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From the Departments of * Animal Science,
Veterinary Anatomy and Public Health, and
Large Animal Medicine, Texas A&M
University, College Station, Texas.
| Correspondence to: Dr Nancy H. Ing, Department of Animal Science, Texas A&M University, Room 410, Kleberg Center, College Station, TX 77843 (e-mail: ning{at}cvm.tamu.edu). |
| Received for publication October 7, 2003; accepted for publication February 17, 2004. |
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Abstract |
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Key words: Gene regulation, testis, spermatogenesis, puberty
We hypothesize that the induction of spermatogenesis in the light testis tissue of the 1.5-year-old horse is the result of the regulation of 1) hormone receptors that confer abilities for hormone response and/or 2) paracrine growth factors that generate local signals that differ between dark and light testis tissues. Initially, 12 gene products were chosen that were predicted to be differentially expressed in dark and light testis tissues; these gene products included androgen receptor, progesterone receptor, estrogen receptors alpha and beta, steroid hormone receptor coactivator 1, high-density lipoprotein binding protein, transforming growth factor (TGF) beta 1, 2, and 3, oxytocin receptor, glyceraldehyde phosphate dehydrogenase, and c-fos. Of the 12, androgen receptor and TGF beta 1 appeared to be differentially expressed, but this was not confirmed on in situ hybridization or Northern blots.
For this reason we turned to microarrays on glass slides in order to simultaneously and efficiently compare levels of expression of 9132 gene products between matched dark and light testis tissues of 3 colts (Rockett et al, 2001b; Schultz et al, 2003). This powerful and unbiased approach identified differential expression of 93 genes in dark and light testis tissues from 2 or more colts. In situ hybridization and Northern blot analysis confirmed and localized the differential regulation of 4 genes chosen for these analyses. These discoveries open investigations into novel pathways that regulate spermatogenesis. Importantly, these data also demonstrate that analysis of equine gene expression can be performed using human cDNA microarrays because of a high level of sequence conservation. This conservation is evident in analyses of 193 horse cDNAs from a testis library that displayed an average of 92% identity between horse and human sequences (Skow, personal communication).
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Materials and Methods |
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Microarrays
Poly A+ RNA samples from matched dark and light left testis samples from 3
horses appeared of high quality and purity on an ethidium
bromide–stained Northern gel (results not shown). Dark and light testis
RNA samples (1.8 µg each) were reverse transcribed and labeled with
5'Cy3 and Cy5 fluors, respectively; hybridized to immobilized human
cDNAs of the UniGEM Human V 2.0 microarray; and analyzed by Incyte (St Louis,
Mo). Probe labeling reactions were incubated at 37°C with 200 ng of polyA
RNA, 200 units M-MLV reverse transcriptase (Life Technologies, Gaithersburg,
Md), 4 mM DTT, 1 unit RNase Inhibitor (Ambion, Austin, Tex), 0.5 mM dNTPs, and
2 µg of labeled 9-mers for 2 hours. Sample amplification was not used in
order to decrease artifacts in expression ratios. The probe samples were then
combined, purified, precipitated, and allowed to competitively hybridize to a
single microarray. Briefly, the hybridization protocol consisted of the
following: resuspension of probe solution with a 5-minute incubation at
65°C, application to array, cover slipping, and sealing in an evaporation
chamber for 6.5 hours at 60°C. After hybridization, the glass slides were
washed in 3 consecutive washes of decreasing ionic strength. The microarrays
consisted of 9132 genes generated from polymerase chain reaction (PCR)
products (15% were expressed sequence tags) and included 190 controls. The
fluorescence data was converted to "balanced difference" values
(balanced to account for differences in fluorescence labeling between the cDNA
pools) with GEMTools 2.4 (Incyte Pharmaceuticals Inc, Palo Alto, Calif).
Balanced differences with positive values are ratios of signals from the dark
testis tissue to light testis tissue (Cy3/Cy5). Negative balanced difference
values indicate that the ratio is inverted, with the larger light testis
tissue (Cy5) signals divided by the lesser dark tissue signals (Cy3).
Normalization was performed per chip to a series of internal controls
including yeast cDNAs and fluorescent standards by gridding and region
detection algorithms. Gene products with balanced difference values of equal
to or more than 1.7 or less than -1.7 were considered differentially expressed
between dark and light testis tissues (P < .05)
(Yue et al, 2001;
Moody et al, 2002;
Reynolds, 2002). Spots failing
to meet minimal criteria (signal strength 2.5 times that of background and
covering at least 40% area of the spot) were given balanced difference values
of 0 and were excluded from evaluation. Differentially expressed genes in two
or more horses were identified with GeneSpring 4.0.2 software (Silicon
Genetics, Redwood City, Calif). Complete technical and analytical information,
including normalization and ratio determination, is available at
(http://animalscience.tamu.edu/ning/microarraydata/hoprimer.html).
Cloning of Down-Regulation of Ovarian Cancer 1 (DOC1), Golgi Apparatus Protein 1 (GLG1), and CDC2 cDNAs From Horse Testis
To make high-specificity probes for androgen receptor (AR), down-regulation
of ovarian cancer 1 (DOC1), Golgi apparatus protein 1 (GLG1), and CDC2 mRNAs
of the horse, cDNAs were cloned from horse testis, as previously described
(Ing et al, 1996). PolyA+ RNA
(200 ng) was reverse transcribed at 42°C with Superscript II (Life
Technologies) and random hexamer primers. PCR was performed twice with 2 sets
of primers designed from human cDNA sequences (see website), with the set of
primers used for the second amplification nested inside the first. cDNAs were
cloned into the PCR2.1 vector (Invitrogen), and multiple clones were
sequenced. The horse DOC1, GLG1, and CDC2 cDNA sequences are in GenBank
(accession numbers AY349169, AF547432, and AF547431, respectively).
Northern Analyses of Dysferlin (DYS), DOC1, Outer Dense Fiber of Sperm Tails (ODF2), Phosphodiesterase 3B (PDE3B), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs
PolyA+ RNA samples (5 µg) were analyzed on a denaturing (Northern) gel,
as previously described (Ing et al,
1996). RNA Millennium Markers from Ambion (Austin, Tex) were run
alongside. The RNA on the gels was transferred to nitrocellulose membranes and
the blots were hybridized to radiolabeled antisense cRNA probes produced by in
vitro transcription (T3 RNA polymerase) with the Maxiscript kit (Ambion) using
[32P]-UTP (3000 Ci/mmol; New England Nuclear, Boston, Mass). The
cDNA template for DOC1 was a PCR product (described above), while those for
dysferlin (DYS), outer dense fiber of sperm tails (ODF2), phosphodiesterase 3B
(PDE3B), and GAPDH were constructed from linearized plasmids as follows. Human
DYS plasmid (pINCY-4462162 vector; Incyte Genomics, Palo Alto, Calif) was
linearized with EcoR1 and transcribed with T7 RNA polymerase; rat
ODF2 plasmid (pBluescript II vector) (GenBank accession number U62821) was
linearized with Nco1 and T7 RNA polymerase; human PDE3B plasmid
(pBluescript II vector) (GenBank accession number U38178) with BamH1
and T3 RNA polymerase; and ovine GAPDH plasmid restricted with BamH1
and T7 RNA polymerase. After stringent washing, the blots were exposed to
x-ray film (XAR, Kodak, Rochester, NY). Densitometry with Intelligent
Quantifier software (Bio Image, Ann Arbor, Mich) was used to compare
hybridization signals between samples and reported as an average fold change
in densitometric units across 4 horses. Individual gene expression units were
normalized to GAPDH to account for loading differences.
In Situ Hybridization
The mRNA for DYS, ODF2, and DOC1 were localized on serial cross sections
from the left testes of 1.5-year-old horses by in situ hybridization analysis,
as described previously (Ing et al, 1997). Tissue sections were hybridized
with radiolabeled antisense or sense cRNA probes generated using in vitro
transcription with 
[35S]-UTP (1250 Ci/mmol, New England
Nuclear). The human DYS plasmid (pINCY-4462162 vector, Incyte) linearized with
EcoRI and transcribed with T7 RNA polymerase to produce antisense
cRNA. In addition, negative control sense cRNA from DYS was produced from
Not1 and SP6 RNA polymerase. Rat ODF2 and human PDE3B cDNAs (GenBank
accession numbers U62821 and U38178) were restricted within the cDNAs with
Nco1 for ODF2 and with BamH1 for PDE3B and transcribed with
T7 or T3 RNA polymerase for antisense and sense cRNAs. Antisense and sense
horse DOC1 cRNAs were produced from PCR products (described above) with T3 and
T7 RNA polymerases, respectively. After hybridization, washing, and
ribonuclease A digestion, slides were coated with photographic emulsion
(Eastman Kodak) and developed 2 or 4 weeks later, depending on hybridization
signal strength. Cell nuclei were counterstained with 1% toluidine blue.
Micrographs were captured on a Zeiss Axioplan 2 Microscope (Gottingen,
Germany) using Adobe Photoshop (Adobe Systems, Seattle, Wash).
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Results |
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The microarray results indicated differential expression of many interesting genes. In the cell-signaling group, the insulin system was implicated, with insulin-like growth factor 2 and insulin-like growth factor binding protein 7 being predominantly expressed in dark tissue, whereas PDE3B was expressed more in light tissue. Gene products that regulate cell cycle progression were also differentially expressed. These included parathymosin and CDC-like kinase in dark tissue and cyclin A1, CDC2, CDC28 protein kinase 2, MAD3L, and PRKC prostate apoptosis response protein 4 in light tissue.
There were many differences in the expression of genes whose products had cytoskeletal or cell adhesion functions. For example, dark testis tissue expressed higher levels of desmoplakin, keratins, and cadherin mRNAs, the products of which are associated with intermediate filaments, as well as myosin-like DOC1, sarcoglycan, and calponin gene products, which are associated with large cytoskeletal filaments. Cell adhesion proteins laminin and GLG1 were also predominantly expressed in dark testis tissue, as were 13 ribosomal proteins and 2 crystallin genes. Light testis expressed higher levels of gene products associated with sperm-specific structures (ODF2 and protamine 1) and microtubules (kinesin-like 1, stathmin, and the testis-specific tubulin a1). In addition, metabolic enzymes, kinases, protease inhibitors, and transcription factor genes were differentially expressed between dark and light tissues, as were novel gene products of unknown function (such as expressed sequence tags).
Northern Blots Confirm Differential Expression of DYS, DOC1, ODF2, and PDE3B Genes— Northern blots were used to confirm the differential expression of DYS, DOC1, ODF2, and PDE3B genes between light and dark testis tissues, utilizing GAPDH mRNAs as a control. Representative Northern blots are depicted in Figure 1. In order to critically analyze and verify these results, horses evaluated were not used in the microarray. Both DYS (8.0 kb) and DOC1 (3.5 kb) mRNA concentrations are greater in dark testis tissue compared to light. Previous Northern blot experiments indicate similar sizes for those mRNAs (Mok et al, 1994; Britton et al, 2000). In contrast, ODF2 and PDE3B mRNAs hybridized weakly in dark tissue but showed strong expression in the light tissue. Sizes of these transcripts (2.5 and 6.5 kb, respectively) were similar to those observed in other studies (Miki et al, 1996; Schalles et al, 1998). Similar Northern blot analyses of light and dark testis tissue RNA samples of 4 horses were quantitated. Since there were no differences in GAPDH mRNA concentrations found between light and dark tissues of other stallions in macroarray, in situ hybridization, or microarray analyses, each lane was normalized to its GAPDH mRNA signal and is reported as mean fold change for each mRNA (±SE). Fold differences on Northern blots were comparable to signal intensities for microarrays. DYS and DOC1 were 3.2 (±0.44) and 2.4 (±0.27), whereas ODF2 and PDE3B both exhibited fold increases over dark tissue at 4.6 (±1.6) and 3.9 (±1.2), respectively.
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In Situ Hybridization Confirms and Localizes Differential Expression of Genes Within Dark and Light Testis Tissues— DOC1, human DYS and PDE3B, and rat ODF2 cDNA localization of horse mRNA in light and dark testis from 1.5-year-old horses by in situ hybridization indicate similar patterns to those obtained by microarray and Northern blot analysis. Like that of the horse AR cDNA (GenBank accession number AY032721), the horse cDNA for DOC1 was highly conserved across species, being 92% identical to the human. Representative bright-field and dark-field views of the in situ hybridization of DYS and DOC1 mRNAs are shown in Figure 2. The bright-field views illustrate the histology of the testes; dark testis tissue has small seminiferous tubules with closed lumina and light tissue has larger seminiferous tubules and open lumina. Hybridization signals (small, black silver grains) for DYS and DOC1 mRNAs were markedly stronger in dark testis compared to the light tissue (Figure 2) and were concentrated over the seminiferous tubules. In contrast, an intense accumulation of signal for ODF2 and PDE3B mRNAs (Figure 3) was found in the light tissue over maturing germ cells (Figure 3). Thus, in situ hybridization confirmed the differential expression between dark and light testis tissue for 4 genes analyzed from the 93 genes listed in the Table and Northern blot hybridization.
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Discussion |
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The large number of gene products analyzed on the microarrays provided an unbiased and feasible method of identifying genes that are regulated during the initiation of spermatogenesis. The microarray data yielded expected results that validated the analyses. For example, AR and other genes analyzed previously were not differentially expressed between dark and light tissues of the horse testes. However, we were able to identify genes regulated during the initiation of spermatogenesis, including DYS, DOC1, ODF2, and PDE3B.
The genes preferentially expressed in dark testis tissue included DYS and DOC1. DYS is a protein involved in scaffolding and aids in membrane repair between intracellular and extracellular components (Ellis, 2003). Mutations result in the human autosomal recessive limb girdled muscular dystrophy disease. A DYS homolog, the FER-1 gene found in Caenorhabditis elegans (Britton et al, 2000), is expressed in spermatic vesicles, and FER-1 mutants produced sperm that were abnormal and were infertile (Achanzar and Ward, 1997). Results demonstrate that DYS gene expression is predominately in spermatogenically inactive tubules that do not have a full complement of germ cells. It is plausible that DYS serves as part of a priming mechanism for spermatogenesis within the seminiferous tubules, enabling them to repair themselves after lumen expansion and compaction during waves of divisions.
The DOC1 gene, a candidate tumor suppressor gene with a myosin-like product, was also found to be more highly expressed in dark tissue. Interestingly, DOC1 is expressed by normal ovarian epithelial cells but not by ovarian cancer cell lines (Mok et al, 1994). Other than involvement with cytoskeletal and adhesion molecules like that of DYS, its role in the initiation of spermatogenesis is still unclear.
Expression of ODF2 and PDE3B genes is predominant in the spermatogenically
active horse testis tissue and is well described in other species, including
the mouse, rat, and human (Hoyer-Fender et
al, 1998; Wiersma et al,
1998; Peterson et al,
1999; Nakagawa et al,
2001). The ODF2 gene is expressed exclusively in the testis and is
part of the cytoskeletal structure of the sperm tail. The function of ODF2
involves maintaining elastic recoil and providing protection from shear forces
during sperm transport in the female reproductive tract. Equine ODF2 mRNA was
localized to mature tubules with round and elongated spermatids, which
coincides with the results of other studies in the rat and bull
(Schalles et al, 1998). PDE3B
also showed a definite expression difference in light tissue in all
experiments conducted and functions to regulate cAMP levels. In the mammalian
ovary, meiotic arrest of oocytes is regulated by cAMP levels. PDE3B acts to
decrease these levels, and when inhibited with products such as hypoxanthine
and milrinone, cAMP levels continue to increase and prohibit maturation
(Wiersma et al, 1998). Also,
PDE3B acts as a mediator in the insulin pathway
(Harndahl et al, 2002). PDE3B
mRNA was expressed only in secondary spermatocytes and round spermatids, but
not in elongated spermatids. These results imply that PDE3B may play an
important role in meiotic division in the testis. Other genes discovered by
the microarrays that have greater expression levels in spermatogenically
active light testis tissue specific to developing male germ cells include
protamine, calmegin, tubulin a1, and phosphorylase kinase 
2
(Amat et al, 1990;
Prigent et al, 1996;
Mochida et al, 1998;
Yoshinaga et al, 1999;
Steger et al, 2001).
Upon analysis, it is obvious that cross-species hybridization in cDNA microarrays is a strong tool to elucidate important genes involved in spermatogenesis. Although initial predictions of which hormone receptors and paracrine factors would be regulated during the initiation of spermatogenesis did not prove to be true, other genes were identified by the microarray analyses. One example is the CBP/p300 transactivator that participates in steroid hormone receptor and c-fos transactivation pathways and was preferentially expressed in light testis tissue (Fronsdal et al, 1998). Another example is the identification of 3 genes in the insulin-like growth factor–signaling system, which is consistent with the proposed role of this system in testis development and seasonal induction of spermatogenesis (Achanzar and Ward, 1997; Fiszer and Kurpisz, 1998; Liu et al, 1998; Wagoner et al, 2000).
Other groups have indicated that some of the gene products identified here are important to the biology of the testis (Yu et al, 2003). For example, ODF2, cholecystokinin, cyclin A1, calmegin, glucose transporters, and stathmin, predominately expressed in light testis tissues, are involved in meiosis and developing male germ cells (Persson et al, 1989; Weiss et al, 1997; Angulo et al, 1998; Flores et al, 1998; Ohsako et al, 1998; Rouiller-Fabre et al, 1998; Schalles et al, 1998; Bushby, 1999; Ravnik and Wolgemuth, 1999; Yoshinaga et al, 1999; Doege et al, 2000; Muller et al, 2000; Tsuruta et al, 2000; Guillaume et al, 2001). Additionally, placental cadherin and laminin are dark testis tissue–specific gene products that others have localized to peritubular cells and the basement membranes of seminiferous tubules, respectively (Virtanen et al, 1997; Johnson et al, 2000). Importantly, altered expression of DYS, kinesin, laminin, and protamine genes has been associated with reductions in male fertility and spermatogenesis in man and other species (Boekelheide et al, 1989; Lee et al, 1995; Virtanen et al, 1997; Mochida et al, 1998; Yoshinaga et al, 1999; Cho et al, 2001; Steger et al, 2001).
In conclusion, this use of human cDNA microarrays to study gene expression in the horse testis was highly successful. These Incyte microarrays have generated data in both human and animal systems that have proven to be reproducible and reliable (Pomp et al, 2001; Yue et al, 2001; Moody et al, 2002). The success of the technique is due to the high level of sequence conservation between horse and human mRNAs. Cross-species hybridization is common in Northern blot and in situ hybridization analyses, as demonstrated here even for species as diverse as rat and horse. Thus, microarrays composed of long human cDNAs (not oligonucleotides) provide a valuable tool for gene expression analyses in domestic animal species. Genetic analysis of equine testis was uniquely powerful in generating a nonbiased list of new gene targets to explore in the developing testis of the prepubertal colt. By understanding the regulation of the identified gene products within testicular cells of immature and mature stallions of high and low sperm, fertility parameters may elucidate the gene networks critical to efficient spermatogenesis as well as novel therapeutic approaches for improving male fertility.
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Acknowledgments |
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Footnotes |
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