Concentrations of oestradiol and testosterone in peripheral and spermatic venous blood of dogs with unilateral cryptorchidism

Plasma testosterone and oestradiol concentrations were measured in peripheral and spermatic venous blood of 13 dogs with unilateral inguinal cryptorchidism, 9 dogs with unilateral abdominal cryptorchidism, and in a control group of 36 mature normal dogs. The hormone concentrations were similar in the three groups, both in the peripheral and in the spermatic venous blood. The weight of the testes in the control group was correlated with the body weight and there was no significant difference between the weight of the right and the left testes. The weight of the abdominal testes was lower than that of the inguinal testes, and there was no compensatory enlargement of the contralateral scrotal testis. The cryptorchid testes showed little or no histological evidence of spermatogenesis, and spermatogenesis was usually normal in the scrotal testes.     

Immunohistochemical study of osteopontin in boar testis

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.     

Prenatal and neonatal exposure to flutamide affects function of Leydig cells in adult boar

In this study, flutamide, an androgen receptor antagonist, was used as a tool to better understand the role of androgen receptor signaling and androgen signaling disruption during fetal and neonatal periods on porcine Leydig cell development and function. Flutamide, 50 mg kg(-1) d(-1) was administered into pregnant gilts during gestational days 20 to 28 and days 80 to 88 and into male piglets on postnatal days 2 to 10 (PD2). Leydig cells of flutamide-exposed boars, especially those of PD2 males, displayed morphologic alterations, increased size, and occupied increased area (P < 0.001) of the testes when compared with the control. Despite this, testosterone concentrations were reduced significantly in comparison with those of controls (P < 0.05, P < 0.001). Reduced testosterone production in response to flutamide exposure appeared to be related to changes in testosterone metabolism, as shown by increased aromatase mRNA (P < 0.05, P < 0.01), protein expression (P < 0.01, P < 0.001), and elevated estradiol concentrations (P < 0.001). Moreover, impaired Leydig cell responsiveness to LH was indicated by the decreased expression of LH receptor (P < 0.05, P < 0.001). No significant effect of flutamide was found on LH and FSH concentrations. Taken together, our data indicate that flutamide when administered during prenatal or neonatal period have a long-term effect on Leydig cell structure and function, leading to androgen-estrogen imbalance. Leydig cell failure was most evident in adult boars neonatally exposed to flutamide, suggesting that androgen action during neonatal development is of pivotal importance for the differentiation and function of porcine adult Leydig cell population.     

性激素受体在子午岭黑山羊隐睾及正常睾丸中的分布比较

旨在研究性激素受体[雄激素受体（androgen receptor,AR）、雌激素受体（estrogen receptor,ER）]在子午岭黑山羊正常睾丸与隐睾组织中的分布与隐睾症的关系,应用免疫组织化学及免疫荧光技术方法结合形态计量学统计软件,比较了正常睾丸与隐睾的组织化学特点。特殊染色结果显示：与正常组相比,隐睾组间质组织疏松,管腔面积明显减小,糖原含量明显减少,胶原纤维及网状纤维含量增多。免疫组化及免疫荧光结果显示：1） AR在正常睾丸组Leydig细胞呈高密度强阳性表达,在各级生精细胞呈中等强度阳性表达,隐睾组中Leydig细胞及各级生精细胞表达明显减弱,且在精原细胞偶见表达;2） ER在正常睾丸Leydig细胞呈高密度强阳性表达,管周肌样细胞呈中等强度阳性表达,Sertoli细胞偶见表达,各级生精细胞无表达;3） ER在隐睾Leydig细胞、初级精母细胞、精原细胞和Sertoli细胞中均呈中等强度阳性表达;4）统计结果显示,隐睾组AR的平均光密度较正常组显著降低（P<0.05）,而ER的平均光密度则显著高于正常组（P<0.01）,且隐睾组AR与ER表达量比值基本接近1：1。子午岭黑山羊隐睾组织胶原纤维和网状纤维分布较正常睾丸多,生精小管基膜主要成分以中性糖蛋白为主,酸性糖蛋白含量明显降低影响精子的正常形成;隐睾组织精原细胞及Sertoli细胞ER与AR表达失常尤为明显,可为哺乳动物隐睾的相关研究提供一定参考。     

Castration of the Vietnamese pot-bellied boar: 8 cases

Surgical techniques for castration of the Vietnamese pot-bellied boar and outcome are described. Vietnamese pot-bellied pig (VPBP) boars (n = 8) were admitted for castration. Data retrieved from medical records (2002–2011) for these pigs included signalment, history, reason for castration, perioperative management, surgical technique, and complications. Follow-up information was obtained from owners. A scrotal approach with closed technique was used for 6 boars with normally descended testes. A scrotal approach and open technique was used in 1 inguinal cryptorchid boar. In a hemicastrated abdominal cryptorchid boar an ipsilateral parainguinal approach was used. No complications occurred. Castration of the Vietnamese pot-bellied boar is associated with minimal complications and a satisfactory cosmetic outcome. We recommend the routine closure of the external inguinal rings, a simple and fast procedure that may prevent post-castration inguinal herniation.     

Relationships of testicular iron and ferritin concentrations with testicular weight and sperm production in boars

The inverse relationship of testicular size and circulating follicle-stimulating hormone (FSH) concentrations has been documented, and accompanying this relationship is the change in color of the parenchymal tissue of the testes. Large testes (300 to 400 g) are pink to light red and small testes (100 g) are dark maroon with color gradations for weights in between. It was hypothesized that this color most likely represented an iron protein. Chromatographic analysis of testicular tissue indicated that the Fe was associated primarily with ferritin, and immunohistochemistry showed that Leydig cells were the primary location of ferritin storage within the testes. Concentrations of Fe and ferritin were higher in small testes and decreased as testes weight increased (P < 0.05). As testicular Fe concentrations increased, daily sperm production (DSP) and total DSP declined (P < 0.05). Genotyping six generations of Meishan x White composite boars (n = 288) for a quantitative trait locus that is indicative of elevated FSH and small testes in boars indicated that the Meishan genotype had elevated testicular iron concentrations and darker color in conjunction with reduced total DSP (P < 0.01). It is not thought the elevated iron concentrations affect testicular weights but are probably a result of elevated FSH and FSH inducement of Fe transport. The storage of Fe in Leydig cells may provide a reservoir of Fe for easy access by Sertoli and germ cells, but still provide a degree of protection to germ cells from ionic iron.     

Reducing endogenous estrogen during development alters hormone production by porcine Leydig cells and seminiferous tubules

High levels of estrogen produced by boar testes and the presence of estrogen receptors in both interstitial and tubular compartments are consistent with a direct role for estrogen in regulation of testicular cell function. This study investigated the importance of estrogen on hormone production by Leydig cells and seminiferous tubules in the developing boar. Thirty-six 1-week-old littermate pairs of boars were treated weekly with vehicle or 0.1 mg/kg BW Letrozole, an aromatase inhibitor, until castration at 2, 3, 4, 5, 6, 7, or 8 months. Tissue was collected and Leydig cells and seminiferous tubules were isolated. In a separate study, five untreated boars (ages 1.5-4 months) were castrated and Letrozole was added in vitro to Leydig cell and seminiferous tubule cultures. Leydig cells were cultured for 24h with and without porcine LH. Media were assayed for estradiol (E(2)) and testosterone (T) concentrations by RIA. Seminiferous tubules were cultured for 4h with and without porcine FSH; media were assayed for E(2) and immunoreactive inhibin (INH). In vivo aromatase inhibition decreased basal E(2) and increased basal T production by cultured Leydig cells. Basal seminiferous tubule production of E(2) but not INH was reduced. Decreasing estrogen synthesis in vivo did not alter LH-induced Leydig cell E(2) production or FSH-induced seminiferous tubule INH production. INH production decreased with advancing age regardless of treatment. In conclusion, in vivo aromatase inhibition altered baseline steroid production by cultured Leydig cells and seminiferous tubules but had little effect on response to gonadotropins.     

Morphological and immunohistochemical studies in testes of normal and cryptorchid boars

Scrotal (n = 66), inguinal (n = 7) and abdominal (n = 36) testes from pigs differing in age (from 1 day to 4 years) were collected after slaughtering or by castration, fixed in 4% formalin and examined by light microscopy. The expression of vimentin, desmin, alpha-actin and PCNA was detected by immunohistochemical technique. Histologically no differences between scrotal and cryptorchid testes are obvious during the prepubertal period, until the age of 10-12 weeks, but with the onset of puberty degenerative alterations (Sertoli-cell-only morphology, giant cells) in dystopic testes occur. Additionally the differentiation of the interstitial cells is disturbed in abdominal testes resulting in a deviating expression pattern of alpha-actin. The Sertoli cells show different distribution patterns (basal, perinuclear, apical) of vimentin depending on the position of the testis and the age of the animal.     

Expression of connexin 43 protein in testes, epididymides and prostates of stallions

REASONS FOR PERFORMING STUDY: Connexin 43 (Cx43) is a ubiquitously distributed gap junction protein in testes and other reproductive tissues. Adjacent cells share ions and small metabolites through intercellular channels, which are present in gap junctions. Previously, Cx43 has not been reported in testes, epididymides and prostates either in healthy stallions or cryptorchid horses. OBJECTIVES: To demonstrate the expression pattern of Cx43 in the reproductive tissues of stallions and examine whether naturally occurring bilateral cryptorchidism has any influence on distribution and expression of Cx43. METHODS: The expression and the presence of Cx43 protein were detected by means of immunohistochemistry and Western blot analysis using a polyclonal rabbit anti-Cx43 antibody. RESULTS: In stallions, gap junctions appeared as structures localised to cell-cell contacts between adjacent cells. In testes, Cx43 expression was detected in the interstitial tissue and seminiferous tubules, between Leydig and Sertoli, as well as Sertoli and germ cells. In epididymides, Cx43 was localised between epithelial cells, whereas in prostates, between secretory cells of the glandular epithelium. In the cryptorchid, a clear reduction of Cx43 signal was observed in all reproductive tissues. CONCLUSIONS: Coupling of Leydig cells via gap junctions may suggest that steroidogenic function of the testis is under the influence of these intercellular channels. Within seminiferous tubules, the expression was found to be stage-specific, pointing to its role in coordinating spermatogenesis. Differential distribution of Cx43 protein in the reproductive tract of normal and cryptorchid stallions indicates that expression is clearly dependent on the physiological status of the horse. POTENTIAL RELEVANCE: Detection of Cx43 expression in equine testicular, epididymal, and prostatic cells is important for a better understanding of the role of intercellular membrane channels in direct cell communication within the reproductive tract of stallions.     

Electroejaculation and artificial insemination in Vietnamese potbellied miniature pigs

A 15-month-old Vietnamese miniature boar was examined because of suspected infertility. A breeding soundness examination was conducted, using electroejaculation under anesthesia for semen collection. Semen values were normal despite a subpubic location of the testes. Artificial insemination of a gilt with extended semen resulted in the birth of a litter 111 days later. Vietnamese potbellied boars have small and sometimes nearly undetectable scrotal pouches, which may cause the producer to question the fertility of the boars.     

Endocrine and sperminogenic testicular function. 7. Relationship between testicular and plasma androgen concentrations in boars of different ages]

Testicular tissue and blood samples (V. spermatica interna) were taken from 32 boars during castration. The animals were of different age groups. Against this background, comparative studies were conducted into the relationships between testicular and plasma testosterone. A very close correlation was found to exist between the androgen values in testicular tissue and those in the blood plasma of V. spermatica interna (r = 0.9795), wich appeared to support the conclusion that by determination of blood plasma the androgen content in the testes can be established with high probability.     

A lectin histochemical study of the thoracic respiratory air sacs of the fowl

The lectin-binding characteristics of the epithelial lining of the thoracic air sacs of the chicken were determined. Con A, LCA and PSA bound to the apical membrane as well as to the cytoplasm distal to the nucleus of the surface epithelium, indicated the presence of a-linked mannose as well as N-acetylchitobiose-linked alpha-fucose residues in the glycoproteins. GSL I bound to the apical membrane and cytoplasm distal to the nucleus, but not to the cilia of the epithelium, where-as MPL, DBA and RCA120 bound to the apical membrane, cilia and cytoplasm, indicated the presence of a-linked N-acetylgalactosamine residues. However, neither SJA or SBA showed any binding, indicating the absence of beta anomers of galactosyl (beta1.3)N-acetylgalactosamine and beta-linked N-acetylgalactosamine residues. UEA I bound to the apical membrane and cilia, as well as to the cytoplasm of a few cells, indicated the presence of alpha-linked fucose residues. PNA bound to the apical membrane of some, but not all, surface epithelium cells, indicated the presence of galactosyl (beta1.3)N-acetylgalactosamine residues. WGA bound to the apical membrane and cilia, as well as to the cytoplasm of a few cells, indicated the presence of neuraminic acid residues.     

Effects of experimental cryptorchidism on sperm motility and testicular endocrinology in adult male rats

The effect of induced cryptorchidism on testicular function and sperm motility was investigated. Bilateral cryptorchidism was created surgically in adult male rats (treated group), and sham-operated rats were used as a control group. Five rats from each group were sacrificed on days 1, 3, 5, and 7 after surgery. The percentage of motile spermatozoa began to decrease 1 day after the operation, followed by an abrupt decline 3 and 5 days later in cryptorchid rats. Furthermore, there were significant decreases in the other sperm motility parameters 5 days after inducement of cryptorchidism. In cryptorchid rats, plasma concentrations of LH, FSH, testosterone, and inhibin B were significantly lower than in the control group 1 day after the operation. Thereafter, plasma concentrations of LH, FSH, and testosterone gradually increased in the cryptorchid rats. On the other hand, plasma concentrations of inhibin B showed a further decline from day 3 after the operation onward. Concentrations of immunoreactive (ir)-inhibin, but not testosterone, in testicular interstitial fluid were remarkably increased until 3 days after surgery in the cryptorchid rats, and declined thereafter. Testicular response to human chorionic gonadotropin (hCG) for testosterone release was decreased in the cryptorchid rats compared with the control rats, indicating that heat stress to testes resulted in a reduction of the activity of Leydig cells and Sertoli cells. These results clearly indicate that heat stress to the testes resulted in a significant reduction of sperm activity within 3 days, and this was followed by changes in testicular endocrine function.     

Histological and sex steroid hormone receptor changes in testes of immature, mature, and aged chickens

Sex steroid hormone receptors play a central role in the regulation of reproduction in male chickens. In this work, we evaluated by histomorphometric methods and Western blot analysis changes in the number of the different cell populations and in the content of sex steroid hormone receptors in testes from immature (1.5-month-old), mature (12-month-old), and aged (48-month-old) chickens. The number of Sertoli cells, germ cells, and Leydig cells per area of testicular tissue markedly changed according to chicken age. The highest number of Sertoli and Leydig cells was found in testes of immature chickens, with a dramatic decrease in those of mature chickens; however, the number of germ cells was the highest in mature chickens in comparison with other ages. The content of androgen receptor diminished in testes of mature and aged animals in comparison with that of immature chickens. In contrast, the content of estrogen receptor alpha and progesterone receptor was higher in testes of mature animals than in other ages. Both progesterone receptor isoforms were expressed in a similar proportion in testes of immature and mature animals. Interestingly, progesterone receptor isoform A was the predominant isoform in aged animals. These results suggest that there are marked age-dependent changes in chicken testes histology and in sex steroid hormone receptors content that should contribute to sex steroid hormone actions, in this tissue throughout the lifespan of chickens.     

Use of Contrast‐Enhanced Ultrasonography in Chronic Pathologic Canine Testes

Contrast‐enhanced ultrasound with sulphur hexafluoride microbubbles was performed in seven healthy dogs without a history of reproductive pathology and with histologically confirmed normal testes and in 42 dogs with chronic scrotal anomalies. All dogs underwent orchiectomy and histological examination. Enhancement patterns and perfusion parameters (peak intensity and regional blood flow) of testes of healthy dogs and testes with chronic lesions were compared. Fourteen non‐pathologic and 60 pathologic testes were considered. Forty testes were neoplastic (24 interstitial cell tumours, 9 seminomas, 7 Sertoli cell tumours), 20 were non‐neoplastic (16 testicular degenerations, 2 chronic orchitis, 1 testicular atrophy, 1 interstitial cell hyperplasia). In healthy dogs, the contrast medium flow had a rapid homogeneous wash‐in and wash‐out, with a short peak phase. With contrast ultrasound, testes that were inhomogeneous with a hyperenhancing pattern were associated with neoplasia (sensitivity: 87.5%, specificity: 100%). Lesions with persistent inner vessels and a hypo‐to‐isoechoic background were significantly associated with seminomas (sensitivity: 77.8%, specificity: 100%). Testes with non‐neoplastic lesions were characterized by a scant/moderate homogeneous enhancement. Perfusion parameters were higher in neoplastic lesions. Contrast ultrasound was a feasible diagnostic tool in the assessment of testicular lesions, with hyperenhancement being an important feature in the diagnosis of malignancy.     

INSL-3 protein expression in normal and cryptorchid testes of Ziwuling black goats

Ziwuling black goats are typically found in loess plateaus regions and the Ziwuling Nature Reserve. Cryptorchidism is a common disease in this inbred goat, and its pathogenesis has been linked with the expression of insulin-like factor 3 (INSL-3). Therefore, this study aimed to investigate anatomical alterations caused by cryptorchism and the expression and distribution of INSL-3 in normal and cryptorchid testicular tissues. The testicular tissues of 6-month-old Ziwuling black goats were collected for microscopic analyses using histochemical, immunohistochemical, immunofluorescence and biometrical methods, as well as Western blotting to compare the expression and distribution of INSL-3. A lower expression of INSL-3 was observed in cryptorchid compared with normal testicular tissues (p < .01). Cryptorchidism caused a significant reduction in layers of spermatogenic epithelium and tubule areas in Ziwuling black goat (p < .01). The interstitial to seminiferous tubule area ratio was larger in cryptorchid than in normal group. Periodic Acid-Schiff (PAS) staining revealed pronounced positive bands in the interstitial tissue, while positive Alcian blue (AB) staining was not clear, and AB-PAS staining revealed a positive red band in the basement membrane of cryptorchid group. Immunofluorescence revealed a strong signal of INSL-3 expression in Sertoli and peritubular myoid cells, and moderate signal in Leydig and spermatogenic cells in the normal group. However, in cryptorchid testicular tissues, the signal of INSL-3 expression was strong in primary spermatocytes, occasional in Sertoli cells, limited in Leydig cells and absent in peritubular myoid cells. Furthermore, immunohistochemistry showed that INSL-3 expression was higher in normal testes compared with cryptorchid testicular tissues (p < .05), especially in primary spermatocytes and Sertoli cells. Collectively, our results indicate that cryptorchidism is closely related to the disorder of acid glycoprotein metabolism and the reduction in release of INSL-3 from Leydig cells. Moreover, Sertoli and peritubular myoid cells are crucial for INSL signalling and could underpin further research on the mechanism of cryptorchidism in animal.     

Decision Making for Cryptorchid Castration; a Retrospective Analysis of 280 Cases

The location of an undescended testicle influences the choice of surgical technique for efficient cryptorchid castration. We review a standardized protocol for preoperative examination to dictate surgical approach to cryptorchidism. Cases are split into two periods: 2004–2006 and 2007–2014. In 2004–2006, conventional cryptorchidectomy and laparoscopic cryptorchid castration (standing) were both offered, but the choice of technique was based primarily on owners' preference for a recumbent or standing procedure. In 2007–2014, ultrasonography was used to locate the testes and dictate the preferred surgical approach; for abdominal testes, laparoscopic intraabdominal spermatic cord ligation without orchidectomy was preferred and for inguinal testes, conventional open orchidectomy. The numbers of animals requiring a second procedure to complete castration were compared between the two periods. In addition, failure rates for individual testes grouped by location were determined separately for the different techniques, and the value of preoperative ultrasonography to locate the retained testes was assessed. In 2004–2006, 15.3% (20/131) of the cryptorchids needed more than one surgery to complete castration, compared to 0.7% (1/144) in 2007–2014. Failure rates for laparoscopic castration were 0/168 (0%) for abdominal, 3/40 (7.5%) for inguinal, and 9/55 (16.4%) for scrotal testes; for conventional castration, failure was recorded for 3/12 (25%) abdominal and 0/92 (0%) inguinal testes. For 94% (156/166) of retained testes, ultrasound-based preoperative advice on surgical approach was correct. Using a standardized preoperative examination to determine choice of surgical technique significantly (P < .001) reduced the number of second surgeries needed to complete castration. Preoperative ultrasound is therefore a useful aid to determining the surgical approach to cryptorchid castration.     

Bilateral Sertoli and Interstitial Cell Tumours in Abdominal Testes of a Goat with Polled Intersex Syndrome (PIS)

An 8‐year‐old, mixed breed, polled goat was presented for evaluation of male‐like behaviour. Clinical findings included clitoromegaly, a heavily muscled neck, pronounced beard, and erect dorsal guard hairs, which are phenotypic characteristics commonly observed in intersex animals. Transrectal ultrasonography revealed the presence of two abdominal masses caudolateral to the uterine horns. Serum concentration of estradiol was elevated. Genetic evaluation was compatible with polled intersex syndrome defined by an XX karyotype without a Y chromosome or SRY gene. Based on gross and histologic evaluation, the abdominal masses were determined to be intra‐abdominal testes, each of which was effaced by Sertoli cell and interstitial (Leydig) cell tumours. The Sertoli cell tumours (SCTs) represented two unique histologic patterns. Regardless of pattern, neoplastic Sertoli cells were consistently lipid laden and positive for vimentin. Interstitial cell tumours (ICTs) were negative for vimentin. Clinical and histopathologic findings suggest that prolonged exposure to steroids secreted by neoplastic Sertoli cells contributed to virilization. In addition, results from immunohistochemistry indicated that vimentin may be a valuable immunodiagnostic tool for differentiation between interstitial and Sertoli cell tumours in goats.     

Immunolocalization of steroidogenic enzymes P450scc, 3betaHSD, P450c17, and P450arom in Göttingen miniature pig testes

The objective of this study was to investigate immunolocalization of steroidogenic enzymes in G?ttingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3betaHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.