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Division of Pediatric Endocrinology, Department of Pediatrics, University of Miami School of Medicine (E.M.P., T.S., G.D.B.), Miami, Florida 33136; and Department of Biological Sciences, Florida International University (E.M.P., L.K.), Miami, Florida 33199; Department of Pediatrics, University of Cambridge (H.M., I.A.H., J.R.H.), Cambridge CB2 2QQ, United Kingdom
Address all correspondence and requests for reprints to: Dr. E. M. Perera, Division of Pediatric Endocrinology, Department of Pediatrics, University of Miami School of Medicine, 1601 NW 12th Avenue, MCCD 3044A, Miami, Florida 33136. E-mail: eperera{at}med.miami.edu
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3 (Col9a3) and
Renin. Col9a3 expression was present at
low levels in male and female gonads at 11.5 dpc. Thereafter, it was
markedly up-regulated in the male, but remained very low in the female.
Expression of Col9a3 was restricted to testicular cords
and was also detected in testis of KitW/W-v mice.
Renin mRNA was first detected in testis at 12.5 dpc,
increased thereafter, and reached a peak at 16.5 dpc.
Renin mRNA was localized in cells of the interstitium
and cells at the border between the gonad and mesonephros. Expression
of Renin in the ovary was not detected using standard
conditions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies in mice and humans have also established a role for other genes such as Sox9 (10, 11), Wilms tumor suppressor-1 (Wt-1) (12, 13), and steroidogenic factor-1 (Sf-1) (14) in the earliest stages of gonadal differentiation. Nonetheless, mutations in these genes and in Sry account for relatively few subjects with 46,XY gonadal dysgenesis (15, 16, 17), implying that there must be other genes involved in this process.
Various approaches have been used to identify genes involved in male sex differentiation. In some cases, the search was directed by association of chromosomal abnormalities and sex reversal (18). In other cases, disruption of known genes resulted in abnormal gonadal development, implicating a role for the gene in normal gonadal determination and/or differentiation (12, 19). Analysis of differential expression of genes also provides a way of identifying genes that are specifically involved in testis development. Such techniques are particularly applicable to the study of testis determination in mammals, as the gonads of genetically male and female individuals are otherwise identical until the expression of Sry. Representational difference analysis (RDA) of complementary DNA (cDNA) is one approach that has been used to isolate differentially expressed genes in different types of cells and tissues (20, 21, 22). In RDA, cDNA from two populations of cells are digested with a restriction endonuclease and amplified to generate what are termed representations. Successive cycles of hybridization and amplification of the representations result in the enrichment of unique sequences and the loss of common sequences (23).
By using RDA of cDNA from mouse fetal gonads at 13.5 dpc, we have isolated a novel gene, tescalcin, and identified two additional genes, Col9a3 and Renin, that are differentially expressed during the early stages of gonadal differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RDA
Total RNA was isolated from the urogenital ridges and attached
mesonephros of 13.5 dpc male and female mouse embryos using the RNAce
method (BIOLINE, London, UK). Synthesis of cDNA was accomplished using
1 µg total RNA and the SMART PCR cDNA synthesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The RDA method was performed
essentially as described by Hubank and Schatz (25), with
minor modifications: 1) the starting amount of cDNA used to generate
the male and female representations was reduced to 200 ng, with the
overall procedure scaled down appropriately; 2) excess and digested
adaptors were removed using Microcon 30 filters (Amicon, Beverly, MA)
(26). To identify genes expressed specifically in the
male, the male representation was used as tester, and the female
representation was used as driver. DpnII-digested difference
products from the third round of RDA were separated by gel
electrophoresis on 1.5% agarose. DNA from distinct bands was purified
using the QIAEX II gel extraction kit (QIAGEN, Valencia,
CA) and was cloned into the BamHI site of pBluescript
KS+ II (Stratagene, La Jolla, CA).
Plasmid DNA was prepared from at least 10 clones of each distinct band
using the QIAprep Spin Miniprep kit (QIAGEN), and the
inserts were sequenced using the T7 Sequenase version 2.0 kit
(Amersham Pharmacia Biotech, Cleveland, OH).
Southern blot analysis
Amplified male and female representations (1 µg) were
subjected to gel electrophoresis and then transferred to Hybond
N+ (Amersham Pharmacia Biotech)
nylon membranes in 0.4 M NaOH using standard protocols
(27). The membranes were hybridized overnight at 65 C with
32P-labeled cDNA sequences isolated by RDA (TE-1
to TE-5) in 0.5 M sodium phosphate (pH 7.2), 7% SDS, and
100 µg/ml herring sperm DNA (28). Membranes were washed
at high stringency [0.1 x SSC (standard saline citrate) and
0.1% SDS, 65 C] and subjected to autoradiography.
In another experiment, PCR products from the third round of RDA (1 µg) were separated by gel electrophoresis and transferred to Hybond N+. Sox9 and Amh cDNA probes were generated by PCR amplification of cDNA prepared from 13.5 dpc mouse testis using Sox9-specific primers 9.5b and 9.5c (29) and Amh-specific primers AMH-F (5'-TGCTGAAGTTCCAAGAGCCT-3') and AMH-R (5'-AGAGCACGAACCAA-GCGAGT-3'). Hybridization was carried out with 32P-labeled Sox9 or Amh cDNA probes using the conditions described above.
Northern blot analysis
Total RNA was extracted from urogenital ridges plus mesonephros
of 13.5 dpc male and female mouse embryos using TRIzol reagent
(Life Technologies, Inc., Grand Island, NY). Aliquots (2
µg) from male and female gonads were subjected to denaturing agarose
gel electrophoresis and then transferred to Hybond
N+ nylon membranes using standard procedures
(27). Membranes were hybridized with
32P-labeled TE-1, TE-2, and TE-3 cDNA probes as
described above. A mouse multiple tissue Northern blot (CLONTECH Laboratories, Inc., Palo Alto, CA) containing 2 µg
polyadenylated [poly(A)+] messenger RNA
(mRNA)/lane was also used for hybridization. The membrane was
sequentially hybridized with 32P-labeled TE-1 and
TE-2 cDNA probes in express hybridization solution (CLONTECH Laboratories, Inc.).
Semiquantitative RT-PCR
Total RNA from male and female urogenital ridges plus
mesonephros (10.5–17.5 dpc) as well as that from newborn, prepubertal,
and adult gonads were extracted using TRIzol reagent (Life Technologies, Inc.). Synthesis of cDNA was performed with 0.5
µg total RNA using the ThermoScript RT-PCR System (Life Technologies, Inc.) and oligo(deoxythymidine) [oligo(dT)] as a
primer. Reactions without reverse transcriptase were performed in every
total RNA preparation to detect genomic DNA contamination. Multiplex
PCR reactions were performed using 0.25 pmol/µl each of
hypoxanthine-guanine phosphoribosyl transferase (HPRT)-specific primers
HPRTG-L (5'-AGGACTGAAAGACTTGCTCGA-3)' and HPRTG-R
(5'-GTAGCTCTTCAGTCTGATA-AA-3') and one of the following primer
pairs at 0.5 pmol/µl: c9–1F [5'-AGCCGACTCCTTTTCAATGTGAG-3',
nucleotides (nt) 462–440] and c9–1R (5'-CCTGACTATCATGTCCTACTTCC-3',
nt 264–286) for TE-1, c9–2F (5'-CTCTTAGATAACACCATCCCTCC-3', nt
1012–990) and c9–2R (5'-CTGAAATACGAGAGCACTCCAGC-3', nt 774–796) for
TE-2, c2–1F (5'-ATAGAACTTGCGGATGAAGGTGG-3', nt 1155–1133) and c2–1R
(5'-CTGGGAGCCAAGGAGAAGAGAAT-3', nt 916–936) for TE-3, and Y11A and
Y11B for Sry. The amount of cDNA added to the PCR reactions
was formerly normalized using the HPRT signal. PCR reactions were
subjected to 20 cycles of amplification using a 60 C annealing
temperature. PCR amplification products from each stage and sex were
subjected to agarose gel electrophoresis, transferred to a Hybond
N+ nylon membrane, and hybridized with
32P-labeled internal oligonucleotides using the
conditions described above. After washes, radiograms were exposed for
2 h with amplifying screens unless otherwise specified. The HPRT
signal was also used as a control of amplification and to determine
transfer efficiencies.
In situ hybridization
Whole mount in situ hybridization experiments were
performed using gonads with attached mesonephros from 13.5 dpc male and
female mouse embryos as described by Wilkinson (30).
Digoxigenin-labeled antisense and sense RNA probes were prepared by
in vitro transcription of linearized pGEM/TE-1, pGEM/TE-2,
and pGEM/TE-3 plasmids using a DIG RNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). For preparation of
tissue sections, whole mounts were overstained, embedded in JB-4 medium
(Polysciences, Warrington, PA), and cut to 10 µm thickness. The
embryos were observed with a Leica Corp. MZ6 stereoscope,
and the sections were viewed with a Leitz DMRB microscope
and photographed on Kodak Ektachrome 160T film
(Eastman Kodak Co., Rochester, NY).
Rapid amplification of cDNA ends (RACE)
RACE was performed following Frohman’s protocol with
modifications (31). Total RNA was isolated from gonads
plus mesonephros of 13.5 dpc mouse embryos using TRIzol reagent
(Life Technologies, Inc.). For 3'RACE, the first strand
cDNA was prepared from 2 µg total RNA using the ThermoScript RT-PCR
System (Life Technologies, Inc.) and an oligo(dT) primer
modified at the 3'-end to avoid priming at random points along the
poly(A) tail; the primer sequence was
5'-ACGAGGACTCGAGCTCAAGC(T)17VN-3' (V = A, C,
or G; n = A, C, G, or T) (32). PCR amplification was
performed using a PCR anchor primer (5'-GAGGACTCGAGCTCAAGC-3') and a
gene-specific primer for TE-1 or TE-2 (see above). For 5'RACE, the
first strand cDNA was prepared from 2 µg total RNA with a
gene-specific primer using the ThermoScript RT-PCR system. Before the
tailing reaction of the 3'-end, the first strand cDNA was washed using
a Microcon 30 filter (Amicon). PCR amplifications were performed using
the modified oligo(dT) primer, the PCR anchor primer, and a
gene-specific primer for TE-1 or TE-2. PCR products of 3'- and 5'RACE
were separated by gel electrophoresis, transferred to Hybond
N+ nylon, and hybridized with a
32P-labeled internal oligonucleotide, using the
conditions described above, to identify specific bands. PCR products
from specific bands were purified from agarose using QIAEX II kit
(QIAGEN) and cloned into pGEM using the pGEM-T Easy Vector
System (Promega Corp., Madison, WI). Plasmid DNA was
prepared using a QIAprep Spin Miniprep kit (QIAGEN), and
the inserts were sequenced using the BigDye terminator cycle sequencing
kit (PE Biosystems, Foster City, CA).
Radiation hybrid mapping
PCR primers were designed that included the 3'-end of the coding
cDNA sequence of TE-1 and part of the 3'-untranslated region (3'UTR) to
ensure that there would be no intronic sequences in the PCR product.
The PCR primers c9–1Fb (5'-CACATTCGTTTCCTCAACCATGGA-3', nt 601–623)
and c9–1Rd (5'-GATTGTCACAGAAGCCCAGGCAT-3', nt 742- 720) amplified a
142-bp fragment comprising the last 42 nucleotides of the coding
sequence and part of the 3'UTR. PCR reactions were performed using the
conditions recommended by The Jackson Laboratory Mouse
Radiation Hybrid Database
(http://www.jax.org/resources/documents/cmdata/rhmap/). The 142-bp PCR
product was analyzed by agarose gel electrophoresis. One hundred clones
from the T31 Mouse/Hamster Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) were analyzed by PCR
amplification in duplicate to identify clones containing TE-1 locus.
The gene was localized accessing the Mouse T31 RH database at
htpp://www.jax.org/resources/documents/cmdata/rhmap/rhsubmit.html.
RT-PCR using gonadal tissue from germ cell-deficient mice
Adult germ cell-deficient mice (KitW/W-v)
and wild-type mice (+/+) from the same colony were obtained from
The Jackson Laboratory (Bar Harbor, ME). Total RNA was
extracted from testis of KitW/W-v and wild-type
mice using TRIzol reagent (Life Technologies, Inc.).
Synthesis of cDNA was performed with 1 µg total RNA as described
above. Amplification of TE-1, Col9a3, and HPRT was
accomplished using the primers c9–1Fb/c9–1Rd, c9–2F/c9–2R, and
HPRTG-L/HPRTG-G, respectively, as described above. PCR products were
analyzed by gel electrophoresis.
Database analysis
Homology searches were performed using BLAST (33).
The predicted amino acid sequence for the mouse gene encoding TE-1 was
analyzed using computer programs and databases available over the
worldwide web (ExPASy Molecular Biology Server: http://www.expasy.ch/,
SignalP: http://www.cbs.dtu.dk/services/SignalP/, Pfam: http://pfam.
wustl. edu/cgi-bin/nph-hmmsearch).
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Results |
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3 (Col9a3;
GenBank accession no. NM 001853), TE-3 corresponded to part of exons 7
and 9 and all of exon 8 of Renin (GenBank accession no.
J00621), TE-4 represented the S8 ribosomal protein (GenBank accession
no. X73829), and TE-5 represented the LLRep-3 protein (GenBank
accession no. M20632). Southern blot analysis indicated that the
sequences corresponding to TE-1, Col9a3, and
Renin were present only in the male representation. By
contrast, when cDNA sequences encoding the S8 and LLRep-3 proteins were
used as probes, the signal intensities in male and female
representations were similar (data not shown).
Differential expression of TE-1, Col9a3, and
Renin genes in the testis was confirmed by Northern blot
analysis of total RNA isolated from gonads of 13.5 dpc mouse embryos
(Fig. 1
). When sequences encoding TE-1
were used as a probe, there was a single transcript of approximately
0.95 kb in the male. No signal was detected using total RNA from gonads
of female mouse embryos. Col9a3 and Renin
hybridized with single transcripts of 2.5 and 1.4 kb, respectively, in
total RNA from males, but neither hybridized with total RNA from gonads
of female embryos.
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Additional sequences from TE-1 and Col9a3
We used RACE to determine the complete coding cDNA sequence of
TE-1 from mouse testis. RACE products of approximately 0.6 kb (3') and
0.4 kb (5') that overlapped by 120 bp at the 3'-ends were sequenced.
The mouse TE-1 cDNA isolated consisted of 864 bp and included 12
nucleotides of the 5'-untranslated region (5'-UTR) and 210 nucleotides
of the 3'-UTR. The 3'-UTR contained a consensus polyadenylation signal,
AATAAA, located 12 nucleotides upstream from the poly(A) tail (Fig. 2A). Sequence analysis indicated that the
open reading frame contained 642 nucleotides encoding a novel protein
with 214 amino acids and a theoretical calculated molecular mass of
24,599 Da (GenBank accession no. AF234783). Further analysis of the
predicted amino acid sequence revealed several putative phosphorylation
sites for cAMP- and cGMP-dependent protein kinase (34),
protein kinase C (35), and casein kinase 2
(36). An N-myristoylation consensus sequence
(37), GAAHSA, and a region (residues 114–142) that
matches the consensus sequence of the EF-hand
Ca2+-binding domain (38) were also
identified (Fig. 2B).
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We also used 3'RACE and 5'RACE to extend information about TE-2 and
confirm that it corresponded to Col9a3. RACE strategy
resulted in the isolation of a 1521-bp fragment that included 732 nt of
the coding cDNA sequence and the 3'UTR (GenBank accession no.
AF237721). Comparison of the nucleotide and amino acid sequences of
mouse Col9a3 with the chicken and human sequences confirmed
that the sequence isolated corresponded to Col9a3. Figure 3 shows the alignment of the
carboxyl-terminal 244 amino acid residues of the mouse, human, and
chicken Col9a3 protein. The amino acid sequence analyzed had
higher homology with human (93.5% identity, 96% similarity) than with
chicken (80% identity, 90% similarity).
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). However, a signal was detected in
RNA of adult testis using RT-PCR (see below). Regarding
Col9a3, a transcript of approximately 2.5 kb was detected at
low levels in adult testis and brain, but no signal was observed in
other tissues (Fig. 4B).
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). Expression of TE-1 was first
detected at 11.5 dpc. An increase in TE-1 expression occurred by 12.5
dpc, with peak levels at 14.5 dpc. TE-1 expression declined slightly
thereafter, but then remained constant until at least 17.5 dpc. A
significant level of expression was detected in the newborn and adult
testis, although there was little expression in the prepubertal testis.
In the female gonad, TE-1 expression was first detected at low levels
in the newborn and was also observed in the adult.
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Expression of Renin in the testis was first detected at very low levels at 12.5 dpc. It increased at 13.5 dpc and reached a peak at 16.5 dpc. Thereafter, Renin expression declined progressively to minimal levels in the adult testis. No expression of Renin was detected in the embryonic or postnatal ovary using standard conditions. However, when films were exposed for 16 h, a weak signal was detected in total RNA from ovaries of fetal and postnatal mice.
Pattern of expression in embryonic gonads
Whole mount in situ hybridization experiments with 13.5
dpc male and female gonads were performed to provide additional
evidence for the roles of TE-1, Col9a3, and Renin
in testis development. TE-1 and Col9a3 were expressed in
testis, but the hybridization signal was restricted to the developing
sex cords. By contrast, mesonephros and kidney did not express these
genes (Fig. 6, A and B). No expression of
TE-1 or Col9a3 was detected in the embryonic ovary (Fig. 6, D and E). In addition, when the sense riboprobes for TE-1 and
Col9a3 were used, no staining was observed in male or female
gonads (data not shown). Sections of the whole mount confirmed that the
expression of TE-1 and Col9a3 was limited to and distributed
throughout the sex cords (Fig. 6, G and H, J, and K, respectively).
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) and was also observed in cells located in the border between
the gonad and the mesonephros (Fig. 6I). Sections of the whole mount
confirmed that the expression of Renin was limited to cells
within the interstitium (Fig. 6, I and L). No expression of
Renin was detected in the ovary or surrounding tissues or
when the Renin sense riboprobe was used (Fig. 6, C and
F).
Chromosomal localization of TE-1
PCR amplification of mouse and hamster DNA showed that the
expected 142-bp product was amplified from mouse alone, whereas a
single faint band of approximately 900 bp was amplified from hamster
DNA (data not shown). One hundred radiation hybrid clones from the T31
Mouse/Hamster Radiation Hybrid Panel were tested for the occurrence of
the 142-bp PCR product. The TE-1 locus was mapped to chromosome 5, 5
cR proximal to D5Mit188 (LOD = 19.4) and 6 cR distal to
D5Mit406 (LOD = 17.2). In the mouse chromosome 5 cytogenetic
map, this location fits in the distal part of the F band and is
positioned at 64 cM in the mouse chromosome 5 integrated linkage and
physical map.
Expression of TE-1 and Col9a3 in germ cell-deficient gonads
Expression of TE-1 and Col9a3 in gonads of
KitW/W-v mutant mice was also analyzed by RT-PCR
using specific primers (Fig. 7).
Amplification products for TE-1 and Col9a3 were
detected in total RNA extracted from gonads devoid of germ cells
(KitW/W-v) as well as from normal
gonads.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 (Col9a3) and Renin. Genes such as Amh, Sox9, Sf-1, and Wt-1, which are also expressed in the testis at 13.5 dpc (18), were not identified among the bands sequenced after the third round of hybridization and amplification. This may have occurred because those sequences were underrepresented and/or were overlapped by major bands. It could also have resulted from poor enrichment of those specific sequences, because the RDA conditions chosen were not optimal for them. Furthermore, it is possible that some sequences were lost by the stringency imposed by the third round of hybridization and amplification (23). However, Southern blot analysis of PCR products from the third round of RDA using Sox9 cDNA as a probe did demonstrate that Sox9 sequences were indeed present in the male representation, but were overlapped by the band corresponding to the tescalcin sequence.
The tescalcin protein is a member of the EF-hand family of
Ca2+-binding proteins, as its predicted amino
sequence contained an EF-hand Ca2+ binding motif
(residues 114–142). EF-hand Ca2+-binding domains
are characterized by a loop with 12 residues that coordinate
Ca2+ in a pentagonal bipyramidal configuration
plus -helical domains of approximately 9 residues, 1 on either side
of the loop (38). The amino acid sequence of the
tescalcin EF-hand motif perfectly matched the positions most
highly conserved in the loop (1, 3, 5, 6, 8, 12), 5 of which are
directly involved in Ca2+ binding (1, 3, 5, 8, 12) (39). EF-hand Ca2+-binding
proteins are typically divided into 2 groups. EF-hand proteins with low
Ca2+ binding affinity generally undergo small
conformational changes upon Ca2+ binding and are
usually Ca2+ buffers. By contrast, those with
high Ca2+ binding affinity usually undergo
greater conformational changes after Ca2+
binding. These proteins are called Ca2+ sensors,
and they are involved in a myriad of functions. Some of them modulate
enzyme activity, ion channels, and muscle contraction. Other functions
that are consistent with a role for tescalcin in testis development
include without question, signal transduction, control of cell cycle,
and differentiation (40). It is difficult to predict the
function of the tescalcin gene product from its amino acid
sequence alone. However, the presence of an N-myristoylation
site and several putative phosphorylation sites at least raises the
possibility that tescalcin operates as a protein subjected
to a myristoyl switch to modulate Ca2+-related
processes associated with the cellular membrane (37).
Tescalcin expression was first detected at a time that corresponds to the peak of Sry expression, as well as to the time when Sox9 expression is up-regulated (18). This raises the possibility that tescalcin expression is linked to that of Sry or Sox9. Tescalcin expression continued until at least 17.5 dpc of the embryonic period, suggesting that additional genes may be involved in maintenance of its expression. Tescalcin was expressed in the newborn and adult testis, but was only minimally expressed in the prepubertal testis, consistent with a role in testicular function.
In situ hybridization studies demonstrated that tescalcin expression was specific to the testis and restricted to testicular cords. However, its expression was not dependent on the presence of germ cells, implying that it may be expressed in Sertoli cells.
The similarity between the size of the tescalcin transcript
(
0.95 kb) on Northern analysis and the size of the cDNA obtained by
RACE (0.864 kb) indicated that the cDNA isolated comprised nearly the
entire transcript. Northern analysis of poly(A)+
mRNA from adult mouse tissues indicated that tescalcin is
likely to have a role in heart and may also have a role in brain and
kidney. The locus of the tescalcin gene maps to the distal
part of mouse chromosome 5, in a region that has not been associated
with abnormal gonadal development in the mouse (7).
Our studies also indicated a role for the Col9a3 gene
product during the early stages of testis differentiation. With respect
to related proteins, collagen II is the major component of cartilage
and is directly regulated by Sox9 protein during chondrocyte
differentiation. Furthermore, it has been proposed that Sox9
might be a regulator of other downstream genes that encode other types
of cartilage matrix components (41). By contrast, collagen
IX is a minor component of cartilage and is covalently cross-linked to
collagen II. Collagen IX itself is a heterotrimeric molecule composed
of three polypeptides (1 to 3) combined in a 1:1:1 ratio
(42). In mice, the absence of the Col9a1 gene
results in a functional loss of collagen IX. Affected mice have
degenerative joint disease but no apparent abnormality of testicular
function (43). This could result if Col9a3 were
playing a role in the testis independent of its role in collagen IX
formation.
Col9a3 expression was restricted to testicular cords, and its timing of expression was consistent with it being a target gene of Sox9. The Col9a3 product could play a role in forming and maintaining the architecture of the sex cords. However, mutations in Col9a3 in humans are associated with multiple epiphyseal dysplasia, but not with abnormalities of testicular function (44, 45).
The third gene that we identified was Renin. However, we did not determine whether the Renin gene detected was Renin 1 and/or Renin 2 (46). Both the timing and the tissue distribution of Renin mRNA were distinct from those of tescalcin and Col9a3. Renin was up-regulated 24 h later than the other genes, peaked at 16.5 dpc, and then declined until low levels in the adult. Renin expression was not detected at any stage in the female gonad using standard conditions. In situ hybridization studies indicated that Renin mRNA was located in cells outside the testicular cords, suggesting a possible role in Leydig cells. In addition, there was a marked intensity of staining in cells at the junction of the testis and mesonephros.
Renin is a protease and as such may be involved in the process of tissue reorganization that occurs in the early stages of testis differentiation. It is intriguing to consider that Renin may be involved in cleaving the connection between the gonad and the mesonephros after the migration of mesonephric cells into the developing testis. Nonetheless, mice lacking either the Renin 1 or Renin 2 gene have previously been studied, and no abnormalities of testis differentiation or testis function have been reported (47, 48). It is possible that there is overlap in function of Renin 1 and Renin 2 with respect to sex differentiation. Hence, silencing of both genes may be necessary to adversely influence testis differentiation.
In summary, our studies have identified three differentially expressed genes in the testis, indicating that representational difference analysis can identify genes that function in the testis during the early stages of differentiation. Variations in the representational difference analysis parameters, such as different tester to driver ratios, stringency of hybridization and PCR conditions, as well as melt depletion of representation, may be necessary to isolate additional differentially expressed genes. Representational difference analysis of mRNA of gonads at other embryonic stages including those closer to the time of Sry expression may help to identify genes more likely to be involved in the earliest stages of testis determination and/or differentiation.
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Acknowledgments |
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Footnotes |
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Received May 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(IX) chain leads to a
functional knock-out of the entire collagen IX protein in mice. J
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