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From the * Nemours Biomedical Research and
Division of Urology, A.I. duPont Hospital for Children, Wilmington, Delaware;
and the National Center for Computational
Toxicology, US Environmental Protection Agency, Research Triangle Park, North
Carolina.
| Correspondence to: Dr Julia Barthold, Division of Urology, A.I. duPont Hospital for Children, 1600 Rockland Rd, Wilmington, DE 19803 (e-mail: jbarthol{at}nemours.org). |
| Received for publication August 30, 2007; accepted for publication December 17, 2007. |
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Abstract |
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Key words: Gubernaculum, undescended testis, gene expression profiling, fetus
Completion of testicular descent in mammals is dependent on the gubernaculum, which is an appendage of the anterior abdominal wall comprising a core of mesenchymal cells with associated extracellular matrix and localized striated muscle (Radhakrishnan et al, 1979; Costa et al, 2002). In the rat fetus, the gubernaculum is visible at gestational day 14 (GD14) in both sexes (Radhakrishnan et al, 1979). The female gubernaculum contains both mesenchymal and poorly organized muscle cells, and further growth fails to occur after GD16. In males, the gubernaculum enlarges after GD16, increases dramatically in size between GD18 and 20, then becomes exteriorized by everting into an extra-abdominal location around the time of birth (GD22). The mesenchymal portion of the gubernaculum disappears, leaving an outer layer of muscle, which persists as a sac of cremaster muscle and surrounds the scrotal testis. Eversion of the gubernaculum-cremaster complex occurs rapidly, but the mechanisms controlling its development and motility are poorly understood.
In vitro studies of gubernacular development and phenotypic analysis of cryptorchid genetic mouse models suggest that the testis is required for proper development of the ipsilateral gubernaculum and implicate secretion of the Leydig cell hormones insulin-like factor 3 (Insl3) and, to a lesser degree, testosterone (Emmen et al, 2000) in gubernacular development. Targeted deletion of either Insl3 or Rxfp2 is associated with high intra-abdominal testes in homozygous male mice and delayed testicular descent in heterozygotes (Zimmermann et al, 1999; Overbeek et al, 2001). Development of the fetal gubernaculum is feminized in homozygous Insl3/Rxfp2 mutants (Tomiyama et al, 2003). By contrast, mice and rats with spontaneous androgen receptor defects or that have been exposed to the antiandrogen flutamide (Spencer et al, 1991) show a milder phenotype. Although a model of testicular descent separates INSL3- and androgen-dependent phases into distinct events (Hutson and Hasthorpe, 2005), both hormones stimulate proliferation of fetal gubernacular cells. Moreover, generalized expression of both the INSL3 receptor RXFP2 (relaxin/insulin-like family receptor peptide 2, also known as LGR8 or GREAT) and the androgen receptor is present in the fetal gubernaculum (Emmen et al, 2000; Scott et al, 2005). Canonical Insl3/Rxfp2 signaling involves the cAMP/protein kinase A (PKA) pathway via activation of the cAMP response element (CRE; Halls et al, 2005), but information regarding downstream effectors is limited.
The Long Evans orl rat strain is an inbred colony at high risk for spontaneous cryptorchidism (Mouhadjer et al, 1989). Approximately two-thirds of offspring are affected, and up to 75% of cases occur unilaterally, with the left side more frequently affected (unpublished observations); overall, approximately 35%–40% of testes fail to descend (Barthold et al, 2006). The orl gubernaculum is reduced in size between GD18 and 20, but the testis descends normally during this time (Barthold et al, 2006). By the first day of life, however, normal eversion fails to occur in about half of orl gubernacula, and subsequent aberrant lateral migration occurs with final localization of the ipsilateral testis in the superficial inguinal pouch, anterior to the rectus muscle. This is a unique animal model of cryptorchidism in that the phenotype is similar to that seen most commonly in the human population.
Because of the complexity of genetic pathways and their interactions that are known in the various models of cryptorchidism, a comprehensive screen of transcript profiles is useful to address the changes associated with the gubernaculum and testis in orl rats. In this study, we use microarray analysis to study gene expression in the developing fetal gubernaculum and testis. Our data indicate that expression of genes involved in energy pathways and in the functionally related categories of muscle development, cytoskeleton organization and biogenesis, and small GTPase-mediated signal transduction is altered in normal and orl fetuses during prenatal growth of the gubernaculum. By contrast, we found fewer strain-specific differences in fetal testicular gene expression, suggesting that genetic variants with gubernaculum-specific effects predispose orl rats to cryptorchidism.
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Materials and Methods |
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RNA Extraction
Total RNA was purified from single gubernacula or testes with the RNeasy
Mini Kit (Qiagen, Valencia, California) and the RNase-free DNase Set (Qiagen).
RNA was quantified on the basis of A260 with the use of an ND-1000
Ultraviolet-visible spectrophotometer (NanoDrop Technologies, Wilmington,
Delaware). Overall integrity of the total RNA was verified with a 2100
Bioanalyzer (Agilent Technologies, Santa Clara, California) before processing
for microarrays to assure consistency across samples.
Microarray Sample Processing
RNA samples (Table 1) were
assessed with Affymetrix Rat Expression Array 230A (Affymetrix, Santa Clara,
California). This microarray contains 15 866 probe sets representing
approximately 10 500 genes and 2700 ESTs. Some genes are represented by more
than 1 probe set. Whereas NCBI Entrez Gene lists approximately 38 000 genes
for Rattus norvegicus, the 230A GeneChips interogates nearly
one-third of known rat genes. For testes, 1 µg of total RNA from single
organs was labeled with the One-Cycle cDNA Synthesis Kit (Affymetrix). This
involved cDNA synthesis followed by in vitro transcription with T7-RNA
polymerase and biotinylated nucleotide. Because of the smaller yield of RNA
from gubernacula, 30 ng of total RNA from single organs was amplified and
labeled with the GeneChip Two-Cycle cDNA Synthesis Kit (Affymetrix). After
cDNA synthesis and in vitro transcription with T7-RNA polymerase, the
resulting cRNA was used as a template for a second round of cDNA synthesis,
which was followed by in vitro transcription in the presence of biotinylated
nucleotide. Biotinylated cRNA was hybridized to 230A GeneChips. Arrays were
washed, stained with strepavidin phycoerythrin conjugate, and scanned at
DuPont Haskell Laboratory for Health and Environmental Sciences in a
Hybridization Oven 640 (Affymetrix), GeneChip Fluidics Station (Affymetrix),
and GeneArray Scanner (Affymetrix) with Affymetrix protocols and reagents.
Standard Affymetrix quality control measures were consistent across all
hybridizations of the same tissue type.
Analysis of Microarray Data
As a measure of the quality of hybridization, the raw and normalized probe
intensity distributions for each GeneChip were determined with histogram plots
within the AffylmGUI interface for the limma package of Bioconductor
(Wettenhall et al, 2006).
Representations of 5' and 3' regions of transcripts in the labeled
cRNA were examined with the Affy package of Bioconductor to verify consistency
within tissue types. Expression values were calculated with the MAS 5
algorithm from raw probe intensities using GCOS (Affymetrix). Expression
values were also calculated with the GC robust multiarray average (GC-RMA)
algorithm (Wu et al, 2004)
from raw probe intensities within AffylmGUI. All further analyses were
performed with the GC-RMA expression values unless otherwise noted. Global
gene expression patterns and overall variability between samples were examined
by principal component analysis (PCA), which was performed in MeV version 3.1
(Saeed et al, 2003). Two
methods were used to identify differentially expressed genes: 1) the LIMMA
linear models approach with the empirical Bayes statistic (B 
3)
and the multiple testing adjustment method of Holm, used within AffylmGUI
(referred to as linear analysis), and 2) calculation of MAS5 expression ratios
using GD18 wt as a reference denominator followed by the scaling of
log2-transformed values to a median of 0.0 and standard deviation
of 0.50 with statistical analysis in GeneSpring version 7.2 using analysis of
variance with a Benjamini and Hochberg false discovery rate of .001 (referred
to as reference denominator method). Differentially expressed genes were
filtered for a GC-RMA average expression value of greater than or equal to 50
in at least 1 sample group in the analysis and then separated into groups
according to the log2 ratio of average expression values for groups
of samples. Some groups of genes were further separated with K-means
clustering in MeV. Plots of expression profiles were created with the
statistical package R
(http://www.r-project.org/).
Gene groups with mean expression levels as well as the entire data set are
available via Accession number GSE7755 at the Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo).
Groups of differentially expressed probe sets were examined for statistical
overrepresentation of Gene Ontology (GO) Biological Function categories and
biological pathways as defined in the Kyoto Encyclopedia of Genes and Genomes
(KEGG;
http://www.genome.jp/kegg/)
with the Database for Annotation, Visualization, and Integrated Discovery
(DAVID) 2007 (Dennis et al,
2003;
http://david.abcc.ncifcrf.gov/).
Real-Time Reverse Transcription Polymerase Chain Reaction
Real-time reverse transcription polymerase chain reaction (RT-PCR) was used
to validate trends in selected array-derived data from gubernaculum. cDNA was
synthesized from 150 ng of total RNA (n > 6 samples per group) with the
High-Capacity cDNA Archive Kit (Applied Biosystems). Amplifications were
performed in triplicate using TaqMan Gene Expression Assays (see
Table 2 for details) and TaqMan
Universal PCR Master Mix in an ABI Prism 7900HT. Levels of target mRNA
expression were determined by the 2–
CT method
(Livak and Schmittgen, 2001)
with tripeptidyl peptidase 2 (Tpp2) as control and total rat embryonic RNA
(Agilent Technologies) as calibrator. Nonparametric statistical analyses of
differences between strains were performed in SPSS (version 14.0; SPSS Inc) as
indicated.
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Results |
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Because of minimal differences between GD18 and 20, we analyzed strain-specific differences at GD18 and 20 only. Linear analysis identified 2401 probe sets that were differentially expressed between wt and orl males at these time points. The reference denominator method, which takes into account all time-, gender-, and strain-specific differences in expression relative to GD18, returned 3707 probe sets. After filtering for low expression, these lists were combined to generate a final list of 3589 probe sets associated with wt versus orl differences.
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Testis— Comparable linear analysis of GD17 and 19 wt samples identified fewer (n = 818) differentially regulated genes at these 2 time points in testis compared with gubernaculum. More significantly, few genes were differentially expressed in the testicular samples of wt compared with orl fetuses. Linear analysis of strain differences at GD17 or 19 yielded only 349 differentially expressed probe sets when combining the 2 lists. Of these, expression was higher in wt males in 248 sets and lower in 101.
Functional Annotation of Differentially Expressed Genes
We performed functional analysis of groups of genes using DAVID and
analyzed common GO Biological Process annotations. Two groups of probe sets
were analyzed separately: genes differentially expressed between wt and orl in
gubernaculum (n = 3589; converted to 3428 unique DAVID IDs) and the group
differentially expressed between wt and orl in testis (n = 349, converted to
347 DAVID IDs). The analyses from each group were compared for recurring
functional themes.
Selected nonredundant categories are shown in Table 3, and expression data for selected genes in these groups are shown in Table 4. Multiple categories related to general physiologic processes such as metabolism and biosynthesis were identified. When analyzing all genes differentially expressed between wt and orl, small GTPase signal transduction was the most significantly represented signaling pathway in GO. We also identified categories related to small GTPase signaling, including muscle development and cytoskeletal organization and biogenesis. These data are consistent with the known morphological changes occurring in the gubernaculum during this time frame, including significant growth and maturation of muscle (Radhakrishnan et al, 1979; Cain et al, 1995). Comparatively few GO annotations were identified in the differentially expressed testis gene group. However, these include muscle development, which might indicate selective effects of the mutation in myoid cells.
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With the use of DAVID, we identified overrepresented KEGG pathways for the 2 groups of genes differentially expressed in wt and orl gubernaculum and testis. Selected pathways are shown in Table 5, with the most genes associated with focal adhesion in both gubernaculum and testis. We separately analyzed overrepresentation of 755 known androgen-regulated and androgen-signaling pathway genes (http://www.netpath.org/pathways?path_id=NetPath_2; Bolton et al, 2007) not included in DAVID with a Fisher's exact test patterned after EASE (Expression Analysis Systematic Explorer) methodology (Hosack et al, 2003). Of 622 present on the microarray, 199 (P = .039) and 30 (P = .004) androgen-associated genes are differentially expressed in gubernaculum and testis, respectively. Together, the functional category and pathway analyses suggest altered regulation of related processes and pathways linked to energy and metabolism, muscle and cytoskeleton organization, and altered expression of androgen-regulated genes.
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Expression of Genes Linked to Cryptorchidism or Testicular Descent
We analyzed the expression patterns of candidate genes annotated on the
230A chip. Hoxa10, Epha4, and Arid5B, genes associated with
cryptorchidism in mice with spontaneous or targeted mutations
(http://www.informatics.jax.org/)
are present in the list of differentially expressed genes
(Table 4). Of the previously
reported tyrosine kinases expressed in the fetal GD16.5 mouse gubernaculum
(Verma-Kurvari and Parada,
2004), several failed to show significant gubernacular expression
during the interval studied (Rous sarcoma virus [c-Src], spleen
tyrosine kinase [Syk], v-Erb erythroblastic leukemia viral oncogene
homolog 4 [Erbb4], and Eph receptor B4 [Ephb4]) using the
microarray methodology. Others were expressed during this time frame at
comparable levels in females and both male strains (platelet-derived growth
factor receptor alpha [Pdgfra], insulin-like growth factor 1 receptor
[Igf1r], c-src tyrosine kinase [Csk], kinase insert domain
protein receptor [Kdr, also known as Vegfr2 or
Flk1], and v-abl Abelson murine leukemia viral oncogene homolog 1
[Abl1; data not shown]). Expression of protein tyrosine kinase 2
(Ptk2, encoding FAK or focal adhesion kinase) is sexually dimorphic
at GD18 and increases significantly in wt males between GD18 and 20 but is not
differentially expressed between strains. None of the functional annotation
analyses of testicular gene expression identified patterns suggestive of
altered hormone synthesis in orl fetal testis and representative Leydig
cell–specific genes, including probe sets for isoforms of Cyp11a1,
Cyp17a1, Hsd3b, and Hsd17b, which were highly, and not
differentially, expressed in both strains (data not shown). Insl3
expression was higher in orl fetal GD17 and 19 testis, but the differences
were not significant on the basis of our linear analysis.
Comparison of Differentially Expressed Gubernaculum and Testis Genes
Of the 349 testis genes differentially expressed between the 2 strains, 117
were also differentially expressed in wt compared with orl gubernaculum. Few
of these genes were down- or up-regulated in both testis and gubernaculum of
orl fetuses. These include insulin-like growth factor–binding protein 5,
interferon-induced transmembrane proteins 1 and 3, osteoglycin, and
calcium/calmodulin-dependent protein kinase II; delta (lower expression in
orl); and Epha4 (higher expression in orl). Many genes encoding ECM
proteins, including several procollagens, laminins, basigin, matrix Gla
protein, chondroitin sulfate proteoglycan 2, and spondin 1, show reduced
expression in orl testis.
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Validation of Array-Derived Expression Profiles
To determine whether the expression profiles obtained from the microarrays
were consistent with the relative amounts of mRNA present in parallel samples,
real-time RT-PCR validation was carried out. Expression levels of selected
genes in specific pathways and functional groups
(Table 4, bold) were analyzed.
We identified 2 candidate control genes, tripeptidyl peptidase 2 (Tpp2) and
lumican, with mean GC-RMA expression levels showing minimal variation across
all groups; real-time RT-PCR results showed differences in raw Ct values of
less than 0.5 for both genes with more consistency seen in Tpp2 expression
across samples (data not shown). We analyzed target genes related to
Tgfβ/Wnt/Hedgehog (Bmp4, Id2, Msx1, Wnt4, Fst), MAPK (Dusp6,
Nfkb1), and insulin-like growth factor signaling (Igf1, Igfbp5);
neurogenesis (Olfm1) and myogenesis (Myog, Tnnt2, Des) in
gubernacula, testes (6–12 samples/group from 2–3 litters), or both
relative to Tpp2. Compared with the microarray data, we identified similar
expression patterns for most wt and orl samples at the 2 time points, and
differences in mRNA levels by RT-PCR were statistically different for many of
these genes at GD20 but not GD18 (Figure
5). The most significant differences between strains were noted
for gubernacular genes associated with neuromuscular development (Olfm1,
Msx1, Myog, Tnnt2, and Id2).
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Discussion |
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By contrast, GD18–20 gene expression in orl fetuses is substantially less varied, with significant overlap in the function of genes that are more highly expressed in orl males and in females, suggesting that loss of male-specific signaling is already present at GD18. The observation that strain-specific expression profiling of fetal testis shows relatively few differences also supports a model of cryptorchidism in the orl strain in which delayed or incomplete development of the gubernaculum is a major contributing factor. Our functional analysis suggests that many general pathways related to metabolism, energy and growth are altered in the orl gubernaculum, consistent with the decreased size of the fetal orl gubernaculum (Barthold et al, 2006). We also identified several specific, related pathways and biological processes represented by differentially expressed gubernacular genes, including small GTPase signal transduction, focal adhesion, actin-filament–based process, cytoskeleton organization and biogenesis, and muscle development. Although no database of androgen-regulated genes in the fetal gubernaculum exists, we observed that genes regulated by androgens, involved in androgen receptor signaling in other cell types, or both were overrepresented in our lists of differentially expressed genes from both testis and gubernaculum. These data suggest that androgen receptor signaling may be altered in the orl fetus, although additional studies are needed.
The respective roles of Insl3 and androgen in cell-specific development of
the gubernaculum remains undefined. In vitro, Rxfp2 activation increases cAMP
production via the stimulatory G-protein G
s, activates the
CRE reporter, and, in cooperation with testosterone, stimulates proliferation
of fetal gubernacular cells (Emmen et al,
2000; Halls et al,
2007). Rxfp2 is one of many G-protein–coupled receptors that
activates cAMP/PKA, a response that regulates multiple developmental
processes, including neurogenesis and myogenesis
(Lonze and Ginty, 2002;
Chen et al, 2005). However,
little is known of the downstream effects of cAMP/PKA signaling in fetal
gubernaculum beyond cellular proliferation. In vivo, hyperplasia and
extracellular matrix production in the fetal gubernaculum is followed by
maturation of muscle precursors that become peripherally oriented to form the
striated cremaster muscle (Radhakrishnan
et al, 1979; Wensing,
1986). Cultured GD17 rat gubernacula enlarge in response to
synthetic androgen R1881 without Insl3 but contain poorly organized
myosin-positive cells within the mesenchymal core. However, when cultured with
testis, they contain a defined outer layer of muscle
(Emmen et al, 2000). Marked
atrophy of the fetal gubernaculum with loss of the inner mesenchymal core is
characteristic of both Insl3 and Rxfp2 null mice
(Kubota et al, 2001). These
data suggest that Insl3 may play a role in the regulation of both matrix
remodeling and muscle development within the gubernaculum.
Other rodent data support a role for Insl3/Rxfp2 and additional candidate genes in regulation of myogenesis during development of the gubernaculum. Rxfp2 mRNA is present throughout the gubernaculum at GD16 in rat (Scott et al, 2005), but by GD19, binding sites for Insl3 are localized to the outer muscle layer (McKinnell et al, 2005), whereas the androgen receptor continues to be expressed in both mesenchyme and muscle (Staub et al, 2005). A cell-specific developmental role for androgens in the gubernaculum is not clear; however, after prenatal exposure to the antiandrogen flutamide prior to GD17, both mesenchymal and muscular compartments of the GD20 rat gubernaculum are reduced in size (Cain et al, 1995) and embryonic muscle isoforms persist in adult cremaster muscle (Tobe et al, 2002). Hoxa10 transcripts are also expressed throughout the GD15.5 gubernaculum, and histological studies of the postnatal cremaster muscle in Hoxa10 (–/–) males show disordered myogenesis (Satokata et al, 1995).
Expression patterns of specific genes that are involved in muscle development, contraction, or both (Table 4; Figure 5) support our global functional analysis results and suggest that terminal differentiation of muscle is delayed or disrupted in orl gubernaculum. Expression levels of Myog and Myf6, myogenic regulatory factors that control later stages of muscle differentiation (Sartorelli and Caretti, 2005), are reduced, whereas several genes that are down-regulated during or inhibit terminal differentiation of muscle (Melnikova et al, 1999; Ohkawa et al, 2006), including Igf1, Id2, Msx1, and representatives of the fibroblast growth factor family, show increased expression in the orl fetal gubernaculum. We also identified altered expression of several genes that promote skeletal muscle development, including Igfbp5, Ilk, Bmp7, and Fst (Huang et al, 2000; Amthor et al, 2002). Expression of Rps6kb1, Eif4e, and Eif4ebp1 are reduced in orl gubernaculum. These genes are effectors of insulin and a mammalian target of rapamycin (mTOR) signaling that regulate protein synthesis and cell size (Ruvinsky and Meyuhas, 2006); mTOR signaling is also critical for myoblast fusion (Park and Chen, 2005). Reduced expression of these genes is consistent with the global reduction in protein synthesis, as well as the reduced expression of muscle-specific genes that we observed in orl gubernaculum, with previous microarray data showing increased expression of energy and metabolism genes and decreased expression of genes involved in DNA replication and transcription during skeletal myotube maturation (Park and Chen, 2005).
In addition to muscle-specific genes, we identified altered expression of genes related to small GTPase signal transduction, cytoskeleton organization and biogenesis, and focal adhesion. The Rho GTPases encode proteins that are responsive to G-protein–coupled receptor and receptor tyrosine kinase signaling and are critical for cytoskeletal reorganization, cell motility, axon guidance, and myogenesis (Kjoller and Hall, 1999; Bishop and Hall, 2000; Bryan et al, 2005). Several, including RhoA, Rac1, Cdc42, and RhoC, are differentially expressed between strains (Table 4). Focal adhesions are sites of cell attachment to the extracellular matrix comprising integrins, cytoskeletal proteins, and signaling molecules (Sastry and Burridge, 2000). Possible roles for focal adhesion signaling in the developing gubernaculum include regulation of myoblast maturation (Clemente et al, 2005), migration (Mitra et al, 2005), or both; formation of costameres (Z-bands anchoring myofibrils to the sarcolemma) (Quach and Rando, 2006); and axon pathfinding (Robles and Gomez, 2006). Expression of the mRNA for several genes that participate in focal adhesion signaling, including Ptk2, Kdr (Flk1), Src (v-src), and Csk (Sastry and Burridge, 2000; Mitra et al, 2005) is present in the GD16.5 mouse gubernaculum (Verma-Kurvari and Parada, 2004). Csk encodes a tyrosine kinase linked to focal adhesion turnover and regulation of the actin cytoskeleton (McGarrigle et al, 2006), and the corresponding protein is localized to both mesenchymal and muscle layers in GD16.5 mouse gubernaculum. Ilk, a key component of integrin-mediated signaling that plays a role in the switch from myogenic proliferation to differentiation (Huang et al, 2000), is expressed at lower levels in orl gubernaculum.
Although the genetic basis for human cryptorchidism remains largely unknown, review of gene defects associated with cryptorchidism as compiled in Online Mendelian Inheritance in Man (OMIM) supports our present data. Several syndromes that include cryptorchidism are linked to genes that participate in small GTPase signaling (SOS1, KRAS, FGD1), actin cytoskeleton regulation (FLNA, FLNB), muscle development (ACTA1), or muscle contraction (RYR1). Expression of some of these genes is altered in orl gubernaculum (Table 4). In humans, the gubernaculum is comprised primarily of mesenchyme, but striated muscle is present within its distal portion in addition to the surrounding cremaster muscle (Tayakkanonta, 1963; Barteczko and Jacob, 2000; Costa et al, 2002), whose role, if any, in testicular descent is unclear. It is notable, however, that cryptorchidism is present in multiple forms of congenital myopathy (OMIM) and is also present in males with Prune-Belly syndrome (Jennings, 2000) and at a higher frequency in males with abdominal wall defects (Kaplan et al, 1986). Moreover, altered structure and function of the cremaster muscle has been reported in cryptorchid boys (Tanyel et al, 2000). These observations taken in combination with our present study suggest that muscle patterning might play a more important role in development and function of the gubernaculum than previously recognized.
Limitations of this study include our analysis of tissue-specific compared with cell-specific gene expression and the requirement for amplification of gubernaculum but not testis. Although the amplification, analysis, or both could be biased toward a particular cell type, we have been unable to identify any clear differences in cellular composition of wt compared with orl gubernaculum using cell-specific immunostaining (data not shown). Because of differences in RNA processing, we avoided direct comparisons of gene expression in testis and gubernaculum. Also, the possibility exists that early transcriptosomal changes associated with male specific gubernacular development were missed because gene expression is already sexually dimorphic in wt at GD18. Therefore, although we can identify global expression profiles that reflect development of wt and orl gubernacula, we cannot determine whether differences in gene expression are the cause or result of abnormal development. Moreover, because testicular descent does not occur until the postnatal period in the rat, we are unable to determine at the fetal stage which gubernacula (less than half) will be associated with cryptorchid testes. Despite this, the expression profiles of individual orl samples are highly similar and markedly different from wt in gubernaculum but not testis and therefore likely phenotype-specific as opposed to strain-specific. The basis for reduced penetrance of the phenotype remains unknown at this time but might be related to a dosage effect determined by environmental factors, modifying loci, or both. To date, we have no evidence that maternal- or paternal-specific factors determine phenotype because the frequency of cryptorchidism in offspring does not appear to be related to paternal phenotype or identity of the dam (unpublished observations).
Analysis of gene expression in fetal tissues of wild-type and cryptorchid orl mutant rats suggests that a primary gubernacular defect that directly or indirectly affects muscle function might predispose to cryptorchidism in the affected strain. Further studies will be necessary to elucidate the mechanism of gubernacular dysfunction in the orl rat.
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Footnotes |
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± 95% confidence interval, *
indicates P = .06; ** P < .05; *** P < .0.01
for differences between strains at respective time points. Wnt4
indicates wingless-related mouse mammary tumor virus (MMTV) integration site
4; Fst, follistatin; Bmp4, bone morphogenetic protein 4;
Msx1, msh-like homeobox 1; Olfm1, olfactomedin 1;
Dusp6, dual-specificity phosphatase 6; Nfkb1, nuclear factor
of kappa light-chain gene enhancer in B-cells 1, p105; Igf1,
insulin-like growth factor 1; Igfbp5, insulin-like growth
factor–binding protein 5; Myog, myogenin; Tnnt2,
troponin T2; Des, desmin; Id2, inhibitor of DNA binding
2.