BMP signaling inhibition in Drosophila secondary cells remodels the seminal proteome and self and rival ejaculate functions.

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.

Novel role of proton-secreting epithelial cells in sperm maturation and mucosal immunity.

Epithelial cells are immune sensors and mediators that constitute the first line of defense against infections. Using the epididymis, a model for studying tubular organs, we uncovered a novel and unexpected role for professional proton-secreting 'clear cells' in sperm maturation and immune defense. The epididymal epithelium participates in the maturation of spermatozoa via the establishment of an acidic milieu and transfer of proteins to sperm cells, a poorly characterized process. We show that proton-secreting clear cells express mRNA transcripts and proteins that are acquired by maturing sperm, and that they establish close interactions with luminal spermatozoa via newly described 'nanotubes'. Mechanistic studies show that injection of bacterial antigens in vivo induces chemokine expression in clear cells, followed by macrophage recruitment into the organ. Injection of an inflammatory intermediate mediator (IFN-γ) increased Cxcl10 expression in clear cells, revealing their participation as sensors and mediators of inflammation. The functional diversity adopted by clear cells might represent a generalized phenomenon by which similar epithelial cells decode signals, communicate with neighbors and mediate mucosal immunity, depending on their precise location within an organ.

Pattern recognition receptor-mediated innate immune responses in seminal vesicle epithelial cell and their impacts on cellular function†.

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.

Quantitative (stereological) study of the epididymis and seminal vesicle in the rat from young to old.

There is a scarcity of morphometric data on the developmental and ageing changes in the epididymis and seminal vesicle in young and old rats. Eighty-six normal male Sprague-Dawley rats were randomly sampled from a cohort of animals aged 1-36 months (7-9 animals each age group). The epididymis and seminal vesicle (with the closely attached coagulating gland) were removed, and methacrylate resin-embedded sections were prepared for quantitative study of key histological structures by light microscopy. Stereological methods (point counting and optical disector) were used to estimate the total volumes of sperm mass, secretion (glandular lumen) and other structures and the number of spermatozoa. The results showed that the rapid growth of the reproductive organs was between 1 and 4 months of age. The epididymis stored the largest volume of sperm mass or number of spermatozoa at 12 months of age, but thereafter until 36 months of age, the sperm storage did not markedly diminish. The volume of secretion stored in the seminal vesicular gland declined by more than 35% from a plateau at 12-18 months until 36 months of age while that in the coagulating gland declined by more than 30% from a plateau at 18-24 months until 36 months of age.

Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.

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.

Effect of Melatonin on Telocytes in the Seminal Vesicle of the Soay Ram: An Immunohistochemical, Ultrastructural and Morphometrical Study.

Telocytes (TCs) are a special type of interstitial cell with characteristic cellular processes that are described in many organs. The current study aimed to investigate TCs in seminal vesicles of the Soay ram responding to melatonin treatment during the nonbreeding season by conventional immunohistochemical stains, and to detect the ultrastructural and morphometrical changes of TCs. TCs in the control group showed a broad range of staining affinity and also reacted positively to CD117/c-kit, CD34, desmin, S-100 protein, and progesterone and estrogen receptors alpha, while after melatonin treatment a strong reaction against these 6 antibodies was recorded. Electron microscopically, TCs in the control group were characterized by a small cell body with distinct long cytoplasmic extensions called telopodes (Tps). Tps had alternation of the thin segment (podomers) and dilated segments (podoms), in which the latter accommodate mitochondria, rough endoplasmic reticulum and caveolae. TCs and their Tps were interconnected by homo- and heterocellular junctions and form a wide network to communicate between different cell types. Tps showed close contact with immune cells, progenitor stem cells, smooth muscle cells and other interstitial cells. Melatonin caused a significant increase in the number of TCs, length of Tps, and number and diameter of secretory vesicles. Also, the melatonin-treated group showed exaggerated secretory activity in the form of a massive release of secretory vesicles from Tps. Moreover, Tps showed an increase in their contact with blood and lymphatic capillaries, nerve endings and Schwann cells. In addition, the shedding of secretory structures (exosomes, ectosomes, and multivesicular bodies) was greater from Tps, which were involved in paracrine signaling in the melatonin-treated group. The length and ramifications of Tps together with the intercellular junctions and the releasing of shed vesicles or exosomes assumed an essential role of TCs in intercellular signaling and coordination. On the basis of their distribution and morphology, we investigated whether the different locations of TCs could be associated with different roles.

Comparative analysis of the male reproductive accessory glands of bats Noctilio albiventris (Noctilionidae) and Rhynchonycteris naso (Emballonuridae).

In eutherian mammals, the male reproductive accessory glands (RAGs) comprise the prostate, bulbourethral glands, ampullary glands, and the seminal vesicles. Their composition, anatomy and function vary widely between species. This study aimed to characterize histologically and compare the RAGs of bats. The RAGs of Noctilio albiventris (Noctilionidae) and Rhynchonycteris naso (Emballonuridae) were studied using anatomical and histological methods, and were reconstructed three dimensionally. The RAGs of N. albiventris and R. naso are composed of a compact glandular complex that surrounds the urethra and a pair of bulbourethral glands, which are extra-abdominally located in the inguinal region. In both species, the glandular complex is composed of two well-defined prostatic regions (ventral and dorsal). The ventral region showed an atypical epithelium (holocrine), where no obvious cellular limits were observed, and PAS-positive secretion. The dorsal region had a pseudostratified cuboidal epithelium, with basal and secretory cells, and PAS-negative secretion. Noctilio albiventris also had urethral glands (Littre glands) surrounding the urethra, however, R. naso had only muscles. Both species had bulbourethral glands, with simple columnar epithelium and PAS-positive secretion. In conclusion, the RAGs of N. albiventris and R. naso comprised a pair of bulbourethral glands and an intra-abdominal complex, composed of a prostate with two different regions (ventral and dorsal), while the ampullary glands and seminal vesicles were missing in both species. This morphology was more closely related between N. albiventris and R. naso, and to species of the family Phyllostomidae than to families Molossidae and Vespertilionidae. J. Morphol. 277:1459-1468, 2016. © 2016 Wiley Periodicals, Inc.

Analysis of the cytoskeleton organization and its possible functions in male earthworm germ-line cysts equipped with a cytophore.

We studied the organization of F-actin and the microtubular cytoskeleton in male germ-line cysts in the seminal vesicles of the earthworm Dendrobaena veneta using light, fluorescent and electron microscopy along with both chemically fixed tissue and life cell imaging. Additionally, in order to follow the functioning of the cytoskeleton, we incubated the cysts in colchicine, nocodazole, cytochalasin D and latrunculin A. The male germ-line cells of D. veneta are interconnected via stable intercellular bridges (IB), and form syncytial cysts. Each germ cell has only one IB that connects it to the anuclear central cytoplasmic mass, the cytophore. During the studies, we analyzed the cytoskeleton in spermatogonial, spermatocytic and spermatid cysts. F-actin was detected in the cortical cytoplasm and forms distinct rings in the IBs. The arrangement of the microtubules changed dynamically during spermatogenesis. The microtubules are distributed evenly in whole spermatogonial and spermatocytic cysts; however, they primarily accumulate within the IBs in spermatogonia. In early spermatids, microtubules pass through the IBs and are present in whole cysts. During spermatid elongation, the microtubules form a manchette while they are absent in the cytophore and in the IBs. Use of cytoskeletal drugs did not alter the general morphology of the cysts. Detectable effects-the occurrence of nuclei in the late spermatids and manchette fragments in the cytophore-were observed only after incubation in nocodazole. Our results suggest that the microtubules are responsible for cytoplasmic/organelle transfer between the germ cells and the cytophore during spermatogenesis and for the positioning of the spermatid nuclei.

Neuroimmunomodulation by allogeneic seminal vesicle fluid in CNS is sex-independent.

OBJECTIVES: Thymus-arisen FoxP3 regulatory T cells (Tregs) are one of the most important immunoregulatory mechanisms in the central nervous system (CNS) and pregnancy. Multiple sclerosis (MS) is an inflammatory disease of CNS associated with a reduced frequency and/or function of Tregs. Previous works have shown that seminal vesicle fluid affects female immune system and causes expansion of Tregs pool in the female reproductive tissue upon mating. Accordingly, it has been demonstrated that intra-CSF administration of seminal vesicle fluid from Wistar rats can ameliorate clinical sign of a female Lewis rat model of experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The results indicated an up-regulation of FoxP3 expression in the brain and spinal cord. However, there are sex-based differences in the CNS structure & composition, sex hormones influence immune system, and gender-based differences affect treatment response. Therefore, we decided to find out if anti-inflammatory effect of seminal vesicle fluid is sex-dependent or -independent. METHODS: EAE was induced in male Lewis rats using guinea pig spinal cord and complete Freund's adjuvant. Intra-CSF injection was done on day 7 after EAE induction and the animals were followed-up until on day 14 after EAE induction when sacrificed. Then, brain and spinal cord of the animals were isolated, total RNA was extracted, and expression of mRNA for IFN-γ, IL-4, IL-17, and FoxP3 was determined using real-time PCR where ß-actin was used as reference gene. RESULT: demonstrated that intra-CSF administration of seminal vesicle fluid from male Wistar rats ameliorated EAE in male Lewis rats and increased FoxP3 in the brain and spinal cord. CONCLUSION: This study suggests that seminal vesicle fluid from Wistar rats has anti-inflammatory effect on Lewis rats in a sex-independent manner. In addition, seminal vesicle fluid from Lewis rats had not beneficial effect on EAE in male Lewis rats. This is consistent with Tregs increase in allogeneic mating. More research is required to find out the immunologic aspect of allogeneic versus syngeneic administration of seminal vesicle fluid.

Sperm traits differ between winged and wingless males of the ant Cardiocondyla obscurior.

Size and shape of sperm cells vary tremendously throughout the animal kingdom. The adaptive significance of this variation is not fully understood. In addition to sperm-female interactions and the environmental conditions, the risk of sperm competition might affect number, morphology and other "quality" traits of sperm. In the male-diphenic ant Cardiocondyla obscurior, winged sneaker males have limited sperm number, because their testes degenerate shortly after adult emergence, as is typical for males of social Hymenoptera. In contrast, wingless fighter males continuously replenish their sperm supply due to their exceptional lifelong spermatogenesis. While winged males usually have to compete with several other winged males for virgin queens, wingless males are able to monopolize queens by killing all other rivals. Hence, this presents a unique system to investigate how alternative reproductive tactics and associated physiology affect sperm morphology and viability. We found that sperm-limited males invest into sperm number instead of sperm size. Variance in sperm length is smaller in winged males, probably reflecting that they have to compete with several other males. Finally, sperm viability is equally high in both male phenotypes.

Expression of androgen receptor and cyclooxygenase-2 in the vesicular glands of castrated and intact goat.

This study was conducted to demonstrate the effect of castration on the structure of vesicular glands of the Egyptian Nubian (Zaraibi) goat. Vesicular glands of castrated (n=4) and intact (n=6) goat were used for histological and immunohistochemical evaluations. In this study, we report the difference in cell specific expression of androgen receptor (AR) and cyclooxygenase-2 (COX-2) in the vesicular glands of castrated and intact goats. In both castrated and intact goats, the present study revealed no immunopositive cells for AR or COX-2 in the fibromuscular stroma meanwhile, AR and COX-2 containing immunoreactive cells were restricted only to the epithelium of the secretory acini of the vesicular gland. Such finding suggests androgen and COX-2 as important regulators for the growth and secretory activity of epithelial cells in the vesicular gland of goats. Overall, the vesicular gland of castrated goats showed significantly (P<0.05) lower AR and COX-2 immuno-expression than intact goats indicating that both AR and COX-2 are androgen dependent.

Role of testicular luminal factors on Basal cell elongation and proliferation in the mouse epididymis.

A subset of basal cells (BCs) in the initial segment (IS) of the mouse epididymis has a slender body projection between adjacent epithelial cells. We show here that these projections occasionally cross the apical tight junctions and are in contact with the luminal environment. Luminal testicular factors are critical for the establishment of the IS epithelium, and we investigated their role in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Cytokeratin 5 (KRT5) labeling showed a time-dependent reduction of the percentage of BCs with intercellular projections from 1 to 5 days after EDL, compared to controls. Double labeling for caspase-3 and KRT5 showed that a subset of BCs undergoes apoptosis 1 day after EDL. Ki67/KRT5 double labeling showed a low rate of BC proliferation under basal conditions. However, EDL induced a marked increase in the proliferation rate of a subset of BCs 2 days after EDL. A 2-wk treatment with the androgen receptor antagonist flutamide did not affect the number of BCs with intercellular projections, but reduced BC proliferation. Flutamide treatment also reduced the increase in BC proliferation induced 2 days after EDL. We conclude that, in the adult mouse IS, 1) luminal testicular factors play an important role in the ability of BCs to extend their body projection towards the lumen, and are essential for the survival of a subset of BCs; 2) androgens play an important role in the proliferation of some of the BCs that survive the initial insult induced by EDL; and 3) the formation and elongation of BC intercellular projections do not depend on androgens.

Sperm production responds to perceived sperm competition risk in male Drosophila melanogaster.

Postcopulatory sexual selection arising from female multiple mating leads to the evolution of ejaculates that maximize a male's reproductive success under sperm competition. Where the risk of sperm competition is variable, optimal fitness may be achieved by plastically altering ejaculate characteristics in response to the prevailing sperm competition environment. In the model species Drosophila melanogaster, males expecting to encounter sperm competition mate for longer and transfer more accessory proteins and sperm. Here we show that after being housed with a single rival for one week, the seminal vesicles of male D. melanogaster contain a significantly greater proportion of live sperm than those of males maintained alone, indicating adaptive adjustment of sperm quality in response to the perceived risk of sperm competition. This effect is due to an increase in the number of live sperm produced, indicating that males upregulate sperm production in response to the presence of rivals. Our data suggest that males show plasticity in the rate of spermatogenesis that is adaptive in the context of a fluctuating sperm competition environment.

Sperm bundles in the seminal vesicles of sexually mature Lasius ant males.

In many insects, sperm cells are produced in bundles with their heads being held together by a glycoprotein matrix secreted by a cyst cell. Mature sperm cells in the seminal vesicles are usually free, but in sawflies and several other insects, such structures (spermatodesmata) remain intact and sperm cells may be ejaculated as bundles. Here we report the occurrence of spermatodesmata in mature males of the ant Lasius pallitarsis. Microscopic investigations of the abdominal contents of males immediately prior to their nuptial flights showed that the anterior ends of numerous sperm cells were embedded in an oval-shaped 20 by 30 micrometer extracellular fibrous cap. Individual sperm ranged in length from 55 to 75 micrometers with an average overall length of 65 micrometers. The bulb-shaped heads of the sperm were relatively small, only about 1.5 micrometers in length and about 1.1 micrometers in diameter. The diameter of the sperm tails was approximately 1 micrometer. Observations of live preparations of the spermatodesmata showed increasingly active undulating wave-like movement of the sperm tails as the slide preparations aged. This appears to be the first case of sperm bundles being present in the seminal vesicles of mature ant males--males that are immediately poised to complete their nuptial mating flight.

Selenoprotein P in seminal fluid is a novel biomarker of sperm quality.

Hepatically-derived selenoprotein P (SePP) transports selenium (Se) via blood to other tissues including the testes. Male Sepp-knockout mice are infertile. SePP-mediated Se transport to Sertoli cells is needed for supporting biosynthesis of the selenoenzyme glutathione peroxidase-4 (GPX4) in spermatozoa. GPX4 becomes a structural component of sperm midpiece during sperm maturation, and its expression correlates to semen quality. We tested whether SePP is also present in seminal plasma, potentially correlating to fertility parameters. Semen quality was assessed by sperm density, morphology and motility. SePP was measured by an immunoluminometric assay, and trace elements were determined by X-ray fluorescence spectroscopy. SePP levels were considerably lower in seminal plasma as compared to serum (0.4±0.1 mg/l vs. 3.5±1.0 mg/l); Se concentrations showed a similar but less pronounced difference (48.9±20.7 µg/l vs. 106.7±17.3 µg/l). Se and Zn correlated positively in seminal fluid but not in serum. Seminal plasma SePP concentrations were independent of serum SePP concentrations, but correlated positively to sperm density and fraction of vital sperm. SePP concentrations in seminal plasma of vasectomized men were similar to controls indicating that accessory sex glands are a testes-independent source of SePP. This notion was corroborated by histochemical analyses localizing SePP in epithelial cells of seminal vesicles. We conclude that SePP is not only involved in Se transport to testes supporting GPX4 biosynthesis but it also becomes secreted into seminal plasma, likely important to protect sperm during storage, genital tract passage and final journey.

Semaphorin 4F as a critical regulator of neuroepithelial interactions and a biomarker of aggressive prostate cancer.

BACKGROUND: Semaphorin 4F (S4F) has roles in embryologic axon guidance and is expressed in adults. S4F is involved in cancer-induced neurogenesis. METHODS: Prostate cells were transfected with S4F retrovirus. Cells and controls were used for a bromodeoxyuridine (BrdUrd) incorporation assay (proliferation) and in vitro scratch and Matrigel Transwell chamber invasion assay (migration). Monoclonal antibodies were developed using baculovirus-expressed recombinant GST-S4F and used to immunostain tissue microarrays. Slides were imaged using deconvolution and analyzed using tissue segmentation. Data were correlated with clinicopathologic parameters, other biomarkers and survival analysis conducted. Heterogeneity of S4F expression was analyzed with unsupervised clustering algorithms. RESULTS: Proliferation rates measured by BrdUrd incorporation were higher in all S4F-transfected cells. S4F overexpression was associated with increased motility of the cancer cells. S4F expression was overexpressed in high-grade prostatic intraepithelial neoplasia/prostate cancer than normal epithelium. S4F expression correlated with seminal vesicle invasion. Patients with high values of S4F in prostate cancer cytoplasm are at significantly higher risk of biochemical recurrence, by univariate and multivariate analyses. S4F cytoplasmic expression in prostate cancer cells also correlates with nerve density in prostate cancer and perineural invasion diameter. Correlations were identified with NF-κB and inversely with apoptosis in perineural invasion. CONCLUSION: These data show that S4F is significantly involved in human prostate cancer progression. S4F is a key regulator of the interactions between nerves in the tumor microenvironment and cancer cells. Because of the importance of cancer nerve interaction in the biology of cancer and its clinical implication, S4F can be considered a major therapeutic target. Clin Cancer Res; 19(22); 6101-11. ©2013 AACR.

Female-biased dimorphism underlies a female-specific role for post-embryonic Ilp7 neurons in Drosophila fertility.

In Drosophila melanogaster, much of our understanding of sexually dimorphic neuronal development and function comes from the study of male behavior, leaving female behavior less well understood. Here, we identify a post-embryonic population of Insulin-like peptide 7 (Ilp7)-expressing neurons in the posterior ventral nerve cord that innervate the reproductive tracts and exhibit a female bias in their function. They form two distinct dorsal and ventral subsets in females, but only a single dorsal subset in males, signifying a rare example of a female-specific neuronal subset. Female post-embryonic Ilp7 neurons are glutamatergic motoneurons innervating the oviduct and are required for female fertility. In males, they are serotonergic/glutamatergic neuromodulatory neurons innervating the seminal vesicle but are not required for male fertility. In both sexes, these neurons express the sex-differentially spliced fruitless-P1 transcript but not doublesex. The male fruitless-P1 isoform (fruM) was necessary and sufficient for serotonin expression in the shared dorsal Ilp7 subset, but although it was necessary for eliminating female-specific Ilp7 neurons in males, it was not sufficient for their elimination in females. By contrast, sex-specific RNA-splicing by female-specific transformer is necessary for female-type Ilp7 neurons in females and is sufficient for their induction in males. Thus, the emergence of female-biased post-embryonic Ilp7 neurons is mediated in a subset-specific manner by a tra- and fru-dependent mechanism in the shared dorsal subset, and a tra-dependent, fru-independent mechanism in the female-specific subset. These studies provide an important counterpoint to studies of the development and function of male-biased neuronal dimorphism in Drosophila.

Distribution of androgen and oestrogen receptors-α in the seminal vesicle-related spinal neurones in male rats.

The seminal vesicles are male accessory sex glands that contribute much of the seminal fluid volume. Previous studies have suggested that the majority of autonomic innervations to the rat seminal vesicles originate from the bilateral major pelvic ganglia. Many preganglionic autonomic neurones innervating the pelvic ganglion were expressed androgen receptors (AR) or oestrogen receptor (ER)-α immunoreactivity. However, direct neuroanatomic data regarding the distribution of AR and ER-α in seminal vesicle related-spinal neurones are lacking. In the present study, a nonvirulent pseudorabies virus (PRV-152 strain) was used in a retrograde tracing experiment. Four days after PRV injection into the seminal vesicles of male rats, spinal cord sections were prepared. Double- and triple-fluorescence techniques using AR and ER-α with choline acetyltransferase (ChAT) and PRV were used to investigate the AR and ER-α distribution in the seminal vesicles related spinal neurones in male rats. In lamina X, 14% of the PRV-labelled neurones in the L1-L4 segments and 43% in the L5-S1 segments were double-labelled with AR. In the L1-L4 segments, 6% of PRV-labelled neurones and 26% in the L5-S1 segments were double-labelled with ER-α. In the intermedial cell column area, 10% of PRV-labelled neurones in the L1-L4 segments and 47% of PRV-labelled neurones in the L5-S1 segments were double-labelled with AR. Up to 16% of PRV-labelled neurones in the L5-S1 segments were double-labelled with ER-α. No PRV-labelled neurones in the L1-L4 segments were double-labelled with ER-α. However, for the AR and ER-α/PRV/ChAT triple-fluorescence experiments, very few seminal vesicle preganglionic neurones expressed AR or ER-α. Our data suggests that many spinal interneurones but not preganglionic neurones involved in the seminal vesicle control in male rats were double-labelled with AR or ER-α, and they were mainly located at the parasympathetic level in the spinal cord.

Distribution of vitamin D receptor and 1α-hydroxylase in male mouse reproductive tract.

Vitamin D has been introduced as one of the main regulators of spermatogenesis. Here, for the first time, we evaluated the expression of vitamin D receptor (VDR) and 1α-hydroxylase in all organs of male mice reproductive tract by immunohistochemistry and Western blotting. Epithelial cells of epididymis, seminal vesicle, coagulating gland, ductus deferens, preputial gland, and prostate were the prominent cell types that concomitantly expressed VDR and 1α-hydroxylase. Nearly all cell types in testis expressed both proteins. Interestingly, VDR intensity in epididymis epithelial cells was reduced toward cauda, in which only strong staining of stereocilia was observed. Although been positive in caput epididymis, sperms lost their VDR expression in cauda region. In all organs, sperms failed to express 1α-hydroxylase. Specific bands of the VDR and 1α-hydroxylase were determined in all tissues, except testis in which novel unprecedented isoforms of 1α-hydroxylase were observed. Our findings could provide further convincing evidence of pivotal role of this hormone in male reproductive biology.

Suppression of spermatogenesis before grafting increases survival and supports resurgence of spermatogenesis in adult mouse testis.

OBJECTIVE: To test whether absence of complete spermatogenesis in mature testicular tissue before grafting will increase graft survival. DESIGN: Prospective experimental study. SETTING: Laboratory. ANIMAL(S): Donor testes were obtained from adult untreated mice, adult mice rendered cryptorchid, and adult mice treated with a GnRH antagonist (acyline). INTERVENTION(S): Donor testes were ectopically grafted to nude mice and recovered at three time points. MAIN OUTCOME MEASURE(S): Most advanced germ cell type and presence of spermatogonia were assessed. Donor testes and grafts were analyzed by histology and by immunocytochemistry for ubiquitin C-terminal hydrolase-L1 to mark germ cells. RESULT(S): Suppression of spermatogenesis by inducing cryptorchidism or acyline treatment resulted in improved survival of grafted tissue compared with controls and recovery of complete spermatogenesis, whereas control testis grafts mostly degenerated and did not restore complete spermatogenesis. CONCLUSION(S): These results indicate that complete spermatogenesis at the time of grafting has a negative effect on graft survival. Grafting of adult testis tissue from donors with suppressed spermatogenesis leads to spermatogenic recovery and may provide a tool to study and preserve fertility and for conservation of genetic resources in individuals that lack complete germ cell differentiation.