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Reproductive Endocrinology |
Department of Reproductive Medicine, University of California-San Diego School of Medicine, La Jolla, California 92093-0633
Address all correspondence and requests for reprints to: Dr. S. S. C. Yen, Department of Reproductive Medicine, University of California-San Diego School of Medicine, La Jolla, California 92093-0633. E-mail: dnye{at}ucsd.edu
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Abstract |
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IrCRF and its mRNA were localized in thecal cells of small antral and
mature follicles. A low abundance of IrCRF and mRNA was also detected
in stromal cells of both stages of follicles. Expression of the gene
encoding CRF was more prominent in mature follicles than in small
antral follicles. CRF-R1 mRNA signal was found exclusively in thecal
cells of mature follicles and moderately in small antral follicles.
Granulosa cells were devoid of CRF and CRF-R1 mRNAs and proteins. The
IrCRF-BP, but not its transcript, was detected in thecal cells and
lumen of capillary vessels of the thecal/stromal compartment of mature
follicles. The absence of CRF-BP gene transcript in human ovarian
follicles was confirmed by reverse transcription-PCR, indicating that
the IrCRF-BP detected is not derived from the ovarian transcript and
suggesting that the presence of IrCRF-BP and luman of capillary vessels
in the thecal compartment originates from the peripheral circulation.
Thecal cells of mature follicles, relative to those of small antral
follicles, exhibited an intensive immunostaining and mRNA signal for
17
-hydroxylase (P450c17) indicative of androgen biosynthesis. We
conclude that the thecal compartment of the human ovary contains a CRF
system endowed with CRF and CRF-R1 and the blood-derived CRF-BP.
Granulosa cells are devoid of the CRF system. The parallel increases in
intensity of CRF, CRF-R1, and 17-hydroxylase proteins and gene
expression with follicular maturation suggest that the intraovarian CRF
system may play an autocrine role in androgen biosynthesis with a
downstream effect on estrogen production by the granulosa cells. The
functionality of the ovarian CRF system may be conditioned by the
relative presence of circulating CRF-BP by virtue of its ability to
compete with CRF for the CRF receptor.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixteen normal ovaries were obtained from 10 normally cycling women (32–44 yr of age) undergoing benign gynecological surgery during the first 9 days of the follicular phase of the menstrual cycle. The phases of the menstrual cycle were determined by the recorded last menstrual period. Thirty-seven small antral follicles (0.5–5 mm in diameter) and 8 mature antral follicles (>6 mm in diameter) were identified. Those follicles found to be in the stages of atresia assessed by morphological criteria as previously described (25) were excluded. Four small antral follicles (2–3.5 mm) and two mature follicles (7 and 8 mm) were selected for analysis. None of the patients had received any hormone therapy within 3 months before surgery. After removal, ovarian specimens were snap-frozen on dry ice and stored at -70 C for ribonucleic acid (RNA) extraction. The procedures for immunohistochemistry and in situ hybridization were described in detail previously (25). Ovarian tissues were snap-frozen in O.C.T. mounting medium (Miles, Elkhart, IN), cryosectioned at 8 µm using a freezing microtome (International Equipment Co., Needham Heights, MA), and mounted on Vectabond (Vector Laboratories, Burlingame, CA)-coated slides. Sections were then air-dried and stored at -70 C with desiccant until used. The study was approved by the committee of investigations involving human subjects at the University of California-San Diego.
Immunohistochemistry
Frozen sections were thawed and fixed for 20 min in 10% neutral formalin buffer and processed for immunohistochemical staining using the Vectastatin Elite ABC alkaline phosphatase system (Vector Laboratories). Sections were washed twice with phosphate-buffered saline (PBS), pH 7.4, and treated with 0.1% Triton X-100 in PBS for 5 min at room temperature. To block the endogenous alkaline phosphatase, sections were treated with 33% acetic acid in ethanol for 5 min at room temperature. Avidin/biotin-blocking reagents (Vector Laboratories) were used to block the endogenous binding sites of avidin and biotin in the tissues. After blocking nonspecific binding of the antibody by incubation for 30 min with 10% goat serum in PBS, sections were incubated for 1 h at room temperature with specific polyclonal antibodies against rat/human CRF, human CRF-BP, or human P450c17 proteins prepared in PBS at a final dilution of 1:1000. Using a monospecific antibody to CRF that exhibits no cross-reactivity with urocortin (Biotechnology, Santa Cruz, CA), the presence of IrCRF in thecal cells was identical to that detected by the polyclonal antibodies. After extensive washing with PBS, sections were successively incubated for 30 min with biotinylated goat antirabbit IgG at 7.5 mg/mL in PBS. Thereafter, sections were washed and incubated for 45 min with avidin/biotinylated complex (Vector Laboratories) in PBS. IrCRF, CRF-BP, and P450c17 were identified by Vector Red Substrate reagents (Vector Laboratories) containing Levamisol (Zymed, South San Francisco, CA). Sections were then counterstained with hematoxylin, dehydrated, cleared with xylene, and permanently mounted with DPX mountant (Gallard Schlesinger, Long Island, NY). Negative control experiments were performed by incubating sections with nonimmune rabbit IgG instead of the primary antibodies.
In situ hybridization
Frozen sections were thawed and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After three washes with PBS, sections were treated with 0.1% Triton X-100 for 10 min at room temperature. To block the positive charges in tissues, sections were acetylated for 10 min in 0.25% acetic anhydride in 0.1 mol/L triethanolamine, pH 8.0. Sections were then rinsed in 2 x standard sodium citrate (SSC), dehydrated in ascending ethanol concentration solutions, and dried under vacuum with desiccant. Hybridization was performed by incubating sections in humidified chamber for 18 h at 42 C in hybridization buffer containing 50% formamide, 0.3 mol/L NaCl, 10 mmol/L Tris (pH 8.0), 1 mmol/L ethylenediamine tetraacetate, 0.1% SDS, 1 x Denhardt’s solution, 200 mg ribosomal RNA, 200 mg/mL denatured salmon sperm DNA, 10 mmol/L dithiothreitol, 10% (wt/vol) dextran sulfate, and 1 x 106 cpm/mL [35S]UTP-labeled CRF, CRF-BP, and type 1 CRF receptor antisense complementary RNA (cRNA) probes. Negative control experiments were performed by hybridization of sections with [35S]UTP-labeled cRNA sense probes. Both antisense and sense cRNA probes were generated by linearization of plasmid constructs with the appropriate restriction enzymes and by transcription with SP6, T7, or T3 polymerases in the presence of cold ATP, CTP, GTP, and [35S]UTP (Amersham, Arlington Heights, IL) using a riboprobe in vitro transcription system (Promega, Madison, WI). The nonincorporated isotope was removed by chromatography on Sephadex G-50 columns. After hybridization, sections were washed four times for 5 min each time with 4 x SSC and treated for 30 min at 37 C with 50 mg/mL ribonuclease A in 10 mmol/L Tris (pH 8.0) containing 0.5 mol/L NaCl and 1 mmol/L ethylenediamine tetraacetate. Slides were then washed twice in 2 x SSC for 5 min each time at room temperature, once in 1 x SSC for 10 min at room temperature, once in 0.5 x SSC for 10 min at room temperature, and once in 0.1 x SSC for 30 min at 60 C. To stabilize the 35S attachment in the riboprobes and reduce nonspecific probe binding, 1 mmol/L dithiothreitol was included in the washing buffer. Slides were dehydrated, dried, dipped in autoradiography emulsion (NTB-2, Eastman Kodak, Rochester, NY), and exposed for 5–10 days at 4 C. After developing (Kodak D19) and fixation (Kodak Fixer), sections were counterstained with hematoxylin and permanently mounted. The in situ hybridization for P450c17 was performed using oligonucleotide probes as previously described (26).
Reverse transcription-PCR (RT-PCR)
Total RNA from a 5-mm ovarian follicle was extracted with RNA
Stat-60 reagents using a procedure provided by the manufacturer
(Tel-Test B, Friendswood, TX). Total RNA of human term placenta was
obtained from Clontech (Palo Alto, CA) and used as positive
experimental control. The total RNA was reverse transcribed by AMV
reverse transcriptase using antisense primers. Briefly, 10 mg total RNA
were incubated for 30 min at 42 C with 5 mmol/L MgCl2
solution; one-strength PCR buffer; 1 mmol/L each of deoxy (d)-GTP,
dATP, dTTP, and dCTP; and 1 U/mL murine leukemia virus reverse
transcriptase in a final volume of 20 mL. Half of the RT reaction
product was used as a template and amplified by PCR using 2 mmol/L
MgCl2 solution, one-strength PCR buffer, 0.15 mmol/L of the
sense and antisense primers, and 2.5 U AmpliTaq DNA polymerase in a
final reaction volume of 100 mL along with three drops of mineral oil
to prevent sample evaporation. Thirty-five cycles of PCR were performed
in a Perkin-Elmer DNA thermal cycler (Norwalk, CT) with the following
cycling parameters: denaturation for 1 min at 95 C, annealing for 1 min
at 60 C, and extension for 1 min at 72 C. The oligonucleotides primers
were designed and synthesized according to the published nucleotide
sequence of complementary DNA for rat/human CRF (27), human CRF-R1
(28), and CRF-BP (22). The primers used for amplification are
summarized in Table 1
. The amplified PCR products were
fractionated by 2% agarose gel and detected by ethidium bromide
staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The gene expression and protein localization of a
2-mm small antral follicle are compared with those of an 8-mm mature
follicle.
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IrCRF and its messenger RNA (mRNA) were both localized in thecal
cells of small antral (Fig. 1A
) and mature (Fig. 1B) follicles.
However, the relative abundance of CRF and mRNA was higher in the
mature follicle than in the small antral follicle. Low levels of the
protein and mRNA were also observed in stromal cells of the mature
follicle, but not in the small antral follicle. Granulosa cells of both
types of follicle exhibited negative immunostaining and no mRNA signal
for CRF.
CRF-R1
A moderate CRF-R1 mRNA signal was detected in both thecal and
stromal cells, but not in granulosa cells of small antral follicles
(Fig. 1A), whereas in the mature follicle abundant signal restricted to
thecal cells was found (Fig. 1B). Thus, in the mature follicle both
stromal cells and granulosa cells were devoid of CRF-R1 transcript.
Immunohistochemical analysis of CRF-R1 was not performed in this study
due to the unavailability of specific antibody against CRF-R1.
CRF-BP
In the small antral follicle, both IrCRF-BP and its mRNA were
absent (Fig. 1A). In contrast, abundant immunostaining, but not mRNA,
for IrCRF-BP was detected in the mature follicles of thecal and stromal
cells (Fig. 1B) and in the lumen of capillary vessels in the
thecal/stromal compartments (data not shown). The presence of IrCRF-BP
unaccompanied by mRNA signal in thecal-stromal cells of mature
follicles suggested that IrCRF-BP may not be derived from the ovarian
transcript and could be delivered to the thecal compartment from the
peripheral circulation (see below).
P450c17
As thecal cells have been shown to contain CRF and CRF-R1,
parallel studies of the gene expression and immunolocalization of
P450c17 were conducted. Thecal cells, but not stromal cells, of the
small antral follicle showed weak immunostaining and mRNA signal for
P450c17 (Fig. 1A). By comparison, the mature follicle exhibited strong
immunostaining and mRNA signal for P450c17 in the theca interna and in
a subset of cells in the stromal compartment (Fig. 1B).
PCR analysis
To further evaluate the absence of CRF-BP transcript by in
situ hybridization, RT-PCR was used to amplify the mRNA signal in
tissue obtained from the wall of mature follicles. Placental RNA was
used as a positive control for PCR amplification. As shown in Fig. 2, using specific primers for CRF, CRF-R1, CRF-BP, and
amplified products for CRF and CRF-R1 were evident in both follicular
and placental tissues, PCR amplification of RNA encoding CRF-BP was not
observed in the follicular tissues, whereas the expected placental PCR
amplification product was evident. When the RT-PCR proceeded in the
absence of reverse transcriptase, there were no amplification products
for CRF, CRF-R1, or CRF-BP in both tissues. Thus, CRF-BP localized in
the thecal layer of the follicles represents an extraovarian
source.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although an intense immunostaining for CRF-BP in the thecal-stromal compartment of mature follicles and in the lumen of capillary vessels was evident, mRNA encoding CRF-BP was not detected in the human ovary by either in situ hybridization or RT-PCR. Thus, the IrCRF-BP found in the thecal-stromal layer is not derived from the ovarian transcript, and in all probability, it originated from peripheral circulation. CRF-BP was initially isolated from human plasma (23), and its complementary DNA was subsequently cloned from a liver DNA library (22). The binding affinity of CRF-BP to CRF exhibited a Kd an order of magnitude lower than that displayed by the CRF receptors (24). CRF-BP was shown to inhibit CRF-induced ACTH secretion from the pituitary (22) and placental tissue (29) in vitro. Although CRF-BP is expressed in many areas of the rat brain, CRF-BP has not been found in the peripheral plasma of several species studied (24). Thus, CRF-BP in plasma appears to be unique to humans, and women have higher concentrations than men (139 ± 10.2 vs. 101 ± 9.3 ng/mL) (30). It is likely, therefore, that the circulating CRF-BP may play an endocrine role in the modulation of the ovarian CRF system by controlling the amount of free CRF to interact with CRF receptors in the thecal compartment. This quenching effect of circulating CRF-BP on the ovarian CRF system is consistent with the higher binding affinity of CRF-BP than the CRF receptor for CRF and the localization of CRF-BP at the same cellular site and surrounding capillaries.
The regulation and functional role of the human ovarian CRF system are unclear. Previous studies in rodents have demonstrated the presence of IrCRF and CRF mRNA in the Leydig cells of the rat testis (16, 17). Whereas CRF inhibits testosterone production by rat Leydig cells (17), it stimulates steroidogenesis in mouse Leydig cells in vitro (18). The reason for this disparity is not known. In the rat ovary, IrCRF has been localized in thecal-stromal cells, and it has been proposed that CRF may participate in the regulation of ovarian steroidogenesis (19). In the human ovary, IrCRF was also localized in thecal-stromal cells of the follicles and was detectable in follicular fluid (20). Thus, these earlier studies have uniformly localized CRF immunoreactivity in the androgen-producing cells. Recently, an inhibitory effect of CRF in FSH-stimulated estradiol production by human luteinized granulosa-luteal cells in vitro has been reported (21). This finding is inconsistent with the absence of CRF receptor on granulosa cells of maturing follicles in vivo. However, the possibility that acquisition of CRF receptor after luteinization of granulosa-luteal cells may occur cannot be excluded. As the site(s) of CRF gene expression was not determined, and the cellular localization of protein and transcript of CRF receptors were not conducted, these findings offer limited information in the context of function and regulation. Based on our present findings, it is highly probable that the regulation of ovarian CRF bioactivity differs between human and rodents by virtue of the presence of an extraovarian modulator, the CRF-BP, in women, but not in rodents (see review in 24 .
In conclusion, the present study has established the presence of a CRF
system replete with ligand and receptor in the thecal-stromal cell
compartment with enhanced expression of both proteins and transcripts
in parallel with the steroidogenic enzyme, 17-hydroxylase in the
maturing follicle. Moreover, the localization of IrCRF-BP (but not its
transcript) at the same cellular sites and surrounding capillaries
implicates an extraovarian modulator that determines the net effect of
intraovarian CRF bioactivities. Thus, the ovarian CRF system may be
viewed as an autocrine and paracrine regulator of steroidogenesis with
an endocrine component of modulation. As CRF is expressed as a
proinflammatory regulator in vivo (8), the possibility that
the ovarian CRF system may participate in the aseptic inflammatory
processes of ovulatory events should be considered (31). Additional
studies are required to further characterize its functionality.
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Acknowledgments |
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Footnotes |
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2 Former Fellow in Reproductive Endocrinology and Infertility.
3 Investigator with the Clayton Foundation.
Received March 10, 1997.
Revised April 21, 1997.
Accepted April 29, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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