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Antimüllerian hormone regulates stem cell factor expression in human granulosa cells

  • Rong Hu
    Affiliations
    Reproductive Medicine Center, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People's Republic of China

    Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia
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  • Fei-miao Wang
    Affiliations
    Reproductive Medicine Center, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People's Republic of China
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  • Liang Yu
    Affiliations
    Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia
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  • Yan Luo
    Affiliations
    Reproductive Medicine Center, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People's Republic of China
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  • Xin Wu
    Affiliations
    Reproductive Medicine Center, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People's Republic of China
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  • Juan Li
    Affiliations
    Reproductive Medicine Center, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People's Republic of China
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  • Xiao-mei Zhang
    Affiliations
    Department of Obstetrics and Gynecology, Reproductive Medicine Center, Northern Jiangsu People's Hospital, Yangzhou University, Yangzhou, People's Republic of China
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  • Sergio Oehninger
    Correspondence
    Reprint requests: Sergio Oehninger, M.D., Ph.D., 601 Colley Ave, Norfolk, VA 23507.
    Affiliations
    Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia
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  • Silvina Bocca
    Affiliations
    Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia
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Published:September 18, 2014DOI:https://doi.org/10.1016/j.fertnstert.2014.08.012

      Objective

      To determine whether there is a correlation between antimüllerian hormone (AMH) and stem cell factor (SCF) in serum, follicular fluid (FF), and granulosa cells (GCs), and to investigate a possible regulatory mechanism of AMH on SCF in human granulosa cells.

      Design

      Prospective clinical and experimental study.

      Setting

      Academic center.

      Patient(s)

      163 women undergoing IVF.

      Intervention(s)

      Serum, FF, and GCs obtained in all women, primary cultures of human GCs.

      Main Outcome Measure(s)

      AMH and SCF were analyzed in serum, FF, and GCs, using enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and immunoblotting.

      Result(s)

      There was a significant negative correlation between AMH and SCF protein level in FF, and in the mRNA expression of AMH and SCF in GCs. Conversely, there was no correlation between AMH and SCF levels in serum. In primary cultures of human GCs, SCF was down-regulated by treatment with recombinant human AMH and was increased by cyclic adenosine 3′:5′ monophosphate (cAMP) in a dose-dependent manner. A protein kinase A (PKA) inhibitor (H89) significantly reversed the effects of recombinant human AMH and cAMP on SCF mRNA and protein expression.

      Conclusion(s)

      This is the first report on a modulatory role for AMH as an ovarian/follicular autocrine/paracrine factor controlling SCF expression via the cAMP/PKA pathway.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/hur-amh-scf-expression-granulosa-cells/
      The number of follicles in the primordial follicle pool is an important determinant of the reproductive life span of a female (
      • Skinner M.K.
      Regulation of primordial follicle assembly and development.
      ,
      • Rajah R.
      • Glaser E.M.
      • Hirshfield A.N.
      The changing architecture of the neonatal rat ovary during histogenesis.
      ). Follicular development and differentiation are sequential events that are tightly regulated by hormones, intraovarian regulators, and cell-cell interactions (
      • Suh C.S.
      • Sonntag B.
      • Erickson G.F.
      The ovarian life cycle: a contemporary view.
      ). The balance of inhibitory signals and stimulatory growth factors determine whether follicle maturation will proceed. Antimüllerian hormone (AMH) is an important regulator of primordial follicle assembly that appears to mediate a stromal-epithelial interaction in the developing ovary to inhibit follicle assembly (
      • Nilsson E.
      • Rogers N.
      • Skinner M.K.
      Actions of anti-müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition.
      ). Also known as Müllerian inhibitory substance (MIS), AMH is a member of the transforming growth factor β (TGF-β) family and binds to AMH type II receptors (AMHRII).
      In the adult human ovary, the expression of immunoreactive AMH is restricted to the granulosa cells of primary, secondary, preantral, and antral follicles; the expression is down-regulated in the atretic follicles. The granulosa cells of the primordial follicles, the oocytes, or the ovarian stroma do not stain positive for AMH (
      • Weenen C.
      • Laven J.S.
      • Von Bergh A.R.
      • Cranfield M.
      • Groome N.P.
      • Visser J.A.
      • et al.
      Anti-müllerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment.
      ). Modi et al. (
      • Modi D.
      • Bhartiya D.
      • Puri C.
      Developmental expression and cellular distribution of müllerian inhibiting substance in the primate ovary.
      ) studied the expression profiles of AMH mRNA in the developing and adult human and monkey ovaries by in situ hybridization. The results revealed that, in the adult human and monkey ovary, AMH mRNA is expressed at low levels in the primordial follicles, maximally in the primary and secondary follicles, and the expression is down-regulated in the antral and atretic follicles. Antimüllerian hormone expression is extinguished in the granulosa cells only after ovulation. It is interesting that by using more sensitive and quantitative methods such as electrochemiluminescent immunoassay (ECLIA) it is possible to detect AMH in the follicular fluids of large antral follicles after ovulation (
      • von Wolff M.
      • Kollmann Z.S.
      • Flück C.
      • Stute P.
      • Marti U.
      • Weiss B.
      • et al.
      Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.
      ).
      In vivo and in vitro studies have shown AMHRII expression colocalized with AMH in the granulosa and the theca cells (
      • Ingraham H.A.
      • Hirokawa Y.
      • Roberts L.M.
      • Mellon S.H.
      • McGee E.
      • Nachtigal M.W.
      • et al.
      Autocrine and paracrine müllerian inhibiting substance hormone signaling in reproduction.
      ). There is lack of AMHRII in oocytes. Antimüllerian hormone produced by preantral and small antral follicles in the postnatal ovary has two sites of action in the postnatal ovary. It inhibits initial follicular recruitment, and it inhibits the stimulatory effect of follicle-stimulating hormone (FSH) on the growth of preantral and small antral follicles (
      • Durlinger A.L.
      • Gruijters M.J.
      • Kramer P.
      • Karels B.
      • Kumar T.R.
      • Matzuk M.M.
      • et al.
      Anti-müllerian hormone attenuates the effects of FSH on follicle development in the mouse ovary.
      ). On the basis of the pattern of expression of the receptors described herein, it is much more likely that AMH exerts its effect on ovarian follicles via the granulosa and theca cells, but not via the oocyte. Antimüllerian hormone also influences transcription factors in the signaling pathways of granulosa cells, mainly through the Smad protein, and then regulates gene transcription of other cytokines to maintain primordial follicles in their arrested state (
      • Ratteralli J.L.
      • Levi A.J.
      • Miller B.T.
      A prospective novel method of determining ovarian size during in vitro fertilization cycles.
      ,
      • Visser J.
      Role of antimüllerian hormone in follicular recruitment and maturation.
      ).
      Stem cell factor (SCF, also called Steel factor or Kit ligand), a granulosa-derived growth factor, binds to oocyte c-Kit receptor and its signal is transduced through the phosphoinositide 3-kinase (PI3K) pathway (
      • Reddy P.
      • Shen L.
      • Ren C.
      • Boman K.
      • Lundin E.
      • Ottander U.
      • et al.
      Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development.
      ); this effect appears to be important for the regulation of early follicular development and enhancement of the production of oocyte factors, which in turn stimulates the proliferation and differentiation of the surrounding granulosa cells (
      • Liu K.
      • Rajareddy S.
      • Liu L.
      • Jagarlamudi K.
      • Boman K.
      • Selstam G.
      • et al.
      Control of mammalian oocyte growth and early follicular development by the oocyte PI3 kinase pathway: new roles for an old timer.
      ). When ovaries treated with AMH in in vitro cultures were examined via microarray chips, the results indicated that AMH suppresses the stimulatory effects of basic fibroblast growth factor (bFGF), SCF, and keratinocyte growth factor (KGF) on primordial to primary follicles (
      • Nilsson E.
      • Rogers N.
      • Skinner M.K.
      Actions of anti-müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition.
      ). It is unknown whether AMH exerts an autocrine/paracrine effect on SCF expression.
      A number of in vitro studies have shown that aromatase activity stimulated by FSH and cyclic adenosine 3′:5′ monophosphate (cAMP) is significantly reduced after AMH treatment (
      • Gruijters M.J.G.
      • Visser J.A.
      • Durlinger A.L.L.
      • Themmen A.P.
      Antimüllerian hormone and its role in ovarian function.
      ). Also, AMH reduced the sensitivity of the follicles to FSH via cAMP. Broekmans et al. (
      • Broekmans F.J.
      • Visser J.A.
      • Laven J.S.E.
      • Broer S.L.
      • Themmen A.P.N.
      • Fauser B.C.
      Anti-müllerian hormone and ovarian dysfunction.
      ) reported that AMH treatment decreased the expression of aromatase mRNA and estradiol production by FSH-stimulated activity in human GCs using a cAMP/protein kinase A (PKA) signaling pathway. Vigier et al. (
      • Vigier B.
      • Forest M.G.
      • Eychenne B.
      • Bézard J.
      • Garrigou O.
      • Robel P.
      • et al.
      Anti-müllerian hormone produces endocrine sex reversal of fetal ovaries.
      ) reported that AMH completely blocked the effect of cAMP/PKA on the aromatase expression in rat ovaries. In addition, some reports have indicated that the cAMP/PKA pathway could enhance the transcription of SCF (
      • Chang L.C.
      • Guo C.L.
      • Lin Y.S.
      • Fu H.
      • Wang C.S.
      • Jauh G.Y.
      Pollen-specific SKP1-like proteins are components of functional SCF complexes and essential for lily pollen tube elongation.
      ,
      • Da Silva C.A.
      • Kassel O.
      • Lebouquin R.
      • Lacroix E.J.
      • Frossard N.
      Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditions.
      ). Antimüllerian hormone inhibited the effect of cAMP/PKA pathway, and it remains to be determined whether a similar mechanism exists in the AMH regulation of SCF.
      Our preliminary study showed that recombinant human AMH treatment decreased SCF expression in human GCs (
      • Hu R.
      • Lou Y.
      • Wang F.M.
      • Ma H.M.
      • Wu X.
      • Zhang X.M.
      • et al.
      Effects of recombinant human AMH on SCF expression in human granulosa cells.
      ). Here, we tested the hypothesis that AMH down-regulates SCF via the cAMP/PKA (protein kinase A) pathway. Within this context, we measured AMH and SCF mRNA and protein in the serum, follicular fluid (FF) of small follicles, and granulosa cells of women undergoing in vitro fertilization (IVF) treatment. Subsequently, SCF mRNA (by real-time reverse transcription polymerase chain reaction [RT–PCR]) and protein expression (by immunocytochemistry and immunoblotting) were evaluated after pretreatment with recombinant human AMH and cAMP (alone or in combination) for 48 hours in the presence or absence of a PKA inhibitor (H89) to gain insight into the signaling pathway that might be involved in AMH regulation of SCF.

      Materials and methods

       Reagents

      The following reagents were used: recombinant human AMH (R&D Systems), dibutyril(DB)-cAMP (Sigma-Aldrich), H89 dihydrochloride (R&D Systems UK), Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12) medium (GIBCO/Invitrogen), fetal bovine serum (FBS; GIBCO), Ficoll-Hypaque (Sigma-Aldrich), hyaluronidase (Sigma-Aldrich), 100% Percoll gradient (Sigma-Aldrich), and anti-SCF rabbit monoclonal antibody (Abcam).

       Patient Selection

      We enrolled a total of 163 patients who underwent IVF therapy: 112 patients aged 23 to 37 years from the Reproductive Medicine Center of General Hospital of Ningxia Medicine University in China from 2011 to 2012, and 51 patients aged 27 to 43 years from the Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia, from April to November 2013. The study was approved by the local institutional review boards of both institutions. Written informed consent was obtained from each participating patient.

       Ovarian Stimulation

      Follicular aspirates were collected during oocyte retrieval following published procedures (
      • Yalçınkaya E.
      • Cakıroğlu Y.
      • Doğer E.
      • Budak O.
      • Cekmen M.
      • Calışkan E.
      Effect of follicular fluid NO, MDA and GSH levels on in vitro fertilization outcomes.
      ), and ovarian stimulation was performed with the use of a long protocol after down-regulation with a gonadotropin-releasing hormone agonist (GnRH-a, leuprolide acetate; Abbott) followed by gonadotropins (
      • Liu B.
      • Zhang L.
      • Guo R.W.
      • Wang W.J.
      • Duan X.Q.
      • Liu Y.W.
      The serum level of C-reactive protein in patients undergoing GnRH agonist protocols for in vitro fertilization cycle.
      ). According to the patient's age, body mass index, serum basal FSH levels, and antral follicle count, initial doses of 150–450 IU/d of recombinant human FSH (Gonal F; Serono, or Follistim; Organon) were used starting on the 3rd day of their menstrual cycle. The dose of recombinant human FSH was adjusted according to ovarian response as monitored by serum estradiol levels and vaginal ultrasound. Administration of human chorionic gonadotropin (hCG) at a dose of 10,000 IU was performed when at least three follicles had reached a size of 17 mm in diameter, followed by ovum pick up 34 to 36 hours later. Couples with tubal, unexplained, or male factor infertility were included. Patients with endometriosis, polycystic ovary syndrome, or a prior history of poor response were excluded from this study.

       Sample Collection

      Serum and FF samples were collected during oocyte retrieval. To study the correlation between AMH and SCF, patients' follicles were categorized into two groups according to follicular size at the time of retrieval: one group with the larger follicles (diameter >12 mm), and the other with small follicles (diameter ≤12 mm). In each ovary, the larger follicles were aspirated first, followed by aspiration of the smaller follicles (all individually aspirated), and then the line was flushed with 2.0 mL of phosphate-buffered saline (PBS). The follicular fluids and the purified granulosa cells of the small follicles were pooled for each patient, and both were used to test the concentrations of AMH and SCF. Any follicular aspirate that was not clear or showed gross contamination with blood was discarded. After oocyte identification, samples from each patient's follicles with diameter ≤12 mm were pooled, placed in sterile tubes, and immediately centrifuged at 3,000 rpm for 10 minutes. Supernatants were aspirated, divided into aliquots, and frozen at −80°C for future analysis.

       Granulosa Cells Collection and Culture

      Harvested follicular fluid cell pellets were resuspended in PBS then transferred to a 50% (volume fraction) Percoll gradient (Sigma-Aldrich); they were centrifuged at 2,500 rpm for 20 minutes to purify human GCs from any red blood cells. After washing and recentrifugation, sheets of hGC cells were digested with hyaluronidase at a 2:1 ratio for 30 minutes to separate them. The GCs were removed using a pipette and washed with PBS. After a final centrifugation at 1,000 rpm twice, the pellet was resuspended in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin, and the cell number was determined using a hemocytometer. The cells were then plated at a concentration of 10,000 cells per well in 35-mm culture dishes at 37°C in a 95% air, 5% CO2 humidified environment for 24 hours. After seeding, the cells were rinsed with PBS and then subjected to treatments with recombinant human AMH (
      • Nilsson E.
      • Rogers N.
      • Skinner M.K.
      Actions of anti-müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition.
      ) and/or cAMP in the presence or absence of an inhibitor of PKA (H89, 1 mM) for 48 hours.

       ELISA for AMH and SCF Measurements

      Concentrations of AMH and SCF in serum and FF were determined with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (AMH ELISA Kit; Antibodies-Online; and SCF ELISA Kit; R&D Systems). Intra-assay and interassay coefficients of variation were 4.5% and 4.8% for AMH, and 5.3% and 5.7% for SCF, respectively. All of the procedures were performed according to the manufacturers' instructions.

       RNA Isolation and Real-time RT-PCR

      Total RNA was extracted using an RNeasy kit (Qiagen) according to the manufacturer's instructions. The quality of total RNA extracted was analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies), and the quantification of total RNA was performed on a NanoDrop spectrophotometer (Thermo Scientific).
      Real-time PCR analysis used a Lightcycler Fastart DNA Master plus SYBR green I and a Lightcycler 2.0 instrument (Roche Applied Science) in a 10-μL total reaction volume containing 1 μL cDNA and 0.5 μM of each sense and antisense primers. The relative mRNA levels were calculated by normalizing to the levels of endogenous 18s mRNA (used as an internal control) using Microsoft EXCEL. Antimüllerian hormone and SCF were amplified by PCR from cDNA derived from human GCs by using the following primers: AMH sense 5′-CGC CTG GTG GTC CTA CAC-3′; antisense primer: 5′-GAA CCT CAG CGA GGG TGTT-3′. Stem cell factor was amplified by using the following primers: sense 5′-CAC TAA ATT GGT GGC AAA TCT TCC-3′; antisense 5′-TGT GAC ACT GAC TCT GGA ATC TTT-3′. For each experiment, quantitative PCR was performed in triplicate.

       Immunoblotting

      After 48 hours of treatment with in AMH and/or cAMP in the presence or absence of the inhibitor of PKA (H89), human GCs were washed and lysed to prepare nuclear and cytosol extracts using the ProteoExtract subcellular proteome extraction kit (Pierce Chemical). Protein concentration was determined using the bicinchoninic acid assay (Pierce Chemical) according to the manufacturer's instructions. Protein lysates (20 μg total protein per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore Corp.). The membranes were incubated with a polyclonal rabbit anti-SCF antibody at 1:2,000 (ab9716; Abcam) at 4°C overnight.
      After they were washed in Tris-buffered saline-Tween 20, the blots were probed with a peroxidase-linked anti-rabbit Ig-G-HRP at 1:5,000 (Invitrogen) for 60 minutes at room temperature. Bands were visualized with the enhanced chemiluminescence detection kit (Perkin Elmer). Stem cell factor protein levels from the experiments were analyzed by quantification of the bands on the Western blots using Image-J software (available at http://rsb.info.nih.gov/ij) and expressed as arbitrary intensity units relative to the control (100%).

       Immunocytochemistry of Human GCs

      Granulosa cells were stained for presence of FSH-R (rabbit polyclonal, sc-13935, Santa Cruz Biotechnology) to evaluate the purity of the cell cultures. In all experiments, >85% of the GCs stained positive for the FSH-R. Immunocytochemistry was performed for SCF protein on six groups of human GCs treated for 48 hours with control medium, recombinant human AMH (20 ng/mL), cAMP (3 mM), recombinant human AMH plus cAMP, recombinant human AMH plus H89 (1 mM), and cAMP plus H89 (1 mM). Briefly, endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 minutes, and nonspecific binding sites were blocked with 2% normal goat serum for 60 minutes at room temperature. The primary antibodies for SCF (rabbit polyclonal, ab9716; Abcam) were diluted in a solution of PBS-2% normal goat serum to optimize sensitivity and specificity and were used at a dilution of 1:1,000.
      After primary antibody incubation, sections were washed three times PBS and incubated with biotinylated goat anti-rabbit secondary antibody (sc-45090; Santa Cruz Biotechnology) at a dilution of 1:200 for 30 minutes at room temperature. After rinsing with PBS, the immunoreactive antigen was visualized by incubating with avidin-biotinylated horseradish peroxidase (1:100) complex for 30 minutes and 3,3′-diaminobenzidine (0.5 mg/mL) as chromogen for 3 minutes to complete the reaction. Negative controls included sections that were treated with a similar dilution of a nonimmune mouse IgG1 (isotype control; eBioscience). Slides were counterstained with Mayer's hematoxylin (Sigma-Aldrich) followed by dehydration in a graded series of ethanol, then cleared in xylene and mounted with mounting media.
      Representative fields were photographed at ×200 and ×400 magnification with an Olympus microscope (Olympus Corporation) using an Olympus Q-color 3 camera. The intensity of SCF immunolabeling was evaluated using a semiquantitative index (
      • McCarty Jr., K.S.
      • Szabo E.
      • Flowers J.L.
      • Cox E.B.
      • Leight G.S.
      • Miller L.
      • et al.
      Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors.
      ) with the HSCORE: Σ(i + l) × Pi, where i = the intensity of staining with a value of 0 to 3 (negative, weak, positive, or strong, respectively), and Pi = the percentage of stained human GCs (0 to 100%). The evaluations were recorded as percentages of positively stained target cells in each of four intensity categories which were denoted as 0 (negative), 1+ (weak but detectable above control), 2+ (positive), and 3+ (strong), Two investigators scored the slides blindly and counted three fields per slide to assess the percentage of target cells. All the assessments and results were averaged and formed the HSCORE. Reproducibility of the scoring method between two observers was greater than 90%. The component HSCORE was derived as described earlier.

       Statistical Analysis

      Data were analyzed using SPSS 16.0 software (IBM), and results are presented as mean ± standard deviation (SD). Correlation analysis between AMH and SCF was performed by Spearman's rank analysis method. Responses to the different recombinant human AMH and cAMP doses were analyzed by repeated-measures analysis of variance (ANOVA) and the post hoc Dunnet's test. Results of different treatment conditions were compared by ANOVA followed by the Student-Newman-Keuls test. P<.05 was considered statistically significant.

      Results

       Correlation between AMH and SCF in Human Serum, FF and Human GCs

      We first analyzed 50 patients' serum samples (and 83 FF samples) and 31 patients' human GCs samples from 78 patients undergoing IVF and embryo transfer (ET) at the Reproductive Medicine Center of General Hospital of Ningxia Medicine University in the People's Republic of China (Supplemental Fig. 1, available online). We investigated the levels of AMH and SCF in serum and FF, and the expression of AMH and SCF mRNA and protein by RT-PCR and ELISA, respectively, from human GCs (after hCG administration). The mean AMH concentration in serum was 9.47 ± 3.18 ng/mL and in FF was 5.63 ± 0.65 ng/mL. The mean SCF concentration in serum was 2.33 ± 0.16 ng/mL and in FF was 0.46 ± 0.03 ng/mL. As shown in Figure 1, there was a statistically significant negative correlation between the concentration of AMH and SCF in FF (r = −0.65; P<.05, Fig. 1A). Similar results were obtained for the mRNA expression of AMH and SCF in human GCs (r = −0.79; P<.01, Fig. 1B). No statistically significant correlation was observed for the protein levels of AMH and SCF in the serum (r = −0.24; P=.09).
      Figure thumbnail gr1
      Figure 1(A) Correlation between protein expression levels of antimüllerian hormone (AMH) and stem cell factor (SCF) in follicular fluid (FF) from small follicles. ELISA was used to analyze AMH and SCF concentration in FF from IVF patients (n = 50). Results showed that there was a statistically significant negative correlation (r = −0.65; P<.05). (B) Correlation between the mRNA expression levels of AMH and SCF in GCs from small follicles of IVF patients (n = 31). Real-time PCR was used to analyze AMH and SCF mRNA in human GCs. Results showed there was a statistically significant negative correlation (r = −0.79; P<.01).

       Effect of Recombinant Human AMH on the Expression of SCF in Human GCs

      The negative correlation between AMH and SCF found in FF and human GCs suggested that SCF is down-regulated by AMH in human GCs. Next, we obtained human GCs from 34 patients undergoing IVF at the Reproductive Medicine Center of General Hospital of Ningxia Medicine University in the People's Republic of China. After seeding, human GCs were cultured for 48 hours in control medium and in the presence of recombinant human AMH, and then the SCF mRNA and protein expression were examined (Fig. 2A and B). We used three different concentrations of recombinant human AMH (10, 15, and 20 ng/mL) to treat human GCs from same patient. Recombinant human AMH treatment resulted in a statistically significant and dose-dependent inhibition of SCF mRNA and protein expression (n = 3 experiments per patient's sample, overall effect P<.01 for both). The inhibition was statistically significantly different for 15 ng/mL and 20 ng/mL groups versus control (P<.05 and P<.001, respectively, for gene expression; and P<.05 and P<.01, respectively, for protein expression).
      Figure thumbnail gr2
      Figure 2Stem cell factor (SCF) mRNA and protein expression were regulated by recombinant human antimüllerian hormone (AMH) in human granulosa cells (GCs) in a dose-dependent fashion. (A) Real-time PCR was used to analyze mRNA expression of SCF. Each bar indicates the fold decrease of mRNA expression relative to the housekeeping gene 18S. Results showed SCF mRNA expression was down-regulated by recombinant human AMH (overall effect P<.01). At 15 ng/mL and 20 ng/mL, the results were statistically significantly lower than control (P<.05 and P<.001, respectively). (B) SCF protein expression in the cell lysates after 48 hours of treatment with recombinant human AMH were demonstrated by immunoblotting. Stem cell factor protein expression was down-regulated by recombinant human AMH (overall effect P<.01); at 15 ng/mL and 20 ng/mL, the recombinant human AMH results were statistically significantly lower than control (P<.05 and P<.01, respectively).

       Recombinant Human AMH Down-regulates SCF mRNA and Protein Expression via the cAMP/PKA Pathway

      In addition, for the following experiments human GCs were obtained from 51 patients undergoing IVF at the Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. The results are shown in Figures 3 and 4. We first tested the effect of cAMP on SCF expression in human GCs. The cAMP statistically significantly stimulated SCF mRNA expression in a dose-dependent fashion (1–4 nM, n = 3 experiments per patient's sample, overall effect P<.01) (see Fig. 3A). We found that 3 nM was the optimal concentration of cAMP at which SCF expression was maximally increased. Stem cell factor protein expression was confirmed by immunohistochemistry (see Fig. 3B). The immunohistochemical studies showed that SCF protein strongly stained in the human GCs treated with cAMP (P<.01 vs. control) and was statistically significantly weaker in human GCs treated with recombinant human AMH (P<.05 vs. control). There were no statistically significant differences when comparing SCF expression in human GCs treated with recombinant human AMH + cAMP and the control group (Fig. 3B).
      Figure thumbnail gr3
      Figure 3(A) Stem cell factor (SCF) mRNA regulation by cyclic adenosine 3′:5′ monophosphate (cAMP) in human granulosa cells (GCs). After seeding, cells were cultured for 48 hours in control medium or in the presence of different cAMP concentrations (1–4 nM). Real-time PCR results showed that cAMP statistically significantly stimulated SCF mRNA expression in a dose-dependent fashion (overall effect P<.01). At 3 and 4 nM, cAMP strongly stimulated SCF mRNA expression (both P<.001 vs. control). (B) Immunolocalization of SCF in human GCs cultured for 48 hours with control medium (i), cAMP (ii), recombinant human antimüllerian hormone (AMH) (iii), and combination of recombinant human AMH and cAMP (iv) (magnification: ×400). NC = negative control (nonimmune mouse IgG). Stem cell factor was expressed in the cytoplasm of human GCs. Stem cell factor had limited and lower expression in the recombinant human AMH group (P<.05 vs. controls). In contrast, a very strong and statistically significantly higher staining was observed in the human GCs treated with cAMP (P<.01 vs. control). There was no difference in SCF expression in the recombinant human AMH and cAMP group compared with control conditions.
      Figure thumbnail gr4
      Figure 4(A) Human granulosa cells (GCs) were cultured for 48 hours in control medium or in the presence of antimüllerian hormone (AMH) (20 ng/mL), cyclic adenosine 3′:5′ monophosphate (cAMP) (3 nM), recombinant human AMH and cAMP, recombinant human AMH and H89 (1 mM), or cyclic adenosine 3′:5′ monophosphate (cAMP) and H89 (1 mM). The results of real-time PCR (gene expression) and immunoblotting (protein expression) were consistent (A and B, respectively). As expected, recombinant human AMH significantly reduced mRNA and protein expression of stem cell factor (SCF) (both P<.01 vs. control), and cAMP statistically significantly increased mRNA and protein expression of SCF (P<.001 and P<.01, respectively, vs. control). Furthermore, recombinant human AMH statistically significantly reduced the effect of cAMP on SCF mRNA and protein expression (cAMP vs. recombinant human AMH and cAMP, P<.001 and P<.01). Using an inhibitor of protein kinase A (PKA) (H89), we then tested whether activation of the cAMP/PKA pathway regulating SCF was suppressed by recombinant human AMH. H89 significantly inhibited the effects of both recombinant human AMH and cAMP on SCF gene and protein expression (recombinant human AMH vs. recombinant human AMH and H89, both P<.05; cAMP vs. cAMP and H89, P<.01 and P<.001, respectively).
      To determine whether recombinant human AMH suppressed the stimulatory effect of cAMP on SCF expression, we then cultured human GCs and treated them with control medium, recombinant human AMH (20 ng/mL), cAMP (3 nM), or the combination of AMH and cAMP. The results of real-time PCR (gene expression) and immunoblotting (protein expression) were consistent (Fig. 4A and B, respectively). As expected, recombinant human AMH reduced (both P<.01) and cAMP increased (P<.001 and P<.01 respectively) SCF mRNA and protein expression. Furthermore, recombinant human AMH abrogated the effect of cAMP on SCF mRNA and protein expression.
      Using an inhibitor of PKA (H89), we then tested whether activation of the cAMP/PKA pathway regulating SCF was suppressed by recombinant human AMH. We found that H89 partially but statistically significantly reversed the effects of recombinant human AMH (inhibitory) and cAMP (stimulatory) on SCF mRNA and protein expression (recombinant human AMH vs. recombinant human AMH and H89, both P<.05; cAMP vs. cAMP and H89, P<.01 and P<.001, respectively). There were no statistically significant differences when comparing SCF expression among recombinant human AMH + H89, cAMP + H89, and the control group (both P>.05).

      Discussion

      Both AMH and SCF are cytokines secreted by granulosa cells, and both play important roles in the recruitment of the primordial follicle pool and oocyte/follicular development (
      • Hoyer P.E.
      • Byskov A.G.
      • Mollgard K.
      Stem cell factor and c-kit in human primordial germ cells and fetal ovaries.
      ,
      • Carlsson I.B.
      • Laitinen M.P.
      • Scott J.E.
      • Louhio H.
      • Velentzis L.
      • Tuuri T.
      • et al.
      Kit ligand and c-kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture.
      ,
      • Brankin V.
      • Hunter M.G.
      • Horan T.L.
      • Armstrong D.G.
      • Webb R.
      The expression patterns of mRNA-encoding stem cell factor, internal stem cell factor and c-kit in the prepubertal and adult porcine ovary.
      ). Studies have reported on two oocyte-derived transforming growth factor β family members, growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15, also known as growth differentiation factor 9B), that appear to regulate SCF expression in a specific manner. GDF-9 suppresses the SCF expression in mouse preantral granulosa cells, whereas in bovine antral granulosa cell cultures GDF-9 increases SCF transcript levels (
      • Joyce I.M.
      • Clark A.T.
      • Pendola F.L.
      • Eppig J.J.
      Comparison of recombinant growth differentiation factor-9 and oocyte regulation of KIT ligand messenger ribonucleic acid expression in mouse ovarian follicles.
      ,
      • Nilsson E.E.
      • Skinner M.K.
      Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development.
      ). BMP-15 stimulates SCF expression in rat antral granulosa cells while SCF is able to negatively regulate BMP-15 transcripts in a paracrine manner (
      • Otsuka F.
      • Shimasaki S.
      A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand: its role in regulating granulosa cell mitosis.
      ). There are differences in rodent and human follicular function (
      • Liu B.
      • Zhang L.
      • Guo R.W.
      • Wang W.J.
      • Duan X.Q.
      • Liu Y.W.
      The serum level of C-reactive protein in patients undergoing GnRH agonist protocols for in vitro fertilization cycle.
      ), and little is known about the regulation of SCF in the human follicle.
      We therefore first investigated the in vivo correlation of AMH and SCF in human serum, FF, and human GCs. The results showed that in serum there was no statistically significant correlation for the protein levels of AMH and SCF. This lack of correlation could be due, in part, to the differences in the cells/organs that produce each factor, each with very different spatial and temporal regulations. Stem cell factor is a growth factor with a broad range of biological activities. It is expressed by fibroblasts and endothelial cells throughout the body, promoting proliferation, migration, survival, and differentiation of hematopoietic progenitors, melanocytes, and germ cells (
      • Lennartsson J.
      • Rönnstrand L.
      Stem cell factor receptor/c-kit: from basic science to clinical implications.
      ). However, the serum AMH level is an acknowledged marker of ovarian reserve (
      • Freour T.
      • Mirallie S.
      • Bach-Ngohou K.
      • Denis M.
      • Barriere P.
      • Masson D.
      Measurement of anti-müllerian hormone by Beckman-Coulter ELISA and DSL ELISA: comparison and relevance in assisted reproduction technology (ART).
      ,
      • Seifer D.B.
      • MacLaughlin D.T.
      Müllerian inhibiting substance is an ovarian growth factor of emerging clinical significance.
      ), mainly produced by granulosa cells.
      On the other hand, we found a statistically significant and negative correlation between AMH and SCF in FF and human GCs (Fig. 1). This result suggests that there may be opposite regulatory roles of these cytokines within the follicular microenvironment. We then tested the effect of AMH on SCF expression in vitro using cultured human GCs. Previous reports have shown that the highest expression of AMH is found in the preantral and the small antral follicles and that it becomes very low or undetectable in the larger ones (
      • Weenen C.
      • Laven J.S.
      • Von Bergh A.R.
      • Cranfield M.
      • Groome N.P.
      • Visser J.A.
      • et al.
      Anti-müllerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment.
      ,
      • Modi D.
      • Bhartiya D.
      • Puri C.
      Developmental expression and cellular distribution of müllerian inhibiting substance in the primate ovary.
      ). The follicular AMH levels were reported to be three times higher in the small follicles (<12 mm) than in the large follicles (>16 mm) (
      • Talebian S.
      • Licciardi F.
      • Liu M.
      • Grifo J.A.
      • Krey L.C.
      Assessing anti-müllerian hormone (AMH) as a marker of ovarian response in anonymous oocyte donors: quantity or quality?.
      ,
      • Nardo L.
      • Gelbaya T.
      • Wilkinson H.
      • Roberts S.A.
      • Yates A.
      • Pemberton P.
      • et al.
      Circulating basal anti-müllerian hormone levels as predictor of ovarian response in women undergoing ovarian stimulation for in vitro fertilization.
      ), which is consistent with our preliminary experiments (data not shown), and guided us to select the smaller ≤12 mm follicles for analysis.
      Nilsson et al. (
      • Nilsson E.
      • Rogers N.
      • Skinner M.K.
      Actions of anti-müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition.
      ) reported in a microarray study of whole rat ovaries that AMH did not change SCF mRNA levels. Our results demonstrated, for the first time, that the expression of SCF mRNA and protein were decreased by recombinant human AMH treatment in human GCs. These effects are consistent with the finding that human FF concentrations of SCF are negatively correlated with AMH concentrations. Our results also confirmed that AMH down-regulates SCF gene expression at cellular level (Fig. 2), which may explain why AMH treatment inhibits the SCF-stimulated primordial to primary follicle transition in rat ovaries (
      • Nilsson E.
      • Rogers N.
      • Skinner M.K.
      Actions of anti-müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition.
      ).
      In this study, we found that the expression of SCF mRNA was highest during the 48 hours of culture and then decreased with the culture time of untreated primary cultures of human GCs with an apparent degradation phenomenon after 72 hours of culturing (data not shown). Stem cell factor expression fell dramatically after 72 hours of culture in the control medium, and that is why we tested SCF expression after 48 hours of treatment.
      We then designed experiments to determine the direct and indirect actions of AMH on SCF expression. Antimüllerian hormone could inhibit the phosphorylation of cAMP responsive element (CRE)-binding protein (CREB) in the cAMP pathway through autocrine and paracrine mechanisms, which results in reducing FSH-stimulated transcription of estrogen synthase (
      • Kanakkaparambil R.
      • Singh R.
      • Li D.
      • Webb R.
      • Sinclair K.D.
      B-vitamin and homocysteine status determines ovarian response to gonadotropin treatment in sheep.
      ). In addition, several studies have investigated the cAMP/PKA responsive element (CRE) that exists on the promoter of the human SCF gene (
      • Gruijters M.J.G.
      • Visser J.A.
      • Durlinger A.L.L.
      • Themmen A.P.
      Antimüllerian hormone and its role in ovarian function.
      ). In the rat seminiferous epithelium, SCF gene expression was up-regulated by the cAMP/PKA pathway (
      • Taylor W.E.
      • Najmabadi H.
      • Strathearn M.
      • Jou N.T.
      • Liebling M.
      • Rajavashisth T.
      • et al.
      Human stem cell factor promoter deoxyribonucleic acid sequence and regulation by cyclic 39, 59-adenosine monophosphate in a Sertoli cell line.
      ,
      • Yan W.
      • Linderborg J.
      • Suominen J.
      • Toppari J.
      Stage-specific regulation of stem cell factor gene expression in the rat seminiferous epithelium.
      ). Based upon those studies, we speculated that AMH might reduce the SCF transcription acting through the cAMP/PKA pathway.
      To understand the regulatory mechanism in detail, we then tested the effects of DB-cAMP, a cAMP analog and PKA stimulator, on SCF gene expression in human GCs. We first investigated how the luteinized GCs (after hCG administration) respond to DB-cAMP. We found that DB-cAMP statistically significantly increased the SCF mRNA in a dose-dependent manner (Fig. 3). The effect of cAMP on the SCF mRNA level indicated that the up-regulation of SCF gene expression is mediated through the cAMP pathway.
      To confirm the hypothesis that the effect of recombinant human AMH on SCF mRNA and protein expression is mediated by the cAMP/PKA pathway, we then treated human GCs with recombinant human AMH and/or DB-cAMP in the presence or absence of an inhibitor of cAMP/PKA pathway (H89). The results showed that the effects of recombinant human AMH (inhibition) and DB-cAMP (stimulation) on the expression of SCF mRNA and protein are opposing, and that there was no difference of SCF mRNA and protein expression in the recombinant human AMH and cAMP group as compared with control conditions. Furthermore, H89 partially but statistically significantly reversed the effects of recombinant human AMH and DB-cAMP on SCF expression in human GCs (Fig. 4). These observations strongly indicate that cAMP/PKA is a major pathway involved in SCF down-regulation in response to recombinant human AMH.
      The doses of recombinant human AMH that we used were based on previously published studies (
      • Nilsson E.
      • Rogers N.
      • Skinner M.K.
      Actions of anti-müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition.
      ). Nilsson et al. (
      • Nilsson E.
      • Rogers N.
      • Skinner M.K.
      Actions of anti-müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition.
      ) studied the inhibitory action of AMH on the ovarian primordial to primary follicle transition. Ovaries from 4-day-old rats were placed into organ culture and incubated in the absence or presence of AMH. An AMH dose curve was run in this study showing that 50 ng/mL was the optimal dose. The doses of recombinant human AMH we used probably do not differ significantly from the physiological condition. The concentration of AMH in follicular fluid of large follicles (>18 mm) is higher in nonstimulated or natural cycles (4.6 ng/mL) compared with stimulated cycles (1.49 ng/mL) (
      • von Wolff M.
      • Kollmann Z.S.
      • Flück C.
      • Stute P.
      • Marti U.
      • Weiss B.
      • et al.
      Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.
      ). It is interesting that the expression of AMH in the smaller follicles (antral and preantral) has mainly been studied by measuring either AMH mRNA by in situ hybridization or by Western blot of whole ovarian tissues, rather than directly measuring AMH in the follicular fluid of these small follicles as we have done. So even though the doses used of 10, 15, and 20 ng/mL seem slightly higher than the AMH concentration found in the follicular fluid by von Wolff et al. (
      • von Wolff M.
      • Kollmann Z.S.
      • Flück C.
      • Stute P.
      • Marti U.
      • Weiss B.
      • et al.
      Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.
      ), it must be noted that the follicles aspirated were the large ones, at least 18 mm, which are known for having a lower concentration of AMH than the smaller follicles we studied.
      The observed down-regulation of SCF gene expression in vitro by recombinant human AMH can help us better understand the potential mechanisms by which AMH plays a role in folliculogenesis. One limitation of our study is the use of luteinized GCs from the smaller/stimulated antral follicles. We reported that AMH down-regulates SCF expression in human luteinized GCs in our initial study. In our experiments in which we attempted to mimic the early events in folliculogenesis, we used FF and luteinized GCs from the small stimulated follicles to study down-regulation; these were a surrogate for GCs and FFs from nonstimulated small antral follicles because of the lack of accessibility of that material.
      As mentioned earlier, most of the literature on AMH in smaller follicles involves quantification of AMH in whole ovaries by either AMH mRNA in situ hybridization in human fetal ovaries (
      • Modi D.
      • Bhartiya D.
      • Puri C.
      Developmental expression and cellular distribution of müllerian inhibiting substance in the primate ovary.
      ) or by immunohistochemistry in adult human ovaries (
      • Weenen C.
      • Laven J.S.
      • Von Bergh A.R.
      • Cranfield M.
      • Groome N.P.
      • Visser J.A.
      • et al.
      Anti-müllerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment.
      ) rather than measuring its concentration in FF. Further studies are needed to address whether the SCF down-regulation by AMH also occurs during the time of primordial follicular assembly.
      The use of superstimulated and luteinized GCs and FFs to study SCF down-regulation by AMH may not correlate with natural cycles. In fact, there are reports showing differences in the intrafollicular concentration of AMH and other factors from follicles >18 mm in natural versus IVF cycles (
      • von Wolff M.
      • Kollmann Z.S.
      • Flück C.
      • Stute P.
      • Marti U.
      • Weiss B.
      • et al.
      Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.
      ). It is interesting that in those studies the AMH concentration was higher (32.8 pml/L) for the natural cycle FFs than for the stimulated FFs (10.7 pml/L). We also chose to discard the FF samples grossly contaminated with blood, even though by doing this we decreased the number of samples available; we felt that because both AMH and SCF are present in serum, we needed to minimize cross contamination.
      A positive feedback loop has been identified between theca and granulosa cells that is mediated by keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and SCF (
      • Parrott J.A.
      • Skinner M.K.
      Direct actions of kit-ligand on theca cell growth and differentiation during follicle development.
      ). Our current study has shown that AMH might interact with a signaling pathway that promotes follicle transition. We speculate that SCF might mediate interactions between AMH and oocytes or follicular cells.
      In summary, our study demonstrated that AMH down-regulates the expression of SCF mRNA and protein in human GCs in a dose-dependent manner. The down-regulation of AMH is mediated, at least partially, through the cAMP/PKA pathway. The regulation of SCF expression at the gene level is very complex and shows species specificity (
      • Yoshida H.
      • Takakura N.
      • Kataoka H.
      • Kunisada T.
      • Okamura H.
      • Nishikawa S.I.
      Stepwise requirement of c-kit tyrosine kinase in mouse ovarian follicle development.
      ,
      • Parrott J.A.
      • Skinner M.K.
      Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis.
      ). In future studies, we plan to [1] determine which SCF form, membrane-bound or soluble SCF, is involved in the down-regulation of AMH, and [2] characterize the results of stimulating overexpression of the AMH gene in GCs as well as the effects of gene down-regulation by cloning and RNA silencing manipulations, and the subsequent changes in the intracellular cAMP-response elements in the SCF promoter in human GCs.

      Appendix

      Figure thumbnail fx1
      Supplemental Figure 1Study flow chart for first experiments. A total of 197 follicular fluid (FF) samples from 78 patients who underwent IVF-ET were collected for the first experiments. Among them, 83 FF samples from 50 patients were not grossly contaminated with blood and were used for ELISA. We discarded 114 FF samples because of blood contamination (37 FF samples and 77 samples from 50 patients and 28 patients, respectively). Thirty-one GCs samples were collected for RT-PCR to study the relationship between the antimüllerian hormone (AMH) and stem cell factor (SCF) mRNA.

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