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Biology of Reproduction 64, 231-241 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Cloning of Complementary Deoxyribonucleic Acids Encoding Quail (Coturnix coturnix japonica) Retinoic Acid Receptor ß Isoforms and Changes in Their Gene Expression During Gonadotropic Growth1

Zhengwei Fua, Tatsuo Kubob, Kunio Sugaharab, Tadashi Noguchia, and Hisanori Kato2,,a

a Laboratory of Nutritional Biochemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan b Faculty of Agriculture, Utsunomiya University, Mine-machi, Utsunomiya-shi, 321-8505, Japan

ABSTRACT

Retinoids have important effects on the development of the reproductive system, where they act via their specific nuclear receptors: retinoic acid receptors (RAR{alpha}, ß, {gamma}) and retinoid X receptors (RXR{alpha}, ß, {gamma}). The research reported here was conducted in an effort to clone quail RARß cDNA (qRARß) and to evaluate the expression of qRARß mRNAs in different tissues and during the development of gonadotropic organs. Two complete cDNAs of qRARß1 and qRARß2 were isolated by a combination of reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends techniques. An RNase protection assay revealed the widespread expression of qRARß1 and ß2 with large tissue-specific variations. The qRARß1 isoform was predominant in the testis, whereas qRARß2 was dominant in the other tissues examined with the exception of the brain, where both isoforms were almost equally expressed. In the developing testes, the qRARß1 mRNA level was high between 30 and 40 days of age, the period during which the testes grew rapidly. The level declined thereafter to its initial level. In contrast, qRARß2 mRNA did not exhibit obvious changes. In the developing oviducts, both qRARß1 and ß2 mRNAs reached their peak levels by 30 days of age, just before the rapid development of the oviduct occurred, and then decreased to almost undetectable levels when the oviduct developed to the laying stage (over 2.88 g in weight). Similar expression patterns of qRARß1 and ß2 were also observed in the developing follicles from the prehierarchical (<2-mm diameter) to the largest preovulatory follicle. In contrast, neither qRARß1 nor ß2 mRNA exhibited developmental changes in the brain. These results suggest that RARß may play an important role in the development of the reproductive systems of birds.

developmental biology, female reproductive tract, follicle, ovary, oviduct, testes

INTRODUCTION

The essential nature of vitamin A for growth, vision, reproduction, embryonic and fetal development, and cellular differentiation has been well established in numerous nutritional studies. It becomes clear that vitamin A is required in the reproduction system when vitamin A-deficient animals are observed. In rats, vitamin A deficiency has resulted in testicular atrophy, reduction of germinal epithelium [1], and fetal death or abnormalities [2]. Similar morphological changes and functional defects have also been observed in birds that have been fed a vitamin A-deficient diet [3]. These lesions can be reversed by administration of retinol, a reduced form of vitamin A. The biologically active form of vitamin A for most of its functions is retinoic acid (RA), a metabolite converted from retinol. Retinoic acid can maintain normal spermatogenesis and egg production in birds as retinol does [3]. However, early studies revealed that retinol-deficient, RA-fed rats failed to reproduce [1, 2]. Histological examination of the testes in these animals revealed lesions similar to those observed in retinol-deficient rats. Female rats given the same diet could mate with normal males and conceive, but they frequently resorbed their fetuses during the period of pregnancy [2]. It is also reported that repeated administration of high doses of RA can restore spermatogenesis in male rats fed the retinol-deficient diet [4].

It is now widely accepted that RA mediates its effects by binding to its specific nuclear receptors [5, 6] that are members of the steroid-thyroid nuclear superfamily [7]. There are two known distinct classes of retinoid receptors: the retinoic acid receptors (RARs) that are activated by both all-trans and 9-cis RA, and the retinoid X receptors (RXRs) that bind to and are activated by the 9-cis RA only. Each class consists of three subtypes of receptors (RAR{alpha}, RARß, and RAR{gamma}; and RXR{alpha}, RXRß, and RXR{gamma}). These receptors act as inducible transcription factors that regulate the transcription of target genes containing appropriate RAR response elements (RARE) or RXR response elements (RXRE) in their promoter regions [8, 9].

The importance of RARs and RXRs in the reproductive system has been widely studied in mammals. All three RARs and all three RXRs are present in the mouse testis [10]. The RAR{alpha} transcripts are expressed both in Sertoli cells and in germ cells, and the level of these transcripts is increased about threefold by the administration of retinol during reinitiation of spermatogenesis in the vitamin A-deficient rat [11]. Additionally, the level of mRNA for RAR{alpha} varied dramatically during the spermatogenic cycle in normal rats, with the highest expression detected at stage VIII [12]. The mRNA expression of RARß in the mouse testis is influenced by the retinoid status [10]. Furthermore, RAR{alpha} [13] and RXRß [14] null mutant mice are sterile due to severe degeneration of the germinal epithelium or abnormal spermatogenesis; and RAR{gamma} [15] null mutant mice are also sterile due to squamous metaplasia of the seminal vesicles and prostate. Interestingly, the morphology of the testes in these mice was similar to that observed in the male mice fed the vitamin A-deficient diet. Thus, these studies strongly suggest that RARs and RXRs play important roles in the development of testes and spermatogenesis.

Recently, we were first to report that Japanese male quail fed diets depleted of vitamin A but supplemented with RA (4 mg/kg diet) exhibited faster growth of their testes than vitamin A-fed controls (retinyl acetate, 14 000 IU/kg diet) [16]. The cloacal gland growth, an external indicator of testicular development in Japanese quail [17], was also advanced by RA [16]. Furthermore, we also found that RA exerts the same effect on the maturation of the female reproductive system in Japanese quail as it does in the male (unpublished data). These results suggest that RA has an accelerative effect on the maturation of the reproductive organs in Japanese quail. The mechanisms underlying this accelerative effect are still not known. As mentioned above, RARs and RXRs are essential for normal testicular growth and spermatogenesis in the mammal. Therefore, determining the developmental changes of expression of different RARs/RXRs in the reproductive organs could shed light on the molecular mechanisms of RA actions in birds. Because RARß is highly dependent on vitamin A nutrition in mammals [10, 18–21], we first focused on RARß as a promising mediator of retinoid action in gonadotropic development. In this study, we cloned the quail RARß (qRARß) cDNAs and examined the developmental patterns of RARß transcripts in testes, oviducts, and ovaries of Japanese quail. In addition, we also examined the tissue-specific distribution of qRARß mRNAs and the developmental patterns of qRARß transcripts in tissues other than the reproductive organs.

MATERIALS AND METHODS

Complementary DNA Cloning of Quail RARß Isoforms

Partial cDNA cloning A partial cDNA of qRARß2 mRNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from quail embryo limbs (embryo Day 8) and reverse-transcribed into first-strand cDNA using a First Strand Synthesis kit (Gibco BRL, Rockville, MD). Sequences for the oligonucleotide primer pairs were designed on the basis of the published chicken RARß2 cDNA sequence. The forward primer was 5'-GGGGGAATCATGTTTGACTG-3' and reverse primer was 5'-GTCCCAGAGCCCAAGGTCCAG-3' corresponding to base pairs (bp) 112–131 and bp 777–757 of the chicken RARß2 cDNA sequence (GenBank accession no. X57340), respectively.

The PCR procedure consisted of a 10-min pre-PCR heat step at 95°C followed by a 30-cycle program with 1 min denaturing at 95°C, 30 sec annealing at 58°C, and an extension for 1 min at 72°C using AmpliTaq Gold polymerase (Perkin Elmer, Branchburg, NJ) in a Perkin-Elmer GeneAmpR PCR system 2400. The PCR product was subcloned into the pCR vector (Invitrogen, Carlsbad, CA) using a TA Cloning kit (Invitrogen).

Plasmid DNA was purified using a Wizard Preps DNA purification system (Promega, Madison, WI). Sequence analysis was performed with a DSQ-1000 automated fluorescent DNA sequencer (Shimadzu, Tokyo, Japan) using a thermo sequenase fluorescent-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham International plc, Buckinghamshire, England), according to the manufacturer's protocols. Sequence data were analyzed using the GeneTYX-MAC (version 10) and databases.

The 5' and 3' rapid amplification of cDNA ends (5' RACE and 3' RACE) The 5' RACE was employed in an attempt to obtain the complete 5' ends of qRARß2 cDNA and different isoforms of qRARß cDNA, and 3' RACE was applied to obtain complete 3' ends of qRARß cDNA. Briefly, first-strand cDNA and second-strand cDNA were synthesized from 3 µg total RNA extracted from quail embryo limbs (embryo Day 8), and a library of adaptor-ligated double-strand cDNA was constructed using the Marathon cDNA amplification kit (Clontech, Palo Alto, CA), according to the product protocol. The qRARß gene-specific primers were 5'-CTGTAGTCTGCAGTACTGGC-3' for the 5' RACE and 5'-GTTCCAAGCCCTCCTTCACCT-3' for the 3' RACE designed according to the partial cDNA sequence of qRARß2 obtained above by RT-PCR.

The PCR procedure consisted of a 1-min pre-PCR heat step at 94°C followed by a 30-cycle program with 30 sec denaturing at 94°C, 30 sec annealing at 60°C, and an extension for 2 min at 68°C using Advantage KlenTaq Polymerase Mix (Clontech) in a Perkin-Elmer GeneAmpR PCR system 2400. The later processes that included subcloning into the pCR vector, purification of plasmid DNA, and analysis of nucleotide sequences were the same as described above.

Animals and Sampling

One-day-old male and female Japanese quail (Coturnix coturnix japonica) were kindly provided by Ebihara Quail Farm (Tochigi, Japan). They were housed under a temperature-controlled brooder with continuous illumination for the first 3 days, and then were subjected to a 14L:10D cycle. A commercial diet (Nihon Nosan, Aichi, Japan) and water were available at all times. The sampling was carried out in the daytime.

To determine tissue distribution and tissue-specific expressions of qRARß transcripts, five male quail at 5 wk of age were killed by decapitation, and their tissues, including brain, liver, heart, lung, and testis, were dissected and frozen in liquid nitrogen immediately, then stored at -80°C until analysis.

To elucidate the developmental expression patterns of qRARß transcripts in the testis and brain, five quail were killed at different intervals ranging from 14 days to 150 days of age. Their testes and brains were removed and frozen in liquid nitrogen immediately and then stored at -80°C until analysis for qRARß mRNA levels.

In the same way, oviducts at different growing stages ranging from 20 days to 50 days of age were collected for analysis of the developmental changes of qRARß transcripts. Quail hens were kept in individual cages, and those laying four or more eggs in a sequence were selected and killed by decapitation. Follicles were removed and ranked from F1 (the largest preovulatory follicle) to F4 (the fourth largest one) and SF (small white follicles). The filled yolk was washed out in ice-cold 0.1 M PBS (pH 7.2), and both granulosa and theca tissues were frozen in liquid nitrogen immediately and then stored at -80°C until analysis for qRARß mRNA levels.

Measurement of mRNA Levels of qRARß Isoforms

Preparation and radiolabeling of antisense cRNA probe A 425-bp segment of qRARß2 cDNA that consisted of 9 bp of a 5'-untranslated region, all of the A and B domains and a part (177 bp) of the C domain was amplified by PCR (underlined in Fig. 1). To generate the antisense RARß cRNA probe, this 425-bp cDNA was subcloned into the pCR II vector (Invitrogen), linearized with HindIII restriction endonuclease and transcribed by T7 RNA polymerase in the presence of a {alpha}-32P-UTP as described previously [22]. Therefore, the undigested cRNA probe will be about 570 nucleotides in length.



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  		FIG. 1. Nucleotide and deduced amino acid sequences of qRARß1 and qRARß2 cDNAs amplified by 5' RACE. Nucleotides are numbered on the right side of the figure. The 5' untranslated region is indicated by lowercase letters. A1 and A2 on the left side of the figure indicate the isoform-specific A domains of RARß1 and ß2, respectively. B and C indicate the common domains of RARß1 and ß2. The nucleotides with a single underline in RARß2 are amplified to synthesize the cRNA probe for the RNase protection assay, and those with a single underline in RARß1 represent the protected region when the cRNA probe of RARß2 is used

Ribonuclease protection assay Total RNA was extracted from the brain, liver, heart, lung, and testis according to the method described previously [23] using TRIzol Reagent (Gibco BRL). Sample quality and quantity were assessed by measuring the optical density of each sample at 260 nm and 280 nm. Sample quality was also checked by ethidium bromide staining of denatured agarose gels and the intensity of 18S and 28S rRNA bands was analyzed using ImagerMaster VDS (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Equal amounts of total RNA (20 µg) from each sample were then used for determining the tissue-specific patterns of RARß isoform mRNA expression by an RNase protection assay as described previously [22] with some modification. Briefly, total RNA was hybridized with 200 000 cpm of probe overnight, then digested with RNase A and RNase T1. The RNase A and RNase T1 were inactivated by proteinase K solution (RNA grade; Gibco BRL). Protected mRNA was directly precipitated by adding isopropanol at the same volume and 4 µl of tRNA (5 mg/ml). Therefore, all the processes can be finished in the same tube from total RNA to electrophoresis.

Lysate RNase protection assay The qRARß mRNA levels in the developing tissues were analyzed by lysate RNase protection assay as described previously [23, 24]. Briefly, tissues were homogenized in five volumes (w/v) of lysis buffer (5 M guanidinium thiocyanate, 0.1 M EDTA, 0.1 M Tris, pH 7.0, 1% ß-mercaptoethanol). The homogenate was centrifuged at 10 000 x g for 5 min at room temperature, and aliquots of 50 µl of the supernatant were mixed with 5 µl lysis buffer containing 250 000 cpm of probe. After overnight hybridization at 37°C, 500 µl digestion buffer containing RNase A and RNase T1 was added, and excess probes were digested at 37°C for 30 min. Twenty microliters of 10% sodium N-lauroyl-sarcosinate and 5 µl of proteinase K solution (RNA grade; Gibco BRL) were added, and tubes were incubated at 37°C for 30 min. The tubes were centrifuged for 2 min at room temperature. The supernatant and 500 µl isopropanol were mixed, kept at -20°C for 30 min, and then centrifuged for 15 min. The precipitates were dried and resuspended in a sample buffer. Hybridized RNA was size-separated on an 8% acrylamide gel.

Data Analyses

Hybridized blots were imaged and analyzed using MacBAS-2500 (FujiPhoto Film, Tokyo, Japan). All values for mRNA levels are given as a relative value in an experiment. In cases where the SEM is given, the means reflect five samples, and the statistical significance was determined by one-way ANOVA followed by multiple comparisons of means with Duncan's new multple range test.

RESULTS

Cloning and Characterization of qRARß Isoforms

We obtained a 666-bp cDNA fragment by RT-PCR (positions 18–683 of GenBank accession no. AF110730), and found that only three nucleotides in this cDNA were different from the corresponding region of chick RARß2 cDNA, meaning that these two cDNAs were 99% identical [25]. However, the differences in the nucleotide sequence did not cause a difference in the deduced amino acid sequences of these two cDNAs. Therefore, we concluded that the obtained cDNA is a part of qRARß2 cDNA. Two cDNA fragments (850 and 442 bp) were amplified by the 5' RACE. They differed completely in their 5' untranslated regions and amino-terminal regions (A domain). The 850- and 442-bp cDNA fragments showed a high degree of homology in their nucleotide sequences with the corresponding regions of chicken RARß1 [26] and RARß2 [25], respectively (Fig. 1). However, no clones corresponding to the chicken RARß4 [27] and to the mouse RARß3 isoform [28] were obtained. Among the several clones obtained from the 3' RACE, the longest one had 1424 bp that included the partial coding region of RARß and 269 bp of its 3' untranslated region. By combining the overlapped parts of the cDNAs from the 5' and 3' RACE, two complete cDNAs for qRARß1 (2049 bp) and qRARß2 (1642 bp) were obtained. These have been deposited in the GenBank database (accession nos. AF110729 and AF110730 for qRARß1 and qRARß2, respectively). The deduced amino acid (aa) sequences of qRARß1 (455 aa) and qRARß2 (448 aa) were, respectively, 99% and 100% identical to those of chicken RARß1 and RARß2.

Differential Expression of qRARß Isoforms

To investigate how each of the qRARß isoforms is expressed among tissues, we performed an RNase protection assay using 32P-labeled qRARß2 probe and 20 µg of total RNA extracted from various quail tissues. Because isoforms of qRARß1 and qRARß2 only differ in their 5' untranslated region and in the A domain (Fig. 1), an assay using the antisense qRARß2 probe is expected to give a fully protected band derived from qRARß2 mRNA (425 bp) and a partially protected band from qRARß1 mRNA (260 bp), providing us with quantitation of both transcripts (underlined parts in Fig. 1).

Both qRARß1 and qRARß2 mRNAs were present in all of the tissues examined, but the distribution patterns were clearly distinct between these two isoforms (Fig. 2). Because both ß-actin and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels were dramatically fluctuated among tissues examined, we measured the intensities of 28S and 18S rRNAs to normalize the mRNA levels of qRARß isoforms. The qRARß1 mRNA was present in the testis at levels apparently much higher than that of qRARß2, while qRARß2 mRNA was the predominant transcript in the heart, kidney, and lung. The liver also predominantly contained qRARß2 isoform mRNA, although at a lower level than the lung and kidney. In the brain, qRARß1 and qRARß2 mRNAs were expressed at approximately equal levels. Furthermore, both qRARß1 and qRARß2 mRNA levels varied greatly between the tissues examined. The expression of qRARß1 mRNA was high in the testis, moderate in the brain, lung, and kidney, low in the heart, and nearly undetectable in the liver. In contrast, qRARß2 mRNA was present at low levels in the testis and liver, at intermediate levels in the brain, heart, and kidney, and at a higher level in the lung. In addition, a fragment was also presented between qRARß1 and qRARß2 fragments in all tissues examined.



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  		FIG. 2. Tissue distribution of qRARß1 and qRARß2 mRNAs analyzed by RNase protection assay. Total RNA (20 µg) isolated from different tissues of Japanese quail at 5 wk of age was hybridized to qRARß2 cRNA probe as indicated in the legend of Figure 1. A typical result of the assay for RARß isoforms, ß-actin, and GAPDH is shown in A, and responsive 28S and 18S rRNA bands are also presented (A). The quantitative representation of multiple results expressed as values relative to the abundance in the brain is shown in B. Data are means ± SEM (n = 5)

Developmental Patterns of Gene Expression of qRARß Isoforms

Testicular weight increased progressively from 14 to 30 days of age, and increased markedly thereafter (Fig. 3). At 50 days of age, quail had reached their sexual maturation with similar testicular weight of their 150-day age and also had big red cloacal glands that secreted white foam, which are the external indicators of sexual maturity in birds [17].



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  		FIG. 3. Changes in the testicular weight with age. Japanese quail were maintained under controlled conditions with a light-dark cycle (14L:10D). Data show the means ± SEM (n = 5)

Age-dependent changes in the expression of qRARß isoform transcripts in the quail testes were examined using a lysate RNase protection assay. A representative autoradiograph of the result is presented in Figure 4A. Both qRARß1 and ß2 transcripts were detected in the testes of all age groups. As is already seen in Figure 2, qRARß1 was the predominant transcript in all stages of testicular development. A transient rise in the expression of qRARß1 mRNA was clearly observed in the course of testicular development. The qRARß1 mRNA began to increase at 21 days of age and increased abruptly beginning at 30 days of age (Fig. 4B), just when the testes commenced rapid growth (Fig. 3). The level of qRARß1 mRNA was about two times higher (P < 0.05) in the testes from 30–40-day-old quail than in testes from 14-day-old quail. By 50 days of age, when sexual maturation was almost complete, the level of qRARß1 mRNA had returned to the level observed at 14 days of age (Fig. 4B). In contrast, no obvious change of qRARß2 mRNA was observed during testicular development (Fig. 4C).



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  		FIG. 4. Expression of RARß isoform transcripts in the developing testes of Japanese quail. One representative result of the lysate RNase protection assay is presented in A. The protected bands of qRARß1 and ß2 were quantitated and integrated in B and C, respectively. Bars represent values relative to that at 14 days old. Days of age are indicated at the bottom. The data are represented as the means ± SEM (n = 5). Means not bearing the same letter are significantly different (P < 0.05)

We next examined the expression profile of qRARß isoforms in the developing oviducts from 20 to 50 days of age. Due to the dramatic change in the oviduct weight between 30 and 40 days of age (data shown in the corresponding columns of Fig. 5C), the oviducts were divided into two groups at this period according to their weights (see the legend for Fig. 5). Both qRARß1 and qRARß2 mRNAs exhibited a temporal rise during oviduct development (see representative autoradiograph, Fig. 5A). Prior to the onset of extensive oviduct growth, the qRARß2 transcript steadily increased to the maximum level by 25 to 30 days of age, then started to decrease gradually before the oviduct grew rapidly. When the average oviduct weight reached 2.88 g (40 days), qRARß2 mRNA became almost undetectable (Fig. 5C). A similar expression pattern was observed in the RARß1 mRNA (Fig. 5B). In contrast to the testes shown in Figure 4, qRARß2 was the major transcript of qRARß isoforms in the oviduct (Fig. 5A).



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  		FIG. 5. The developmental expression patterns of qRARß1 and ß2 transcripts in the oviducts of Japanese quail. Oviducts at the various ages indicated were sampled for measuring the abundance of qRARß1 and ß2 by using a lysate RNase protection assay. The individual variance of oviduct weight was extremely great between 30 and 40 days of age because of the rapid growth of oviduct in this period. So we found a practical way to subdivide the oviducts according to their weights. The mean oviduct weights of the lighter half (L) and the heavier half (H) are shown in the corresponding columns (g/bird) of C. A representative autoradiograph is shown in A. Quantitation of the protected qRARß1 and ß2 as the values relative to the 20-day-old value are presented in B and C, respectively. The data are presented as the means ± SEM (n = 5). Means not bearing the same letter are significantly different (P < 0.05)

Furthermore, to elucidate whether the developmental changes of qRARß isoforms expression also occur during the course of egg formation, we analyzed the expression of qRARß isoform mRNAs during follicle development in the ovary. As shown in Figure 6, the follicle, including both granulosa and theca tissues, contained qRARß1 and ß2 mRNAs with qRARß2 as the major transcript, and exhibited transient rises of qRARß1 and ß2 expression as the follicle developed from the prehierarchical (SF, small white ovarian follicles <2-mm diameter) to the largest preovulatory follicle (F1) with a pattern similar to that seen in oviduct development.



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  		FIG. 6. Expression of RARß isoform transcripts at different stages of the follicular growth of Japanese quail. F1, F2, F3, and F4 indicate the largest, second-, third-, and fourth-largest follicles, respectively; SF refers to small white follicles (in diameter <2 mm). The data are the means ± SEM (n = 5). Different letters indicate statistically significant differences (P < 0.05)

Finally, we analyzed the developmental changes of qRARß isoforms expression in brain tissue in addition to tissue from the gonad organs. Unlike in the testis and oviduct, qRARß1 and qRARß2 mRNA levels in the developing brain remained almost constant throughout the entire experimental period (Fig. 7). In addition, qRARß1 and qRARß2 mRNAs were expressed at approximately equal levels at all ages examined.



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  		FIG. 7. Expression of RARß1 and ß2 transcripts in the brains of Japanese quail at different ages. Brains sampled at the different ages indicated at the bottom were used for measuring the abundance of qRARß1 and ß2 mRNA by the lysate RNase protection assay. Bars represent values relative to that at 0 days old. Days of age are indicated at the bottom. The data are represented as the means ± SEM (n = 5)

DISCUSSION

The cloning of cDNAs encoding RARs and RXRs has greatly advanced our understanding of the mechanism through which RA exerts its multiple effects on development and differentiation. Therefore, to understand how RA exerts its stimulating effects on the sexual maturation in Japanese quail [16], the cloning of quail cDNA of these receptors also became inevitable. Six subtypes of these receptors [29] in the mammal and four subtypes (three RARs and RXR{gamma}) in the chicken [25, 30–32] have been identified. None of the RARs and RXRs in quail have yet been reported. In the present study, two complete cDNAs corresponding to the qRARß1 and qRARß2 isoforms were cloned using RT-PCR and the RACE techniques, and their overall sequences were determined to have high homology with the chick RARß1 and RARß2, respectively. The amino acid sequence of qRARß1 and ß2 can be divided into six domains (A–F) based on homology with other members of the hormone nuclear receptor superfamily [33, 34]. As in other species [27, 28], isoforms of quail RARß1 and RARß2 also diverge from each other in their 5' untranslated region and in the A domain, with their other regions being common. Several transcripts of RARß that are generated by the use of two promoters and alternative splicing [28, 35] have been ascertained in mammals (ß1–4) [28, 35–37], and in the chicken (ß1, 2, and 4) [27]. However, we could not obtain the quail orthologue of mouse RARß3 [28] and chicken RARß4 isoform [27] by the methods used in this study.

We carried out the RNase protection assay to detect the expression of different isoforms using one cRNA probe that spanned the qRARß2-specific A domain, and the common B and C domains. Two main bands with lengths of about 420 bp and 260 bp were protected in all of the tissues examined. Because the 260-bp band represented the B and C regions common among all isoforms of RARß, it is possible that this 260-bp band represents the total of qRARß isoforms with the exception of qRARß2. There are two sources of evidence suggesting that the 260-bp band might represent only the expression of qRARß1 mRNA. First, chick RARß4 that shares a part of the A domain with RARß2 was a splice variant of RARß2 [26, 27]. Therefore, if this were the case in Japanese quail as well, qRARß4 mRNA would appear to be longer than 260 bp in this study. In fact, a band did exist between the two main bands in all tissues examined. Additionally, the expression of this band could be induced by RA administration in a similar manner as in qRARß2 (unpublished data). Thus, this band may represent the expression of qRARß4, although we did not succeed in cloning this isoform. Second, it has been reported that RARß3 expressed more abundantly than RARß1 in the adult mouse lung and skin, and almost at the same level in the brain. Thus, it could be assumed that RARß3 will also be expressed in the Japanese quail, giving rise to the possibility that the 260-bp band may include the total expression of RARß1 and ß3. Mouse RARß3, having an additional 81-bp sequence just upstream of the A/B junction in contrast to mouse RARß1, was a splice variant of RARß1 and had a 5' upstream sequence in common with RARß1 [28]. Therefore, to identify whether RARß3, a corresponding isoform of the mammal, also exists in the Japanese quail, we carried out PCR analysis, using 5' and 3' oligonucleotide primers assumed to be common to both isoforms according to the information from mouse RARß1/ß3 (positions 446–465 and 602–621 of qRARß1 in Fig. 1). Complementary DNAs were synthesized from RNA samples containing relatively high transcripts of the 260-bp band as judged from the RNase protection assay. These samples were from the brain, testis, lung, and kidney in this case. The anticipated lengths of the RT-PCR products that were derived from qRARß1 and qRARß3 were 173 and 254 bp, respectively. We could find only one band with a length near 180 bp among all of the PCR products (Fig. 8), and sequence analysis showed that this band was a 173-bp cDNA fragment representing the designed part of qRARß1. Thus, this result suggests that qRARß3, or at least a similar splice form of mouse RARß3, might not exist in the quail or may be expressed in a more restricted spatiotemporal manner.



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  		FIG. 8. The result of RT-PCR using RNA from brain, testis, lung, and kidney. The sense and antisense oligonucleotide primers, corresponding to positions 446–465 and 602–621 of qRARß1 in Figure 1, respectively, were used. Total RNA isolated from brain, testis, liver, and kidney (5 wk of age) was reverse-transcribed into first-strand cDNA. The PCR was carried out as described in Materials and Methods for obtaining the partial cDNA of qRARß2

Tissue distribution analysis revealed that qRARß isoforms expressed widely in various tissues with a distinct expression pattern. In general, one of the two qRARß isoforms was much more abundant than the other, except in the brain. The tissues examined in the present study could be classified into three groups with respect to the relative expression of qRARß isoforms: qRARß1-dominant tissues such as testis, qRARß2-dominant tissues such as heart, kidney, lung, liver, oviduct, and follicle, and evenly expressing tissues such as brain. These differential expression patterns of RARß isoforms were generally in accordance with the patterns observed in the adult human and mouse tissues by Northern blot analysis [28, 38]. Some differences, however, existed between quail and mammal tissues. In the quail lung, qRARß2 was the predominant transcript, while RARß1 was predominant in the mouse lung. Additionally, RARß1 isoform exceeded ß2 in the mouse brain, while both existed evenly in the quail brain. Furthermore, RARß1 mRNA could not be detected in the mouse liver, kidney, and heart, whereas it presented in all of these tissues in the present study. This difference may be partly attributable to the different methods used (Northern blot analysis vs. RNase protection assay). Although the expression of RARß isoforms during chick embryo development has been reported [27, 39, 40], no data are available on the tissue distribution of RARß isoforms in the avian species after hatching. Recently, we found that gene expression of RAR{alpha} and RXR{gamma} also manifested their unique tissue-specific pattern in Japanese quail (unpublished data), which was quite different from that of qRARß isoforms. Therefore, the differential expression of qRARß1 and qRARß2 isoforms in various tissues strongly suggests that these two isoforms may assume, at least, distinct functions among tissues.

Nuclear run-on studies of different human cell lines revealed that the mRNA levels of expression of RARß isoforms in these cell lines are positively related to their transcription rates [38]. This result suggests that some of the variations in the RARß expression between human tissues might be due, at least partly, to differences in the transcription rates of the RARß gene. Analysis of RARß gene structure in mammals has shown that the expression of RARß1 and RARß2 are controlled by two different promoters (P1 and P2) [28, 35]. Therefore, it is possible that the regulation of each promoter by many factors, including retinoids and transcription factors, probably determines the tissue-specific profile of RARß gene expression. We recently analyzed the structure of the qRARß gene and found that the DNA sequence of the qRARß2 5' untranslated region was highly conserved compared to that in human and mouse RARß2. Furthermore, RARE and TATA box existed in the qRARß2 promoter (P2) region (unpublished data), as is seen in mammals. Further characterization of the qRARß gene promoter will surely help elucidate the mechanism of tissue-specific expression.

The lysate RNase protection assay [24, 41, 42] that enables us to directly estimate the mRNA levels per wet-tissue weight, eliminating the necessity to measure control mRNA such as actin and GAPDH, was applied to analyze the developmental patterns of qRARß isoforms expression. Because mRNA levels of even actin and GAPDH undergo drastic changes during development, there seems to exist no satisfactory control mRNA where the developmental change of gene expression is concerned. In addition to the usefulness of measuring target mRNA levels in very small organs [24, 42], this method has been proved to apply well to the precise analysis of mRNA during development.

The developmental changes of RAR{alpha} and RAR{gamma} expression have been detected in the postnatal rat testis [12, 43]. No studies have been made of the expression of RARß mRNA during testicular development in either mammals or birds, although the presence of RARß mRNAs in the murine testis has been reported by many researchers [10, 20, 44]. In the present study, we report for the first time the developmental patterns of the different qRARß transcripts in the quail testis. Two transcripts of qRARß (ß1 and ß2) have been detected in the quail testis, and their expressions were found to be completely different from each other. The dominant transcript, qRARß1 mRNA, demonstrated a temporal change with testis growth. By contrast, qRARß2 mRNA that was expressed at a lower level did not display any obvious change with testis growth. These findings suggest that RARß isoforms may function differently from each other, and that RARß1 may play a more important role in the process of the growth of the quail testis than does RARß2. Kim and Griswold [44] reported that the RARß transcript was present exclusively in Sertoli cells in the adult rat testis. Recently, Dufour and Kim [45] analyzed the developmental changes of RARß protein in the rat testis during postnatal growth using an immunohistochemical method and found that the presence of RARß protein in Sertoli cells of rats aged 15 days to adulthood coincided with the rapid growth of the rat testis. In the present study, we also found that the expression of qRARß1 mRNA increased just when the testis weight underwent a dramatic increase. In Japanese quail, the rapid increase in testicular weight is due to the proliferation and hypertrophy of Sertoli cells [46]. Thus, it could be speculated that qRARß1 may also be expressed mainly in Sertoli cells and may be necessary for Sertoli cell proliferation. Though we have not yet identified the type of cells that express the RARß gene, the high expression of qRARß1 mRNA before the rapid increase of testicular growth and after sexual maturation also suggest that qRARß1 may be important for Sertoli cell differentiation and for spermatogenesis. In fact, the expression of RARß protein was also detected in rat spermotocyte and spermotogonium [45], although mice lacking all RARß isoforms developed normally and were fertile [47]. This finding does not necessarily mean, however, that RARß is not involved in spermatogenesis, because there is evidence of functional redundancy among the RARs [48].

The present study is also, to our knowledge, the first report on the developmental changes of qRARß isoforms in the female reproductive organs. Both qRARß1 and ß2 mRNAs exhibited clear changes during oviduct development and during follicular growth. In the oviduct, the expression of both qRARß mRNAs reached their peak levels just before the rapid change in the oviduct occurred, meaning that the rapid growth of the oviduct was preceded by an increase in the expression of two RARß isoforms. In addition, the expression of both qRARß mRNAs declined to almost undetectable levels when the oviduct developed to the laying stage (over 2.88 g in weight). The rapid growth in oviduct size was caused by the cellular proliferation and differentiation of the magnum (the main part of the oviduct) [49]. It also has been reported that the further development of the magnum (above 2.3 g weight) is caused solely by the accumulation of secretory products such as ovalbumin [50]. Thus, these results suggest that RARß might play an important role in the cellular proliferation and differentiation of the quail oviduct. From the developmental pattern of qRARß isoforms during follicular growth, we may speculate that RARß is also necessary for follicle development. In contrast to the testis, RARß2 may play a more important role in the process of quail oviduct and follicle growth than does RARß1, because RARß2 was the major transcript in both cases.

The gene expression of RARs and/or RXRs is regulated by many hormonal factors. It was reported that administration of exogenous testosterone resulted in an increase in transcripts of RAR{alpha}, but a decrease in transcripts of RAR{gamma} in rat testis [43]. Furthermore, FSH inhibited the transcriptional activation of RAR{alpha} in mouse Sertoli cell lines [51]. Both testosterone and FSH have been reported to stimulate an induction of cAMP in testis and ovary [52, 53]. The cAMP-dependent protein kinase (PKA) decreased the transcriptional activity of RAR{alpha} and inhibited the RA-induced expression of mRNAs for RAR{alpha}, ß, and {gamma} in several cell lines [54–56]. It is well known that both vitamin A and FSH play very important roles in the development and functions of testis. Testosterone synthesis in mammalian testes is vitamin A dependent. In the development of oviduct, vitamin A is involved in estrogen-induced cell proliferation [57]. Ovarian follicular development depends upon FSH acting on its cognate receptor (FSHR) expressed by granulosa cells. It is also reported that ovarian insulin-like growth factor-I, which is a growth factor involved in the cell proliferation and differentiation, serves to enhance the responsiveness of granulosa cell to FSH by augmenting FSHR expression [58]. Therefore, it could be speculated that all these hormone and growth factors or others might influence the expression pattern of the RARß gene during the gonadotropic growth reported in the present study.

The expression of qRARß isoforms was relatively high and maintained almost a constant level in the brain after hatching. This result further supports the hypothesis mentioned above that RARß may be important in the regulation of target gene expression during the growth of the quail reproductive organs.

Using birds as experimental model has provided a great body of information in the function of vitamin A. For example, the quail embryo model has been widely used to study the function of vitamin A in embryonic development and has elucidated the important role of vitamin A in the very early stages of embryonic development, which is difficult to obtain in mammals [59]. Thus, the results presented here may also give new insights into the function of the RARß gene in the reproductive system of birds and mammals as well. It is well known that RARs or RXRs must form heterodimers and homodimers with RXRs in order to function as transcription factors. Complete RAR{alpha} [13], RAR{gamma} [15], and RXRß [14] mutant mice are sterile. Compound RXR and RAR mutants reveal defects that are either absent or less severe in the single mutants and also belong to the vitamin A deficiency syndrome [48]. These genetic studies also suggest that RXR{alpha} may be the main RXR implicated in the developmental functions of RARs [60]. Immunohistochemical studies suggest that RAR{alpha} may potentially dimerize with RXR{alpha} in Sertoli cells and with RXR{gamma} in germ cells in the rat testis [45]. Therefore, further studies of the expression of other retinoid receptors, the regulation of these receptors by vitamin A status, and the identification of RA-responsive genes during the development of the reproductive organs are necessary for a better understanding of the molecular mechanisms that underlie the sexual maturation of Japanese quail.

ACKNOWLEDGMENTS

The Japan Society for Promotion of Science (JSPS) is greatly acknowledged for providing a postdoctoral fellowship to Z.F. We thank Mr. Teruo Ebihara (Ebihara Quail Farm, Tochigi, Japan) for kindly providing day-old Japanese quail.

FOOTNOTES

First decision: 21 April 2000.

1 This work was supported partly by a Grant-in-Aid for Exploratory Research (9876071) to T.K. and by a Grant-in-Aid for JSPS Fellows (98218) to H.K. and Z.F. from the Japanese Ministry of Education, Science, Sports and Culture. Back

2 Correspondence. FAX: 81 3 5841 5114; akatoq{at}mail.ecc.u-tokyo.ac.jp Back

Accepted: August 22, 2000.

Received: March 27, 2000.

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