Canine Testes Research Articles

Study on the expression of testin in the testes of dogs.

Testin is a protein involved in cell mobility, adhesion and colony formation. In rats, testin presence has been reported in the testes, and its possible role in spermatogenesis has been suggested. Studies in humans also suggest a possible role of testin as a cancer suppressor protein. In the dog, which represents both an important pet species and a good animal model for studying biological and pathological testicular processes, the presence of testin has never been reported. In the present study, the expression of testin in foetal, prepubertal, adult and aged canine testes was investigated. Testes from 5 adult and 3 aged dogs, from 2 one-month-old puppies and from 2 foetuses miscarried at the end of pregnancy were immunohistochemically examined with a commercial antibody against testin. Testin was intensely expressed in Sertoli cells in every testis examined. Spermatids were also positive for testin in mature dogs and in the testicular areas of the aged ones which were not atrophic. Weak expression of testin was also detected in all testes examined. The present study, the first demonstrating the presence of testin in canine testes, provides the basis for further dog-human comparative research and for studies on the role of this protein in canine physiology, reproduction and testicular pathologies.

Detection of spermatogonial stem cells in testicular tissue of dogs with chronic asymptomatic orchitis.

Chronic asymptomatic idiopathic orchitis (CAO) is an important but neglected cause of acquired infertility due to non-obstructive azoospermia (NOA) in male dogs. The similarity of the pathophysiology in infertile dogs and men supports the dog's suitability as a possible animal model for studying human diseases causing disruption of spermatogenesis and evaluating the role of spermatogonial stem cells (SSCs) as a new therapeutic approach to restore or recover fertility in cases of CAO. To investigate the survival of resilient stem cells, the expression of the protein gene product (PGP9.5), deleted in azoospermia like (DAZL), foxo transcription factor 1 (FOXO1) and tyrosine-kinase receptor (C-Kit) were evaluated in healthy and CAO-affected canine testes. Our data confirmed the presence of all investigated germ cell markers at mRNA and protein levels. In addition, we postulate a specific expression pattern of FOXO1 and C-Kit in undifferentiated and differentiating spermatogonia, respectively, whereas DAZL and PGP9.5 expressions were confirmed in the entire spermatogonial population. Furthermore, this is the first study revealing a significant reduction of PGP9.5, DAZL, and FOXO1 in CAO at protein and/or gene expression level indicating a severe disruption of spermatogenesis. This means that chronic asymptomatic inflammatory changes in CAO testis are accompanied by a significant loss of SSCs. Notwithstanding, our data confirm the survival of putative stem cells with the potential of self-renewal and differentiation and lay the groundwork for further research into stem cell-based therapeutic options to reinitialize spermatogenesis in canine CAO-affected patients.

Presence and localization of apelin and its cognate receptor in canine testes using immunohistochemical and RT-PCR techniques

Apelin, a member of the adipokine family, is a novel endogenous peptide which regulates the male reproductive system of mammals by interacting with a specific receptor. Recent studies have highlighted that apelin may play a role in the regulation of reproduction by reducing testosterone production and inhibiting LH secretion. To the best of our knowledge, there is no available data on the presence of the apelin and its receptor in canine testes. Therefore, the aim of this study was to reveal the presence of apelin and evaluate its distribution in the canine testes using immunohistochemical and RT-PCR techniques. For this purpose, five fertile and healthy male dogs were subjected to elective orchiectomy. The immunohistochemical reaction revealed the presence of apelin and its receptor in the canine testes. Apelin was localized in spermatids and spermatozoa with a positive signal in the “acrosomal bodies”. As regards the apelin receptor, a positive immunoreaction was detected in the cytoplasm of the cells localized near to the basal membrane of the seminiferous tubules and in the cytoplasm of Leydig cells. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all of the samples under study. A 35kDa band confirmed apelin receptor protein expression in all of the samples analysed. In conclusion, the paracrine and endocrine role of apelin and its cognate receptor on male reproduction reported in humans and laboratory animals could also be hypothesized in dogs.

Heat stress and hypoxia inhibit the secretion of androgens and induce epithelial-to-mesenchymal transition associated with activated TGF-β/Smad signalling in canine cryptorchidism.

Cryptorchidism, as a common congenital disease of canine testes, is mainly caused by factors leading to endocrine abnormalities in testes and infertility in a heat stress and hypoxia microenvironment. Moreover, heat stress and hypoxia, as critical microenvironmental factors, promote epithelial-mesenchymal transition (EMT), which occurs during adult tissue remodelling responses including carcinogenesis and fibrosis and is the main cause of testicular tumours. In this study, we found by haematoxylin-eosin staining that the canine cryptorchid tissue produced a lot of collagen fibres. Also, the quantitative PCR and Western blot results showed that the mRNA and protein levels of the heat stress makers HSP70 and HO-1 and the hypoxia maker HIF-1α are significant higher compared with normal testes. Moreover, we found the expression levels of TGF-βs and its two receptors TGF-βRI and TGF-βRII increased in case of cryptorchidism. From the study in vitro, we found both heat stress and COCl2 mimic hypoxia inhibited the secretion of testosterone (T) and androstenedione (A4) and promoted the expression of the EMT maker α-SMA and vimentin in Leydig cells, and also that heat stress and COCl2 stimulated with the TGF-β signalling promoted the expression of TGF-βs and its two type receptors and also the active phosphorylation of Smad2 and Smad3. The use of LY2109761, a receptor inhibitor of TGF-βs/Smad signalling pathway, was associated with heat stress and COCl2 suppression of androgens' secretion and stimulated EMT in Leydig cells. These findings characterized a novel pathogenesis of cryptorchidism and provided a new idea for therapeutics.

Use of Contrast-Enhanced Ultrasound of the Testes after Non-Surgical Sterilization of Male Dogs with CaCl2 in Alcohol.

Simple SummaryThe uncontrolled stray dog population presents a serious threat to public health due to the increased risk of transmitting zoonotic diseases and inflicting human injury. There is a growing interest in non-surgical chemical sterilization via intratesticular injection of chemical neutering agents such as calcium chloride, which causes sterility due to azoospermia. In this study, we monitored immediate vascular alterations in dogs from chemical sterilization, using contrast-enhanced ultrasound (CEUS) before and after intratesticular inoculation with calcium chloride. The CEUS is an imaging technique used to obtain a quali-quantitative assessment of tissue perfusion by intravenously injecting gas-filled microbubble contrast agent. The CEUS evaluation was performed in canine testes before and after intratesticular inoculation with calcium chloride. Our results showed that the hardening agent caused a drastic reduction of the intratesticular blood flow, which was reflected by sonographic findings. CEUS can help clinicians define immediate testicular vascular alterations achieved by chemical castration. Nevertheless, more studies are required to apply this methodology to more subjects in a broader weight range among stray dogs.Sterilization by intratesticular injection of chemical agents is a non-surgical alternative to neutering male companion animals. We used contrast-enhanced ultrasound (CEUS) to monitor vascular alterations to testes immediately after the intratesticular injection of CaCL2 in alcohol. We evaluated the CEUS features of normal and damaged testes in 20 dogs after the intratesticular injection of CaCl2. The CEUS evaluation was performed at the site of the chemical agent inoculation. In treated testes, qualitative CEUS showed a lower intensity enhancement of the parenchyma than pre-treatment normal testes with a predominantly anechoic pattern and only a few hyperechoic vascular focal spots. Quantitative CEUS showed significantly lower values of time-intensity curve (TIC) parameters, including signal intensity (Peak: 4.72 ± 2.1), regional blood volume (RBV: 134.3 ± 63.7), and regional blood flow (RBF: 4.36 ± 2.18) than normal testes (p < 0.001). Sonographic findings from CEUS showed hypovascularization of the canine testicular parenchyma caused by the hardening agent. This diagnostic technique helps clinicians define testicular vascular alterations achieved by chemical castration more efficiently. Nevertheless, more studies are required to apply this methodology to more subjects with a broader weight range and stray dogs.

Glyoxal: a proposed substitute for formalin in H&E and special stains

ABSTRACT Neutral-buffered formalin (NBF) has been used as the primary fixative in anatomic pathology laboratories for decades. Although it yields excellent morphologic and staining results, NBF poses significant health hazards requiring tissue to be grossed under a grossing/chemical fume hood. Glyoxal fixatives offer far less toxic alternatives and do not necessitate use of a grossing hood. Using freshly extracted canine and feline testes, ovaries, and uteri, the effects of glyoxal and NBF fixation were compared. While NBF is still considered the gold standard, some glyoxal fixatives perform as well as NBF in regards to morphology, H&E staining properties, and histochemical staining properties.

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.

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice

BackgroundXenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis.MethodsSSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L−1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis.ResultsSupplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice.ConclusionsIn conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

Aging and Senescence in Canine Testes.

Senescent cells accumulate with age but tissue-based studies of senescent cells are limited to selected organs from humans, mice, and primates. Cell culture and xenograft studies have indicated that senescent cells in the microenvironment may play a role in tumor proliferation via paracrine activities. Dogs develop age-related conditions, including in the testis, but cellular senescence has not been confirmed. We hypothesized that senescent cells accumulate with age in canine testes and in the microenvironment of testicular tumors. We tested the expression of the established senescence markers γH2AX and p21 on normal formalin-fixed, paraffin-embedded testes from 15 young dogs (<18 months of age) and 15 old dogs (7-15 years of age) and correlated the findings with age-dependent morphological changes. A statistically significant age-dependent increase in the percentage of p21-expressing cells was observed for testicular fibroblasts (4-fold) and Leydig cells (8-fold). However, p21-expressing cells were still a rare event. In contrast, the percentage of γH2AX-positive cells did not increase with age. P21- and γH2AX-expressing cells were rare in the microenvironments of tumors. Age-dependent morphological changes included an increased mean number of Leydig cells per intertubular triangle (2.95-fold) and a decreased spermatogenesis score. To our surprise, no age-related changes were recorded for interstitial collagen content, mean tubular diameter, and epithelial area. Opposed to our expectations based on previous in vitro data, we did not identify evidence of a correlation between age-associated accumulation of senescent cells and testicular tumor development. Understanding the role of the microenvironment in senescence obviously remains a challenging task.

Abstract 5527: Characterizing the role of WWOX dysregulation in canine osteosarcoma

Abstract Introduction: The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that is frequently deleted or reduced in human osteosarcoma (OS) tumors and loss of WWOX in OS cells promotes a highly tumorigenic phenotype. The purpose of this study was to investigate the functional consequences of WWOX deficiency in canine OS, a well-established spontaneous large animal model of human OS. Methods: Real-time PCR and Western blotting was performed to evaluate WWOX expression was in primary canine OS tumors and canine OS cell lines. Full-length canine Wwox cDNA was amplified from normal canine testes and cloned into the pCDH-cop-GFP lentiviral vector (Systems Biosciences). Canine OS cell lines expressing low basal levels of WWOX were transduced with either empty or WWOX lentiviral vectors and stably transduced cells were sorted based on GFP positivity. Overexpression of canine WWOX lentiviral constructs was confirmed by Western blotting and OS cell lines were evaluated for differences in proliferative capacity, expression of bone differentiation markers, and the ability to migrate through matrigel. Transgenic mice (Tg) carrying a conditional allele of Wwox (Wwoxfl/fl-Tg) were crossed with mice expressing Cre recombinase under the osteoblast-specific Collagen1α1 promoter (Col1a1-Cre-Tg), restricting deletion of WWOX to osteoblasts. Calvarial osteoblast cultures were generated from Wwoxfl/fl and Col1a1-Cre; Wwoxfl/fl mice and the effects of WWOX deletion on osteoblast functions was evaluated using assays described above. Results and Conclusions: Western blotting demonstrated that WWOX protein is absent or reduced in primary canine OS tumors and canine OS cell lines. To assess the functional consequences of WWOX deletion on normal osteoblast behavior, the canine OSA8 and Abrams OS cell lines which express low levels of WWOX were transduced with WWOX lentiviral constructs resulting in high levels of WWOX expression as demonstrated by real time PCR and Western blotting. Restoration of WWOX in canine OS cells resulted in decreased cellular proliferation and invasion. To more critically evaluate the consequences of Wwox deficiency in normal osteoblast biology without the confounding influence of altered Wwox expression in other tissues, Col1a1-Cre; Wwoxfl/fl-Tg mice were generated in which Wwox deletion is restricted to osteoblasts. Consistent with our findings in canine OS cell lines, deletion of WWOX in osteoblasts derived from Col1a1-Cre; Wwoxfl/fl-Tg mice resulted in enhanced cellular proliferation. Studies are underway to identify WWOX binding partners and investigate regulatory pathways altered by WWOX that promote cell proliferation and invasion in normal and malignant osteoblasts. In summary, these data provide evidence supporting a role for WWOX in canine OS cell proliferation and invasion, and suggest that dysregulation of WWOX may be fundamental to the disease process in both human and canine OS. Citation Format: Justin Breitbach, Feng Xu, F. Kay Huebner, Cheryl A. London, Joelle M. Fenger. Characterizing the role of WWOX dysregulation in canine osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5527.

Senescence Markers in Canine Testes: P21, But Not H2ax, Is Increased With Age

Senescence Markers in Canine Testes: P21, But Not H2ax, Is Increased With Age

Testicular gene expression of steroidogenesis-related factors in prepubertal, postpubertal, and aging dogs

Developmental and aging changes in testicular factors related to steroidogenesis are unknown in dogs. Using reverse transcription quantitative real-time PCR, this study examined testicular mRNA levels of CYP11A1 (P450 cholesterol side-chain cleavage enzyme, P450scc), CYP17A1 (P450 17α-hydroxylase/C17–20 lyase, P450c17), HSD3B2 (3β-hydroxysteroid dehydrogenase, 3β-HSD), CYP19A (P450 aromatase, P450arom), STAR (steroidogenic acute regulatory protein, StAR), cyclooxygenase (COX) -1 and COX-2 in prepubertal (4–6 months of age), postpubertal (1 year of age), and aging (2–18 years of age) dogs. Testicular mRNA levels for P450scc, 3β-HSD, StAR, COX-1, and COX-2 did not change from prepubertal to postpubertal stages, whereas that for P450arom markedly and abruptly increased and that for P450c17 gradually decreased. In postpubertal and aging dogs, a negative correlation was found between aging and testicular P450arom mRNA levels. Based on the rapid testicular growth observed during puberty, these results suggested that total testis gene expression for steroidogenesis-related factors, in particular for P450arom, increases during puberty in dogs. In addition, the decline in P450arom gene expression during aging may affect the ability to synthesize steroids in canine testes.

Immunolocalization of proteins in the spermatogenesis process of canine.

Spermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine. Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

Immunohistochemical expression of SOX9 protein in immature, mature, and neoplastic canine Sertoli cells

Sex-determining region Y box9 gene (SOX9) protein plays a pivotal role in male sexual development. It regulates the transcription of the anti-Müllerian hormone gene promoting development of testis cords, multiplication, and maturation of Sertoli cells (SCs) and maintenance of spermatogenesis in adult testis. The immunohistochemical expression of SOX9 in normal testes has been reported in humans, mice, and rats. The present study aimed to investigate the expression of SOX9 in canine SCs during testicular maturation and neoplastic transformation. Canine testicular samples derived from three fetuses, four newborns, four prepubertal puppies, five adult dogs, 31 Sertoli cell tumors (SCTs) (one metastasizing), and five Leydig cell tumors (LCTs) were selected from departmental archive and tested immunohistochemically with a polyclonal antibody against SOX9 (1:150). All SCs from fetal, neonatal, and adult testes had a strong and exclusively nuclear labeling for SOX9. In SCs from prepubertal testes, SOX9 staining was highly variable with one negative sample (one of four), two samples with exclusively nuclear staining (two of four), and one with both nuclear and cytoplasmic labeling (one of four). Leydig cells (LCs) and LCTs were always negative. All 31 SCTs were positive for SOX9. The expression of SOX9 was nuclear, nuclear and cytoplasmic, and exclusively cytoplasmic in 18 of 31, 11 of 31, and two of 31 SCTs, respectively. This first report on the immunohistochemical expression of SOX9 in canine testes reports that in normal SCs from fetal, neonatal, and adult testes SOX9 labeled the nucleus, as in humans and laboratory animals. The cytoplasmic labeling observed in one prepubertal pairs of testes and in 11 SCTs could reflect SC immaturity or dedifferentiation, paralleling results observed in rat testes. The expression of SOX9 in SCs and SCTs and its absence in LCs and LCTs suggests that SOX9 is a reliable diagnostic marker for both normal and neoplastic SCs.

Expression analyses of insulin-like peptide 3, RXFP2, LH receptor, and 3β-hydroxysteroid dehydrogenase in testes of normal and cryptorchid dogs

Insulin-like peptide 3 (INSL3) plays a key role in testicular descent in rodents, whereas in domestic animals, many aspects of the roles of INSL3 in reproductive organs after puberty are still unknown. This study was undertaken to (1) determine the quantitative changes of gene expression of testicular INSL3, its receptor (RXFP2), LH receptor, and 3β-hydroxysteroid dehydrogenase during and after puberty in normal male dogs; (2) compare the expressions of these substances in normal and cryptorchid dogs; and (3) localize the cells expressing INSL3 in normal and retained canine testes. Testes were obtained from small-breed normal male dogs (n = 56) and cryptorchid dogs (n = 22). Normal scrotal testes from the normal dogs (normal testes), retained testes from both the unilateral and bilateral cryptorchid dogs (retained testes), and scrotal testes of the unilateral cryptorchid dogs (cryptorchid scrotal testes) were used. We measured the concentrations of these testicular messenger RNAs (mRNAs) by quantitative real-time reverse transcription polymerase chain reaction, and an enzyme immunoassay was used for measuring INSL3 peptide. Immunohistochemistry for INSL3 peptide was done in paraformaldehyde-fixed frozen testicular tissue. In the normal dogs, total amount of INSL3 mRNA per testis tended to decrease (P = 0.05) from pubertal (6–12 months) to postpubertal (1–5 years) and decreased (P < 0.01) to middle age (5–10 years), but total amount of INSL3 peptide per testis did not change among age groups. Concentrations of INSL3 mRNA were higher (P < 0.01) in retained testes than those in the normal testes and cryptorchid scrotal testes, and similar differences were observed for INSL3 peptide. Reversely, total amounts of INSL3 mRNA and peptide per retained testis were lower (P < 0.01) than those per normal testis because of smaller weight of retained testes. Concentrations and total amount of RXFP2 mRNA in the retained testes were almost nil and lower (P < 0.01) than those in the normal testes and in the cryptorchid scrotal testes. Total amount of LH receptor mRNA per retained testis was lower (P < 0.01) than that per normal testis. The immunohistochemical analysis revealed that INSL3 was expressed only in Leydig cells of both the normal and retained canine testes. These results suggest that INSL3 in retained testes of cryptorchid dogs is substantially expressed per unit-weight basis but may be produced with lower amount as a whole testis. Also, this study provides findings that RXFP2 gene is expressed scarcely in the retained testes but normally in cryptorchid scrotal testes.

Identification and in vitro derivation of spermatogonia in beagle testis.

BackgroundIn vitro culture of spermatogonial stem cells (SSCs) is important for exploration of SSCs self-renewal, differentiation, and manipulation. There are several reports on rodent SSC cultures; however, data on SSC cultures in domestic animals are limited. To provide basic scientific information on canine SSC cultures, we report canine testes development, and the development of spermatogonia-derived colonies (SDCs) for in vitro cultures.Methodology/Principal FindingsTestes from 2-, 3-, and 12-month-old beagles were used for histology, immunohistochemistry, in vitro culture, immunocytochemistry, and PCR. Protein gene product 9.5 (PGP9.5)-positive spermatogonia, both single and paired, were found to be abundant in the testes of 2-month-old beagles. stempro-34 and Dulbecco's modified Eagle medium with 5% fetal bovine serum provided as useful substrates for culture of SDCs, and fibroblast growth factor (FGF) played a key role in colony formation. Colonies were positive for alkaline phosphatase and anti-PGP9.5 staining. The early spermatogonia and stem cell markers such as octamer binding protein 4 (Oct4), Nanog homeobox (Nanog), promyelocytic leukemia zinc finger (PLZF), PGP9.5, and GDNF family receptor alpha-1 (GFRα-1) were expressed in the colonies at higher levels than in the testis tissue.ConclusionsTestes of the 2-month-old beagles had abundant single and paired spermatogonia, which can be used for derivation of SDCs, and FGF was important for colony formation.

Differential expression of microRNAs in sexually immature and mature canine testes

Mammalian testis exhibits spatiotemporal gene expression patterns that are essential for successful and continuous spermatogenesis. Although microRNAs (miRNAs) modify gene expression through translational repression and degradation of target messenger RNAs, the precise molecular mechanisms of these regulatory processes are unclear. We used canine miScript miRNA polymerase chain reaction (PCR) Array technology to elucidate the repertoire of canine testis miRNAs and compared their expression patterns between sexually immature (prepubertal) and mature (adult) dog testes. Eighty-four well-characterized canine miRNAs were customized in this study. The data were analyzed by RT2 Profiler PCR Array Data Analysis (version 3.5). Results identified upregulation of 32 and considerable downregulation of 12 miRNAs in adult dog testis. In conclusion, the two developmental stages had significantly different miRNAs expression patterns. The finding provides fundamental information of miRNAs which may help to elucidate their role in spermatogenesis and male infertility in this species. To the best of our knowledge, this study is the first to offer comparative profile of the miRNA transcriptome in prepubertal and adult canine testes using miRNA PCR array approach.

Effect of Short‐Term Scrotal Hyperthermia on Spermatological Parameters, Testicular Blood Flow and Gonadal Tissue in Dogs

The objective was to assess the effect of a short-term scrotal hyperthermia in dogs on quantitative and qualitative ejaculate parameters, testicular blood flow and testicular and epididymal histology. After a control period, the scrotum of seven normospermic adult beagle dogs was insulated with a self-made suspensory for 48h. Nine weeks later, two animals were castrated, while in five animals, scrotal hyperthermia was repeated. Dogs were castrated either 10 or 40days thereafter. In each phase of scrotal insulation, average scrotal surface temperature increased by 3.0°C. Semen was collected twice weekly throughout the experiment. Total sperm count did not change after the first hyperthermia, but it slightly decreased after the second (p<0.05). Profiles of sperm morphology and velocity parameters (CASA) rather indicated subtle physiological variations in sperm quality than effects of a local heat stress. Chromatin stability of ejaculated spermatozoa as indicated by SCSA remained constant throughout the experiment. Perfusion characteristics of the gonads, that is, systolic peak velocity, pulsatility and resistance index at the marginal location of the testicular artery, did not change due to hyperthermia (p>0.05). Histological examination of excised testes and epididymides for apoptotic (TUNEL and activated caspase-3) and proliferating cells (Ki-67 antigen) indicated only marginal effects of scrotal insulation on tissue morphology. In conclusion, a mild short-term scrotal hyperthermia in dogs does not cause substantial changes in sperm quantity and quality. In contrast to other species, canine testes and epididymides may have a higher competence to compensate such thermal stress.

Immunohistochemical evaluation of lymphocyte types infiltrate into the canine seminomas

BACKGROUNDS: Seminoma is frequently observed in human and canine testes especially in cryptorchids. Canine and human seminomas are typically associated with leukocytic infiltration. OBJECTIVES: We aimed to identify the type of lymphocytes that infiltrate to seminomas. METHODS: Tumor infiltrating lymphocytes were evaluated by immunohistological techniques in 5 dogs with diffuse seminoma. Routine pathological examination of these specimens showed diffuse seminoma with tumor infiltrating lymphocytes in all cases. RESULTS: Lymphocytes in seminomas were predominantly T cytotoxic (CD3+, CD8+) cells. These cells were distributed diffusely, but patchy around the vessels. B-cells were also identified as some rare single cells diffusely scattered in the tumoral parenchyma. CONCLUSIONS: The results of this study showed that the lymphocyte infiltrating cells are mainly T lymphocytes particularly CD8 cytotoxic type. BACKGROUNDS: Seminoma is frequently observed in human and canine testes especially in cryptorchids. Canine and human seminomas are typically associated with leukocytic infiltration. OBJECTIVES: We aimed to identify the type of lymphocytes that infiltrate to seminomas. METHODS: Tumor infiltrating lymphocytes were evaluated by immunohistological techniques in 5 dogs with diffuse seminoma. Routine pathological examination of these specimens showed diffuse seminoma with tumor infiltrating lymphocytes in all cases. RESULTS: Lymphocytes in seminomas were predominantly T cytotoxic (CD3+, CD8+) cells. These cells were distributed diffusely, but patchy around the vessels. B-cells were also identified as some rare single cells diffusely scattered in the tumoral parenchyma. CONCLUSIONS: The results of this study showed that the lymphocyte infiltrating cells are mainly T lymphocytes particularly CD8 cytotoxic type.