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BOR - Papers in Press, published online ahead of print December 11, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008235
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BIOLOGY OF REPRODUCTION 68, 1742–1747 (2003)
DOI: 10.1095/biolreprod.102.008235
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Type II and Type IX Collagen Transcript Isoforms Are Expressed During Mouse Testis Development

Peter J. McClive1, and Andrew H. Sinclair

Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Melbourne, Victoria 3052, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the transcription factor SOX9 give rise to campomelic dysplasia, a syndrome characterized by skeletal abnormalities and XY sex reversal. Sox9 is expressed at sites of chondrogenesis and in the developing testis, and, thus, it plays a role in two overtly different pathways of differentiation. Previous studies have identified the gene for type II collagen, Col2a1, as a target of Sox9 in mouse chondrocytes and implicated Col9a3 as a Sox9 target in testis. Using differential expression analysis combined with reverse transcription-polymerase chain reaction and whole-mount in situ hybridization, we have identified nonchondrocytic collagen transcript isoforms that are expressed in the early male mouse gonad. Male-specific, gonadal expression of nonchondrocytic Col2a1 was first seen at 11.5 days postcoitum (dpc) and was undetectable by 13.5 dpc. This was accompanied by increasing expression of nonchondrocytic Col9a1, Col9a2, and Col9a3, first detected at 11.5 dpc. Expression was analyzed in testes that had been depleted of germ cells by the cytotoxic drug busulfan. These studies showed Col9a3 and Col2a1 to be expressed in Sertoli cells within the developing testis cords. Nonchondrocytic type II collagen contains a cysteine-rich domain that has been shown to bind members of the transforming growth factor ß superfamily of signaling molecules. Thus, this interaction may play a role in the morphogenesis and differentiation of the testis.

developmental biology, early development, embryo, Sertoli cells, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sex is determined in vertebrates when the bipotential gonad is switched to become either a testis or an ovary. In mammals, the expression of the architectural transcription factor SRY initiates testis determination and, if mutated, results in XY sex reversal [1, 2]. The transgenic expression of Sry causes sex reversal in mice that are chromosomally female [3]. Sry is first expressed in the mouse gonad at 10.5 days postcoitum (dpc) [3]. Its normal expression is thought to induce cell migration into the gonad from the underlying mesonephros and to promote the increased cellular proliferation responsible for defining the early structure of the testis [4, 5].

SRY exerts its actions by binding DNA with sequence specificity [6, 7]. Although the protein has not been shown to directly transactivate genes, its binding and subsequent bending of DNA may bring together otherwise distant sites [8]. In certain mouse strains, a repeat sequence at the N-terminal is required for Sry to function, possibly forming a transactivation complex with other proteins. The repeat domain is absent from most mammals, and it is hypothesized that a linker protein may play a substituting role in these cases [9].

Soon after Sry is expressed, at 11.5 dpc, the transcription factor Sox9 is upregulated; however, no direct link between Sry and Sox9 has been shown. The overexpression of Sox9 is also sufficient for female-to-male sex reversal [10, 11]. Gene-targeting studies have demonstrated that anti-Müllerian hormone (Amh) is one of the direct transcriptional targets of Sox9 in testis [12]. SOX9 mutations give rise to campomelic dysplasia, a syndrome characterized by skeletal abnormalities and XY sex reversal [13]. Expression studies in chondrogenic tissues have shown the coincident expression of Sox9 and Col2a1, the gene encoding type II collagen [14, 15]. Furthermore, SOX9 has been shown to bind motifs found in the COL2A1 first intron and can drive the expression of a COL2A1 intron enhancer-driven reporter gene in vivo [16].

Sox9 also regulates Aggrecan [17], CD-RAP [18], and Col11a2 [19] in chondrocytes in addition to Amh in testis. Recent studies have implicated Col9a3 in testis development and suggested its regulation by Sox9 [20]. Target genes of Sox9 must be expressed in the same spatiotemporal window. Sox9 is expressed at a low level in male and female gonads at 10.5 dpc. From 11.5 dpc, male expression is upregulated and female expression downregulated. By 12.5 dpc, male expression is 5-fold the level of female expression [3]. We have used a subtractive hybridization and suppression polymerase chain reaction (PCR) method to identify genes expressed in the testis at 12.5 dpc in an attempt to find targets of Sox9 in the mouse.

To minimize confusion, we will adopt the following nomenclature when describing both RNA and protein isoforms of type II and type IX collagen: The type II collagen gene, Col2a1, encodes two isoforms. The long form is found in noncartilage tissues and has been also termed collagen type IIA [21–23]. We propose to call this the nonchondrocytic form of type II collagen. The short form is found in mature cartilage and is also known as collagen type IIB [21–23]. We propose to call this the chondrocytic form.

Isoforms of type IX collagen are also either long or short. In this case, however, the short form predominates in noncartilage tissue. We propose to call this the nonchondrocytic form. Regarding the long form that is found in mature cartilage, we propose to call this the chondrocytic form.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Swiss CD1 (OLA) mice and embryos were used for all experiments. All experiments were conducted with the approval of the Royal Children's Hospital Animal Experimentation Ethics Committee.

PCR Subtraction and Screening

One-hundred pairs of gonads were dissected free from mesonephroi at 12.0–12.5 dpc, sexed by morphological criteria or PCR [24], and pooled for RNA extraction [25]. PolyA RNA was extracted from 100 µg of total RNA of each sex using oligo-dT-coated Dynabeads (Dynal, Oslo, Norway). This served as the starting material for PCRSelect (Clontech, Palo Alto, CA) subtractive hybridization and suppression PCR following the recommended protocol. On cloning into pGEM-T (Promega, Annandale, Australia) and sequencing, a fragment corresponding to Col9a3 was identified.

Whole-Mount and Section In Situ Hybridization

The pGEM-T plasmid containing a 371-base pair (bp) RsaI Col9a3 fragment was linearized with NotI or NcoI and transcribed with T7 or SP6 RNA polymerase to generate sense or antisense riboprobes, respectively. The Col2a1 PCR fragment (amplified using primers A and B described below) was cloned into PCRScript (Stratagene, La Jolla, CA), linearized with NotI or EcoRI, and transcribed with T7 or T3 RNA polymerase to generate sense or antisense riboprobes, respectively. The insert from a Sox9 subclone was amplified using M13 forward and reverse universal primers (including 4% dimethyl sulfoxide [DMSO]), and antisense riboprobe was transcribed using T7 RNA polymerase. Primers used to amplify and clone the 249-bp Oct4 riboprobe template were 5'-CCCCCACTTCACCACACTC-3' and 5'-GCATCACTGAGCTTCTTTCCC-3'; antisense riboprobe was transcribed using T7 RNA polymerase.

Hybridization was performed as previously described [26]. Briefly, tissue was fixed in 4% paraformaldehyde (PFA) and then dehydrated through a methanol series, subjected to limited proteinase K digestion, hybridized with riboprobe in formamide buffer at 65°C, incubated with {alpha}-digoxygenin alkaline phosphatase antibody, and visualized with BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/4-nitro blue tetrazolium chloride). Individual experiments contained at least two embryos per time point and sex, and expression patterns were further confirmed in independent triplicate experiments.

Stained tissues were prepared for sectioning by equilibrating overnight in 20% sucrose/PBS at 4°C. Tissues were transferred to freezing molds containing OCT freezing compound (Tissue-Tek, Torrance, CA) and snap-frozen in isopentane/liquid nitrogen. Blocks were sectioned (thickness, 12 µm) using a cryostat machine.

Treatment of Developing Embryos with Busulfan

The cytotoxic drug busulfan has been shown to preferentially target migrating and proliferating germ cells in the rat [27, 28]. Based on these studies and on those involving adult mice [29], we injected pregnant mice to target embryonic germ cells. Busulfan (Sigma, Castle Hill, Australia) was dissolved in a 50% DMSO solution, and a single dose of 40 mg/kg was given by i.p. injection to pregnant female mice at 9.5 dpc. Embryos were removed at 12.5–13.5 dpc and analyzed by whole-mount in situ hybridization (WISH).

Semiquantitative Reverse Transcription-PCR Analysis of Collagen Transcript Isoforms in Developing Mouse Gonads

For all reverse transcription (RT)-PCR reactions, products were sampled with increasing cycle number, and those detectable at the lowest cycle number (thus falling within the linear range) were used for subsequent analysis. Each analysis time point was performed on pooled gonads dissected free of mesonephroi (n = 6). Southern blots were washed at high stringency (0.2x SSC [1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate]) to minimize nonspecific binding.

The RT-PCR (30 cycles) and hybridization were used to define the Col9a1 transcript isoform that was differentially expressed in the developing gonad. Amplification was carried out as previously described [30] using the primers indicated in Figure 2a. Specifically, the chondrocytic isoform was amplified using primer 1 (5'-GTAGACTTCAGGATTCCA-3') and primer 3 (5'-CCGGAACTCCAGGAGGC-3'), and the nonchondrocytic form was amplified using primer 2 (5'-ATGGCCTGGGCTGCCTGG-3') and primer 3. The probe for Southern blot analysis spanned exons 6–8 and was amplified using corrected primer 4 (5'-TGAACTCCAGTGGATGC-3'; the published primer 4 did not completely match the genomic sequence [30]) and primer 3 from the plasmid template YM-128 (containing the Col9a1 cDNA; obtained from Y. Muragaki, Wakayama Medical College, Japan). Products at the same cycle number in the same experiment, blot, and gel image are collated in Figure 3b.



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  		FIG. 2. Primer annealing sites for discriminating collagen transcript isoforms. Black boxes indicate shared exons; unfilled boxes indicate alternative exons. a) Col9a1 structure. Primer 1 and primer 3 amplify the chondrocytic isoform (583 bp); primer 2 and primer 3 amplify the nonchondrocytic isoform (136 bp). Probe for Southern blot analysis was amplified using primer 3 and primer 4. b) Col2a1 structure. Primers A and B amplified both the chondrocytic isoform (380 bp) and the nonchondrocytic isoform (584 bp)



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  		FIG. 3. RT-PCR analysis of Col9a2 and Col9a1 expression in developing mouse gonads. a) Col9a2 expression in male (M) and female (F) gonadal RNA and Hprt loading control. Col9a2, 179 bp; Hprt, 351 bp. b) Col9a1 isoforms detected by Southern blot analysis in male (M) and female (F) gonadal RNA (ch., Chondrocytic isoform; non, nonchondrocytic isoform) and Hprt loading control. Col9a1 ch., 583 bp; Col9a1 non, 136 bp; Hprt, 351 bp

The analysis of the invariant Col9a2 transcript was performed using cDNA-specific primers bridging a splice junction and, thus, were incapable of generating a genomic DNA product. Primer sequences were 5'-CGCCCTGGCTCAGATCAGAG-3' and 5'-GTTTCCCATCTGGACCATCTG-3'; 28 cycles of PCR were carried out.

Analysis of Col2a1 was also cDNA-specific, because the product spanned five exons and genomic amplification was not detected. Primers for Col2a1 flanked the alternative exon and included exons 1–5B [14]: primer A, 5'-TCTCCTGCCTCCTCCTGCTC-3'; and primer B, 5'-CTCCATCTCTGCCACGGGGT-3' (see Fig. 2b). The PCR was carried out for 27 cycles. The Col2a1 probe for Southern blot analysis corresponded to the cloned PCR fragment, as was used for whole-mount in situ analysis.

The RNA loading was estimated using primers to Hprt (hypoxanthine guanine phosphoribosyl transferase), 5'-CCTGCTGGATTACATTAAAG-3' and 5'-TCAAGGGCATATCCAACAAC-3', which were added to aliquots of each reaction mix in separate tubes and subjected to 27 cycles of PCR. Purified Hprt products were subsequently used as probes on Southern blots.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Analysis of Col9a3

The identification of Col9a3 expression in the testis by subtractive suppression hybridization was unexpected; thus, the identity and specificity of the 371-bp RsaI fragment that was isolated was confirmed by library screening and RT-PCR. A 12.5-dpc mouse gonad cDNA library was constructed and screened. Four clones were characterized and defined 1306 bp of incomplete Col9a3 coding sequence and 363 bp of 3'-untranslated region. A polyadenylation signal was detected 23 bp 5' of the poly(A) tail. This sequence was used to screen GenBank, and additional 5' sequences were identified. The RT-PCR was carried out on male 13.5-dpc gonad RNA, defining a total coding sequence of 2052 bp (accession no. AF349718; the 371-bp fragment corresponded to bases 1906–2276 and spanned the stop codon). The alignment of the mouse and human (accession no. L41162 [31]) sequences revealed 83% nucleotide identity and suggested that the mouse coding sequence lacked 19 bp at the 5' end. Alignment of the predicted translation products showed the human and mouse proteins shared 91% identity and 93% similarity over 677 amino acids and 100% concordance in their Gly-X-Y repeat units definitive of collagen structure.

In Situ Analysis of Col9a3 Transcript Expression

The spatial and temporal profile of Col9a3 expression was studied using RNA whole-mount in situ analysis. Analysis of male and female urogenital systems (Fig. 1) revealed detectable expression in testis only; sense controls were negative (data not shown). At 11.5 dpc, expression was first observed in a band of cells at the center of the male gonadal ridge (Fig. 1a, arrow). By 12.5 dpc, Col9a3 was expressed within the forming testis cords (Fig. 1b). This expression was maintained through 13.5 dpc (Fig. 1c). This pattern of expression was similar to that described for Sox9. It is known that Sox9 is expressed within the Sertoli cells that lie within the testis cords [32]. Sections taken from stained gonads also showed that Col9a3 was clearly expressed within the cords (Fig. 1d); however, it was not possible to discriminate between expression in Sertoli or germ cells. The cytotoxic drug busulfan was thus used in vivo to deplete developing embryos of germ cells. Subsequent analysis confirmed that this procedure significantly reduced germ cell gene expression (as shown for the germ cell marker Oct4 in Fig. 1e). However, Sertoli cell gene expression and, in particular, that of Sox9 was essentially unchanged (Fig. 1f). The similar maintenance of Col9a3 expression following treatment (Fig. 1g) thus demonstrated the gene to be expressed (at least) in Sertoli cells.



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  		FIG. 1. In situ analysis of Col9a3 expression in developing mouse gonads. a) An 11.5-dpc male urogenital ridge (expression indicated by arrow). b) A 12.5-dpc male (left) and female (right) urogenital ridge showing expression in testis cords (c). Note the gonads (g) and mesonephros (m). c) Positive 13.5-dpc male (left) and negative female (right) urogenital ridges. d) Transverse section showing expression within the testis cord at 13.0 dpc. e) Untreated (-) and busulfan-treated (+), 13.0-dpc male urogenital ridges stained for Oct4 expression. f) Untreated (-) and busulfan-treated (+), 13.0-dpc male urogenital ridges stained for Sox9 expression. g) Untreated (-) and busulfan-treated (+), 13.0-dpc male urogenital ridges stained for Col9a3 expression. Original magnification: a–c, e–g, x50; d, x400

RT-PCR Analysis of Collagen Transcript Expression in Developing Mouse Gonads

Col9a3 encodes one chain of the type IX collagen heterotrimer. Col9a1 and Col9a2 encode the other two chains. Collagen IX protein is expressed as two different isoforms, and these arise from alternative promoter usage in the chain encoded by Col9a1 [30, 33] (Fig. 2a). The RT-PCR was used to define the isoform that was differentially expressed in the developing gonad. Despite some variations in starting material, both the chondrocytic and nonchondrocytic forms of Col9a1 were detected (Fig. 3b). The chondrocytic form showed a similar level of expression in both sexes at 12.5 dpc and became restricted to the male at 13.5 dpc. The nonchondrocytic form showed male-specific expression at both 12.5 and 13.5 dpc (Fig. 3b). The expression of the invariant Col9a2 gene was shown to be upregulated testis-specifically at 13.5 dpc (Fig. 3a).

Type IX collagen has been found to colocalize with type II collagen at many sites of expression [34]. Therefore, it was decided to use a similar RT-PCR and hybridization strategy to look for transcript expression of the gene encoding the collagen II homotrimer, Col2a1, in early gonads. Type II collagen occurs as two isoforms arising from the alternative splicing of its second exon [21] (Fig. 2b). The PCR conditions defined previously [14] were used on 12.5-dpc testis cDNA and amplified both Col2a1 and a similarly sized background band present equally in males and females that, on cloning, was identified as Fto (accession no. U237917). Multiple Col2a1 clones were sequenced and contained the nonchondrocytic form second exon. The sequences were identical to published genomic sequence spanning exons 1–5B [35], except that the first 9 bp from the third exon were absent (Fig. 4b). The cloned Col2a1 cDNA was used as a probe on Southern blots, thus eliminating Fto background bands (Fig. 4a). Little chondrocytic Col2a1 expression was detected in gonads. The nonchondrocytic form was male-upregulated and was expressed at 11.5 dpc, decreasing to undetectable levels by 13.5 dpc. In contrast, cultured chondrocytes showed much higher expression of the chondrocytic form of Col2a1 than the nonchondrocytic form.



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  		FIG. 4. RT-PCR analysis of Col2a1 expression in developing mouse gonads and cultured chondrocytes. a) Col2a1 isoforms detected by Southern blot analysis in cultured chondrocytes (C) and in male (M) and female (F) gonadal RNA (ch., Chondrocytic isoform; non, nonchondrocytic isoform) and Hprt loading control. Col2a1 ch., 380 bp; Col2a1, non, 584 bp; Hprt, 351 bp. b) Alignment of Col2a1 third exon in chondrocyte genomic sequence [35] with testis cDNA

Whole-Mount In Situ Analysis of Col2a1 Transcript Expression

To further define the expression profile of nonchondrocytic Col2a1, the cloned cDNA was used to generate sense and antisense digoxygenin-labeled riboprobes. Whole-mount expression analysis in male and female urogenital systems (Fig. 5) revealed detectable, albeit weak, expression in testis only. At 11.5 dpc, diffuse expression was observed within the anterior pole of the male gonadal ridge (Fig. 5, b and e). This was not detected in male sense controls (Fig. 5, a and d) or female gonadal ridges (Fig. 5, c and f). By 12.5 dpc, expression was localized to the forming testis cords (Fig. 5g). At 13.5 dpc, expression was undetectable in either sex (Fig. 5h). Sections through 13.0-dpc testis showed Col2a1 expression throughout the testis cords (Fig. 5i). Analysis of busulfan-treated embryos demonstrated that expression within testis cords was independent of germ cells, indicating expression within the Sertoli cell compartment (Fig. 5j).



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  		FIG. 5. In situ analysis of Col2a1 expression in developing mouse gonads. a) 11.5 dpc, negative male sense control. b) 11.5 dpc, male expression at anterior pole. c) 11.5 dpc, negative female. d) Higher-power view of boxed area in a. e) Higher-power view of boxed area in b. f) Higher-power view of boxed area in c. g) 12.5 dpc, positive male (left) and negative female (right). h) 13.5 dpc, negative male (left) and negative female (right). i) Transverse section of 13.0-dpc male gonad showing expression throughout the testis cords. j) Untreated (-) and busulfan-treated (+), 13.0-dpc male urogenital ridges stained for Col2a1 expression. Original magnification: a–c, g, h, j, x50; d–f, x100; i, x400


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our unexpected finding of Col9a3 expression in the developing testis (recently also demonstrated by Perera et al. [20] using RT-PCR) has prompted the analysis of type II and type IX collagen transcript expression during testis differentiation. Using whole-mount in situ and RT-PCR analyses, the sequential, male-upregulated expression of type IX collagen transcripts was demonstrated. Col9a3 was first expressed at 11.5 dpc, followed by Col9a1 and then by Col9a2, such that by 13.5 dpc, all three chains were expressed male-specifically. Detailed expression analyses showed that Col9a3 localized to Sertoli cells in a wave of expression similar to that reported for Sry [36, 37] and that the nonchondrocytic transcript isoform of type IX collagen was the earliest isoform to show male-specific expression. The male-specific upregulation of type IX collagen transcripts from 11.5 dpc and expression within Sertoli cells correlated with both the time and place of Sox9 expression. These results were therefore consistent with a regulatory role for Sox9 (or Sry) in activating and maintaining type IX collagen transcript expression in testis.

Further to these studies, we have documented, to our knowledge for the first time, Col2a1 testis expression in detail. The demonstration of type II collagen transcripts in testis was perhaps more surprising than that of type IX collagen, given that they had been specifically tested for in previous work. Significantly, we showed that it was the rarer, nonchondrocytic isoform that was present. Our studies also showed that this isoform in testis was 9 bp shorter than the same isoform present at low abundance in chondrocytes (Fig. 3b). One explanation for this shorter form was the use of an alternative testis splice acceptor site, which was thus defined as ggaaaattag/GGCCAAAG. The four bases before and the single base after this alternative splice junction conformed to the splice acceptor consensus [38]. Alternative splicing may provide an additional level of gene regulation for Col2a1 within the testis. In contrast to the type IX collagen genes, Col2a1 transcripts decreased over time and went undetectable by 13.5 dpc. Given that the gene paralleled Sox9 expression at 12.5 dpc in its male-specificity and localization to Sertoli cells, this subsequent downregulation may imply an early role for Sox9 in Col2a1 activation, but not a later role in its maintenance of expression. Because the testis Col2a1 isoform differed in exon composition from any forms found in chondrocytes, it further suggested that additional testis-specific cofactors may regulate its expression via splicing and mRNA turnover mechanisms.

The low level of nonchondrocytic Col2a1 testis expression mentioned above may explain why it has not previously been detected in the mouse using section in situ methods [14, 15]. In the present study, the diffuse staining at 11.5 dpc was more readily detected by RT-PCR than by WISH; however, at 12.5 dpc, expression was more striking in whole-mount tissue than by RT-PCR. The difference between time points and methods probably reflects the shift in Col2a1 expression from being relatively homogenous at 11.5 dpc to a distinct subset of cells at 12.5 dpc. Whole-mount studies also have an advantage compared to section studies in that weaker signals are enhanced when multiple tissue layers are superimposed. In human studies, however, sections of embryonic gonads (sex unknown) have been shown to express nonchondrocytic COL2A1 transcripts [39], and this greater sensitivity may result from expression differences between the two species. Other studies using COL2A1 sequences driving a lacz reporter gene failed to see expression in the gonad [16]. This also suggests that the required regulatory sequences for testis expression lie outside those used in the transgene (i.e., over 6.1 kb 5' of the start codon or further 3' than 309 bp of the first intron).

Could there be specific developmental roles for collagens? The nonchondrocytic form of type IX collagen has been found to play a role in bone repair and is postulated to contribute to osteoblast homing to the preliminary bone matrix [40]. The alternative exon present in nonchondrocytic Col2a1 encodes a cysteine-rich domain showing significant homology to domains within short gastrulation protein (Sog, Drosophila melanogaster, accession no. Q24025) and chordin (Xenopus laevis, accession no. Q91713). Both these proteins play important roles in dorsoventral patterning through their ability to bind members of the transforming growth factor (TGF) ß superfamily of growth factors. Messenger RNA injection studies in X. laevis embryos have shown that type II collagen can functionally substitute for chordin, binding BMP4 and inducing dorsalization [41]. A number of other TGFß superfamily members are present in testis and show a phenotype when mutated: inhibin, activin, Bmp-8a, Bmp-8b, and Amh (for review, see [42]). Bmp8a and Bmp8b are expressed in germ cells and play a role in spermatogenesis [43]; Amh is expressed in Sertoli cells and regulates Leydig cell growth and regression of the female Müllerian ducts [44]. Type II collagen/TGFß family interactions may thus have an impact on testis patterning and development.

Like Col2a1, the gene Crim1 has been shown to encode a cysteine-rich domain and is expressed male-specifically in testis from 12.5 dpc [45]. Interestingly, the Crim1 cysteine domain is more closely related to Sog than chordin (BLASTP E value = 0.005 vs. 0.73, respectively), whereas the nonchondrocytic type II collagen cysteine domain is more closely related to chordin than Sog (BLASTP E value = 0.007 vs. 0.013, respectively). Col2a1 is expressed earlier than Crim1 in the testis; however, the two genes overlap significantly in their timing and domains of expression and may play similar or shared roles.


   ACKNOWLEDGMENTS
 
The Co9a1 plasmid YM-128 was obtained from Y. Muragaki (Wakayama Medical College, Japan), and cultured chondrocytes were obtained from S. Golub and Prof. J. Bateman (Murdoch Children's Research Institute, and Dept. of Paediatrics, University of Melbourne, Melbourne, Australia). The Col9a3 sequence has been submitted to GenBank (accession no. AF349718).


   FOOTNOTES
 
1 Correspondence. FAX: 61 3 9345 6000; mcclivep{at}cryptic.rch.unimelb.edu.au Back

Received: 23 June 2002.

First decision: 29 July 2002.

Accepted: 2 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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