Endocrinology Vol. 144, No. 4 1612-1619
Copyright © 2003 by The Endocrine Society
Androgen Down-Regulated and Region-Specific Expression of Germ Cell Nuclear Factor in Mouse Epididymis
Yuanxin Hu,
Zongyao Zhou,
Chen Xu,
Quan Shang,
You-Duan Zhang and
Yong-Lian Zhang
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Y.H., Q.S., Y.-D.Z., Y.-L.Z.), Shanghai 200031, China; and Department of Histology and Embryology, Shanghai Second Medical University (Z.Z., C.X.), Shanghai 200025, China
Address all correspondence and requests for reprints to: Dr. Yong-Lian Zhang, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China. E-mail: yonglz{at}sunm.shcnc.ac.cn.
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Abstract
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Germ cell nuclear factor (GCNF), a nuclear orphan receptor, involved in spermatogenesis, neurogenesis, differentiation, and embryo development, was highly expressed with two transcripts (7.4 and 2.3 kb) in mouse testis and with only one transcript (7.4 kb) slightly expressed in brain, liver, and kidney. The 2.3-kb transcript was restricted to round spermatids at stages VII and VIII of the spermatogenic cycle. The present report demonstrated its expression in epididymis as well, but at a very low level. Northern blot analysis showed two transcripts: a common 7.4-kb transcript and a unique 3.1-kb transcript. The expression levels of both GCNF transcripts in epididymis were down-regulated by androgen, as observed in castrated animals and aged mice. Polyclonal antisera against GCNF protein were raised. Western blot analysis showed the presence of only one band in total protein extracts from either mouse testis or epididymis. It indicated that the two mRNAs (7.4 and 3.1 kb) encode for the same protein as in testis. Fluorescent immunohistochemical staining and in situ hybridization showed that its expression was in the principal cell abundant in the corpus region. It implies that some androgen-regulated gene expressions located at the corpus principal cells might be controlled by GCNF.
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Introduction
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GERM CELL nuclear factor (GCNF), also named retinoid receptor-related testis-associated receptor (RTR), neuronal cell nuclear factor, or NR6A1, is an orphan member of the nuclear receptor superfamily that is also a putative transcription repressor (1, 2, 3, 4, 5). The GCNF gene was initially reported to be predominantly expressed as two transcripts (7.4 and 2.3 kb) in germ cells of adult mice, but only its 7.4-kb mRNA was detectable in brain, liver, and lung (2, 6). Further analysis revealed that the 2.3-kb mRNA was restricted to round spermatids at stages VII–VIII of the spermatogenic cycle (7). As a transcription factor, its DNA-binding site (responsive element) and its downstream targets have been identified, such as protamine-1 and -2 (8). The 7.4-kb mRNA was also found in the embryo and embryonic carcinoma cells (3, 9). Furthermore, knockouts of the GCNF gene were embryonically lethal (10). This suggested that the receptor might participate in the process of gametogenesis and neurogenesis. This complex temporal and spatial expression pattern suggested that GCNF might play fundamental roles in different genesis (3, 4, 7, 10, 11, 12, 13). We were interested in its roles in spermatogenesis and sperm maturation because it is highly expressed in testes with an extra 2.3-kb transcript, and its expression in epididymis has not been reported.
Mammalian spermatozoa are highly differentiated by the time they leave the testis (14, 15). Nonetheless, they lack motility and the ability to fertilize, which is not intrinsic to sperm cells themselves, but depends on the epididymis (16). The epididymis is a long convoluted tubule in which spermatozoa migrate from the testis to the vas deferens. Along the tubule, with distinct morphological changes and gradients in gene expression and secretion of specialized proteins, the epididymis is distinguished into three major regions: caput, corpus, and cauda (17, 18, 19). As spermatozoa move along the epididymis, the sperm surface undergoes a series of physical, chemical, and morphological alterations resulting from the unique and ever-changing luminal fluid microenvironment provided by different, well organized epididymal regions (17). The regional specificity of gene expression in epithelial cells is crucial to establish the luminal fluid microenvironment in the epididymis (20). The spatial and temporal patterns of gene expression are critical to the development and maintenance of a fully functional epididymis, and there must be some stringent regulation involved in this series of complex events (21). Thus, the identification and analysis of the expression pattern in epididymis may provide valuable information about the role of the gene in spermatozoa maturation. Here we report the expression and characterization of GCNF in mouse epididymis determined by improved Northern blot analysis and using polyclonal antisera against GCNF protein.
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Materials and Methods
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Animals
Animals (BALB/c mice and Sprague Dawley rats) were purchased from the Animal Center of the Chinese Academy of Sciences. At 24 d after birth, mice were regarded as "immature," at 56 d after birth as "adult," and at 20 months after birth as "old."
RNA isolation and Northern blot analysis
Adult (56 d old) male BALB/c mice were castrated by surgery. The mice were classified into seven groups (six mice per group), each killed at different times after castration (0, 3, 5, and 7 d) and 1, 3, and 5 d after a single injection of testosterone propionate (5 µg/g body weight) applied to the 7-d castrated mice. Epididymis samples for each group were pooled for RNA extraction. Pooled serum samples from every group were sent to Shanghai Zhong-Shan Hospital for measurement of testosterone content by RIA. The other samples of testis, epididymis, and spleen were from six immature (24 d), six adult (56 d), and two old (20 months) BALB/c mice and three adult Sprague Dawley rats. Total RNA extraction and Northern blot analysis were performed as described previously (22). Total mRNAs were prepared according to the instructions of the manufacturer (Promega Corp., Madison, WI). The probe used was a 271-bp cDNA fragment of GCNF (nucleotides 1535–1806) that was 
-32P-labeled (2). The films were exposed with two intensifying screens at -80 C for 10 d for autoradiography. RNA expression levels were quantified using a Gel-Pro Analyzer 3.0 (Microsoft Corp., Redmond, WA) and were normalized by 18S ribosomal RNA.
RT-PCR and sequencing
A reverse transcription (RT) kit from Invitrogen (San Diego, CA) was used. Oligo(deoxythymidine)17MN was annealed to 2 µg of old mouse epididymis total RNA, and the reaction was initiated by adding reverse transcriptase kept at 42 C for 50 min. The enzyme was inactivated at 70 C for 15 min. The resulting first strand cDNAs were used as template for first round PCR amplification using oligo(deoxythymidine)17MN and GCNF-specific (SP9, 5'-ATACACACATAAGCCAAACC-3') primers. The cycling conditions were 94 C for 1 min, 50 C for 30 sec, and 68 C for 2 min for 35 cycles. The PCR products corresponding to a band size of approximately 1.1 kb (see Fig. 4B
, area between the two arrows) were cut and extracted for the second round PCR amplification using two GCNF-specific primers (SP9 and 3TT, 5'-TTTATTCTTTCAGCAGCCTGG-3'). Cycling conditions were 94 C for 30 sec, 57 C for 30 sec, and 68 C for 2 min for 35 cycles. The sample in the secondary PCR product size of 1.1 kb was cut and extracted from gel, then sequenced with primer SP9 (United GENE Holdings, Ltd., Shanghai, China).
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Figure 4. Identification of the sequence of GCNF smaller transcript in epididymis by RT-PCR. A, The structure of two transcripts. The entire 3'-untranslated region and part of its sequence (2661–2860 bp) for the 7.4-kb cDNA were quoted (26 ). The predicted polyadenylase adding signal (AAUAAA) for 3.1-kb mRNA is shown with shading and in bold type. The predicted size of the RT-PCR products was 4.7 kb for 7.4-kb mRNA and 1.1 kb for the 3.1-kb mRNA. The probe used in the Northern blot is shadowed and was amplified with primers sp9 and sp6. The nested reverse primer (3TT) for the second round PCR is framed. B, PCR results with different templates and primer combinations. The results of the first round PCR were obtained using the RT mixture as template and sp9 and oligo(deoxythymidine)17MN as primers. The results of the second round PCR were obtained using the first PCR products of approximately 1.1 kb recovered from the agarose gel as template and sp9 and 3TT as primers. M, DNA marker.
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Protein extracts preparation and Western blot analysis
The testis nuclear protein extracts were prepared according to the published method (23) with minor modification [adding 100 µl 0.1 M phenylmethylsulfonylfluoride and 50 µl protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) to buffer II instead of adding various proteases].
The total protein extracts of mouse epididymis were prepared as follows. Epididymis samples were homogenized directly in 1x sodium dodecyl sulfate sample buffer [2% sodium dodecyl sulfate, 62.5 µM Tris (pH 8.0), 0.4 M dithiothreitol, and 10% glycerol tissue weight (g)/buffer volume (ml), 1:5] on ice, then left at 80–85 C for 5 min. The mixture was centrifuged at 12,000 rpm for 15 min. The supernatant was assayed for protein concentration.
Western blot analysis
Protein samples (80 µg proteins of epididymis extracts/lane) were separated on a 10% SDS-PAGE gel and electron-blotted to a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The anti-GCNF antiserum was used as primary antibody (24), and goat antirabbit immunoglobulin G-horseradish peroxidase was used as secondary antibody with diaminobenzidine for color development.
Immunohistochemical staining
Tissues were fixed in Bouin’s fluid and then embedded in paraffin. For immunohistochemical staining of mouse testis and epididymis and to visualize GCNF for confocal microscopy, the fluorescein isothiocyanate-conjugated AffiniPure goat antirabbit IgG Indirect kit (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used. For cell discrimination, the immunofluorescence-stained testis and epididymis sections were then restained with hematoxylin and eosin. Photographs of hematoxylin and eosin-stained sections were made (Leica Corp., Wetzlar, Germany), and digital photographs of fluorescent sections were taken using a 410 laser scanning confocal microscope and software (Carl Zeiss, Jena, Germany). The whole epididymis image with immunofluorescent staining was edited with Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA).
In situ hybridization
Single-stranded RNA probes for in situ hybridization were synthesized in the presence of digoxigenin-UTP (Roche, Mannheim, Germany) and T3/T7 RNA polymerase (MBI Fermentas, St. Leon-Rot, Germany) from linear pBluescript KS+ containing 3'-untranslated region sequences of GCNF (nucleotides 2448–2602) (2). The antisense probe was generated by T3 RNA polymerase, and complementary sense probe was generated by T7 RNA polymerase. Both probes have less than 13% homology with other genes according to Blast in GenBank.
Tissues were fixed in 4% paraformaldehyde/PBS and then embedded in paraffin. In situ hybridization was performed as previously described (22, 25).
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Results
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GCNF was expressed in mouse epididymis with two transcripts
Hirose et al. (1) and Chen et al.(2) showed that GCNF mRNAs were predominant in mouse testis and were just detectable in brain, liver, and lung. For detecting the low level mRNA in Northern blot analysis, three parameters were modified i.e. enhancing the specific activity of the probe, loading total mRNAs instead of the total RNAs, and extending the exposure time for the x-ray film. GCNF expression patterns in mouse and rat epididymis were studied with testis as the positive control and spleen as the negative control. As shown in Fig. 1, in rats two transcripts (7.4 and 2.3 kb) were observed in both testis and epididymis, although the signals in epididymis were much lower than those in testis and could only be seen in lane Em where 2.5 µg total mRNAs was used instead of 20 µg total RNAs (Fig. 1A). The proportions of the two transcripts in the two tissues were different (i.e. 7.4/2.3 kb = 1/5 in testis and 3/1 in epididymis). In mouse epididymis, however, besides the common 7.4-kb mRNA, a unique 3.1-kb mRNA appeared (Fig. 1B). Using our sensitive assay, GCNF mRNA could also be detected in spleen at low levels, although it was undetectable in the Northern analysis performed by Hirose’s group (1). The sizes of the two transcripts were exactly the same as those in epididymis. Moreover, the 3.1-kb mRNA was also found in mouse brain (data will be shown in another report). This suggested that the expression pattern of GCNF might have species specificity. Although we reported previously that the two transcripts (7.4 and 2.3 kb) were expressed in testis conservatively in diverse species representative of three mammalian orders, the 2.3-kb mRNA was the predominant one in all of the species tested. Here we found that the 7.4-kb mRNA was the predominant one in epididymis. Testing the expression patterns in mouse epididymis at different ages further confirmed the presence of this 3.1-kb mRNA (Fig. 1C). It also showed that both of the transcripts were slightly increased in adult mice and were greatly increased in old animals (Fig. 1D).
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Figure 1. Northern blot analysis of GCNF total RNA/mRNA from different tissues of mouse and rat. All blots contained 20 µg total RNAs/lane, except for lane Em (2.5 µg total mRNAs/lane). A, Total RNAs from rat testis (T) and epididymis (E), respectively. Em, Total mRNA from rat epididymis. B, Samples from adult mouse testis (AT), spleen (AS), and epididymis (AE) and immature mouse testis (IT) and epididymis (IE) are shown. C, Samples from two old mouse testes (OT) and epididymes (OE), six adult mouse testes (AT) and epididymes (AE), and six immature mouse testes (IT) and epididymes (IE). D, The quantitative comparison (the relative hybridization intensity expressed as mGCNF/18S ribosomal RNA) of GCNF transcripts expressed at different ages in mouse epididymis. 3.1kb and 7.4kb, The relative amounts of the 3.1- and 7.4-kb transcripts, respectively; 3.1 + 7.4, the sum of the two transcripts.
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Down-regulation of GCNF mRNAs in epididymis by androgen
The expression of genes in the epididymis responds differentially to androgens and/or testicular factors (17). The effects of androgen manipulation on GCNF mRNA expression were studied by Northern blot analysis (Fig. 2A). The expression levels of both GCNF transcripts in epididymis increased gradually and reached a plateau 5–7 d after castration. After a single injection of testosterone to the 7-d castrated animals, the steady state of the two mRNAs gradually decreased toward normal in parallel with the rapid increase in the serum testosterone level. It increased again while the serum testosterone level fell back to zero (Fig. 2B). This androgen down-regulated expression pattern provides a logical explanation for why levels of GCNF mRNA in the aged mouse epididymis were much higher than those in adults.
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Figure 2. The expression profiles of GCNF in mouse epididymis after androgen manipulation. A, Northern blot analysis of adult mouse epididymis RNA from precastration (PC); bilaterally castrated for 3, 5, and 7 d (C3, C5, C7); and 1, 3, and 5 d after a single injection of testosterone propionate applied to the 7-d castrated mice (C7 + 1, C7 + 3, C7 + 5). There were six mice per group. B, Diagram showing the changes in the steady state of GCNF transcripts in mouse epididymis and the serum testosterone level (nanograms per milliliter) during androgen manipulation. Y1, The relative expression levels of transcripts (hybridization density of GCNF mRNA/18S ribosomal RNA); 3.1 and 7.4, the levels of 3.1- and 7.4-kb mRNAs, respectively; 3.1 + 7.4, the sum of the two transcripts; Y2, the serum testosterone concentration (nanograms per milliliter).
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GCNF expressed one protein in testis and epididymis
To identify the endogenous GCNF protein forms in epididymis, we raised polyclonal antisera against an Escherichia coli-expressed GCNF protein with a six-histidine tag (24). For testing the quality of the antisera, an immunohistochemical experiment was performed on mouse testis. The results shown in Fig. 3A are in agreement with the data reported previously and verified the specificity of the antiserum (11). On the Western blot only a single band of approximately 55 kDa was detected in testis nuclear extracts and epididymis total protein extracts (Fig. 3B), which well matched the expected size of the deduced GCNF open reading frame (1, 2). This result was also in keeping with the same protein we found in testis using a polyclonal antiserum against a synthetic GCNF peptide (26). No matter how many different forms of transcripts were found in mouse testis or epididymis, only one protein form could be detected. Therefore, different transcripts were translated to the same protein. This also suggested that the 3.1-kb mRNA must result from different alternative initiation or termination.
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Figure 3. Immunostaining and immunoblot analysis using polyclonal antisera against GCNF protein. A, Immunostaining of mouse testis sections with GCNF polyclonal antisera (I) and with preimmunized serum (PI) as the negative control. The I section was then restained by hematoxylin and eosin (HE). Staining was restricted to the nuclei of the primary and secondary spermatocytes and the round spermatocytes in the mouse testis section. E, Elongated spermatid; L, Leydig cell; P, pachytene spermatocyte; R, round spermatid. B, Western blot analysis of GCNF protein in adult mouse testis and epididymis. GP, Purified GCNF protein with a size of 55 kDa expressed in E. coli; TN, nuclear protein extract of adult mouse testis; EE, total protein extract of epididymis.
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The 3.1-kb mRNA was transcribed from the GCNF gene by alternative termination
To verify that the 3.1-kb transcript was not a nonspecific hybridization signal, RT-PCR was used to detect its 3' end. Using the complete sequence of the entire 3'-untranslated region (1874–6302 bp) of the 7.4-kb mRNA (26), a typical polyadenylase-adding signal, AAUAAA, was coincidentally found at 2815 bp, which might be the end of the 3.1-kb mRNA. Thus, using oligo(deoxythymidine) as a primer and old mouse epididymis total RNA as a template, RT was performed. As the probe used in the Northern blot analysis was a 271-bp cDNA fragment of GCNF (nucleotides 1721–1992) resulting from PCR with a pair of primers (sp9, nucleotides 1721–1740; sp6, nucleotides 1973–1992), sp9 and oligo(deoxythymidine)17MN were selected as primers for the PCR in the RT mixture to obtain the 4.7- and 1.1-kb bands expected for the 7.4- and 3.1-kb transcripts, respectively (Fig. 4A
). Due to the great disparity in annealing temperature for the two primers, it was difficult to obtain the distinct PCR products in this run as shown in Fig. 4B. Cutting off the PCR products about 1.1 kb from the agarose gel as template, a second round PCR was performed with sp9 as the forward primer and 3TT (with polyadenylase adding signal) as the nested reverse primer. A 1.1-kb fragment was recovered from the gel and sequenced. The sequencing results were as predicted and proved that the 3.1-kb mRNA was transcribed from the GCNF gene by alternative termination (sequence data not shown).
GCNF is region- and cell-specifically expressed in epididymis
GCNF expression in epididymis was further characterized by immunohistochemistry and in situ hybridization assays. With immunofluorescence staining, the expression pattern of GCNF exhibited regional specificity. As shown in Fig. 5, A and B, the distribution of the signal was as follows: initial segment and intermediate zone (-+), caput (+), proximal and middle corpus (2+), distal corpus (+), and cauda (-+). Such a region-specific expression pattern was further confirmed by in situ hybridization assay at the mRNA level (Fig. 6). The immunostaining of GCNF protein was restrictedly localized in the nuclei of epithelial cell (Fig. 5, C and D). The positive signal in the cauda lumen was due to round spermatids that highly expressed both transcripts of GCNF. Several reports mentioned that lymphocytes and round spermatids dropped from testis can be seen in epididymis cauda lumen (27, 28, 29). By hematoxylin and eosin staining of the same sections and confocal microscopy, we found that not all the principal/clear cells and halo cells were immunostained by the anti-GCNF antiserum. The percentage of stained principal/clear cells was low in the initial segment and intermediate zone, higher in the caput region, highest in the middle corpus, and then decreased gradually in the distal corpus and cauda (Fig. 5B). Moreover, we found that most of the halo cells in cauda and some halo cells in corpus were also immunostained by anti-GCNF antiserum, and there was no staining in apical cells, narrow cells, and basal cells throughout the epididymis (Fig. 5, C and D). It was uncertain whether GCNF was expressed in clear cells because of the difficulty in distinguishing principal cells and clear cells by hematoxylin and eosin staining (Fig. 5).
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Figure 5. Immunocytochemical localization of GCNF in mouse epididymis sections. A, The whole expression pattern of GCNF in adult mouse epididymis. B, The magnified photos of some parts of A; the table represents the types and numbers of cells expressing GCNF protein. nc, Immunostaining results with preimmunized serum as the negative control. C, The magnified photos of some related parts of B (B = b, D = d, and F = f). The immunofluorescence localization of GCNF in the nuclei of epithelial cells: principal cells and/or clear cell (thin arrows), and halo cells (thick arrows). D, Hematoxylin and eosin staining of the same sections as those in C, showing principle cells and/or clear cells (thin arrows) and halo cell (thick arrows).
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Figure 6. In situ hybridization analysis of GCNF mRNAs in adult mouse epididymis. B, D, and F, Hybridized with GCNF antisense probe; NB, ND, and NF, hybridized with the corresponding sense probe. B, D, and F correspond to those regions marked b, d, and f in Fig. 5. The red framed area in D was magnified by x100.
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Discussion
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Reports from different laboratories showed that the GCNF gene predominantly expressed two transcripts (2.3 and 7.4 kb) in testis, whereas only the 7.4-kb mRNA was detectable in other tissues (brain, liver, kidney, and lung; Ref. 2). By improving the sensitivity of the Northern blot analysis, we obtained the following results. We confirmed that the 2.3-kb mRNA only occurred in testis, but found it to be present in rat epididymis, although not in mouse epididymis (2). Epididymis and spleen were added to the list of tissues with low expression of GCNF (3). In addition to the 7.4-kb mRNA, there was also a faintly detectable 3.1-kb mRNA found in the tissues tested, including testis. We have demonstrated previously that the two transcripts (7.4 and 2.3 kb) in testis have same open reading frame and thus translated into one protein (26). Thus, according to the Western blot results, the 7.4- and 3.1-kb transcripts in epididymis should be the same as those in testis. In other words, this minor 3.1-kb mRNA must be transcribed from the same GCNF gene by alternative initiation and termination. This was further confirmed by the sequence of the 3.1-kb mRNA amplified by RT-PCR. Why the GCNF gene has to express two or three transcripts to encode a single protein is an interesting subject for further studies.
Most genes in the testis and epididymis are regulated by androgen (17). Although GCNF is a transcription factor, we wondered whether its expression in testis is also regulated by androgen. As GCNF, especially the 2.3-kb mRNA, is predominantly expressed in round spermatids, the number of which will be affected by androgen, it is difficult to design a convincing experiment to address this topic. Here we were able to show that androgen down-regulates GCNF mRNAs (7.4 and 3.1 kb) in the epididymis of the castrated mice. As we used only a single injection of testosterone for the hormone replacement, the serum testosterone level increased rapidly on the first day after injection, but dropped to nil on the third day. The change in GCNF mRNA levels, on the other hand, was not synchronous, but delayed. This is not surprising because we did not measure its transcription rate and only showed the steady state of its mRNA, which represented the total balanced amount of mRNA during its synthesis and degradation. Whether the 2.3-kb mRNA in testis is also regulated by androgen still needs to be ascertained. Although the majority of the gene expression in epididymis was up-regulated by androgen, some of the gene expression was reported to be down-regulated by androgen (17, 30, 31).
Regional expression is one of the most intriguing aspects of gene expression in epididymis (17). The GCNF gene also exhibited a region-specific pattern of expression at the protein level. It was mainly expressed in the nuclei of epithelial cells located at the caput, proximal, and middle corpus and had a very weak signal in the distal corpus and the entire cauda. In general, the acquisition of mobility and fertility for spermatozoa is essentially completed in most species only by the time sperm enter the cauda region of the epididymis (32, 33). The regional expression pattern of GCNF in mouse epididymis suggested that it plays a role in regulating the expression of its target genes, which might be essential for sperm maturation. There have been reports of several genes specifically expressed in the corpus region of the epididymis that might be candidates for the downstream targets of GCNF action (17, 34, 35, 36). The expression of GCNF in halo cells suggested that it might be involved in immune actions in the epididymis. Thus, GCNF may be involved in spermatozoa maturation as well as gametogenesis, neurogenesis, cell differentiation, and embryo development (1, 2, 3, 10).
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Acknowledgments
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We thank Qiangsui Guo for confocal microscopy performance.
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Footnotes
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This work was supported by the National Natural Sciences Foundation of China (Grant 39893320), the 973 Basic Research Funding Scheme of China (Grant G1999055901), Director’s initiating funding of Chinese Academy of Sciences, Shanghai Science and Technology Development funding, and the CAS Knowledge Creative Program (Grant KSCX2-SW-201).
Abbreviations: GCNF, Germ cell nuclear factor; RT, reverse transcription; RTR, retinoid receptor-related, testis-associated receptor.
Received September 3, 2002.
Accepted for publication December 19, 2002.
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References
|
|---|
- Hirose T, O’Brien DA, Jetten AM 1995 RTR: a new member of the nuclear receptor superfamily that is highly expressed in the murine testis. Gene 152:247–251[CrossRef][Medline]
- Chen F, Cooney AJ, Wang Y, Law SW, O’Malley BW 1994 Cloning of novel orphan receptor (GCNF) expressed during germ cell development. Mol Endocrinol 8:1434–1444[Abstract]
- Bauer U-M, Schneider-Hirschs S, Reinhardt S, Pauly T, Maus A, Wang F, Heiermann R, Rentrop M, Maelicke A 1997 Neuronal cell nuclear factor, a nuclear receptor possibly involved in the control of neurogenesis and neuronal differentiation. Eur J Biochem 249:826–837[Medline]
- David R, Joos TO, Dreyer C 1998 Anteroposterior patterning and organogenesis of Xenopus laevis require a correct dose of germ cell nuclear factor (xGCNF). Mech Dev 79:137–152[CrossRef][Medline]
- Cooney AJ, Hummelke GC, Herman T, Chen F, Jackson KJ 1998 Germ cell nuclear factor is a response element-specific repressor of transcription. Biochem Biophys Res Commun 245:94–100[CrossRef][Medline]
- Agoulnik IY, Cho Y, Niederberger C, Kieback DG, Cooney AJ 1998 Cloning, expression analysis and chromosomal localization of the human nuclear receptor gene GCNF. FEBS Lett 424:73–78[CrossRef][Medline]
- Zhang Y-L, Akmal KM, Tsuruta JK, Shang Q, Hirose T, Jetten AM, Kim KH, O’Brien DA 1998 Expression of germ cell nuclear factor (GCNF/RTR) during spermatogenesis. Mol Reprod Dev 50:93–100[CrossRef][Medline]
- Hummelke GC, Meistrich ML, Cooney AJ 1998 Mouse protamine genes are candidate targets for the novel orphan nuclear receptor, germ cell nuclear factor. Mol Reprod Dev 50:396–405[CrossRef][Medline]
- Heinzer C, Susens U, Schmitz TP, Borgmeyer U 1998 Retinoids induce differential expression and DNA binding of the mouse germ cell nuclear factor in p19 embryonal carcinoma cells. J Biol Chem 379:349–359
- Chung ACK, Katz D, Pereira FA, Jackson KJ, Demayo FJ, Cooney AJ, O’Malley BW 2001 Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation. Mol Cell Biol 21:663–677[Abstract/Free Full Text]
- Bauer U-M, Schueider-Hirsch S, Reinhard S, Benavente R, Maelicket A 1998 The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes. FEBS Lett 208:208–214
- Kata D, Niederberger C, Slaughter GR, Cooney AJ 1997 Characterization of germ cell-specific expression of the orphan nuclear receptor, germ cell nuclear factor. Endocrinology 138:4364–4372[Abstract/Free Full Text]
- Turner TT 1995 on the epididymis and its role in the development of the fertile ejaculate. J Androl 16:292–298[Free Full Text]
- Steinberger E, Steinberger A 1975 Spermatogenic function of the testis. In: Greep RO, Astwood EB, eds. Handbook of physiology, vol 5, sect 7. Baltimore: Waverly Press; 1–21
- Robaire B, Hermo L 1988 Efferent ducts, epididymis and vas deferens: structure, functions and their regulation. In: Knobil E, Neill J, eds. The physiology of reproduction. New York: Raven Press; 999–1080
- Yanagimachi R 1994 Mammalian fertilization. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 89–317
- Cornwall GA, Hann SR 1995 Specialized gene expression in the epididymis. J Androl 16:379–383[Free Full Text]
- Hamilton DW 1975 Structure and function of the epithelium lining the ductuli efferentes, ducts epididymidis and ductus defferens in the rat. In: Greep RO, Astwood EB, eds. Handbook of physiology, vol 5. Baltimore: Waverly Press; 259–383
- Miller RJ, Killian GJ 1987 Morphometric analysis of the epididymis from normal and vasectomized rats. J Androl 8:279–291[Abstract/Free Full Text]
- Wong PYD, Au CL, Ngai HK 1978 Electrolyte and water transport in rat epididymis: its possible role in sperm maturation. Int J Androl 1:600–628
- Rodriguez CM, Kirby JL, Hinton BT 2001 Regulation of gene transcription in the epididymis. Reproduction 122:41–48[Abstract]
- Li P, Chan HC, He B, So SC, Chung YW, Shang Q, Zhang Y-D, Zhang Y-L 2001 An antimicrobial peptide gene found in the male reproductive system of rats. Science 291:1783–1785[Abstract/Free Full Text]
- Heberlein U, Tjian R 1988 Temporal pattern of alcohol dehydrogenase gene transcription reproduced by Drosophila stage-specific embryonic extracts. Nature 331:410–415[CrossRef][Medline]
- Hu Y, Guo J, Shen L, Chen Y, Zhang Z, Zhang Y-L 2002 Get effective polyclonal antisera in one month. Cell Res 12:57–161
- Tauz U, Pfeifle C 1989 A non-radioactive in situ hybridization method for localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81–85[CrossRef][Medline]
- Yang G, Zhang Y-L, Buchold GM, Jetten AM, O’Brien DA, Analysis of germ cell nuclear factor (GCNF) transcripts and protein expression during spermatogenesis. Biol Reprod, in press
- Pogany GC 1987 Effects of x-irradiation on the kinetics of abnormal sperm production and sperm loss in the mouse. J Reprod Fertil 80:1–12[Abstract]
- Nashan D, Malorny U, Sorg C, Cooper T, Nieschlag E 1989 Immuno-competent cells in the murine epididymis. Int J Androl 12:85–94[Medline]
- Serre V, Robaire B 1999 Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content. Biol Reprod 61:705–714[Abstract/Free Full Text]
- Hsu SH, Shyu HW, Hsieh-Li HM, Li H 2001 Spz1, a novel bHLH-Zip protein, is specifically expressed in the testis. Mech Dev 100:177–187[CrossRef][Medline]
- Quarmby VE, Yarbrough WG, Lubahn DB, French FS, Wilson EM 1990 Autologous down-regulation of androgen receptor messenger ribonucleic acid. Mol Endocrinol 4:22–28[Abstract]
- Hamilton DW 1972 The mammalian epididymis. In: Balin H, Glassner S, eds. Reproductive biology. Amsterdam: Excerpta Medica; 268–337
- Hermo L, Dworkin J, Oko R 1988 Role of epithelial clear cells of the rat epididymis in the disposal of the contents of cytoplasmic droplets detached from spermatozoa. Am J Anat 183:107–124[CrossRef][Medline]
- Jervis KM, Robaire B 2001 Dynamic changes in gene expression along the rat epididymis. Biol Reprod 65:696–703[Abstract/Free Full Text]
- Kaunisto K, Fleming RE, Kneer J, Sly WS, Rajaniemi H 1999 Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis. Biol Reprod 61:1521–1526[Abstract/Free Full Text]
- Mahony MC, Swanlund DJ, Billeter M, Roberts KP, Pryor JL 1998 Regional distribution of 5-reductase type 1 and type 2 mRNA along the human epididymis. Fertil Steril 69:1116–1121[CrossRef][Medline]
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Abstract
Introduction
