Canine Testes Research Articles

Localization of tumor necrosis factor in the canine testis, epididymis and spermatozoa

Tumor necrosis factor (TNF), formerly known as Tumor necrosis factor alpha is now regarded as a natural component of the mammalian seminal plasma (SP). Although not completely clarified, its functions in the SP have been associated with paradoxal roles, such as sperm survival in the female genital tract, while at high levels negatively affect sperm survival and fertility potential. Recently, it has been discovered that canine inseminated spermatozoa display a strong immunoreaction for TNF when lining the female endometrium. As a continuation of this finding, the present work aimed at documenting TNF localization in the canine testes and epididymis and in freshly ejaculated spermatozoa (SPZ) through immunohisto- or cytochemistry.Immunoreaction for TNF was found in all samples used. In the dog testis, TNF immunoexpression was limited to the seminiferous tubules, where late round spermatids (SPD) showed weak intensity of immunostaining, while elongating and elongated SPD evidenced moderate and the residual bodies a strong intensity. In the epididymis, a gradual progressive increase of TNF immunolabelling was found throughout the epididymal regions, ranging from a weak intensity at the caput epididymis to a moderate intensity at the cauda. TNF immunolabelling was found in mature SPZ during the epididymal transit and also in freshly ejaculated SPZ, which showed a strong midpiece immunolabelling. Data presented here provide important information on expression of TNF in spermatozoa, which is acquired by the SPZ during their formation at the testis. It further provides the basis for subsequent studies on the physiological importance of cytokines in sperm function.

Impact of Fine‐ or Large‐Needle Aspiration on Canine Testes: Clinical, In Vivo Ultrasonographic and Seminological Assessment

The safety and consequences of fine- (FNA) and large-needle aspiration (LNA) to the testicular parenchyma and its normal function have not been thoroughly established. This study was performed to accurately assess, by serial clinical, in vivo ultrasonographic and seminological examinations, the type and extent of the effect of FNA or LNA on canine testes. Eighteen sexually mature, 1-2 years old, healthy laboratory Beagles were used. One of their testes was aspirated using a 23-G butterfly needle (FN) and the other using a 19-G butterfly needle (LN). Two dogs at a time were orchiectomized 10, 60 min, 2, 14, 29, 63, 76, 90 or 180 days post-aspiration. Five and 2 days and 1 h before aspiration (in all dogs), immediately post-aspiration, and 1, 2, 4, 7, 9, 14, 19, 29, 35, 43, 49, 56, 63, 70, 76, 90, 111, 132 and 180 days post-aspiration (in the remaining intact dogs), evaluation of scrotal surface temperature over each testis, evaluation of scrotum-testis volume by electronic sliding callipers, ultrasonographic evaluation of testicular volume and texture and clinical and semen examination were performed. Following FNA and LNA, the clinical and ultrasonographic appearance of the testis were normal. Sperm production nearly always remained unchanged, with the exception of a slight decrease in spermatozoal motility 2-14 days post-aspiration. However, even then, with the exception of six samples, spermatozoal motility was above normal values. Within the parameters of this experiment, testicular FNA and LNA have no ill effect on sperm production or clinical and ultrasonographic appearance of the canine testis, and therefore, both FNA and LNA should be considered safe.

The effect of donor age on progression of spermatogenesis in canine testicular tissue after xenografting into immunodeficient mice

The objective of this study was to examine the effect of donor age on progression of spermatogenesis in dog (Canis lupus familiaris) testis tissue after xenografting. In Experiment 1, canine testes were obtained by surgical castration. Based on developmental pattern of spermatogenesis at the time of grafting, donors were categorized as immature, young, and adult (<4, 4 to 6, and >6 mo old, respectively). Fragments of testis tissue were implanted subcutaneously on the back of immunodeficient mice; xenografts were retrieved and analyzed 4, 6, or 8 mo later. At 4 mo postgrafting, immature and young groups had higher graft recovery rates, graft weights, vesicular gland indices, seminiferous tubule numbers, and larger seminiferous tubular diameters compared with those of adult donor xenografts. At 8 mo postgrafting, immature donor xenografts had maintained growth and development as exhibited by greater graft weights, vesicular gland indices, seminiferous tubule numbers, and tubular diameters compared with those of adult donor xenografts. At this time point, growth and development of xenografts did not differ between immature and young donors, whereas those from young donors had greater seminiferous tubule numbers and diameters compared with those of adult donor xenografts. Elongated spermatids were the most advanced germ cell type present at 4 and 8 mo postgrafting in xenografts of immature age groups. In Experiment 2, the longer-term efficiency of spermatogenesis and the potential sperm production in xenografts from immature donor dogs were determined. Testis tissue from 2-mo-old donor dogs were grafted into recipient mice, and xenografts were retrieved after 13 mo. Complete spermatogenesis was present in 5 of 29 recovered xenografts, with isolation of fully formed sperm (up to 36.3 × 10 6 per gram tissue). In conclusion, immature and young donors (<6 mo of age) were the most promising donors for dog testis tissue xenografting. This strategy may offer an alternative for male germ-line preservation for canids that die prematurely or must be castrated before maturation.

Immunohistochemical Expression of the KIT Protein (CD117) in Normal and Neoplastic Canine Testes

The aim of the present study was to characterize the expression of the KIT protein (CD117) in normal and neoplastic canine testes. Archival samples of normal testis (n=5), interstitial cell tumours (ICTs; n=10), Sertoli cell tumours (SCTs; n=10) and seminomas (n=10) were selected. Seminomas were subclassified on the basis of expression of placental alkaline phosphatase (PLAP) as classical seminoma (SE; PLAP positive; n=5) or spermatocytic seminoma (SS; PLAP negative; n=5). In normal testes, KIT expression was observed in Leydig cells and in spermatogonia. All ICTs expressed KIT, but no SCT was positively labelled. Seven of 10 seminomas expressed KIT and these tumours were reclassified on this basis as SS (KIT negative) or SE (KIT positive). These findings are consistent with observations of SE in man where many of the neoplastic cells reach the stage of spermatogonia where PLAP expression is lost and that of KIT is maintained. It would therefore appear that immunolabelling for KIT expression is a more appropriate means of distinguishing between canine SE and SS.

Expression of Connexin 43 in normal canine testes and canine testicular tumors

In human testis, gap junctions containing connexin(Cx)43 are located within the seminiferous epithelium between Sertoli cells and between Sertoli and germ cells. Cx43 is known to play a role in the differentiation and proliferation of these cell types. It can further be associated with human seminoma development. The dog has been proposed as a model for studies of the male reproductive system, because of the frequent occurrence of testicular neoplasms. Thus, we investigated Cx43-mRNA and -protein expression in testes of normal prepubertal dogs, adult dogs, and in canine testicular tumors. Sertoli cells in prepubertal cords express Cx43 mRNA, but do synthesize only less Cx43 protein. Within the seminiferous tubules, Cx43 mRNA was detected in Sertoli cells, spermatogonia, and spermatocytes. Cx43 protein was mainly present in the basal compartment. In canine testicular tumors Cx43 mRNA was detectable in both seminoma and neoplastic Sertoli cells, whereas Cx43 protein was only found in neoplastic Sertoli cells. Our data indicate that Cx43 is regulated differentially in testicular tumors and that alterations of Cx43 expression may be involved in the pathogenesis of canine testicular malignancies. This study represents the first morphological work on the spatiotemporal expression pattern of Cx43 in normal and neoplastic canine testis.

Evidence of testicular dysgenesis syndrome in the dog

Human male reproductive disorders comprising testicular dysgenesis syndrome (TDS) have become more prevalent during the last 50 years. These disorders include cryptorchidism, hypospadias, decreased semen quality, and the development of seminomas. Based on experimental evidence, it has been suggested that environmental pollutants with oestrogen-like or anti-androgenic activities play a role in the pathogenesis of TDS. In humans, histological lesions associated with TDS have been well characterized; this includes seminomas as well as their precursors, carcinoma in situ (CIS) lesions. CIS are seminiferous tubules lined by gonocytes and are a sign of testicular maldevelopment. Such CIS have recently been described in canine species, and an increased frequency of testicular tumours in dogs has also been reported. In this study, we investigated the presence of TDS lesions in canine testes submitted to routine histological examination. Histological features considered typical of human TDS were observed in 8/38 dogs examined; as in humans, individual dogs presented with various TDS features with a range of severity. In all eight dogs, CIS and at least one of the histological feature of TDS was observed in combination with seminoma. These findings suggest that as in humans, TDS may predispose canines to develop testicular cancer. A larger study is needed to better evaluate the actual incidence of TDS in canines, its clinical consequences, and the possible underlying pathogenic factors.

Isolation, culture, and characterization of canine Sertoli cells

Primary Sertoli cell cultures have been established from several animals including the sheep and rhesus monkey; however, not for the domestic dog, Canis familiaris. Sertoli cells are the only readily accessible cell type in the body which expresses all six type IV collagens. These collagens play key roles in tissue structure, basement membrane formation, and filtration. The study of these genes is necessary to determine their exact roles and regulation in the aforementioned functions and to investigate diseases associated with mutations in these genes. For such studies, a cell culture system is a requisite tool. Therefore, Sertoli cells were targeted, and a culture was established from cells isolated from canine testes. Cultures maintained consistent morphology and steady growth for up to seven passages. Cultured cells were identified as Sertoli cells through positive Western blot results for SOX9 and Clusterin B proteins and transcript sequence verification of SOX9 as well as the presence of type IV collagen transcripts. Primary cultures of canine Sertoli cells will provide a useful tool for study of the function and regulation of collagen genes and will permit new research pertaining to canine health while also serving as a model for the study of human diseases.

Pathohistological study of tumors in canine testes and ovarian in the period 1999-2003

Testes and ovarian tumors are tumors which appear in small percentages in dogs. They rarely lead to a lethal outcome in animals, but, due to the different clinical manifestations that can accompany these tumors, their timely diagnosis is significant because it often determines the treatment of the animal in which such tumors were diagnosed. During the period from 1999 until 2003, the Department for Pathological Morphology of the Belgrade Faculty of Veterinary Medicine examined macroscopically and pathohistologically 5 ovaries with macroscopic signs of neoplastic changes from 5 female and 25 neoplastically altered testes from 18 male dogs. In the testes, seminom, collision tumor (Sertoli cell tumor), Sertoli cell tumor, Leydig cell tumor, and lymphosarcoma were diagnozed. In the ovaries, granulosa cell tumor and dysgerminoma were diagnozed.

353 EFFECTS OF CULTURE DURATION AND CUMULUS CELL REMOVAL ON CANINE OOCYTE IN VITRO MATURATION AND FERTILIZATION USING VASAL SPERM

The objective of this study was to test the hypothesis that in vitro maturation (IVM) and fertilization (IVF) rates of canine oocytes could be improved by increasing culture duration or decreasing/increasing cumulus cell contact with the oocytes when using sperm retrieved from the vas deferens. The canine oocyte is ovulated at the germinal vesicle stage, and maturation of the oocyte occurs in the oviduct and requires up to five days. Therefore, an increase in the culture duration may cause an increase in oocyte nuclear maturation. Canine ovaries and testes were collected from a local clinic, placed in warm saline solution, and transported to the laboratory. Two distinct experiments were carried out, one involving IVM (M-II) after cumulus cell removal at 72 h and 96 h for nuclear maturation evaluation, and the second experiment the same but continued up to IVF. The oocytes were recovered from the ovaries by mincing them in warm Medium-199 with Hanks salts, L-glutamine, and HEPES (GIBCO, Grand Island, NY, USA; Invitrogen Co., Carlsbad, CA, USA). Canine oocytes with a dark cytoplasm and at least 2 layers of cumulus cells were cultured in Medium-199 supplemented with Earle&amp;apos;s salts, 2200 mg mL&amp;minus;1 sodium bicarbonate, 25 mM HEPES, 2 mM sodium pyruvate, 5 &amp;micro;g mL&amp;minus;1 progesterone, 100 ng mL&amp;minus;1 epidermal growth factor, 10 IU mL&amp;minus;1 human chorionic gonadotropin (HCG), 5 &amp;micro;g mL&amp;minus;1 insulin, 0.50 mM epinephrine, 10&amp;percnt; estrus bitch serum, 0.01 mM nonessential amino acids, and 20 &amp;micro;g mL&amp;minus;1 gentamicin. The oocytes were cultured for 72, 96, 120, or 144 h at 38.5&amp;deg;C in 5&amp;percnt; CO2 in humidified air. The cumulus cells were removed after either a 72- or 96-h culture period. For the first 48 h, the cumulus&amp;ndash;oocyte complexes were cultured in the modified Medium-199 containing 10 IU mL&amp;minus;1 HCG and then cultured in the same medium free of HCG. The oocytes were denuded by pipetting, stained with Hoechst 33342, and examined for nuclear maturation. ANOVA was used for statistical analysis of the data. The IVM rate (MII) was significantly higher (P &amp;lt; 0.05) at 72 and 96 h compared to 48, 120, and 144 h (15.1&amp;percnt; and 16.9&amp;percnt; vs. 6&amp;percnt;, 12.4&amp;percnt;, and 9.1&amp;percnt;, respectively). The removal of cumulus cells at 72 h and 96 h resulted in 17.9&amp;percnt; (43/240) and 14.8&amp;percnt; (35/236) IVM rate (MII), respectively (P &amp;gt; 0.05). The sperm motility index (SMI &amp;equals; motility percentage &amp;times; sperm activity grade) was significantly higher in sperm retrieved from the vas deferens (vasal sperm) compared to epididymal and testicular sperm (259 vs. 95 and 19.2, respectively, P &amp;lt; 0.05). The mature oocytes were inseminated by vasal sperm following in vitro hyperactivation with HEPES solution supplemented with 3 mg mL&amp;minus;1 bovine serum albumin. The IVF rates of the oocytes following 72 and 96 h of maturation in vitro were 48.2&amp;percnt; and 40&amp;percnt;, respectively (P &amp;gt; 0.05). Sperm penetration was significantly higher at 96 h compared to 72 h, and the number of sperm heads inside the ooplasm was 3.2 for the 72 h group vs. 4.8 for the 96 h group (P &amp;lt; 0.05). In conclusion, increasing the IVM culture period beyond 72 h did not increase the oocyte maturation rates, and increasing the culture time to 96 h without cumulus cells present increased the rate of sperm penetration.

Molecular Cloning and Characterization of Three β-Defensins from Canine Testes

ABSTRACTMammalian β-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine β-defensin isoforms (cBD-1,-2, and-3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the β-defensin consensus six-cysteine motif. The two longer isoforms,cBD-2and-3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine β-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenesandStaphylococcus aureus; MICs of 6 and 100 μg/ml, respectively), gram-negative bacteria (Escherichia coli,Klebsiella pneumoniae, andNeisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 μg/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 μg/ml) and lower activity againstUreaplasma urealyticumandU. canigenitalium(MIC of 200 μg/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine β-defensins were highly expressed in testis. In situ hybridization indicated thatcBD-1was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast,cBD-2was located primarily within Leydig cells. The longest isoform,cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine β-defensins play an important role in host defense and other physiological functions of the male reproductive system.

Sry and Sox9 expression during canine gonadal sex determination assayed by quantitative reverse transcription-polymerase chain reaction.

Testis induction is associated with gonadal Sry and Sox9 expression in mammals, and with Sox9 expression in vertebrates where Sry is absent. In mammals, Sry might initiate testis induction by upregulating Sox9 expression; however, direct evidence supporting this hypothesis is lacking. Models of Sry-negative XX sex reversal (XXSR), in which testes develop in the absence of Sry, could provide the link between Sry and Sox9 in testis induction. To define the stages at which testis determination occurs in the canine model, Sry and Sox9 expression were measured in normal urogenital ridges (UGR) and gonads by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Testicular Sry expression rose continuously during canine developmental ages comparable to human carnegie stages (CS) 16-18, with maximal expression at CS 18. Sox9 was expressed in both male and female canine UGR up to CS 17, at which time testis expression became tenfold greater than in the ovary. Although Sox9 was detected by qRT-PCR in ovaries and mesonephroi of both sexes, expression was detected only in canine testes by whole mount in situ hybridization (WMISH). The timing of Sry and Sox9 expression is consistent with a role in testis determination: Sry expression begins at CS 16 in testes, followed by upregulation of Sox9 expression at CS 17. The quantity and temporal and spatial patterns of Sry and Sox9 expression in normal canine gonads are similar to those in humans, sheep, and pigs. These studies should provide the basis for understanding the mechanism of testis induction in the canine model of Sry-negative XXSR.

HOMOLOGOUS TRANSPLANTATION OF PREPUBERTAL TESTES IN THE DOG1

The current standard of care for anorchia, the absence of both testes, is testosterone replacement therapy. Although transplantation of a single testis has been successful in a case of an identical twin (1), transplants in nonidentical siblings have not been performed. Homologous testis transplantation has been performed in sexually mature dogs, however, only one testis was transplanted, and histopathology alone was used to evaluate sperm production (2). The donor and recipient dogs in our experiment were two prepubertal (18-week-old) Beagle/Brittany littermates; the sire and dam of which shared a common ancestor. Based on results of a previous study (2), major histocompatability com- plex testing was not performed. The testes were harvested from the donor and transplanted into the recipient within 1 hour, via a prescrotal incision, by end-to-end anastamosis of the testicular artery, vein, and vas deferens. Immediately before anastamosis of the first testis, the recipient received 1000 U of heparin i.v. to reduce the risk of thrombosis. After anesthetic recovery, the dog was maintained on a course of previously successful immunosuppressive therapy and anticoagulants (2). The recipient lacked signs of graft-versus-host disease, or deleterious effects of the cyclosporine and prednisone therapy. Serum testosterone levels were monitored weekly by radioimmunoassay (Diagnostic Systems Laboratories, Webster, TX) from 18 to 39 weeks of age (see Fig. 1). Testosterone levels may fluctuate by as much as 2.5 ng/ml during a 24hr period in an individual dog, however, levels in castrated animals are consistently <0.20 ng/ml (3). A gradual increase in baseline testosterone, with mean levels approaching the normal range for a sexually mature animal (0.5–5.0 ng/ml), signals the onset of puberty (4). Figure 1: Longitudinal serum testosterone levels (ng/ml) from the day of transplantation, 18 weeks of age, through 39 weeks of age. Minimum testosterone level for a sexually mature dog is 0.20 ng/ml (indicated by dashed line).When the recipient reached 7 months of age, a human chorionic gonadotrophin (hCG) stimulation test was performed in order to determine the responsiveness of Leydig cells, which produce testosterone. The dog received 44 μg/kg hCG i.m. and serum was collected 4 and 24 hr later. Baseline testosterone was 0.83 ng/ml and increased to 1.35 ng/ml at 4 hr and 2.46 ng/ml at 24 hr, consistent with functional Leydig cells (3, 4). At 8 months of age, serum testosterone remained consistently within the normal range for a sexually mature dog. Semen was collected at 8 months of age, using an artificial vagina, in the presence of a bitch in estrus. A total of 300×106 sperm/ejaculate were present, 80% of sperm were progressively motile, and 85% were morphologically normal. All parameters are known indicators of fertility and were within normal limits for fertile dogs (3). Lack of graft-versus-host disease did not guarantee normal sperm production, as many factors and functional interactions are involved in organ maturation and spermatogenesis. At 17 months of age, the dog was bred to a bitch in estrus, which was confirmed by ultrasound to be pregnant with a litter of 4 pups. This case is the initial report of bilateral transplantation of prepubertal canine testes with subsequent maturation, semen evaluation, and proven fertility. These findings suggest that the dog may be a suitable animal model to evaluate microsurgical techniques and immunosuppressive therapies for use in prepubescent humans receiving a testis transplant. If successful, homologous transplantation among siblings would provide stable testosterone levels and restore fertility. Jennifer K. Pullium2 Peter H. Lin, and Martin J. Pinter

Characterization of angiotensin-converting enzyme in canine testis

The aim of this study was to characterize angiotensin-converting enzyme (ACE) in canine testis. Detergent-extracted canine testes were sonicated in the presence of protease inhibitors and purified on an affinity column with the ACE inhibitor, lisinopril, as an affinity ligand for ACE. The fractions recovered were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based on the hydrolysis of Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8 min incubation. The specific activity of ACE in the starting testicular extracts was 3.53 +/- 0.99 mU mg(-1) protein with a 1588 times enrichment in ACE activity after lisinopril affinity chromatography (4239 +/- 2600 mU mg(-1) protein). The recovery efficiency of ACE after lisinopril affinity chromatography was 71.2%. The ACE activity in the detergent extracts and the purified fractions was inhibited significantly by 10 micromol captopril l(-1), a specific ACE inhibitor, and was restored to 88% of normal activity by the addition of the thiol-alkylating agent N-ethylmaleimide (0.5 mmol l(-1)) in the detergent extracts and the purified fractions incubated with captopril. The treatment of testicular extracts with 10 mmol EDTA l(-1) reduced the ACE activity significantly (5.40 +/- 1.26 versus 0.58 +/- 0.23 mU mg(-1)). The ACE activity was restored fully in the presence of zinc (5.28 +/- 0.70 mU mg(-1)). The anti-ACE antibody (raised against a 70 kDa protein from the periacrosomal plasma membrane of equine spermatozoa) recognized a 65-70 kDa protein in the detergent-extracted testes as well as in the affinity-purified fractions. This antibody also recognized a protein of similar molecular mass in ejaculated spermatozoa. ACE was localized in the periacrosomal area of the ejaculated spermatozoa and in spermatids in the seminiferous tubules. The results of this study demonstrate that ACE is present in canine testis and retains its enzyme activity after purification with lisinopril affinity chromatography. Activity of canine ACE is inhibited by captopril and EDTA and is restored in the presence of N-ethylmaleimide and zinc.

Characterization of angiotensin-converting enzyme in canine testis

The aim of this study was to characterize angiotensin-converting enzyme (ACE) in canine testis. Detergent-extracted canine testes were sonicated in the presence of protease inhibitors and purified on an affinity column with the ACE inhibitor, lisinopril, as an affinity ligand for ACE. The fractions recovered were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based on the hydrolysis of Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8 min incubation. The specific activity of ACE in the starting testicular extracts was 3.53 +/- 0.99 mU mg(-1) protein with a 1588 times enrichment in ACE activity after lisinopril affinity chromatography (4239 +/- 2600 mU mg(-1) protein). The recovery efficiency of ACE after lisinopril affinity chromatography was 71.2%. The ACE activity in the detergent extracts and the purified fractions was inhibited significantly by 10 micromol captopril l(-1), a specific ACE inhibitor, and was restored to 88% of normal activity by the addition of the thiol-alkylating agent N-ethylmaleimide (0.5 mmol l(-1)) in the detergent extracts and the purified fractions incubated with captopril. The treatment of testicular extracts with 10 mmol EDTA l(-1) reduced the ACE activity significantly (5.40 +/- 1.26 versus 0.58 +/- 0.23 mU mg(-1)). The ACE activity was restored fully in the presence of zinc (5.28 +/- 0.70 mU mg(-1)). The anti-ACE antibody (raised against a 70 kDa protein from the periacrosomal plasma membrane of equine spermatozoa) recognized a 65-70 kDa protein in the detergent-extracted testes as well as in the affinity-purified fractions. This antibody also recognized a protein of similar molecular mass in ejaculated spermatozoa. ACE was localized in the periacrosomal area of the ejaculated spermatozoa and in spermatids in the seminiferous tubules. The results of this study demonstrate that ACE is present in canine testis and retains its enzyme activity after purification with lisinopril affinity chromatography. Activity of canine ACE is inhibited by captopril and EDTA and is restored in the presence of N-ethylmaleimide and zinc.

Detection of keratinocyte growth factor (KGF) messenger ribonucleic acid and immunolocalization of KGF in the canine testis

Keratinocyte growth factor (KGF) was originally discovered in human embryonic lung fibroblasts and is a member of the fibroblast growth factor (FGF) family. Members of the FGF family have been shown to regulate testicular function. However, the recently discovered KGF has not been studied in the testis. KGF has been detected in many other tissues, including the prostate, an organ whose development and function have been associated with presence of the testis. In this study, KGF mRNA was detected in the whole testis using reverse transcription polymerase chain reaction (RT-PCR). The 575-bp KGF-specific product was detected along with a 594-bp β-actin-specific product. To identify the cell types in which KGF mRNA was predominantly expressed, interstitial cells were physically separated from seminiferous tubules. The interstitial cells were then sorted on a discontinuous Percoll gradient and total cellular mRNAs isolated. Using RT-PCR and Southern hybridization with specific cDNA probes, the KGF mRNA was detected in interstitial cells. KGF expression levels were then evaluated semiquantitatively with a competitive RT-PCR assay. KGF expression levels were highest in interstitial cells that equilibrated between 20 and 30% Percoll. Enriched Leydig cells and seminiferous tubules expressed low levels of KGF. Finally, immunohistochemical analysis was performed on canine testes using a rabbit anti-KGF polyclonal antibody. The KGF protein was localized predominantly to peritubular cells of the canine testis. These results suggest that KGF is synthesized in the canine testis.

The influence of temperature on changes in pH, lactate and morphology during testicular ischaemia.

To examine the role of temperature in the irreversible changes that occur within 4-6 h as ischaemic atrophy develops in the testis during testicular torsion, by determining effects on testicular pH, lactate accumulation and morphology. Tissue acidification (pH), tissue lactate and structural changes were measured at temperatures of 35, 25, 15 and 5 degrees C in 34 human testes obtained as orchidectomy specimens from patients with metastatic prostatic cancer, and in 19 testes taken from young dogs. At a normal testicular temperature of 35 degrees C, the pH decreased to 6.0 within 2 h of the onset of ischaemia; cooling to 15 degrees C extended this delay to 6 h. Tissue lactate increased from 25 mumol/g dry weight to about 200 mumol/g at 35 degrees C. Semi-thin sections of the canine testes showed swelling of the intratubular tissue with loss of interstitial space; lower temperature delayed these changes. In the present study, 6 h of torsion was relatively prolonged, in that the pH decreased to 5.8 and testicular tissue was destroyed. Acidification and histological damage can be prevented by cooling. The critical pH of the testis beyond which irreversible changes occur is unknown; a pH of < 6.0 is likely to provoke such changes.

Ultrasonic measurement of canine testes

The goals of the present study were to determine if ultrasonic measurement of testicular dimensions (length, width, and height) would provide an accurate assessment of canine testicular size (weight) and to determine the relationship of these measurements to animal body weight. The bodies of 30 intact male dogs of unknown health, breed or breeding history were obtained after the dogs were humanely killed at the local animal shelter. Total scrotal width (TSW) was measured by calipers and the length, width and height of each scrotal testis, excluding the epididymis, were measured by sonography. The testes were then excised and weighed, again excluding the epididymis. Multiple regression was used to predict total testicular weight from 1) only sonographic measurements (Model 1), 2) all testicular measurements (Model 2), 3) only total scrotal width (Model 3), and 4) only body weight (Model 4). In addition, stepwise multiple regression was used to identify models (Models 5 and 6) using external measurements of the testes which seemed most useful in predicting total testicular weight. Models 1, 2, 3, 4, and 6 yielded r 2 values 0.90, 0.94, 0.88, 0.48 and 0.95 respectively. Model 5 yielded an r 2 of 0.90, but the additional accuracy achieved by using the testicular height was minimal. Although sonographic testicular measurement accurately predicted testicular weight, the small degree of additional accuracy achieved over TSW measurement by calipers does not justify the use of sonography in each case. However, if a testicular ultrasound scan is being performed, the ultrasonic measurements could be used to predict testicular weight.

Production and Action Mechanism of Sex Hormones in Male Dogs

The present study was undertaken to assess the production and the action mechanism of sex hormones in male dogs. The following results were obtained. 1) Plasma concentrations of LH, androstenedione, testosterone, 5 α-dihydrotestosterone (DHT) and estradio1-17 β were measured by the radioimmunoassay method. The level of LH increased with days of age and stayed constant from 8 years through 13 years of age in male dogs. The level of testosterone increased with days of age and stayed constant from 1 year through 10 years of age. Thereafter, the level of testosterone declined gradually. In contrast, there was no age-related increase in the level of estradiol-17β in mature dogs. 2) By measuring androstenedione, testosterone and DHT simultaneously in peripheral and spermatic vein plasma, and determining the temporal relationship between the two vein plasma levels of these hormones, it was suggested that plasma testosterone and DHT were derived mainly from the testis, while plasma androstenedione was derived from the testis and other organs like the adrenal. 3) Plasma concentrations of LH, androstenedione, testosterone and DHT in mature dogs fluctuated episodically, but neither diurnal rhythm nor annual variation of LH and testosterone concentrations was observed. 4) It was suggested that the pathway for activation of steroidogenesis in canine testes by LH was through cAMP formation. 5) In canine testes, major C19-metabolites of pregnenolone were dehydroepiandrosterone, androstenedione and testosterone. Following the addition of a 17α-hydroxylase inhibitor to the testis caused a significant increase in pregnenolone production, indicating that the formation of testosterone from pregnenolone by the Δ 5-pathwaywas dominant in androgen biosynthesis of the testis. 6) Hexestrol dicaprylate (H8), a long-acting estrogen, was used in an attempt to induce temporary sterilization of male dogs. H8 treatment caused significant decreases in the gonadotrophic activities of the pituitary gland and testosterone formation by the testis with the inhibition of spermatogenesis in the dog. It was suggested that H8 interfered with testosterone production by blocking the pituitary function, and also directly inhibited the testicular steroidogenesis. 7) In canine prostates, major metabolites of testosterone were DHT and 5α-androstanediols. DHT had a high binding affinity for androgen receptor in the prostate, while 5α-androstanediols had a low affinity for the receptor. The prostate was shown to contain estrogen receptor. Following injection of estrogen to dogs, it bounded to the estrogen receptor in the prostate gland and caused the enlargement of the gland.

Assessment of testicular metabolic integrity with P-31 MR spectroscopy.

To evaluate the reliability of phosphorus-31 magnetic resonance (MR) spectroscopy in the assessment of acute testicular ischemia, vascular integrity, and spermatogenesis, the authors studied in vivo canine and primate testicles grouped as follows: group 1 testes (n = 8), in situ canine controls; group 2 (n = 11), canine testes subjected to warm ischemia; group 3, canine (n = 4) and primate (n = 4) testicles from hormone-treated animals. Group 1 control testicles showed high monophosphoester (MP) levels; low levels of inorganic phosphate (Pi), phosphodiester (PD), and phosphocreatine; and high levels of adenosine triphosphate (ATP). Group 2 testes revealed a time-dependent decay of MP/Pi ratios (from 2.1 to 0.70). Regeneration of ATP was noted in the acute reperfusion period. After 6 weeks of pituitary gonadotropin suppression, group 3 testes showed a significant decrease (P less than .05) in MP/PD ratios from a control level of 2.6 +/- 0.3 and a decrease in the MP/beta-ATP ratio from 2.4 +/- 0.1 to 1.8 +/- 0.3. P-31 MR spectroscopy appears to be a potential method for noninvasively assessing testicular ischemic injury and the metabolic integrity of spermatogenesis.

Inhibition of testosterone synthesis in the canine testis in vitro

Testicular 17β-hydroxysteroid oxidoreductase (17β-HOR) is one of the several enzymes necessary for the synthesis of testosterone, but is not required for either glucocorticoid or mineralocorticoid synthesis. Since specific inhibition of the testicular enzyme has potential contraceptive and experimental uses, the inhibitory effect of twenty steroids on 17β-HOR activity was examined in microsomal preparations of canine testes. Six steroids inhibited testosterone formation, but only 4-estrene-3,17-dione (K I=2.4 μM) and 5-androstene-3,17-dione (K I=6.8 μM) had significant inhibitory activity. The data suggest the following molecular characteristics necessary for competitive inhibition of 17β-HOR activity: (a) requirement for 17-keto group, (b) relative requirement for 3-keto group, (c) decreased inhibition with unsaturation in position 5–6, and (d) marked loss of inhibitory activity with 6β-, 11β or 19- hydroxylation, and A-ring aromatization.