From the Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510
Received for publication, March 7, 2003 , and in revised form, April 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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The gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25),1 cloned from the rat Leydig cell, mouse and human testis cDNA libraries, is a novel member of the DEAD-box protein family of RNA helicases and is the first member found to be regulated by a hormone (3). Purified recombinant GST-GRTH displayed ATPase and ATP-dependent bi-directional RNA helicase activities. It also increased in vitro translation of luciferase RNA templates (3). Northern analysis indicated that this helicase is highly expressed in rat, mouse, and human testes and is weakly expressed in the pituitary and hypothalamus. In vivo/in vitro studies demonstrated that GRTH is transcriptionally up-regulated by human chorionic gonadotropin at doses that cause down-regulation of luteinizing hormone/human chorionic gonadotropin (hCG) receptors, steroidogenic enzymes, and androgen formation (4). Furthermore, in vitro studies revealed that induction of GRTH mRNA by hCG is mediated via second messenger and androgen in Leydig cells. Inhibition of androgen production by inhibitors of steroidogenic enzymes or blockade of androgen action by a receptor antagonist abrogated the stimulation of GRTH mRNA by gonadotropin or cAMP. In situ hybridization analysis demonstrated that GRTH is predominantly expressed in the testis in both somatic Leydig cells and meiotic spermatocytes and haploid germinal cells of the seminiferous epithelium and is developmentally regulated. In the present study, we identified GRTH protein species, evaluated their protein levels, and studied their cellular distribution within the testicular compartments and their hormonal regulation in the adult rats testis. The regulation of this enzyme by gonadotropin and androgen and its stage-specific localization in germ cells indicate that GRTH could participate in the regulation of androgen-dependent steroidogenesis and spermatogenesis.
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EXPERIMENTAL PROCEDURES |
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Testicular Leydig and Germ Cells PreparationLeydig cells were prepared by collagenase dispersion and purified by centrifugal elutriation (5). After collagenase dispersion, seminiferous tubules were minced and incubated in Medium 199 containing 0.1% bovine serum albumin, 0.1% trypsin (Sigma), and 17 µg/ml DNase (Sigma) for 30 min in a rotary water bath (80 rpm, 35° C). After the addition of soybean 0.04% trypsin inhibitor, the sample was filtered through a 300-, 90-, 40-µm mesh screen, and glass wool and cells were pelleted and resuspended in elutriation buffer containing 2 µg/ml DNase. The relevant types of germ cells were subsequently separated and purified by centrifugal elutriation using Beckman Avanti 21B centrifuge with elutriator rotor model J 5.0 as described previously (6). The first 2 fractions (1 and 2) were collected with flow rates of 31.5 and 41.4 ml/min at 3000 rpm, and 2 additional fractions (3 and 4) were obtained with flow rates of 23.2 and 40 ml/min at 2000 rpm. Cells were identified on air-dried smears, fixed in Bouin's fixative, and stained with hematoxylin and periodic acid-Schiff. Fractions 2 and 4 containing round spermatids and pachytene spermatocytes at a purity of 84 and 86%, respectively, were used for Western blot analyses.
Western Blot and Immunohistochemistry AnalysisA polyclonal
antibody was raised in rabbits against a GRTH peptide (amino acids
465477) and purified by protein A-Sepharose (Amersham Biosciences).
Protein extracts from testicular Leydig, germ cells, and various tissues
including adrenal, ovary, pancreas, brain, and pituitary were assessed by
Western blot analysis using the purified GRTH antibody (1/400 dilution, 1
µg of IgG/µl). The specificity of the antibody-GRTH interaction was
evaluated in the presence of 60 µM GRTH peptide 465477.
Immunosignals were detected by a super-signal chemiluminescent system
(Pierce). GRTH values were normalized by the corresponding -actin
signals. For immunochemistry analysis, testes from adult rats were fixed in 4%
paraformaldehyde and embedded in paraffin. Serial sections were incubated with
GRTH antiserum at a 1/500 dilution (1 µg IgG/µl) and developed with a
peroxidase-labeled avidin biotin detection system. Sections of seminiferous
tubules were staged according to the method of Leblond and Clermont
(7). Quantitation of relative
immunostaining intensity was performed by three independent observers using
scores 05 assigned for the signal varying from absent, weak, medium,
high, to maximum by vision inspection. At least 50 pachytene spermatocytes and
round spermatids per stage in 10 different seminiferous tubules were evaluated
by two observers. Maximum intensity observed at the stage IX pachytene
spermatocyte was defined as score 5.
Overexpression of GRTH-GFP and GRTH-PBK Protein in COS-1 CellsThe full length of the GRTH cDNA-coding region including the first ATG codon (newly identified) or from the second ATG codon (first ATG in our previous report (3)) were subcloned into the pEGFP-N2 (GRTH-GFP) or pBK (GRTH-PBK) (2 µg) and transfected into COS-1 cells with LipofectAMINE Plus (Invitrogen). After 36 h, cells were harvested for Western blot analyses by using a GFP monoclonal antibody (Clontech, Palo Alto, CA) or purified GRTH antibody.
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RESULTS |
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To further analyze the expression of this full-length cDNA we performed Western analysis using either specific GRTH antiserum or GFP monoclonal antibody for GRTH-GFP fusion protein overexpressed in COS-1 cells. Western blot analyses using a GFP monoclonal antibody showed expression of GRTH-GFP fusion proteins with construct A, which contains both first and second ATG (new clone), and construct B, our previous clone with only the 2nd ATG (Fig. 1A, left-most panel, lanes A and B). Expression of construct A revealed a predominant 83-kDa band (56-kDa GRTH, 27-kDa GFP) and a weak 70-kDa protein band (43-kDa GRTH, 27-kDa GFP) and that of construct B, a 70-kDa band. This indicated utilization of both ATGs in the translation process. A 27-kDa GFP band was detected in cells transfected with the GFP construct without the GRTH insert (Fig. 1A, left-most panel, lane GFP). An additional 88-kDa species (61-kDa GRTH, 27-kDa GFP) was also observed. All protein species corresponding to the overexpressed GRTH-GFP fusion protein were also detected with a specific GRTH polyclonal antibody (Fig. 1A, second panel, lanes A and B). In addition to the predominant band of 88/83 kDa, a second less prominent protein band of 70 kDa was detectable by the GRTH antibody from the expression of the full-length GRTH-GFP fusion construct A (Fig. 1A, second panel, lane A). This corresponded to the deduced protein size derived from the amino acid sequence reported in our early studies (3) (construct B, lane B), suggesting that the 2nd ATG in the coding region can also function in the translation process.
Also, the expressed protein species resulting from transfecting the construct containing full-length GRTH in PBK vector (construct C) were evaluated. We observed 61/56-kDa (predominant) and 43-kDa (minor) bands of size comparable with that calculated from the expressed GRTH-GFP construct (A) that corresponded to usage of the 1st and 2nd ATG codons, respectively (Fig. 1A, second panel, lane C).
These findings demonstrate that newly identified ATG is the major translation initiation codon in overexpression studies in COS1 cells. However, the 2nd ATG codon appears to function in a cell-specific manner in Leydig cells (Fig. 1A, fourth panel), and it may also play a significant role under hormonal stimulation (see Fig. 6). There are specific endogenous 56 61-kDa protein complexes detected in the whole testis (third panel) and purified adult rat testicular germ cells (round spermatids and pachytene spermatocytes) (Fig. 1A, fourth panel, lanes RS and P), whereas the 43-kDa protein is the major protein species in Leydig cells. Other minor protein bands of 48 and 56 kDa are also expressed in Leydig cells (Fig. 1A, fourth panel, lane LC). An additional minor protein band 33 kDa was also noted in germ cells (Fig. 1A, fourth panel, lanes RS and P). The later may result from utilization of ATG codons at +568 or +598. The specificity of the endogenous GRTH protein bands was confirmed by inhibition of the antibody-GRTH complex by the peptide used to prepare the antibody (Fig. 1A, right-most panel).
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Developmental studies revealed that endogenous testicular GRTH protein was expressed predominantly as the 61/56-kDa form observed in the testes of pubertal and adult animals but was not present in immature animals (Fig. 2A), whereas a weak 43-kDa protein was expressed in the adult testis. This is reasonable because Leydig cells, which are the major source of the 43-kDa species, represent about 4% of the total testis cell volume (10). GRTH was not observed in other organs examined including adrenal, ovary, heart, hypothalamus, or pituitary (Fig. 2B).
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Immunocytochemical Analysis of GRTH in Adult Rat TestisImmunohistochemistry studies showed that GRTH immunoreactive protein was predominantly present in the interstitial cells of the adult rat testis and weakly expressed in the seminiferous tubules (Fig. 3A, a and b). Subsequent studies revealed that the intensity of positively stained germ cells varied during the spermatogenic cycle (see Figs. 3B and 4).
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To determine the cellular expression patterns of GRTH protein in the seminiferous tubules during germ cells development, we performed a detailed analysis of immunoreactive staining present in different types of germ cells during the spermatogenic cycle of the adult rat testis (Fig. 3B). Both pachytene spermatocytes and round spermatids expressed GRTH protein, and the intensity of staining in the individual cell types varied at different stages of the spermatogenic cycle. GRTH immunoreactive protein staining was significantly higher in round spermatids when compared with pachytene spermatocytes only at early stages (I-III). GRTH levels in round spermatids reached peak levels at stages VIII and IX and were diminished in late stages of elongating spermatids. In pachytene spermatocytes, signals were minimally detectable in stage I and gradually increasing at stages II through IX. The strongest immunoreactive signals were observed in stage VIII and IX, where levels were significantly higher compared with round spermatids. GRTH immunoreactivity in pachytene spermatocytes was gradually decreased from stages X through stage XIII. It was noted that immunoreactive signals were prominent in stage XIV of spermatocytes when cells entered the metaphase of meiotic division. GRTH was not expressed in Sertoli cells or other types of germ cells.
Analysis of the intensity of GRTH immunoreactive signals at different stages of round spermatids and pachytene spermatocytes during the spermatogenic cycle and the corresponding pattern of individual cell types are shown in Fig. 4, A and B. The specific cell and stages of GRTH protein expression during germ cells development suggests that GRTH may play an important role in spermatogenesis.
Gonadotropin Up-regulation of GRTH Expression in Adult Rat TestesThe GRTH protein levels were significantly increased in adult rat Leydig cells (43 kDa) at 12 h after gonadotropin treatment of adult rats, further increased at 24 h, and returned to near control level at 96 h (Fig. 5A). The weaker 48- and 56-kDa species were unchanged at 12 h but were not detectable at 24 h and were again observed 96 h after treatment (Fig. 5B). The increase of the 43-kDa GRTH species in Leydig cells caused by gonadotropin was prevented by flutamide treatment (Fig. 5C). Flutamide treatment per se also reduced GRTH protein to 56% that of controls (p < 0.01). These findings indicated that GRTH protein expression is up-regulated by the action of androgen induced by gonadotropin stimulation in the Leydig cells.
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In germ cells the GRTH protein (61/56 kDa) were markedly affected by gonadotropin stimulation in a cell-specific manner (Fig. 6). Although there were no changes observed in pachytene spermatocytes, gonadotropin treatment significantly altered the pattern of protein expression in round spermatids. In these cells the 61-kDa species was significantly decreased to barely detectable levels, whereas only minor reduction in the 56-kDa species were observed. Most notably was the marked induction of the 48/43-kDa species that were undetectable in round spermatids of control animals. Moreover, treatment with flutamide prevented the induction of these species by hCG in the round spermatids. This indicated alternative utilization of GRTH translation initiation codon governed by direct or indirect action of androgen in these cells.
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DISCUSSION |
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The primary antibody used in this study is a rabbit polyclonal antibody
raised against a synthetic peptide corresponding to the unique C-terminal
amino acid sequence (amino acids 465477), with no similarity to any
other member of RNA helicase gene family or known proteins. Western blot
analyses of transiently transfected COS-1 cells with various constructs
(Fig. 1) confirmed the antibody
specificity since both GRTH and GFP antibodies revealed major 88/83-kDa
species and a minor 70-kDa protein band from expression of GRTH-GFP fusion
construct A. Moreover, the 70-kDa species was detected from expression of the
5'-truncated GRTH-GFP, construct B. In cells transfected with construct
C, both the 61/56- and 43-kDa species were present. The observed sizes of the
endogenous proteins (61/56- and 43-kDa species) were consistent with those
derived from the deduced amino acid sequences and the expressed GRTH-GFP
fusion proteins corrected for the GFP contribution. The 5-kDa difference
observed in the 61 versus 56-kDa and 48 versus 43-kDa
species may result from post-translational modification (i.e.
N-glycosylation at amino acid site 426 or 472). The difference of the
61/56- and 48/43-kDa species can be accounted for by codon usage at ATG +1 and
+343 nt, respectively. The intensity of the 61-kDa species is stronger than
the 56-kDa species in the testis and round spermatids, whereas the pachytene
spermatocytes do not the contain 56-kDa species. In Leydig cells, the 43-kDa
protein is the dominant species. The mechanism involved in the formation of
these various GRTH protein species and the significance of their respective
function in specific cell types remain to be resolved. GRTH was present in
testis and not detected in other tissues examined, including ovary,
hypothalamus, and pituitary. The lack of detectable protein expression in
these tissues is of interest since our previous study demonstrated mRNA
expression at these sites (3)
and indicates a lack of translatability or very low levels of the protein,
undetectable by the method employed.
Production of multiple protein isoforms could result from transcriptional
and/or translational mechanism. Different promoter usage, alternate mRNA
splicing, utilization of alternative translation initiation codon, and
proteolytic cleavage could generate protein diversity and ultimately fulfill
unique biological functions. Because GRTH mRNA is a single transcript of 1.6
kilobases, one of the mechanisms for the differential expression of GRTH
protein species in the testis is the alternative initiation of translation
from different ATG codons. Initiation of translation at alternative ATG codons
in a single transcript has been documented for a number of genes including
intron-less members of C/EBP family, C/EBP
(11), and C/EBP
(12) and intron-containing
Egr3 gene and eukaryotic translation initiation factor 4GI
(13,
14). In the case of the GRTH
gene, the usage of alternative ATG codons is supported by findings from
overexpression studies of engineered constructs containing different GRTH
translational initiation codons. It is also indicated by the size of the
specific GRTH protein species endogenously expressed in the testis, which
corresponded to the proposed ATG initiation codons
(Fig. 1). The overexpression
studies indicated that both ATG codons at +1 and +343 nt of GRTH actively
initiated the synthesis of different protein species. Testicular germ cells
preferentially utilize the +1 ATG codon, whereas Leydig cells use the 2nd ATG
codon at nt +343 in the translation process. In addition, there is a 3rd
functional ATG codon at nt 568 or 598 that is utilized in germ cells
exclusively yielding a 33-kDa species. All the sequences surrounding these
different initiation codons contain consensus Kozak sequences
(GCC(A/G)CCATGG) with 24 nt mismatches 5' to ATG
(Fig. 1). Although the 1st ATG
codon at +1 nt position appears to better match the consensus Kozak sequence,
our study indicated that all the proposed ATG codons are functional depending
on the cell type.
Although proteolytic cleavage could also be an alternative mechanism for generation of different protein isoforms, we believe this is unlikely in the case of GRTH gene products. The rationale is based on the fact that ATG codons are not only utilized in a cell-specific manner but also appear to be hormonally sensitive in a specific type of germ cells (round spermatids). The switch between 1st and 2nd ATG usage in round spermatids is dependent on the gonadotropin stimulation and androgen production. In the presence of hormone, 48/43-kDa protein species were generated presumably using the translation initiation codon located at nucleotide 343. The possibility of generating identical 48/43-kDa protein species as the Leydig cells through a proteolytic process induced by gonadotropin would be very low. It is important to note that the Leydig cell is tonically regulated by gonadotropin, a hormone and that promotes androgen (testosterone) formation in these cells. Autocrine actions of testosterone in Leydig cells and paracrine actions at tubule sites (round spermatids) exert increases in GRTH and promote the utilization of the 2nd ATG codon in both cell types. The usage of an alternative ATG codon by a stimulant was also reported for isoform formation of the murine interleukin-12 gene during lipopolysaccharide stimulation (15).
The action of androgen on GRTH protein expression in germ cells appears to be cell-specific, although germinal cells do not exhibit either gonadotropin or androgen binding activity (16, 17). In contrast to the up-regulation of GRTH gene expression caused by hCG-induced androgen action in the Leydig cells, GRTH protein levels in purified pachytene spermatocytes were not affected by hormonal stimulation. However, the 48/43-kDa protein species were induced by gonadotropin treatment in round spermatids, whereas the 61-kDa protein species was markedly diminished in this cell type. We propose that a specific androgen response factor(s) that is present in both Leydig cell and round spermatids but not in pachytene spermatocytes promotes utilization of the 2nd ATG codon of GRTH mRNA for the synthesis of 48/43-kDa GRTH protein.
The up-regulation of GRTH expression by gonadotropin/androgen Leydig results from transcriptional regulation (3), and this also applicable to the GRTH induction observed during development in the various cell types. In preliminary studies we have determined that the GRTH gene, which lacks consensus androgen/glucocorticoid-responsive element, is not modulated by androgen in COS-1 cells co-transfected with androgen receptor and GRTH promoter/5'-flanking construct.3 This indicated indirect cell-specific actions of androgen on GRTH transcription in the testis. On the other hand, the prevalence of the 48/43-kDa species in Leydig cells and the hormonal switch of expressed species in round spermatids is reflective of alternative promoter usage. Thus, this indicates that the 48/43-kDa protein is an androgen-responsive protein that may participate in both metabolic function of the Leydig cells and spermatogenesis.
Studies on the distribution of the GRTH immunoreactive protein in the seminiferous epithelium of the adult rat testis have clearly demonstrated that GRTH expression is cell- and stage-specific during germ cell development. Immunocytochemical analysis showed the presence of high levels of GRTH in pachytene spermatocytes and haploid spermatids at stages VIII to IX. High levels of GRTH immunostaining was also observed in the metaphase of primary and secondary spermatocytes at stage XIV when chromosomes are condensed. The cell- and stage-specific GRTH expression in the germ cell maturation process suggests that GRTH may act either directly or indirectly on germ cells to regulate spermatogenesis.
Several candidate genes for the regulation of spermatogenesis have been reported. Within the DEAD-box protein helicase family, Mvh (18), a mouse homolog of the Drosophila maternal gene Vasa that is required for the completion of oogenesis (19), is expressed in male premeiotic germ cells and appears to be associated with the meiotic process. Moreover, homozygous knockout Mvh mice produced no sperm in the testis due to the failure of premeiotic cells to complete meiosis (18). Two other evolutionary DEAD-box RNA helicase genes distantly related to GRTH, mouse PL10 (20) and P68 (21), displayed testis-selective mRNA expression restricted to late pachytene spermatocytes and round spermatids in mouse. In other studies, male germ cells were reported to contain protein(s) that is able to specifically bind to 3'-untranslated region of mouse P68 helicase (22). Based on its structural characteristics and in vitro function (3) we proposed that GRTH might function as a translational activator to promote protein expression of crucial gene(s) at selective stages of the spermatogenic cycle. It is not known whether the above three distantly related RNA helicases have synergistic, independent, or substitutive roles in the regulation on spermatogenesis. The similarity of these protein sequences with GRTH is only limited to the conserved motifs of the RNA helicase gene family and displayed very low overall similarities (3235%). The physiological significance and role of GRTH during the spermatogenic cycle remains to be determined.
The distribution and regulatory pattern of GRTH gene expression at the protein level is similar to its mRNA expression in tissues and cells examined (3). The testicular cell-specific and hormonal-dependent usage of translation codons for GRTH protein synthesis add complexity and diversity to regulatory mechanism(s) that may participate during testicular development. The up-regulation of GRTH protein by gonadotropin in the Leydig cells follows closely the down-regulation of receptors, steroidogenic enzymes, and acyl-CoA synthetase (gonadotropin-regulated long chain acyl-CoA synthetase) (4, 23), and its return to control levels is concomitant to its recovery from down-regulation. These changes could be reflective of the existence of a regulated mechanism responsible for recovery of down-regulated transcriptional events through increases in translatability of the reduced relevant messages. The findings presented in this study demonstrate a cell-, stage-, and compartment-specific expression of GRTH protein in testicular cells and provide further evidence for potential roles of GRTH in the control of steroidogenesis and spermatogenesis in the testis. Development of GRTH null mice will establish the physiological function of the protein.
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FOOTNOTES |
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To whom correspondence should be addressed: Bldg. 49, Rm. 6A-36, 49 Convent
Dr., MSC 4510, National Institutes of Health, Bethesda, MD 20892-4510. Tel.:
301-480-8010; E-mail:
dufau{at}helix.nih.gov.
1 The abbreviations used are: GRTH, gonadotropin-regulated testicular RNA
helicase; hCG, human chorionic gonadotropin; GFP, green fluorescent protein;
nt, nucleotide(s); PBK, Bluescript plasmid.
2 C. H. Tsai-Morris, Y. Sheng, and M. L. Dufau, unpublished information.
3 Q. Jiang, C.-H. Tsai-Morris, and M. L. Dufau, unpublished observations.
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REFERENCES |
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