Canine Testis Research Articles

Ultrasonography of Testicular Maturation and Correlation with Body Growth and Semen Evaluation in Beagle Dog Model.

This prospective study investigated the ultrasonographic appearance of the canine testis from birth to adulthood. Eight purpose-bred laboratory Beagle-breed dogs were monitored from 4 to 40 weeks of life. The following parameters were evaluated every two weeks: bodyweight and height, scrotal and testicular volume, ultrasonographically measured testicular volume, echogenicity, heterogeneity, blood-flow score, ratio of the grayscale intensity value of the testis to the capsule, ejaculate volume, motility, viability, and number of spermatozoa. A correlation analysis was carried out between the various measurements obtained. Fertility was achieved in the 36th week of life. The echogenicity of the testicular parenchyma increased with age, and subsequently to the 30th week of life remained constant. The heterogeneity of the testicular parenchyma, as was evaluated by the standard deviation of the values of grayscale intensity of the parenchyma, also increased with age and was >19 at the onset of fertility. The ratio of grayscale intensity of testicular parenchyma had values < 200 at maturity. A colour Doppler evaluation first detected blood flow in the testis in the 22nd week. After the 32nd week, distinct signals were visible. In the 36th week, >80% of the testes imaged had visible vessels. A significant correlation was found between all the evaluation methods. The findings of the study may help clinicians detect the onset of fertility in dogs, especially when semen evaluation is not feasible; however, their applicability in all breeds or individuals might possibly vary due to genetic, physiological, and developmental differences. In summary, the study ultrasonographically explores the testicular maturity in dogs, with the aim to improve clinical assessments and health management in these animals.

In Silico Analysis of miRNA-Mediated Genes in the Regulation of Dog Testes Development from Immature to Adult Form.

High-throughput in-silico techniques help us understand the role of individual proteins, protein-protein interaction, and their biological functions by corroborating experimental data as epitomized biological networks. The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. Differentially expressed (DE) canine testis miRNAs between healthy immature (2.2 ± 0.13 months; n = 4) and mature (11 ± 1.0 months; n = 4) dogs were utilized in this investigation. In silico analysis was performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The results showed protein-protein interaction for the upregulated miRNAs, which revealed 978 enriched biological processes GO terms and 127 KEGG enrichment pathways, and for the down-regulated miRNAs revealed 405 significantly enriched biological processes GO terms and 72 significant KEGG enrichment pathways (False Recovery Rate, p < 0.05). The in-silico analysis of DE-miRNA's associated genes revealed their involvement in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes' specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control.

Integrated miRNA Changes in Canine Testis and Epididymis According to Age and Presence of Cryptorchidism.

In the present study, we aimed to investigate age-, cryptorchidism-, and testicular tumor-related changes in miRNAs in the testis and epididymis of dogs. Twelve healthy male dogs were divided into two groups: young (<1 year, n = 8) and old (>3 years, n = 4). Five dogs with unilateral cryptorchidism, one with a Sertoli cell tumor, and one with seminoma were referred to a veterinary hospital. After surgery, the testes and epididymis tails were collected. A high-throughput miRNA array analysis was performed to identify miRNAs affected by age, cryptorchidism, and testicular tumors. The expression of only cfa-miR-503 was downregulated in the epididymis of younger dogs, whereas the expression of 64 miRNAs was upregulated. Among them, the top five miRNAs were cfa-miR-26a, cfa-miR-200c, cfa-let-7c, cfa-let-7b, and cfa-let-7a. The expression of cfa-miR-148a and cfa-miR-497 was considerably lower in cryptorchid testis than in healthy dog testis. In the epididymis, the cfa-miR-1841 level was significantly decreased. We observed a significant difference in the expression of 26 cfa-miRNAs between testicular tumors and normal tissues. This study demonstrated that aging and cryptorchidism have a causal relationship with miRNA expression. The identified miRNAs may be candidate genes for male reproductive traits and could be applied in molecular breeding programs.

Testicular Expression of Antioxidant Enzymes and Changes in Response to a Slow-Release Deslorelin Implant (Suprelorin® 4.7 mg) in the Dog.

Simple SummaryWe investigated the expression of the testicular antioxidant enzyme system, i.e., superoxide dismutase (SOD1 and 2), catalase (CAT), glutathione peroxidase (GPx1), and glutathione disulfide reductase (GSR), in healthy adult and prepubertal dogs and after slow-release GnRH superagonist deslorelin (Suprelorin® 4.7 mg subcutaneous implant, Virbac, France) treatment in adult dogs. qPCR and immunohistochemistry were used to detect gene expression and protein expression and localization, respectively. These antioxidant enzymes were variably expressed within cellular compartments in the seminiferous epithelium and the interstitium. SOD1, CAT, and GSR protein expression were also dependent on the stage of the seminiferous epithelium cycle in normal adult dogs. Deslorelin treatment and prepubertal status affected the expression and localization of the investigated antioxidant enzymes. Our results confirmed the presence of a functional antioxidant enzyme system in the canine testis, which was also observed during deslorelin-induced spermatogenic and steroidogenic arrest, as well as in prepubertal dogs. These results improve our current understanding of the local antioxidant protective mechanisms within the testis in the dog. Spermatogenesis takes place in a hypoxic environment, and antioxidant enzymes protect germ and somatic cells from free radical-mediated damage. Expression of the antioxidant enzyme system in the canine testis has not yet been investigated. We hypothesized that the slow-release GnRH superagonist deslorelin 4.7 mg implant, which induces temporary reversible suppression of endocrine and germinative testicular function, would affect the testicular expression of antioxidant enzymes compared to untreated adult and prepubertal dogs. The goal of this study was to investigate and compare gene (by qPCR, in whole-tissue homogenates) and protein expression (by immunohistochemistry) of superoxide dismutase (SOD1, SOD2), catalase (CAT), glutathione peroxidase (GPx1), and glutathione disulfide reductase (GSR) in the testes of untreated adult (CON, n = 7), prepubertal (PRE, n = 8), and deslorelin-treated (DES, n = 5, 16 weeks after implantation) dogs. We found that in DES dogs, the gene expression of SOD1 was significantly (p < 0.05) lower and GPx1 was higher than in CON, and SOD2 was higher than in PRE. Expression of all, except for the SOD2 mRNA, differed between the CON and PRE dogs. Immunohistochemistry showed distinct cell-specific localization and expression patterns for the antioxidant enzymes in each experimental group. Additionally, in the CON animals, cell-specific SOD1, CAT, and GSR expression was dependent on the stage of the seminiferous epithelium cycle. These findings confirm that members of the antioxidant enzyme system are present in normal adult and prepubertal testis as well as in the deslorelin-treated downregulated adult canine testis, and that this local antioxidant system protects developing germ cells and somatic cells from oxidative damage. Different expression patterns of antioxidant enzymes in various germ cell populations and stages of the seminiferous epithelium cycle may indicate differences in their susceptibility to oxidative stress depending on their developmental and maturation stage. The continued presence of the antioxidant enzymes in the testis of DES dogs offers protection to spermatogonia as well as Sertoli and Leydig cells from oxidative stress during temporary infertility, potentially contributing to ensure the reversibility of suppression and the return of normal spermatogenesis and steroidogenesis after the end of deslorelin treatment.

Sertoli, Leydig, and Spermatogonial Cells' Specific Gene and Protein Expressions as Dog Testes Evolve from Immature into Mature States.

Simple SummarySertoli-, Leydig-, and spermatogonial-cells proliferate and differentiate from birth to puberty and then stay stable in adulthood. We hypothesized that expressions of spermatogenesis-associated genes are not enhanced with the increase of these cells’ numbers. To accomplish this postulation, we investigated the abundances of Sertoli cell-specific FSHR and AMH, Leydig cell-specific LHR and INSL3, and spermatogonia-specific THY1 and CDH1 markers in immature and mature canine testis. Four biological replicates of immature and mature testes were processed, and RT-PCR was performed to elucidate cells’ specific markers. Results showed that the gene expressions of all the studied cells’ specific markers were downregulated in adult testis compared with immature testis. Western blot and immunohistochemistry showed the presence of these proteins in the testis. Protein expressions of these markers were greater in immature compared with mature testis. The results support the postulation that the gene expressions do not directly correlate with the increase of the cell numbers during post-natal development but changes in gene expressions show functional significance.Sertoli, Leydig, and spermatogonial cells proliferate and differentiate from birth to puberty and then stay stable in adulthood. We hypothesized that expressions of spermatogenesis-associated genes are not enhanced with a mere increase of these cells’ numbers. To accept this postulation, we investigated the abundances of Sertoli cell-specific FSHR and AMH, Leydig cell-specific LHR and INSL3, and spermatogonia-specific THY1 and CDH1 markers in immature and mature canine testis. Four biological replicates of immature and mature testes were processed, and RT-PCR was performed to elucidate the cells’ specific markers. The data were analyzed by ANOVA, using the 2−∆∆Ct method to ascertain differences in mRNA expressions. In addition, Western blot and IHC were performed. Gene expressions of all the studied cells’ specific markers were down-regulated (p < 0.05) in adult testis compared with immature testis. Western blot and immunohistochemistry showed the presence of these proteins in the testis. Protein expressions were greater in immature testis compared with mature testis (p < 0.05). Despite the obvious expansion of these cells’ numbers from immature to adult testis, the cells’ specific markers were not enriched in mature testis compared with immature dog testis. The results support the postulation that the gene expressions do not directly correlate with the increase of the cell numbers during post-natal development but changes in gene expressions show functional significance.

Canine DUXC: implications for DUX4 retrotransposition and preclinical models of FSHD.

Mis-expression of DUX4 in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). Human DUX4 and mouse Dux are retrogenes derived from retrotransposition of the mRNA from the parental DUXC gene. Primates and rodents have lost the parental DUXC gene, and it is unknown whether DUXC had a similar role in driving an early pluripotent transcriptional program. Dogs and other Laurasiatherians have retained DUXC, providing an opportunity to determine the functional similarity to the retrotransposed DUX4 and Dux. Here, we identify the expression of two isoforms of DUXC mRNA in canine testis tissues: one encoding the canonical double homeodomain protein (DUXC), similar to DUX4/Dux, and a second that includes an in-frame alternative exon that disrupts the conserved amino acid sequence of the first homeodomain (DUXC-ALT). The expression of DUXC in canine cells induces a pluripotent program similar to DUX4 and Dux and induces the expression of a similar set of retrotransposons of the ERV/MaLR and LINE-1 families, as well as pericentromeric satellite repeats; whereas DUXC-ALT did not robustly activate gene expression in these assays. Important for preclinical models of FSHD, human DUX4 and canine DUXC show higher conservation of their homeodomains and corresponding binding motifs compared with the conservation between human DUX4 and mouse Dux, and human DUX4 activates a highly similar transcriptional program in canine cells. Together, these findings show that retrotransposition resulted in the loss of an alternatively spliced isoform and that DUXC containing mammals might be good candidates for certain preclinical models ofFSHD.

Reduced translation efficiency due to novel splicing variants in 5\u2032 untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2

BackgroundBreast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination.ResultsIn this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1.ConclusionsThis study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

Senescence and adiponectin signaling — Studies in canine testis

BackgroundThe meaning of senescence for tissue physiological and pathological conditions is poorly known. Based on initial reports especially proteins and mechanisms that regulate this process are necessary to be determinate. MethodsThe main aim of the study was to investigate the presence of senescent cells in canine testicular tissue (mixed breed testes; n = 60) in relation to adiponectin signaling. In detail, new information on the senescence cell number, as well as senescence and adiponectin signaling mechanisms in cryptorchid and germ cell tumor testes were provided with the use of immunohistochemical and colorimetric analyses. ResultsComparison of immunohistochemical results, in cryptorchid and tumor testes revealed increased number of senescent cells (p16 and γH2AX markers). Increased expression of adiponectin and adiponectin receptor 1, as well as extracellular signal-activated kinase (ERK1/2) in pathological testes were detected. In addition, decreased cholesterol and increased testosterone levels in tumor testis were found. ConclusionThe present study is the first to demonstrate the presence as well as the differences that exist in senecent cell number in mixed breed dog testes with cryptorchidism and germ cell tumor. Altered expression of adiponectin signaling and ERK1/2 signaling pathways together with altered cholesterol and testosterone levels reflect important senescence role in disturbed functions of canine testis. Moreover, the application of studied here senescence regulating molecules for detection and prevention against pathologies of the male gonad should be furtherly considered.

Pulse-wave Doppler pulsatility and resistive indexes of the testicular artery increase in canine testis with abnormal spermatogenesis

Pulse-wave Doppler (PWD) ultrasonography of the testicular artery was recently used to compare parameters in fertile and infertile males, or males with normal and abnormal semen quality, while no studies verified the concomitant histological feature of the testis. The experimental hypothesis of the present study was that the blood flow parameters measured by PWD at different levels of the testicular artery reflect sperm attributes evaluated on spermatozoa collected from the tail of the epididymis and testicular histological characteristics. Pulse-wave Doppler parameters were recorded on both testes from 16 dogs of unknown fertility. After routine castration, epididymal spermatozoa were collected and evaluated for sperm total and progressive motility, membrane integrity, and sperm morphology. Testes were submitted to histological and immunohistochemical evaluations to quantify microscopic attributes and connective tissue and vascular area extent, respectively. The findings showed that the increased PWD indexes were suggestive of testicular histological alteration since negative correlations were found between pulsatility index and resistive index, measured in the marginal testicular artery, and histological attributes such as the seminiferous epithelial thickness (R = −0.528, P = 0.016 and R = −0.554, P = 0.005, respectively) and the tubular area/total area ratio (R = −0.494, P = 0.024 and R = −0.563, P = 0.004, respectively). Some histological characteristics, such as tubular diameter, seminiferous epithelium thickness, and the tubular area/total area ratio of the testis are correlated with the percentage of primary abnormalities (R = −0.874, P = 0.001, and R = −0.847, P = 0.001, and R = - 0.865, P = 0.001, respectively). In testes with increased sperm primary abnormalities, higher pulsatility index was found compared with testes with normal spermatogenesis in supratesticular artery (1.07 ± 0.06 vs 0.82 ± 0.23, P < 0.05), marginal artery (0.73 ± 0.04 vs 0.61 ± 0.07, P < 0.05), and intratesticular artery (0.67 ± 0.03 vs 0.43 ± 0.1, P < 0.05). Similarly, the resistive index was found significantly higher in the same vessel tracts in testes with abnormal and normal spermatogenesis (0.62 ± 0.02, 0.53 ± 0.02, and 0.46 ± 0.02 vs 0.51 ± 0.06, 0.44 ± 0.04, and 0.32 ± 0.06, respectively; P < 0.05). Our data demonstrated for the first time the relationship between PWD indexes and the contextual testicular architecture, suggesting that the pulsatility and resistive indexes could be an effective tool in andrology. In light of different histological alterations in the testis, caution is warranted in the definition of thresholds for normal and abnormal testicular blood flow indexes.

Expression of claudin-11 in canine prepubertal testes, and in canine adult testes showing normal spermatogenesis, impaired spermatogenesis, or testicular neoplasia

The blood-testis barrier (BTB) consists of different cell-to-cell connections, including tight junction proteins like claudin-11 (CLDN11). For dogs, only limited data is published dealing with these proteins in general. Therefore, their physiological relevance, their postnatal expression, and their distribution pattern in pathological conditions, e.g. in altered spermatogenesis and testicular neoplasia were assessed. Canine testes from routine castrations, and those sent in for diagnostic purposes were investigated. Based on morphological evaluation, the dogs and testes were divided into groups: (1) dogs with normal spermatogenesis, (2) four months old prepubertal dogs, (3) intratubular seminoma, (4) diffuse seminoma, (5) Sertoli cell tumours (SCT), (6) Leydig cell tumours (LCT), and (7) dogs with impaired spermatogenesis (e.g. mixed atrophy). In order to examine possible alterations of the BTB components, immunohistochemistry (IHC) and immunofluorescence using a commercial antibody against CLDN11 was performed. Sertoli cell (SC) nuclei (SOX9) and peritubular myoid cells (smooth-muscle-actin, SMA) were also assessed using IHC. Additionally, semi-quantitative Western-blot (WB) and RT-PCR analyses of CLDN11 were conducted. In tubules with normal spermatogenesis, IHC of CLDN11 revealed a basolateral staining at BTB localisation. In prepubertal cords, CLDN11 was diffusely expressed along the cytoplasmic extensions of SCs supposing that the BTB was neither built up nor functional, yet. A shift from weakly expressed CLDN11 between/in residual SCs in intratubular seminoma to only small CLDN11 immunopositive stained spots in the cytoplasm of remaining SOX9-positive SCs in diffuse seminoma was detectable. Reduction or even loss of CLDN11 expression in diffuse seminoma was confirmed using RT-PCR and WB analyses, thus indicating that in seminoma, CLDN11 was downregulated at transcriptional level and completely lost its sealing function. Basal SCs in SCT still showed a CLDN11/SOX9 co-localisation, suggesting that luminal neoplastic SCs undergo de-differentiation during tumour progression. In LCT, no CLDN11 was detectable. Dogs with mixed atrophy showed an upregulation of CLDN11 in tubules with spermatogenic arrest on mRNA and protein level, leading to the conclusion that within these tubules regulatory mechanisms lost their equilibrium. For the first time, the spatial expression of CLDN11 in prepubertal canine testis, impaired spermatogenesis, intratubular seminoma and its absence in diffuse seminoma and LCT was shown. Since altered CLDN11 levels could be part of adaptive mechanisms to modify BTB integrity, further functional investigations to characterize the canine BTB need to be conducted.

Expression of prostaglandin (PG) D synthase lipocalin and hematopoietic type and PG D receptor during restart of spermatogenesis following downregulation using a slow release GnRH agonist implant in the dog.

Prostaglandin D and the associated prostaglandin D synthases (PGDS) and receptor (DP) are considered to be involved in spermatogenesis. However, the interplay of the PGDS-DP system in male reproduction is far from being understood. The expression of PGDS lipocalin (L) and hematopoietic (H) type and DP was studied in the GnRH agonist-downregulated canine testis (week, w 0) and during recrudescence of spermatogenesis after implant removal (w 3, 6, 9, 12). H-PGDS, L-PGDS and DP were present in the adult (CG), juvenile (JG) and downregulated canine testis at the mRNA level. PGDS immunohistochemistry revealed positive staining in the cytoplasm of Leydig cells (LCs) of all samples i.e., no difference between groups. mRNA expression (ratio) of L-, H-PGDS and DP did not differ between groups w 0-12 and CG. In contrast, significant differences were found for L-PGDS (p = 0.0388), H-PGDS (p < 0.001) and DP (p < 0.001) for the groups at downregulation (w0, suprelorin group, SG, profact group, PRG) compared with the control groups (JG, CG). L-PGDS expression was lowest in JG, whereas H-PGDS was significantly lower in CG compared with JG and at downregulation (p < 0.001 to p < 0.01). The highest ratio for H-PGDS and DP was observed in the dogs treated with buserelin acetate (PRG). Our data show that the PGDS-DP system is expressed in juvenile and adult canine testes and that downregulation of the testicular endocrine and germinative function significantly affects H-PGDS, L-PGDS and DP mRNA expression indicating a role in the regulation of spermatogenesis.

Age-related changes in interstitial fibrosis and germ cell degeneration of the canine testis.

Fifty-five healthy medium-sized dogs were divided into four groups; young (1-3years old, n=14), adult (>3 to 6years old, n=12), old (>6 to 9years old, n=14) and senile (>9years old, n=15). After routine orchiectomy, testes were collected, and the degree of white streak areas on cut surfaces was subjectively assessed. Later, testicular tissue sections were stained with haematoxylin and eosin and Masson's trichrome for evaluation of germ cell degeneration and the proportion of interstitial connective tissue, respectively. Semiquantitative severity scoring of germ cell degeneration and quantitative analysis of spermatogenic cells for spermatic index (SI) and Sertoli cell index (SEI) was performed. The score of white streak on cut surface area of the testes increased with age, being higher (p<0.05) in senile dogs than other age groups; no difference was found between adult and old dogs. The proportion of testicular interstitial fibrosis was highest (p<0.05) in senile dogs. Positive correlations between age and white streak area (rho=0.77, p<0.01) as well as age and interstitial fibrosis (rho=0.63, p<0.01) were observed. The severity of germ cell degeneration gradually increased with age and differed among age groups (p<0.05). Age positively correlated with atrophy of seminiferous tubules (rho=0.93, p<0.01). The SI was lower (p<0.05) in senile dogs compared to other age groups, and SI was not different among young, adult and old dogs. Conversely, SEI was significantly higher in senile dogs compared to young, adult and old dogs. A negative correlation between age and SI (rho=-0.69) and a positive correlation between age and SEI (rho=0.68) were significant (p<0.01). In conclusion, influence of age on testicular interstitial fibrosis and germ cell degeneration/depletion were pronounced in dogs over 9years old.

Expression of PTGS2, PGFS and PTGFR during downregulation and restart of spermatogenesis following GnRH agonist treatment in the dog.

Prostaglandins (PGs) and prostaglandin endoperoxide synthase (PTGS) are considered to be relevant for spermatogenesis and steroidogenesis. PTGS2, prostaglandin F synthase (PGFS) and PGF receptor (PTGFR) are investigated in the adult male dog using the model of the GnRH-agonist implant downregulated canine testis and its subsequent restart of spermatogenesis following abolition of treatment (3, 6, 9, 12weeks after implant removal). On the mRNA level (ratio), PTGS2, PGFS and PTGFR expression did not differ between downregulation, different stages of recovery of spermatogenesis and untreated adult controls (CG). On the protein level, Sertoli and Leydig cells in all samples and some peritubular cells stained immunopositive for PTGS2. In the tubular compartment, the percentage of the PTGS2-immunopositive area (PIA) and the mean PTGS2-staining intensity (gray scale, GS) did not differ between groups but in the interstitial compartment, the PIA (p = 0.0494) and the GS (p < 0.0001) were significantly upregulated during early recrudescence (week 3/6). Comparing downregulation by two GnRH-agonist implants with juvenile controls (JG) and CG, the mRNA expression (ratio) did not differ. In the tubular compartment, the GS (p = 0.0321) was significantly higher at downregulation compared to CG and in the interstitial compartment, the PIA (p = 0.0073) and the GS (p = 0.0097) were significantly higher in JG compared to downregulation/CG. PTGS2, PGFS and PTGFR mRNA and PTGS2 protein are regularly expressed in the adult, juvenile and downregulated canine testis and downregulation and subsequent recrudescence affect PTGS2 protein expression mainly in Leydig cells. PTGS2 expression in the downregulated testis resembles the one in seasonal Syrian hamster but not juvenile canine testis.

Potential role of orexin A binding the receptor 1 for orexins in normal and cryptorchid dogs

BackgroundCryptorchidism is one of the most common birth disorders of the male reproductive system identified in dogs and other mammals. This condition is characterised by the absence of one (unilateral) or both (bilateral) gonads from the scrotum. The peptides orexin A (OxA) and B (OxB) were obtained by post-transcriptional proteolytic cleavage of a precursor molecule, called prepro-orexin. These substances bind two types of G-coupled receptors called receptor 1 (OX1R) and 2 (OX2R) for orexins. OX1R is specific to OxA while OX2R binds the two peptides with equal affinity. Orexins modulate a great variety of body functions, such as the reproductive mechanism. The purpose of the present research was to study the presence of OxA and its receptor 1 and their possible involvement in the canine testis under healthy and pathological conditions.MethodsThis study was performed using adult male normal dogs and male dogs affected by unilateral cryptorchidism. Tissue samples were collected from testes and were divided into three groups: normal, contralateral and cryptic. The samples were used for immunohistochemistry, Western blot and in vitro tests for testosterone evaluation in normal and pathological conditions.ResultsOxA-immunoreactivity (IR) was described in interstitial Leydig cells of the normal gonad, and Leydig, Sertoli cells and gonocytes in the cryptic gonad. In the normal testis, OX1R-IR was described in Leydig cells, in pachytene and second spermatocytes and in immature and mature spermatids throughout the stages of the germ developing cycle of the male gonad. In the cryptic testis OX1R-IR was distributed in Leydig and Sertoli cells. The presence of prepro-orexin and OX1R was demonstrated by Western blot analysis. The incubation of fresh testis slices with OxA caused the stimulation of testosterone synthesis in the normal and cryptic gonad while the steroidogenic OxA-induced effect was cancelled by adding the selective OX1R antagonist SB-408124.ConclusionsThese results led us to hypothesise that OxA binding OX1R might be involved in the modulation of spermatogenesis and steroidogenesis in canine testis in healthy and pathological conditions.

Characterization of male germ cell markers in canine testis

Spermatogenesis begins at puberty and continues throughout a male’s life. This process is initiated and maintained by spermatogonial stem cells in the seminiferous tubules, and these cells produce haploid spermatozoa. Markers of male germ cells have been fully identified in rodents, including mice and rats, but not in canines. To characterize the canine male germ cells, histological and immunohistochemical analyses were performed, using prepubertal (1–3-month-old), early pubertal (4-month-old), and postpubertal (7-month-old) dog testes. Expression of protein gene product 9.5 (PGP9.5), deleted in azoospermia-like (DAZL), synaptonemal complex protein (SCP3), tyrosine-protein kinase Kit (C-kit), and acrosin was confirmed by immunohistochemical analysis. PGP9.5 and DAZL were detected in spermatogonia and co-localized near the basement membrane of seminiferous tubules. Some SCP3-positive cells expressed PGP9.5 but not C-kit, and most of these cells were located near the basement membrane. C-kit is a marker of differentiated spermatogenic cells. In addition, acrosin was detected in C-kit-positive spematocytes and mature spermatozoa, whereas C-kit was detected in Sertoli cells in all stages of canine testis development. We suggest that male germ cell markers detected in other species are conserved in canines. PGP9.5, DAZL, SCP3, and acrosin expressions were conserved among various species, but C-kit expression varied. This study might facilitate the identification of stage-specific canine germ cell markers and cellular mechanisms of spermatogenesis.

Expression of retinoic acid-metabolizing enzymes, ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1 in canine testis during post-natal development.

Mammalian spermatogenesis involves highly regulated temporal and spatial dynamics, carefully controlled by several signalling processes. Retinoic acid (RA) signalling could have a critical role in spermatogenesis by promoting spermatogonia differentiation, adhesion of germ cells to Sertoli cells, and release of mature spermatids. An optimal testicular RA concentration is maintained by retinaldehyde dehydrogenases (ALDHs), which oxidize RA precursors to produce RA, whereas the CYP26 class of enzymes catabolizes (oxidize) RA into inactive metabolites. The objective was to elucidate gene expression of these RA-metabolizing enzymes (ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1) and their protein presence in testes of young, peripubertal and adult dogs. Genes encoding RA-synthesizing isozymes ALDH1A1, ALDH1A2 and ALDH1A3 and RA-catabolizing isomers CYP26A1, CYP26B1 and CYP26C1 were expressed in testis at varying levels during testicular development from birth to adulthood in dogs. Based on detailed analyses of mRNA expression patterns, ALDH1A2 was regarded as a primary RA-synthesizing enzyme and CYP26B1 as a critical RA-hydrolysing enzyme; presumably, these genes have vital roles in maintaining RA homeostasis, which is imperative to spermatogenesis and other testicular functions in post-natal canine testis.

Evaluation of Human Semenogelin Membrane Strip Test for Species Cross-reactivity in Dogs.

Semenogelins are proteins originating in the seminal vesicle and are useful markers for the presumptive identification of human semen. Detection of semenogelin can be done with a commercially available membrane test. In this study, a commercially available membrane test for human semenogelin proteins was used to assess for cross-reactivity in dog bodily fluids to allow for the potential utilization for detection of human semen in dog bodily fluids. The authors analyzed canine semen and other bodily fluids, including urine, saliva, vaginal secretions, fecal material, and blood. They also examined the distribution of human semenogelin I transcripts in the canine testis, prostate, and several bodily fluids by reverse transcription polymerase chain reaction. No cross-reactivity was observed in the canine bodily fluids tested except for a single rectal swab, which was negative on a second test. Further testing should be done to validate the use of this kit for screening samples from dogs suspected to have been victims of sexual abuse.

PIWIL1 Is Expressed in the Canine Testis, Increases with Sexual Maturity, and Binds Small RNAs.

Spermatogenesis is a highly regulated process leading to the development of functional spermatozoa through meiotic division and subsequent maturation. Recent studies have suggested that a novel class of Argonaute proteins, known as the PIWI clade, plays important roles in multiple stages of spermatogenesis. PIWI proteins bind specific small noncoding RNAs, called PIWI-interacting RNAs (piRNAs). These piRNAs guide the PIWI-piRNA complex to retrotransposon targets that become expressed during meiosis. Retrotransposons are subsequently silenced, either through PIWI "slicer" activity or through PIWI-directed methylation of the retrotransposon locus. Most mammalian studies have employed mouse models where sterility follows PIWI inactivation. The goal of this study was to characterize canine PIWIL1 to determine whether expression pattern and functional characteristics support a similar function in that species. Canine PIWIL1 cDNA is a 2.6-kb transcript that encodes an 861-amino acid protein showing high homology to other mammalian PIWIL1 proteins and containing features consistent with PIWI family members (PAZ, PIWI domains). Analysis of PIWIL1 protein and transcript levels revealed that PIWIL1 expression is limited to the testes and is associated with sexual maturity, with mature dogs showing higher levels of PIWIL1 expression. Immunohistochemistry demonstrated expression primarily in seminiferous tubules and confirmed higher levels of PIWIL1 in mature dogs. Functional characterization by RNA immunoprecipitation demonstrated that canine PIWIL1 binds short RNAs consistent in size with piRNAs (27-32 nucleotides). Together, these studies represent the first characterization of a PIWI protein in the dog and suggest that it is a functional piRNA-binding protein most highly expressed in the mature testes.

Expression and localization of epidermal growth factor, transforming growth factor-α and epidermal growth factor receptor in the canine testis.

Gene expression of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and EGF receptor (EGF-R) and the localization of the corresponding proteins in the canine testis were studied. Levels of mRNA expressions were determined by semiquantitative reverse transcription polymerase chain reaction in the testes of the peripubertal (4–6 months), young adult (3–4 years), advanced adult (7–8 years) and senescent (11–16 years) groups. The EGF-R mRNA level in the testes of the peripubertal group was significantly higher than those in the other groups, whereas there was no difference in EGF and TGF-α mRNA levels among groups. Immunohistochemical stainings for EGF, TGF-α and EGF-R in the testis revealed that immunoreactivity in the seminiferous epithelium and Sertoli cell was weak and nonspecific for the stage of spermatogenesis, and distinct staining was found in Leydig cells. These results suggest that the EGF family of growth factors may be involved in testicular maturation and function in the dog.

Expression of matrix metalloproteinases (MMP-2, MMP-9) and their inhibitors (TIMP-1, TIMP-2) in canine testis, epididymis and semen.

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n=21) and abnormal (n=25) semen samples showed a significant, sixfold increase in proMMP-2 and MMP-2 activity in high than low sperm concentration samples (p<0.001). ProMMP-9 and MMP-9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p<0.001). High levels of proMMP-2 and MMP-2 were associated with high sperm motility (≥70%, p<0.001). Sperm-rich fraction showed significantly (eight-fold) higher proMMP-9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP-2-specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP-9, TIMP-1 and TIMP-2 were absent in these cells. Matrix metalloproteinase-9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP-1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP-2 localization along acrosomal region of sperm, while MMP-9, TIMP-1 and TIMP-2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP-2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP-2 and MMP-9 may serve as an alternative biomarker in determining semen quality.