not start appearing at the periphery of developing follicles until Day 14 PN (Fig. 2D). The intensity of CYP17 immunoreactivity increased steadily from Days 14 through 17 PN (Fig. 2, D and E). By Days 18–20 PN development, CYP17 immunoreactivity was restricted to cells that were in distinct thecal layers surrounding preantral follicles (Fig. 3, C and D).
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Amplification of Hamster CYP11A1 and CYP17 by RT-PCR
Polymerase chain reaction amplification resulted in a predicted 509-base pair (bp) CYP11A1, 360-bp CYP17, and 398-bp S4 cDNA. The hamster CYP11A1 fragment was 88% and 80% identical to the corresponding rat and human enzyme cDNA, respectively. On the other hand, the hamster CYP17 cDNA fragment was 79%, 86%, and 81% identical to the rat, mouse, and human enzyme cDNA, respectively. The S4 cDNA sequence was identical to that of the hamster because the primers were selected from a hamster S4 cDNA sequence.
Expression of CYP11A1 and CYP17 Messenger RNA
The steady-state levels of CYP11A1 mRNA (Fig. 4) were low in the hamster ovary from Day 15 prenatal through Day 9 PN. The CYP11A1 mRNA level increased on the day of birth but declined significantly by Day 2 PN. The mRNA levels showed unique 3- to 4-day fluctuations with a sharp increase followed by a steady decline to the basal level until Day 9 PN. CYP11A1 mRNA levels increased significantly by Day 10 PN followed by a 50% decrease by Day 11 PN and remained relatively steady until Day 16 PN (Fig. 4). A further increase occurred by Day 17 through 20 PN, corresponding to the increase in enzyme protein (Fig. 3). The expression pattern of CYP11A1 mRNA was consistent with expression patterns observed for CYP11A1 protein. The CYP17 mRNA maintained a low basal profile similar to CYP11A1 until Day 9 PN (Fig. 5); however, an increase occurred on Day 1 PN. In contrast to CYP11A1 mRNA, CYP17 mRNA level did not present any fluctuation (Fig. 5). The CYP17 mRNA levels began to increase slowly by Days 10 through 12 PN, and then increased 2.5-fold by Day 13 PN and remained steady through Day 20 PN (Fig. 5). The steady increase in CYP17 mRNA from Day 12 PN coincided with CYP17 protein expression.
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DISCUSSION
In these studies, we show for the first time the spatiotemporal expression patterns of CYP11A1 and CYP17, two key steroidogenic enzymes that signify the differentiation of thecal cells [16] in the developing hamster ovary. Thecal cell differentiation is critical to successful follicular development [1]. The expression of CYP11A1 and CYP17 mRNA without any detectable enzyme protein suggests that the activation of post-transcriptional mechanisms in presumptive theca cells may require additional factors that appear with granulosa cell maturation and follicle formation, and the activation of enzyme protein synthesis may initiate the differentiation process. Moreover, the delay in CYP17 protein expression compared to that of CYP11A1 clearly indicates that mechanisms controlling CYP17 enzyme synthesis are not activated until multilayered secondary follicles develop in the hamster ovary. The unique wave of CYP11A1 mRNA levels in the early postnatal period may indicate the maturation of the regulatory system for CYP11A1 gene expression or mRNA stability before the onset of primary stimulus, i.e., LH. The appearance of CYP11A1 protein in the hamster ovary before any detectable increase in serum LH highlights the fact that mRNA, when it reaches a certain level, can be translated to enzyme protein. The significance of the increase in CYP17 mRNA levels in hamsters on Day 1 PN is unclear at this time; however, it is clear from the results that an increase in enzyme mRNA without a consequent protein synthesis does not necessarily reflect the onset of theca cell differentiation. The expression of CYP17 mRNA and protein have been shown to occur in the rat [17, 18] and sheep [19, 20] placenta during labor. Gray and coworkers [3] have detected CYP17 mRNA expression in newborn mice; however, the increase in mRNA levels occur from Day 10 and onward. On the contrary, the activities of CYP11A1 and aromatase maximize by Days 7–10 of age [21, 22]. It needs to be appreciated that due to a shorter gestation period in the hamster (16 days) compared to that of the mouse (19 days), the 7-day-old mouse ovary will be comparable to a 10- to 11-day-old hamster ovary, and the newborn mouse ovary will be comparable to a 3- to 4-day-old hamster ovary. Besides, follicular development in the mouse occurs at a faster pace than that in the hamster. Despite the species differences, the onset of appreciable activities of CYP11A1 and CYP17 in the mouse ovary correlates very well with the spatiotemporal expression of steroidogenic enzymes in the hamster ovary. Keeney et al. [23] have documented that CYP17 gene transcripts are transiently expressed in the fetal mouse adrenal between embryonic Days 12.5 through 14.5 and are lost by embryonic Days 16.5 through 18.5, and this pattern of gene expression occurs long before pituitary ACTH secretion is activated. Despite the onset of CYP17 mRNA expression at embryonic Day 12.5, 17-OH-progesterone production (a small amount) does not occur until embryonic Day 14.5, when the mRNA expression reaches its peak, indicating that there is a 24–48-h lag before enzyme protein can be detected. These results corroborate the present findings on CYP17 mRNA and protein expression in perinatal hamsters, in which CYP17 mRNA levels begin to increase by Day 10 PN but protein first appears on Day 12 PN in conjunction with a sustained increase in the mRNA level. The differential expression pattern of CYP11A1 and CYP17 protein in the hamster ovary during the early postnatal period leads to speculation that translation of these proteins occurs when a critical amount of mRNA is accumulated in the cell, and all factors regulating translation are present.
The unique spatial expression of CYP17 protein in cells adjacent to multilayered follicles by Day 12 PN and their gradual organization in the thecal layer by Day 19 PN suggests that differentiating thecal cells may migrate toward follicles to form morphologically distinct thecal layers. The results of the present studies, however, cannot distinguish whether the sharp increase in CYP17-positive cells in the surrounding thecal layer is due to multiplication of a few initial CYP17-positive cells or more cells expressing CYP17 as the ovary develops. The arrangement of CYP17-positive (differentiated) thecal cells around the follicle may result from one of two possible mechanisms: 1) interstitial cells near multilayered secondary follicles may receive a paracrine signal either from granulosa cells, the oocyte, or both, which induces CYP17 protein synthesis in those cells that then migrate closer to follicles and eventually surround them forming the thecal layer; 2) the paracrine signal emanating from large multilayered secondary follicles from Days 12 through 18 postnatal is received at random by the interstitial cells in the vicinity of developing follicles. However, cells juxtaposed to granulosa cells receive maximum signal and differentiate into thecal cells with a corresponding increase in CYP17 protein synthesis. Eventually, the thecal differentiating signal remains localized to adjacent thecal cells, and cells elsewhere remain as undifferentiated interstitial cells. Magoffin and Magarelli [24] have demonstrated that conditioned medium of rat preantral follicle culture can induce androgen production in rat theca-interstitial cell preparation in vitro. The results of the present studies also suggest that the window of onset of thecal differentiation may lie between Days 12 through 18 PN in the hamster.
The first induction of CYP11A1 and CYP17 mRNA and protein in hamster ovaries occurs prior to the increase in serum LH levels on Day 15 PN [5, 6], suggesting that the onset of steroidogenesis in interstitial cells may be indirectly influenced by FSH rather than LH. Relatively high levels of FSH that begin to rise around age 9–10 days are present in newborn hamsters [5, 6]. Because granulosa cells are the exclusive sites of FSH action, FSH may induce follicular synthesis of a putative paracrine factor whereby regulating the onset of steroidogenesis in interstitial cells. That FSH can induce such a putative factor in preantral follicles has been demonstrated in the rat [25]. That growth factors influence CYP17 mRNA expression and enzyme activity in rat theca-interstitial cells (TIC) in vitro has been documented [16]. Moreover, insulin-like growth factor I potentiates the effect of LH on CYP17 mRNA expression in cultured rat TIC [26, 27], whereas in the same culture system, transforming growth factor-ß (TGF-ß) attenuates LH-stimulated androsterone production by directly inhibiting CYP17 enzyme activity, while inducing CYP17 mRNA [28]. Gelety and Magoffin [29] showed that cultured interstitial cells from 5-day-old rats respond to hCG; however, no LH binding exists in the rat ovary before 8–10 days of age [30–33]. In the hamster, consistent hCG binding in the interstitium and theca occurs only from 15 days of age and onward [34]. Moreover, interstitial cells from 10-day-old neonatal hamsters do not respond to LH in vitro (unpublished observations). These lines of evidence support our contention that the differentiation of thecal cells may occur due to an FSH-induced paracrine mechanism that originates in granulosa cells, oocytes, or both.
In summary, the results of the present studies provide a spatiotemporal developmental expression pattern of CYP11A1 and CYP17 mRNA and protein in postnatal hamster ovaries. While the exact factor responsible for interstitial cell differentiation remains to be identified, a correlative induction of enzyme protein in the interstitial cells and multilayered secondary follicle development is evident.
FOOTNOTES
First decision: 26 April 2000.
1 This work was supported by a grant from NIHCHD (HD28165) and the Olson Foundation (Omaha, NE). 
2 Correspondence: Shyamal K. Roy, Departments of OB/GYN and Physiology and Biophysics, 984515 University of Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu
Accepted: July 5, 2000.
Received: March 15, 2000.
REFERENCES
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4- and 5-steroid-17,20-lyase reactions. Mol Endocrinol 1989; 3:968–975.[CrossRef][Medline]-hydroxylase/C17–20 lyase. Genomics 1991; 10:270–275.[CrossRef][Medline]-hydroxylase protein and mRNA in the testis of the testicular feminized (Tfm) mouse. J Mol Endocrinol 1993; 11:77–82.This article has been cited by other articles:
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  P. Yang, J. Wang, Y. Shen, and S. K. Roy Developmental Expression of Estrogen Receptor (ER) {alpha} and ER{beta} in the Hamster Ovary: Regulation by Follicle-Stimulating Hormone Endocrinology, December 1, 2004; 145(12): 5757 - 5766. [Abstract] [Full Text] [PDF]
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