High‐resolution imaging of the actin cytoskeleton and epithelial sodium channel,CFTR, and aquaporin‐9 localization in the vas deferens

Vas deferens is a conduit for sperm and fluid from the epididymis to the urethra. The duct is surrounded by a thick smooth muscle layer. To map the actin cytoskeleton of the duct and its epithelium, we reacted sections of the proximal and distal regions with fluorescent phalloidin. Confocal microscopic imaging showed that the cylinder‐shaped epithelium of the proximal region has a thick apical border of actin filaments that form microvilli. The epithelium of the distal region is covered with tall stereocilia (13–18 µm) that extend from the apical border into the lumen. In both regions, the lateral and basal cell borders showed a thin lining of actin cytoskeleton. The vas deferens epithelium contains various channels to regulate the fluid composition in the lumen. We mapped the localization of the epithelial sodium channel (ENaC), aquaporin‐9 (AQP9), and cystic fibrosis transmembrane conductance regulator (CFTR) in the rat and mouse vas deferens. ENaC and AQP9 immunofluorescence were localized on the luminal surface and stereocilia and also in the basal and smooth muscle layers. CFTR immunofluorescence appeared only on the luminal surface and in smooth muscle layers. The localization of all three channels on the apical surface of the columnar epithelial cells provides clear evidence that these channels are involved concurrently in the regulation of fluid and electrolyte balance in the lumen of the vas deferens. ENaC allows the flow of Na⁺ ions from the lumen into the cytoplasm, and the osmotic gradient generated provides the driving force for the passive flow of water through AQP channels.     

Cyclic changes in the testis of the brook stickleback Eucalia inconstans (Kirtland)

The cyclic changes in the testis of the five-spined stickleback Eucalia inconstans (Kirtland) were studied histologically. Specimens were trapped between July 1965 and July 1967 in a shallow pond near London, Ontario. A three-dimensional microscopic study showed a main vas deferens and a system of primary, secondary and tertiary tubules. The testis cycle was divided into seven arbitrary stages. Spawning takes place from mid-April to mid-July. This is followed by the division of primary spermatogonia which are located along the walls of the tubules, producing cysts of spermatogonia enclosed in connective tissue which is surrounded by a thin epithelium. Both primary and secondary spermatocytes develop within these cysts. Breakdown of the cysts occurs with the development of spermatids and spermiogenesis occurs while spermatids are free in the tubules. Over-wintering of mature sperm takes place. Development of mature sperm from primary spermatogonia takes about 156 days. Germinal epithelium is absent but primary germ cells are believed to be those cells occupying the spaces between the tubules of the testis. No tissue which might be implicated in hormone production was observed. Phagocytic invasion of the testis has been studied. Massive infiltration by phagocytes is believed to be responsible for the sudden increase in testis weight observed during spawning. These cells ingest sperm nuclei and groups of them have been observed in the lumen of the tubules and the vas deferens, probably on their way out of the body.     

Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and DeltaF508 CFTR

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.     

Circadian rhythm of sperm release in the gypsy moth,Lymantria dispar: ultrastructural study of transepithelial penetration of sperm bundles

Release of mature bundles of spermatozoa from the testis into the vas deferens is a critical but poorly understood step in male insect reproduction. In moths, the release of sperm bundles is controlled by a circadian clock which imposes a temporal gate on the daily exit of bundles through the terminal epithelium-a layer of specialized epithelial cells separating testis follicles from the vas deferens. The sequence of cellular events associated with the daily cycle of sperm release was investigated by scanning and transmission electron microscopy. In the hours preceding sperm release, there is a solid barrier between the testis and the vas deferens formed by the interdigitation of cytoplasmic processes of adjacent terminal epithelial cells. At the beginning of the sperm release cycle, sperm bundles protrude through this barrier while the terminal epithelial cells change their shape and position relative to the bundles. Subsequently, the cyst cells enveloping the sperm bundles break down and spermatozoa move out of the testis through the exit channels formed between the epithelial cells. Afterwards, cyst cell remnants and other cellular debris are released into the vas deferens lumen, and the epithelial barrier is reconstructed due to phagocytic activity of its cells. These data provide a foundation on which to build an understanding of the cellular mechanisms of clock-controlled sperm release in insects.     

Circadian rhythm of glycoprotein secretion in the vas deferens of the moth, Spodoptera littoralis

Background  

Reproductive systems of male moths contain circadian clocks, which time the release of sperm bundles from the testis to the upper vas deferens (UVD) and their subsequent transfer from the UVD to the seminal vesicles. Sperm bundles are released from the testis in the evening and are retained in the vas deferens lumen overnight before being transferred to the seminal vesicles. The biological significance of periodic sperm retention in the UVD lumen is not understood. In this study we asked whether there are circadian rhythms in the UVD that are correlated with sperm retention.     

Ultrastructural evidence for secretion from the epithelium of ampulla ductus deferentis of the fan-throated lizard Sitana ponticeriana Cuvier

Among reptiles, an ampulla ductus deferentis has been reported only in Squamata. Fairly detailed studies are available only for two species, the lizard Calotes versicolor (Fam: Agamidae) and the snake Seminatrix pygaea (Fam: Colubridae). The light microscopic study on C. versicolor revealed the ampulla to be a prominent organ, whereas the light and transmission electron microscopic study in S. pygaea revealed it to be discernable only in histological preparations. Further, the epithelium of the ductal portion of vas deferens as well as the ampulla of C. versicolor appears to contribute to the seminal plasma and can also phagocytose dead sperm, whereas in S. pygaea neither of these roles has been established. Thus, we hypothesize that there may be variations in the anatomy, histology, and the role of the vas deferens in general, and the ampulla in particular, of the squamate reptiles. In this study, the ductus deferens of the small fan-throated lizard Sitana ponticeriana (Fam: Agamidae) was subjected to light and transmission electron microscopic analysis. In this lizard the ampulla is more prominent than in C. versicolor. The epithelium of the ductal portion of vas deferens consists of principal cells (with features reflecting roles in endocytosis and phagocytosis of dead sperm), dark cells (which are absent in the epithelium of the ductal portion of vas deferens of snakes), and basal cells. The ampulla of S. ponticeriana is differentiated into storage and glandular portions. The epithelium of the storage portion is like that in the ductal portion of the vas deferens, whereas that of the glandular portion, consisting of dark and light principal cells and foamy cells, is tall and forms into smooth villous folds. All three cell types show evidence for a role in secretion, in all likelihood different from each other, for release into the lumen to contribute to seminal plasma. These cells do not provide evidence of a role in phagocytosis of dead sperm. It appears that within the Squamata, the ductal ampulla differs in structure as well as function. We suggest that the ductal ampulla of agamid lizards is a composite gland of the ampulla ductus deferentis and seminal vesicles of mammals.     

A study of the effect of high concentrations of fluoride on the reproductive organs of male rabbits, using light and scanning electron microscopy

Fluoride was orally administered to rabbits at 10 mg NaF/kg body weight for 18 or 29 months. The animals were then killed and the structure of the testis, epididymis and vas deferens studied under light and scanning electron microscopes. In animals treated for 29 months, the spermatogenic cells in the seminiferous tubules were disrupted, degenerated and devoid of spermatozoa. In animals treated for 18 or 29 months, loss of cilia on the epithelial cells lining the lumen of the ductuli efferentes of the caput epididymidis and of stereocilia on the epithelial cells lining the lumen of the vas deferens was observed. In some regions of the epithelial lining of the lumen of the ductuli efferentes and vas deferens, the boundaries of the cells were not clear and appeared to be peeled off. Mucus droplets were abundant in the vas deferens of control animals, but absent in both the treated groups. Spermatogenesis ceased only in animals treated for 29 months. The difference in the structural changes observed in the testes of the 2 treated groups may have been due to the blood-testis barrier. It is concluded that ingestion of high concentrations of fluoride has harmful effects on the male reproductive system.     

Difference of fertilizing capacity between testicular sperm and vas deferens sperm in Cynops pyrrhogaster

The fertilizing capacity was compared between testicular and vas deferens sperm in Cynops pyrrhogaster. The testicular sperm was not capable of fertilizing jelly eggs. In contrast, the vas deferens sperm was already capable of fertilizing the newt jelly eggs. There was no inhibitory factor for fertilizing jelly eggs in the testis. These results suggest that the testicular sperm is immature as to the fertilizing capacity. The testicular sperm gained the fertilizing capacity for the jelly eggs by treatment with Holtfreter's solution or 1/20 strength Holtfreter's solution. The treatment may promote the step of maturation to achieve the fertilizing capacity. The treated testicular sperm did not fertilize dejellied eggs, although vas deferens sperm fertilized dejellied eggs. Therefore, the maturation state of the treated testicular sperm is different from that of vas deferens sperm. Newt sperm may be matured within the vas deferens, as the newt does not have an organ like the mammalian epididymis.     

Light and electron microscopic studies on the seminal secretions and the vas deferens of the penaeiodean shrimp,Sicyonia ingentis

Unlike the other penaeiodean shrimp, the ridge back shrimp, Sicyoniaingentis does not produce a spermatophore, but transfers sperm suspended in seminal plasm. This paper reports on the histomorphology and ultrastructure of the vas deferens with reference to its functional role in secreting the sperm bearing materials. The vas deferens is divisible into proximal secretory, mid storage and distal ejaculatory regions. The epithelial cells lining the proximal vas deferens are comprised of secretory and absorptive cell types. The loose sperm cells found in the lumen of this region are in an immature condition, and are agglutinated into a compact mass with signs of spermiogenesis in the mid vas deferens. The epithelial cells lining the mid vas deferens are short flattened cells. The distal vas deferens is ensheathed by muscular fibres. The inner epithelial cells are highly secretory and contain numerous microvilli at the luminal end. The sperm cord gets liquefied in this region facilitating the transfer of sperm in liquid form to the female during mating.     

Anatomical and histophysiological characterization of the male cloacal protuberance of the polygynandrous Alpine Accentor Prunella collaris

This paper describes the morphology of the male cloacal protuberance (CP) of the polygynandrous Alpine Accentor Prunella collaris , with special reference to intense sperm competition in the mating system. The fully developed CP consisted of dorsal (DL) and ventral (VL) lobes. The DL was found to be a massive part formed by a highly convoluted extension of the distal vas deferens with a fibro-muscular sheath. Here, the vas deferens contained abundant spermatozoa, round cells of unknown origin, and secretory droplets in the tubular lumen. The epithelium of the vas deferens was lined with columnar cells that showed numerous microvilli and apocrine processes in their periluminal portion, frequent engulfing of spermatozoa, and many mitochondria and vesicular endoplasmic reticula in their cytoplasm. The VL was a smaller subdivision distal to the DL and further connected to a terminal papilla. The VL was organized similarly to the DL, but showed different features: it lay beneath the thick skin; the fibro-muscular sheath was more muscular; the epithelial cells were cuboid and showed poorly developed microvilli; and the figures of secretion and engulfed spermatozoa were infrequent. Thus, the CP of the male Alpine Accentor showed a clear regional differentiation, i.e. the DL for storing/maintaining spermatozoa and the VL for storing/ejaculating spermatozoa.     

Ultrastructure and role of the lobster vas deferens in spermatophore formation: The proximal segment

We have examined the anatomy of the vas deferens of the lobster Homarus americanus and have described the structure of the proximal vas deferens (segments one and two). The two tubes of segment one descend from the testes and gradually merge into segment two. The epithelium of segment one has synthetic activity and appears to contribute to the sperm-supporting matrix by exocytotic release of granules through its apical surface. The epithelium of segment two is also highly synthetic and secretes the primary spermatophore layer and part of the intermediate layer that surround the sperm mass. The trifoil shape of the extruded spermatophore is established through a change in height of some of the cells lining the lumen in segment two. Connective tissue and circular bands of striated muscle surround the epithelium of both segments.     

Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function

Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant DeltaF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-kappaB. G551D mutants also lack Cl- channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-kappaB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked DeltaF508 allele caused a sevenfold activation of NF-kappaB compared with wild-type CFTR or the G551D mutant (P < 0.001). NF-kappaB was also activated in 9/HTEo-/pCep-R cells and in 16HBE/pcftr antisense cell lines, which lack CFTR Cl- channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-kappaB was associated with elevated interleukin-8 expression. Impaired CFTR Cl- channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-kappaB in cells with the CFTR mutation.     

Post-translational disruption of the delta F508 cystic fibrosis transmembrane conductance regulator (CFTR)-molecular chaperone complex with geldanamycin stabilizes delta F508 CFTR in the rabbit reticulocyte lysate

The DeltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) is a trafficking mutant, which is retained and degraded in the endoplasmic reticulum by the ubiquitin-proteasome pathway. The mutant protein fails to reach a completely folded conformation that is no longer a substrate for ubiquitination ("stable B"). Wild type protein reaches this state with 25% efficiency. In this study the rabbit reticulocyte lysate with added microsomal membranes has been used to reproduce the post-translational events in the folding of wild type and DeltaF508 CFTR. In this system wild type CFTR does not reach the stable B form if the post-translational temperature is 37 degrees C, whereas at 30 degrees C the behavior of both wild type and mutant proteins mimics that observed in the cell. Geldanamycin stabilizes DeltaF508 CFTR with respect to ubiquitination only when added post-translationally. The interaction of wild type and mutant CFTR with the molecular chaperones heat shock cognate 70 (hsc70) and heat shock protein 90 (hsp90) has been assessed. Release of wild type protein from hsc70 coincides with the cessation of ubiquitination and formation of stable B. Geldanamycin immediately prevents the binding of hsp90 to DeltaF508 CFTR, and after a delay releases it from hsc70. Release of mutant protein from hsc70 also coincides with the formation of stable B DeltaF508 CFTR.     

Morphology and immunolocalization of aquaporins 1 and 9 in the agouti (Dasyprocta azarae) testis excurrent ducts

This study investigated the morphology and immunoexpression of aquaporins (AQPs) 1 and 9 in the rete testis, efferent ducts, epididymis, and vas deferens in the Azara’s agouti (Dasyprocta azarae). For this purpose, ten adult sexually mature animals were used in histologic and immunohistochemical analyses. The Azara’s agouti rete testis was labyrinthine and lined with simple cubic epithelium. Ciliated and non-ciliated cells were observed in the epithelium of the efferent ducts. The epididymal cellular population was composed of principal, basal, apical, clear, narrow, and halo cells. The epithelium lining of vas deferens was composed of the principal and basal cells. AQPs 1 and 9 were not expressed in the rete testis. Positive reaction to AQP1 was observed at the luminal border of non-ciliated cells of the efferent ducts, and in the peritubular stroma and blood vessels in the epididymis, and vas deferens. AQP9 was immunolocalized in the epithelial cells in the efferent ducts, epididymis and vas deferens. The morphology of Azara’s agouti testis excurrent ducts is similar to that reported for other rodents such as Cuniculus paca. The immunolocalization results of the AQPs suggest that the expression of AQPs is species-specific due to differences in localization and expression when compared to studies in other mammals species. The knowledge about the expression of AQPs in Azara’s agouti testis excurrent ducts is essential to support future reproductive studies on this animal, since previous studies show that AQPs may be biomarkers of male fertility and infertility.     

A histological description of intersexuality in the roach

This paper is an illustrative guide to intersex in the roach Rutilus rutilus , based on 150 intersex individuals. Most intersex roach had female germ cells, or oocytes, within a predominantly male gonad (testis), and/or malformed/intersex reproductive ducts. The number, pattern and developmental stage of oocytes within testicular tissue in intersex fish varied greatly. In most intersex fish, a few primary oocytes, or numerous primary and secondary oocytes, were scattered randomly throughout the testicular tissue (multifocal intersex). In other, more severely feminized individuals, large areas of ovarian tissue were separated clearly from testicular tissue (focal intersex). Almost all intersex individuals had a female-like reproductive duct (ovarian cavity). In mild cases of intersex (in which the majority of the germ cells were male) the ovarian cavity was present together with the male sperm duct/vas deferens, whilst in certain severe cases, the sperm duct was absent or vestigial.     

Effects of a single ethanol injection into the vas deferens on the testicular function of rats.

1. A new approach to rapid male sterilization has been studied by giving a single injection of 95% ethanol directly into the vas deferens. It produced an effective block in the lumen. The mating exposure test showed that the males were sterile. The sperm granulomas at the site of vas injection were confirmed. 2. Ethanol injection in the vas deferens caused an atrophy of the testes. Extensive necrosis and exfoliation of the seminiferous elements were conspicuous. 3. Decrease in testicular weight, seminiferous tubule diameter and RNA and sialic acid levels after 4 weeks of vas injection were associated with the histological evidence for severe degeneration of spermatogenic elements. The protein contents of testis, epididymides and seminal vesicles did not change. 4. Testicular cholesterol, total lipids and alkaline phosphatase activity was increased. 5. Low sialic acid levels in the testis, epididymides and seminal vesicles of vas-injected rats indicated an inhibition of androgen production, which was further reflected in reduced nuclear diameters of leydig cells.     

Immunolocalization of clusterin in the ram testis, rete testis, and excurrent ducts

Clusterin, a glycoprotein that elicits cell aggregation, has previously been isolated from ram rete testis fluid, and has been partially characterized. In experiments reported, we have used monoclonal antibodies against clusterin in combination with indirect immunofluorescence microscopy to investigate the distribution of clusterin in the adult ram testis, rete testis, and excurrent ducts. Tissue blocks (5 mm3) were fixed in periodate/lysine/paraformaldehyde containing 0.1% glutaraldehyde and, after embedding, 5-microM sections were prepared for immunolocalization. In the testis, 2 basic patterns were observed: 1) strong to moderate staining for clusterin in the adluminal region with little staining in the basal region of the seminiferous epithelium and germinal cells; and 2) moderate staining throughout the seminiferous epithelium between germinal cells. In the rete testis, strong clusterin staining was localized intracellularly in the rete epithelial cells, most often associated with the luminal surface. In the epididymis, intracellular clusterin was localized in some principal cells of the caput epididymidis. The luminal surfaces and spermatozoa within the lumen were strongly positive. In the vas deferens, clusterin staining was associated with the luminal surface only. The presence of clusterin was clearly detected in unwashed isolated epididymal spermatozoa, but not in spermatozoa washed with phosphate-buffered saline containing 0.05% Tween 20.