| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Article |
and ß Expression in Theca and Granulosa Cells from Women with Polycystic Ovary Syndrome
Department of Obstetrics and Gynecology (S.R.W., H.-W.Y., D.A.M.), Cedars-Sinai Burns and Allen Research Institute, Cedars-Sinai Medical Center/David Geffen School of Medicine at UCLA, Los Angeles, California 90048; and Department of Obstetrics and Gynecology (A.J.J., M.B.), Second Clinic of Surgical Gynecology, University School of Medicine, 20-090 Lublin, Poland
Address all correspondence and requests for reprints to: Denis A. Magoffin, Ph.D., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Davis 2066, Los Angeles, California 90048. E-mail: magoffin{at}cshs.org.
Abstract
A defining characteristic of dominant follicles is high estradiol concentrations. Abnormal expression of estrogen receptors (ERs) could contribute to poor follicular development and ovulatory failure in polycystic ovary syndrome (PCOS). The aim of this study was to determine whether there are differences in ER
and ERß expression in granulosa cells (GC) and theca cells (TC) from women with PCOS, compared with regularly cycling women. GC and TC were obtained by microdissection from 12 polycystic and 23 normal ovaries. ER and ERß mRNA and protein expression were measured by semiquantitative RT-PCR and Western blot, respectively. In control ovaries, both GC and TC ER mRNAs were higher in small antral (SA) than in dominant follicles. ER mRNA was similar in PCOS and size-matched control follicles. In control follicles, ER protein concentrations were higher in GC than in TC. In GC, the ER concentrations were comparable among SA, dominant, and PCOS follicles. In TC, ER concentrations were lower in dominant follicles but were markedly increased in PCOS. In control ovaries, GC and TC expression of ERß mRNA was higher in SA, compared with dominant follicles. In PCOS, ERß mRNA was intermediate between SA and dominant follicles in both GC and TC. In GC, the ERß protein concentrations followed the same pattern as mRNA expression; but in TC ERß, protein in PCOS was equivalent to that in dominant follicles. The results of this study demonstrate that there are significant alterations in the expression of ER and ERß in PCOS that may be related to abnormal follicular development.
THE OVARY IS the principal source of estrogen biosynthesis in women; yet, the role of estrogen in regulating follicle development in the human ovary is largely unknown. During normal follicle development, a single follicle is selected for ovulation when the follicles reach 7–9 mm in diameter (1). When a dominant follicle is selected, the expression of aromatase mRNA increases in the granulosa cells (GC) (2), and the follicular microenvironment is characterized by an androstenedione to estradiol ratio of 4 or less (3, 4). Animal studies have demonstrated a variety of estrogenic effects on ovarian cells, including stimulating proliferation of GC from small follicles (5), enhancing gonadotropin receptor levels (6, 7), progesterone production (8), gap junction formation in GC (9), and inhibition of androgen production by theca cells (TC) (10, 11).
Despite the overwhelming evidence for a role of estrogen in regulation of follicle development in animal species, the evidence for an important role of estrogen in human follicle development is lacking. Although estrogen is important for regulating gonadotropin secretion and stimulating the midcycle LH surge, when human follicles are given exogenous gonadotropin stimulation, they are capable of developing and producing viable oocytes in the absence of estradiol production (12). In natural cycles, however, a defining characteristic of the dominant follicle is the high concentration of estradiol in the follicular microenvironment (3, 4), but the local effects of estrogen in dominant follicle development remain unclear.
Estradiol is known to cause many of its effects by binding to a specific, high-affinity receptor that is a ligand-activated transcription factor. Early studies demonstrated estrogen binding in the ovary (13) and, specifically, GC (14). Immunohistochemical studies localized estrogen receptors (ERs) in the GC of small antral (SA) and dominant follicles but not in TC (15, 16). After the LH surge, ER staining was shown to decline (15). Thus, it seemed that ERs were expressed exclusively in the GC in developing follicles.
Before 1996, all studies regarding ERs investigated the -form of the receptor (ER). In 1996 a second (ß) ER gene (ERß) was cloned (17, 18). Both ER and ERß are expressed in tissue-specific patterns during human development, with ER predominantly expressed in the uterus and less in the skin, colon, stomach, heart thymus, and ovary, and ERß predominantly expressed in the ovary, testis, spleen, brain, and skin and (to a lesser extent) in kidney, thymus, and adrenal (19). Recent studies demonstrated differential expression of ER and ERß in rat ovaries (20, 21). ER was localized exclusively to the TC, whereas ERß was exclusively localized in the GC of maturing follicles. In the human ovary, ERß was localized in the GC of follicles at all stages of development, whereas ER was absent from GC but present in TC (22). These data are consistent with the hypothesis that estradiol may play a role in regulating the growth and development of the dominant follicle in women.
In polycystic ovary syndrome (PCOS), dominant follicles fail to develop consistently. During the anovulatory cycles, there is a failure to up-regulate the expression of the aromatase enzyme in GC, and the estradiol concentration in the follicular microenvironment fails to increase adequately (2). These observations raise the question of whether abnormal ER expression in polycystic ovaries might contribute to the failure of dominant follicle selection in PCOS. The purpose of the present study was to measure the expression of ER and ERß mRNAs and proteins in GC and TC from women with polycystic ovaries and to determine whether there are significant differences, compared with developing antral follicles from regularly cycling women.
Subjects and Methods
Subjects
Ovarian tissue specimens were obtained from 12 women with PCOS, who were 44 yr of age or younger, undergoing electrocauterization of the ovarian surface or wedge resection for the treatment of their infertility. Women with PCOS were identified based on a history of oligo/amenorrhea, hirsutism, and typical morphological appearance of polycystic ovaries (normal or enlarged ovarian volume with multiple subcapsular cysts <8 mm in diameter) at laparotomy or laparoscopy with no evidence of hyperprolactinemia, Cushing’s syndrome, congenital or nonclassical adrenal hyperplasia, thyroid disease, or hormone-secreting tumors. Control tissues were obtained from 25 regularly cycling, age-matched premenopausal women in the follicular phase of their menstrual cycle, undergoing total abdominal hysterectomy and bilateral oophorectomy for uterine leiomyoma or cervical cancer. All subjects had not received hormonal treatment or ovarian suppression for at least 3 months before obtaining the specimens. Informed consent was obtained from all subjects participating in the study as approved by the Ethics Committee at the University School of Medicine in Lublin and the Institutional Review Board at Cedars-Sinai Medical Center.
GC and TC collection
The ovarian specimens were immediately placed into ice-cold Medium-199 (Life Technologies, Inc., Gaithersburg, MD) containing 25 mM HEPES and 1 mg/ml BSA. After washing off the blood, we placed the ovaries under a dissecting microscope, and the follicular fluid was completely aspirated from the visible follicles using a Hamilton syringe. The follicular fluid volume was measured, and the GC were collected by centrifugation for 5 min at 250 x g. The follicular fluid was frozen at -80 C until androstenedione and estradiol were measured by RIA (Diagnostic Products, Los Angeles, CA). The follicle diameter was calculated from the volume of aspirated fluid. The follicle was opened with microscissors, and the GC were gently scraped from the follicle wall with a platinum loop and collected by flushing with medium. The GC were centrifuged, and the pellet was pooled with the GC collected from the follicular fluid. The theca interna was microdissected from the follicle wall after the GC had been removed. The isolated GC and TC were frozen at -80 C until nucleic acids were extracted. Twenty-one control follicles (3–7 mm in diameter) and 23 dominant follicles (8–23 mm in diameter) from regularly cycling women and 37 follicles (3–7 mm in diameter) from women with PCOS were analyzed. Dominant follicles were defined as the largest follicle of at least 8 mm in diameter, in a subject’s pair of ovaries, during the follicular phase of the cycle, having an A4/E2 ratio of at least 4 (3, 4). No ovary of any subject with PCOS contained a dominant follicle.
RT-PCR analysis of CYP17 and CYP19 expression
To determine whether there was significant contamination of the TC preparation with GC and whether there was significant contamination of the GC preparation with TC, CYP17 and CYP19 mRNA expression was examined by RT-PCR, as previously described (2, 23) but omitting the mutant control template. Cloned cDNAs for CYP17 and CYP19 were used as positive controls.
DNA assay
Total cellular DNA was isolated from the GC and TC of individual follicles, using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. The DNA pellet was resuspended in 50 µl of 8 mM NaOH at 37 C for 10 min, then the pH was adjusted to 7.4 with 1 M HEPES. The DNA concentration of the samples was measured using a sensitive fluorescence assay (PicoGreen dsDNA Quantitation Kit; Molecular Probes, Inc., Eugene, OR). Briefly, 20 µl of sample were diluted with 2 ml PicoGreen solution, and the fluorescence was measured in a fluorometer (Turner Designs, Sunnyvale, CA). Sample concentrations were interpolated from a standard curve calculated by linear regression of the fluorescence of known concentrations of 
DNA standard.
Measurement of ER and ERß mRNA
ER and ERß mRNAs were measured by semiquantitative assays based on reverse transcription of the mRNA and PCR amplification of the cDNA. Total RNA was isolated with TriReagent according to the manufacturer’s instructions. GC RNA and TC RNA were resuspended in 10 µl and 20 µl diethylpyrocarbonate-treated water, respectively, and then frozen at -80 C. Aliquots of the RNA (10 µl) were transcribed into cDNA by incubating (37 C) for 30 min in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM deoxy-ATP, 1 mM deoxy-CTP (dCTP), 1 mM deoxy-GTP, 1 mM thymidine 5'-triphosphate, 5 µg oligo(dT)12–18 (Amersham Pharmacia Biotech, Piscataway, NJ), 40 U RNAsin (Promega Corp., Madison, WI), and 400 IU Maloney murine leukemia virus-reverse transcription (Life Technologies, Inc., Rockville, MD) in a total volume of 40 µl. The reaction was then heated to 95 C (5 min) and cooled to 4 C. Aliquots (4 µl) of the reverse transcription reaction were frozen (-80 C) until the PCR was performed. One picogram of mutant control DNA, 50 pmol of each PCR primer, 8 µl of 10x PCR buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl], 9.6 µl of 25 mM MgCl2, 10 µCi [32P]-dCTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA), and 2.5 U Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT) were added to an aliquot of cDNA, and the volume was adjusted to 100 µl. ER and ERß cDNAs were amplified individually for 25 cycles (94 C for 60 sec; 55 C for 60 sec; and 72 C for 60 sec). The amplification products were ethanol-precipitated and digested with BamH I to cut the control products, then separated on a 2% agarose gel. The DNA was visualized with ethidium bromide staining, and the bands were cut from the gel and counted in a scintillation counter. The counts per minute in the bands amplified from the cellular mRNA were normalized to the counts per minute in the bands amplified from the mutant DNA to control for procedural variations. The data were also normalized to total cellular DNA to control for variations in the number of GC and TC in each sample. All TC or GC samples were amplified separately in single experiments.
The oligonucleotide primers were purchased from Life Technologies, Inc. (Grand Island, NY). A specific 505-bp fragment of ER cDNA was amplified using primers corresponding to bases 803–822 and 1288–1307 of the published sequence (GenBank accession no. M1674). The primers for ERß cDNA corresponding to bases 628–647 and 1052–1071 of the published sequence (GenBank accession no. AF051427) amplified a 444-bp fragment. To control for PCR variations, a C was substituted for an A at base 1057 in the ER sequence and a C was substituted for a G at base 854 in the ERß sequence, respectively, by site-directed mutagenesis, (24) to introduce a unique BamH I site. The control template (1 pg) was included in each PCR amplification.
Western blot analysis
Protein pellets were solubilized in 1% sodium dodecyl sulfate, and the concentrations were determined by a sensitive fluorescent assay (NanoOrange; Molecular Probes, Inc.). Twenty to 30 µg protein from each sample were separated by 4–12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride membranes (Hybond-P; Amersham Pharmacia Biotech). Membranes were blocked overnight at 4 C with TBST [10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] containing 5% (wt/vol) nonfat dry milk. Blots were incubated with anti-ER (1:1,000 dilution) or anti-ERß (1:1,000 dilution) rabbit polyclonal antibody for 4 h at room temperature in blocking buffer. The anti-ER antibody (HC-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) is a rabbit polyclonal antiserum raised against a C-terminal epitope of human ER and displays no cross-reactivity with expressed recombinant human ERß. The anti-ERß antibody (Alexis Biochemicals, San Diego, CA) is a rabbit polyclonal antiserum raised against a synthetic peptide corresponding to the C-terminal amino acid residues 467–485 of human ERß, conjugated to keyhole limpet hemocyanin, and displays no cross-reactivity with expressed recombinant human ER. Membranes were washed three times with TBST and incubated with donkey antirabbit antibody conjugated to alkaline phosphatase (1:10,000 dilution; Chemicon International, Inc., Temecula, CA) for 1 h at room temperature in blocking buffer. Blots were washed three times at room temperature with TBST, and the immunoreactive proteins were detected using DDAO-phosphate [1:3,000 dilution in 10 mM Tris (pH 9.5) and 1 mM MgCl2] as substrate. Blots were scanned and quantified with a Typhoon fluorescence imaging system (Molecular Dynamics, Inc., Sunnyvale, CA).
Statistical analysis
Multiple comparisons were performed using one-way ANOVA with post hoc comparisons employing Tukey’s test. The unpaired t test was used to compare results between control and PCOS subjects. The paired t test was used to compare ER mRNA levels between TC and GC from the same follicles. Statistical significance was considered to be P 
0.05.
Results
As shown in Fig. 1
, the RT-PCR assays for ER and ERß mRNA expression yielded linear increases in amplified cDNA over approximately 2 orders of magnitude of standard concentrations. Both ER and ERß mRNAs were detectable in GC and TC from regularly cycling women and women with PCOS (Figs. 2 and 3). The overall concentrations and patterns of ER mRNA expression were similar in both GC and TC (Fig. 2). ER mRNA expression was higher (P < 0.001) in SA (3–7 mm) control follicles than in dominant (8–23 mm) follicles, but there was no difference between size-matched control and PCOS follicles.
|
|
|
). In both GC and TC, ERß mRNA expression was approximately 4-fold higher (P < 0.01) in SA control follicles, compared with dominant follicles. In GC from polycystic ovaries, ERß mRNA expression was significantly less (P < 0.015) than size-matched control follicles but was similar to dominant follicles (Fig. 3). In TC from polycystic ovaries, ERß mRNA expression was less (P < 0.016) than size-matched control follicles and greater (P < 0.02) than dominant follicles (Fig. 3).
Previous studies (20, 21, 22) have localized ER exclusively to TC and have demonstrated high ERß expression in GC. To determine whether our results could be explained by cross-contamination of the cell preparations, we examined CYP17 and CYP19 expression in the TC and GC by RT-PCR. As shown in Fig. 4, CYP17 was highly expressed in TC but was undetectable in GC. Conversely, CYP19 was highly expressed in the GC but not in TC. These data indicate that there was no significant cross-contamination of the TC preparation with GC and vice versa.
|
mRNA expression in dominant follicles, compared with SA follicles at least 7 mm in diameter, Western blot analysis revealed interesting differences in protein concentrations. In regularly cycling women, ER protein concentrations were approximately 4-fold higher (P < 0.05) in GC than in TC (Fig. 5). Although the GC ER mRNA expression was lower in dominant follicles, the ER protein concentrations were unchanged in each of the classes of follicles measured. In TC, the ER protein concentration declined (P < 0.001) in dominant follicles, similar to mRNA expression (Fig. 5). In contrast to the mRNA expression pattern, thecal ER protein concentrations were approximately 4-fold higher (P < 0.004) in PCOS, compared with size-matched controls.
|
). In dominant follicles, however, TC contained significantly higher (P < 0.001) concentrations of ERß protein than did GC. As with mRNA expression, the concentration of ERß in GC was markedly lower (P < 0.001) in dominant follicles, compared with SA follicles, in regularly cycling women (Fig. 6). In PCOS follicles, GC ERß protein concentrations were lower (P < 0.04) than in size-matched control follicles but higher (P < 0.001) than in dominant follicles. In TC, the ERß protein concentrations were slightly lower (P < 0.02) in dominant follicles than in SA control follicles, and the concentrations in PCOS follicles were similar to dominant follicles (Fig. 6).
|
There have been inconsistencies regarding the expression of ER and ERß in animal and human ovaries. In the rat, expression of ERß was localized exclusively to GC, whereas ER was found only in TC (20, 21). Similarly, in ovine, bovine, and rodent ovarian follicles, ERß was detected in GC but not in TC (25, 26, 27). These data indicated that the ER isoforms were compartmentalized in the ovarian follicle.
In the human ovary, however, a different pattern was apparent. Immunohistochemical studies performed before the discovery of ERß, using antibodies that presumably detected ER, revealed the presence of ERs only in GC of antral follicles (15, 28). Recently, ERß was localized in the GC of human follicles at all stages of development, whereas ER was absent from GC but present in TC (22). In the present study, the expression of ER and ERß at the mRNA and protein levels was detected in GC and TC from regularly cycling control ovaries as well as polycystic human ovaries. Our data indicate that ER is more highly expressed in GC than in TC, but ERß is expressed at similar levels in both cell types.
The differences between our data and the previous immunohistochemical data may be attributable to differences in the sensitivities of the techniques used. By testing for CYP17 and CYP19 expression in our cell preparations, we eliminated the possibility that TC and GC were cross-contaminating the cell preparations. Although immunohistochemistry is excellent for localizing proteins, it is not quantitative, and it is difficult to detect low concentrations of antigen or to prove that a protein is absent. In the present study, highly sensitive Western blotting and semiquantitative RT-PCR techniques were used to measure ER and ERß protein and mRNA expression directly in isolated human GC and TC. Both techniques yielded similar results, suggesting that the previous failure to detect ER in human GC and ERß in human TC was attributable to inadequate sensitivity of the immunohistochemical procedures used.
Interestingly, there was a differential pattern of ER and ERß concentrations between SA and dominant follicles. Dominant follicles expressed less ERß, both in GC and TC, in comparison with 3- to 7-mm follicles from control ovaries. There is evidence indicating that ERß can be down-regulated in response to the preovulatory surge of LH (15), and in vitro studies have shown that hCG can decrease the levels of ER and ERß mRNAs in human granulosa-luteal cells (29). The dominant follicles in this study were not collected at midcycle and are unlikely to have been influenced by the LH surge. Although our study does not address regulation of ER mRNA and protein expression, it is intriguing to note that several mRNAs, including those of CYP11A and CYP17, seem to be down-regulated in dominant follicles, relative to SA follicles (30). Although the mRNA expression is reduced, protein concentrations may remain high, as we observed for ER in GC and ERß in TC.
Studies on ER knockout animals revealed that the presence of both ERs is a prerequisite for the proper functioning of the hypothalamic-pituitary-ovarian axis and successful ovulation. Mice with a disrupted ER gene (ERKO mice) exhibit a phenotype similar to PCOS, with high circulating LH concentrations and ovaries characterized by the presence of multiple hemorrhagic and cystic follicles with no evidence of ovulation (31). Although ERß knockout (ßERKO) mice are fertile, the ovaries show morphological signs of abnormal follicular development and a reduced ovulatory capacity. There are more early atretic follicles and fewer corpora lutea than in wild-type mice (32). These changes are almost certainly not caused exclusively by the deficiency of ERs in the ovary but also may be secondary to the lack of pituitary ERs, resulting in disarrangement of feedback mechanisms. Nevertheless, it is reasonable to propose that in conditions characterized by impaired ovulation, such as PCOS, alterations in the expression of ovarian ERs could play a role.
The expression of ERß mRNA and protein was lower in both GC and TC from follicles derived from subjects with PCOS, in comparison with size-matched control follicles. With respect to ER, the only difference was a striking increase in protein expression in the TC of polycystic ovaries. These data indicate that there are alterations in the ratio of ER isoforms in both GC and TC in PCOS. The consequences of these changes remain to be determined; but because ER and ERß have different sensitivities to 17ß-estradiol (33, 34) and there is the possibility for cross-talk between receptors or heterodimer formation to occur (35), even small changes in the ER/ERß ratio may disturb normal follicle development.
In PCOS, TC overexpress the mRNAs for androgen biosynthesis (30) and secrete increased amounts of androgen, compared with TC, from control ovaries (36). Alterations in TC responsiveness to estrogens may play a role in ovarian hyperandrogenism, because estrogen is a potent inhibitor of thecal androgen production. Estrogens have been shown to inhibit ovarian androgen production (10, 11), by a direct mechanism in TC that seems to be mediated through ERs (37). In addition, estrogens are exquisitely potent inhibitors of TC TGF-ß secretion, by an ER-mediated mechanism (38). Because TGF-ß is a potent inhibitor of thecal androgen production (39, 40), a decrease in estrogen responsiveness by the TC could lead to increased androgen production. Thus, it is reasonable to propose that changes in ER/ERß ratios could decrease TC responses to estrogens that can inhibit excessive androgen production in developing follicles, by direct and indirect mechanisms. Further studies will be required to determine the role of the ER isoforms in regulating ovarian androgen biosynthesis.
Acknowledgments
Footnotes
This work was supported by NICHD Grant HD-33907 (to D.A.M.) and a Kosciuszko Foundation Fellowship and University School of Medicine Grant 122/01 (both to A.J.J.).
Abbreviations: dCTP, Deoxy-CTP; ER, estrogen receptor; GC, granulosa cell(s); PCOS, polycystic ovary syndrome; SA, small antral; TC, theca cell(s).
Received March 1, 2002.
Accepted August 20, 2002.
References
-hydroxylase deficiency despite extremely low intrafollicular 17ß-estradiol. J Clin Endocrinol Metab 68:693–697[Abstract]
(ER-) and ß (ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol Metab 82:3509–3512 in the rat ovary. Endocrinology 140:963–971, oestrogen receptor ß and androgen receptors in the rat reproductive organs. J Endocrinol 165:359[Abstract]
and -ß in the human reproductive organs. J Clin Endocrinol Metab 85:4835–4840 and ß gene expression in human granulosa-luteal cells in vitro. J Clin Endocrinol Metab 85:3828–3839 gene in female mice: characterization of ovarian responses and phenotype in the adult. Endocrinology 140:2733–2744. Mol Endocrinol 11:1486–1496-hydroxylase activity by a direct non-competitive mechanism. Endocrinology 137:166–174[Abstract]This article has been cited by other articles:
 |
|
 |
|
  H. Malekinejad, E. J Schoevers, I. J.J.M Daemen, C. Zijlstra, B. Colenbrander, J. Fink-Gremmels, and B. A.J Roelen Exposure of Oocytes to the Fusarium Toxins Zearalenone and Deoxynivalenol Causes Aneuploidy and Abnormal Embryo Development in Pigs Biol Reprod, November 1, 2007; 77(5): 840 - 847. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Taniguchi, J. F. Couse, K. F. Rodriguez, J. M. A. Emmen, D. Poirier, and K. S. Korach Estrogen receptor-{alpha} mediates an intraovarian negative feedback loop on thecal cell steroidogenesis via modulation of Cyp17a1 (cytochrome P450, steroid 17{alpha}-hydroxylase/17,20 lyase) expression FASEB J, February 1, 2007; 21(2): 586 - 595. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.R. Barnett, C. Schilling, C.R. Greenfeld, D. Tomic, and J.A. Flaws Ovarian follicle development and transgenic mouse models Hum. Reprod. Update, September 1, 2006; 12(5): 537 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.V. Younglai, Y.J. Wu, T.K. Kwan, and C.-Y. Kwan Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells Hum. Reprod., September 1, 2005; 20(9): 2383 - 2390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Spicer Effects of Estradiol on Bovine Thecal Cell Function In Vitro: Dependence on Insulin and Gonadotropins J Dairy Sci, July 1, 2005; 88(7): 2412 - 2421. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
