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Abstract: Seminal fluid extracellular vesicles (SFEVs) have previously been shown to interact with spermatozoa and influence their fertilisation capacity. Here, we sought to extend these studies by exploring the functional consequences of SFEV interactions with human spermatozoa. SFEVs were isolated from the seminal fluid of normozoospermic donors prior to assessing the kinetics of sperm-SFEV binding in vitro, as well as the effects of these interactions on sperm capacitation, acrosomal exocytosis, and motility profile. Biotin-labelled SFEV proteins were transferred primarily to the flagellum of spermatozoa within minutes of co-incubation, although additional foci of SFEV biotinylated proteins also labelled the mid-piece and head domain. Functional analyses of high-quality spermatozoa collected following liquefaction revealed that SFEVs did not influence sperm motility during incubation at pH 5, yet SFEVs induced subtle increases in total and progressive motility in sperm incubated with SFEVs at pH 7. Additional investigation of sperm motility kinematic parameters revealed that SFEVs significantly decreased beat cross frequency and increased distance straight line, linearity, straightness, straight line velocity, and wobble. SFEVs did not influence sperm capacitation status or the ability of sperm to undergo acrosomal exocytosis. Functional assessment of both high- and low-quality spermatozoa collected prior to liquefaction showed limited SFEV influence, with these vesicles inducing only subtle decreases in beat cross frequency in spermatozoa of both groups. These findings raise the prospect that, aside from subtle effects on sperm motility, the encapsulated SFEV cargo may be destined for physiological targets other than the male germline, notably the female reproductive tract., Lay Summary: A male's influence over the biological processes of pregnancy extends beyond the provision of sperm. Molecular signals present in the ejaculate can influence the likelihood of pregnancy and healthy pregnancy progression, but the identity and function of these signals remain unclear. In this study, we wanted to understand if nano-sized particles present in the male ejaculate, called seminal fluid extracellular vesicles, can assist sperm in traversing the female reproductive tract to access the egg. To explore this, we isolated seminal fluid extracellular vesicles from human semen and incubated them with sperm. Our data showed that seminal fluid extracellular vesicles act to transfer molecular information to sperm, but this resulted in only subtle changes to the movement of sperm.

Despite the significant morphological changes that occur in the seminal vesicles with aging, the transcriptomic characteristics remain largely unexplored. To address this, we performed bulk RNA sequencing on seminal vesicle samples from mice aged 3, 13, and 21 months to uncover transcriptomic alterations. Our findings reveal that aged seminal vesicles display cystic dilatation, epithelial hypoplasia, disordered muscle layers, fibrosis, and reduced proliferation capability. A comparison between 3-month-old and 21-month-old mice indicated that leukocyte-mediated immunity and leukocyte migration were the most significantly upregulated biological processes among differentially expressed genes (DEGs). Notably, several DEGs associated with "leukocyte migration," such as Vcam1 , Cxcl13 , and Ccl8 , exhibited an increasing trend in transcriptomic and protein expression at three different time points in the seminal vesicles of mice. Additionally, we identified multiple aging-associated DEGs, including P21 and Tnfrsf1b . Two genes ( Cd209f and Ccl8 ) were consistently upregulated across all six regions of the male reproductive glands (testis, epididymis, and seminal vesicle) in the comparison of bulk RNA datasets from 3-month-old and 21-month-old mice. These analyses highlight an enhanced state of immune and inflammatory response in aged seminal vesicles. This study represents the first exploration of the overall transcriptome landscape of seminal vesicles in a murine model of natural aging, offering new insights into the mechanisms underlying aging-related seminal vesicle dysfunction.

Aims: To evaluate the basal release of 6-nitrodopamine (6-ND) from human isolated seminal vesicles (HISV) and to characterize its action and origin., Main Methods: Left HISV obtained from patients undergoing prostatectomy surgery was suspended in a 3-mL organ bath containing warmed (37 °C) and gassed (95%O 2 :5%CO 2 ) Krebs-Henseleit's solution (KHS) with ascorbic acid. An aliquot of 2 mL of the supernatant was used to quantify catecholamines by LC-MS/MS. For functional studies, concentration-responses curves to catecholamines were obtained, and pEC 50 and E max values were calculated. Detection of tyrosine hydroxylase and S100 protein were also carried out by both immunohistochemistry and fluorescence in-situ hybridization assays (FISH)., Key Findings: Basal release of 6-ND was higher than the other catecholamines (14.76 ± 14.54, 4.99 ± 6.92, 3.72 ± 4.35 and 5.13 ± 5.76 nM for 6-ND, noradrenaline, adrenaline, and dopamine, respectively). In contrast to the other catecholamines, the basal release of 6-ND was not affected by the sodium current (Nav) channel inhibitor tetrodotoxin (1 μM; 10.4 ± 8.9 and 10.4 ± 7.9 nM, before and after tetrodotoxin, respectively). All the catecholamines produced concentration-dependent HISV contractions (pEC 50 4.1 ± 0.2, 4.9 ± 0.3, 5.0 ± 0.3, and 3.9 ± 0.8 for 6-ND, noradrenaline, adrenaline, and dopamine, respectively), but 6-ND was 10-times less potent than noradrenaline and adrenaline. However, preincubation with very low concentration of 6-ND (10 -8  M, 30 min) produced significant leftward shifts of the concentration-response curves to noradrenaline. Immunohistochemical and FISH assays identified tyrosine hydroxylase in tissue epithelium of HISV strips., Significance: Epithelium-derived 6-ND is the major catecholamine released from human isolated seminal vesicles and that modulates smooth muscle contractility by potentiating noradrenaline-induced contractions., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Neurokinin B; an endogenous decapeptide, mediates its reproductive physiological actions through gonadotropin releasing hormone. Despite the potential role of Neurokinin B on seminal vesicles, its effects on seminal vesicles in adult male mammals remain elusive. We aimed to investigate the potentials of variable doses of Neurokinin B, its agonist and antagonist on histomorphology and expression of NK3R on seminal vesicles, and secretory activity of seminal vesicles in adult male rats., Methods: Adult male Sprague Dawley rats (n=10 in each group) were administered intraperitoneally with Neurokinin B in three variable doses: 1 μg, 1 ηg and 10 ρg while, Senktide (Neurokinin B agonist) and SB222200 (Neurokinin B antagonist) in 1 μg doses consecutively for 12 days. After 12 days of peptide treatment, half of the animals (n=05) in each group were sacrificed while remaining half (n=05) were kept for another 12 days without any treatment to investigate treatment reversal. Seminal vesicles were dissected and excised tissue was processed for light microscopy, immunohistochemistry and estimation of seminal fructose levels., Results: Treatment with Neurokinin B and Senktide significantly increased while SB222200 slightly decrease the seminal vesicles weight, epithelial height and seminal fructose levels as compared to control. Light microscopy revealed increased epithelial height and epithelial folding as compared to control in all Neurokinin B and Senktide treated groups while decreased in SB222200. Effects of various doses of Neurokinin B, Senktide and SB222200 on seminal vesicles weight, epithelial height, seminal fructose levels and histomorphology were reversed when rats were maintained without treatments. Immuno-expression of Neurokinin B shows no change in treatment and reversal groups., Conclusion: Continuous administration of Neurokinin B and Senktide effect positively while SB222200 have detrimental effects on cellular morphology, epithelial height and seminal fructose levels in seminal vesicles. Effects of peptide treatments depicted a reversal towards control group when rats were kept without any treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Seminal vesiculitis is a common inflammation in the male genital tract. Etiologically, microbial infection and non-infectious factors can be responsible for seminal vesiculitis. The pathogenic triggers and mechanisms underlying non-infectious seminal vesiculitis remain unclear., Objectives: To demonstrate that spermatozoa can induce seminal vesiculitis in mice, which could be attributable to spermatozoa-induced innate immune responses in seminal vesicular epithelial cells., Material and Methods: Spermatozoa from epididymis were injected into seminal vesicles at the tail of the gland. Histopathology of seminal vesicles were examined by hematoxylin-eosin staining. Infiltration of leukocytes were identified by immunohistochemistry. Seminal vesicular epithelial cells were isolated from 5-week-old mice and cell types were detected by immunofluoresence. Western blot and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to detect protein and gene expression levels., Results: In vivo, local injection of epididymal spermatozoa into seminal vesicles resulted in seminal vesiculitis characterized by tissue swelling and leukocyte infiltration. In vitro, spermatozoa induced the expression of pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, CXCL10, and MCP1, and the activation of NF-κB in seminal vesicular epithelial cells., Discussion and Conclusion: Spermatozoa may induce seminal vesiculitis through the activation of innate immune responses in seminal vesicular epithelial cells, which provide novel insights into the mechanisms underlying non-infectious seminal vesiculitis., (© 2023 American Society of Andrology and European Academy of Andrology.)

It was known that diabetes may affect the male reproductive function by inhibiting the secretion of male accessory glands including seminal vesicles. Increased cell apoptosis induced by oxidative stress is thought to be an important pathological change in the seminal vesicles in diabetic patients. Quercetin is a potent anti-oxidative bioflavonoid. In this study, we explore the effect of quercetin on cell apoptosis of seminal vesicles and its underlying mechanism. The STZ-induced type 1 diabetic rat model was established. Three doses (low, medium and high) of quercetin were administrated to the STZ-induced type 1 diabetic rats for 4 months. Fasting blood glucose, the fructose in seminal plasma, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in seminal vesicles were determined by colorimetric method. Nuclear transcription factor- Nrf2 was observed by immunofluorescent staining. Biomarkers related to cell apoptosis, such as Bcl-2, Bax and cleaved -Caspase3 were measured by Western blotting and immumohistochemical staining. The body weight and seminal vesicle weight indexes were also determined. The results showed that T-AOC and Nrf2 were decreased, the levels of MDA were increased, the cleaved Caspase-3 was increased and the ratio of Bax to BCL-2 was decreased in seminal vesicles of diabetic rats, along with the severe hyperglycemia. When diabetic rats were treated by quercetin for 4 months, all the indexes were reversed at different degree except the fasting blood glucose. Our results suggested that quercetin could ameliorate oxidative stress‑induced cell apoptosis of seminal vesicles via inhibiting Nrf2 in type 1 diabetic rats, which indicated that quercetin could be used for preventing lesions of seminal vesicles in type 1 diabetes., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology., (© 2022 The Authors. Proteomics published by Wiley-VCH GmbH.)

Lysozyme (LYZ) c-like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c-type LYZ family, the seminal vesicle-secreted LYZ c-like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N-linked glycans. SVLLP is structurally similar to c-type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c-type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin-induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm-oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor., (© 2021 Wiley Periodicals LLC.)

Male subfertility causes are very varied and sometimes related to post-gonadic maturation disruption, involving seminal plasma constituents. Among them, extracellular vesicles are involved in key exchanges with sperm in mammals. However, in birds, the existence of seminal extracellular vesicles is still debated. The aim of the present work was first to clarify the putative presence of extracellular vesicles in the seminal plasma of chickens, secondly to characterize their size and protein markers in animals showing different fertility, and finally to make preliminary evaluations of their interactions with sperm. We successfully isolated extracellular vesicles from seminal plasma of males showing the highest differences in semen quality and fertility by using ultracentrifugation protocol (pool of 3 ejaculates/rooster, n =3/condition). Size characterization performed by electron microscopy revealed a high proportion of small extracellular vesicles (probably exosomes) in chicken seminal plasma. Smaller extracellular vesicles appeared more abundant in fertile than in subfertile roosters, with a mean diameter of 65.12 and 77.18 nm, respectively. Different protein markers of extracellular vesicles were found by western blotting (n = 6/condition). Among them, HSP90A was significantly more abundant in fertile than in subfertile males. In co-incubation experiments (n = 3/condition), extracellular vesicles enriched seminal fractions of fertile males showed a higher capacity to be incorporated into fertile than into subfertile sperm. Sperm viability and motility were impacted by the presence of extracellular vesicles from fertile males. In conclusion, we successfully demonstrated the presence of extracellular vesicles in chicken seminal plasma, with differential size, protein markers and putative incorporation capacity according to male fertility status.

Dendritic cells (DCs) are innate immune cells which engulf, process and present antigens to the naïve T-lymphocyte cells. However, little is known about the effect of melatonin on the DCs. The present study aimed to investigate the morphology and distribution of the DCs by transmission electron microscopy and Immunohistochemistry after melatonin administration. A total of 8 out of 15 adult ram was randomly selected to receive the melatonin implant and the remaining 7 animals received melatonin free implants. DCs showed positive immunoreactivity for CD117, S-100 protein and CD34. There is an obvious increase in the number of the positive immunoreactive cells to CD3, estrogen receptor alpha and progesterone in the treated groups. The expression of CD56 and MHCII in the DCs was abundant in the treated groups. The ultrastructure study revealed that melatonin exerts a stimulatory effect on the DCs which was associated with increment in the secretory activity of DCs. The secretory activity demarcated by an obvious increase in the number of mitochondria, cisternae of rER and a well-developed Golgi apparatus. The endosomal- lysosomal system was more developed in the treated groups. A rod-shaped Birbeck granule was demonstrated in the cytoplasm of the melatonin treated group. DCs were observed in a close contact to telocytes, T-Lymphocytes, nerve fibers and blood vessels. Taken together, melatonin administration elicits a stimulatory action on the DCs and macrophages through increasing the size, the number and the endosomal compartments which may correlate to increased immunity.

Seminal vesicles are an integral part of the male reproductive accessory gland system. They produce a complex array of secretions containing bioactive constituents that support gamete function and promote reproductive success, with emerging evidence suggesting these secretions are influenced by our environment. Despite their significance, the biology of seminal vesicles remains poorly defined. Here, we complete the first proteomic assessment of mouse seminal vesicles and assess the impact of the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or control daily for five consecutive days prior to collecting seminal vesicle tissue. A total of 5013 proteins were identified in the seminal vesicle proteome with bioinformatic analyses identifying cell proliferation, protein synthesis, cellular death, and survival pathways as prominent biological processes. Secreted proteins were among the most abundant, and several proteins are linked with seminal vesicle phenotypes. Analysis of the effect of acrylamide on the seminal vesicle proteome revealed 311 differentially regulated (FC ± 1.5, p ≤ 0.05, 205 up-regulated, 106 downregulated) proteins, orthogonally validated via immunoblotting and immunohistochemistry. Pathways that initiate protein synthesis to promote cellular survival were prominent among the dysregulated pathways, and rapamycin-insensitive companion of mTOR (RICTOR, p = 6.69E-07) was a top-ranked upstream driver. Oxidative stress was implicated as contributing to protein changes, with acrylamide causing an increase in 8-OHdG in seminal vesicle epithelial cells (fivefold increase, p = 0.016) and the surrounding smooth muscle layer (twofold increase, p = 0.043). Additionally, acrylamide treatment caused a reduction in seminal vesicle secretion weight (36% reduction, p = 0.009) and total protein content (25% reduction, p = 0.017). Together these findings support the interpretation that toxicant exposure influences male accessory gland physiology and highlights the need to consider the response of all male reproductive tract tissues when interpreting the impact of environmental stressors on male reproductive function., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent evidence entail paternal factors as plausible contributors in spontaneous recurrent pregnancy loss (RPL). Seminal extracellular vesicles secreted from cells of male reproductive tract carry regulatory proteins and RNAs. They are proposed to regulate sperm maturation and function while their fusion to endometrial stromal cells helps in decidualization. Nevertheless, the mechanism(s) involved in these processes are poorly understood. This study aims at elucidating the molecular basis of paternal contribution by comparative proteomics (label-free LC-MS/MS) of isolated seminal extracellular vesicles from fertile men and partners of patients with RPL (n = 21 per group). Bioinformatics analysis revealed the identified differentially expressed proteins to be involved in DNA replication, recombination and repair, gene expression, cellular assembly and organization, cell death, and survival. Major disease pathways affected were identified as developmental, hereditary, and immunological disorders. Of the three identified hub genes regulating the above disease pathways, two (HNRNPC and HNRNPU) are overexpressed while RUVBL1 is underexpressed along with over expression of HIST1H1C, DDX1, surmising defective chromatin packaging, and histone removal in spermatozoa resulting in improper expression in paternal genes thereby leading to abnormal embryo development. Besides, alteration in GSTP1 expression points oxidative predominance in RPL group. Differential expression of C3, C4a/C4b, CFB, and GDF 15 may be involved in altered maternal immune response to paternal antigens resulting in impaired decidualization., (© 2020 Wiley Periodicals LLC.)

Puberty is a transitional period from juvenile stage to adulthood, followed by the functional maturation of gonads and reproductive organs. This period is sensitive to environmental pollutants like cadmium (Cd), a heavy metal that represents a serious health risk. Cd is an endocrine disruptor that interferes with reproduction by causing oxidative stress in the reproductive organs, affecting the sexual function and decreasing testosterone (T) levels. However, little research has been done on the effects of Cd on puberty markers and antioxidant systems. In this study, we evaluated the effects of Cd on puberty markers: preputial separation, testes descent and T levels, and the antioxidant activity (SOD, CAT, GSH/GSSG and TAC) in the seminal vesicles, testis and epididymis. Male Wistar pups were treated with 1 mg/kg Cd or saline solution by i.p. injection from day 1 to 35; the other treatment was administrated for 49 days. At the end of treatment, the animals were sacrificed, and the tissues of interest dissected, weighed and prepared for the respective assays. Cd treated rats from birth to puberty showed a delay onset in the puberty markers and a low weight in reproductive organs. Also, Cd induced differential effects on the redox system in reproductive organs and decreased T levels, these effects played a pivotal role in the delay of puberty markers onset (testes descent and preputial separation), affecting the development and sexual maturity of the male rats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

NKX3.1 is considered a reliable immunohistochemical marker of prostatic origin with high specificity and sensitivity. However, NKX3.1 positivity has been described in other neoplastic and non-neoplastic tissues, such as mesenchymal chondrosarcoma, sex-cord stromal tumors, rete testis adenocarcinoma, lobular and ductal carcinoma of the breast, salivary glands, peribronchial submucosal glands, and Sertoli cells. We analyzed expression of two antibodies (mono and polyclonal) of NKX3.1 in a total of 63 non-neoplastic seminal vesicles. We used 52 resection materials (12 seminal vesicles without prostatic adenocarcinoma, 26 seminal vesicles with prostatic adenocarcinoma infiltration, and 14 cases of seminal vesicles infiltrated by urothelial carcinoma) and 11 prostatic core needle biopsies with incidentally sampled fragment of seminal vesicles. In all cases, tissues from seminal vesicles were completely negative for NKX3.1, despite using polyclonal and monoclonal NKX3.1 antibodies, and regardless of the detection system utilized (diaminobenzidine (DAB) versus alkaline phosphatase (AF)). However, prostatic adenocarcinoma was negative in several cases (n = 6), when AF detection system was used. Reaction with DAB was strong and robust in all cases. Based on our data, we can recommend NKX3.1 as a negative immunohistochemical marker of seminal vesicles., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Purpose: The fate of subcutaneously transplanted urogenital sinus (UGS) and seminal vesicle (SV) was investigated in the present study., Materials and Methods: Fetal UGS and SV extracted from 20-embryonic-day-old male normal and GFP transgenic rats were subcutaneously transplanted into 7-week-old male immunologically inhibited rats. The transplants were then examined at 2, 4, 8, and 16 weeks after transplantation. We analyzed the survival ratio, weight, and histopathology as well as the immunohistochemical characteristics of the transplanted tissues. For control experiments, 2-, 4-, 8-, and 16-week-old normal male rats were used., Results: Almost all of the transplanted tissues survived under the skin, and the tissue weights increased over time after transplantation. The histopathological characteristics and immunohistochemical staining pattern with certain antibodies of the transplanted tissues were similar to those of normal adult rat prostate and seminal vesicle. The transplanted GFP transgenic tissues demonstrated spontaneous growth and organ formation under the skin, showing distribution and movement of transplanted cells and tissues., Conclusion: Subcutaneously transplanted fetal UGS and SV were able to develop into mature adult organs., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

A 64-year-old man with lung cancer underwent F-FDG PET/CT for restaging, which demonstrated intense F-FDG uptake in the right lobe of prostate gland and seminal vesicles, indicating a potential prostate cancer. In Ga-PSMA PET/CT, intense uptake in the right lobe of prostate gland and seminal vesicles was also observed but decreased in postmictional delayed images. Magnetic resonance imaging showed high signal intensity of urine in the same areas of uptakes. F-FDG and Ga-PSMA PET/CT findings in the prostate gland and seminal vesicles were considered to be a result of urinary reflux possibly because of the patient's previous transurethral resection.

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.

Mucopolysaccharidosis type I (MPS I) is a genetic disease caused by a deficiency of the lysosomal hydrolase α-L-iduronidase (IDUA). IDUA degrades two types of glycosaminoglycans (GAGs): heparan and dermatan sulfates, important components of extracellular matrix, with signaling and structural functions. The accumulation of GAGs results in progressive physiological impairments in a variety of tissues, making MPS I a complex and multisystemic disease. Due the advent of therapeutic strategies which have increased patients' life expectancy, our group have been investigating the effect of IDUA deficiency on the reproductive system. In the present study, we aimed to characterize some of the accessory glands of the male reproductive tract in an MPS I mouse model. We used 6-month-old Idua+/+ and Idua-/- male mice to evaluate the histology of the seminal vesicles and prostate. Interstitial deposits of GAGs and collagen fibers were also observed. Seminal vesicles were smaller in the Idua-/- group, regardless of the normal staining pattern of the epithelial cells, marked with antiandrogen receptor. The prostate of Idua-/- mice presented necrotic acini and increased deposition of collagen fibers in the interstitium. All glands presented evident deposits of GAGs in the extracellular matrix, especially inside vacuolated interstitial cells. We concluded that, at this stage of the disease, the prostate is the most damaged accessory gland and may therefore, be the first to manifest functional impairments during disease progression.

Infections of the genital tract can perturb the fertility in humans and animals. Pathogen recognition and activation of innate immunity onset through the pattern recognition receptor activation, such as Toll-like receptor 4 (TLR4), leading to the production of proinflammatory cytokines and mediators. TLR4 is expressed both on leukocytes and nonimmune cells. Rabbit TLR4 shows great similarity to its human counterpart. Moreover, the TLR4 signalling pathway could be modulated by long-chain polyunsaturated fatty acids (LC-PUFA). The objectives of this study were (i) to determine the expression levels of TLR4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit and (ii) to evaluate if the n-3 PUFA-enriched diets can modify their expression levels in the tissues and LC-PUFA profiles in seminal plasma. Fifteen rabbit bucks ( n = 5/experimental group) were fed with different diets: commercial standard (group C), rich in extruded linseed (10%, group L), and in fish oil (3%, group FO) for 110 days. TLR4, TNF- α , and IL-1 β mRNA were ubiquitously expressed throughout the hypothalamic-gonadal axis. However, TLR4 mRNA expression was lower in the hypothalamus than the epididymis ( P < 0.01), seminal vesicles ( P < 0.01), and pituitary gland ( P < 0.05). Dietary enrichment in PUFA did not modify the gene expression profile nor the histological characteristics of the tissues. Conversely in seminal plasma, rabbits fed with L and FO had lower n-6 ( P < 0.05), LC-PUFA n-6 ( P < 0.05), and n-6/n-3 ratio ( P < 0.05) but higher n-3 ( P < 0.001) and LC-PUFA n-3 ( P < 0.01) compared to the control group. Our study builds a map of the gene expression of TRL4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit, fundamental step for understanding the immune defence mechanisms. Diets enriched in LC-PUFA did not affect basal gene expression but modulated sperm fatty acid composition. Finally, rabbit may be an excellent animal model to study the relationship between inflammation and infertility, and the nutritional modulation of immune functions., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Laura Menchetti et al.)

Seminal fluid proteins (SFPs) exert potent effects on male and female fitness. Rapidly evolving and molecularly diverse, they derive from multiple male secretory cells and tissues. In Drosophila melanogaster , most SFPs are produced in the accessory glands, which are composed of ∼1,000 fertility-enhancing "main cells" and ∼40 more functionally cryptic "secondary cells." Inhibition of bone morphogenetic protein (BMP) signaling in secondary cells suppresses secretion, leading to a unique uncoupling of normal female postmating responses to the ejaculate: refractoriness stimulation is impaired, but offspring production is not. Secondary-cell secretions might therefore make highly specific contributions to the seminal proteome and ejaculate function; alternatively, they might regulate more global-but hitherto undiscovered-SFP functions and proteome composition. Here, we present data that support the latter model. We show that in addition to previously reported phenotypes, secondary-cell-specific BMP signaling inhibition compromises sperm storage and increases female sperm use efficiency. It also impacts second male sperm, tending to slow entry into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high female refractoriness and sperm competitiveness are mutually exclusive. Using quantitative proteomics, we reveal changes to the seminal proteome that surprisingly encompass alterations to main-cell-derived proteins, indicating important cross-talk between classes of SFP-secreting cells. Our results demonstrate that ejaculate composition and function emerge from the integrated action of multiple secretory cell types, suggesting that modification to the cellular make-up of seminal-fluid-producing tissues is an important factor in ejaculate evolution., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The seminal vesicles can be infected by microorganisms, thereby resulting in vesiculitis and impairment in male fertility. Innate immune responses in seminal vesicles cells to microbial infections, which facilitate vesiculitis, have yet to be investigated. The present study aims to elucidate pattern recognition receptor-mediated innate immune responses in seminal vesicles epithelial cells. Various pattern recognition receptors, including Toll-like receptor 3, Toll-like receptor 4, cytosolic ribonucleic acid, and deoxyribonucleic acid sensors, are abundantly expressed in seminal vesicles epithelial cells. These pattern recognition receptors can recognize their respective ligands, thus activating nuclear factor kappa B and interferon regulatory factor 3. The pattern recognition receptor signaling induces expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (Tnfa) and interleukin 6 (Il6), chemokines monocyte chemoattractant protein-1 (Mcp1) and C-X-C motif chemokine 10 (Cxcl10), and type 1 interferons Ifna and Ifnb. Moreover, pattern recognition receptor-mediated innate immune responses up-regulated the expression of microsomal prostaglandin E synthase and cyclooxygenase 2, but they down-regulated semenogelin-1 expression. These results provide novel insights into the mechanism underlying vesiculitis and its impact on the functions of the seminal vesicles., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transgenic adenocarcinoma mouse prostate (TRAMP) model is established to mimic human prostate cancer progression, where seminal vesicle lesions often occur and has been described as phyllodes-like epithelial-stromal tumors. However, the molecular mechanism regulating tumorigenesis and progression in seminal vesicles of TRAMP mice remains largely unknown. In this study, C57BL/6 TRAMP mice were found to have a significantly shorter lifespan than wild-type (WT) mice and all of the seminal vesicles were markedly increased in size and weight with age from 24 weeks exhibiting a clearly papillary-phyllode pattern, though no obvious difference was observed in multiple organs including heart, liver, spleen, lungs, kidneys, testicles and bone between TRAMP and WT mice, and less than 10% of TRAMP mice developed prostate tumors. Western blotting showed Cyclin (CCN) B1 and CCND1 were remarkably overexpressed in seminal vesicle tumors of TRAMP mice at 24 weeks of age and increased with age till the end of trial, which was confirmed by Immunohistochemistry (IHC). P21 and P27 were also significantly augmented, whereas P53 and phosphorylated P53 (p-P53) were constantly expressed in normal controls and P53 did not appear to be mutated. Not only cyclin-dependent kinase (CDK) 1 and phosphorylated forkhead box protein (FOX) O1 but also CDK4, CDK6 and phosphorylated retinoblastoma-associated protein (RB) had similar increase trends, so did epidermal growth factor receptor (EGFR), AKT serine/threonine kinase (AKT), and their respective phosphorylation levels. Signal transducer and activator of transcription (STAT) 3, p-STAT3, enhancer of zeste homolog 2 (EZH2) and EZH2 mediated trimethylation of histone H3 lysine 27 (H3K27me3) were considerably elevated, too. Taken together, this finding suggests P21 and P27 promote carcinogenesis and development in seminal vesicles of TRAMP mice via accelerating cell cycle progression, in which oncogenic transformation of P21 and P27 might be through regulation of EGFR-AKT signaling., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Previous studies reported the effects of valproic acid (VPA) on tyrosine-phosphorylated (TyrPho) protein expression in the testis and epididymis, but its effects on that in the seminal vesicle (SV) have never been demonstrated. This study attempted to investigate the expressions of TyrPho proteins in SV treated with VPA. Sixteen rats were divided into control and VPA-treated groups. The control rats were injected with normal saline, whereas the treated animals were intraperitoneally injected with VPA (500 mg/kg BW) for 10 consecutive days (n = 8/each). The biochemical parameters in blood plasma and SV fluid were analysed. The SV tissues and fluid were investigated for the presence and expression of TyrPho proteins, androgen receptor (AR) proteins and heat shock protein 70 (Hsp70). Significantly, VPA caused SV atrophy and reductions in secretion and biochemical parameter levels. There were significant increases in many TyrPho proteins in the plasma (a 95 kDa) and SV tissue (a 40 kDa) of the VPA rats. However, the expressions of seminal AR, Hsp70 and TyrPho proteins (50 and 48 kDa) were significantly lower in VPA rats. Recent results have indicated that VPA affected SV morphology and decreased biochemical fluid substances including TyrPho proteins associated with decreased expressions of AR and Hsp70., (© 2019 Blackwell Verlag GmbH.)

We aimed to establish a novel rat model of seminal vesiculitis that would provide an effective approach to investigate the pathogenesis of this disease in the future. Eight male rats received the same operation, during which the root of one of the two seminal vesicles was partly ligatured with sutures and the other vesicle was left intact. The samples of seminal vesicles were harvested on the 8 th day following the operation. Hematoxylin and eosin and Masson's trichrome stains were used to observe the histopathology and the presence of fibrous tissue in seminal vesicles, respectively. Immunoblotting and immunohistochemistry were applied to determine the tumor necrosis factor-alpha and cyclooxygenase-2 levels in seminal vesicle tissues. Real-time fluorescence quantitative polymerase chain reaction was performed to measure the gene expression levels of proinflammatory cytokines. H 2 O 2 levelsin the seminal plasma from the seminal vesicle were also measured. Hematoxylin and eosin staining suggested that there was inflammatory cell infiltration into the seminal vesicles treated by partial root ligation. The tumor necrosis factor-alpha and cyclooxygenase-2 proteins were significantly upregulated in the treated seminal vesicles. The tumor necrosis factor-alpha, cyclooxygenase, interleukin 6, and inducible nitric oxide synthase mRNA expression levels were also upregulated in the treated seminal vesicles. The H 2 O 2 levels in the seminal plasma from seminal vesicles with partial root ligation were significantly elevated compared with those from vesicle left intact. In conclusion, partially ligating the root of the seminal vesicle via sutures in rats is an effective method to establish a seminal vesiculitis rat model.

Purpose: No comprehensive information is available about uterus fatty acid (FA) change during implantation period and possible effects of the seminal vesicle secretion on it., Materials and Methods: In this study, we evaluated FA composition of uterus phospholipids during the implantation period in intact and seminal vesicle-excised (SVX) mated female mice. Forty NMRI female mice were divided into control (mated with intact male) and seminal vesicle excised (SVX)-mated (mated with SVX-male) groups. The phospholipid fatty acids composition was monitored during the fi rst fi ve days of pregnancy using gas chromatography and also implantation rate was evaluated on fi fth day of pregnancy., Results: We found that levels of linoleic acid (LNA) and arachidonic acid (ARA) showed a decreasing trend from the fi rst to the third day of pregnancy and then started to increase on the fourth day and peaked on the fi fth day. In contrast, the level of saturated FA (SFA) increased on the second and third day of pregnancy compared to the fi rst (p<0.05) and then decreased on the fourth and fi fth. We also found that the seminal vesicle secretion could affect the levels of LNA, ARA, SFA, and PUFA in uterine phospholipids especially on second and third day. Moreover, there was a positive correlation between ARA level and implantation rate in control but not SVX-mated groups., Conclusions: It can be concluded that several uterus FA that have important roles in early pregnancy could be affected by seminal vesicle secretion., Competing Interests: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Direct treatment of ER (+) breast cancer with Formestane diminishes the tumor within weeks. This is unlikely due to lack of estrogens alone. We proposed that it is the negative influence of androgens on the growth of ER(+) breast cancer. We investigated the influence of Formestane and Exemestane and of their major androgenic metabolites 4-hydroxytestosterone and 17-hydroexemestane on the proliferation of MCF-7 cells and ZR-75-1 cells. Inhibitory effects could be prevented by antiandrogens and siRNA. Activation of the AR in MCF-7 and U2-OS cells was tested by reporter gene assays. In vivo androgenicity was evaluated using the Hershberger assay. Influence on the cell cycle was demonstrated by flow-cytometry. Influence of androgens on the activity of CCND1 was demonstrated by Chip-qPCR. Antitumor activity was determined by topical treatment of DMBA tumors. We found that breast cancer cells can metabolize Formestane and Exemestane to androgenic compounds which inhibit proliferation. This can be explained by hindering the accessibility of CCND1 by histone modification. Androgenic metabolites can abolish the growth of DMBA-tumors and prevent the appearance of new tumors. The lack of cross-resistance between steroidal and nonsteroidal aromatase inhibitors is due to inhibitory effects of androgenic steroidal metabolites on the production of cyclin D1. These sterols not only inhibit proliferation of cancer cells but can also stop the growth of DMBA cancers upon direct absorption into the tumor. The quick and considerable effect on ER(+) tumors may open a new avenue for neodjuvant treatment.

The development of highly differentiated sperm cells that are specialized for navigating to and fusing with an oocyte is essential for sexual reproduction. As a major part of differentiation, sperm undergo extensive post-meiotic maturation en route to the oocyte. This is regulated largely by soma-derived cues. In Caenorhabditis elegans , this process is called sperm activation, and it transforms immotile spermatids into migratory fertilization-competent cells. Here, we show that the negative regulator of sperm activation, SWM-1, is produced in an unexpected cell type: body wall muscle. SWM-1 is secreted into the body cavity and enters the gonad; there, it is present with its likely target, TRY-5, a spermiogenesis activator. We show that, in addition to SWM-1, the somatic gonad and body fluid can exchange other factors, suggesting that soma-germ line transfer could affect other reproductive processes. In addition, we show that SWM-1 may have a separate role in the sperm migratory environment, to which it is contributed by both males and hermaphrodites. These findings reveal that late stages in gamete differentiation can be regulated at the whole-organism level by broadly secreted factors.This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Pigment epithelium derived factor (PEDF) expression has been described in many organs as showing neurotrophic, anti-angiogenic, anti-apoptotic, anti-inflammatory, anti-oxidant and pro-cell survival properties. However, references to its activity in the male reproductive system are scarce. We aimed to characterize the expression of PEDF in the male reproductive tract of Wistar rats by using RT-PCR, western blot and immunostaining and also evaluate the effect of flutamide in PEDF expression. We found that PEDF is expressed in the epididymis, prostate and seminal vesicles in Wistar rats, but notably not in the testes. Under the effect of flutamide PEDF expression decreased, recovering by suppressing the antiandrogen. The epididymis is an essential organ in sperm maturation-storages. The role of PEDF in this physiological process has not been fully elucidated yet, but considering that in other systems PEDF has anti-apoptotic, anti-oxidants and pro-cell survival properties, its expression along the epididymis could play a role in the protection of spermatozoa while they are stored., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Currently, no genomic signature exists to distinguish men most likely to progress on adjuvant androgen deprivation therapy (ADT) after radical prostatectomy for high-risk prostate cancer. Here we develop and validate a gene expression signature to predict response to postoperative ADT. Experimental Design: A training set consisting of 284 radical prostatectomy patients was established after 1:1 propensity score matching metastasis between adjuvant-ADT (a-ADT)-treated and no ADT-treated groups. An ADT Response Signature (ADT-RS) was identified from neuroendocrine and AR signaling-related genes. Two independent cohorts were used to form three separate data sets for validation (set I, n = 232; set II, n = 435; set III, n = 612). The primary endpoint of the analysis was postoperative metastasis. Results: Increases in ADT-RS score were associated with a reduction in risk of metastasis only in a-ADT patients. On multivariable analysis, ADT-RS by ADT treatment interaction term remained associated with metastasis in both validation sets (set I: HR = 0.18, P interaction = 0.009; set II: HR = 0.25, P interaction = 0.019). In a matched validation set III, patients with Low ADT-RS scores had similar 10-year metastasis rates in the a-ADT and no-ADT groups (30.1% vs. 31.0%, P = 0.989). Among High ADT-RS patients, 10-year metastasis rates were significantly lower for a-ADT versus no-ADT patients (9.4% vs. 29.2%, P = 0.021). The marginal ADT-RS by ADT interaction remained significant in the matched dataset ( P interaction = 0.035). Conclusions: Patients with High ADT-RS benefited from a-ADT. In combination with prognostic risk factors, use of ADT-RS may thus allow for identification of ADT-responsive tumors that may benefit most from early androgen blockade after radical prostatectomy. We discovered a gene signature that when present in primary prostate tumors may be useful to predict patients who may respond to early ADT after surgery. Clin Cancer Res; 24(16); 3908-16. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERα) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERα mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in αERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health., Competing Interests: The authors declare no conflict of interest.

Objective: To investigate the effect of finasteride on the microvascular density (MVD) and the expression of the vascular endothelial growth factor (VEGF) in the seminal vesicle of rats., Methods: Forty male SD rats were randomly and equally divided into groups A, B, C and D, those in groups A and B fed with normal saline as the control and those in C and D with finasteride at 40 mg per kg of the body weight per day, A and C for 14 days and B and D for 28 days. Then the seminal vesicles of the animals were harvested for HE staining, measurement of MVD, determination of the expressions of CD34 and VEGF by immunohistochemistry, and observation of histomorphological changes in the seminal vesicle., Results: The expressions of CD34 in groups C and D were decreased by 6.7% and 15.8% as compared with those in A and B (P<0.01), and that in group D decreased by 9.3% in comparison with that in C (P<0.01). The expression indexes of VEGF in groups C and D were decreased by 6.9% and 14.1% as compared with those in A and B (P<0.01), and that in group D decreased by 9.0% in comparison with that in C (P<0.01)., Conclusions: Finasteride can inhibit the expression of VEGF in the seminal vesicle tissue of the rat and hence suppress the angiogenesis of microvessels of the seminal vesicle.

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell-cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles., (Copyright © 2018 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.)

Prostaglandin E 2 (PGE 2 ) has been introduced as an important factor for embryo implantation. So the present study was carried out to evaluate the effect of seminal fluid (SF) on PGE 2 pathway in uterus tissues of mice during window of preimplantation. The messenger RNA (mRNA) expressions of microsomal PGE synthase (mPGES) and cytosolic PGE synthase (cPGES) as well as protein expression of PGE receptor 2 and 4 (EP2 and EP4) were determined in uterine tissue of control and seminal vesicle-excised (SVX)-mated female mice during days 1 to 5 of pregnancy using real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. We found that mRNA expression of mPGES at day 1 and 2 of pregnancy was significantly higher in the control group than the SVX-mated group ( P < .05), but such result was not obtained for cPGES expression. The protein levels of EP2 at day 1 to 4 of pregnancy were significantly higher in the control group compared with the SVX-mated group ( P < .05), also the EP4 levels were significantly different between the control and SVX-mated groups at the first day of pregnancy ( P < .05). Implantation rate was higher in the control group and also there were positive correlations between mPGES and EP2 expressions in the fifth day of pregnancy with implantation rate. Our results demonstrated significant effect of SF on uterine expressions of the evaluated factors, especially mPGES and EP2. Regarding the correlations between levels of these factors and implantation rate, we suggest that possibly one of the important mechanisms of SF in affecting female pregnancy is through mPGES and EP2.

The present study was conducted to investigate the effect of incorporating Cicer arietinum in the diet on the testicular functions of the male mice. Seventy-two mice were divided equally into four groups that were daily fed a diet containing 0, 20, 30 and 50% of C. arietinum seeds, respectively. After 7, 14 and 21 days of starting the experiments, the mice were anesthetized and euthanized to collect the blood, testes, epididymis and seminal vesicles. The present results showed that the increased percentage of C. arietinum in the diet caused significant elevations in the serum levels of testosterone and luteinizing hormone (LH), sperm concentration, sperm motility as well as the testicular levels of antioxidants including glutathione (GSH), glutathione peroxidase (GPx) and catalase (CAT), in comparison to the controls. On the other hand, marked reductions in the sperm abnormality, testicular levels of malondialdehyde (MDA), the percentage of DNA damage in tail and tail moment (TM) were observed in the mice that received a diet containing C. arietinum as compared to the controls. Both the sperms and testes of the mice fed a diet containing C. arietinum in the diet showed a normal intact appearance of the electrophoresed genomic DNA on agarose, as those of the controls. In conclusion, C. arietinum is not only a safe ingredient in the fast-food but also an enhancer of the testicular functions.

The endocannabinoid system (ECS), comprising the cannabinoid receptors (CBR), their ligands, and enzymes controlling the turnover of endocannabinoids, has been suggested to be involved in male reproductive function. As information is scarce on the expression of the ECS in human male reproductive tissues, this study aimed to investigate by means of molecular biology (RT-PCR) and immunohistochemistry/immunofluorescence the expression and distribution of CB1 and CB2, GPR55 (an orphan G protein-coupled receptor that recognises cannabinoid ligands) and FAAH (isoforms 1 and 2) in the human seminal vesicles (SV). The specimens expressed PCR products corresponding to CB1 (66 bp), CB2 (141 bp), GPR55 (112 bp), FAAH1 (260 bp) and FAAH2 (387 bp). Immumohistochemistry revealed dense expression of CB1, CB2 and GPR55 located to the pseudo-stratified columnar epithelium and varicose nerves (also characterised by the expression of vasoactive intestinal polypeptide and calcitonin gene-related peptide). Cytosolic staining for FAAH1 and FAAH2 was seen in cuboidal cells of all layers of the epithelium. No immunoreactivity was detected in the smooth musculature or nerve fibres. CB1, CB2, GPR55, FAAH1 and FAAH2 are highly expressed in the human SV. Considering their localisation, the ECS may be involved in epithelial homeostasis, secretory function or autonomic mechano-afferent signalling., (© 2017 Blackwell Verlag GmbH.)

The present study aimed at investigating age-related changes in epididymal sperm motility, morphology, DNA integrity and androgen receptor (AR) expression in canine reproductive tissues. Fifty-five healthy medium-sized male dogs were divided into four groups: young (1 - 3 years old, n = 14), adult (>3 - 6 years old, n = 12), old (>6 - 9 years old, n = 14) and senile (>9 years old, n = 15). After routine castration, testes, epididymides (head, body and tail) and vas deferens were collected. Spermatozoa were flushed from epididymal tails and their motility, morphology and DNA integrity were examined. Localization of AR was investigated by immunohistochemistry and the positive immunostaining cells were evaluated using image analysis software (NuclearQuant, 3DHISTECH). We found significantly lower percentages of epididymal sperm motility, sperm vigour and viability in adult, old and senile dogs in comparison with young dogs (p < 0.05). Animal's age negatively correlated with epididymal sperm motility, sperm vigour and viability. The primary, secondary, major and minor epididymal sperm defects were significantly higher in senile dogs compared to young dogs. There were positive correlations between age and epididymal sperm defects (p < 0.01). The percentage of sperm with fragmented DNA did not differ between age groups. Testicular AR was expressed in the nucleus of Sertoli cells, Leydig cells and peritubular myoid cells, except for germ cells. Expression of AR was found in all epithelium, lamina propria and smooth muscle cells of the epididymis and vas deferens. Expression levels of AR in testis, epididymis and vas deferens did not differ between age groups (p > 0.05). In conclusion, the present study clearly demonstrated that senescence in dogs was associated with decreased epididymal sperm quality. An age-related increase in the incidence of poor epididymal sperm quality may promote subfertility, especially in senile dogs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Millions of people consume betel nut for increased capacity to work and for stress reduction. The nut contains arecoline, which has multiple side effects on endocrine functions. Objective of the work is to investigate pineal-testicular responses to noise and after arecoline treatment in noise in rats. Noise exposure (100 dB, 6 h daily, 10 days) caused pineal stimulation ultrastructurally and at indoleamines level. Leydig cell dysfunction with fall of testosterone level and suppression of sex accessories were noticed. In contrast, pineal activity was inhibited and reproductive functions were stimulated after arecoline administration, confirmed from reversed changes to those of noise. Arecoline treatment in noise exposure showed same results as in noise both in pineal and in reproductive functions. It is concluded that noise causes testicular dysfunction probably by gonadotropin suppression induced by pineal melatonin in noise. Furthermore, arecoline cannot prevent it in noise in rats.

Diabetes mellitus significantly affects the male reproduction and sexual function. In the present study, we investigated the diabetes-induced dysfunction of seminal vesicles (SVs) in the diabetes-rat model and the role of antioxidants. Streptozotocin-induced diabetes after 4 weeks caused smaller size of the organs, hypercontractility, histological abnormalities, increased concentrations of malondialdehyde in the serum and tissue, overexpression of oxidative stress markers, and cleaved caspase-3 as identified by immunohistochemistry in the SVs. In addition, diabetes resulted in deceased levels of serum testosterone and no newborns after the mating studies. Antioxidants significantly normalized all the above parameters, except for the severely decreased serum testosterone levels and the negative outcome of the mating studies. The present study gives evidence for the important role of diabetes-induced oxidative stress in the function and structure of these androgen-dependent organs. Antioxidants may be a promising supplementary therapy for diabetic male patients to alleviate ejaculatory disorders but alone is not efficient treatment for the mitigation of infertility.

Objective: To evaluate whether preoperative urinary prostate cancer antigen 3 (PCA3) scores predict for adverse pathologic features (APFs) or progression-free survival (PFS) in men with intermediate- or high-risk prostate cancer (PCa) undergoing radical prostatectomy (RP)., Materials and Methods: One hundred nine men with intermediate- (n = 52) or high-risk (n = 57) PCa who underwent RP were retrospectively identified. Logistic regression analysis was performed to evaluate the association of PCA3 score with various APFs (eg, extracapsular extension, seminal vesicle invasion, etc.). Among 78 men with ≥1 year of follow-up, the association between PCA3 score and PFS was assessed using Cox regression analysis., Results: At RP, 52% of patients had at least 1 APF, and with median follow-up of 2.3 years, overall 3-year PFS was 70%. PCA3 was not a significant predictor of any APF on multivariate analysis (MVA), whereas canonical predictors (eg, biopsy Gleason score and initial prostate-specific antigen) remained predictive of various APFs. No significant predictors for PFS were found on MVA, although certain canonical predictors (eg, National Comprehensive Cancer Network risk group) were significant predictors of PFS on univariate analysis (UVA). PCA3 score was not a significant predictor of PFS on either UVA or MVA., Conclusion: Unlike in lower risk cohorts, increasing PCA3 score was not associated with any APF in this higher risk cohort, despite enrichment for APFs, nor was it associated with PFS. Notably, multiple known preoperative predictors for APFs were significant on MVA, and multiple predictors were associated with PFS on UVA. Therefore, PCA3 may not be a useful adjunct predictive marker in men with intermediate- or high-risk PCa., (Copyright © 2017 Elsevier Inc. All rights reserved.)

VASA, also known as DDX4, is reported to be specifically expressed in cells belonging to the germ cell lineage, both in males and females. Therefore, it could be an informative protein biomarker to be applied on semen to differentiate between obstructive and nonobstructive azoospermia (OA and NOA, respectively). In addition, it could be of value to predict sperm retrieval based on testicular sperm extraction. Immunocytochemistry of proven OA semen using both polyclonal and monoclonal antibodies against VASA showed positive staining of both cells and cell sized particles. This is spite of being the absolute negative controls, completely lacking germ lineage derived cells and material. In order to identify the source of the VASA-positive material, a detailed screen of different anatomical parts of the whole male urogenital tract was performed of multiple cases using immunohistochemistry.The polyclonal antibody stained, besides the expected germ cells in the testis, epithelium of the bladder and the seminal vesicles. The monoclonal antibody only stained the latter. To investigate whether the immunohistochemical staining is associated with the presence of the corresponding VASA mRNA, samples of seminal vesicles, bladder, testis, and semen (with and without germ cells) were investigated using the specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on 42 samples. A positive result was detected in testis and semen containing germ cells (n = 10 and 8), being negative in semen without germ cells (n = 11), bladder (n = 3), and seminal vesicles (n = 10).Two commercially available VASA antibodies (mono- and polyclonal) are not specific. In contrast, VASA-mRNA evaluation, using qRT-PCR, is specific for the presence of germ cells, therefore, is an interesting molecular biomarker for germ cell detection in semen.

Background: Post-spermiogenesis membrane surface modifications rely on molecules present in the reproductive tracts. Two isoforms (isoform 1 and 2) from Quiescin Q6-Sulfydryl Oxidase protein family have been identified in the male reproductive tract of rodent species. However, unlike isoform 1, scarce information is available for isoform 2, likely due to its lower expression level and lack of proper purification methods to obtain sufficient protein quantity for further assays., Results: This study demonstrated the presence of short and long forms of Quiescin Q6-Sulfydryl Oxidase 2 in boar, likely representing the secretory (short form) and transmembrane (long form) forms of Quiescin Q6-Sulfydryl Oxidase 2. Immunohistochemistry studies revealed the presence of Quiescin Q6-Sulfydryl Oxidase 2 in a broad range of porcine tissues; the pronounced vesicle-contained Quiescin Q6-Sulfydryl Oxidase 2 at the apical region of epididymis and seminal vesicles epithelium suggested its involvement in sperm physiology and its participation in semen formation. The majority of porcine Quiescin Q6-Sulfydryl Oxidase 2 could be purified via either antibody affinity column or be salted out using 10%-40% ammonium sulfate. Higher amount of low molecular weight Quiescin Q6-Sulfydryl Oxidase 2 observed in the seminal vesicle likely represents the secretory form of Quiescin Q6-Sulfydryl Oxidase 2 and reflects an exuberant secretory activity in this organ., Conclusions: We demonstrated for the first time, the presence of Quiescin Q6-Sulfydryl Oxidase 2 in porcine species; moreover, two forms of Quiescin Q6-Sulfydryl Oxidase 2 were identified and exhibited distinct molecular weights and properties during protein purification processes. This study also provided feasible Quiescin Q6-Sulfydryl Oxidase 2 purification methods from slaughterhouse materials that could potentially allow obtaining sufficient amount of Quiescin Q6-Sulfydryl Oxidase 2 for future functional investigations.

We evaluated the effects of dutasteride on the genitourinary tract using fifteen 8-week-old male Sprague-Dawley rats. Animals were divided into three groups comprising five animals each and treated as follows. Group A was a control group, members of Group B received oral administration of dutasteride 0.1 mg/kg/day from the age of 8 to 16 weeks, and members of Group C were castrated at the age of 8 weeks. All rats were killed at the age of 16 weeks for the sample collection of blood, bladder, prostate, seminal vesicles, and penis. Then, we evaluated the pathological examination for evaluating the tissue fibrosis and hormonal receptor expression. The results showed that the mean size of the prostate and seminal vesicles was smaller in Group B and Group C than in Group A. Serum and tissue concentrations of both testosterone and dihydrotestosterone were remarkably reduced in serum and all tissues in Group C compared with Group A. On the other hand, in Group B, only dihydrotestosterone was reduced in serum and penis. Histopathological examination revealed that Group C showed statistically significant histological changes, such as an increase in fibrotic tissue in the bladder, prostate, and penis. Similarly, Group B showed fibrotic changes in the prostate and penis compared with the Group A. Immunofluorescent staining revealed that the androgen receptor was more strongly expressed than the estrogen receptor beta in Group A. On the other hand, in Group C, weak expression of the androgen receptor and strong expression of the estrogen receptor beta was noted. In Group B, these changes were noted in the prostate and penis. These findings suggest that dutasteride cause morphological changes not only in prostate but also in penis. These changes are associated with altered expression patterns of androgen receptor and estrogen receptor., (© 2016 American Society of Andrology and European Academy of Andrology.)

With increased industrial utilization of iron oxide nanoparticles (Fe 2 O 3 -NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe 2 O 3 -NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe 2 O 3 -NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe 2 O 3 -NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe 2 O 3 -NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe 2 O 3 -NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe 2 O 3 -NPs could be an environmental risk factor for reproductive disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Study Question: Is junctional adhesion molecule A (JAM-A), a sperm protein essential for normal motility, expressed in the murine post-testicular pathway and involved in sperm maturation?, Summary Answer: JAM-A is present in the prostate and seminal vesicles and in all three regions of the epididymis where it is secreted in epididymosomes in the luminal fluid and can be delivered to sperm in vitro., What Is Known Already: JAM-A shares with the plasma membrane Ca2+ATPase 4 (PMCA4, the major Ca2+ efflux pump in murine sperm) a common interacting partner, CASK (Ca2+/CaM-dependent serine kinase). JAM-A, like PMCA4, plays a role in Ca2+ regulation, since deletion of Jam-A results in significantly elevated intracellular Ca2+ levels and reduced sperm motility. Recently, PMCA4 was reported to be expressed in the epididymis and along with CASK was shown to be in a complex on epididymosomes where it was transferred to sperm. Because of the association of JAM-A with CASK in sperm and because of the presence of PMCA4 and CASK in the epididymis, the present study was performed to determine whether JAM-A is expressed in the epididymis and delivered to sperm during their maturation., Study Design, Size, Duration: The epididymides, prostate and seminal vesicles were collected from sexually mature C57BL/6J and Institute for Cancer Research mice and antibodies specific for JAM-A and Ser285 -phosphorylated JAM-A (pJAM-A) were used for the analysis. Tissues, sperm and epididymal luminal fluid (ELF) were studied. Epididymosomes were also isolated for study. Caput and caudal sperm were co-incubated with ELF individually to determine their abilities to acquire JAM-A in vitro., Participants/materials, Setting, Methods: Sections of all three regions of the epididymis were subjected to indirect immunofluorescence analysis. Epididymal tissues, fluid, sperm, prostate and seminal vesicle tissues were analyzed for JAM-A and/or pJAM-A via western blotting analysis. The relative amounts of JAM-A and pJAM-A among epididymal tissues, ELF and sperm were detected by western blot via quantification of band intensities. Epididymosomes were isolated by ultracentrifugation of the ELF after it was clarified to remove cells and tissue fragments, and the proteins western blotted for JAM-A and pJAM-A, and exosomal biochemical markers. FACS analysis was used to quantify the amount of JAM-A present on caput and caudal sperm, as well as the amount of JAM-A acquired in vitro after their co-incubation with ELF., Main Results and the Role of Chance: Western blots revealed that JAM-A is expressed in all three regions of the epididymis, the prostate and seminal vesicles. As confirmed by indirect immunofluorescence, a western blot showed that JAM-A has a higher expression in the corpus and caudal regions, where it is significantly (P < 0.01) more abundant than in the caput. Both JAM-A and Ser285-phosphorylated JAM-A (pJAM-A) are secreted into the ELF where it is highest in the distal regions. In the ELF, both JAM-A and pJAM-A were detected in epididymosomes. Western blotting of sperm proteins showed a significant (P < 0.01) increase of JAM-A and pJAM-A in caudal, compared with caput, sperm. Flow-cytometric analysis confirmed the increase in JAM-A in caudal sperm where it was 1.4-fold higher than in caput ones. Co-incubation of caput and caudal sperm with ELF demonstrated ~2.3- and ~1.3-fold increases, respectively, in JAM-A levels indicating that epididymosomes transfer more JAM-A to caput sperm that are less saturated with the protein than caudal ones., Large Scale Data: Not applicable., Limitations, Reasons for Caution: First, although the ELF was clarified prior to ultracentrifugation for epididymosome isolation, we cannot rule out contamination of the epididymosomal proteins by those from epididymal epithelial cells. Second, the JAM-A detected in the prostate and seminal vesicles might not necessarily be secreted from those organs and may only be present within the tissues, where it would be unable to impact sperm in the ejaculate., Wider Implications of the Findings: Although performed in the mouse the study has implications for humans, as the highly conserved JAM-A is a signaling protein in human sperm. There is physiological significance to the finding that JAM-A, which regulates sperm motility and intracellular Ca2+, exists in elevated levels in the cauda where sperm gain motility and fertilizing ability. The study suggests that the acquisition of JAM-A in the epididymal tract is involved in the mechanism by which sperm gain their motility during epididymal maturation. This increased understanding of sperm physiology is important for aspects of ART., Study Funding and Competing Interest(s): The work was supported by NIH-RO3HD073523 and NIH-5P20RR015588 grants to P.A.M.-D. The authors declare there are no conflicts of interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Prostate cancer is a leading cause of cancer-related death in males worldwide. Indeed, advanced and metastatic disease characterized by androgen resistance and often associated with neuroendocrine (NE) differentiation remains incurable. Using the spontaneous prostate cancer TRAMP model, we have shown that mast cells (MCs) support in vivo the growth of prostate adenocarcinoma, whereas their genetic or pharmacologic targeting favors prostate NE cancer arousal. Aiming at simultaneously targeting prostate NE tumor cells and MCs, both expressing the cKit tyrosine kinase receptor, we have tested the therapeutic effect of imatinib in TRAMP mice. Imatinib-treated TRAMP mice experience a partial benefit against prostate adenocarcinoma, because of inhibition of supportive MCs. However, they show an unexpected outgrowth of prostate NE tumors, likely because of defective signaling pathway downstream of cKit receptor. Also unexpected but very effective was the inhibition of epithelial-stromal tumors of the seminal vesicles achieved by imatinib treatment. These tumors normally arise in the seminal vesicles of TRAMP mice, independently of the degree of prostatic glandular lesions, and resemble phyllodes tumors found in human prostate and seminal vesicles, and in breast. In both mice and in patients, these tumors are negative for cKit but express PDGFR-β, another tyrosine kinase receptor specifically inhibited by imatinib. Our results imply a possible detrimental effect of imatinib in prostate cancer patients but suggest a promising therapeutic application of imatinib in the treatment of recurrent or metastatic phyllodes tumors. Mol Cancer Ther; 16(2); 365-75. ©2016 AACR., (©2016 American Association for Cancer Research.)

Estrogen related receptors (ERRs), categorized as orphan nuclear receptors, are critical for energy homeostasis and somatic development. However, significance of ERRs in the development of reproductive organs/organelles/cells remain poorly understood, albeit their homology to estrogen receptors. In this context, here, we show that knockdown of ERR in the testes leads to improperly developed testes with mis-regulation of genes (aly, mia, bruce, bam, bgcn, fzo and eya) involved in spermatogenesis, resulting in reduced male fertility. The observed testicular deformity is consistent with the down-regulation of SOX-E group of gene (SOX100B) in Drosophila. We also show dispersion/disintegration of fusomes (microtubule based structures associated with endoplasmic reticulum derived vesicle, interconnecting spermatocytes) in ERR knockdown testes. A few ERR knockdown testes go through spermatogenesis but have significantly fewer sperm. Moreover, flagella of these sperm are defective with abnormal axoneme and severely reduced mitochondrial derivatives, suggesting a possible role for ERR in mitochondrial biogenesis, analogous to mammalian ERRα. Interestingly, similar knockdown of remaining seventeen nuclear receptors did not yield a detectable reproductive or developmental defect in Drosophila. These findings add newer dimensions to the functions envisaged for ERR and provide the foundation for deciphering the relevance of orphan nuclear receptors in ciliopathies and testicular dysgenesis.

The Sudan-Black-B (SBB) histochemical stain is well known to specifically react against lipofuscin, an aggregate of oxidized proteins, lipids, and metals. Lipofuscin is related to many ageing processes. It is also known to accumulate in senescent cells. We recently proved that lipofuscin detection, when applying the SBB staining, is highly specific for the visualization of senescent cells. Here, we present in detail this SBB method that can detect senescent cells in any material, irrespective of its preparation. This provides unique advantages not only in understanding physiological processes and the pathophysiology of various diseases but also in estimating the response to therapeutic interventions.

Introduction: Elevated plasma homocysteine (Hcy) levels have been associated with several tissue injuries including heart and liver fibrosis. In these diseases, hyperhomocysteinemia (Hhcy) plays a major role in modulating the alteration of the balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMPs), leading to the pathological accumulation of extracellular matrix (ECM) proteins. Since the effect of Hhcy on ECM of seminal vesicle was not studied, the aim of our research was to check if Hcy can induce a remodeling within seminal vesicles ECM., Material and Methods: The study was conducted in 22 adult male Wistar rats. The rats were divided into two groups: a control group, which received standard diet and tap water; the treated group received the same diet and water supplemented with solution of L-methionine (200 mg/kg b.w./day) for 6 months. Plasma homocysteine concentration was measured. Histological changes were observed with light microscope. The presence of collagen I and III and metalloproteinases (2, 3, 7 and 9) in the seminal vesicles was examined using immunohistochemistry and Western blotting., Results: Plasma Hcy levels increased significantly after methionine treatment and interfered significantly with body weight in treated rats. The content of fibrillar collagens (I and III) in the wall of seminal vesicles was elevated in hyperhomocysteinemic rats. Moreover, we found that hyperhomocysteinemia increased the expression of MMP-2, -3, -7 and -9 in seminal vesicles of experimental rats., Conclusions: Increased plasma concentration of Hcy accompanied by the accumulation of collagen and upregulation of MMPs in rat seminal vesicles might contribute to the remodeling of seminal vesicles.