Quantification of Inhibin/Activin and ß_A Subunit Messenger Ribonucleic Acid by Competitive Reverse Transcription-Polymerase Chain Reaction in Chicken Granulosa Cells during Follicular Development

^a Laboratory for Physiology and Immunology of Domestic Animals, Catholic University of Leuven, B-3001 Leuven, Belgium

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The very sensitive quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate the expression of inhibin/activin subunits in the granulosa cells of developing ovarian follicles of the hen. Two competitors specific to inhibin and ß_A subunits were constructed. In one study, the expression of inhibin and ß_A genes was determined in the granulosa cells of the five largest yellow follicles (F1, F2, F3, F4/5), the small yellow follicles (SYF), and the large white follicles (LWF) of a layer strain of chickens. Competitive RT-PCR for inhibin subunit revealed 10.35 ± 2.15 pg/µg total RNA in the LWF. The expression increased 40-fold in the SYF and remained at that level in the F4/5 but decreased markedly thereafter up to the F1 stage. Inhibin/activin ß_A subunit was also detected in the LWF in low amounts and showed no significant increase until the F2 stage. The highest level was found in the F1. The pattern of the mRNA for and ß_A subunits in the five largest follicles (F1, F2, F3, F4/5) of a broiler breeder strain of chicken was compared with that in the layer strain. Expression of the subunit was significantly higher in the three largest follicles (F1, F2, F3) of the broiler breeder hens, but only in the F2 for the ß_A. The results suggest that inhibin may play an important role in the recruitment and differentiation of follicles and that differences between broiler breeders and layers may have consequences at both the pituitary and ovarian levels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Inhibin is a dimeric protein hormone composed of an and a ß subunit joined by disulfide bonds. The ß subunit exists in two forms, the ß_A and ß_B. Thus inhibin can be secreted as either inhibin A (-ß_A) or inhibin B (-ß_B) depending on which of the ß subunits forms a combination with the subunit [1]. A combination of two ß subunits gives rise to the hormone activin, which can be secreted in either of three forms, activin A (ß_A-ß_A), activin B (ß_B-ß_B), or activin AB (ß_A-ß_B) [1].

The ovary is the main site for the synthesis of inhibins and activins. In mammalian females, the roles played by inhibin/activin in modulating FSH secretion by the pituitary gland in vivo and in vitro have been well documented. Inhibins selectively suppress [2–7] while activins stimulate [6–10] the release of FSH. In addition to their endocrine role in gonadotropin regulation, inhibins and activins exert paracrine/autocrine effects in the ovary. They have been shown to modulate steroidogenesis and folliculogenesis [8–15].

Recent studies have shown that the avian ovary also produces immunoreactive inhibin, which plays important roles in the regulation of pituitary FSH [16–21]. However, the intraovarian roles of the inhibins have received little attention in birds. As in mammals, the main site of production has been localized, through cell cultures, to the follicular granulosa cells [18, 19], and results suggest that production varies with follicular development. Further studies by Johnson and Wang [22] and Chen and Johnson [23], using Northern blot analysis, demonstrated that granulosa cells of the five largest follicles in layers express the gene for both the and ß_A subunits of inhibin and that expression varies with follicular maturation.

However, elucidation of the full physiological roles of these subunits in the avian species is hindered by the absence of specific assays. As Northern analysis is a less sensitive method, and because understanding the regulation of gene expression depends, in part, on the ability to accurately measure mRNA species in defined cell populations, we have developed a very sensitive quantitative competitive reverse transcription-polymerase chain reaction (QC RT-PCR) to detect, quantify, and compare the expression of inhibin and inhibin/activin ß_A mRNAs in the granulosa cells of the developing follicles of layers. We show, for the first time, that the ovarian follicles of broiler breeder female strains also express the mRNAs for these subunits, and we compare the levels of expression with those in the follicles of the layer strain.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Experimental Animals

ISA Brown layers and Ross broiler breeder hens at 35–40 wk of age were used for this study. Layer hens with a laying sequence of 5-6 eggs and Ross broiler breeder hens of 4 eggs or more were used. The birds were housed in cages individually and had free access to water. They were maintained in a lighting regime of 16L:8D. Layers and broiler breeders were fed ad libitum.

Collection of Ovarian Granulosa Cells and RNA Isolation

Hens of similar age were killed by cervical dislocation at 2-4 h after oviposition, and the preovulatory follicles were removed and kept in Hanks' Balanced Salt Solution (HBSS; Gibco BRL, Paisley, Scotland). The granulosa layer was either separated from the theca layer of each follicle according to the method of Gilbert et al. [24] or dissected out, as in the case of the smaller follicles. The granulosa layer was rinsed clean of yolk materials with HBSS and tissues were pooled, from 3–4 hens, for similar size of follicles for independent experiments. Tissues were dispersed in Ca²⁺/Mg²⁺-free HBSS (Gibco BRL) containing 0.1% collagenase (Sigma Chemical Co., St Louis, MO) and 0.5% BSA (Sigma). Cells were recovered by centrifugation at 400 x g for 10 min. The cell pellets were washed twice with PBS. Cells were prepared from the hierarchy of yellow follicles including F1, F2, F3, F4+F5 combined (F4/5), and 7- to 10-mm-diameter small yellow follicles (SYF) and the 4- to 6-mm-diameter large white follicles (LWF).

Total RNA was extracted from cells using a commercial RNA isolation kit (High Pure RNA Isolation Kit; Boehringer Mannheim, Mannheim, Germany). The RNA concentration was estimated by reading the absorbance at 260 nm and was checked for purity at 280 nm in a spectrophotometer (Kontron, Rotkreuz, Switzerland). Samples were stored at -80°C for subsequent use for RT-PCR.

Primer Sequences

The oligonucleotide primers used for RT-PCR of both inhibin and inhibin/activin ß_A were selected by a computer program. The upstream and downstream primers were chosen at equal distance from a unique restriction site (HinfI and HpaI for the and ß_A subunits, respectively). The upstream sequence for the primer used for inhibin was 5'-TTG AGC CCC CCG GCC ATG CA-3' and the downstream sequence was 5'-GCT CAG GGC GTG CGC CGA GG-3' based on the sequence reported by Wang and Johnson [25]. The sequence predicts native PCR product of 225 base pairs (bp) corresponding to bases 239-464. The upstream sequence for the primer used for the inhibin/activin ß_A was 5'-GAG CTC CTT GGA TGT GCG GAT TG-3' while the downstream sequence was 5'-ATG GGC CGC AGT TTG GTG GG-3' based on the sequence reported by Chen and Johnson [26]. The primer predicts a native product of 485 bp corresponding to bases 885–1370. All primers were synthesized for our use by Gibco BRL.

Reverse Transcription

Complementary DNA was synthesized by the extension of respective downstream primers. Nineteen microliters of RT master mix for each sample (500 ng total RNA) was prepared by using single-strength first-strand buffer (Gibco BRL), 10 U RNasin (Promega, Leiden, The Netherlands), 10 U Moloney murine leukemia virus reverse transcriptase (Gibco BRL), 1 mM of each dNTP (dATP, dTTP, dCTP, dGTP; Boehringer Mannheim), 10 mM dithiothreitol (Gibco BRL), and 2 µmol of the downstream primer separately. The RT reaction was carried out in a DNA thermal cycler (MJ-PTC-200; Biozym, Landgraaf, The Netherlands) at 37°C for 45 min followed by 5 min at 99°C. The samples were then placed on ice immediately and used for amplification.

PCR Amplification

PCR was performed for each inhibin/activin subunit by adding the following components to the reaction mixture: PCR buffer (200 mM (NH₄)₂SO₄, 750 mM Tris-HCl, 0.1% Tween), MgCl₂ (1.5 mM for inhibin , 2 mM for inhibin/activin ß_A), 1 mM of each dNTP (dATP, dTTP, dCTP, dGTP), 1 pmol of each corresponding primer, and 0.4 U red hot DNA polymerase (Westburg, Leusden, The Netherlands) in a total volume of 80 µl. The reaction was started with an initial denaturation for 3 min at 94°C followed by 35 cycles of denaturation at 93°C for 30 sec, annealing (at 60°C for subunit and 65°C for ß_A subunit) for 1 min, and extension at 72°C for 45 sec. The last reaction cycle to make full-size copies lasted 5 min at 72°C and was followed by quenching at 4°C. Twenty microliters of PCR products was electrophoresed on 2% agarose gel (Gibco BRL) containing 1% ethidium bromide. A 100-bp DNA ladder (Gibco BRL) was used as molecular weight standard. After electrophoresis, the gel was scanned using a densitometer (Image Master VDS; Pharmacia Biotech, Uppsala, Sweden). Negative control RT-PCR with diethyl pyrocarbonate-treated water was included in all experiments.

Preparation of Internal Standards

The plasmid, pBluescriptIISK, containing clones of inhibin and inhibin/activin ß_A subunit cDNA was kindly provided by P. Johnson of Cornell University, Ithaca, NY. These clones were used for the construction of the respective internal standards.

For ß_A internal standard, a fragment containing a unique NcoI restriction site instead of an HpaI was chosen. The plasmid pBluescriptIISK was linearized with HpaI (Gibco BRL) and dephosphorylated. An oligonucleotide of 8 bases containing an NcoI restriction site (TCCATGGA) was synthesized, phosphorylated with T4 kinase (Gibco BRL), and ligated with T4 ligase (Gibco BRL) into the HpaI site of the pBluescript plasmid as shown schematically in Figure 1A. The clone was named pinhßA mutant.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Schematic view of generation of internal standards for inhibin and ßA subunits with subsequent QC RT-PCR. A) Generation of internal standard ßA. The gray regions on the cDNA are complementary to primers used for the PCR. B) Generation of internal standard . Two pairs of primers were used for the mutation. A1 and A4 contain the complementary region to restriction sites NarI and MunI. A2 and A3 contain the restriction site BclI.

The construction of the internal standard for the inhibin subunit was not possible by cassette mutagenesis as for ß_A or inverse PCR because of the multiple presence of the HinfI restriction sites in the vector. Therefore the internal standard was developed using PCR. In brief, a fragment of the coding sequence containing the HinfI restriction sequence was cut out of the plasmid using NarI and MunI endonucleases (Boehringer Mannheim). The "empty" vector was separated from these fragments by gel electrophoresis, dephosphorylated, and stored at -20°C until used. Two primer pairs (depicted below as A1:A2 and A3:A4) were chosen using primer design software. For each pair, one primer contained a specific tail containing the BclI recognition sequence that we wished to be built in. The two primer pairs were used in separate PCR reactions to synthesize two subfragments, together spanning the same sequence as the fragment cut out of the vector. After digestion of the fragments with endonucleases BclI, MunI, and NarI (Boehringer Mannheim) to generate 5' sticky ends, the fragments were ligated and then cloned back into the empty vector. As a result, the HinfI restriction site was replaced with a BclI recognition sequence. After transfection, correctly mutated plasmids were selected by direct PCR clone analysis of colonies and restriction digests [27]. This clone was named pinh mutant (Fig. 1B). The primer pairs are A1: 5'-TGG GGC CGG CGC CGA CCG AC-3'; A2: 5'-TAT CAA TGA TCA CAC CTG GGA TGT GTC CTC CTG-3'; A3: 5'-TAT CAA TGA TCA CTC TTC CCT TCC ACA GAC GTG-3'; A4: 5'-GTG CAC GAT CCA ATT GTC CCA GCC-3'.

In Vitro Transcription of Mutant RNA

The two mutants pinh and pinhßA were used as target DNA for the in vitro transcription of mutant RNAs, which was performed by the method described by Stofflet et al. [28]. DNA was first linearized with restriction enzyme: SacI for pinh and PstI (Gibco BRL) for pinhßA. The enzyme was then removed by extraction with phenol-chloroform-isoamylalcohol (25:24:1), and the DNA was desalted over a Sephadex G50 column (Pharmacia). The transcription reaction mixture (20 µl), containing 5-strength transcription buffer, 0.5 mM of each NTP (Boehringer Mannheim), 1.6 U RNase inhibitor (Perkin Elmer, Foster City, CA), 10 U T7 RNA polymerase (Amersham International, Buckinghamshire, UK), 10 mM dithiothreitol, and 3 µg of target DNA (pinh/SacI or pinhßA/PstI), was prepared. The mixture was incubated for 30 min at 37°C and subsequently treated with 34 U RNase-free DNase (Boehringer Mannheim) to fragment the DNA. The reaction was stopped by the addition of EDTA to a final concentration of 5 mM.

QC RT-PCR

Three validating trials were conducted before conditions were established for ideal quantitative analysis. The optimum PCR conditions were determined for each set of primers used. The amounts of internal standards (measured by spectrophotometry at 260 nm), the starting amount of RNA, and the serial dilutions were refined to permit adequate quantification of mRNA. Serial dilutions of competitor pinh cRNA (400, 200, 100, 50, 25, 12.5, and 6.25 pg) were established for the subunit. Similarly, serial dilutions of competitor pinhßA (400, 100, 25, 6.25, 1.562, and 0.390 pg) were used for ß_A subunit. Five hundred nanograms total RNA from granulosa cells was required for adequate quantification. Total RNA was mixed with an aliquot of a given dilution of standard RNA (in vitro-transcribed mutant RNA). Mixtures were transcribed into cDNA and coamplified. Digestion of PCR products by BclI for and HpaI for ß_A selectively cuts DNA fragments derived from the internal standard in the case of the subunit and from the native RNA in the case of the ß_A subunit. Samples were run on 3% agarose containing ethidium bromide. The relative amount of DNA in each band was measured by densitometry (Image Master VDS; Pharmacia). Using the Image Master densitometer software, the percentage digested PCR product relative to the total product (100%) was calculated as a function of the initial added concentration of mutant RNA. A sigmoid curve was fitted to the plot using the Slide-Write Plus program (Advanced Graphics Software Inc., Carlsbad, CA); and from the 50% level of PCR product originating from each initial RNA, the initial concentration of mutant RNA was calculated.

Quantification of Inhibin Subunit mRNAs in the Granulosa of Developing Follicles

Decreasing amounts of internal standards for either the or ß_A subunit were added to 500 ng of total RNA isolated from granulosa cells of each follicle (1 µg LWF was used for the two subunits); each subunit was reverse transcribed and coamplified separately with the corresponding mutant for 35 cycles. PCR products were digested and electrophoresed. The band size and intensity were determined: the upper band corresponding to the PCR product derived from the native RNA in the case of (or derived from the internal standard in the case of ß_A) and the lower band from the internal standard in the case of (or from the native RNA in the case of ß_A). The amount of mRNA was calculated. Experiments were repeated four times with at least three replications for each independent experiment.

The expression of inhibin and ß_A genes was determined in F1, F2, F3, F4/5, SYF, and LWF of the laying hens and in F1, F2, F3, and F4/5 of the broiler breeders. Expression was compared between layer and broiler breeder strains for the five largest follicles. The total RNA that was extracted from granulosa cells of layers and used for the first experiment was also used for comparison with that in samples collected from broiler breeders.

Data Analysis

Data on amount of mRNA are presented as pg RNA/µg extracted total RNA. Data were examined by ANOVA, and differences between follicles of each strain were determined by Duncan's multiple range test. Differences between strains were also examined by ANOVA (Duncan's multiple range test or t-test).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
RT-PCR

RT-PCR was tested on individual RNA samples from granulosa cells, obtained from both layer and broiler breeder hens, using primers selected to generate the and ß_A subunit cDNA fragment. All samples showed the expression of inhibin and inhibin/activin ß_A. The primer selected for subunit generated a PCR product of 225 bp, while that selected for the ß_A subunit gave a native product of 485 bp (Fig. 2).

View larger version (44K): [in this window] [in a new window]   FIG. 2. RT-PCR products of and ßA subunits from layer (A) and broiler breeder (B) granulosa cell RNA. Lanes 1, 2, 3, 4, 5, 6: RT-PCR of started from 1 µg total RNA of granulosa cells of F1, F2, F3, F4/5, SYF, and LWF, respectively. Lanes 8, 9, 10, 11, 12, 13: RT-PCR of ßA started from 1 µg total RNA of granulosa cells of F1, F2, F3, F4/5, SYF, and LWF, respectively. Lanes 7 and 14: 100-bp DNA ladder.

Mutant clones were prepared as described in Materials and Methods. Figure 3 shows restriction digestion of the products of PCR clone analysis of transformed colonies. Mutant RNAs prepared from both pinh and ßA generally yielded 12 µg from 3 µg of clones.

View larger version (53K): [in this window] [in a new window]   FIG. 3. Restriction digestion of products of the PCR clone analysis of and ßA subunits. Lanes 2-12: PCR clone analysis products of subunit digested with BclI. Colonies in lanes 3, 4, 5, 6, 7, and 10 contain the pinh mutant since their PCR products were cut with BclI. Lanes 14–21: PCR clone analysis products of ßA subunit digested with NcoI. Colonies in lanes 15, 17, and 19 contain the pinhßA mutant since their PCR products were cut with NcoI. Lanes 1 and 13: Puc 78/AluI.

QC RT-PCR

The primers used for cDNA synthesis and for PCR defined a 225-bp fragment as a product from granulosa RNA and a 113-bp fragment (after digestion by BclI) as a product from the mutant RNA. For the ß_A subunit, the PCR product was a 485-bp fragment for the mutant RNA and 243 bp (after digestion by HpaI) for the native RNA. Scans showed that digestion by the appropriate enzyme selectively cuts DNA fragments derived from the mutant RNA in the case of (Fig. 4A) or from the native RNA in the case of ß_A (Fig. 4B) into two subfragments with approximately similar sizes, which comigrate on the gel, resulting in a series of double bands. The upper band represents the nondigested DNA fragment originating from native RNA for the or from the mutant RNA for the ß_A, while the lower band corresponds to the cleaved DNA fragment originating from the mutant RNA for or from the native RNA for the ß_A (in granulosa cells).

View larger version (28K): [in this window] [in a new window]   FIG. 4. QC PCR analysis of granulosa cell RNA of the F1 follicle. A) Quantification of inhibin mRNA in F1 follicle. Lanes 1–7: Samples containing 500 ng total RNA and a 2-fold dilution of mutant RNA (internal standard ) subjected to RT-PCR, digested with BclI, and analyzed on 3% agarose gel. The upper band corresponds to the native RNA, and the lower band corresponds to the internal standard . Lane 8: 100-bp DNA ladder. B) Quantification of inhibin ßA mRNA in F1 follicle. Lane 1: 100-bp DNA ladder. Lanes 2–7: Samples containing 500 ng total RNA and a 4-fold dilution of mutant RNA (internal standard ßA) subjected to RT-PCR, digested with HpaI, and analyzed on 3% agarose gel. The upper band corresponds to the internal standard ßA, and the lower band corresponds to the native RNA. Similar results were obtained in three separate experiments. The same protocol was used for the other hierarchical follicles. The graphics correspond to the standard curves for the quantification of the amount of inhibin and ßA mRNA. Note that the internal standard was loaded in decreasing concentrations from left to right on the gel.

Comparative Expression of and ßA Subunits in the Hierarchy of Follicles in Layer Hens

The results of QC RT-PCR of inhibin and inhibin/activin ß_A mRNA levels in the granulosa cells obtained from the F1, F2, F3, F4/5, SYF, and LWF of layer hens are shown in Figure 5 (A and B, white bars). Figure 5A shows a very low level of inhibin mRNA in the granulosa of LWF. Recruitment into the hierarchy of yellow follicles was accompanied by a significant increase in the mRNA of the subunit in the granulosa of SYF. Similar levels of mRNA were found in both the SYF and the F4/5 granulosa cells. The level of mRNA decreased significantly after transition to the F3 stage, with a further decrease in the larger F2 and F1 follicle granulosa cells. The level of mRNA at any stage in the hierarchy of yellow follicles was significantly higher than that in the LWF, which showed the lowest amount of mRNA. The F4/5 and SYF showed a 4-fold higher expression of the gene than the F1 or F2.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Quantitative analysis of inhibin (A) and ßA (B) subunits in the granulosa cells of layers (white bars) and broiler breeders (gray bars). Both subunits were analyzed in the F1-LWF in layers, and comparison with broiler breeder granulosa cells was determined in the F1-F4/5. The results represent the mean quantity of mRNA (± SEM) as determined by competitive RT-PCR. Data were analyzed by ANOVA and mean differences determined by multiple range test or Student's t-test where applicable. Significant differences between the follicles of each strain are denoted by the different letters: a, b, c, d < 0.05. *Denotes significant difference between broilers and layers within follicles, p < 0.05.

Data on the expression of the inhibin/activin ß_A mRNA are shown in Figure 5B (white bars). In contrast to the inhibin mRNA levels, the ß_A subunit did not show any significant increase after the recruitment of the LWF into the hierarchy of yellow follicles until the F2 stage (4-fold). A very high level of the subunit was found in the F1, and this represented a 4-fold increase over that in the F2. Thus the highest expression of the gene was found in the F1 and the lowest in the LWF.

Comparative Expression of and ßA Subunits in Layer and Broiler Breeder Hens

The PCR products obtained show that broiler breeder granulosa follicles also express the genes for both the and ß_A subunits of inhibin/activin (Fig. 2B). Inhibin gene expression in the granulosa cells of F1-F4/5 follicles decreased with follicular maturation (Fig. 5A, gray bars). The level of mRNA expressed was highest in the F4/5 and decreased significantly in the F3. Levels decreased significantly (p < 0.05) further in both F2 and F1 compared to F3.

In comparison with the level of mRNA in the granulosa of layer hens, inhibin in broiler breeder hen granulosa cells was similar in the F4/5 but showed a significantly (p < 0.05) higher level in the F1, F2, and F3 follicles.

Inhibin/activin ß_A mRNA in the granulosa cells of broiler breeder hens again showed a trend similar to that found in the layers. Levels increased significantly only at the F2 stage, with an even greater increase in the F1 (Fig. 5B, gray bars). Comparison of the levels of mRNA between the layer and the broiler breeder hens showed no significant differences in the expression of ß_A in all follicles except the F2, where the granulosa of the broiler breeder hen showed a significantly higher mRNA level (p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have demonstrated, using RT-PCR, that chicken granulosa cells express the genes for inhibin and inhibin/activin ß_A subunits. RT-PCR is a powerful method for analyzing very low-abundance mRNAs derived from cells or tissues and is now a well-established technique whose sensitivity provides a major advantage over Northern blot [29] or RNase protection analysis. To adequately quantify these messages, we have developed a QC RT-PCR in which specific internal standard molecules (pinh mutant and pinhßA mutant), differing in one restriction site in the amplified portions of the specific target molecules, are amplified simultaneously with the target samples. We have used these internal standards as controls instead of another control molecule such as ß-actin. This method sets up a competition between the target molecule and the internal standard within the same PCR reaction. Extracted granulosa cell RNA—reverse transcribed, coamplified in the presence of varying amounts of the developed internal standards, and digested with the appropriate enzyme—adequately produced for inhibin a 225-bp band corresponding to the predicted target cDNA and a 113-bp band corresponding to the internal standard cDNA. For the ß_A subunit, a 485-bp product corresponding to the internal standard cDNA and a 243-bp band corresponding to the predicted target cDNA were obtained.

This validated method was used to determine the absolute level of expression of the inhibin/activin subunits in the developing follicles of the chicken. The results showed that the expression of inhibin and inhibin/activin ß_A was related to the level of maturation of the preovulatory follicles. There was an inverse relationship between the expression of the two subunits in the SYF through to the F1. Expression of the subunit mRNA decreased markedly with follicular maturation whereas that of the ß_A increased, but not significantly until about 48 h to ovulation (F2 stage). The quantitative analysis of competitive RT-PCR for inhibin also revealed that follicles acquired a dramatic 40-fold increase in the expression of the subunit for recruitment to the hierarchy of yellow follicles. Then as follicles approached ovulation and as granulosa cells became more differentiated, the expression decreased significantly. A small amount of inhibin/activin ß_A subunit mRNA was detected in the LWF. The inverse relationship between the two subunits indicates that each of the inhibin/activin subunit mRNAs is differentially regulated in preovulatory follicles. This has been suggested by studies done in chickens [23] and in rats [30]. Moreover, Fenz et al. [31] identified different response elements in the promoter regions of the rat inhibin and ß_A genes. It should be noted that the comparison of estimates of mRNA between the and ß_A subunits may not be totally adequate because 1) the kinetics of the two subunits (e.g., annealing temperature) were different and 2) we used different dilution series in the competitive RT-PCR, which resulted in different standard curves. The detection of the expression of the genes for the and ß_A subunits of inhibin in the granulosa cells of the yellow follicles of layers in this study is consistent with a previous investigation [23] using Northern analysis. Similarly, the patterns of expression reported by the previous authors were confirmed in our study. However, in the previous study, the method of analysis gave only crude quantification of mRNA compared to the present study, in which absolute amounts of steady-state mRNA were calculated. Moreover, absolute quantification allows comparison of levels of expression of each subunit between follicles and within follicles from different strains.

We have shown in our study that the SYF and LWF granulosa also express the genes for the and ß_A subunits. In an abstract form, Davis et al. [32] reported that the SYF category expresses the subunit gene but not the ß_A. The authors also did not detect expression of either of the two subunits in the LWF. Data presented here provide evidence that both subunits are expressed in the LWF but at low levels. Attempts to detect inhibin and ß_A subunits in the LWF by Northern blotting have up to now failed, suggesting amounts of and ß_A gene transcripts in this follicle size that are low and that are beyond the detection limit of Northern analysis. The detected amounts of 10.35 pg for the and 0.52 pg for the ß_A subunit confirm this assumption.

Our results show that the granulosa cells of broiler breeder ovarian follicles also express inhibin and ß_A subunits. The data indicate differential levels of expression between broiler breeders and layers. While the overall pattern of expression during follicular maturation is similar, the absolute levels differ within some follicles of similar hierarchy. Broiler breeder females showed significantly higher level of expression for the subunit in the three largest follicles, whereas the level of expression of ß_A is similar for all follicles except the F2.

Recent studies in female chickens have shown that inhibins are involved in the regulation of their reproductive function [18, 19, 32]. Immunoreactive inhibins in plasma have been shown to increase after sexual maturation [19], and it has been reported that granulosa immunoreactive inhibin content and secretion into culture medium is correlated with plasma levels [33]. There is evidence that inhibin exerts an endocrine function in the regulation of FSH release [19–21, 33]. Up to now the specific roles of the subunits of inhibin have not been clearly delineated at the ovarian level. The changing expression level of subunits with follicular size, however, suggests changing roles for the different subunits as the follicle develops. The present data suggest that inhibin may be important for follicular recruitment into the yellow category, since expression dramatically increases as follicles are selected from the LWF to be SYF. It may also be necessary for follicular differentiation, since substantial expression was still present in the follicles up to the F1 stage although it was decreasing rapidly. Wang and Johnson [34] found a correlation between laying rate and expression of the subunit, thus suggesting that the different subunits may have specific roles in the regulation of ovarian function. Short-sequence layers expressed higher levels of inhibin compared with long-sequence layers. Since broiler breeders have shorter egg sequences, it is therefore not surprising that our data comparing broiler breeders with layers showed a higher expression of subunit for broiler breeders in the largest three follicles. The ß_A levels also indicate that this subunit may be of importance at the final 48 h before ovulation, as this is the period when levels increase dramatically. This subunit is possibly important for follicular differentiation and ovulation processes. Chen and Johnson [35, 36] found dramatic changes during the ovulatory cycle of the laying hen. Expression in the F1 and F2 showed significant decreases a few hours before ovulation, which coincided with the period of LH increases, suggesting a negative regulation by LH. The full significance of each subunit and the differences in their expression require further study. The present data, however, suggest that broiler breeder females may be producing higher amounts of inhibins than layers, which may alter FSH/LH profiles and the sensitivity of the follicles to gonadotropins.

	FOOTNOTES

 
¹ Correspondence: Eddy Decuypere, Laboratory for Physiology&Immunology of Domestic Animals, Catholic University Leuven, Kardinaal Mercierlaan 92 (Blok E), B-3001 Heverlee, Belgium. FAX: 32 16 32 19 94; eddy.decuypere{at}agr.kuleuven.ac.be

Accepted: June 18, 1998.

Received: February 23, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Burger HG, Igarashi M. Inhibin: definition and nomenclature, including related substances. Endocrinology 1988; 122:1701–1702.[Medline]
2. Findlay JK, Robertson DM, Clarke IJ. Influences of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31000) on plasma FSH concentration in ovariectomized ewes. J Reprod Fertil 1987; 80:455–461.[Abstract]
3. Mercer JE, Clements JA, Funder JW, Clarke IJ. Rapid and specific lowering of pituitary FSHb mRNA levels by inhibin. Mol Cell Endocrinol 1987; 53:251–254.[CrossRef][Medline]
4. Ling N, Ueno N, Ying S-Y, Esch F, Shimasaki S, Hotta M, Cuevas M, Guillemin R. Inhibins and activins. Vitam Horm 1988; 44:1–46.[Medline]
5. Rivier C, Schwall R, Maso A, Burton L, Vaughan J, Vale W. Effects of recombinant inhibin on luteinizing hormone and follicle-stimulating hormone secretion in the rat. Endocrinology 1991; 128:1548–1554.[Abstract]
6. Carroll RS, Kowash PM, Lofgren JA, Schwall RH, Chin WW. In vivo regulation of FSH synthesis by inhibin and activin. Endocrinology 1991; 129:3299–3304.[Abstract]
7. Ying S-Y. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle stimulating hormone. Endocr Rev 1988; 9:267–293.[Abstract]
8. Ling N, Ying S-Y, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R. Pituitary FSH is released by a heterodimer of the ß-subunits from the two forms of inhibin. Nature 1986; 321:779–782.[CrossRef][Medline]
9. Schwall RH, Nikolics K, Szonyi E, Gorman C, Mason AJ. Recombinant expression and characterization of human recombinant activin-A. Mol Endocrinol 1988; 2:1237–1242.[Abstract]
10. Muttukrishna S, Knight PG. Inverse effects of activin and inhibin on the synthesis and secretion of FSH and LH by ovine pituitary cells in vitro. J Mol Endocrinol 1991; 6:171–178.[Abstract]
11. Hsueh AJW, Dahl KD, Vaughan J, Tucker F, Rivier J, Batdin CW, Vale W. Heterodimers and homodimers of inhibin subunits have different paracrine actions in modulation of luteinizing hormone-stimulated androgen synthesis. Proc Natl Acad Sci USA 1987; 84:5082–5086.[Abstract/Free Full Text]
12. Woodruff TK, Lyon RJ, Hassen SE, Rice GC, Mather JP. Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinology 1990; 127:3196–3205.[Abstract]
13. Rabinovici J, Spencer SJ, Jaffe RB. Recombinant human activin-A promotes proliferation of human luteinized preovulatory granulosa cells in vitro. J Clin Endocrinol Metab 1990; 71:1396–1398.[Abstract]
14. Hillier SG, Yong EL, Illingworth PI, Baird DT, Schwall RH, Mason AJ. Effect of recombinant inhibin on androgen synthesis in cultured human theca cells. Mol Cell Endocrinol 1991; 75:R1–R6.
15. Findlay JK. An update of the roles of inhibin, activin and follistatin as local regulators of folliculogenesis. Biol Reprod 1993; 48:15–23.[Abstract]
16. Akashiba H, Taya K, Sasamoto S. Secretion of inhibin by chicken granulosa in vitro. Poult Sci 1988; 67:1625–1631.[Medline]
17. Tsonis CG, Sharp PJ, McNeilly AS. Inhibin bioactivity and pituitary cell mitogenic activity from cultured chicken ovarian granulosa and theca/stroma cells. J Endocrinol 1988; 116:293–299.[Abstract]
18. Vanmontfort D, Rombauts L, Decuypere E, Verhoeven G. Source of immunoreactive inhibin in the chicken ovary. Biol Reprod 1992; 47:977–983.[Abstract]
19. Vanmontfort D, Berghman LR, Rombauts L, Verhoeven G, Decuypere E. Developmental changes in immunoreactive inhibin and FSH in plasma of chickens from hatch to sexual maturity. Br Poult Sci 1995; 36:779–790.[Medline]
20. Johnson PA, Brooks C, Wang S-Y, Chen C-C. Plasma concentrations of immunoreactive inhibin and gonadotrophins following removal of ovarian follicles in the domestic hen. Biol Reprod 1993; 49:1026–1031.[Abstract]
21. Johnson PA, Brooks C. Developmental profile of plasma inhibin and gonadotrophins from hatch to sexual maturity in male and female chickens. Gen Comp Endocrinol 1996; 102:56–61.[CrossRef][Medline]
22. Johnson PA, Wang S-Y. Characterization and quantitation of mRNA for the inhibin subunit in the granulosa layer of the domestic hen. Gen Comp Endocrinol 1993; 90:43–50.[CrossRef][Medline]
23. Chen C-C, Johnson PA. Expression of inhibin and inhibin/activin ßA-subunits in the granulosa layer of the large preovulatory follicles of the hen. Biol Reprod 1996; 55:450–454.[Abstract]
24. Gilbert AB, Evans AC, Perry MM, Davidson MH. A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic hen (Gallus domesticus). J Reprod Fertil 1977; 50:179–181.[CrossRef][Medline]
25. Wang S-Y, Johnson PA. Complementary deoxyribonucleic acid cloning and sequence analysis of the -subunit from chicken ovarian granulosa cells. Biol Reprod 1993; 49:453–458.[Abstract]
26. Chen C-C, Johnson PA. Molecular cloning of inhibin/activin ßA-subunit complementary deoxyribonucleic acid and expression of inhibin/activin and ßA subunits in the domestic hen. Biol Reprod 1996; 54:429–435.[Abstract]
27. Verhasselt P, Voet M, Volckart G. DNA sequencing by a subcloning-walking strategy using a specific and semi-random primer in the polymerase chain reaction. J DNA Seq Map 1992; 2:281–287.
28. Stofflet ES, Koebel DD, Sakar G, Sommer SS. Genomic amplification with transcript sequencing. Science 1988; 239:491–494.[Abstract/Free Full Text]
29. Kiyoshi K, Hiroshi O, Yuriko Y, Kei N. Regulation of Na+, K+-ATPases. II. Cloning and analysis of the 5'-flanking region of the rat NKAB2 gene encoding the ß2 subunit. Gene 1990; 91:271–277.[CrossRef][Medline]
30. Woodruff TK, Meunier H, Jones PBS, Hsueh AJW, Mayo KE. Rat inhibin: molecular cloning of and inhibin/activin ß subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 1987; 1:561–568.[Abstract]
31. Fenz Z-M, Li Y-P, Chen C-LC. Analysis of the 5'-flanking regions of rat inhibin and ßB subunit genes suggests two different regulatory mechanisms. Mol Endocrinol 1989; 3:1914–1925.[Abstract]
32. Davis AJ, Wu JM, Johnson PA. Expression pattern of mRNA for follistatin and the inhibin/activin subunits during follicular and testicular development in Gallus domesticus. Biol Reprod 1997; 56(suppl 1):144 (abstract 247).
33. Vanmontfort D, Room G, Bruggeman V, Rombauts L, Berghman LR, Verhoeven G, Decuypere E. Ovarian and extragonadal sources of immunoreactive inhibin in the chicken; effects of dexamethasone. Gen Comp Endocrinol 1997; 105:333–343.[CrossRef][Medline]
34. Wang S-Y, Johnson PA. Increase in ovarian -inhibin gene expression and plasma immunoreactive inhibin level is correlated with a decrease in ovulation rate in the domestic hen. Gen Comp Endocrinol 1993; 91:52–58.[CrossRef][Medline]
35. Chen C-C, Johnson PA. Expression of inhibin ßA and -subunits in the granulosa of the two largest preovulatory follicles throughout the ovulatory cycle of the hen. Biol Reprod 1995; 52(suppl 1):197 (abstract).
36. Chen C-C, Johnson PA. Expression and regulation of mRNA for inhibin/activin - and ßA-subunits in the granulosa layer of the two largest preovulatory follicles during the hen ovulatory cycle. Gen Comp Endocrinol 1997; 107:386–393.[CrossRef][Medline]