Abstract
Both the viability of hen prehierarchal follicles and subsequent differentiation associated with the selection of a single follicle per day into the preovulatory hierarchy depend on circulating FSH and the expression of FSH receptor (FSH-R) in granulosa cells. The present study addresses mechanisms that mediate both basal expression plus selective up-regulation of FSH-R mRNA in granulosa cells from prehierarchal follicles. Results demonstrate that FSH-R mRNA is both expressed and functional in granulosa cells collected from growing prehierarchal follicles as small as those of 1–2 mm in diameter, as indicated by rapid induction of steroidogenic acute regulatory (StAR) protein expression by FSH in vitro. Real-time polymerase chain reaction determined that relative FSH-R expression within the granulosa layer from individual prehierarchal follicles of 6–8 mm in diameter was similar among the 8–13 follicles within this cohort, with the notable exception that the granulosa layer from a single follicle (presumably the selected follicle) showed elevated expression. Levels of FSH-R mRNA expression were enhanced by both recombinant human (rh) transforming growth factor (TGF) β1 and, to a lesser extent, rh-activin A after 20 h of culture. This stimulatory effect was effectively blocked by mitogen-activated protein (MAP) kinase signaling induced by TGFα treatment. Finally, inhibition of MAP kinase signaling, using the selective inhibitor U0126, promoted FSH-R expression and further enhanced TGFβ1-induced FSH-R expression in vitro. Collectively, results suggest that premature granulosa cell differentiation normally is suppressed by tonic MAP kinase signaling. At the time of follicle selection, a release from inhibitory MAP kinase signaling is proposed to occur, which enables the full potentiation of FSH-R expression mediated by intrafollicular factors.
Introduction
It is well accepted that FSH plays a pivotal role in the control of ovarian follicular development and that both follicular viability and associated differentiation following selection are dependent on FSH stimulation and intrafollicular factors that modulate the actions of FSH. Studies in mammals have demonstrated the importance of various members of the transforming growth factor (TGF) β superfamily and for insulin-like growth factors in regulating FSH receptor (FSH-R) expression within granulosa cells from primary through antral-stage follicles [1–3]. For instance, insulin-like growth factor (IGF)-I treatment in vitro enhances levels of FSH-induced FSH-R mRNA in cultured rat and bovine granulosa cells [4, 5], at least in part by enhancing mRNA stability [4]. In turn, the bioavailability of IGF appears to be determined by the relative availability of IGF-binding proteins [6]. Moreover, both TGFβ and activin directly induce FSH-R mRNA expression as well as potentiate FSH-induced differentiation [2, 7, 8]. The actions of activin, but not of TGFβ, can be modulated by the presence of an activin-binding protein, follistatin (FS) [2, 7].
In the Leghorn hen ovary, one follicle from a cohort of 8–13 follicles of 6–8 mm in diameter is selected each day to enter the preovulatory hierarchy. Similar to the situation in mammals, FSH is proposed to be essential not only for maintaining hen prehierarchal follicular viability, in part by supporting granulosa cell survival [9, 10], but also for the initiation of granulosa cell differentiation following follicle selection [11]. Circulating levels of FSH in the hen have been characterized during the laying cycle, yet some discrepancy exists regarding whether a discrete surge occurs at any time relative to ovulation [12–14], as occurs in mammals [3, 15]. Nevertheless, whereas all hen prehierarchal follicles, irrespective of size, are exposed to circulating FSH, only a single follicle per day is selected from the cohort of follicles of 6–8 mm in diameter into the preovulatory hierarchy to begin rapid growth and final differentiation.
As in mammals, the signals and cellular mechanisms responsible for follicle selection and differentiation in the hen have yet to be elucidated. Within the granulosa layer, Northern blot analysis has demonstrated that levels of FSH-R mRNA are highest in pooled granulosa layers collected from follicles of 6–8 mm in diameter and that such levels progressively decline following follicle selection [16, 17]. This pattern is consistent with the decline in FSH binding observed during the final stages of preovulatory follicular development [18]. Subsequent to selection, a follicle (specifically, the granulosa layer) undergoes a transition from largely FSH-dependence to LH-dependence [16, 19, 20]; thus, LH becomes the primary gonadotropin responsible for promoting progesterone production in preovulatory follicles [11, 21].
To date, however, and to our knowledge, no studies have addressed possible endocrine and/or intrafollicular factors capable of regulating FSH-R mRNA expression in hen prehierarchal follicles. Recently, we determined that TGFβ, but not the related family member, activin, up-regulates LH-receptor (LH-R) mRNA expression in cultured granulosa cells from follicles of 6–8 mm in diameter [22]. Notably, TGFβ, but not activin A, also induces FSH-R mRNA expression, yet neither TGFβ nor activin A directly induces steroidogenic acute regulatory (StAR) protein or P450 side-chain cleavage (P450scc) mRNA expression or promotes progesterone production. By comparison, whereas FSH signaling does not up-regulate its own receptor, it induces expression of components critical to initiating progesterone production (e.g., StAR and P450scc), primarily by protein kinase A signaling [11, 23].
It has been established in the hen that mitogen-activated protein (MAP) kinase signaling via Erk phosphorylation (Erk-P) can negatively regulate FSH-induced granulosa cell differentiation in vitro. For instance, TGFα-induced Erk-P prevents FSH-mediated LH-R mRNA and StAR protein expression, and it inhibits P450scc mRNA expression and progesterone production [11, 23]. These effects can be replicated by the use of epidermal growth factor (EGF) [24]. Conversely, inhibition of tonic MAP kinase signaling using a specific pharmacologic inhibitor, U0126, promotes LH-R, StAR, and P450scc expression, which subsequently enables gonadotropin-induced progesterone production [11, 23].
Accordingly, experiments described herein were designed to characterize the expression and evaluate the functional signaling of the FSH-R during early follicle growth (e.g., follicles of 1–8 mm in diameter). A second objective was to identify endocrine and intrafollicular factors that regulate FSH-R mRNA expression in a positive or negative fashion within the granulosa layer of follicles of 6–8 mm in diameter. Results of the present study provide evidence that the TGFβ family members (TGFβ and, to a lesser extent, activin A) play a role in promoting FSH-R expression in hen granulosa cells, whereas the MAP kinase signaling pathway represents a potent negative modulator of such expression. These data support the working hypothesis that a release from the inhibitory actions of MAP kinase signaling is a prerequisite for the full potentiation of FSH-R expression and, subsequently, follicle selection into the preovulatory hierarchy.
Materials and Methods
Animals and Reagents
Single-comb white Leghorn hens (age, 25–35 wk; Creighton Bros., Warsaw, IN) laying regular sequences of six eggs or more were used in the present study. Hens were housed individually in laying batteries with free access to feed (Purina Layena Mash; Purina Mills, St. Louis, MO) and water and were exposed to a 15L:9D photoperiod, with lights-on at midnight. Individual lay patterns were monitored daily. Hens were killed by cervical dislocation 16–18 h before a midsequence ovulation, and the ovary was removed and placed in ice-cold sterile 1% NaCl saline solution for immediate use. All procedures described herein were reviewed and approved by the University of Notre Dame Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Recombinant human (rh) TGFα, TGFβ1, and IGF-I were obtained from PeproTech (Rocky Hill, NJ); rhFSH was provided by the National Hormone and Pituitary Program (Torrance, CA). The cell-permeable cAMP analogue, 8-bromo-cAMP (8-br-cAMP), was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human activin A, rh-follistatin 300 (FS), and anti-human follistatin antibody were from R&D Systems (Minneapolis, MN). The selective MAP kinase/Erk inhibitor, U0126, was purchased from BioMol (Plymouth Meeting, NJ).
Granulosa Cell Cultures
Prehierarchal follicles (diameter, 1–8 mm) were removed from the ovary and grouped according to size (diameter: 1–2, 3–5, or 6–8 mm). Granulosa layers from each follicle were collected and combined within their respective group and then dispersed for culture as previously described [11, 25]. An aliquot of cells was frozen at −70°C (T0; control), whereas the remaining cells were cultured at 40°C in an atmosphere of 95% air: 5% CO2 in six-well polystyrene culture plates (Beckton Dickinson Labware, Franklin Lakes, NJ), with a density of approximately 1 × 106 per well in 2 ml of Dulbecco modified Eagle medium (DMEM) plus 2.5% fetal bovine serum (FBS) with 0.1 mM nonessential amino acids and 1% antibiotic-antimycotic mixture (Invitrogen, Carlsbad, CA).
To demonstrate functional coupling of the FSH-R in granulosa cells during early follicular development, StAR protein was evaluated following a short-term incubation of 1 × 106 granulosa cells from follicles of 1–2, 3–5, or 6–8 mm in diameter in 12- × 75-mm polypropylene tubes (Fisher Scientific, Pittsburgh, PA) for 4 h in DMEM medium (control), 100 ng/ml of FSH, or 1 mM 8-br-cAMP [11]. Dispersed cells from the second plus third largest preovulatory follicles (F2 and F3, respectively) were included within the experimental design as a positive control. In a second experiment, granulosa cells from follicles of 6–8 mm in diameter were cultured in medium alone and collected after 1, 3, 6, 12, and 20 h to establish the extent and time frame under which FSH-R mRNA levels decline on plating.
We recently established that rhTGFβ1 and rh-activin A treatments are effective in promoting increased LH-R and FSH-R mRNA levels alone and/or in combination with FSH [22]. To examine the effects of MAP kinase signaling on FSH-R mRNA expression, granulosa cells from follicles of 6–8 mm in diameter were precultured in the absence or presence of 50 ng/ml of TGFα [11] for 30 min, then treated with TGFβ1 (10 ng/ml) or activin A (25 ng/ml) [22] in the absence or presence of FSH. To evaluate further the effects of MAP kinase/Erk signaling on FSH-R expression, granulosa cells were pretreated with 50 μM U0126 [11] for 1 h, then cultured in the presence or absence of FSH, TGFβ1, or activin A for an additional 20 h. In a separate experiment, FSH-R mRNA levels were evaluated following a 20-h culture in the absence or presence of FSH, 50 ng/ml of IGF-I [26], or a combination of these factors.
To investigate the ability of FS to modulate activin A-induced FSH-R mRNA expression and progesterone production in vitro, cells were pretreated for 1 h without or with FS (100 and 150 ng/ml). Cells were subsequently treated with activin A (25 ng/ml) plus FSH or with TGFβ1 (10 ng/ml) plus FSH, then cultured for an additional 20 h. In a related experiment, cells were pretreated with anti-human follistatin serum (FS Ab; 6 μg/ml) for 1 h, then treated with activin A plus FSH. For comparative purposes, cells were treated with TGFβ1 plus FSH in the absence of FS Ab.
Two-Step Real-Time Polymerase Chain Reaction for FSH-R
Primers and TaqMan probes specific for FSH-R and β-actin were generated using Primer Express software (Applied Biosystems, Foster City, CA) (Table 1). The FSH-R probe was labeled at the 5′ end with FAM (6-carboxyfluorescein) as the reporter dye, whereas β-actin was labeled with VIC (Applied Biosystems). Each probe had TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) quencher dye at the 3′ end. Primers and probes generated by this method were validated for real-time polymerase chain reaction (PCR) by determining the optimal amplification efficiency and primer/probe concentrations as described by the manufacturer (Applied Biosystems).
Parameters for oligonucleotide primer pairs and probes.
| Target gene | Oligonucleotide sequence | Product size (bp) |
|---|---|---|
| FSH-R | Sense:5′-TTA ATT CCT GTG CTA ACC CTT TCC-3′ | 85 |
| Antisense:5′-CCA AAC TTG CTG AAC AGA ATG AAG-3′ | ||
| Probe:5′-CCA TTT TCA CAA AGA CTT TTC GCA GGG AT-3′ | ||
| β-Actin | Sense:5′-CGA GAG AGA AAT TGT GCG TGA C-3′ | 85 |
| Antisense:5′-GCC ATC TCC TGC TCG AAA TC-3′ | ||
| Probe:5′-TCA AGG AGA GAA GCT GTG CTA CGT CGC A-3′ | ||
| TGF-β4 | Sense:5′-TGA GGA TGC TGC GGC AAA AG-3′ | 85 |
| Antisense:5′-GGT GCT TCT TGG CAA TGC TCT G-3′ | ||
| TGFβ-RI | Sense:5′-GAC AGA TGG GCT TTG TTT TAC CTC-3′ | 85 |
| Antisense:5′-TCT TCT TCA CTT GGC ACA CGA TG-3′ | ||
| TGFβ-RII | Sense:5′-TGG ACA TTG TTG TTG GCA AAG G-3′ | 85 |
| Antisense:5′-TTC TCA AGG TTC ATC CTG GAC TCC-3′ |
| Target gene | Oligonucleotide sequence | Product size (bp) |
|---|---|---|
| FSH-R | Sense:5′-TTA ATT CCT GTG CTA ACC CTT TCC-3′ | 85 |
| Antisense:5′-CCA AAC TTG CTG AAC AGA ATG AAG-3′ | ||
| Probe:5′-CCA TTT TCA CAA AGA CTT TTC GCA GGG AT-3′ | ||
| β-Actin | Sense:5′-CGA GAG AGA AAT TGT GCG TGA C-3′ | 85 |
| Antisense:5′-GCC ATC TCC TGC TCG AAA TC-3′ | ||
| Probe:5′-TCA AGG AGA GAA GCT GTG CTA CGT CGC A-3′ | ||
| TGF-β4 | Sense:5′-TGA GGA TGC TGC GGC AAA AG-3′ | 85 |
| Antisense:5′-GGT GCT TCT TGG CAA TGC TCT G-3′ | ||
| TGFβ-RI | Sense:5′-GAC AGA TGG GCT TTG TTT TAC CTC-3′ | 85 |
| Antisense:5′-TCT TCT TCA CTT GGC ACA CGA TG-3′ | ||
| TGFβ-RII | Sense:5′-TGG ACA TTG TTG TTG GCA AAG G-3′ | 85 |
| Antisense:5′-TTC TCA AGG TTC ATC CTG GAC TCC-3′ |
Parameters for oligonucleotide primer pairs and probes.
| Target gene | Oligonucleotide sequence | Product size (bp) |
|---|---|---|
| FSH-R | Sense:5′-TTA ATT CCT GTG CTA ACC CTT TCC-3′ | 85 |
| Antisense:5′-CCA AAC TTG CTG AAC AGA ATG AAG-3′ | ||
| Probe:5′-CCA TTT TCA CAA AGA CTT TTC GCA GGG AT-3′ | ||
| β-Actin | Sense:5′-CGA GAG AGA AAT TGT GCG TGA C-3′ | 85 |
| Antisense:5′-GCC ATC TCC TGC TCG AAA TC-3′ | ||
| Probe:5′-TCA AGG AGA GAA GCT GTG CTA CGT CGC A-3′ | ||
| TGF-β4 | Sense:5′-TGA GGA TGC TGC GGC AAA AG-3′ | 85 |
| Antisense:5′-GGT GCT TCT TGG CAA TGC TCT G-3′ | ||
| TGFβ-RI | Sense:5′-GAC AGA TGG GCT TTG TTT TAC CTC-3′ | 85 |
| Antisense:5′-TCT TCT TCA CTT GGC ACA CGA TG-3′ | ||
| TGFβ-RII | Sense:5′-TGG ACA TTG TTG TTG GCA AAG G-3′ | 85 |
| Antisense:5′-TTC TCA AGG TTC ATC CTG GAC TCC-3′ |
| Target gene | Oligonucleotide sequence | Product size (bp) |
|---|---|---|
| FSH-R | Sense:5′-TTA ATT CCT GTG CTA ACC CTT TCC-3′ | 85 |
| Antisense:5′-CCA AAC TTG CTG AAC AGA ATG AAG-3′ | ||
| Probe:5′-CCA TTT TCA CAA AGA CTT TTC GCA GGG AT-3′ | ||
| β-Actin | Sense:5′-CGA GAG AGA AAT TGT GCG TGA C-3′ | 85 |
| Antisense:5′-GCC ATC TCC TGC TCG AAA TC-3′ | ||
| Probe:5′-TCA AGG AGA GAA GCT GTG CTA CGT CGC A-3′ | ||
| TGF-β4 | Sense:5′-TGA GGA TGC TGC GGC AAA AG-3′ | 85 |
| Antisense:5′-GGT GCT TCT TGG CAA TGC TCT G-3′ | ||
| TGFβ-RI | Sense:5′-GAC AGA TGG GCT TTG TTT TAC CTC-3′ | 85 |
| Antisense:5′-TCT TCT TCA CTT GGC ACA CGA TG-3′ | ||
| TGFβ-RII | Sense:5′-TGG ACA TTG TTG TTG GCA AAG G-3′ | 85 |
| Antisense:5′-TTC TCA AGG TTC ATC CTG GAC TCC-3′ |
Reverse-transcription (RT) cDNA synthesis reactions were performed using the TaqMan Gold RT-PCR Kit (Applied Biosystems) under the conditions described by the manufacturer. For real-time PCR, primers and probes were added to 50 μl of total reaction volume using reagents provided in the TaqMan PCR Core Reagents Kit (Applied Biosystems). Final concentrations of the sense and antisense FSH-R primers were adjusted to 900 nM, whereas β-actin primers were set to 50 nM. Both FSH-R and β-actin probe concentrations were 50 nM. Reactions were completed on the ABI 7700 Thermocycler (Applied Biosystems). Conditions were set to the following parameters: 2 min at 50°C (AmpErase activation), 10 min at 95°C (AmpliTaq Gold DNA polymerase activation), and 40 cycles each of 15 sec at 95°C (melting) and 1 min at 60°C (annealing/extension). The CT (the cycle number at which the fluorescence exceeds the threshold level) was determined for each reaction (run in triplicate) using the Sequence Detection software (version 0.1.6.3, Applied Biosystems), and quantification was completed using the ΔΔCT method [27]. Briefly, the FSH-R CT was determined for each sample, then normalized to the β-actin CT from the same sample (β-actin CT subtracted from the FSH-R CT yields the ΔCT). These values were then compared to control levels using the |${2^{ - \Delta \Delta \;{C_{\rm{T}}}}}$| method and expressed as the fold-difference compared to an appropriate reference tissue.
Amplification of Chicken TGFβ4, TGFβ Receptor Type I (ALK5), and TGFβ Receptor Type II from Ovarian Tissues by RT-PCR
To establish the potential for paracrine and/or autocrine signaling by TGFβ within the ovary, the chicken equivalent to mammalian TGFβ1, TGFβ4 [28, 29], was amplified from reversed-transcribed RNA (Reverse Transcription System; Promega, Madison, WI) collected from ovarian stromal tissue plus granulosa and theca tissue from follicles of 6–8 mm in diameter. Primers specific for the chicken TGFβ4 sequence were derived from GenBank submission M31160 (Table 1). Amplification conditions included an initial denaturation for 3 min at 94°C, followed by a 45-sec denaturation at 94°C, a 30-sec annealing step at 56°C, and a 90-sec extension at 72°C for 35 cycles using Taq DNA polymerase (Invitrogen).
Primers for TGFβ receptor type I (TGFβ-RI; also called ALK5; GenBank accession no. D14460) and TGFβ receptor type II (TGFβ-RII; GenBank accession no. NM 205428) were generated to verify expression of both the TGFβ-RI and -RII specifically within the granulosa layer from follicles of 6–8 mm in diameter. Amplification conditions for both products included an initial denaturation for 3 min at 94°C, followed by 45-sec denaturation at 94°C, a 30-sec annealing step at 51°C, and a 90-sec extension at 72°C for 35 cycles. All PCR products were subsequently subcloned using the TOPO TA Cloning Kit for Sequencing (Invitrogen) and sequenced for verification of nucleic acid identity.
Western Blot Analysis of StAR Protein
Analysis of StAR protein was conducted as previously described [11]. The anti-StAR serum was generously provided by Dr. D.B. Hales (University of Illinois, Chicago, IL). The α-tubulin antiserum used for standardization was obtained from Sigma-Aldrich, and the horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody was from Pierce Endogen (Rockford, IL).
Northern Blot Analysis of FSH-R mRNA
Levels of FSH-R mRNA were evaluated by Northern blot analysis using a chicken FSH-R cDNA probe as previously described [16]. Although this gonadotropin receptor has been reported to express multiple transcripts, only the predominant 2.5-kilobase transcript was quantified in the present study [16]. Images were visualized on phosphor screens using the Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and analyzed using the ImageQuant data reduction system (Molecular Dynamics, Inc., Sunnyvale, CA). All FSH-R data were standardized to 18S rRNA.
Progesterone Radioimmunoassay
Progesterone in media samples was measured by radioimmunoassay as previously described [30] and expressed as ng/ml (mean ± SEM) for the combined replicate experiments.
Data Analysis
Experiments were independently replicated a minimum of three times unless otherwise stated. Standardized values were expressed as a fold-difference (mean ± SEM) versus freshly collected (T0) controls. Data were analyzed by one-way ANOVA without including data from the control group (arbitrarily set to 1.0) and the Fisher protected least significant difference multiple-range test.
Results
FSH-R Expression in Prehierarchal Follicles
Real-time PCR analysis determined that FSH-R mRNA is expressed within pooled granulosa layers from slow-growing follicles as early as the 1- to 2-mm stage of growth, that its highest levels occur in follicles of 6–8 mm in diameter and recently selected follicles of 9–12 mm in diameter, and that levels subsequently decrease in large preovulatory follicles (Fig. 1, top). That this transcript is translated into a functional receptor protein and is coupled to an active signaling pathway at each stage of follicular development investigated is demonstrated by the finding that FSH treatment induced StAR protein expression within a 4-h treatment period (Fig. 1, bottom). Analysis of FSH-R mRNA in granulosa layers from individual follicles of 6– 8 mm in diameter determined that the relative levels of expression were similar in all follicles, with the exception that one of such follicles from each hen showed elevated levels (Fig. 2).
Top) Relative expression of FSH-R mRNA within granulosa tissue from prehierarchal and preovulatory follicles as determined by real-time PCR. Freshly collected granulosa cell layers were collected and pooled from prehierarchal follicles of 1–2, 3–5, and 6–8 mm in diameter. Single granulosa layers were collected from follicles of 9–12 mm in diameter (the most recently selected) and from F2 (second largest) and F1 (largest) preovulatory follicles. Data were normalized to β-actin mRNA and expressed as the mean fold-difference ΔΔCT ± SEM (see Materials and Methods) compared to F1. A, B, and C: P < 0.05, n = 3. Bottom) Western Blot analysis of StAR protein in dispersed granulosa cells from follicles of 1–2, 3–5, and 6–8 mm in diameter and from F2 and F3 follicles following a 4-h incubation with rhFSH (100 ng/ml) or 8-br-cAMP (8-br; 1 mM). Levels of α-tubulin are presented to indicate loading of total protein. This experiment was repeated two additional times with similar results
Relative expression of FSH-R mRNA in freshly collected, individual granulosa layers from (prehierarchal) follicles of 6–8 mm in diameter as determined by real-time PCR. Results from a total of 13 follicles from each of three hens are presented. Data were normalized to β-actin mRNA and expressed as the fold-difference versus the smallest follicle in this group (set to 1.0; not shown)
FSH-R mRNA Expression in Culture
Levels of FSH-R mRNA in granulosa cells collected from follicles of 6–8 mm in diameter decreased significantly after 12 h (by 40%) and 20 h (by 60%) in culture (P < 0.05 for both vs. freshly collected cells) (Fig. 3, top). Neither FSH nor IGF-I treatment alone prevented this decline (P > 0.2 compared to control cultured cells); however, the combination of FSH plus IGF-I resulted in increased FSH-R mRNA compared to control cultured cells. These levels were not different from those from freshly collected (T0) cells (Fig. 3, bottom).
Top) Levels of FSH-R mRNA progressively decrease in granulosa cells from follicles of 6–8 mm in diameter cultured for 1–20 h. Data are standardized to 18S rRNA and expressed as the mean fraction ± SEM compared to levels in freshly collected and dispersed (0-h) cells. *P < 0.05 versus 0 h, n = 3. Bottom) Effects of IGF-I plus FSH treatment on follicle granulosa cell FSH-R levels in follicles of 6–8 mm in diameter. The T0 represents freshly collected cells. A and B: P < 0.05; ns: P > 0.35 by paired t-test, n = 3
TGFβ and Receptor Expression in Ovarian Tissues
Chicken TGFβ4 was amplified from ovarian stromal tissue as well as granulosa and theca tissue collected from follicles of 6–8 mm in diameter (Fig. 4, top). Moreover, both the TGFβ-RI and -RII subtype cDNAs were amplified from granulosa cells collected from prehierarchal follicles of 6–8 mm in diameter (Fig. 4, bottom).
Top) Amplification of a 321-base pair (bp) chicken TGFβ4 cDNA from ovarian stromal (Str) tissue and granulosa (Gr) and theca (Th) tissue from follicles of 6–8 mm in diameter by PCR. Bottom) Amplification of TGFβ-RI (ALK5; 360-bp) and -RII (577-bp) cDNA from granulosa cells collected from follicles of 6–8 mm in diameter. Left lane represents DNA size markers in multiples of 100 bp; a minus represents the negative control (absence of reverse-transcribed template) for each respective amplification. Each experiment was independently replicated three additional times with similar results
Modulation of FSH-R mRNA Expression by MAP Kinase Signaling
As recently established [22], treatment with rhTGFβ1 increases FSH-R mRNA levels in cultured granulosa cells from follicles of 6–8 mm in diameter, whereas the combination of TGFβ1 plus FSH promotes a further increase. Significantly, pretreatment with TGFα, an activator of MAP kinase/Erk signaling [23], completely blocks TGFβ1-induced FSH-R mRNA expression in both the absence and the presence of FSH (Fig. 5, top). By comparison, activin A treatment increases FSH-R mRNA levels in the presence of FSH, whereas pretreatment with TGFα prevents activin A plus FSH-induced receptor expression (Fig. 5, bottom). Treatment with a selective MAP kinase/Erk inhibitor, U0126, for 20 h significantly increased levels of FSH-R mRNA compared to those in control cultured cells, and coculture with FSH, activin A, or TGFβ1 further enhanced this effect (Fig. 6).
Northern blot analysis of FSH-R mRNA in granulosa cells from follicles of 6–8 mm in diameter cultured for 20 h without (FBS control) or with rhTGFβ1 (Top) or rh-activin A (Bottom). Additional treatments included FSH (100 ng/ml) and/or TGFα (50 ng/ml). Data are standardized to 18S rRNA and expressed as the mean fold-difference ± SEM compared to levels in freshly collected cells (not shown) from four to six replicate experiments. A, B, and C: P < 0.05
Inhibition of MAP kinase/Erk signaling with the pharmacologic inhibitor, U0126 (U0; 50 μM), increases FSH-R mRNA levels in granulosa cells from follicles of 6–8 mm in diameter after 20 h in culture, and this increase is enhanced by coculture with FSH (100 ng/ml) and TGFβ1 (10 ng/ml) but not with activin A (Act; 25 ng/ml). Data are standardized against 18S rRNA and expressed as the mean fold-difference ± SEM versus levels in freshly collected cells (not shown). A, B, and C: P < 0.01 from three replicate experiments
Follistatin Inhibition of Activin-Induced FSH-R Expression
Follistatin treatment had no effect on FSH-R mRNA levels or progesterone production compared to control cultured cells, but it significantly inhibited activin A plus FSH-induced effects on each parameter at a dose of 150 ng/ml (Fig. 7). By comparison, FS did not alter FSH-R mRNA levels or progesterone production following treatment with TGFβ plus FSH.
Preculture of granulosa cells from follicles of 6–8 mm in diameter with rh-follistatin (FS; 150 ng/ml) inhibits the stimulatory effects of FSH plus activin A (Act) treatment on FSH-R mRNA (Top) and progesterone production (Bottom), but it does not inhibit the stimulatory effects of FSH plus TGFβ1. Data for FSH-R mRNA are standardized to 18S rRNA and expressed as the mean fold-difference ± SEM compared to levels in freshly collected cells (not shown). A, B, C, a, b, and c: P < 0.05, from three replicate experiments
Following pretreatment with an FS antiserum, activin A significantly increased FSH-R mRNA to levels comparable with those of TGFβ-treated cells in both the absence and the presence of FSH. The FS antiserum did not affect TGFβ plus FSH-induced FSH-R (Fig. 8, top). Not unexpectedly, activin A alone failed to induce progesterone production either in the absence or the presence of FS antiserum (data not shown). However, following treatment with activin A plus FSH, the presence of FS antiserum enhanced progesterone production to a level comparable with that induced by TGFβ plus FSH (Fig. 8, bottom).
Immunoneutralization of follistatin with a polyclonal follistatin antiserum (6 μg/ml) promotes activin A-induced FSH-R mRNA expression both alone and when granulosa cells from follicles of 6–8 mm in diameter are cocultured with FSH (Top). In addition, follistatin immunoneutralization (Ab) enhances progesterone production in response to culture with activin A plus FSH (Bottom). Data for FSH-R mRNA are standardized to 18S rRNA and expressed as the mean fold-difference ± SEM compared to levels in freshly collected cells (not shown). *P < 0.05, from three replicate experiments
Discussion
To our knowledge, the present results are among the first to establish endocrine and intrafollicular mechanisms that regulate FSH-R mRNA expression in undifferentiated granulosa cells from hen prehierarchal follicles. Moreover, the release from inhibitory MAP kinase signaling that results in increased FSH-R expression within a single follicle of 6–8 mm in diameter is proposed to represent the earliest event during the process of hen follicle selection described to date. A working model to describe changes in granulosa cell signaling during the prehierarchal to preovulatory follicle transition is presented in Figure 9.
![Working model to describe proposed changes in granulosa cell signaling during the transition from the prehierarchal to preovulatory follicle stage of development. A) Both TGFβ-RI and -RII (see Fig. 4) and activin [2] receptors are expressed within the granulosa cell layer. The autocrine/paracrine factor, TGFβ, can bind to its respective receptors, but the bioactivity of activin is modulated by follistatin. We propose that active induction of FSH-R expression by TGFβ (and, possibly, activin) is precluded in all but one prehierarchal follicle per day by one or more EGF-family ligand binding to one or more ErbB-receptor isoform and the tonic activation of MAP kinase/Erk signaling. The sites of MAP kinase/Erk-mediated inhibition include Smad2 phosphorylation [22] and inhibition of the protein kinase A/cAMP signaling pathway both before and after cAMP formation [35]. B) Pharmacologic inhibition of MAP kinase signaling by the selective inhibitor, U0126, enables TGFβ (and activin) signaling via Smad and results in enhanced FSH-R expression (1). Elevated FSH-R expression subsequently facilitates FSH-induced LH-R expression (2), P450scc expression, and StAR expression and, as a consequence, progesterone production (3). Factors that attenuate MAP kinase/Erk signaling in vivo in the single follicle selected into the preovulatory hierarchy per day are currently under investigation. (See text for supportive information and further explanation of pathways)](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/biolreprod/72/3/10.1095_biolreprod.104.033902/1/m_bire-72-03-18-f09.jpeg?Expires=1713195097&Signature=Vs1SZ1XdAiqDMcez-uarkfmRZ9AnxBb6OUJ3mdpHYKKpJnQHFSL0aA7BQJlSwhxrefPEjeUNm8WJ5E3Yw7XAzvMyQRoOeOHko2t9Yr6sLo4gJcWRSmLSkAzIoj22XqTPUo5f5eJveDi58eoQWcqTyibNy3U~Q5rmGaeqZQeqaLelLaHHb4FLr6JRw~ynQ5uv76pEejyvZNpfqydORtWBw2~UWcY~0TPnEoN4QLaPBaaq5ZchDpGSLwhA91s4h4VHjc1SaLxdVJFMjWy8~EfzZvmpjCq350~S7jrSDS0pOSWkzNrzVuGpdJlIPneYhrLgWxuGKj~Tnb0voeqAtXXT5Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Working model to describe proposed changes in granulosa cell signaling during the transition from the prehierarchal to preovulatory follicle stage of development. A) Both TGFβ-RI and -RII (see Fig. 4) and activin [2] receptors are expressed within the granulosa cell layer. The autocrine/paracrine factor, TGFβ, can bind to its respective receptors, but the bioactivity of activin is modulated by follistatin. We propose that active induction of FSH-R expression by TGFβ (and, possibly, activin) is precluded in all but one prehierarchal follicle per day by one or more EGF-family ligand binding to one or more ErbB-receptor isoform and the tonic activation of MAP kinase/Erk signaling. The sites of MAP kinase/Erk-mediated inhibition include Smad2 phosphorylation [22] and inhibition of the protein kinase A/cAMP signaling pathway both before and after cAMP formation [35]. B) Pharmacologic inhibition of MAP kinase signaling by the selective inhibitor, U0126, enables TGFβ (and activin) signaling via Smad and results in enhanced FSH-R expression (1). Elevated FSH-R expression subsequently facilitates FSH-induced LH-R expression (2), P450scc expression, and StAR expression and, as a consequence, progesterone production (3). Factors that attenuate MAP kinase/Erk signaling in vivo in the single follicle selected into the preovulatory hierarchy per day are currently under investigation. (See text for supportive information and further explanation of pathways)
Analysis of FSH-R mRNA by real-time PCR established that the receptor transcript is expressed in granulosa layers from follicles as small as 1–2 mm in diameter (Fig. 1). The profiles of expression from pooled granulosa layers in prehierarchal follicles of 3–5 and 6–8 mm in diameter and from individual layers of preovulatory follicles were virtually identical to those previously determined by Northern blot analysis [16]. Significantly, the ability of FSH treatment to rapidly induce StAR protein expression indicates both that this transcript is translated and that the receptor is functionally coupled to stimulatory G proteins. We recently established that StAR mRNA and protein expression in hen granulosa cells is up-regulated primarily, if not exclusively, by the protein kinase A signaling pathway [11, 23]. Thus, although a functional FSH-R is expressed in granulosa cells at all stages of prehierarchal follicular development and is capable of inducing the expression of genes associated with differentiation (e.g., LH-R, P450scc, and StAR), the absence of expression for those genes in vivo suggests the presence of tonic inhibitory mechanisms.
In contrast to the significant increase in LH-R mRNA expression that occurs after a 20-h culture of granulosa cells from follicles of 6–8 mm in diameter [11], levels of FSH-R mRNA decline by 60% under comparable conditions (Fig. 3). The increase in LH-R mRNA is attributed to a release from inhibitory signals that block premature LH-R expression [11], but the decline of FSH-R mRNA levels is proposed to reflect the removal or disruption of supportive intrafollicular factors. In mammals, IGF-I represents one such intrafollicular factor that is suggested to “sensitize” granulosa cells to FSH [31]. However, given that IGF-I plus FSH treatment has been reported previously not to enhance LH-R, StAR, or P450scc expression or to promote steroidogenesis in cultured granulosa cells from prehierarchal follicles [11, 32], it is possible that a primary action of IGF-I is to decrease FSH-R mRNA turnover, as has been reported in the rat [4].
It also is hypothesized that the homolog to mammalian TGFβ1, chicken TGFβ4, can serve as an intrafollicular factor to support FSH-R expression. Both TGFβ and the related activins induce their biological effects by forming a heterodimeric complex with two type II and two type I serine and threonine kinase receptors. Activation of the type I receptor results in the phosphorylation and activation of one or more receptor Smads (e.g., Smad2 and Smad3), which then associate with a co-Smad (e.g., Smad4) and are subsequently translocated to the nucleus to initiate gene transcription [2]. Recently, it was established that rhTGFβ1 treatment of hen granulosa cells induces Smad2 phosphorylation [22], yet direct evidence for TGFβ4, TGFβ-RI, and TGFβ-RII expression within hen granulosa cells had yet to be documented. The identification of TGFβ4 mRNA in follicular (granulosa and theca) and stromal tissues combined with the localization of TGFβ-RI and -RII within granulosa cells (Fig. 4) provides support for TGFβ4, in addition to previously identified TGFβ2 and TGFβ3 [33], to regulate follicular development.
The ability of TGFα to block TGFβ-mediated FSH-R mRNA expression (Fig. 5) has been reported in the rat, but the cellular mechanisms of this action were not identified [34]. This ability of TGFα to inhibit the differentiation-inducing activity of TGFβ is consistent with its inhibitory effects on FSH- and TGFβ-induced LH-R mRNA expression in cultured hen granulosa [22]. Also, TGFα prevents the sensitizing effects of TGFβ and activin cotreatments with FSH on FSH-R mRNA expression. The mechanism of TGFα inhibitory effects is attributed to the attenuation of protein kinase A signaling, both before and after cAMP formation [35], as well as the inhibition of Smad2 phosphorylation [22]. Moreover, these inhibitory effects are related specifically to MAP kinase signaling, because the pharmacologic inhibitor, U0126, blocks TGFβ-induced Erk-P (but not protein kinase B signaling [26]) and promotes FSH-R mRNA expression both alone and in combination with FSH or TGFβ treatment (Fig. 6).
Interestingly, activin A treatment failed to reliably induce FSH-R (Figs. 5 and 6) or LH-R [22] mRNA expression in follicle granulosa cells of 6–8 mm in diameter cultured for 20 h. In light of recent data demonstrating that granulosa cells at this stage of follicular development produce the highest levels of FS observed during follicular development, it was reasoned that this binding protein could bioneutralize the actions of activin [36]. This proposal is supported by the combined findings that exogenously added rh-FS prevents the sensitizing effects of activin A plus FSH treatment on both FSH-R mRNA expression and progesterone production (Fig. 7), whereas the addition of an anti-FS serum facilitates the effects of activin (both alone and when combined with FSH) on these end points (Fig. 8). Significantly, because FS binding is specific to the β subunit of activins and inhibins [37], such treatment had no inhibitory effects on TGFβ-mediated differentiation (Fig. 7).
Real-time PCR analysis of FSH-R mRNA in granulosa layers from individual (prehierarchal) follicles of 6–8 mm in diameter also revealed that a single—but not necessarily the largest—follicle within this cohort expresses elevated levels relative to the remaining follicles (Fig. 2). An implication of this finding is that the up-regulation of FSH-R mRNA expression occurs following the alleviation of tonic inhibitory signaling. Taken collectively, the data presented suggest that constitutive MAP kinase signaling within granulosa cells from prehierarchal follicles tonically suppresses FSH-R expression. It is hypothesized that the selective attenuation of MAP kinase signaling within a single follicle of 6–8 mm in diameter per day enables intrafollicular factors, such as TGFβ, activin, and/or IGF-I, to actively promote expression of FSH-R. The mechanisms by which this can occur have not yet been elucidated, but one possibility would include a down-regulation of autocrine/paracrine factors that activate MAP kinase signaling, including TGFα, EGF, or alternative members of the EGF ligand superfamily (e.g., epiregulin and betacellulin). In turn, elevated FSH/FSH-R signaling promotes expression of LH-R, P450scc, and StAR and the initiation of progesterone production. Concomitant with these effects on differentiation, enhanced capacity for FSH-R and, subsequently, LH-R signaling via protein kinase A results in the expression of antiapoptotic proteins that enhance both granulosa cell viability and preovulatory follicle survival.
Finally, the selection of a dominant follicle during the bovine estrous cycle is reported to occur in the absence of detectable changes in gonadotropin receptor (FSH-R or LH-R) expression [38]. Accordingly, results from the cow compared to those in the present study suggest that the observed increase in FSH-R expression in both models clearly results from a previous selection mechanism, which in the hen is proposed to include the alleviation of inhibitory MAP kinase signaling.
Acknowledgments
We thank Dr. B. Hales for the gift of StAR antiserum and acknowledge technical assistance from J. Martin, J. Bridgham, C. Ratajczak, and M. Haugen.
References
Author notes
Supported by the NSF (IBN01-31185 to A.L.J.).
