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RESEARCH |
Departments of Neuroscience and Psychology, Ohio State University, Columbus, OH 43210, USA
Correspondence should be addressed to L M Pyter; Email: pyter.1{at}osu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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6-fold different between long- and short-day testes (i.e. hypoxia-inducible factor 1
(Hif1), plasminogen activator inhibitor 1 (Serpine1), transforming growth factor ß receptor 3 (Tgfßr3) and tumour necrosis factor (Tnf )) were sequenced and expression differences were compared throughout gonadal regression and recrudescence using quantitative RT-PCR. Our results suggest that short days trigger expression of Hif1, Serpine1, and Tgfßr3 to inhibit angiogenesis or promote apoptosis during testicular regression, and also trigger expression of Tnf to promote angiogenesis during testicular recrudescence. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The mass of the reproductive tract of male white-footed mice (Peromyscus leucopus) decreases following 6–12 weeks of exposure to short photoperiods (Lynch et al. 1981). Testicular cells (primarily spermatids and spermatocytes) undergo apoptosis in short days, which may contribute to decreased tissue mass (Young et al. 2000, Young & Nelson 2001). Seasonal changes in testicular capillary blood flow (Joffre & Joffre 1973), permeability of the blood–testis barrier (Pelletier 1986), and volume and density of testicular microvasculature (Mayerhofer et al. 1989) have been reported in several species that breed seasonally. The molecular mechanisms that regulate vascular development and demolition in response to environmental cues remain unspecified. However, the collapse and regrowth of the testes positively correlates with vascular endothelial growth factor (VEGF) protein expression (Young & Nelson 2000). VEGF is a growth factor that is present during blood vessel growth of tumours, wound healing and uterine vascularization (Neufeld et al. 1999).
The photoperiod-driven cycle of VEGF protein levels in the testes of white-footed mice is evidence that changes in tissue mass may be accompanied by changes in the supporting vasculature. Tissue growth may precede growth of the vascular bed and tissue demise may precede vascular regression or vice versa. For example, excessive tumour growth can be inhibited by prevention of angiogenesis suggesting that tissue growth precedes angiogenesis (Brem 1976). However, seasonal changes in testicular blood vessel volume precede measurable changes in testes morphology in Syrian hamsters (Mesocricetus auratus), suggesting that angiogenesis precedes tissue morphology (Mayerhofer et al. 1989). Also, a seasonal increase in testicular blood flow precedes the presence of spermatozoa in testes of dormice (Glis glis), but not ferrets (Mustello furo) and foxes (Vulpes vulpes) (Joffre & Joffre 1973). Regardless of the timing of angiogenesis, the process consists of four main steps: (1) degradation of the epithelial layer of the existing vessel by matrix metalloproteinases; (2) recruitment of growth factors; (3) proliferation and recruitment of new epithelial cells; (4) stabilization of the new vessel by smooth muscle cells (Klagsbrun & Moses 1999). To maintain vasculature, many blood-borne factors are involved and are expressed in a balance between promoting and inhibiting angiogenesis (Klagsbrun & Moses 1999). When the signal to expand the vasculature is initiated, this balance swings in favour of the proangiogenic factors. We hypothesized that expression of angiogenesis genes would differ in the testes between long- and short-photoperiod-treated white-footed mice and that these differences would correspond to the pattern of testicular regression and regrowth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Experimental procedures
Body mass and reproductive responses to photoperiod were assessed bi-weekly under light isoflurane anaesthetization. Reproductive status was assessed by external measurement of the left testis with calipers. The product of testis length times testis width squared provides a measure of estimated testis volume (ETV) that is highly correlated with testis weight (Gorman & Zucker 1995). After 7, 14, 21 and 34 weeks of photoperiod treatment, 4–34 male mice (from each photoperiod) were killed via rapid cervical dislocation and tissues were collected.
Tissue collection
Right testis, epididymides, epididymal fat pads and seminal vesicles were collected from mice under aseptic conditions and submerged in 10 volumes (10 µl per mg tissue) of RNALater RNA stabilization reagent (Qiagen) and stored at 4 °C for 24 h. All surgical instruments were washed consecutively with 70% alcohol, RNAse Away (Fisher, Pittsburgh, PA, USA) and sterile deionized water between mice. Seminal vesicle fluid was expressed prior to storage. Following 24 h of RNALater submergence, tissues were blotted briefly, weighed and frozen at –70 °C until RNA processing. Prior to weighing, connective tissue and fat were cleaned from epididymides. The left testis was collected from each mouse and fixed in 10% neutral buffered formalin for at least 2 weeks until processed for histology. The mean testes mass for long-day (LD) mice was determined and all short-day (SD) males with testes mass equal to or greater than 1.5 standard deviations below this mean were considered reproductively responsive to short days (SDR), whereas those mice that did not differ from the LD mean were considered non-responsive to short days (SDNR).
Histology
Testes were fixed in 10% neutral buffered saline, paraffin embedded and cut into 5 µm sections on a rotary microtome. Sections were progressively stained with eosin Y and Harris haematoxylin, and coverslips were affixed with Permount. Diameters of 10 seminal vesicles chosen semi-randomly using a fixed line across the section were visualized with light microscopy at x 20 or x 30 power and measured on five randomly selected sections of each testes using SpotBasic software (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Diameters were measured only if their length and width differed by < 25%. Seminiferous tubule measurements of SDNR mice at 21 weeks were excluded due to improper embedding processing.
RNA extraction
Total RNA was extracted from 
30 mg of individual testes using a rotor-stator homogenizer with an RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen). Extracted RNA was suspended in 30–50 µl RNase-free water and RNA concentration was determined by spectrophotometer. For gene array analysis, aliquots of equal mass (e.g. 1 µg) from individual RNA samples at the 14-week time point were pooled to create a single pooled sample representative of each photoperiod treatment group. Individual samples with concentrations <100 µg/ml were excluded from the pooled sample (n = 1, LD at week 21). For quantitative PCR (qPCR), individual RNA samples were used. All RNA samples were stored at –80 °C until gene expression analysis.
Microarrays
As a preliminary screen for differences in angiogenesis gene expression, microarrays were hybridized with testicular RNA after 14 weeks of photoperiod treatment. Non-Radioactive Mouse Angiogenesis GEArray Q Series microarrays with the GEArray RT-labeling method (Super-Array, Frederick, MD, USA) were used according to the manufacturer’s protocol with a few modifications. Briefly, 4 µg of each pooled RNA sample were reverse transcribed to cDNA and used for probe synthesis and the reverse transcription step was extended to 4 h. Hybridization of labeled cDNA to the microarray occurred at 59 °C for 24 h. On a sheet of plastic wrap, the chemiluminescent substrate was incubated on the array for 4 min while covered by a piece of Parafilm (Fisher) to equalize the substrate distribution. Immediate, side-by-side chemiluminescent imaging of the microarray from each treatment was made possible by a CCD camera imaging system and detection was stopped upon pixel saturation. Quantity One software (Applied Biosystems, Foster City, CA, USA) was used to capture images and measure relative density of gene spots after background subtraction. Individual gene spot density was quantified following normalization to the density of the Gapdh spot. We plotted the distribution of the fold differences in gene expression against the number of genes and designated a cut-off point based on the mean fold difference (mean = 5.7). Therefore, genes with expression levels that differed 6-fold among treatment groups were examined throughout testicular regression and recrudescence via qPCR.
Sequencing
In order to design species-specific primers and probes for qPCR, a portion of each gene of interest was sequenced. Semi-degenerate primers were designed based on conserved regions between Mus musculus and Rattus norvegicus gene sequences using PrimerExpress software (Applied Biosystems; Table 1
). Forty cycles of PCR were conducted on 1 µl cDNA from pooled P. leucopus RNA with Taq DNA Polymerase enzyme (Invitrogen), according to the manufacturer’s protocol, in a thermocycler with melting temperatures of 59–60 °C. Gene-product amplification was visualized on 2% 40 mM Tris acetate and 2 mM ETDA (TAE)-agarose gels containing ethidium bromide using a CCD camera. To verify amplification of the correct gene, PCR products of the expected molecular size were purified (Centricon-100, Millipore, Billerica, MA, USA), and sequenced at the Plant-Genomics Centre at Ohio State University. Resulting amplicon sequences were >90% homologous to the Mus gene of interest and therefore were assumed to be the correct P. leucopus gene of interest.
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(Hif1), transforming growth factor ß receptor 3 (Tgfßr3), plasminogen activator inhibitor 1 (Serpine1) and tumour necrosis factor (Tnf ) using qPCR. Expression of these genes was quantified for individual animals of each photoperiod group at all four time points in duplicate or triplicate. Individual RNA samples (n = 4 per photoperiod group per time point) were reverse transcribed into cDNA with MMLV Reverse Transcriptase enzyme (Invitrogen) according to the manufacturer’s protocol. Primers and probes for qPCR were designed based on the attained sequencing information using PrimerExpress. Primers and probes were synthesized as follows, with probes labeled with 6-FAM (fluorescent dye) and MGB (non-fluorescent quencher dye) at the 5' and 3' ends respectively: Hif1forward 5'-CTGT-GATGAAAGAATTACTGAGTTGATG-3', Hif1reverse 5'-CATAAATTGAGCGGCCCAAA-3', Hif1probe 5'-TATGA-GCCAGAAGAAC-3'; Tgfßr3 forward 5'-CAGGACCAGCTC-GATGGAA-3', Tgfßr3 reverse 5'-CACCAGGAAGAGGTCT-GTTGTTATACA-3', Tgfßr3 probe 5'-CATCACCTTCAAC-ATGG-3'; Serpine1 forward 5'-CTCACCAGCATCTTGGA-TGCT-3', Serpine1 reverse: 5'-CGAGGCAGTCTGGTCAT-GTTC-3', Serpine1 probe 5'-CTCATCCGACAATGGAA-3'; Tnf forward 5'-CCCCAAAGGGAGAGAAGT-3', Tnf reverse 5'-GCCATGGAGCCGTGATG-3', Tnf probe 5'-CCCAAC-AACCTCC-3'. A TaqMan 18S Ribosomal RNA primer and probe set (labeled with VIC (fluoresent dye); Applied Biosystems) was used as the control gene for relative quantification. Ninety-six-well PCR plates were composed of duplicate or triplicate wells for all five genes of each individual sample. On each plate, batches of samples were randomized for photoperiod treatment and time point, and sample identification was encoded to eliminate potential experimental bias. Amplification was performed on an ABI 7000 Sequencing Detection System by using TaqMan Universal PCR Master Mix. The universal two-step RT-PCR cycling conditions used were: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative gene expression was calculated by comparison to a relative standard curve consisting of serial dilutions of pooled P. leucopus cDNA (1, 1:10, 1:100, 1:1000) followed by normalization to 18S rRNA gene expression.
Statistical analysis
ANOVA repeated measure tests were used to compare all repeated measures among time points. Two-tailed Student’s t-tests compared treatment groups of interest. Data with unequal variances were log transformed and compared by ANOVA (within-week comparisons for PCR data) or compared using the non-parametric Kruskal–Wallis test (between-week comparisons for PCR data). All comparisons were considered significant when P < 0.05. StatView software was used for all analyses (Cary, NC, USA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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; P < 0.05 in all cases). SDR mice also weighed less than SDNR mice after 2, 4, 10, 12 and 14 weeks of photoperiod treatment (Fig. 1; P < 0.05 in all cases).
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; P < 0.05 in all cases). SDR mice displayed decreased ETV as compared with SDNR mice after 0, 6, 8, 10, 12, 14, 17, 19, 21 and 23 weeks of photoperiod treatment (Fig. 2; P < 0.05 in all cases).
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. Testes and epididymides absolute masses decreased in SDR mice as compared with LD mice after 7, 14 and 21 weeks of photoperiod treatment, whereas absolute seminal vesicles mass decreased after 7, 14, 21 and 34 weeks (P < 0.05 in all cases). Calculation of masses of these tissues after adjusting for body mass yielded similar statistically significant differences except for epididymides mass at 7 weeks and testes mass at 14 weeks (see Table 2; P < 0.05). Absolute and relative epididymal fat pad mass also decreased in SDR mice as compared with LD mice, but only after 14 weeks of treatment (P < 0.05). Additionally, relative seminal vesicles and epididymides masses were decreased in SDR mice after 34 weeks as compared with LD mice (P < 0.05). Both absolute and relative testes and epididymides masses of SDR mice also decreased as compared with SDNR mice after 7, 14 and 21 weeks of photoperiod treatment (P < 0.05 in all cases). Absolute seminal vesicle mass also decreased in SDR as compared with SDNR at all time points (P < 0.05 in all comparisons). Both absolute and relative epididymal fat pad mass decreased in SDR mice as compared with SDNR mice, but only after 14 weeks of treatment (P < 0.05).
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; P < 0.05 for both time points). SDR mice had significantly reduced seminiferous tubule diameters as compared with SDNR mice at 14 weeks (Fig. 3A–D; P < 0.05). Tubule diameter did not differ between LD and SDNR mice at any time point (P > 0.05 in all cases). Additionally, stained nuclei found in the centre of seminiferous tubules of LD and SDNR mice depicted active spermatogenesis in these groups (Fig. 3B and D).
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. Based on the distribution of the fold differences, genes with 6-fold differences among photoperiod treatments were examined throughout testicular regression and recrudescence using qPCR.
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, Tgfßr3, Serpine1 and Tnf was increased in all SD mice at week 7 as compared with LD mice (Fig. 4A–D; P < 0.05). At week 14, only SDR mice had increased Hif1, Tgfßr3 and Tnf gene expression (Fig. 4A, B and D; P < 0.05), whereas expression levels of SDNR mice were similar to LD mice (P >0.05). However, the Serpine1 gene expression relationship among photoperiod groups reversed at 14 weeks such that expression in LD mice was numerically higher than in SDNR mice, although this difference was not statistically significant (Fig. 4C; P = 0.17). At week 21, this increase in Serpine1 expression in LD mice was significantly greater than all SD mice (Fig. 4C; P <0.05). Also at week 21, SDR mice maintained their heightened expression level of Tnf, although it was only significantly higher than SDNR mice (Fig. 4D; P <0.05). SDR and LD mice had similar Tgfßr3 expression levels by week 21, although the SDNR level was significantly decreased (Fig. 4B; P <0.05). Finally, LD mice displayed an increase in Tgfßr3 expression after 34 weeks of photoperiod treatment (Fig. 4B; P <0.05). No differences in Hif1, Serpine1 or Tnf expression existed among groups at 34 weeks (Fig. 4A, C and D; P >0.05).
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expression in LD only (P <0.05). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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, Tgfßr3 and Serpine1 during testicular regression and high expression levels of Tnf during testicular recrudescence. These data are consistent with the hypothesis that photoperiod alters expression of angiogenesis genes in adult males. Angiogenesis in adult mammals has been studied in the context of tumour growth, wound healing and the cyclical changes of the female reproductive tract. Studies of angiogenesis specific to adult males are sparse and are limited to models of seasonal breeders including dormice (G. glis), ferrets (M. furo), and foxes (V. vulpes) (Joffre & Joffre 1973). Dormice and foxes increase capillary blood flow in the testes during the breeding season. Short days decrease blood vessel volume and transfer to long days increases vascular permeability in the testes of Syrian hamsters (M. auratus) (Mayerhofer et al. 1989). Similarly, testicular blood vessel volume of adult male red stags (Cervis elaphus) and camels (Camelus dromedaries) was greater when measured in the breeding season than during the non-breeding season (Hochereau-de Reviers & Lincoln 1978, Zayed et al. 1995). In mink (Mustela vison), the blood–testis barrier competency changes across seasons, although the pattern of competency does not completely match the seasonal progression of spermatogenesis (Pelletier 1986). These studies provide evidence for structural changes in the testicular vasculature of seasonally breeding adult males.
The mechanisms by which testicular vasculature varies across seasons have not been established. However, Vegf may be implicated in this process based on the positive relationship between VEGF protein expression and testes mass in SD and LD white-footed mice (Young & Nelson 2000). No differences (2-fold) in Vegf gene expression were found in this study. However, the time points at which gene expression were assessed are ‘snapshots’ of a 34-week process of regression and recrudescence. It is possible that the differential gene expression necessary for morphological changes in the testes was relatively transient, and therefore missed by our sampling schedule. Additionally, gene transcription and translation of gene products may not overlap in time, and therefore, VEGF protein expression may be high at particular time points whereas, Vegf transcription may have already returned to basal levels. Studies using models in which more abrupt changes in testes mass are measured are necessary to examine changes in angiogenesis gene expression over a more concentrated window of time. Our study implicates several other genes in the process of seasonal angiogenesis of the testes.
Directed by our preliminary microarray findings after 14 weeks of photoperiod treatment (the nadir of testicular regression), we investigated expression patterns of four genes involved in angiogenesis. Hif1 is an oxygen-regulated subunit of the HIF transcription factor that has been implicated in both angiogenesis and apoptosis (Piret et al. 2002). The overlap of the timing of testicular apoptosis in SD white-footed mice and the expression pattern of Hif1 suggests that expression of Hif1 in SD mice may be an indicator of apoptosis associated with testicular regression (Young et al. 2001). Tgfßr3, also known as betaglycan, in its soluble form, is a potent antagonist of TGFß, a promoter of angiogenesis. Additionally, expression of the soluble extracellular domain of TGFßr3 inhibits angiogenesis in vivo and in vitro (Bandyopadhyay et al. 2002). Therefore, it is likely that the initial increase in Tgfßr3 expression in SD mice represents an inhibition of testicular angiogenesis. The effect of Serpine1 on tumour growth and angiogenesis depends on the level of expression. High SERPINE1 doses inhibit tumour growth and angiogenesis in vivo, whereas, low doses increase angiogenesis (McMahon et al. 2001). Consistent with this bi-modal function of SERPINE1, SD mice display high expression levels of Serpine1 at week 7 during regression, which suggests an inhibitory role of Serpine1 on angiogenesis. Lastly, Tnf promotes proliferation and differentiation of mesenchymal cells into pericytes or smooth muscle cells that support newly formed vessels during angiogenesis (Distler et al. 2003). The late increase in Tnf in SD testes suggests that Tnf may promote new vessel stabilization during testicular recrudescence. Overall, the timing of the expression of these four genes in SDR mice suggests that Hif1, Tgfßr3 and Serpine1 are involved in testicular regression, whereas Tnf is involved in testicular recrudescence.
The effect of time (weeks) on gene expression in LD testes was unexpected. We predicted that the vasculature in LD animals would be stable. However, many genes (including the ones examined in the present study) have multiple functions and it is possible that the changes measured in LD testes may be associated with extra-angiogenic processes. Alternatively, the trend of increasing gene expression peaking around week 21 in LD mice may be due to a fluctuation in overall body growth (including the testes). Between 14 and 21 weeks, the LD animals display a slight drop in body mass followed by a recovery that may account for slight changes in testicular gene expression at this time.
In this study, microarrays designed for Mus musculus were used for P. leucopus which may have resulted in false positive and negative findings based on possible sequence differences between the species. Therefore, the microarray analysis was solely used as a screening procedure whereas the specificity and sensitivity of qPCR enabled precise examination of specific genes of interest. Common laboratory animals that have been selectively bred to ignore seasonal cues – such as M. musculus and Rattus norvegicus – and for which molecular tools are readily available, are not suitable for this type of comparative study. Because of the genomic homology among species, however, it is possible to work around some of the molecular roadblocks to address ecologically relevant questions in genetically heterogeneous populations.
The SDNR group represents the population of mice that fails to regress their gonads in response to short day lengths. This population is not merely a laboratory anomaly because winter breeding has been reported in field studies and is thought to confer fitness advantages during mild winters (Prendergast et al. 2001). We compared the SDNR animals to the LD and SDR phenotypes. Body mass, testes size and testes morphology of the SDNR group were more similar to LD than to SD mice. With respect to gene expression of our four candidate genes, the SDNR profile initially (Fig. 4A–D
; week 7) was similar to that of the SDR group. However, by week 14, SDNR expression of all four genes switched to resemble that of the LD mice. These data suggest that there is some breakdown between the transcription of the gene and the function of the gene product. Potential differences between SDNR and SDR post-transcriptional pathways may exist. However, in SDNR mice the uncoupling between large, sperm-filled testes (characteristic of LD animals) and their initial SDR-like gene expression profile suggests that testosterone is not the only factor influencing angiogenesis gene expression in the testes. Additionally, long durations of melatonin secretion do not appear sufficient to induce SDR-like angiogenesis expression profiles in the testes because SDNR and SDR white-footed mice display the same long melatonin durations (Carlson et al. 1989) but different gene expression patterns.
The present study is among the first to describe seasonal plasticity of angiogenesis in adult testes on a molecular level. Changes in angiogenesis gene expression differ between testes of LD and SD white-footed mice and correspond to changes in testicular morphology. The gene expression profile of SDNR mice initially resembled that of SDR mice, but subsequently became similar to LD mice. Future studies are necessary to determine the intervening role of testosterone and melatonin in photoperiod-induced changes in testicular angiogenesis gene expression. It is likely that seasonal melatonin secretion exerts its effects on testicular angiogenesis via an indirect central mechanism that may implicate testosterone. Melatonin receptors have not been localized in the testes in this species and melatonin does not affect testosterone secretion from testicular cells in vitro (Knotts et al. 1988). However, binding of melatonin to receptors in the hypothalamus does modulate reproductive hormone secretion (Glass & Dolan 1988). Therefore, melatonin-induced changes in testosterone secretion may regulate seasonal angiogenesis (or possibly gonadotrophin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) or some combination of these peptides). Because non-pathological angiogenesis in adults is rare (particularly in males) understanding the mechanisms underlying this model of seasonal angiogenesis may prove to have therapeutic value.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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