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Biology of Reproduction 62, 1128-1134 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Molecular Cloning and Expression Analysis of the Complementary Deoxyribonucleic Acid for Chicken Inhibin/Activin ßB Subunit1

Danielle J. Hechta, Adam J. Davis3,a, Carrie F. Brooksa, and Patricia A. Johnson2,a

a Department of Animal Science, Cornell University, Ithaca, New York 14853


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibins and activins are dimeric peptide hormones that play an integral role in the intraovarian regulation of folliculogenesis. The domestic hen, with its well-defined follicular hierarchy, provides a unique model in which to study the role of these hormones in follicular development. In the present study, the complete coding sequence and deduced amino acid sequence for the chicken inhibin/activin ßB subunit has been determined from cDNA clones isolated from a chicken ovarian granulosa cell library. This ßB-subunit cDNA predicts a precursor protein of 392 amino acids containing the mature C-terminal 115 amino acid ßB subunit. When compared to the ßB subunit isolated from a variety of species, the chicken cDNA clone showed high nucleotide identity in the full-length coding region (>70%) and in the mature coding region (>80%). In addition, the deduced amino acid sequence of chicken ßB subunit showed greater than 95% identity compared to other species in the mature peptide region. Expression of the ßB-subunit mRNA was detected by reverse transcription-polymerase chain reaction in both gonadal and extragonadal tissues. Northern blot analysis detected expression in the gonadal tissues only, specifically in the granulosa tissue from the F3-F5 follicles, small yellow follicles (SYF), large white follicles, and immature and mature rooster testes. A major transcript of approximately 4.1 kilobases (kb) and three minor transcripts of approximately 8.4 kb, 6.5 kb, and 1.7 kb were detected in the SYF granulosa samples. To examine the expression pattern of the ßB subunit around the stage of follicle selection, the SYF granulosa was subdivided into two groups: 6–8 mm and 9–12 mm. Quantification of RNA expression (n = 3) showed that expression of the ßB subunit was maximal in the 6–8 mm SYF. Activin B, as well as other intraovarian signals, may regulate early follicle selection and/or development in the chicken.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibin and activin are related but distinct dimeric proteins that share a common beta subunit. On the basis of the beta subunit, they are classified as members of the transforming growth factor ß (TGFß) superfamily [1]. Members of this family have a variety of actions on cell growth, differentiation, and function from early embryonic stages through adulthood [2–4]. Inhibin is a heterodimer of the inhibin {alpha} subunit and one of the ß subunits, forming inhibin A ({alpha}ßA) or inhibin B ({alpha}ßB). Activin is formed by the disulfide linkage of two ß subunits, resulting in either activin A (ßAßA), activin AB (ßAßB), or activin B (ßBßB). Two additional forms of the ß subunit have been isolated and cloned from human liver (ßC subunit) [5] and xenopus liver (ßD subunit) [6], although their ability to form dimers has not yet been characterized. The endocrine actions of inhibin and activin upon regulation of FSH release from the pituitary are well documented, and a paracrine role for these ovarian hormones in regulating follicular selection and development is increasingly well accepted [2–4, 7, 8].

The reproductive cycle of the domestic hen is exquisitely regulated. Ovulation occurs on a 24- to 26-h cycle, and the follicles within the ovary are arranged in a size hierarchy. The largest follicle (F1) is destined to ovulate the next day, the second follicle (F2) the following day, and so on. The small yellow follicles (SYF) have a diameter of 6–12 mm and represent a stage of maturity 2–3 wk from ovulation [9]. While many follicles will grow to a 6- to 12-mm stage, some of these will fail to develop further and become atretic [10]. Every 24–26 h, one of the follicles from this pool will begin a period of rapid growth and progress toward the hierarchy [11].

It has been suggested that the ßB subunit may play a role in the early phases of follicular development, since in situ experiments in the primate ovary revealed the presence of mRNA for the ßB subunit, but not the {alpha} or ßA subunit, in small antral follicles [12]. In mammals, much follicular atresia occurs during the transition from the small antral to preovulatory stage [13]. The factors responsible for selection and persistence of follicles at this transition point have yet to be resolved, although a role for activin B has been implicated in mammalian species [14]. Direct comparison between mammalian and avian species is difficult since, unlike the mammalian species, the avian ovarian follicles do not form an antrum nor form a corpus luteum after ovulation. The transition point from small antral to preovulatory follicle in mammalian ovaries, however, seems to be similar to the transition point between the SYF (6–12 mm) and the hierarchy follicles (>12 mm) in avian species, since selection for growth appears critical at this point in both classes. SYF have been partitioned into 6- to 8-mm and 9- to 12-mm size groups based on documented functional characteristics [9, 15]. We previously examined expression of the ßB subunit in SYF [16], but we next wanted to look in detail at the critical stages (6–8 and 9–12 mm) during which follicle selection is believed to occur.

Previously, our lab cloned the inhibin {alpha} subunit and the inhibin/activin ßA subunit from chicken ovarian tissue [17, 18]. By Northern analysis it was shown that the large preovulatory follicles (F1-F5) are the predominant site for expression of the mRNA for these subunits [18, 19]. The objectives of the current study were 1) to clone the chicken inhibin/activin ßB subunit; 2) to quantitate the expression of the ßB-subunit mRNA from developing and hierarchical follicles, particularly at the 6- to 8- and 9- to 12-mm stages; and 3) to characterize the expression of this subunit in gonadal and extragonadal tissues using reverse transcription (RT)-polymerase chain reaction (PCR).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Sequencing

A lambda gt10 custom cDNA library of approximately 3.6 x 106 independent clones was commercially constructed from chicken ovarian granulosa cells from the five largest follicles (Stratagene, La Jolla, CA). The library was screened by colony hybridization using a 32P-labeled 162-base pair (bp) PCR-generated probe. The PCR piece was produced using primers derived from the published partial sequence of the chicken inhibin/activin ßB subunit [20]. Positive clones containing the putative inhibin/activin ßB-subunit cDNA were isolated after screening and excised from the phage by restriction endonuclease (EcoRI) digestion. The cDNA insert was separated by agarose gel electrophoresis, purified (Spin-X; Costar, Cambridge, MA), and subcloned into the EcoRI cloning site of pBluescript (Stratagene) by ligation for sequencing. The cDNA was sequenced by the dideoxy chain termination method (Biotechnology Resource Center at Cornell University) using T3, T7, and five internal oligonucleotide primers of 18–22 bp.

Tissue Collection and RNA Extraction

Single-comb White Leghorn hens approximately 1 yr of age with regular laying cycles were killed, and the follicles, ovaries, and samples from other body tissues were removed for expression studies. Follicles were immediately placed in ice-cold Krebs-Ringer bicarbonate (pH 7.4) for dissection. Follicles with a diameter greater than 12 mm were classified as hierarchical follicles, with the F1 follicle being the largest. Nonhierarchical follicles were classified as either 6- to 8-mm or 9- to 12-mm SYF, or large white follicles (2–5 mm; LWF). The granulosa and theca layers were separated in all but the LWF, and both were frozen in a guanidinium isothiocyanate solution until extraction. Other tissue samples collected included the postovulatory follicle and sections from the magnum, isthmus, shell gland, infundibulum, brain, pituitary, thyroid, bone marrow, breast and leg muscle, heart, intestine, pancreas, liver, spleen, and kidney of adult hens. Additionally, the adrenal and testes of immature roosters (5 wk old), and the testes of mature roosters (greater than 1 yr of age) were also isolated. Tissue homogenization and RNA extraction followed previously described procedures [16].

Northern Blot Analysis

Northern analysis for the inhibin/activin ßB subunit and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control) was performed for all tissues using 40 µg of total RNA as previously described [16] except that 30 µg of total RNA was used in the quantitative analysis of the gonadal tissues. The samples selected for quantitative analysis were granulosa from the F1, F2, F3, F4, and F5 follicles; pooled granulosa from 9- to 12-mm and 6- to 8-mm follicles; combined granulosa and theca layers from the LWF; and mature and immature testes. The Northern blot analysis was repeated three times with separate pools of tissue for each analysis. It was necessary to pool the granulosa layer from small follicles for efficient RNA extraction. Our lab has previously documented differential expression of the inhibin ßB subunit in the mature and immature rooster testes. For this reason, testes were used as an internal positive control to ensure that our probe could detect differential expression of the ßB subunit. Exposure times were experimentally determined to prevent film burnout, which adversely affects the ability to accurately quantitate the density of the strongest signals. The ßB subunit films were exposed for 48 h, and the GAPDH films were exposed for 6 h. The autoradiographs were scanned and quantitated as described previously [16].

RT-PCR and Southern Blot Analysis

As a more sensitive assay to detect the presence of ßB-subunit mRNA, RT-PCR was performed on all tissue samples. Primers for the inhibin/activin ßB subunit were designed on the basis of sequence analysis of our ßB-subunit clones and predicted a product of 279 bp. This piece encompassed the 162-bp PCR product used for Northern and Southern analysis. The primers for GAPDH were the same as those reported previously [18]. PCR primers were synthesized by the Biotechnology Resource Center at Cornell University. The method used for the RT-PCR reactions was the same as that used previously in our laboratory [16].

Twenty microliters of the PCR reaction for the ßB-subunit and GAPDH samples for each tissue were run on a 1.5% agarose gel in single-strength Tris-acetate-EDTA buffer along with a DNA size marker ({phi}X174 RFDNA/HaeIII fragments). Although the ßB-subunit and GAPDH PCR products were visible by ethidium bromide staining, the samples were transferred and UV cross-linked to a GeneScreen Plus nylon membrane and subjected to Southern hybridization using the 32P-labeled probes described previously for the Northern hybridizations to insure that the control samples had no product. Membranes were washed twice at room temperature for 5 min with double-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) and twice at 65°C for 30 min with double-strength SSC in 1% SDS. Blots were exposed to radiographic film with intensifying screens at room temperature for 1.5 min.

Statistics

ANOVA was used to assess differences in RNA expression in Northern blots using the General Linear Model Procedure of Statistical Analysis Systems (SAS) with replicate and tissue as factors. Protected least-significant difference was used to compare differences among tissues.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Sequencing

Eight positive clones containing the putative inhibin/activin ßB subunit were identified from our cDNA library after screening 1.25 million plaque-forming units. The cDNA inserts from two of the largest clones, cßB10.6 (~2200 bp) and cßB3.2 (1330 bp), were selected for sequence analysis and subcloned into the pBluescript vector. The reading frame for the sequences was established by comparison to the published partial sequence of the chicken activin ßB subunit [20] and to the published sequences of the ßB subunit isolated from bovine [21], porcine [22], human [23, 24], ovine [25], and rat [26] sources. The schematic relationship among our sequenced clones and the reported partial chicken activin ßB-subunit sequence [20] is depicted in Figure 1. Sequence analysis revealed that the cßB10.6 clone lacked a small amount (99 bp) of the terminal 3' coding region, while cßB3.2 contained all of the mature coding region, but lacked 402 bp from the 5' end of the open reading frame. The two clones have an overlapping region of 678 bp and together contain the complete open reading frame of 1179 bp for the chicken inhibin/activin ßB subunit. The region of cßB10.6 upstream of -265 bp was highly GC-rich and therefore could not be successfully sequenced.



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  		FIG. 1. Schematic relationship among the chicken inhibin/activin ßB subunit clones (cßB10.6 and cßB3.2) and the partial sequence published by Mitrani et al. [20]. Numbers correspond to nucleotide bases shown on the composite open reading frame in Figure 2. Start site is indicated by position 1 (ATG) and the end of the peptide coding sequence by position 1179 (TGA). The heavy arrow indicates the extent of the region coding for the mature peptide (832–1179). The region upstream to -265 on cßB10.6 (GC-Rich Region) was not sequenced

The complete cDNA and derived amino acid sequence as determined from our chicken inhibin/activin ßB-subunit clones is presented in Figure 2 (GenBank Accession AF055478). The open reading frame of 1179 bp predicts a precursor protein of 392 amino acids. Based on the N-terminal sequencing of porcine ßB subunit [22], the predicted mature chicken peptide begins at base 832 (amino acid 278) and extends for 115 amino acids to base 1176 (amino acid 392). The mature peptide follows a typical proteolytic cleavage site (R-K-R; amino acids 275–277) [27]. Only one potential N-glycosylation site (N-I-T) is present in the precursor protein, from base 232 through 240 (amino acids 78–80), while none is found in the region of the mature peptide (Fig. 2). Fourteen cysteine residues are found in the chicken inhibin/activin ßB-subunit cDNA, nine of which are located within the mature peptide.



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  		FIG. 2. Nucleotide and deduced amino acid (one-letter code) sequence for the chicken inhibin/activin ßB subunit. Nucleotides are numbered on the left; amino acids are also numbered on the left, in bold italics. The 5' and 3' untranslated regions are shown in the small lower-case letters with the exception of the stop signal (nucleotides 1177–1179), which is bold and underlined. Solid boxes contain cysteine residues and their codons. The broken box contains a potential N-glycosylation site (nucleotides 232–240, amino acids 78–80). The arrow with identifying text indicates where the proposed region of the mature inhibin/activin ßB subunit begins (nucleotide 832, amino acid 278). GenBank Accession number AF055478

Two GC boxes with the consensus sequence GGGCGG or CCGCCC for the transcription factor SP1 [28] are present in the 5' region upstream from the ATG start codon at bases -205 to -200 and -101 to -96, respectively. In the 3' untranslated region there are two regions of the purine-pyrimidine repeat (AC)n, from bases 1429–1446 and bases 1476–1503. (AC)n regions can potentially form left-handed helices or Z-DNA [29, 30].

Sequence Homologies

Our clone of the chicken inhibin/activin ßB subunit showed a high degree of nucleotide and amino acid sequence similarity with the ßB subunit of other species, particularly in the region coding for the mature protein. A comparison of the predicted amino acid sequences among several species for which the ßB subunit has been reported is shown in Figure 3. The full-length coding region of the chicken inhibin/activin ßB subunit has the following percentages of nucleotide identity and deduced amino acid identity (in parentheses) with the full-length coding region of the ßB subunit from other species/tissues: pig 74% (88%), sheep 71% (81%), cow 71% (82%), rat 73% (83%), human ovary 76% (88%), and human testis 73% (83%); it also has 48% (45%) identity with the chicken inhibin/activin ßA subunit. In addition, the mature region of our chicken ßB subunit has the following percentages of nucleotide identity and deduced amino acid identity (in parenthesis) with the mature coding region of the ßB subunit from other species/tissues: pig 82% (97%), sheep 81% (96%), cow 81% (96%), rat 80% (95%), human ovary 83% (98%), and human testis 83% (98%); it also has 67% (62%) identity with the mature coding region of chicken inhibin/activin ßA subunit.



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  		FIG. 3. Comparison among species of the deduced amino acid residues (one-letter code) in the mature region of the inhibin/activin ßB subunit. The mature region of the chicken inhibin/activin ßA subunit is shown for comparison as well. A solid line indicates that the amino acid residue at that position is identical to the deduced chicken ßB subunit; differences at a single position are indicated by text. Numbers correspond to the position of the deduced amino acids as determined by the complete coding region. Cysteine residues are shown in the boxes. pßB (porcine) [22]; oßB (ovine) [25]; bßB (bovine) [21]; mßB (murine) [26, 32]; hßB (human ovarian) [23]; cßA (chicken inhibin/activin ßA subunit) [18]

Northern Analysis

By Northern analysis, no signal for the inhibin/activin ßB subunit was detected in any of the extragonadal tissues examined (data not shown). However, as shown in Figure 4, Northern analysis did detect a signal for the inhibin/activin ßB subunit in ovarian tissue. In addition to the 4.1-kb major transcript, our cDNA probe identified three other transcripts in the SYF samples of approximately 8.4 kb, 6.5 kb, and 1.7 kb. A representative Northern blot for the gonadal tissues examined is presented in Figure 5. Quantitation of this analysis (n = 3) is presented in Figure 6. Statistical analysis revealed that ßB-subunit expression was significantly the greatest in the 6–8 mm SYF.



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  		FIG. 4. Autoradiogram from a Northern analysis of inhibin/activin ßB subunit with total RNA from SYF. Approximate sizes of the ßB subunit RNA transcripts are shown with arrows



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  		FIG. 5. Autoradiogram from one of three replicates of the Northern blot analysis of the inhibin/activin ßB subunit. F, Ovarian follicle (subscripted number represents the hierarchical follicle order); 9–12, SYF with a diameter of 9–12 mm; 6–8, SYF with a diameter of 6–8 mm; LW, LWF with a diameter of 2–5 mm; IT, immature testes; MT, mature testes. GAPDH was used as a control for loading and transfer



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  		FIG. 6. Quantitative analysis of the expression pattern of mRNA for chicken inhibin/activin ßB subunit. Significant differences among groups are depicted by the letters above the bar graph. Values with different letters are significantly different from one another (P < 0.05). n = 3 for all groups except F4 and F5 where n = 2. Analysis was done with autoradiograms exposed for 48 h to the chicken ßB subunit and corrected for loading with GAPDH exposed for 6 h. Sample groups are the same as those represented in Figure 5

Southern Analysis of RT-PCR

Amplification and detection of GAPDH in the samples confirmed the integrity of the cDNA samples. The presence of an mRNA transcript for the ßB subunit was detected in every gonadal and extragonadal tissue tested by RT-PCR (Fig. 7). No product was detected in negative control PCR reactions.



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  		FIG. 7. Autoradiograms from the Southern analysis of the RT-PCR for the inhibin/activin ßB subunit and GAPDH products. Films were exposed for 90 sec. FxG, granulosa tissue with "x" denoting the hierarchical order of the follicle; SYG, small yellow follicle granulosa; LWF, large white follicle; Ist, Isthmus; Mag, magnum; ShG, shell gland; Inf, infundibulum; FxT, theca tissue with "x" denoting the hierarchical order of the follicle: SYT, small yellow follicle theca; POF, postovulatory follicle; Brn, brain; Pit, pituitary; Thy, thyroid; BnM, bone marrow; BrM, breast muscle; LgM, leg muscle; Hrt, heart muscle; Int, intestine; Lng, lung; Pcs, pancreas; Liv, liver; Spl, spleen; Kid, kidney; Adr, adrenal; ImT, immature rooster testes; MtT, mature rooster testes


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The cDNA coding for the full-length inhibin/activin ßB subunit was isolated and sequenced from the ovarian follicles of the domestic hen. The cDNA sequence predicted a high percentage of identity (81–88%) in the precursor peptide and an even greater percentage of identity (95–98%) in the mature peptide when compared to that of other species. The identity of the precursor and mature peptide between the chicken inhibin/activin ßB subunit and ßA subunit was lower (45% and 62%, respectively). Thus, we conclude that the clones we isolated truly represent the ßB subunit. We also found that the predominant site for expression of the ßB-subunit mRNA is the granulosa layer of the small preovulatory follicles.

The inhibin/activin ßB subunit is a member of the TGFß superfamily [1]. Members of this family are characterized by a 50–371 amino acid pro-domain followed by a 110–140 amino acid mature domain [1]. The pro-region appears to be necessary for normal protein synthesis and secretion [1]. The mature region is more highly conserved than the pro-region. As shown in Figure 3, there are nine cysteine residues within the mature region of the inhibin/activin ßB subunit that are invariant across species. Six of these cysteine residues form a rigid cysteine knot structure, and others may be involved in dimer formation [1].

When used in Northern analysis of reproductive tissues, the chicken inhibin/activin ßB-subunit cDNA probe hybridized to a major 4.1-kb transcript and three minor transcripts. In the cow, major 3.5- and 4.6-kb bands of ßB-subunit mRNA were detected in RNA isolated from pools of small, medium, or large follicles [31]. Many different transcript sizes for the ßB-subunit mRNA have also been reported in the rat. Esch et al. [32] detected three ovarian transcripts (5.1 kb, 4.3 kb, and 3.1 kb) and four testicular transcripts (5.1 kb, 4.3 kb, 3.1 kb, and 2.3 kb), and Feng et al. [26, 33] detected two transcripts from a genomic library (4.4 kb and 3.3 kb) and two transcripts (4.8 kb and 3.7 kb) in rat ovarian and testicular tissue. Analysis of the 5' flanking regions of the ßB subunit revealed several potential transcription start sites [26], and researchers have demonstrated differential regulation of the various transcript sizes [33, 34].

Of particular interest is the expression pattern of the ßB-subunit mRNA within the gonadal tissues. We have demonstrated that the ßB subunit is abundantly expressed in the SYF, most predominantly at the 6–8 mm stage. We had previously reported that ovarian expression of the ßB subunit was found to be maximal in the SYF and the F6–F8 follicle pool [16]. Interestingly, our finding of maximal production in the 6- to 8-mm follicle corresponds to an important transition point in follicle development. Tilly et al. [9] found that steroidogenic competence in granulosa cells occurs during the transition from the 6- to 8-mm to the 9- to 12-mm stage. Steroidogenic competency is necessary for further follicular maturity. Activin may act as a local regulator of steroidogenesis at this transitional stage [2]. Presumably the dimer formed in these small follicles is activin B, since expression of the ßA subunit was not detected by Northern analysis and the relative expression of the {alpha} subunit is lowest at this stage [16]. It has also been reported, however, that the expression of follistatin, an activin binding protein, is maximal at the SYF stage and is also expressed at the 2- to 5-mm stage [16]. Therefore, the bioactivity of activin B in the small immature follicles remains to be determined. We chose two stages of testis development to examine because of our previous observation that the ßB subunit was expressed in this tissue [16]. We cannot eliminate the possibility that expression of the ßB subunit may be increased at another time, such as around puberty.

Although inhibin and activin were initially isolated from follicular fluid, the subunits for these proteins have been found in a variety of tissues, including the placenta, pancreas, pituitary, and brain [35, 36]. Furthermore, inhibin and activin have been shown to have a variety of diverse functions in these reproductive and nonreproductive tissues as previously reported [2, 36, 37]. Finally, it is important to note that the presence of mRNA does not guarantee that the functional protein will be produced.

In summary, the cDNA for the full-length chicken inhibin/activin ßB subunit was cloned and sequenced. Comparison of the nucleotide and deduced amino acid sequences among species revealed a high percentage of identity, suggesting that the inhibin/activin ßB subunit is highly conserved. Northern blot analysis showed that the granulosa layers from the SYF, particularly the granulosa layers from the 6- to 8-mm-sized follicles of this class, are the major site for expression of the inhibin/activin ßB subunit in the chicken. Several transcript sizes were identified in the SYF, and the major 4.1-kb transcript was quantitated in the granulosa of the follicles as well as in immature and mature rooster testes. RT-PCR analysis revealed the presence of mRNA transcripts across a wide array of extragonadal tissues. Evidence that the inhibin/activin ßB subunit is expressed most strongly by 6- to 8-mm-sized follicles suggests a possible role the for activin B in regulating early follicle selection and/or development in the chicken.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Chih-Chien Chen for providing the PCR-generated chicken ßB-subunit probe (162 bp).


   FOOTNOTES
 
First decision: 4 November 1999.

1 This work was supported by grants from the USDA (94–37203-0791 and 97–35203–4979). Back

2 Correspondence: Patricia A. Johnson, 202 Morrison Hall, Dept. of Animal Science, Cornell University, Ithaca, NY 14853. FAX: 607 255 9829;paj1{at}cornell.edu Back

3 Current address: Dept. of Poultry Science, Univ. of Georgia, Athens, GA 30602. Back

Accepted: December 1, 1999.

Received: October 5, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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