Ageing changes testes and epididymis blood flow without altering biometry and echodensity in dogs.

Senescence leads to deleterious effects in testicular function, sperm quality and fertility in dogs. There, however, are no consistent results of vascular changes in the testes and epididymis during natural ageing in dogs. The aim of this study, therefore, was to compare testes and epididymis blood flow, biometry and echodensity between young and senile dogs. Ten young dogs (1-4 years) and eight senile dogs (over 7 years) were selected and assigned to two experimental groups: Young Group and Senile Group. Dogs were evaluated using testicular and epididymis B-mode (dimensions and echodensity) and Doppler ultrasonography (tissue perfusion parameters and blood flow velocity of the testicular artery). There were no differences between experimental groups for the echographic evaluation of testicular and epididymis parenchyma and biometric variables. The dogs in the Young Group had greater (P = 0.02) testes vascularization score and greater (P = 0.06) testicular artery blood flow velocity than those in the Senile Group. Furthermore, the older dogs had a greater (P = 0.06) pulsatility index of the testicular artery than those in the Young Group. Ageing, therefore, seems to cause natural hemodynamical changes to the testicular artery, resulting in reduced blood flow (ischemia) and tissue damage. Testes and epididymis vascular characteristics, therefore, may represent the causal factors for changes in spermatogenesis and, as a consequence, negatively affect the sperm quality of older dogs. In conclusion, senescence alters testicular artery blood flow and vascularization of the testes, without changing testicular and epididymis ultrasonographic dimensions and echodensity in dogs.

Salmon oil supplementation in dogs affects the blood flow of testicular arteries.

The administration of fish oils is known to cause changes in several reproductive parameters of domestic animals. The ingestion of polyunsaturated fatty acids (PUFAs) of the omega-3 family, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), has been described and correlated with changes in the semen quality, testosterone levels and male fertility. Nevertheless, few studies monitored and registered effects after ceasing supplementation. In the present study, we monitored the Doppler velocimetric and ultrasonographic parameters of nine dogs' testis for 90 days (D90) checking the effect of salmon oil supplementation, and monitoring continued for 60 days more, after ceasing supplementation (D150). Ultrasonographic evaluations comprised determining the Doppler velocimetric parameters, testicular and epididymal volume, and testicular echotexture. Peak systolic velocity (PSV) as well as final diastolic velocity (EDV) in the supratesticular arteries (STA), and marginal artery (MA) increased during the period of treatment and kept that level up to D150. There was no difference between the fish-oil supplementation period and the unsupplemented one regarding the testicular and epididymal volume and echogenicity and heterogeneity characteristics. A negative correlation was found between heterogeneity of testis and sperm production (r = -.41, p = .008). Doppler velocimetry indices were affected by the supplementation, leading to an increase in testicular blood flow.

Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking.

To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p = .027), percentage of progressively motile spermatozoa (p = .002), total sperm count (p = .039), morphology (p = .001) and higher percentage of immotile spermatozoa (p = .03), round cell (p = .045) and neutrophil count (p = .009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette.

Arterial supply to the rabbit male genital organs.

The improvement of veterinary care has prolonged the lifespan of rabbits, and the number of rabbits suffering from age-related, male genital disorders may increase in the near future. This could result in increased opportunities for male genital surgery, requiring knowledge of their arterial anatomy, which, however, has not been sufficiently studied. Therefore, the arteries supplying the genitals were observed in 20 male New Zealand White rabbits. The testis was supplied by the testicular artery originating from the abdominal aorta. The right testicular artery usually emerged at a more cranial level than the left artery (65%). The testicular artery encircled the testis in the sagittal plane and bifurcated (95%) or trifurcated (5%) at the caudal extremity of the testis before entering the parenchyma. The epididymis was supplied by the epididymal branches, either from only the testicular artery (75% of the right and 80% of the left halves) or from both the testicular artery and aorta. The deferent duct was supplied in all halves by the dorsal and ventral branches of the deferential artery, which usually arose from the umbilical artery. The accessory genital glands were supplied by the dorsal branch of the deferential artery and the prostatic artery. The latter, which emerged from the internal iliac artery, exhibited 3 branching types. The most frequent type (55% of the right and 45% of the left halves) had 3 branches supplying the accessory genital glands. These findings will help improve rabbit genital surgery.

Ultrasonographic characteristics of the testes, epididymis and accessory sex glands and arterial spectral indices in peri- and post-pubertal Nelore and Caracu bulls.

Ultrasonography can provide information about the integrity of organs; however, rarely is applied to the reproductive organ evaluation of bulls. The objective of the present study was to characterize and compare values for variables and ultrasonographic characteristics of the testes, epididymis and accessory sex glands, as well as spectral Doppler indices of the testicular and internal iliac arteries, between peri- and post-pubertal Nelore and Caracu bulls. Nelore (n = 203) and Caracu (n = 79) bulls were assigned by age class: peri-pubertal (12-15 months) and post-pubertal (> 22 months). Data were analyzed using SAS's PROC MIXED procedure (P < 0.05). The biometric variables of the testes and cauda epididymis differed between peri- and post-pubertal Nelore and Caracu bulls. There was a difference between breeds for the vesicular glands, ampulla of vas deferens, disseminate portion of the prostate, and craniocaudal dimension of the bulbourethral glands. Echogenicity of the testicular parenchyma differed between breeds and age classes. The pulsatility and resistive indices of the testicular arteries differed between Nelore and Caracu bulls. The biometric and ultrasonographic characteristics of the testes, epididymis and accessory sex glands, as well as of the arterial indices in bulls are affected by genetic group and age class, and when assessed there is useful information regarding the progression of sexual maturation.

Of vessels and cells: the spatial organization of the epididymal immune system.

BACKGROUND: One third of infertility cases in couples worldwide has an exclusive male origin and immune disorders, essentially due to repetitive infections, are emerging an cause of male infertility. As the place of sperm maturation, epididymis must be preserved from excessive immune responses that may arise following infections of the male genital tract. At the same time, epididymis must set and maintain a tolerogenic environment in order not to destroy sperm cells that enter the tissue at puberty, long after the immune system has been taught to recognize self pathogens. The immune cells that populate the epididymis have raised growing interest over the last thirty years but they may be not sufficient to understand the immune balance existing in this organ, between immune response to pathogens and tolerance to spermatozoa. Indeed, immune cells are the most motile cells in the organism and need blood and lymphatic vessels to traffic between lymphoid organs and sites of infection to induce efficient responses. OBJECTIVES: To review the literature on the blood and lymphatic vessels, and on the immune cells present at steady state in the rodent epididymis (rat and mouse). MATERIALS AND METHODS: PubMed database was searched for studies reporting on the spatial organization of the rodent epididymal vasculature and immune cell types at steady state. This search was combined with recent findings from our team. RESULTS: At steady state, the rodent epididymis presents with dense blood and lymphatic networks, and a large panel of immune cells distributed across the interstitum and epithelium along the organ. CONCLUSIONS: The immune system of the rodent epididymis is highly organized. Exploring its functions, especially in an infectious context, is the essential coming step before any transposition to human.

Nestin in the epididymis is expressed in vascular wall cells and is regulated during postnatal development and in case of testosterone deficiency.

Vascular smooth muscle cells (SMCs), distinguished by the expression of the neuronal stem cell marker nestin, may represent stem cell-like progenitor cells in various organs including the testis. We investigated epididymal tissues of adult nestin-GFP mice, rats after Leydig cell depletion via ethane dimethane sulfonate (EDS), rats and mice during postnatal development and human tissues. By use of Clarity, a histochemical method to illustrate a three-dimensional picture, we could demonstrate nestin-GFP positive cells within the vascular network. We localized nestin in the epididymis in proliferating vascular SMCs by colocalization with both smooth muscle actin and PCNA, and it was distinct from CD31-positive endothelial cells. The same nestin localization was found in the human epididymis. However, nestin was not found in SMCs of the epididymal duct. Nestin expression is high during postnatal development of mouse and rat and down-regulated towards adulthood when testosterone levels increase. Nestin increases dramatically in rats after Leydig cell ablation with EDS and subsequently low testosterone levels. Interestingly, during this period, the expression of androgen receptor in the epididymis is low and increases until nestin reaches normal levels of adulthood. Here we show that nestin, a common marker for neuronal stem cells, is also expressed in the vasculature of the epididymis. Our results give new insights into the yet underestimated role of proliferating nestin-expressing vascular SMCs during postnatal development and repair of the epididymis.

Phospholipase Cγ2 Is Required for Luminal Expansion of the Epididymal Duct during Postnatal Development in Mice.

Phospholipase Cγ2 (PLCγ2)-deficient mice exhibit misconnections of blood and lymphatic vessels, and male infertility. However, the cell type responsible for vascular partitioning and the mechanism for male infertility remain unknown. Accordingly, we generated a mouse line that conditionally expresses endogenous Plcg2 in a Cre/loxP recombination-dependent manner, and found that Tie2-Cre- or Pf4-Cre-driven reactivation of Plcg2 rescues PLCγ2-deficient mice from the vascular phenotype. By contrast, male mice rescued from the vascular phenotype exhibited epididymal sperm granulomas. As judged from immunostaining, PLCγ2 was expressed in clear cells in the epididymis. PLCγ2 deficiency did not compromise differentiation of epididymal epithelial cells, including clear cells, and tube formation at postnatal week 2. However, luminal expansion of the epididymal duct was impaired during the prepubertal period, regardless of epithelial cell polarity and tube architecture. These results suggest that PLCγ2-deficient clear cells cause impaired luminal expansion, stenosis of the epididymal duct, attenuation of luminal flow, and subsequent sperm granulomas. Clear cell-mediated luminal expansion is also supported by the observation that PLCγ2-deficient males were rescued from infertility by epididymal epithelium-specific reactivation of Plcg2, although the edematous and hemorrhagic phenotype associated with PLCγ2 deficiency also caused spontaneous epididymal sperm granulomas in aging males. Collectively, our findings demonstrate that PLCγ2 in clear cells plays an essential role in luminal expansion of the epididymis during the prepubertal period in mice, and reveal an unexpected link between PLCγ2, clear cells, and epididymal development.

Single organ variant of polyarteritis nodosa in epididymis.

Polyarteritis nodosa (PAN) is a systemic necrotizing vasculitis that typically affects medium-sized muscular arteries, with occasional involvement of small muscular arteries. Unlike some other vasculitides (e.g. microscopic polyarteritis, Wegener's granulomatosis) PAN is not associated with antineutrophil cytoplasmic antibodies. Patients typically present with systemic symptoms such as fever, weight loss, and malaise. The kidneys, skin, joints, muscles, nerves, and gastrointestinal tract are commonly involved, usually in some combination. PAN can affect any organ, but usually spare the lungs. Clinical variants or subsets of PAN include single-organ disease and cutaneous-only PAN. Scrotal involvement is rarely the first presenting sign. We herein report a case of 36-year-old man who presented with a swelling in the left epididymis, which was surgically removed. The swelling histopathologically showed necrotizing inflammation, fibrinoid necrosis of the medium-sized arteries of the epididymis and was diagnosed to be single organ variant of PAN.

Exploring the role of mononuclear phagocytes in the epididymis.

The onslaught of foreign antigens carried by spermatozoa into the epididymis, an organ that has not demonstrated immune privilege, a decade or more after the establishment of central immune tolerance presents a unique biological challenge. Historically, the physical confinement of spermatozoa to the epididymal tubule enforced by a tightly interwoven wall of epithelial cells was considered sufficient enough to prevent cross talk between gametes and the immune system and, ultimately, autoimmune destruction. The discovery of an intricate arrangement of mononuclear phagocytes (MPs) comprising dendritic cells and macrophages in the murine epididymis suggests that we may have underestimated the existence of a sophisticated mucosal immune system in the posttesticular environment. This review consolidates our current knowledge of the physiology of MPs in the steady state epididymis and speculates on possible interactions between auto-antigenic spermatozoa, pathogens and the immune system by drawing on what is known about the immune system in the intestinal mucosa. Ultimately, further investigation will provide valuable information regarding the origins of pathologies arising as a result of autoimmune or inflammatory responses in the epididymis, including epididymitis and infertility.

Cyclohexane-1,2-dicarboxylic acid diisononyl ester and metabolite effects on rat epididymal stromal vascular fraction differentiation of adipose tissue.

Plastics are generally mixed with additives like plasticizers to enhance their flexibility, pliability, and elasticity proprieties. Plasticizers are easily released into the environment and are absorbed mainly through ingestion, dermal contact, and inhalation. One of the main classes of plasticizers, phthalates, has been associated with endocrine and reproductive diseases. In 2002, 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) was introduced in the market for use in plastic materials and articles intended to come into contact with food, and it received final approval from the European Food Safety Authority in 2006. At present, there is limited knowledge about the safety and potential metabolic and endocrine-disrupting properties of DINCH and its metabolites. The purpose of this study was to evaluate the biological effects of DINCH and its active metabolites, cyclohexane-1,2-dicarboxylic acid (CHDA) and cyclohexane-1,2-dicarboxylic acid mono isononyl ester (MINCH), on rat primary stromal vascular fraction (SVF) of adipose tissue. DINCH and its metabolite, CHDA, were not able to directly affect SVF differentiation. However, exposure of SVF to 50 µM and 100 µM concentrations of MINCH affected the expression of Cebpa and Fabp4, thus inducing SVF preadipocytes to accumulate lipids and fully differentiate into mature adipocytes. The effect of MINCH was blocked by the specific peroxisome proliferator-activated receptor (PPAR)-α antagonist, GW6471. Taken together, these results suggest that MINCH is a potent PPAR-α agonist and a metabolic disruptor, capable of inducing SVF preadipocyte differentiation, that may interfere with the endocrine system in mammals.

Pre-cultivation of adipose tissue-derived microvascular fragments in porous scaffolds does not improve their in vivo vascularisation potential.

Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFP-positive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, non-cultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures.

15(S)-HETE-induced angiogenesis in adipose tissue is mediated through activation of PI3K/Akt/mTOR signaling pathway.

Chronic low-grade inflammation underlies obesity and associated metabolic dysfunctions. Lipoxygenase pathways are activated in adipose tissue during obese conditions. Since adipogenesis is associated with angiogenesis, the present study was designed to examine the role of 15-lipoxygenase metabolite, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] on angiogenesis in adipose tissue. Results showed that 15(S)-HETE induced sprouting in fat pad stromovascular tissues, induced morphological changes relevant to angiogenesis in endothelial cells derived from adipose tissue, upregulated the production of CD31, upregulated the gene level expression and production of vascular endothelial growth factor (VEGF), indicating the pro-angiogenic effect of 15(S)-HETE. LY294002, an inhibitor of PI3K-Akt pathway, and rapamycin, inhibitor of mammalian target of rapamycin (mTOR), significantly reversed the effect of 15(S)-HETE. 15(S)-HETE also induced activation of Akt and mTOR. These observations suggest that 15(S)-HETE stimulates angiogenesis in adipose tissue through activation of PI3K/Akt/mTOR signaling.

The blood-epididymis barrier and human male fertility.

Spermatozoa undergo a posttesticular maturation in the epididymis to acquire motility and the capacity to fertilize. Sperm maturation depends in part upon the creation of a specific microenvironment within the epididymal lumen. This environment is conditioned by proteins secreted by the epithelium and by exchange of molecules between the lumen and the blood circulation. These exchanges are selectively regulated by the blood-epididymis barrier. The blood-epididymis barrier is comprised of apical tight junctions between adjacent principal cells. Adherens junctions, which are necessary for cell adhesion, can also be found at the junctional complex present between adjacent principal cells. Progress has been made on the understanding of cellular interactions in the epididymis as well as the regulation of the luminal microenvironment and its importance for sperm maturation in rodents and humans. Clearly, changes in the function of cellular junctions in the human epididymis are associated with male infertility.

Microsurgical subinguinal varicocelectomy in children, adolescents, and adults: surgical anatomy and anatomically justified technique.

Microsurgical varicocelectomy has become the gold standard in adults because of low recurrence and postoperative hydrocele rates; it is increasingly applied in children and adolescents. This review aims to provide the surgeon with the necessary surgical anatomy of the spermatic cord and with a step-by-step, anatomically justified description of technique, toward clearer comprehension and improved application. The anatomic compartments of the spermatic cord are delineated by the external and internal spermatic fasciae. Venous drainage of testis-epididymis is accomplished by the internal spermatic, deferential, and external spermatic (cremasteric) veins. All 3 anastomose at the caudal pole of testis, and then via gubernacular veins with the posterior scrotal veins. Another anastomosis exists between a cremasteric branch and anterior scrotal veins, which gives the external pudendal vein. Subinguinal approach offers access to varicose spermatic veins and collaterals. Use of surgical microscope offers identification of small veins, preservation of arteries, lymphatics, and nerves, and appreciation of spermatic cord fasciae, which permits the development of two surgical planes. In the surgical plane of internal spermatic vessels, internal spermatic veins are ligated, whereas the testicular artery and innervation, as well as lymphatics, are preserved. In the plane of cremasteric vessels and vas, cremasteric veins are ligated, whereas the cremasteric artery, vas deferens and its vasculature, lymphatics, and the genital branch of genitofemoral nerve are preserved. Delivery of the testis to ligate gubernacular veins is at the discretion of the surgeon. Finally, venous return is effected by deferential and scrotal veins, or, when gubernacular veins are ligated, by deferential veins only.

Prenatal development of the bovine epididymis: light microscopical, glycohistochemical and immunohistochemical studies.

Prenatal development of the epididymis was studied in bovine fetuses ranging from 10 to 90cm crown-rump length (CRL) (75-285 pcd). The studies aimed to apply both glycohistochemistry and immunohistochemistry for the detection of the differentiation of the developing prenatal epididymis. Both conventional histological and histochemical techniques were applied on paraffin sections of the epididymis from different fetal stages. Establishment of the urogenital junction between the extra-testicular rete testis and the mesonephric duct, via the growing efferent ductules (ductuli efferentes) was first evident in fetuses with 10cm CRL. At the fetal age of 110 pcd (24cm CRL), the mesonephric duct began to lengthen and coil forming three distinct regions (caput, corpus and cauda). In addition to the macroscopical modifications in the extra-testicular excurrent duct system, histological differentiation involved both the tubular epithelial and the peritubular mesenchymal cells. The epithelium lining the efferent ductules was differentiated into ciliated and non-ciliated columnar cells. The simple epithelium of the epididymal duct increased in height and developed stereocilia on the apical surface. Additionally, some basal cells first appeared at 185 pcd (56cm CRL), within the epithelium lining the cauda only. Lectin histochemistry (WGA, PNA, GSA-I) showed early immunostaining in epithelium of the efferent ductules and in peritubular mesenchymal structures. Immunoreactivity for different proteins (S-100, fibroblast growth factor-1 and factor-2, angiotensin converting enzyme, laminin, alpha-smooth muscle actin) was evident, both in the epithelial and in the peritubular mesenchymal cells as early as at 75 pcd. On the basis of our histochemical observations, we conclude that both glycohistochemistry and immunohistochemistry are useful tools to demonstrate that the differentiation in the peritubular structures and efferent ductular epithelium begins earlier than other components.

Angioarchitecture of the bovine spermatic cord.

We described the topography and morphometry of the testicular artery, pampiniform plexus veins, and indirect connections between them in the spermatic cord of the bull. Sixty microcorrosive casts of bovine spermatic cords were analyzed macroscopically, by stereomicroscopy, and by scanning electron microscopy. The average size of the testicles was 94.6 × 49.7 × 54.7 mm. The testicular artery formed a superiorly pointed cone-like structure with its base fixed to the proximal part of the gonad. The artery gave off one or two branches to the head of epididymis and to the deferens duct. The pampiniform plexus originated from intra-tunical veins. Veins of the pampiniform plexus were of smaller diameter but larger number than intra-tunical ones. The density of the veins of the pampiniform plexus was 9.37 ± 1.07 mm(-2) . The testicular vein began 90-121 mm above the superior pole of the testis. In 2.9% of specimens, the testicular vein was doubled. Numerous anastomoses among veins of pampiniform plexus were observed. Additionally, indirect anastomoses between the testicular artery and pampiniform plexus veins formed by the capillary network of the vasa vasorum of the testicular artery were visualized by scanning electron microscopy. In all cases, narrowings in the casts of the precapillary vessel were observed. We also documented the vasa vasorum of the testicular artery in bulls. The density of these vessels was 22.87 ± 11.48 mm(-2) . The indirect arteriovenous connections together with the presence of circular constrictions of the lumen in precapillary vessels may play a role in testicular blood flow regulation.

Effects of artery-ligating and artery-preserving varicocelectomy on ipsilateral epididymis of varicocele-induced rats.

OBJECTIVES: To investigate the effects of artery-ligating varicocelectomy (ALV) and artery-preserving varicocelectomy (APV) on the ipsilateral epididymis of varicocele-induced rats. METHODS: A total of 50 adolescent male rats were randomly divided into the 4 groups: control group (n = 8), experimental varicocele (EV) without treatment (EV group, n = 14), EV with ALV (ALV group, n = 14), and EV with APV (APV group, n = 14). The EV was induced by partial ligation of the left renal vein. ALV was performed by total ligation of the left internal spermatic artery and vein. APV was performed by ligation of the left internal spermatic vein only. The microstructure, epithelial ultrastructure, sialic acid and carnitine concentration, and epithelial apoptotic index of the left epididymis were measured. RESULTS: Microstructural and ultrastructural abnormalities of the left epididymis were observed in the EV group and especially in the ALV group. Both the mean epididymal tubular diameter and the concentration of sialic acid, carnitine gradually decreased or increased from the control group to the EV group then to the ALV group (P < .05). However, the epithelial apoptotic index orderly increased for the control group, EV group, and ALV group (P < .05). Furthermore, no significant difference was found between the control and APV groups for these parameters (P > .05). CONCLUSIONS: Varicocele was demonstrated to cause lesions of the ipsilateral epididymis. APV was able to repair the lesions; however, ALV led to additional lesions.