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From the Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington.
| Correspondence to: Dr Michael D. Griswold, 531 Fulmer Hall, School of Molecular Biosciences, Washington State University, Pullman, WA 99164 (e-mail: griswold{at}mail.wsu.edu). |
| Received for publication June 9, 2008; accepted for publication December 10, 2008. |
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Abstract |
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Key words: Heat stress, testis, mouse strain, AKR/N, C57BL/6, microarray analysis
One of the adverse effects of heat on the mammalian testis is germ cell loss. The most vulnerable cell types are primary spermatocytes (meiotic spermatogenic cells) and round spermatids (early haploid cells) (Blackshaw et al, 1973; Parvinen, 1973; Blackshaw and Massey, 1978; Steinberger, 1989; Yin et al, 1997). Spermatogonia have a slightly lower susceptibility to elevated temperature (Moore and Chase, 1923; Chowdhury and Steinberger, 1964; Davis and Firlit, 1966; Perez-Crespo et al, 2007). Cataldo et al (1997) found that the initiation of translation in pachytene spermatocytes and Sertoli cells is inhibited by exposure to abdominal temperature and that elongated spermatids are much more resistant to thermal stress.
Although the physiological and cellular responses of testis to heat stress have been well documented, the molecular mechanisms through which these responses are directed remain largely unknown. Studies indicate that the testicular germ cell loss observed in cryptorchidism occurs via apoptosis (Yin et al, 2002). Rockett et al (2001) found that several groups of genes, such as heat shock genes (HSPs), non-HSP stress response genes, and cell-signaling genes, were induced by heat stress. It has been reported that heat stress impairs DNA, RNA, and protein synthesis and may cause protein denaturation, abnormal chromatin packing, and reduction of DNA integrity (Steinberger, 1991; Sailer et al, 1997; Banks et al, 2005; Perez-Crespo et al, 2007).
Recently, Kon and Endoh (2001) and Kazusa et al (2004) reported heat-shock resistance in the experimental cryptorchid testes of some strains of mice. An increase of apoptotic cells after heat stress was detected in the mouse strains of A/J, BALB/c, CBA/N, C3H/He, C57BL/6, ddY and ICR, whereas mouse strains of AKR/N, MRL/MpJ-+/+, and MRL/MpJ-lpr/lpr showed relative resistance to experimental cryptorchidism (Kon and Endoh, 2001; Kazusa et al, 2004). A mutation of the exonuclease 1 gene was found in MRL/MpJ-+/+, suggesting a possible relation to the heat resistance of spermatocytes in the MRL/MpJ-+/+ mouse (Namiki et al, 2003, 2005).
The heat resistance observed in the testes from a specific strain of mice provides an excellent in vivo model system for studying the underlying mechanisms of heat response. In this study, the gene expression in the testes of 3 different strains of mice was monitored by microarray analysis, and then 2 of these strains, 1 normal (C57BL/6) and 1 reported to be heat-resistant (AKR/N), were subjected to scrotal heat exposure. The effect of hyperthermia was assessed by comparing global gene expression and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses in hope of identifying the key molecular components involving in the heat response.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Heat Treatment
To examine the effects of heat stress on the testis of AKR/N and C57BL/6
strains of mice, the animals were subjected to a short exposure of elevated
temperature. Scrotal heat stress was performed as described previously
(Lue et al, 1999) with minor
modifications. Briefly, mice were anesthetized with an IP injection of
ketamine-xylazine mixture at a dose of 0.1 mg/kg (ketamine) and 0.05 mg/kg
(xylazine). The testes were secured into the scrotum and lowered into a
43°C water bath so that the scrotum and one-fourth of the body were
completely immersed for 15 minutes. Control mice were treated the same way
except that the testes were not immersed. Fourteen mice of each strain were
assigned to 7 groups of 2 mice each. One group of mice was assigned to each of
the following time points: 4, 6, 8, 10, 12, and 24 hours posttreatment and
control (8 hours after manipulation). One testis from each animal was placed
in Trizol reagent (Invitrogen, Carlsbad, California) for RNA extraction, and
the other testis was fixed by immersion in Bouin solution
(Bailey et al, 2002) for
histological analysis. To eliminate the variation caused by minor differences
of temperature, the mice for each time point from both strains were put in the
same water bath at the same time and taken out simultaneously. The experiment
was performed twice.
Immunohistochemistry and TUNEL Analysis
One testis from each mouse was immersed in Bouin solution overnight, then
dehydrated in graded ethanol and embedded in paraffin. The paraffin-embedded
testis was cut into 5-µm sections and mounted on Probe-on-Plus microscope
slides (Fisher Scientific Co, Pittsburgh, Pennsylvania). To assess apoptosis
in germ cells, in situ end labeling of DNA strand breaks was performed on
testis sections by TUNEL techniques using a BD ApoAlert DNA Fragmentation
Assay kit (BD Biosciences, Palo Alto, California) according to the
manufacturer's instructions. Briefly, sections were deparaffinized in xylenes,
rehydrated in a graded series of ethanol solutions, and rinsed in PBS; this
was followed by proteinase K treatment. Terminal transferase was used for the
tailing reaction to label DNA fragments with FITC. Quantitation of apoptosis
was carried out by counting TUNEL-positive germ cells visualized with a Nikon
Microphot-FX (Meridian Instrument Company Inc, Kent, Washington) microscope.
The rate of germ cell apoptosis was expressed as the number of apoptotic germ
cells per tubule and the number of tubules containing increased apoptotic germ
cells. Data are shown as the mean ± SEM from 3 different mice for each
time point of each strain. Photomicrographs were taken with an Olympus OLY-200
digital camera (Olympus America Inc, Melville, New York) using Olympus
MagnaFire Camera Imaging and Control version 1.0.
Preparation of Total Testis RNA and Microarray Processing
Total RNA was extracted from whole testes homogenized in Trizol in
accordance with the manufacturer's protocol (Invitrogen). The quality of the
RNA was assessed by gel electrophoresis and the A260:A280 ratio. A minimum
A260:A280 ratio of 1.8 was required for microarray hybridization. All
microarray hybridization, scanning, and analysis was performed in the LBB1
service unit at Washington State University as described previously
(Shima et al, 2004). Briefly,
5 µg total RNA of posttreatment or control from C57BL/6 or AKR/N or
MRL/MpJ-+/+ testis was used to generate biotinylated cRNA target from an
oligo-deoxythymidine–primed reverse transcription reaction using the
MEGAScript kit (Ambion, Austin, Texas). The cRNA target was fragmented and
hybridized to the Affymetrix Mouse genome 430 2.0 arrays (Affymetrix, Santa
Clara, California) in duplicate, and stained in accordance with the
manufacturer's protocol. The arrays were washed utilizing the Affymetrix
GeneChip Fluidics Station 400 and scanned using a GeneArray Scanner 2500A
(Agilent, Palo Alto, California).
Absolute and Comparison Analysis
After scanning, microarray output was viewed to examine the presence of
excessive background hybridization and physical anomalies. GeneChip Operating
Software (GCOS; Affymetrix) was used to do absolute analysis. The default GCOS
statistical values were used for all analyses, and all probe sets were scaled
to a mean target signal intensity of 125. Relative expression levels of each
transcript (signal) were determined based on signal and detection (present,
absent, and marginal). After the initial analysis, data were exported and
loaded into GeneSpring 6.1 (Silicon Genetics, Redwood City, California) for
further analysis. The data comprising C57BL/6, AKR/N and MRL/MpJ-+/+ with or
without heat treatment were normalized in GeneSpring using the
default/recommended normalization methods
(Shima et al, 2004;
Small et al, 2005;
Zhou et al, 2005). To describe
the differences in transcript levels observed between the normal, untreated
testis of the 3 strains of mice used in this study, the term
differentially expressed gene is used. A differentially expressed
gene was required to have a raw signal value of no less than 50 in at least 1
strain, a 2-fold or higher difference in signal between 2 strains, and
statistical significance based on the 1-way analysis of variance (ANOVA) test.
For example, a transcript described as being differentially expressed between
C57BL/6 and AKR/N required a raw signal value of no less than 50 in either
C57BL/6 or AKR/N, a 2-fold or higher difference in signal between these 2
strains, and a statistically significant difference based on the 1-way ANOVA
test. The term heat-regulated gene describes those transcripts that
are different between the normal testis and the heat-treated testis of the
same strain. Once again, these transcripts must have a raw signal value of no
less than 50 in either the control or the heat-treated samples, a 2-fold or
higher difference in signal between the control and treated samples, and a
statistically significant difference based on the 1-way ANOVA test.
Regulated in both strains includes the transcripts up-regulated or
down-regulated similarly in both strains. Differentially regulated
denotes the transcripts differentially regulated between 2 unique strains. For
example, differentially up-regulated in C57BL/6 by heat
exposure indicates those transcripts that have a raw signal value of no
less than 50 in the heat-treated samples and are up-regulated 2-fold or
greater by heat in C57BL/6 but down-regulated or not regulated in AKR/N mice.
In the tables, listed genes were those with the highest fold change in each
comparison.
Functional Characterization and Cell Type Analysis
Ontological analysis was performed using Onto-Express
(http://vortex.cs.wayne.edu)
from the Intelligent Systems and Bioinformatics Laboratory at Wayne State
University. A list of selected transcripts was classified based on biological
function using the Onto-Express ontology classification. Briefly, the
Affymetrix probe ID of the selected transcripts was saved as a .txt file and
input into the onto-tool for the analysis. The expression levels of particular
transcripts in different cell types were analyzed utilizing the database
available on the Griswold laboratory web page
(http://www.wsu.edu/~griswold/microarray/
under "Mouse germ cells reference samples M430 2.0").
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Comparison Analysis of Gene Expression in Normal Testes of C57BL/6, AKR/N, and MRL/MpJ-+/+ Mice
To examine testis gene expression in the 3 murine strains, the raw data
generated from the microarrays were imported into GeneSpring after initial
analysis. The testes from different strains of mice showed divergent gene
expression. About 416 transcripts were differentially expressed between normal
C57BL/6 testis and normal MRL/MpJ-+/+ testis (228 higher in C57BL/6 testis and
188 higher in MRL/MpJ-+/+ testis). The biological functions of 185 out of
these 416 transcripts are unknown, and 36 are involved in transport, 24 in
DNA-dependent regulation of transcription, 15 in metabolic processes, 13 in
protein amino acid phosphorylation, and 5 in apoptosis. The AKR/N and
MRL/MpJ-+/+ testes revealed 268 differentially expressed transcripts between
them (128 higher in AKR/N testis and 140 higher in MRL/MpJ-+/+ testis). The
biological processes of 134 out of the 268 transcripts are unknown, and 21 are
involved in transport, 6 in cell cycle, 6 in cell differentiation, and 3 in
apoptosis. About 415 transcripts were differentially expressed between normal
C57BL/6 and AKR/N testis, including 229 transcripts higher in C57BL/6 testis
and 186 higher in AKR/N testis. The biological functions of 190 out of these
415 transcripts are unknown, and 25 are involved in DNA-dependent regulation
of transcription, 4 in immune response, and 12 in lipid
metabolism/biosynthetic process. Table
1 contains selected genes differentially expressed between
different strains of mice.
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Genes differentially expressed between C57BL/6 and 2 purported heat-resistant strains, AKR/N and MRL/MpJ-+/+, were also examined. Some 104 transcripts showed significantly higher levels in both AKR/N and MRL/MpJ-+/+ mice than in C57BL/6 mice, and 127 showed significantly lower levels in heat-resistant strains (Table 2 lists selected transcripts showing the greatest differences in expression levels). Within these 231 transcripts, 101 have unknown biological function; 21 are involved in transport, 15 in DNA-dependent regulation of transcription, 14 in oxidation and reduction, and 8 in metabolic processes; and 6 are related to apoptosis or antiapoptosis.
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Different Responses to Single Heat Treatment in Testes of AKR/N and C57BL/6 Mice
To investigate the response of C57BL/6 and AKR/N testes to heat exposure,
germ cell apoptosis was monitored at different time points after heat stress
by TUNEL analysis (Figure 1).
No increase in apoptotic cells was observed in either AKR/N or C57BL/6 mice at
6 hours post–heat stress compared with the untreated control testis
(data not shown). At 8 hours after heat exposure, an increase of
TUNEL-positive cells was detected in C57BL/6 testis, and this increase
continued through 10 and 24 hours. By 24 hours, all tubules in the C57BL/6
showed a dramatic increase in the number of apoptotic cells and were visibly
disrupted. However, in the AKR/N strain, no increase in germ cell apoptosis
was detectable until 10 hours after heat exposure. Even after 24 hours, there
were tubules that did not contain apoptotic cells or spaces in seminiferous
epithelium because of germ cell loss. Although the increase of apoptotic cells
appeared at different time points between the 2 strains, the same cell types
(primary spermatocytes and round spermatids) were the first cell types to
enter into programmed cell death in both strains
(Figure 1B1 and A2).
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Overall Gene Regulation in Testes of AKR/N and C57BL/6 Mice After a Single Heat Exposure
Testicular samples were collected at different times from treated animals
and controls from both strains, and RNA was isolated for microarray analysis
on the 430 2.0 chips. Correlation coefficients between the replicates of
controls or heat-treated samples calculated using Microsoft Excel (Microsoft,
Redmond, Washington) were greater than 0.98 (range, 0.985–0.995).
Therefore, no significant differences were observed between any
replicates.
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The testis samples from 8, 10, 12, and 24 hours after heat exposure and controls were examined by microarrays and analyzed by GeneSpring. As shown in Figure 2, a total of 4609 transcripts exhibited significant changes in expression levels as defined by our parameters between any time points and control in C57BL/6 testes, whereas 3060 transcripts showed differences in AKR/N testes. There were 1564 transcripts that displayed significant changes in expression in both strains, whereas 1496 transcripts showed change only in AKR/N testes and 3045 transcripts in C57BL/6 testes. As many as 68 transcripts that were differentially expressed between C57BL/6 and AKR/N controls also changed in expression levels only in C57BL/6 after heat exposure, whereas 36 showed significant change in expression in AKR/N after heat shock (Figure 2A). Some 23 transcripts that were differentially expressed between C57BL/6 control testis and 2 purported heat-resistant strains also changed in expression levels only in AKR/N after heat treatment, and 42 only in C57BL/6 after heat treatment (Figure 2B).
Gene Regulation in Testes of AKR/N and C57BL/6 Mice 8 Hours After Heat Shock
As shown in Table 3, levels
of germ cell apoptosis in the C57BL/6 and AKR/N strains started to show
differences 8 hours after heat exposure. Many tubules contain apoptotic cells
and spaces in seminiferous epithelium because of germ cell loss within 24
hours after heat shock in both C57BL/6 and AKR/N strains
(Figure 1). To identify those
genes demonstrating regulation as a direct result of heat exposure and not
germ cell loss, the global gene expression data from the 8-hour time point
were utilized. The transcripts similarly heat regulated (both go up or both go
down) in both strains or diametrically heat regulated (1 up, 1 down or not
regulated) between 2 strains were further analyzed using Onto-Express for
biological function. Table 4
shows the number of transcripts in the 2 strains of mice altered by scrotal
hyperthermia. About 15 transcripts were similarly up-regulated in both strains
8 hours after heat shock. These include 9 (60%) heat shock proteins
(Hsp) or HSP-related proteins: Hspa1a (heat shock protein
1A), Hspb1 (Hsp27, heat shock protein 1), Hspe1
(mt-cpn10, heat shock protein 1, chaperonin 10), Hsph1 (heat
shock protein 105/110 kDa protein 1), Hspa8 (Hsc70, heat
shock protein 8), Hspa9a (mortalin, heat shock protein 9A),
Cryab (crystallin, alpha B), Dnaja1 (DnaJ (Hsp40)
homolog, subfamily A, member 1), Dnaja4 (DnaJ (Hsp40)
homolog, subfamily A, member 4); and 3 transcription factors, Egr1
(early growth response 1), Jun (Jun oncogene), and Fos (FBJ
osteosarcoma oncogene). A number of transcripts exhibited different regulation
levels in the 2 strains. For example, Egr1 was up-regulated almost
27-fold by heat in C57BL/6 testes but less than 4-fold in AKR/N mice, although
this gene was up-regulated by heat in both strains. Of the 4 transcripts
similarly down-regulated in both strains, no known biological function could
be assigned. Table 5 lists
selected genes commonly regulated by heat shock in C57BL/6 and AKR/N.
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There were a total of 67 transcripts identified as being heat-regulated in C57BL/6. Of those, 37 were up-regulated and 28 down-regulated. Nineteen (51.3%) of the up-regulated transcripts were up-regulated in C57BL/6 but not in AKR/N. Of these 19 differentially up-regulated transcripts, 4 are involved in steroid or sterol biosynthesis: Sc4mol (Sterol-C4-methyl oxidase-like), Sc5d (sterol-C5-desaturase [fungal ERG3, delta-5-desaturase] homolog [S cerevisae]), Star (steroidogenic acute regulatory protein), and Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1). Two are transcription factors: Btg2 (B-cell translocation gene 2, antiproliferative) and Stat3 (the signal transducer and activator of transcription 3). One gene was involved in FASL biosynthesis: Phlda1 (pleckstrin homology-like domain, family A, member 1). About 20% of the transcripts were unknown or their biological function was unknown. Of the 28 transcripts that were down-regulated in C57BL/6 after heat shock, 20 (71.5%) were differentially down-regulated in C57BL/6 and not in AKR/N. Twelve (60%) of the transcripts were unknown or without a known function.
There were 87 heat-regulated transcripts identified in the AKR/N testis. Of those, 48 were up-regulated and 39 down-regulated. About 30 (62.5%) of the up-regulated transcripts were up-regulated only in AKR/N and not in C57BL/6. Four were transcription factors, and 4 of the transcripts respond to unfolding proteins and apoptosis: Hspb8 (heat shock protein 8), Hsp90ab1 (Hspcb, heat shock protein 1, beta), Cebpb (CCAAT/enhancer binding protein, beta), and Gadd45b (the growth arrest and DNA-damage–inducible 45 beta). Approximately 40% of the differentially up-regulated transcripts were unknown or their biological functional was unknown. Thirty-one transcripts were differentially down-regulated in AKR/N, with over 50% being unknown transcripts. About 13% were transcription factors, and 10% were related to protein folding, including Fkbp3 (FK506 binding protein3) and Fkbp5 (FK506 binding protein 5). Table 6 lists some selected genes differentially regulated by heat shock in C57BL/6 and AKR/N.
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Comparison Analysis of Gene Expression and Gene Regulation
Six transcripts differentially expressed between untreated C57BL/6 and
AKR/N testes were found to be differentially regulated in C57BL/6 and AKR/N 8
hours after heat exposure. These genes included Strbp (spermatid
perinuclear RNA binding protein), Sc4mol, D3Ertd300e (p38 interacting
protein), Tra2a (transformer 2 alpha homolog [Drosophila]),
Rerg (RAS-like estrogen-regulated growth inhibitor), and 1 gene with
unknown biological function. Five out of these 6 genes (except Rerg)
were found also differentially expressed between C57BL/6 control testis and 2
heat-resistant strains.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Our results confirm that primary spermatocytes and round spermatids are the most vulnerable to heat stress in C57BL/6 and AKR/N, because DNA damage examined by TUNEL assay was detected first in these cells. However, DNA damage in AKR/N testis was delayed compared with C57BL/6, and a large portion of spermatocytes and spermatids in AKR/N mice didn't show obvious cell damage even 24 hours after heat shock (Figure 1). Although a large number of apoptotic cells were seen among the first metaphase spermatocytes in stage XII of seminiferous tubules in normal MRL/MpJ-+/+ mice (not heat treated) but not in AKR/N mice, similar heat-resistant pachytene spermatocytes and round spermatids were detected in MRL/MpJ–+/+ mice after cryptorchidism (Kazusa et al, 2004). This result suggests that the same mechanisms may be involved in heat resistance in AKR/N and MRL/MpJ-+/+ mice. Therefore, some of the transcripts differentially expressed between C57BL/6 and 2 heat-resistant strains, AKR/N and MRL/MpJ-+/+, may play very important roles in this process. For example, 6 genes were involved in the apoptosis or antiapoptosis processes, with some of them highly expressed in the heat-resistant strains, including caspase 9 (Casp9), vanin 1 (Vnn1), tumor necrosis factor, and alpha-induced protein 8 (Tnfaip8), whereas some of the genes were highly expressed in C57BL/6, including serine/threonine kinase 17b, apoptosis-inducing (Stk17b), peroxiredoxin 2 (Prdx2), and RNA binding motif and ELMO domain 1 (Rbed1). Vnn1 was one of the antiapoptosis genes that showed dramatic differences between C57BL/6 and heat-resistant strains and is highly expressed in pachytene spermatocytes and round spermatids in the 129 mouse strain according to our array data. Further studies are needed to determine which genes and related proteins in this list are involved in heat sensitivity in the testes of different strains.
To identify the factors associated with different heat sensitivity between 2 strains of mice, we examined gene expression profiles of controls and heat-treated testes. Numerous transcripts exhibited differential regulation between 2 strains 24 hours after heat treatment. Because many tubules were visibly disrupted at 24 hours in both strains, the changes in gene expression most likely include many secondary responses to heat stress and mask the responses of the genes directly affected by the insult. Therefore, the data from the 8-hour time point was emphasized because the difference in response between the 2 strains as indicated by the TUNEL assay was measurable but no obvious morphology change was detectable. A relatively small number of transcripts were observed to be differentially regulated by heat stress between testes of C57BL/6 and AKR/N strains. Twenty transcripts were found to be differentially down-regulated and 19 genes differentially up-regulated in C57BL/6 but not in AKR/N following hyperthermia. Out of 19 differentially up-regulated genes, only 6 have a raw signal above 100 in pachytene spermatocytes, 5 in round spermatids, and 12 in Sertoli cells, according to the cell-specific array data available from the Griswold lab. This suggests that somatic cells may play an important role in early response to hyperthermia in heat-sensitive strain C57BL/6 testis. Among 30 differentially up-regulated transcripts in AKR/N, 19 have a raw value above 100 in pachytene spermatocytes and 14 in round spermatids, whereas 18 are present in Sertoli cells. One of the most differentially up-regulated genes in AKR/N was Hsp90ab1, which has high expression levels in all 3 cell types. The data generated indicate that both somatic cells and germ cells contribute to the heat resistance in AKR/N testis. Further effort is needed to determine the importance of a specific cell type in heat resistance and how a unique transcript affects heat sensitivity in these strains.
The biggest gene family commonly up-regulated in both strains was the heat shock proteins (Hsp). Expression of as many as 9 different Hsps or Hsp-related proteins was increased at least 2-fold at 8 hours after a single heat exposure. Aguilar-Mahecha et al (2001) reported that HSPs-chaperones were expressed in spermatogenic cells and that a number of Hsps are expressed in a developmentally regulated fashion from pachytene spermatocyte to elongated spermatids. Most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins (for reviews, see Bukau and Horwich, 1998; Garrido et al, 2001). However, the increase in expression of these Hsps didn't prevent testicular cells from apoptosis, at least not in the C57BL/6 testis. The possible reasons are as follows: 1) The expression of Hsps is cell-specific in testis (Meinhardt et al, 1995; Ogi et al, 1999; Aguilar-Mahecha et al, 2001) so that the regulated transcription of Hsps may also be cell-specific and cannot be discerned when whole testis was used for microarray analysis. 2) It has been recognized that HSPs regulate apoptosis differently; for example, HSP27 and HSP70 are antiapoptotic whereas HSP60 and HSP10 are proapoptotic. It has also been shown that HSPs function at multiple points in the apoptotic signaling pathway (for reviews, see Creagh et al, 2000; Lebret et al, 2003; Didelot et al, 2006). Even additional heat-inducible HSP70 in pachytene spermatocytes and round spermatids does not appear to protect male germ cells from the harmful effects of elevated temperature on spermatogenic cells (Chowdhury and Steinberger, 1970). Therefore, different HSPs regulated by heat shock may function through different pathways and either protect cells from apoptosis or induce certain cells into programmed cell death. It seems that regulation of HSPs is not associated with different responses to hyperthermia, because these Hsps were up-regulated in both strains. However, several Hsps exhibited different expression levels and different regulation levels in the 2 strains. For example, Hspa1a (Hsp70) and Egr1 showed differences of over 7-fold between 2 strains, suggesting that some HSPs may be involved in heat resistance seen in AKR/N.
Four genes involved in steroid biosynthesis were differentially up-regulated in C56BL/6 after heat exposure: Star, Cyp11a1, Sc4mol, and Sc5d. Star also showed differential expression levels between controls from C56BL/6 and AKR/N testes, and Sc4mol was also differentially expressed between C57BL/6 and 2 heat-resistant strains. It has been reported that steroid biosynthesis was disturbed in the cryptorchid testis (Damber et al, 1980; Bergh and Damber, 1984; Bergh et al, 1984) and the concentration of testosterone within the cryptorchid testis is reduced compared with the normal scrotal testis (Keel and Abney, 1981; Farrer et al, 1985). This suggests a deleterious effect on the function of Leydig cells by heat stress in heat-sensitive strains. However, Star showed higher expression levels in AKR/N control mouse, and low resting steroid levels in plasma were noticed in this strain (http://web.ncifcrf.gov/research/animal_production_program/strain_information). The reason and significance for the disturbed steroid biosynthesis in heat sensitivity is unknown. Further study is warranted to determine whether the difference in regulation of transcripts associated with steroid biosynthesis in different strains of mice is related to the differences in the sensitivity of germ cells to heat exposure in these strains of mice.
Two major pathways, intrinsic and extrinsic, are involved in the process of caspase activation and apoptosis in mammalian cells (for reviews, see Adams and Cory, 1998; Ashkenazi and Dixit, 1998; Green, 2000; Hengartner, 2000, 2001; Reed, 2000). Members of the BCL-2 family play a major role in governing the intrinsic pathway, which is the mitochondria-dependent apoptotic pathway, with proteins such as caspase 9 (Apaf-3) and BAX functioning as inducers and proteins such as BCL-2 as suppressors of cell death. The extrinsic pathway for apoptosis involves ligation of the death receptor (such as Fas) to its ligand (FasL), and activation of caspase 8 or 10 and caspases 3 and 7, which results in cellular disassembly. The endoplasmic reticulum (ER) has also shown to be involved in apoptotic execution (Nakagawa et al, 2000). As shown in Table 2, several genes involving in the process of apoptosis were more highly expressed in both AKR/N and MRL/MpJ–+/+ than in C57BL/6, such as caspase 9, Mapk14, and Map3k12 (for reviews see Cho and Choi, 2002). High expression levels of these genes are unlikely to contribute to the metaphase-specific apoptosis noticed in the MRL/MpJ–+/+ mouse (Kon, 2005), because no abnormal programmed cell death was observed in the AKR/N control testis when compared with the C57BL/6 testis. Further study is needed to determine whether these genes are involved in the heat resistance in both the AKR/N and MRL/MpJ–+/+ strains. Testicular germ cell loss observed with exposure to abdominal heat stress occurs by apoptosis (Yin et al, 1997). Studies indicated that the mitochondria-dependent and possibly also the ER-dependent pathways are the key apoptotic pathways for heat-induced germ cell death (Hikim et al, 2003) and Fas/FasL signaling may be dispensable for heat-induced germ cell apoptosis (Hikim et al, 2003; Sinha Hikim et al, 2003; Vera et al, 2004). Therefore, the occurrence of apoptosis, at least at the earlier time points after heat exposure, is more likely driven by activation of intrinsic and/or extrinsic signaling pathways involving pre-existing proteins in germ cells than related to global changes in gene expression. In this study, apoptosis was used as an indicator to indicate: 1) germ cell apoptosis in testes of control C57BL/6 and AKR/N mice; 2) different responses to heat stress in different mouse strains; and 3) a relatively early time point of response to heat shock. However, a few genes were noticed to be differentially regulated by heat stress in the testes of either strain. For example, a small increase in expression of the Bcl2a1a gene (B-cell leukemia/lymphoma 2 related protein A1a) was observed in the AKR/N mouse. Phlda1 (pleckstrin homology-like domain, family A, member 1, Tdag51), a gene that plays a key role in Fas up-regulation and apoptosis in T cells (Park et al, 1996; Gomes et al, 1999), was also differentially up-regulated in C57BL/6 testis after heat shock. Bid (BH3 interacting domain death agonist) was slightly up-regulated by heat at 24 hours in C57BL/6 but not in AKR/N. Further studies are needed to address the relationship between the heat resistance and the gene regulation involved in apoptotic pathways.
The microarray data also identified many other groups of genes, as well as numerous unknown transcripts not mentioned above, whose expression was differentially regulated by heat exposure in either strain of mouse, suggesting multiple possible mechanisms contributing to various heat responses from dissimilar strains of mice.
Taken together, the combination of histological and molecular biological analyses of testes from 3 strains and heat response from 2 strains of mice has demonstrated that: 1) diverse genetic backgrounds lead to inherent differences in testis gene expression profiles; 2) germ cells from the AKR/N testis are relatively heat-resistant compared with those of the C57BL/6 testis; and 3) multiple mechanisms are likely involved in the different responses of testes to heat shock between 2 strains of mice. This study represents the first transcriptome analysis and comparison of testes from different strains of mice showing varied heat sensitivity. Genes and gene networks identified as significant by microarrays provide important leads for pursuing a more complete understanding of temperature regulation in spermatogenesis. However, the analysis of the current data is by no means conclusive and final. Because of the stringent nature of the analysis, some genes involved in the regulation of heat sensitivity may be omitted in the present report, either because they have a lesser fold change (less than 2-fold) or because they are unable to pass the statistical tests performed in the present analysis. Another possibility is that certain genes are cell type–specific and the expression levels may be diluted in the assays by examining expression within the whole testis. Thus, further analyses of the array data by employing different approaches and cutoff criteria are necessary to use fully the wealth of information contained in this study. It is also noteworthy that the data obtained from the present experiment represent an integrated information or response by at least 3 major testicular somatic cells and numerous developing germ cells after heat stress. Each testicular cell type could play a unique role in heat sensitivity corresponding to its specialized function. Further work to classify changes in expression levels of a unique transcript into specific cell type is in progress and could provide information about functions of specific cell type in heat response. In addition, further studies on proteins correlating with message levels, in combination with their functions, could deepen our understanding of the mechanism regarding sensitivity of testis to hyperthermia.
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