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RESEARCH |
School of Biological Sciences, University of Reading, Whiteknights, Reading, Berkshire RG6 6AJ, UK
Correspondence should be addressed to P G Knight; Email: p.g.knight{at}reading.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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LH and FSH play key roles in regulating ovarian dynamics through stimulation of steroidogenesis, production of peptide hormones (including Inh and Act), cell proliferation (Bahr & Johnson 1984, Tilly et al. 1991, Johnson 1993, Lovell et al. 2002a) and apoptosis (Johnson et al. 1999) through specific LH and FSH receptors (Johnson et al. 1996, You et al. 1996). Inh and Act have been implicated as direct (paracrine/autocrine) and indirect (endocrine) regulators of ovarian development in birds (Davis et al. 2001, Johnson et al. 2004; review: Knight et al. 2005) and mammals (reviews: Knight & Glister 2003, Philips 2005).
The action of Act is elicited through a well-characterised signal transduction cascade (review Abe et al. 2004). Act can bind with high affinity to a type-II cell-surface receptor (ActRII) of which two have been cloned in avian (Ohuchi et al. 1992, Nohno et al. 1993, Stern et al. 1995) and mammalian species (Donaldson et al. 1992, Stern et al. 1995). Binding promotes the recruitment of activin type-I receptors (ActRI) to the complex which is then able to mediate ligand-dependent signalling by Smads (Knight & Glister 2003, Abe et al. 2004). ßgly (also known as transforming growth factor ß (TGFß) III receptor) is a membrane-bound proteoglycan, which can bind inhibin and increase its affinity for ActRII (Lewis et al. 2000). This Inh/ßgly/ActRII complex is thought to prevent binding of Act to the ActRII and thereby block the recruitment of ActRI and activin-stimulated signal transduction (Phillips & Woodruff 2004).
During follicle development in the laying hen, changes have been observed in Inh/Act subunit mRNA expression (Davis & Johnson 1998, Knight et al. 2005), InhA, InhB, ActA and follistatin protein content (FS; Lovell et al. 1998, 2003) and expression of mRNAs for ActR (type I, IIA, IIB; Lovell et al. 2006) and ßGly (Sweeney & Johnson 2005, Lovell et al. 2006). ActRIIA mRNA expression has also been shown (by Northern blot) to change throughout follicle development in broiler breeder hens (Slappey & Davis 2003). This suggests a functional involvement of the intraovarian Inh–Act system in follicle progression. Similarly, marked differences in steroidogenic capacity (Etches & Duke 1984, Kato et al. 1995) and mRNA expression of gonadotrophin receptors (LH-R, FSH-R; Johnson et al. 1996, You et al. 1996) have been recorded within both GCs and TCs during follicle development in birds.
The observation that treatment of hen preovulatory follicle GCs with ActA greatly enhanced gonadotrophin-induced InhA and progesterone (P4) release suggests a functional interaction between gonadotrophin-dependent and Act-dependent signalling pathways (Lovell et al. 2002a). In support of this, ActA was recently shown to enhance the expression of both FSH-R and LH-R in GC from preovulatory follicles (Johnson et al. 2006). There are other reports (Johnson et al. 2004, 2006, Woods & Johnson 2005) that ActA augments gonadotrophin receptor mRNA expression in granuolsa cells from prehierarchical (small yolky) hen follicles.
With the above findings in mind, the primary aim of the present study was to test the hypothesis that pituitary gonadotrophins differentially modulate granulosal expression of ActRs and ßgly mRNAs at two key stages of folliculogenesis: prehierarchical and preovulatory. In the absence of comparable studies on avian TC, we also conducted parallel experiments to determine the effects of gonadotrophins on TC from both prehierarchical and preovulatory follicles.
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Materials and Methods |
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Isolation of granulosa and theca cells
Granulosa and theca layers were separated under aseptic conditions (Gilbert et al. 1977). In brief, granulosa and thecal tissue derived from a given follicle position in different hens were combined (creating separate granulosa and theca follicle pools from the 6–8 mm and the F1 follicles). Pooled tissues were enzymatically dispersed as described by Lovell et al. (2002b) except that thecal tissue was dispersed for a further 15 min (total: 45 min). It should be noted that the theca tissue collected for dispersion consists of the theca interna and theca externa layers and therefore all the different cell types these layers may contain in vivo. The number of viable cells, estimated by trypan blue exclusion (Sigma UK Ltd), was invariably >90% for both the cell types. Isolated cells were diluted in incubation medium (medium 199 with 25 mM Hepes, 0.01% (v/v) L-glutamine and 1% (v/v) antibiotic solution) containing 2% (v/v) charcoal-stripped fetal calf serum (Sigma UK Ltd) and divided into aliquots in 24-well plates (Falcon 3047; Becton Dickinson Labware, Franklin Lakes, NJ, USA) at 5x105 viable cells per well. The cells were maintained for 24 h at 39 °C and the unattached cells were aspirated off and the adherent cells were washed three times with 1 ml serum-free incubation medium. All further incubations were done in serum-free conditions. Incubation buffer (1 ml) and test treatments were added to the appropriate wells. The cells were incubated for 2x24 h treatment periods with the media replenished with the appropriate test treatments after 24 h. At termination of the culture, the media were removed and stored at –20 °C and the cell monolayers were washed three times with PBS (Oxoid Ltd, Basingstoke, Hampshire, UK) before lysis in Tri-reagent (Sigma UK Ltd).
Treatments
Stock solutions of ovine (o)LH (NIDDK-oLH-25) and oFSH (NIDDK-oFSH-19-SIAPH) were prepared at 10 µg/ml in HBSS containing 0.3% (v/v) BSA (fraction V; Sigma UK Ltd). Before culture, the treatments were diluted in the above incubation medium, to a 40x treatment concentration and filter sterilised using a 0.2 µM Millipore filter (FlowPore D; ICN Biomedicals Ltd, Basingstoke, Hampshire, UK). Both gonadotrophins were tested at final concentrations of 1, 10 and 100 ng/ml.
RNA purification and cDNA synthesis
RNA was purified from cell monolayers according to the standard Tri-reagent protocol (Sigma UK Ltd). The final RNA pellet was resuspended in 100 µl nuclease-free water containing RNA Secure (Ambion, Huntington, Cambridgeshire, UK) and then treated with RNase-free DNAse (15 min at 37 °C; RQ1, Promega) to remove potential genomic DNA contamination. The RNA preparation was re-purified using 15 volumes Tri-reagent. The resultant purified RNA was resuspended in 50 µl nuclease-free water containing RNA Secure.
RNA was quantified on a spectrophotometer (Gene-Quant, GE Healthcare UK Ltd, Amersham) and cDNA was synthesised using ImProm-II Reverse Transcriptase (Promega; used according to the Manufacturers instructions) with 1 µg RNA, 0.5 µg Random Hexamer Primers (MWG-Biotech, Covent Garden, London, UK), dNTPS (0.5 mM Final; Promega) and 0.5 µl RNase-inhibitor (40 U/µl; Ambion) per reaction. cDNA synthesis was terminated by heat-inactivation (15 min at 70 °C). cDNA samples were treated with 1 µl RNase cocktail (0.5 U/µl RNase A and 20 U/µl RNase T1; Ambion) and 0.5 µl RNase H (40 U/µl; Ambion), which specifically degrades the RNA in RNA:DNA hybrids. A 1 µl aliquot of cDNA was removed for estimation using a fluorometric assay (Oligreen ssDNA Quantification assay; Molecular Probes Inc., Paisley, Renfrewshire, UK).
Quantitative PCR
Duplicate Q-PCRs were carried out using 1 µl RT reaction product or 1 µl standard (from 200 to 1.56 amol/µl; standard oligonucleotides were custom-synthesised by Sigma-Genosys), in a volume of 25 µl containing 12.5 µl master mix with 1 µl ROX dye (Abgene, Epsom, Surry, UK), 2 µl forward and reverse primers were each added (final concentration: 300–900 nM), 1 µl probe (final concentration: 100–200 nM) and 5.5 µl nuclease-free water. All forward and reverse primer working concentrations were 900 nM except the forward primer for GAPDH (300 nM). Probe working concentrations were 200 nM except for LH-R (150 nM), FSH-R (150 nM) and GAPDH (100 nM). The samples were processed for 40 cycles using an ABI Prism 7700 Sequence detector (Applied Biosystems, Warrington, Cheshire, UK) with the thermal cycler conditions; stage 1: 50 °C/2 min, stage 2: 95 °C/15 min, stage 3: 40 cycles of 95 °C/15 s and 60 °C/1 min. TaqMan primers and probes were designed to target mRNA sequences based on criteria set by Applied Biosystems. Probes were 5 '-modified with 6-FAM and 3'-modified with TAMRA. Primer and probe sequences and target mRNA accession numbers are presented in Table 1
. Intra- and inter-plate coefficients of variation (CV) for each Q-PCR assay (ActRI, ActRIIA, ActRIIB, LHR, FSHR, betaglycan and GAPDH) were between 0.9–1.5% and 7.4–10.2% respectively. Intra-assay CV values were based on independent (n=30) Ct sample values across a single Q-PCR plate, whereas inter-assay CVs were based on the calculated concentrations of pre-aliquoted quality control samples tested on independent Q-PCR plates (n=4).
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- and ßA-subunits (Muttukrishna et al. 1994). This assay has been validated for domestic fowl as described by Lovell et al.(1998). Recombinant human InhA was used as an assay standard (detection limit 2 pg/well). The ELISA showed minimal (<0.3%) cross-reaction with bovine inhibin -subunit (200 ng/ml), rh inhibin-B (10 ng/ml), activin-A (10 ng/ml), follistatin (20 ng/ml). When tested at 1, 10 and 100 ng/ml, the ovine FSH and LH preparations used in the study showed no detectable cross-reaction (<0.002%) in the inhA ELISA. P4 and oestradiol (E2) concentrations were determined by direct RIA as described by Sauer et al.(1986) and Tannetta et al.(1998) respectively. The detection limits of the assays were 8 and 1.5 pg/ml respectively. Intra- and inter-assay coefficients of variation for each assay were <10%. Attempts were made to quantify secretion of InhB, ActA and FS in selected GC and TC culture experiments (Methods as described in Lovell et al. 1998, 2000, 2003). However, levels were below the detection limit of each assay (15 pg/well, 50 pg/well and 0.6 ng/well respectively).
Statistical analysis
One-way ANOVA was used in conjunction with post hoc Fisher’s protected least significant difference (PLSD) test to evaluate treatment effects on levels of mRNA encoding receptors and on hormone release by each of the different follicle GC and TC populations (6–8 mm and F1 follicle). Two-way ANOVA was used to make comparisons between the effects of treatments on the different cell populations. Levels of mRNA for each receptor were normalised to GAPDH. A value of P<0.05 was considered significant. There was no significant difference (ANOVA; P>0.05) in GAPDH mRNA transcript levels between the GC and the TC cultures of different follicle classes or following treatment with LH or FSH. Hormone secretion results presented are for the final 24 h period of culture. In a given experiment, treatments were tested using triplicate wells which were pooled for RNA isolation within a given culture and each experiment was repeated using at least three independent cultures (utilising independently isolated cell preparations). All values presented are means ± S.E.M. (with n indicating the number of independent cultures).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Effect of LH and FSH on mRNA for ActRI, ActRIIA, ActRIIB, ßgly, LH-R and FSH-R in GCs from 6 to 8 mm follicles (6-8GC)
Changes in the 6-8GC expression of mRNAs encoding ActRs (ActR-I, -IIA, -IIB) and ßgly during culture with LH or FSH are shown in Fig. 1. LH treatment (100 ng/ml) caused a significant (P<0.05; 83%) increase in ActRI mRNA transcript level, whereas FSH (100 ng/ml) promoted a decrease (P<0.05; 65%). FSH (10–100 ng/ml; 3.7–8.5-fold) and LH (100 ng/ml; 8.5–fold) significantly increased the ActRIIA mRNA level. Changes in ActRIIB mRNA with LH and FSH treatment paralleled ActRI transcript; 100 ng/ml LH increased significantly (P<0.01; 3.7-fold), while 100 ng/ml FSH decreased significantly (P<0.05; 57%) ActRIIB transcript. ßgly mRNA level was significantly (P<0.01) increased by LH (10–100 ng/ml; by up to twofold) and FSH (1–100 ng/ml; by up to 3.5-fold). FSH (10–100 ng/ml) significantly increased (4.5-fold; P<0.0001) the LH-R mRNA expression in 6-8GC. All doses of FSH increased FSH-R transcript (eight- to tenfold) when compared with controls. LH also caused a dose-dependent increase in FSH-R mRNA expression (up to eightfold).
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. Neither LH nor FSH significantly altered the mRNA expression of ActRI, ActRIIA or ActRIIB (P>0.05). However, both LH and FSH significantly (P<0.05) increased the ßgly transcript level (7- and 3.5-fold respectively). FSH-R mRNA expression in 6-8TC was stimulated by both LH (threefold) and FSH (fourfold) at 100 ng/ml. LH and FSH also stimulated LH-R transcript levels in 6-8TC (5.6- and 4.7-fold respectively).
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. ActRI mRNA expression was increased by both LH (10–100 ng/ml; twofold) and FSH (100 ng/ml; 2.4-fold). ActRIIA transcript level was not significantly altered by LH or FSH treatment. ActRIIB mRNA expression was increased dose-dependently by FSH (up to 2.7-fold; P<0.01), whereas only 100 ng/ml LH significantly increased expression (3.2-fold). ßgly mRNA level was also significantly (P<0.001) increased by LH (100 ng/ml; sevenfold) and FSH (100 ng/ml; fourfold). The expression of LH-R mRNA in F1 GC was increased approximately twofold by LH (10–100 ng/ml) while FSH had no significant effect. In contrast, FSH-R mRNA expression was slightly increased by FSH (40%; 100 ng/ml; P< 0.05) while LH had no effect.
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. Neither LH nor FSH affected the expression of ActRI and ActRIIB mRNA (P>0.05) although they slightly reduced the ActRIIA mRNA expression by 35–50% (P<0.01). LH and FSH dose-dependently increased ßgly transcript level by 4.5-fold (P<0.05) and 7.6-fold (P<0.0001) respectively. In F1 TC, unlike in F1 GC, expression of FSH-R transcript was increased twofold by LH (at 100 ng/ml) but FSH had no effect. In contrast, LH-R mRNA expression in F1 TC was increased by both LH (up to fivefold) and FSH (up to 2.5-fold).
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ActRIIA mRNA expression in both GC and TC was significantly higher in the F1 than in 6–8 mm follicles. However, only 6-8GC ActRIIA mRNA expression was significantly increased by LH and FSH (P<0.0001). In contrast, ActRIIA mRNA expression in the F1 TC, but not 6–8 mmTC, was significantly (P<0.01) reduced by LH and FSH treatment.
ßgly mRNA expression in 6-8GC+TC and F1 GC+TC was positively regulated by LH and FSH. The levels of ßgly transcript were maximal with LH in F1 GC and with FSH in 6-8G. The ßgly mRNA expression in F1 TC was stimulated by LH to a lesser extent than FSH (4.5-fold vs 7-fold), while in 6-8TC LH was more stimulatory than FSH (7.2-fold vs 3.5-fold; P<0.01).
Analysis using multifactorial ANOVA demonstrated that TC ßgly mRNA expression within 6–8 mm follicles was significantly higher (P<0.05) than in the corresponding GC following LH and FSH stimulation. F1 TC ßgly mRNA expression was also significantly higher than in the corresponding GC following FSH stimulation, although there was no significant difference following LH stimulation.
Effect of LH and FSH on the release of InhA, P4 and E2 by 6–8 mm and F1 GC and TC
The release of InhA, P4 and E2 in response to LH and FSH treatment in 6–8 mm and F1 GC and TC is shown in Fig. 5. Basal InhA levels in 6-8GC media were below the assay detection limit (2 pg/ml); however, following stimulation by LH (all doses) and FSH (10–100 ng/ml), InhA levels increased to detectable levels (Fig. 5B). LH and FSH both elicited marked dose-dependent increases in P4 release by 6-8GC (5.6-fold and 17.3-fold respectively; P<0.001). Conversely, LH promoted a greater P4 increase than FSH in F1 GC (5-fold vs 1.5-fold; P<0.05 by two-way ANOVA). It should be noted that basal P4 release was some 1000 times greater for F1 GC when compared with 6-8GC. LH and FSH treatment of F1 GC also significantly increased InhA release (up to fourfold; P<0.05). E2 was undetectable in GC–CM under all treatment conditions.
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). E2 release by F1 TC was much lower than in 6-8TC and only a slight response to FSH was observed (Fig. 5C). Basal P4 levels in both 6-8TC and F1 TC CM were at or below the assay detection limit (8 pg/ml) but rose to detectable levels following LH or FSH treatment. As in F1 GC, FSH had no effect on P4 release in F1 TC. It should be noted that basal- and LH-stimulated P4 release from F1 GC were ~6500-fold and 15 000-fold respectively, higher than from F1 TC. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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A quantitative assessment of receptor protein expression under the different experimental conditions was beyond the scope of this study but previous in vitro studies on chicken GCs clearly support the presence of functional type-I and -II ActRs that are capable of forming active signalling complexes. Lovell et al. (2002a) shown that ActA (ßA-ßA dimer, which is mainly confined to TC in chickens; Lovell et al. 1998, 2003) enhanced gonadotrophin-induced secretion of InhA (-ßA dimer) and P4 by GC from hen preovulatory follicles. ActA also enhanced FSH-R and LH-R mRNA expression in GC from preovulatory follicles (Johnson et al. 2006). An ActA-induced increase in FSH-R mRNA expression has also been observed in GC from preheirarchical hen follicles (Johnson et al. 2004, 2006, Woods & Johnson 2005).
Within the hen GC layer, there is a developmental shift from an FSH- to an LH-dependent mechanism of regulating follicular development as follicles are selected (Tilly et al. 1991, Johnson et al. 1996, You et al. 1996), similar to that occurring in mammals (Richards 1994). Of key interest in this regard are the findings that FSH reduced and LH increased both ActRIIB and ActRI mRNA transcript levels within GC of ‘unselected’ prehierarchical (6–8 mm) follicles, whereas both LH and FSH stimulated mRNA expression of both receptors in ‘selected’ F1 GC. We hypothesise that it is this shift in the response to FSH and LH of ActR mRNA expression that modulates this responsiveness of cells to intrafollicular activin.
As it has been shown that ActA increased LH-R mRNA expression in 6–8 mmGC (Johnson et al. 2004), the present and previous data lead us to propose that GC of prehierarchical follicles approaching selection, which are primarily FSH-driven, have reduced expression of key ActRs (ActRI and ActRIIB) and therefore a diminished responsiveness to Act. In turn, as follicles are selected and become more responsive to LH, ActRI and ActRIIB mRNA expression is upregulated leading to greater co-stimulation by Act. Indeed, ActA can further stimulate LH-R mRNA expression in 6–8 mm GC (Johnson et al. 2004), which could lead to a further increase in LH-responsiveness aiding ‘selection’.
Once selected, GC from preovulatory follicles primarily secrete InhA and P4. This secretion in response to ActA is greatly enhanced during co-treatment with LH or FSH (greater than the sum of the responses to ActA and LH/FSH alone; Lovell et al. 2002a); the present data suggest that this increased responsiveness to Act may in part be due to a LH/FSH stimulated increase in ActR. Consistent with this and with the above-mentioned functional evidence of synergism between ActA and gonadotrophins, ActA was recently shown to upregulate the expression of LH-R and FSH-R mRNA in preovulatory follicles (Johnson et al. 2006).
Although LH and FSH differentially regulate ActR and ßgly transcript levels (this study) and, reciprocally, ActA can increase LH-R and FSH-R mRNA expression in prehierarchical follicle GC (Johnson et al. 2004, 2006), further work is required to understand how the balance of these, and most likely other, endocrine and paracrine signals allow the promotion of one ‘selected’ follicle into the preovulatory hierarchy.
GC inhibin B (InhB; -ßB dimer) protein levels (Lovell et al. 2003) peak in 6–8 mm prehierarchical follicles before falling steadily from the stage of follicle selection. Although InhB can associate with the ActRIIs, association with co-receptor ßgly, which is also expressed (Sweeney & Johnson 2005, Lovell et al. 2006) can greatly enhance their affinity for ActRIIs (Lewis et al. 2000). However, as granulosal ßgly mRNA expression (in this study) was an order of magnitude lower in prehierarchical follicles when compared with preovulatory follicles, InhB/ßgly may not be as an effective Act antagonist on prehierarchical GCs as InhA/ßgly in preovulatory follicles. Unfortunately, InhB protein levels in GC-CM from 6–8 mm follicles were below the current assay detection limits. In a previous study, however, we were able to detect InhB in CM from 6–8 mm follicle wall explant cultures (Lovell et al. 2003). This suggests that a theca-derived factor present in follicle wall explant cultures (and absent in the GC mono-cultures) may play a role in upregulating InhB production. ActA is a prime candidate here as ActA treatment increased inhibin/ activin ßB-subunit mRNA level in GC from 6–12 mm follicles (Johnson et al. 2006). Further work is required to establish whether this increase in ßB-subunit mRNA leads to increased InhB secretion although, as mentioned above, this is technically challenging due to the limited sensitivity of the current inhibin B assay.
InhA protein levels were below the assay detection limits in ‘basal’ CM from 6–8 mm GC (this study) and 6–8 mm follicle wall explants (Lovell et al. 2003) and in GC extracts from follicles <9mm (Lovell et al. 2003). Following gonadotrophin stimulation of 6–8 mm GC (this study) InhA secretion increased to detectable levels (albeit very much lower than from F1 GCs). The functional significance of the dramatic shift from InhB to InhA production around the point of follicle selection in vivo (Lovell et al. 2003) remains obscure and further work is warranted to investigate this striking phenomenon. Previous data in mammals indicate that ßgly potentiates the binding of both InhA and InhB to ActRIIA, with the complex being resistant to disruption by ActA. In contrast, only InhA binding to ActRIIB was signi-ficantly enhanced by ßgly (Chapman et al. 2002). Therefore, it is tempting to speculate that, at the stage of follicle selection (~6–8 mm) when granulosal InhB levels peak and InhA levels have yet to rise (Lovell et al. 2003), access of thecal-derived ActA to granulosal ActRIIB receptors would not be antagonised, allowing recruitment of ActRI to generate an active signalling complex (see Fig. 6).
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Although only one ActRI subtype to date has been cloned in birds and investigated in this study (Accession number AJ318064 [GenBank] ), it does not exclude the possibility that multiple ActRI subtypes exist, as in mammals (Attisano et al. 1993, Tsuchida et al. 1995) or that ActRIIs may also form signalling complexes with related TGFß-superfamily type-I receptors.
As with all studies investigating mRNA expression, the possibility cannot be excluded that the observed changes in steady-state mRNA transcript levels reflect changes in mRNA stability, rather than effects on gene transcription per se. Further, studies to verify that the changes in AcR and ßgly mRNA transcript levels reported here are accompanied by alterations in receptor protein expression would also be useful. However, as discussed earlier, this seems highly likely considering previous functional studies (Lovell et al. 2002a, Johnson et al. 2004, 2006, Woods & Johnson 2005) which demonstrated interactions between the effects of ActA and gonadotrophins on hen GCs.
In conclusion, this study shows, for the first time in an avian species, that mRNA transcript levels for ActR and ßgly in TC and GC are modulated by gonadotrophins according to the cell-type and stage of follicle development. These findings, together with emerging evidence that follicles express a myriad of ligands, receptors and binding proteins, underscore the complexity of potential interactions between systemic and locally produced factors required to coordinate follicle progression into and through the preovulatory hierarchy.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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