Exploring novel function of Gpx5 antioxidant activity: Assisting epididymal cells secrete functional extracellular vesicles.

Glutathione peroxisomal-5 (Gpx5) promotes the elimination of H2O2 or organic hydrogen peroxide, and plays an important role in the physiological process of resistance to oxidative stress (OS). To directly and better understand the protection of Gpx5 against OS in epididymal cells and sperm, we studied its mechanism of antioxidant protection from multiple aspects. To more directly investigate the role of Gpx5 in combating oxidative damage, we started with epididymal tissue morphology and Gpx5 expression profiles in combination with the mouse epididymal epithelial cell line PC1 (proximal caput 1) expressing recombinant Gpx5. The Gpx5 is highly expressed in adult male epididymal caput, and its protein signal can be detected in the sperm of the whole epididymis. Gpx5 has been shown to alleviate OS damage induced by 3-Nitropropionic Acid (3-NPA), including enhancing antioxidant activity, reducing mitochondrial damage, and suppressing cell apoptosis. Gpx5 reduces OS damage in PC1 and maintains the well-functioning extracellular vesicles (EVs) secreted by PC1, and the additional epididymal EVs play a role in the response of sperm to OS damage, including reducing plasma membrane oxidation and death, and increasing sperm motility and sperm-egg binding ability. Our study suggests that GPX5 plays an important role as an antioxidant in the antioxidant processes of epididymal cells and sperm, including plasma membrane oxidation, mitochondrial oxidation, apoptosis, sperm motility, and sperm-egg binding ability.

Retinoic acid mitigates the NSC319726-induced spermatogenesis dysfunction through cuproptosis-independent mechanisms.

Copper ionophore NSC319726 has attracted researchers' attention in treating diseases, particularly cancers. However, its potential effects on male reproduction during medication are unclear. This study aimed to determine whether NSC319726 exposure affected the male reproductive system. The reproductive toxicity of NSC319726 was evaluated in male mice following a continuous exposure period of 5 weeks. The result showed that NSC319726 exposure caused testis index reduction, spermatogenesis dysfunction, and architectural damage in the testis and epididymis. The exposure interfered with spermatogonia proliferation, meiosis initiation, sperm count, and sperm morphology. The exposure also disturbed androgen synthesis and blood testis barrier integrity. NSC319726 treatment could elevate the copper ions in the testis to induce cuproptosis in the testis. Copper chelator rescued the elevated copper ions in the testis and partly restored the spermatogenesis dysfunction caused by NSC319726. NSC319726 treatment also decreased the level of retinol dehydrogenase 10 (RDH10), thereby inhibiting the conversion of retinol to retinoic acid, causing the inability to initiate meiosis. Retinoic acid treatment could rescue the meiotic initiation and spermatogenesis while not affecting the intracellular copper ion levels. The study provided an insight into the bio-safety of NSC319726. Retinoic acid could be a potential therapy for spermatogenesis impairment in patients undergoing treatment with NSC319726.

[Effects of oral exposure to iron oxide nanoparticles on reproductive system of male rats].

OBJECTIVE: To investigate the effects of oral exposure to iron oxide nanoparticles(Fe_2O_3NPs) on the reproductive system of male rats. METHODS: Forty male SD rats were randomly divided into control group and low, medium, high dose groups, 10 rats in each group, normal saline and 50, 100 and 200 mg/kg Fe_2O_3NPs suspension were given by gavage, respectively. The volume of gavage was 10 mL/kg for 28 days. The body weight was weighed every three days, and the body weight changes of rats were recorded. After intraperitoneal anesthesia with 10% chloral hydrate, the rats were sacrificed by cervical dislocation, and the testis and epididymis were collected. Weigh and calculate the testicular coefficient and epididymal coefficient, the pathological sections of rat testis were observed by hematoxylin-eosin staining, the number of epididymal sperm was counted under an optical microscope and the sperm deformity rate was calculated. The activities of acid phosphatase(ACP), alkaline phosphatase(AKP), lactate dehydrogenase(LDH) and γ-glutamyl transpeptidase(γ-GT), the activity of superoxide dismutase(SOD), and the contents of glutathione(GSH) and malondialdehyde(MDA) in rat testis homogenate were detected by kit method. RESULTS: Compared with control group, there was no significant difference in body weight, testicular coefficient and epididymal coefficient in each dose group. In the medium and high dose groups, the arrangement of spermatogenic epithelium was disordered and spermatogenic cells decreased. The number of sperm in high dose group was decreased, and the sperm deformity rate in medium and high dose groups was increased(P<0.01). The activity of ACP in medium and high dose groups increased(P<0.05), and the activity of γ-GT decreased(P<0.01). There was no significant change in the activity of AKP and LDH in testicular homogenate of rats in each group(P>0.05). The level of GSH in medium dose group was increased(P<0.05), and the content of MDA in medium and high dose groups was increased(P<0.01). There was no significant difference in SOD activity among the groups(P>0.05). CONCLUSION: Under the conditions of this experiment, Fe_2O_3NPs can cause damage to the structure of rat testicular tissue, reduce the number of sperm, increase the rate of sperm deformity, interfere with the activity of marker enzymes in testicular tissue and induce oxidative stress injury, which has a negative impact on the reproductive system of male rats.

Physiological and pharmacological impact of oxytocin on epididymal propulsion during the ejaculatory process in rodents and men.

During the emission phase of ejaculation, the sperm is driven from the cauda epididymidis, where it is stored, through the vas deferens by strong contractions. These contractions are thought of as being mainly induced by the sympathetic nervous system and the neurotransmitter noradrenaline. In the present study, we investigated the effect of oxytocin (suggested to exert effects during ejaculation as well) on defined segments of the rat and human epididymis using live imaging. Our results indicate that it is the very last part of the epididymis, segment 19 (S19) in rat and likewise segment 9 in human, which responds in a uniquely strong and rapid manner to oxytocin (similar to noradrenaline). Because of the complex nature of this contractile response, we developed an imaging analysis method, which allowed us to quantify multidirectional contractions and to display them using heat maps. The reaction of S19 to oxytocin was concentration-dependent and could be inhibited by pretreatment with oxytocin antagonists (atosiban and cligosiban), but not with an arginine vasopressin 1A antagonist (SR49059). In both rat and human tissue, pretreatment with the alpha-1 adrenoreceptor antagonist tamsulosin inhibited the response to noradrenaline, whereas the effect of oxytocin was unimpaired. Our data (from men and rodents) strongly suggest that the hormone oxytocin is involved in the ejaculatory process. Thus, oxytocin-based medications might be a promising non-adrenergic treatment option for ejaculatory disorders. Additionally, we propose that S19 could be an advantageous model (detecting very low concentrations of oxytocin) to test the bioactivity of new oxytocin agonists and oxytocin antagonists.

Seed extract of Thai Mucuna pruriens reduced male reproductive damage in rats induced by chronic stress.

CONTEXT: Thai Mucuna pruriens (L.) DC. var. pruriens (Fabaceae) (TMP) is known to enrich reproduction but preventive effects on stress related adverse reproductive parameters are not documented. OBJECTIVE: This study investigates the protective property of TMP seed extract on reproductive damage under chronic stress (CS). MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into four groups. The control and CS groups received distilled water, whereas the pre-treated rats received the aqueous TMP seed extract at doses of 150 and 300 mg/kg BW for 20 days before co-treatments with CS induction (immobilization and forced swimming) for 81 days. Serum was used to determine the cortisol and testosterone levels. Histology of testis and epididymis was observed with localization of androgen receptor (AR). Sperm parameters and the expression of steroidogenic acute regulatory (StAR), cytochrome P450 family 11 subfamily a member 1 (CYP11A1), AR, HSP70, caspases (3 and 9) and tyrosine phosphorylation (TyrPho) proteins were investigated. RESULTS: TMP extract improved cortisol level (0.84 ± 0.02 µg/dL) and protected against the damage of reproductive tissues and sperm parameters (count [49.78 ± 3.74 million sperm/mL], viability [90.01 ± 1.17%] and precocious acrosome reaction [1.38 ± 0.48%]). Expression of testicular StAR, CYP11A1, AR and HSP70 proteins was improved. Caspase expression was decreased in treated rats. TMP increased AR expression in CS sperm. Moreover, TyrPho protein expression was corrected after TMP administration. CONCLUSIONS: TMP seed protected against adverse reproductive parameters in CS via improvements of functionally testicular markers and reductions of apoptotic proteins. It is possible to develop the TMP beans as an alternative medicine in treating of male subfertility caused by CS.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5 mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300 µm dia.) exhibited SPCs, while distal segments (350-500 µm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1 µM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1 µM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10 µM). SPCs were also inhibited by caffeine (1 µM), 2-APB (100 µM), niflumic acid (100 µM), or by lowering extracellular [Cl-] from 134.4 to 12.4 mM but not by ryanodine (25 µM) or tetracaine (100 µM). Electrical field stimulation (EFS) at 2 Hz for 60 s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

Bee bread mitigates downregulation of steroidogenic genes, decreased spermatogenesis, and epididymal oxidative stress in male rats fed with high-fat diet.

The pituitary-gonadal axis plays an important role in steroidogenesis and spermatogenesis, and by extension, fertility. The aim of this study was to investigate the protective role of bee bread, a natural bee product, against obesity-induced decreases in steroidogenesis and spermatogenesis. Thirty-two adult male Sprague-Dawley rats weighing between 200 and 300 g were divided into four groups (n = 8/group), namely: normal control (NC), high-fat diet (HFD), HFD plus bee bread administered concurrently for 12 wk (HFD + B), HFD plus orlistat administered concurrently for 12 wk (HFD + O) groups. Bee bread (0.5 g/kg) or orlistat (10 mg/kg/day) was suspended in distilled water and given by oral gavage daily for 12 wk. Levels of follicle-stimulating hormone, luteinizing hormone, testosterone, and adiponectin, as well as sperm count, motility, viability, normal morphology, and epididymal antioxidants decreased, whereas levels of leptin, malondialdehyde, and sperm nDNA fragmentation increased significantly in the HFD group relative to the NC group. There were significant decreases in the testicular mRNA transcript levels of androgen receptor, luteinizing hormone receptor, steroidogenic acute regulatory protein, cytochrome P450 enzyme, 3ß-hydroxysteroid dehydrogenase (HSD) and 17ß-HSD in the testes of the HFD group. Furthermore, mount, intromission and ejaculatory latencies increased, and penile cGMP level decreased significantly in the HFD group. Supplementation with bee bread significantly reduced leptin level and increased adiponectin level, enhanced sperm parameters and reduced sperm nDNA fragmentation, upregulated the levels of steroidogenic genes and proteins in HFD-induced obese male rats. Bee bread improved steroidogenesis and spermatogenesis by upregulating steroidogenic genes. Therefore, bee bread may be considered as a potential supplementation to protect against infertility in overweight men or men with obesity.NEW & NOTEWORTHY The high-fat diet utilized in the present study induced obesity in the male rats. Bee bread supplementation mitigated impaired steroidogenesis, spermatogenesis, mating behavior, and fertility potential by counteracting the downregulation of steroidogenic genes, thus increasing testosterone levels and suppressing epididymal oxidative stress. These benefits may be due to the abundance of phenolic and flavonoid compounds in bee bread.

Cadmium induced inflammation and apoptosis of porcine epididymis via activating RAF1/MEK/ERK and NF-κB pathways.

Cadmium (Cd) was a serious heavy metal pollutant. Cd exposure will cause damage to reproductive organs. It was largely unknown whether Cd exposure caused inflammation and apoptosis in epididymis. In this study, we established models of Cd exposure in swine, and the apoptotic level of epididymis was detected by in situ TUNEL fluorescence staining assay, the results showed that Cd exposure significantly increased TUNEL-apoptosis index. Furthermore, the results of qRT-PCR and Western blot showed that Cd activated the proto-oncogenic serine/threonine kinase-1 (RAF1)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signal pathway (RAF1/MEK/ERK) and led to the subsequent up-regulation of the nuclear factor-κB (NF-κB), tumor necrosis factor α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), caused inflammation in epididymis. NF-κB inflammation pathway also mediated the tumor protein P53 (P53) and indirectly activated the Cytochrome c (Cytc), B-cell lymphoma-2 (Bcl-2), Bcl-2-Associated X protein (Bax), Caspase 3, Caspase 9. In summary, we believed that the RAF1/MEK/ERK pathway came into play in the apoptosis of epididymal tissues exposed to Cd by activating the NF-κB Inflammation pathway, followed by activation of the mitochondrial apoptotic pathway. This study provides more abundant data for exploring the reproductive toxicity of Cd.

Testosterone regulation on quiescin sulfhydryl oxidase 2 synthesis in the epididymis.

The epididymis is an androgen-responsive organ, whose structure and functions are modulated by the coordination between androgen and epididymal cues. Highly regulated molecular interaction within the epididymis is required to support viable sperm development necessary for subsequent fertilization. In the present study, we extended our earlier findings on a promising epididymal protein, quiescin sulfhydryl oxidase 2 (QSOX2), and demonstrated a positive correlation between testosterone and QSOX2 protein synthesis through the use of loss- and restore-of-function animal models. Moreover, based on transcriptomic analyses and 2D culture system, we determined that an additional polarized effect of glutamate is indispensable for the regulatory action of testosterone on QSOX2 synthesis. In conclusion, we propose noncanonical testosterone signaling supports epididymal QSOX2 protein synthesis, providing a novel perspective on the regulation of sperm maturation within the epididymis.

The ameliorative effect of ellagic acid on di-(2-ethylhexyl) phthalate-induced testicular structural alterations, oxidative stress, inflammation and sperm damages in adult mice.

BACKGROUND: Phthalates such as di (2-ethylhexyl) phthalate (DEHP) are well known exogenous substances, disrupting reproductive system function and structure. The current research demonstrated the effect of ellagic acid (EA) on DEHP-induced testicular injury in mice. METHODS: Thirty-five healthy adult male mice were randomly divided to five groups; normal saline receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) and EA receiving groups (25, 50 and 100 mg/kg/day, p.o.). Treatment duration of animals was 14 days. Body and testes weights and sperm characteristics and histological changes of testes were evaluated. Serum testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were analyzed. In the testicular tissue, oxidative/nitrosative stress markers and inflammatory cytokine levels were measured. RESULTS: Ellagic acid significantly reduced DEHP-induced reduction of body and testes weights. The DEHP-induced reduction of spermatogonia, primary spermatocyte and sertoli cells numbers as well as reduction of sperm vitality and progressive motility were reversed by EA. Furthermore, EA inhibited DEHP-induced alterations in serum hormone levels. These effects were associated with the reduction of DEHP-induced increased level of oxidative stress and inflammatory responses. CONCLUSIONS: Ellagic acid considerably inhibits testicular toxicity of DEHP through reducing oxidative/nitrosative stress and inflammatory responses. Our data suggest that EA may be considered as a promising agent to inhibit male reproductive toxicity induced by endocrine disrupting chemicals such as DEHP.

Hedgehog signaling pathway regulates gene expression profile of epididymal principal cells through the primary cilium.

Primary cilia (PC) are organelles that sense and respond to dynamic changes of the extracellular milieu through the regulation of target genes. By using the epididymis as a model system, we determined the contribution of primary cilia in the regulation of epithelial cell functions through the transduction of the Hedgehog (Hh) signaling pathway. Both Sonic (SHH) and Indian Hedgehog (IHH) ligands were detected in epididymal epithelial cells by confocal microscopy and found secreted in the extracellular space. Gene expression profiling preformed on ciliated epithelial cells indicated that 153 and 1052 genes were differentially expressed following treatment with the Hh agonist SAG or the Hh antagonist cyclopamine (Cyclo), respectively. Strikingly, gene ontology analysis indicated that genes associated with immune response were the most affected following Hh modulation. The contribution of epididymal PC to canonical Hh pathway transduction was validated by ciliobrevin D treatment, which induced a significant decrease in PC length and a reduction in the expression Hh signaling targets. Such findings bring us closer to a molecular understanding of the subtle immune balance observed in some epithelia, including the epididymis and the intestine, which are organs featuring both tolerance toward autoimmune spermatozoa (or commensal bacteria) and defense against pathogens.

Bisphenol A exposure increases epididymal susceptibility to infection in mice.

Male fertility is linked with several well-orchestrated events including spermatogenesis, epididymal maturation, capacitation, the acrosome reaction, fertilization, and beyond. However, the detrimental effects of bisphenol A (BPA) on sperm maturation compared to spermatogenesis and sperm cells remain unclear. Therefore, this study was to investigate whether pubertal exposure to BPA induces male infertility via interruption of the immune response in the epididymis. CD-1 male mice (5 weeks old) were treated daily with vehicle (corn oil) and 50 mg BPA/kg-BW for 6 weeks by oral gavage. Following BPA exposure, we observed decreased intraepithelial projection of basal cells, indicative of changes to the luminal environment. We also observed decreased projection of macrophages and protrusion of apoptotic cells into the lumen induced by incomplete phagocytosis of apoptotic cells in the caput epididymis. Exposure to BPA also reduced the anti- and pro-inflammatory cytokines IL-10, IL-6, IFN-γ, and IL-7 in the epididymis, while the chemotaxis-associated cytokines CCL12, CCL17, CXCL16, and MCP-1 increased. This study suggests two possible mechanisms for BPA induction of male infertility. First, exposure to BPA may induce an imbalance of immune homeostasis by disrupting the ability of basal cells to perceive environmental changes. Second, exposure to BPA may lead to collapse of macrophage phagocytosis via downregulation of intraepithelial projection and inflammatory-related cytokines. In conclusion, the observed potential pathways can lead to autoimmune disorders such epididymitis and orchitis.

Characterization of Estrogenic Activity and Site-Specific Accumulation of Bisphenol-A in Epididymal Fat Pad: Interfering Effects on the Endocannabinoid System and Temporal Progression of Germ Cells.

The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3ß-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell-cell junction genes (i.e., zonula occcludens protein-1, vimentin and ß-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.

[Antagonistic effect of modified Dahuang Zhechong Granule against the apoptosis of epididymal spermatogenic cells in varicocele rats based on Nrf2/HO-1 pathways].

OBJECTIVE: To investigate the effects of modified Dahuang Zhechong Granule (DZG) on the epididymal tissue of varicocele (VC) rats and the expressions of the nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) and heme oxygenase-1 (HO-1) protein. METHODS: Sixty SD rats were randomly divided into six groups of an equal number: sham operation, VC model control, aescuven forte (AF) and low-, medium- and high-dose DZG. The VC model was established by ligation of the left renal vein with the Turner's method, followed by intragastrical administration of normal saline to the rats in the sham operation and VC model control groups, AF Tablets at 54 mg/kg to those in the AF group, and modified DZG at 0.6, 1.2 and 2.4 g/ml to those in the low-, medium- and high-dose DZG groups respectively, all once daily for 8 weeks. Then, all the animals were sacrificed and their left epididymides harvested for examination of semen quality, observation of local ultrastructural changes, measurement of the apoptosis of spermatogenic cells by Annexin V-FITC, and determination of the expressions of Nrf2 and HO-1 in the epididymal tissue by immunohistochemistry. RESULTS: Evident pathological damage was observed in the left epididymal tissue of the VC model controls, with significantly reduced numbers of spermatogenic cells and sperm at all levels, partially destroyed cellular structure, and disappearance of some subcellular structures such as the lysosome, mitochondrion, endoplasmic reticulum, nucleus and cell membrane, which were all improved to some extent in the DZG and AF group. Sperm concentration and motility in the left epididymis were significantly higher in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05), even more significantly in the high-dose DZG than in the AF group (P < 0.05). The apoptosis rate of spermatogenic cells was markedly higher in the VC model control than in the sham operation group (P < 0.05), but lower in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05). Immunohistochemistry showed positive expressions of Nrf2 and HO-1 proteins, brown, scattered and with a low luminance of the cells, in the left epididymis tissue of the VC model control rats, but with a significantly higher cell luminance in the high-dose DZG and AF groups. CONCLUSIONS: Modified Dahuang Zhechong Granule can effectively repair pathological damage to the epididymis of varicocele rats, increase the expressions of Nrf2 and HO-1 proteins, antagonize the apoptosis of spermatogenic cells and provide a favorable condition for sperm maturation.

Cellular mechanism underlying oxytocin-stimulated Cl- secretion in rat cauda epididymal epithelium.

The neurohypophyseal hormone oxytocin (OT) plays critical roles in lactation and parturition, while its function in male reproduction system is largely unknown. This study aims to investigate the effect of OT on regulating transepithelial ion transport in rat cauda epididymal epithelium. With the use of RT-PCR, Western blot, and immunohistochemical analysis, we found that OT receptor (OTR) was expressed and localized at the basal membrane of rat cauda epididymal epithelium. The short-circuit current (Isc) measurement showed that basolateral application of OT to the primary cultured rat cauda epididymal epithelial cells elicited an increase in Isc, which was abrogated by pretreating the epithelial cells with CFTRinh-172, a blocker of cystic fibrosis transmembrane conductance regulator (CFTR). Pretreatment with the prostaglandin H synthase inhibitors indomethacin and piroxicam, or the nonselective antagonists of prostaglandin E2 (PGE2) receptor EP2 or EP4, AH-6809, and AH-23848, significantly attenuated OT-stimulated Isc response. Furthermore, the generation of PGE2 was measured using enzyme-linked immunosorbent assay, demonstrating that OT induced a substantial increase in PGE2 release from primary cultured rat cauda epididymal epithelial cells. In conclusion, activation of OTR by OT triggered PGE2 release, resulting in CFTR-dependent Cl- secretion through paracrine/autocrine pathways in rat cauda epididymal epithelium.

S100A4 promotes the progression of lipopolysaccharide-induced acute epididymitis in mice†.

S100A4 has been suggested to be a critical regulator of tumor metastasis and is implicated in the progression of inflammation. The aim of this study is to investigate the expression and possible role of S100A4 in epididymitis. Using a mouse model of epididymitis induced by the injection of lipopolysaccharide (LPS) in the deferent duct, we found that LPS administration induced an upregulation of S100a4 transcription (P < 0.05) and a recruitment of S100A4 positive cells in the epididymal interstitium of wild type (WT) mice. Co-immunofluorescence showed that S100A4 was mainly expressed by granulocytes, CD4 lymphocytes, and macrophages. Deficiency of S100A4 reduced epididymal pathological reaction and the mRNA levels of the pro-inflammatory cytokines IL-1ß and TNF-α (P < 0.01), suggesting that S100A4 promotes the progression of epididymitis. Furthermore, S100A4 deficiency alleviated the decline of sperm motility and rectified the abnormal expression of sperm membrane protein AMAD3, which suggested that in the progression of epididymitis, S100A4 aggravates the damage to sperm vitality. In addition, both Ki-67 marked cell proliferation and transferase-mediated dUTP-biotin nick end labeling detected cell apoptosis were reduced in S100a4-/- mice compared with WT mice after LPS treatment, indicating that S100A4 promotes both cell proliferation and cell apoptosis in epididymitis. Overall, these results demonstrate that S100A4 promotes the progression of LPS-induced epididymitis and facilitates a decline in sperm vitality, and its function may be related to the process of cell proliferation and apoptosis during inflammation.

Androgens are essential for epithelial cell recovery after efferent duct ligation in the initial segment of the mouse epididymis†.

Efferent duct ligation (EDL) induces epithelial cell degeneration followed by regeneration in the epididymal initial segment. We tested here the role of androgens in the recovery phase. EDL was performed at post-natal weeks (PNW) 3, 4, 5, 6, and 7, and apoptotic and proliferating epithelial cells were quantified 24 h, and at days 2 and 2.5 post-EDL, respectively. A progressive increase in the number of apoptotic basal cells (BCs) and principal cells (PCs) was detected from PNW3 to 6, 24 h after EDL. Two days after EDL, no increase in proliferating BCs and PCs was observed at PNW3 and 4, despite the induction of apoptosis by EDL. A progressive increase in the number of proliferating BCs was then observed from PNW5 to 6, while the number of proliferating PCs remained low. 2.5 days after EDL, the number of proliferating BCs and PCs remained low at PNW3, 4, and 5, but a marked increase in the number of proliferating PCs was observed at PNW6. Flutamide pretreatment for 3 weeks followed by EDL at PNW7 dramatically decreased the number of proliferating BCs on EDL day 2, and the number of proliferating PCs on EDL day 2.5, compared to controls. We conclude that (1) BCs are the first to show recovery after EDL, followed by PCs; (2) androgens are essential for BC and PC repair after injury in the postpubertal epididymis; and (3) the prepubertal epididymis lacks repair ability following injury.

Prepubertal exposure to arsenic alters male reproductive parameters in pubertal and adult rats.

Arsenic induces reproductive disorders in pubertal males after prepubertal exposure. However, it is unclear the extent to which those effects remain in testis and epididymis of sexually mature rats after arsenic insult. This study evaluated the effects of prepubertal arsenic exposure in male organs of pubertal rats, and their reversibility in adult rats. Male pups of Wistar rats on postnatal day (PND) 21 were divided into two groups (n = 20/group): Control animals received filtered water and exposed rats received 10 mg L--1 arsenic from PND 21 to PND 51. At PND 52, testis and epididymis of ten animals per group were examined for toxic effects under morphological, functional, and molecular approaches. The other animals were kept alive under free arsenic conditions until PND 82, and further analyzed for the same parameters. Pubertal rats overexpressed mRNA levels of SOD1, SOD2, CAT, GSTK1, and MT1 in their testis and SOD1, CAT, and GSTK1 in their epididymis. In those organs, catalase activity was altered, generating byproducts of oxidative stress. The antioxidant gene expression was unchanged in adult rats in contrast to the altered activity of antioxidant enzymes. Histological alterations of testis and epididymis tissues were observed in pubertal and adult rats. Interestingly, only adult rats exhibited a remarkable decrease in serum testosterone levels. Prepubertal exposure to arsenic caused morphological and functional alterations in male reproductive organs of pubertal rats. In adult rats, these damages disappeared, remained, get worsened, or recovered depending on the parameter analyzed, indicating potential male fertility disorders during adulthood.

Muscular atrophy, impaired epithelial autophagy and UCHL1 increase in androgen-deficient cauda epididymis.

In epididymis, cimetidine induces androgenic failure due to reduced sex hormone-binding globulin stromal levels and blockade of androgen receptor (AR) nuclear import. UCHL1, a hydrolase of ubiquitin-proteasome system (UPS), seems to play a role in autophagy and apoptotic pathway. However, the role of UPS and autophagy in epididymis has not been clarified. We evaluated UCHL1 and autophagy in epididymal cauda epithelium under androgenic deficiency induced by cimetidine, focusing on the interplay among these processes and apoptosis. The integrity of epididymal muscular layer was also evaluated. Male rats received cimetidine (CMTG) or saline (CG). Seminal vesicles were weighed, the expression of androgen-responsive genes Crisp1 and connexin 43 (Cx43) in cauda epididymis was evaluated, and cauda fragments were processed for light and transmission electron microscopy. The epithelium height and muscular thickness were measured. TUNEL, immunohistochemistry for caspase-3 and Cx43, and immunofluorescence for AR, Bcl-2, UCHL1, MAP LC3A, and p62/SQSTM1 (autophagic markers) were performed. Bcl-2, UCHL1, and Cx43 were detected by Western blot. In CMTG, the reduction in seminal vesicles weight accompanied by downregulation of Crisp1 and Cx43 confirmed epididymal androgenic failure. These results were associated with muscular atrophy, apoptosis and weak Cx43 and AR immunoexpression, supporting the androgenic dependence of muscular integrity. The high UCHL1 levels and reduction in Bcl-2 reinforce UCHL1 role in epithelial cells death. The intense immunoexpression of LC3A and p62/SQSTM1 indicates autophagic disturb, which in association with high UCHL1 levels, points to a role of UPS and autophagy in the regulation of epididymal epithelial cells viability under androgenic control.

Changes in porcine cauda epididymal fluid proteome by disrupting the HPT axis: Unveiling potential mechanisms of male infertility.

Male infertility or subfertility is frequently associated with disruption of the hypothalamic-pituitary-testis axis events, like secondary hypogonadism. However, little is known how this condition affects the proteomic composition of the epididymal fluid. In the present study, we evaluated the proteomic changes in the cauda epididymal fluid (CEF) in a swine model of secondary hypogonadism induced by anti-GnRH immunization using multidimensional protein identification technology. Seven hundred and eighteen proteins were identified in both GnRH-immunized and control groups. GnRH immunization doubled the number of proteins in the CEF, with 417 proteins being found exclusively in samples from GnRH-immunized boars. CEF from GnRH-immunized boars presented an increase in the number of proteins related to cellular and metabolic processes, with affinity to organic cyclic compounds, small molecules, and heterocyclic compounds, as well changed the enzymatic profile of the CEF. Also, a significant increase in the number of proteins associated to the ubiquitin-proteasome system was identified in CEF from GnRH-immunized animals. These results bring strong evidence of the impact of secondary hypogonadism on the epididymal environment, which is responsible for sperm maturation and storage prior ejaculation. Finally, the differently expressed proteins in the CEF are putative seminal biomarkers for testicular and epididymal disorders caused by secondary hypogonadism.