Impaired Spermatogenesis in Infertile Patients with Orchitis and Experimental Autoimmune Orchitis in Rats.

Experimental autoimmune orchitis (EAO) is a well-established rodent model of organ-specific autoimmunity associated with infertility in which the testis immunohistopathology has been extensively studied. In contrast, analysis of testis biopsies from infertile patients associated with inflammation has been more limited. In this work, testicular biopsies from patients with idiopathic non-obstructive azoospermia diagnosed with hypospermatogenesis (HypoSp) [mild: n = 9, and severe: n = 11], with obstructive azoospermia and complete Sp (spermatogenesis) (control group, C, n = 9), and from Sertoli cell-only syndrome (SCOS, n = 9) were analyzed for the presence of immune cells, spermatogonia and Sertoli cell (SCs) alterations, and reproductive hormones levels. These parameters were compared with those obtained in rats with EAO. The presence of increased CD45+ cells in the seminiferous tubules (STs) wall and lumen in severe HypoSp is associated with increased numbers of apoptotic meiotic germ cells and decreased populations of undifferentiated and differentiated spermatogonia. The SCs showed an immature profile with the highest expression of AMH in patients with SCOS and severe HypoSp. In SCOS patients, the amount of SCs/ST and Ki67+ SCs/ST increased and correlated with high serum FSH levels and CD45+ cells. In the severe phase of EAO, immune cell infiltration and apoptosis of meiotic germ cells increased and the number of undifferentiated and differentiated spermatogonia was lowest, as previously reported. Here, we found that orchitis leads to reduced sperm number, viability, and motility. SCs were mature (AMH-) but increased in number, with Ki67+ observed in severely damaged STs and associated with the highest levels of FSH and inflammatory cells. Our findings demonstrate that in a scenario where a chronic inflammatory process is underway, FSH levels, immune cell infiltration, and immature phenotypes of SCs are associated with severe changes in spermatogenesis, leading to azoospermia. Furthermore, AMH and Ki67 expression in SCs is a distinctive marker of severe alterations of STs in human orchitis.

Palmitic acid control of ciliogenesis modulates insulin signaling in hypothalamic neurons through an autophagy-dependent mechanism.

Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.

Identification of VEGFR2 as the Histatin-1 receptor in endothelial cells.

Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells. First, we observed that VEGFR2 is necessary for Histatin-1-induced endothelial cell migration, as shown by both pharmacological inhibition studies and siRNA-mediated ablation of VEGFR2. Moreover, Histatin-1 co-immunoprecipitated and co-localized with VEGFR2, associating spatial proximity between these proteins with receptor activation. Indeed, pulldown assays with pure, tagged and non-tagged proteins showed that Histatin-1 and VEGFR2 directly interact in vitro. Optical tweezers experiments permitted estimating kinetic parameters and rupture forces, indicating that the Histatin-1-VEGFR2 interaction is transient, but specific and direct. Sequence alignment and molecular modeling identified residues Phe26, Tyr30 and Tyr34 within the C-terminal domain of Histatin-1 as relevant for VEGFR2 binding and activation. This was corroborated by mutation and molecular dynamics analyses, as well as in direct binding assays. Importantly, these residues were required for Histatin-1 to induce endothelial cell migration and angiogenesis in vitro. Taken together, our findings reveal that VEGFR2 is the endothelial cell receptor of Histatin-1 and provide insights to the mechanism by which this peptide promotes endothelial cell migration and angiogenesis.

Antisperm Antibody Testing: A Comprehensive Review of Its Role in the Management of Immunological Male Infertility and Results of a Global Survey of Clinical Practices.

Antisperm antibodies (ASA), as a cause of male infertility, have been detected in infertile males as early as 1954. Multiple causes of ASA production have been identified, and they are due to an abnormal exposure of mature germ cells to the immune system. ASA testing (with mixed anti-globulin reaction, and immunobead binding test) was described in the WHO manual 5th edition and is most recently listed among the extended semen tests in the WHO manual 6th edition. The relationship between ASA and infertility is somewhat complex. The presence of sperm agglutination, while insufficient to diagnose immunological infertility, may indicate the presence of ASA. However, ASA can also be present in the absence of any sperm agglutination. The andrological management of ASA depends on the etiology and individual practices of clinicians. In this article, we provide a comprehensive review of the causes of ASA production, its role in immunological male infertility, clinical indications of ASA testing, and the available therapeutic options. We also provide the details of laboratory procedures for assessment of ASA together with important measures for quality control. Additionally, laboratory and clinical scenarios are presented to guide the reader in the management of ASA and immunological male infertility. Furthermore, we report the results of a recent worldwide survey, conducted to gather information about clinical practices in the management of immunological male infertility.

Relevance of Leukocytospermia and Semen Culture and Its True Place in Diagnosing and Treating Male Infertility.

The current WHO 2010 manual for human semen analysis defines leukocytospermia as the presence of peroxidase-positive leukocytes at a concentration >1×106/mL of semen. Granular leukocytes when activated are capable of generating high levels of reactive oxygen species in semen resulting in oxidative stress. Oxidative stress has been correlated with poor sperm quality, increased level of sperm DNA fragmentation and low fertility potential. The presence of leukocytes and pathogens in the semen may be a sign of infection and/or localized inflammatory response in the male genital tract and the accessory glands. Common uro-pathogens including Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma hominis, and Escherichia coli can cause epididymitis, epididymo-orchitis, or prostatitis. The relationship between leukocytospermia and infection is unclear. Therefore, we describe the pathogens responsible for male genital tract infections and their association with leukocytospermia. The review also examines the diagnostic tests available to identify seminal leukocytes. The role of leukocytospermia in male infertility and its management is also discussed.

PKD2/polycystin-2 induces autophagy by forming a complex with BECN1.

Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.

[Klippel-Trenaunay-Weber syndrome with vesical and uterine involvement treated by endoscopic and endovascular routes]. / Síndrome de Klippel-Trenaunay-Weber con compromiso vesical y uterino tratado por vía endoscópica y endovascular.

Klippel-Trenaunay-Weber syndrome (KTWS) is a rare venous malformation that generally affects the lower limbs and, more infrequently, the upper limbs. It is characterized by cutaneous angiomatous formations, varicose veins and hypertrophy of the affected limb. The involvement of the genitourinary tract is extremely infrequent. We expose the case of a 14 years old female patient who was admitted for macroscopic hematuria of 48 hours of evolution and metrorrhagia with severe hemodynamic decompensation. The patient was under study for presenting a hemangioma in the lower left limb that extended to the pelvic region. Urethrocystofibroscopy showed the presence of multiple wide-spread angiomatous lesions in the bladder, some of them with active bleeding. The angio-resonance showed a voluminous hypervascular formation in contact with the bladder wall showing several arteriovenous fistulas at the pelvic level and in the left lower limb confirming the etiological diagnosis. A selective arterial embolization of the internal and external iliac territories was performed and then, a laser endocoagulation of the bleeding angiomatous foci was carried out. The hematuria completely stopped within 24 hours later of the procedure. The metrorrhagia associated with KTWS was controlled by the use of LHRH analogs and progestogens.

El síndrome de Klippel-Trenaunay-Weber (SKTW) es una rara malformación venosa que, en general afecta a los miembros inferiores y, más raramente, a los superiores. Se caracteriza por formaciones angiomatosas cutáneas, várices e hipertrofia del miembro afectado. El compromiso genitourinario es sumamente infrecuente. Se presenta el caso de una paciente de 14 años. Ingresó por hematuria macroscópica de 48 h de evolución y metrorragia con grave compromiso hemodinámico. Se encontraba en estudio por presentar un hemangioma en el miembro inferior izquierdo que se extendía hasta la región pelviana. La uretrocistofibroscopía demostró la presencia de múltiples lesiones angiomatosas diseminadas en forma amplia en la vejiga, algunas de ellas con sangrado activo. La angioresonancia mostró una voluminosa formación hipervascularizada en contacto con la pared vesical a la cual desplazaba y fístulas arteriovenosas a nivel pelviano y en el miembro inferior izquierdo confirmando el diagnóstico etiológico. Se realizó una embolización arterial selectiva de los territorios ilíacos interno y externo e inmediatamente después una endocoagulación láser de los focos angiomatosos sangrantes. La hematuria remitió completamente en las 24 h posteriores al procedimiento. La metrorragia asociada al SKTW fue controlada mediante la utilización de análogos LHRH y progestágenos.

Síndrome de Klipper-Trenaunay-Weber con compromiso vesical y uterino tratado por vía endoscópica y endovascular / Klippel-Trenaunay-Weber syndrome with vesical and uterine involvement treated by endoscopic and endovascular routes

El síndrome de Klippel-Trenaunay-Weber (SKTW) es una rara malformación venosa que, en general afecta a los miembros inferiores y, más raramente, a los superiores. Se caracteriza por formaciones angiomatosas cutáneas, várices e hipertrofia del miembro afectado. El compromiso genitourinario es sumamente infrecuente. Se presenta el caso de una paciente de 14 años. Ingresó por hematuria macroscópica de 48 h de evolución y metrorragia con grave compromiso hemodinámico. Se encontraba en estudio por presentar un hemangioma en el miembro inferior izquierdo que se extendía hasta la región pelviana. La uretrocistofibroscopía demostró la presencia de múltiples lesiones angiomatosas diseminadas en forma amplia en la vejiga, algunas de ellas con sangrado activo. La angioresonancia mostró una voluminosa formación hipervascularizada en contacto con la pared vesical a la cual desplazaba y fístulas arteriovenosas a nivel pelviano y en el miembro inferior izquierdo confirmando el diagnóstico etiológico. Se realizó una embolización arterial selectiva de los territorios ilíacos interno y externo e inmediatamente después una endocoagulación láser de los focos angiomatosos sangrantes. La hematuria remitió completamente en las 24 h posteriores al procedimiento. La metrorragia asociada al SKTW fue controlada mediante la utilización de análogos LHRH y progestágenos.

Klippel-Trenaunay-Weber syndrome (KTWS) is a rare venous malformation that generally affects the lower limbs and, more infrequently, the upper limbs. It is characterized by cutaneous angiomatous formations, varicose veins and hypertrophy of the affected limb. The involvement of the genitourinary tract is extremely infrequent. We expose the case of a 14 years old female patient who was admitted for macroscopic hematuria of 48 hours of evolution and metrorrhagia with severe hemodynamic decompensation. The patient was under study for presenting a hemangioma in the lower left limb that extended to the pelvic region. Urethrocystofibroscopy showed the presence of multiple wide-spread angiomatous lesions in the bladder, some of them with active bleeding. The angio-resonance showed a voluminous hypervascular formation in contact with the bladder wall showing several arteriovenous fistulas at the pelvic level and in the left lower limb confirming the etiological diagnosis. A selective arterial embolization of the internal and external iliac territories was performed and then, a laser endocoagulation of the bleeding angiomatous foci was carried out. The hematuria completely stopped within 24 hours later of the procedure. The metrorrhagia associated with KTWS was controlled by the use of LHRH analogs and progestogens.

Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord.

The aim of this work was to study effects of ketotifen fumarate (KF) on prevention of tissue damage in testes of rats with experimental autoimmune orchitis (EAO) and on the contralateral testis in a model of prolonged testicular cord torsion (TCT). Rats with EAO or TCT were injected intraperitoneally once daily with KF or saline solution (vehicle group). Incidence and severity of testicular damage were evaluated by histopathology using an EAO score or a Johnsen score. Mast cells (MC) were identified by histochemistry and quantified. In EAO model, KF significantly reduced severity of histopathological testicular damage compared to rats in the vehicle group. KF also reduced the number of testicular MC compared to vehicle group. Similarly, in TCT model, multifocal damage of the contralateral testis was observed 30 days after testicular torsion characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed. In contrast, sections of contralateral testis of rats injected with KF and killed 30 days after surgery showed normal histological features. A significant decrease in the number of MC was observed in rats treated with KF compared to untreated animals. In conclusion, we demonstrated that treatment with KF reduced testicular inflammatory process and MC infiltrates in both EAO and TCT models. The results suggest a promising treatment for infertile male patients with testicular pathologies associated with inflammation and germ cell loss.

mTORC1 inhibitor rapamycin and ER stressor tunicamycin induce differential patterns of ER-mitochondria coupling.

Efficient mitochondrial Ca2+ uptake takes place at contact points between the ER and mitochondria, and represents a key regulator of many cell functions. In a previous study with HeLa cells, we showed that ER-to-mitochondria Ca2+ transfer increases during the early phase of ER stress induced by tunicamycin as an adaptive response to stimulate mitochondrial bioenergetics. It remains unknown whether other types of stress signals trigger similar responses. Here we observed that rapamycin, which inhibits the nutrient-sensing complex mTORC1, increased ER-mitochondria coupling in HeLa cells to a similar extent as did tunicamycin. Interestingly, although global responses to both stressors were comparable, there were notable differences in the spatial distribution of such changes. While tunicamycin increased organelle proximity primarily in the perinuclear region, rapamycin increased organelle contacts throughout the entire cell. These differences were paralleled by dissimilar alterations in the distribution of regulatory proteins of the ER-mitochondria interface, heterogeneities in mitochondrial Ca2+ uptake, and the formation of domains within the mitochondrial network with varying mitochondrial transmembrane potential. Collectively, these data suggest that while increasing ER-mitochondria coupling appears to represent a general response to cell stress, the intracellular distribution of the associated responses needs to be tailored to meet specific cellular requirements.

ER-to-mitochondria miscommunication and metabolic diseases.

Eukaryotic cells contain a variety of subcellular organelles, each of which performs unique tasks. Thus follows that in order to coordinate these different intracellular functions, a highly dynamic system of communication must exist between the various compartments. Direct endoplasmic reticulum (ER)-mitochondria communication is facilitated by the physical interaction of their membranes in dedicated structural domains known as mitochondria-associated membranes (MAMs), which facilitate calcium (Ca(2+)) and lipid transfer between organelles and also act as platforms for signaling. Numerous studies have demonstrated the importance of MAM in ensuring correct function of both organelles, and recently MAMs have been implicated in the genesis of various human diseases. Here, we review the salient structural features of interorganellar communication via MAM and discuss the most common experimental techniques employed to assess functionality of these domains. Finally, we will highlight the contribution of MAM to a variety of cellular functions and consider the potential role of MAM in the genesis of metabolic diseases. In doing so, the importance for cell functions of maintaining appropriate communication between ER and mitochondria will be emphasized.

MARCKS protein is phosphorylated and regulates calcium mobilization during human acrosomal exocytosis.

Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.