Testicular Gene Expression Research Articles

Renin mRNA is upregulated in testes and testicular cells in response to treatment with aflatoxin B 1

Aflatoxin B 1 (AFB 1) has been shown to affect fertility in many species; however, the exact molecular mechanisms associated with the disruption are not known. Our objectives were to determine changes in testicular gene expression due to exposure to AFB 1 and to investigate which cell types were affected by treatment with AFB 1. Male mice 4 wk of age were administered a daily placebo (control; N = 9) or 50 μg/kg AFB 1 (AFB 1 treated; N = 10) daily for 45 days. Males were then mated to four females each for 8 days. Male mice were characterized as being “Tolerant” (N = 3) or “Intolerant” (N = 3) to the effects of AFB 1 based on positive terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in the testes and the number of pups sired. Tolerant males produced a similar average number of fetuses (mean ± SEM) (12.5 ± 1.2) per male as selected control males (13.4 ± 1.2), but more fetuses (P = 0.01) than Intolerant males (7.6 ± 1.2). The number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in Intolerant males tended to be (P = 0.10) greater (136.5 ± 27.2) than in Tolerant (55.0 ± 22.2) and selected control (54.3 ± 22.2) males. Affymetrix microarray (Sunnyvale, CA, USA) analysis revealed differential expression (P < 0.05) of 193 extra cellular space and signaling genes, 49 signal transduction genes, 45 immune regulation genes, and 230 cell differentiation genes in the testis. Renin was commonly represented amongst many clusters and was chosen for further analyses. Upregulation (P < 0.001) of Renin in Tolerant mice (N = 3) compared with Intolerant mice (N = 3) was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR) (P = 0.05). This upregulation (P = 0.01) was also observed in representative AFB 1 treated males (N = 8) compared with control males (N = 8) selected for real-time reverse transcription polymerase chain reaction analysis. Spermatogonia cultured in vitro and treated with 0, 5, 10, or 20 μg/mL AFB 1 (N = 6 per treatment) resulted in a 10-fold upregulation (P = 0.01) of Renin message at the 20 μg/mL level, whereas Leydig tumor cells showed similar differences (P = 0.03) in message for Renin in treated (10 and 20 μg/mL) versus control cell cultures. Based on these results, we inferred a role for Renin at the molecular level in the response to the adverse effects of AFB 1 in male mice.

Characterization of testicular expression of P450 17α-hydroxylase, 17,20-lyase in zebrafish and its perturbation by the pharmaceutical fungicide clotrimazole

The aim of the present study was to characterize P450 17α-hydroxylase/17,20-lyase ( cyp17a1) expression in zebrafish and to assess the effect of the pharmaceutical clotrimazole, a known inhibitor of various cytochrome P450 enzyme activities, on testicular gene and protein expression of this enzyme as well as on the testicular release of 11-ketotestosterone (11-KT), a potent androgen in fish. We first showed that cyp17a1 is predominantly expressed in gonads of zebrafish, notably in male. In vivo, clotrimazole induced a concentration-dependent increase of cyp17a1 gene expression and Cyp17-I protein synthesis in zebrafish testis. Using zebrafish testicular explants, we further showed that clotrimazole did not directly affect cyp17a1 expression but that it did inhibit 11-KT release. These novel data deserve further studies on the effect of azole fungicides on gonadal steroidogenesis.

1, 3-dinitrobenzene誘発精巣毒性モデルラットにおける遺伝子発現変動

1, 3-dinitrobenzene誘発精巣毒性モデルラットにおける遺伝子発現変動

The X and Y chromosome in meiosis: how and why they keep silent

The X and Y chromosome in meiosis: how and why they keep silent

Dose-Response Assessment of Fetal Testosterone Production and Gene Expression Levels in Rat Testes Following InUtero Exposure to Diethylhexyl Phthalate, Diisobutyl Phthalate, Diisoheptyl Phthalate, and Diisononyl Phthalate

Several phthalate esters have been linked to the Phthalate Syndrome, affecting male reproductive development when administered to pregnant rats during in utero sexual differentiation. The goal of the current study was to enhance understanding of this class of compounds in the Sprague Dawley (SD) fetal rat following exposure on gestational days (GDs) 14-18 by determining the relative potency factors for several phthalates on fetal testes endpoints, the effects of a nine phthalate mixture on fetal testosterone (T) production, and differences in SD and Wistar (W) strain responses of fetal T production and testicular gene expression to di(2-ethylhexyl) phthalate (DEHP). We determined that diisobutyl phthalate (DIBP) and diisoheptyl phthalate (DIHP) reduced fetal testicular T production with similar potency to DEHP, whereas diisononyl phthalate (DINP) was 2.3-fold less potent. DINP was also less potent at reducing StAR and Cyp11a gene expression levels, whereas DIBP was slightly more potent than DEHP. We observed that administration of dilutions of a mixture of nine phthalates (DEHP, DIHP, DIBP, dibutyl-, benzyl butyl-, dicyclohexyl-, diheptyl-, dihexyl-, and dipentyl phthalate) reduced fetal T production in a dose-dependent manner best predicted by dose addition. Finally, we found that the differential effects of in utero DEHP treatment on epididymal and gubernacular differentiation in male SD and W rats (0, 100, 300, 500, 625, 750, or 875 mg DEHP/kg/day) are likely due to tissue-specific strain differences in the androgen and insl3 signaling pathways rather than differential effects of DEHP on fetal testis T and insl3 production.

Differential expression of non-coding RNAs and continuous evolution of the X chromosome in testicular transcriptome of two mouse species.

BackgroundTight regulation of testicular gene expression is a prerequisite for male reproductive success, while differentiation of gene activity in spermatogenesis is important during speciation. Thus, comparison of testicular transcriptomes between closely related species can reveal unique regulatory patterns and shed light on evolutionary constraints separating the species.Methodology/Principal FindingsHere, we compared testicular transcriptomes of two closely related mouse species, Mus musculus and Mus spretus, which diverged more than one million years ago. We analyzed testicular expression using tiling arrays overlapping Chromosomes 2, X, Y and mitochondrial genome. An excess of differentially regulated non-coding RNAs was found on Chromosome 2 including the intronic antisense RNAs, intergenic RNAs and premature forms of Piwi-interacting RNAs (piRNAs). Moreover, striking difference was found in the expression of X-linked G6pdx gene, the parental gene of the autosomal retrogene G6pd2.Conclusions/SignificanceThe prevalence of non-coding RNAs among differentially expressed transcripts indicates their role in species-specific regulation of spermatogenesis. The postmeiotic expression of G6pdx in Mus spretus points towards the continuous evolution of X-chromosome silencing and provides an example of expression change accompanying the out-of-the X-chromosomal retroposition.

Toxicological effect of emodin in mouse testicular gene expression profile

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a herbal medicine extracted from the rhizomes of Rheum palmatum, and is known as an inhibitor of casein kinase II (CK2). The CK2α' knockout mice are known to be male-infertile; however, there have been no reports on the toxicity of emodin in male reproductive organs/tissues. To evaluate the toxicological effects of emodin on differential gene expression profiles of the testis as compared with acrylamide, mice were orally administered emodin and acrylamide for 5 days at a dose of 1000 and 50 mg kg(-1) per day, respectively, and euthanized 24 h after the final administration. Both chemicals induced hypospermatogenesis, eosinophilic change and apoptosis of germ cell. A DNA microarray analysis showed that the IGF-1 receptor signaling was most closely related to the above testicular toxicity induced by emodin, and the RhoA regulation, TGF/WNT and cytoskeletal remodeling, TNFR1 signaling and adenosine A2A receptor signaling were commonly associated with the two chemicals. We selected 36 genes associated with CK2, apoptosis and spermatogenesis and determined their expression by quantitative reverse transcription-polymerase chain reaction (qPCR). Both chemicals perturbed the expression of genes associated with CK2. Genes related to spermatogenesis were also affected, as evidenced by hypospermatogenesis, and eosinophilic change and apoptosis of germ cell. The results suggest that emodin causes testicular toxicity, including apoptosis with related the IGF-1 receptor signaling pathway, and the two chemicals commonly affect CK2, spermatogenesis and sperm motility via four pathways, such as TNFR1 signaling.

Effects of a mild heat treatment on mouse testicular gene expression and sperm quality

The decrease in sperm quality under heat stress causes a great loss in animal husbandry production. In order to reveal the mechanism underlying the sperm quality decrease caused by heat stress, we first established a mild heat-treated mouse model. Then, the sperm quality was identified. Further, the testicular proteome profile was mapped and compared with the control using 2D electrophoresis and mass spectrometry. Finally, the differential expressed proteins involved in the heat stress response were identified by real-time PCR and Western blotting. The results showed that heat stress caused a significant reduction in mouse sperm quality (P<0.05). Further, 52 protein spots on the 2D gel were found to differ between the heat-shocked tissues and the control. Of these spots, some repair proteins which might provide some explanation for the influence on sperm quality were found. We then focused on Bag-1, Hsp40, Hsp60 and Hsp70, which were found to be differently expressed after heat shock (P<0.05). Further analysis in this heat-shocked model suggests numerous potential mechanisms for heat shock-induced spermatogenic disorders.

Plasticity of the Reproductive Axis Caused by Social Status Change in an African Cichlid Fish: II. Testicular Gene Expression and Spermatogenesis

Reproduction in all vertebrates is controlled by the brain-pituitary-gonad (BPG) axis, which is regulated socially in males of the African cichlid fish Astatotilapia burtoni. Although social information influences GnRH1 neurons at the apex of the BPG axis, little is known about how the social environment and dominance affects the cellular and molecular composition of the testes to regulate reproductive capacity. We created an opportunity for reproductively suppressed males to ascend in status and then measured changes in gene expression and tissue morphology to discover how quickly the perception of this opportunity can influence the testes. Our results show rapid up-regulation of mRNA levels of FSH receptor and several steroid receptor subtypes in the testes during social ascent. In contrast, LH receptor was not elevated until 72 h after ascent, but this increase was coincident with elevated circulating androgens and early stages of spermatogenesis, suggesting a role in steroidogenesis. The spermatogenic potential of the testes, as measured by cellular composition, was also elevated before the overall increase in testes size. The presence of cysts at all stages of spermatogenesis, coupled with lower levels of gonadotropin and steroid receptors in subordinate males, suggests that the BPG axis and spermatogenesis are maintained at a subthreshold level in anticipation of the chance to gain a territory and become reproductively active. Our results show that the testis is stimulated extremely quickly after perception of social opportunity, presumably to allow suppressed males to rapidly achieve high reproductive success in a dynamic social environment.

Waterborne fluoxetine disrupts the reproductive axis in sexually mature male goldfish, Carassius auratus

Fluoxetine (FLX) is a pharmaceutical acting as a selective serotonin reuptake inhibitor and is used to treat depression in humans. Fluoxetine and the major active metabolite norfluoxetine (NFLX) are released to aquatic systems via sewage-treatment effluents. They have been found to bioconcentrate in wild fish, raising concerns over potential endocrine disrupting effects. The objective of this study was to determine effects of waterborne FLX, including environmental concentrations, on the reproductive axis in sexually mature male goldfish. We initially cloned the goldfish serotonin transporter to investigate tissue and temporal expression of the serotonin transporter, the FLX target, in order to determine target tissues and sensitive exposure windows. Sexually mature male goldfish, which showed the highest levels of serotonin transporter expression in the neuroendocrine brain, were exposed to FLX at 0.54 μg/L and 54 μg/L in a 14-d exposure before receiving vehicle or sex pheromone stimulus consisting of either 4.3 nM 17,20β-dihydroxy-4-pregnene-3-one (17,20P) or 3 nM prostaglandin F 2α (PGF 2α). Reproductive endpoints assessed included gonadosomatic index, milt volume, and blood levels of the sex steroids testosterone and estradiol. Neuroendocrine function was investigated by measuring blood levels of luteinizing hormone, growth hormone, pituitary gene expression of luteinizing hormone, growth hormone and follicle-stimulating hormone and neuroendocrine brain expression of isotocin and vasotocin. To investigate changes at the gonadal level of the reproductive axis, testicular gene expression of the gonadotropin receptors, both the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were measured as well as expression of the growth hormone receptor. To investigate potential impacts on spermatogenesis, testicular gene expression of the spermatogenesis marker vasa was measured and histological samples of testis were analyzed qualitatively. Estrogen indices were measured by expression and activity analysis of gonadal aromatase, as well as liver expression analysis of the estrogenic marker, esr1. After 14d, basal milt volume significantly decreased at 54 μg/L FLX while pheromone-stimulated milt volume decreased at 0.54 μg/L and 54 μg/L FLX. Fluoxetine (54 μg/L) inhibited both basal and pheromone-stimulated testosterone levels. Significant concentration-dependent reductions in follicle-stimulating hormone and isotocin expression were observed with FLX in the 17,20P- and PGF 2α-stimulated groups, respectively. Estradiol levels and expression of esr1 concentration-dependently increased with FLX. This study demonstrates that FLX disrupts reproductive physiology of male fish at environmentally relevant concentrations, and potential mechanisms are discussed.

Cadmium alters testicular expression of genes regulating the structure and function of the actin cytoskeleton

OBJECTIVE: Actin dynamics affects cell cycle progression, cell junction function, spermiogenesis and spermiation. Actin immunoreactivity is low in testis of infertile men with varicocele. Varicocele actin loss may lead to sloughing of germ cells, increased apoptosis, and abnormal sperm head shape. Testes cadmium (Cd) is elevated in infertile men with varicoceles. Athough Cd can directly disassemble and degrade actin, it may also reduce actin by altering gene expression. In this study, we asked if Cd changed actin-related gene expression in mammalian testes.

Suppression of Radiation-Induced Testicular Germ Cell Apoptosis by 2,5-Hexanedione Pretreatment. II. Gene Array Analysis Reveals Adaptive Changes in Cell Cycle and Cell Death Pathways

Sertoli cells are essential for testicular germ cell maintenance and survival. We made the unexpected observation that x-radiation (x-ray)-induced germ cell loss is attenuated by co-exposure with the Sertoli cell toxicant 2,5-hexanedione (HD). The mechanisms underlying this attenuation of germ cell apoptosis with reduced Sertoli cell support are unknown. The current study was performed to examine alterations in testicular gene expression with co-exposure to HD and x-ray. Adult male rats were exposed to HD (0.33 or 1%) in the drinking water for 18 days followed by x-ray (2 or 5 Gy), resulting in nine treatment groups. Testis samples were collected after 3 h and total messenger RNA was analyzed using Affymetrix Rat Genome 230 2.0 arrays. Normalized log2-expression values were analyzed using LIMMA and summarized using linear contrasts designed to summarize the aggregated effect, in excess of x-ray alteration, of HD across all treatment groups. These contrasts were compared with the overall linear trend expression for x-ray, to determine whether HD effects were agonistic or antagonistic with respect to x-ray damage. Overrepresentation analysis to identify biological pathways where HD modification of gene expression was the greatest was performed. HD exerted a significant influence on genes involved in cell cycle and cell death/apoptosis. The results of this study provide insight into the mechanisms underlying attenuated germ cell toxicity following HD and x-ray co-exposure through the analysis of co-exposure effects on gene expression, and suggest that HD pre-exposure reduces Sertoli cell supported germ cell proliferation thereby reducing germ cell vulnerability to x-rays.

Effects of Moderate Calorie Restriction on Testosterone Production and Semen Characteristics in Young Rhesus Macaques (Macaca mulatta)1

We have previously reported a modest influence of moderate calorie restriction (CR) on testicular gene expression in young adult rhesus macaques (Macaca mulatta); however, it is unclear if these modifications correspond to subsequent changes in testicular function or sperm physiology. This study extends our earlier findings to examine potential physiological differences due to this differential gene expression. Animals were subjected to 30% CR (CR, n = 5) or were fed a standard control diet (CON, n = 5) starting during their peripubertal period. Circulating testosterone (T) levels were measured across a 24-h period after 7 yr of dietary treatment and were found to be similar in CR and CON males; however, maintenance of daily minimum T levels was significantly higher in the CR animals. Semen collection was performed on the same cohort of animals three times per male (CR, n = 4; CON, n = 4) after 8 yr of treatment, and samples were assessed by a variety of measures. Parameters, including semen quality and sperm cell viability and function, showed less variability in semen samples taken from CR males, but overall testicular function and sperm quality were comparable regardless of diet. There is mounting evidence that CR may promote health and longevity in a wide range of organisms, including nonhuman primates. Importantly, our data suggest that moderate CR has no obvious lasting detrimental effect on testicular function and sperm parameters in young adult primates and may in fact help maintain higher levels of circulating T.

Testis transcriptome analysis in male infertility: new insight on the pathogenesis of oligo-azoospermia in cases with and without AZFc microdeletion

BackgroundAbout 10% of cases of male infertility are due to the presence of microdeletions within the long arm of the Y chromosome (Yq). Despite the large literature covering this critical issue, very little is known about the pathogenic mechanism leading to spermatogenesis disruption in patients carrying these microdeletions. In order to identify the presence of specific molecular pathways leading to spermatogenic damage, testicular gene expression profiling was carried out by employing a microarray assay in 16 patients carrying an AZFc microdeletion or affected by idiopathic infertility. Hierarchical clustering was performed pooling the data set from 26 experiments (16 patients, 10 replicates).ResultsAn intriguing and unexpected finding is that all the samples showing the AZFc deletion cluster together irrespectively of their testicular phenotypes. This cluster, including also four patients affected by idiopathic infertility, showed a downregulation of several genes related to spermatogenesis that are mainly involved in testicular mRNA storage. Interestingly, the four idiopathic patients present in the cluster showed no testicular expression of DAZ despite the absence of AZFc deletion in the peripheral blood.ConclusionsOur expression profiles analysis indicates that several forms of infertility can be triggered by a common pathogenic mechanism that is likely related to alterations in testicular mRNA storage. Our data suggest that a lack of testicular DAZ gene expression may be the trigger of such mechanism. Furthermore, the presence of AZFc deletions in mosaic or the loss of function of AZFc genes in absence of Yq deletion can perhaps explain these findings. Finally, based on our data, it is intriguing to hypothesize that DAZ gene dysfunctions can account for a larger number of previously thought "idiopathic" infertility cases and investigation of such testicular gene dysfunction can be important to reveal the molecular determinant of infertility than are undetected when only testing Yq deletions in peripheral blood.

Early effects of Sertoli cell‐selective androgen receptor ablation on testicular gene expression

Evidence from several models of hormone depletion and/or replacement and from knockout animals points to a key role of androgens in the control of spermatogenesis. In testes of mice with a Sertoli cell-selective ablation of the androgen receptor (SCARKO), transcriptional profiling, using microarray technology, revealed that, already on postnatal day 10,692 genes are differentially expressed compared with testes of control mice. Further evaluation of a subset of these genes by quantitative RT-PCR suggested that differences in expression may already be evident on day 8 or earlier. As the androgen receptor in mouse Sertoli cells becomes immunologically detectable around day 5, we tried to identify the earliest responses to androgens by a new transcriptional profiling study on testes from 6-day-old SCARKO and control mice. No obvious and novel early androgen response genes, potentially acting as mediators of subsequent indirect androgen actions, could be identified. However, several genes differentially expressed on day 10 already displayed a response to androgen receptor ablation on day 6. Quantitative RT-PCR studies for 12 of these genes on 10 paired SCARKO and control testes from 4-, 6-, 8-, 10-, 20- and 50-day-old mice revealed significant differences in expression level from day 4 onwards for three genes (Eppin, PCI, Cldn11) and from day 6 onwards for one more gene (Rhox5). For at least two of these genes (Rhox5 and Eppin), there is evidence for direct regulation via the androgen receptor. For three additional genes (Gpd1, Tubb3 and Tpd52l1) significantly lower expression in the SCARKO was noted from day 8 onwards. For all the studied genes, an impressive increase in transcript levels was observed between day 4-50 and differential expression was maintained in adulthood. It is concluded that the SCARKO model indicates incipient androgen action in mouse Sertoli cells from day 4 onwards.

Examination of Testicular Gene Expression Patterns in Yorkshire Pigs with High and Low Levels of Boar Taint

Boar taint refers to the objectionable odor and flavor in meat of some uncastrated male pigs, which is primarily due to high levels of androstenone, a steroid produced in the testis, and 3-methylindole (skatole) which is produced by bacterial degradation of tryptophan in the intestinal tract. We determined testicular gene expression patterns of Yorkshire pigs with high and low levels of boar taint using swine DNA microarrays with two-color hybridization. The microarrays contained 19486 annotated probes; the expressions of 8719 genes were detected. Fifty-three genes were significantly up-regulated in the high boar taint group and four were significantly down-regulated (p < 0.05; fold change > ±1.55). Gene ontology (GO) analysis short-listed 11 significant GO terms (p < 0.05), most of which are associated with steroid metabolism and mitochondrial components. Comparing the results of this study with published work on Duroc and Norwegian Landrace boars,1 eleven genes (HSB17B4, FDX1, CYP11A1, DHRS4, PRDX1, CYB5, CYP17A1, FTL, IDI1, SULT2A1, and RDH12) were over-expressed in all three breeds with a high androstenone level. The current findings confirmed a number of candidate genes identified in previous functional studies and suggest several new genes differentially expressed with different levels of boar taint.

The effects of an in utero exposure to 2,3,7,8‐tetrachloro‐dibenzo‐p‐dioxin on male reproductive function: identification of Ccl5 as a potential marker

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like compounds are widely encountered toxic substances suspected of interfering with the endocrine systems of humans and wildlife, and of contributing to the loss of fertility. In this study, we determined the changes in testicular gene expression caused by in utero exposure to TCDD along with the intra-testicular testosterone levels, epididymal sperm reserves, daily sperm production (DSP) and testis histology. To this purpose, female pregnant Sprague–Dawley rats orally received TCDD (10, 100 or 200 ng/kg body weight) or vehicle at embryonic day 15, and the offspring was killed throughout development. Hepatic Cyp1a1 gene expression was measured in the offspring to confirm the exposure to TCDD. The gross histology of the testes and intra-testicular testosterone levels were normal among the studied groups. Sperm reserves were altered in 67-day-old rats of the TCDD-200 group, but not in 145-day-old animals or in the other TCDD-exposed groups. Nonetheless, fertility was not altered in males of the TCDD-200 group, and the F2 males generated had normal sperm reserves and DSP. Microarray analysis permitted the identification of eight differentially expressed genes in the 4-week-old testes of the TCDD-200 compared with that of the control group (cut-off value ± 1.40), including the down-regulated chemokine Ccl5/Rantes. Inhibition of Ccl5/Rantes gene expression was observed throughout development in the TCDD-200 group, and at 67 and 145 days in the TCDD-100 group (animals of younger ages were not examined). Ccl5/Rantes gene expression was mostly confined in Leydig cells. F2 males generated from males of the TCDD-200 group had normal levels of Ccl5/Rantes in testis and Cyp1a1 in liver, which might indicate that Ccl5/Rantes is a marker of TCDD exposure in testis such as Cyp1a1 in liver. In conclusion, we demonstrated a decrease in Ccl5/Rantes RNA levels and a transitory decline in sperm reserves in the testes of rats of TCDD-dosed dams.

Impact of Moderate Calorie Restriction on the Reproductive Neuroendocrine Axis of Male Rhesus Macaques.

The impact of moderate calorie restriction on reproductive neuroendocrine function was investigated in young adult male rhesus macaques (Macaca mulatta). The animals were subjected to either 30% calorie restriction (CR; n=5), or were fed a standard control diet (CON; n=5), starting during their peripubertal period. Plasma LH and testosterone concentrations were examined after 7 years of differential dietary treatment, and were found to be similar in both groups, both during the day and during the night. Microarray profiling of pituitary gland and testicular gene expression was performed after 8 years of treatment, using GeneChip® Rhesus Macaque Genome Arrays (Affymetrix), and showed very little effect of caloric restriction. Using a 1.5-fold difference threshold, our microarray analysis revealed differential expression of only 145 probesets in the pituitary gland and 260 in the testes, out of a total of >54,000. Semi-quantitative RT-PCR performed on pituitary gland mRNA corroborated the microarray findings for selected modulated genes, including TSH receptor (TSHR) and sperm-specific antigen 2 (SSFA2). Most notably, significantly lower expression of TSH receptor mRNA was observed in the pituitary of CR compared to CON animals. Also, significantly lower expression of the glycoprotein hormone alpha subunit (CGA) was observed in CR animals, and this finding was further corroborated using quantitative real-time RT-PCR. No significant diet-induced changes were detected in the testis for genes associated with reproduction, circadian clocks, or oxidative stress. There is mounting evidence that CR may promote health and longevity in a wide range of organisms, including nonhuman primates. Importantly, our data suggest that moderate CR has no obvious lasting detrimental effect on the reproductive neuroendocrine axis of long-lived primates, and has only a modest influence on pituitary and testicular gene expression.

Fhl5/Act, a CREM-binding transcriptional activator required for normal sperm maturation and morphology, is not essential for testicular gene expression

BackgroundThe LIM domain protein Fhl5 was previously found to interact with CREM, a DNA binding transcriptional regulator necessary for spermiogenesis in mammals. Co-transfection experiments using heterologous promoter constructs indicated a role for Fhl5 in transcriptional up-regulation of CREM-dependent testicular genes. Male mice lacking Fhl5 were reported to be fertile but displayed partially abnormal sperm maturation and morphology.MethodsTo identify Fhl5 testicular target genes we carried out two whole-genome expression profiling experiments using high-density oligonucleotide microarrays and total testis samples from Fhl5 wild-type versus homozygous mutant mice first in different and then in isogenic strain backgrounds.ResultsWeak signal differences were detected in non-isogenic samples but no statistically significant expression changes were observed when isogenic Fhl5 mutant and wild-type samples were compared.ConclusionThe outcome of these experiments suggests that testicular expression profiling is extremely sensitive to the genetic background and that Fhl5 is not essential for testicular gene expression to a level detected by microarray-based measurements. This might be due to redundant function of the related and similarly expressed protein Fhl4.

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH

In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n=3) and cut into slices (mean slice weight= 13.85 ± 0.20 mg; mean slice thickness=515.00 ± 2.33 μm) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50, and 500 ng/ml for 6 h at 32 °C. Viability of the tissue was maintained based on histological integrity and lack of appreciable lactate dehydrogenase in the medium. The production and release of testosterone (T) and estradiol-17β (E2) into the medium was measured following in vitro culture. The addition of oLH increased T and E2 at least 400% and 120%, respectively, over the 0-ng oLH control cultures. Testicular gene expression was assessed with in situ hybridization methodology for steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs. In situ hybridization revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA in Leydig cells. No differences were observed for the expression of PDE3B or ODF2 genes in seminiferous tubules among treatment groups as expected. These results demonstrate the value of in vitro culture of the precision-cut tissue slices for studies of the regulation of steroidogenesis and gene expression in the stallion testes.