Zebrafish mesonephric renin cells are functionally conserved and comprise two distinct morphological populations.

Zebrafish provide an excellent model in which to assess the role of the renin-angiotensin system in renal development, injury, and repair. In contrast to mammals, zebrafish kidney organogenesis terminates with the mesonephros. Despite this, the basic functional structure of the nephron is conserved across vertebrates. The relevance of teleosts for studies relating to the regulation of the renin-angiotensin system was established by assessing the phenotype and functional regulation of renin-expressing cells in zebrafish. Transgenic fluorescent reporters for renin (ren), smooth muscle actin (acta2), and platelet-derived growth factor receptor-beta (pdgfrb) were studied to determine the phenotype and secretory ultrastructure of perivascular renin-expressing cells. Whole kidney ren transcription responded to altered salinity, pharmacological renin-angiotensin system inhibition, and renal injury. Mesonephric ren-expressing cells occupied niches at the preglomerular arteries and afferent arterioles, forming intermittent epithelioid-like multicellular clusters exhibiting a granular secretory ultrastructure. In contrast, renin cells of the efferent arterioles were thin bodied and lacked secretory granules. Renin cells expressed the perivascular cell markers acta2 and pdgfrb Transcriptional responses of ren to physiological challenge support the presence of a functional renin-angiotensin system and are consistent with the production of active renin. The reparative capability of the zebrafish kidney was harnessed to demonstrate that ren transcription is a marker for renal injury and repair. Our studies demonstrate substantive conservation of renin regulation across vertebrates, and ultrastructural studies of renin cells reveal at least two distinct morphologies of mesonephric perivascular ren-expressing cells.

The pelvic kidney of male Ambystoma maculatum (Amphibia, urodela, ambystomatidae) with special reference to the sexual collecting ducts.

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum.

Activin A is an endogenous inhibitor of ureteric bud outgrowth from the Wolffian duct.

Development of metanephric kidney begins with ureteric bud outgrowth from the Wolffian duct (WD). GDNF is believed to be a crucial positive signal in the budding process, but the negative regulation of this process remains unclear. Here, we examined the role of activin A, a member of TGF-beta family, in bud formation using an in vitro WD culture system. When cultured with the surrounding mesonephros, WDs formed many ectopic buds in response to GDNF. While the activin signaling pathway is normally active along the non-budding WD (as measured by expression of activin A and phospho-Smad2/3), activin A was absent and phospho-Smad2/3 was undetectable in the ectopic buds induced by GDNF. To examine the role of activin A in bud formation, we attempted to inactivate activin action. Interestingly, the addition of neutralizing anti-activin A antibody potentiated GDNF action. To further clarify the role of activin A, we also tested the effect of activin blockade on the WD cultured in the absence of mesonephros. WDs without mesonephros did not form ectopic buds even in the presence of GDNF. In contrast, blockade of activin action with a variety of agents acting through different mechanisms (natural antagonist, neutralizing antibodies, siRNA) enabled GDNF to induce ectopic buds. Inhibition of GDNF-induced bud formation by activin A was accompanied by inhibition of cell proliferation, reduced expression of Pax-2, and decreased phosphorylation of PI3-kinase and MAP kinase in the WD. Our data suggest that activin A is an endogenous inhibitor of bud formation and that cancellation of activin A autocrine action may be critical for the initiation of this process.

Region-specific inhibition of prostatic epithelial bud formation in the urogenital sinus of C57BL/6 mice exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure causes abnormal ventral, dorsolateral, and anterior prostate development in wild-type but not aryl hydrocarbon receptor (AhR) null mutant C57BL/6 mice. Experiments have now been conducted to test the hypothesis that TCDD causes an AhR-dependent inhibition of the earliest visible stage of prostate development, the formation of prostatic buds by urogenital sinus (UGS) epithelium. A novel method for viewing budding was developed that uses scanning electron microscopy of isolated UGS epithelium instead of three-dimensional reconstruction of serial histological sections of intact UGS. In the initial experiment, the time course for prostatic epithelial bud formation in vehicle- and TCDD-exposed wild-type C57BL/6J mice was determined. A single maternal dose of TCDD (5 mug/kg) on gestation day 13 delayed the appearance of dorsal, lateral, and anterior buds by about one day, reduced dorsolateral bud number, and prevented ventral buds from forming. No such effects were seen in TCDD-exposed AhR null mutant fetuses, while AhR null mutation, alone, had no detectable effect on budding. Treatment of wild-type dams with sufficient 5alpha-dihydrotestosterone (DHT) to masculinize female fetuses failed to protect against the inhibition of budding caused by TCDD. These results demonstrate that in utero TCDD exposure causes an AhR-dependent inhibition of prostatic epithelial bud formation commensurate with its inhibitory effects on ventral and dorsolateral prostate development, and that the inhibition of budding is not due to insufficient DHT. Inhibited bud formation appears to be the primary cause of abnormal prostate development in TCDD-exposed mice.

Ontogeny of calbindin-D28K and calretinin in developing chick kidney.

The ontogeny of two calcium-binding proteins (calbindin-D28k and calretinin) was studied by immunohistochemical techniques in developing chick kidney. This study showed the presence of calbindin on the 5th incubation day and calretinin on the 7th incubation day in mesonephric distal and connecting tubules, and in the medial wall of the Wolffian duct. At later stages, immunostaining for these two proteins, in particular for calretinin, was also demonstrated in some metanephric proximal tubules. Glomeruli and Bowman's capsules were negative both in the mesonephros and metanephros. The presence of calretinin in the developing kidney has thus been demonstrated for the first time. The early expression of calbindin and calretinin in mesonephric distal tubules suggests their role in regulating the final excretion of calcium. The different patterns of immunoreactivity of the walls of the Wolffian duct can be correlated with their different histogenetic and histological features.

Ultrastructure of mullerian and wolffian ducts of fetal rabbit in vivo and in organ culture.

Emphasis is put on programmed cell death in the epithelial cells of mullerian and wolffian ducts, in rabbit fetuses, by observations on the timing and ultrastructural characteristics of cell degeneration. Genital tracts were collected on days 17 to 25 post-coitum and processed for electron microscopy. An organ culture assay was performed on fetal ducts and testis or ovary, for 4 days. Then, ducts and gonads were processed for electron microscopy. The involution of male mullerian and female wolffian ducts in culture and in vivo follows a similar pattern. Cell degeneration is initialized by an increase in the number of lysosomes which are subsequently involved in invading autophagic vacuoles. Cytochemical localizations of acid phosphatase and aminopeptidase indicate the presence of new lysosomes within the cells. In conclusion, this cell degeneration seems to be due to a double lysosome system in the rabbit urogenital ridge: A mullerian one spontaneously inactive when mullerian inhibiting substance is absent, and a wolffian one, spontaneously active when testosterone is absent. The organ culture is reliable for further attempts to study the responses to substances known to act on lysosome formation or activity.

Paravaginal female adnexal tumor of probable wolffian origin.

Female adnexal tumor of wolffian origin (FATPWO) is a lesion that predominantly occurs in the broad ligament. Its occurrence in the right paravaginal area is rare; only one other case has been reported to occur at this site. The authors report a case of paravaginal FATPWO in a 20-year-old woman that recurred twice during a 5-year period. The microscopic and ultrastructural features of this lesion were identical to other cases of FATPWO reported in the literature. This tumor must be recognized, especially at this site, so that it is not confused with vaginal adenocarcinoma related to diethylstilbestrol exposure in utero.

Involution of the Wolffian duct in the rat.

The involution of Wolffian duct in female rat fetuses from 15 to 21 days of gestation was studied in electron microscope. The female Wolffian duct stopped its differentiation on day 17 and the onset of duct regression was found in 18 days old fetuses. The involution was accompanied by degeneration and disintegration of epithelial cells, removal of basal lamina and elimination of altered cells into periductal mesenchyme. Although the mechanism of programmed cell death contributed to the WD regression the findings of non-altered epithelial cells supported the opinion of possible migration of some WD cells into the mesenchymal cell compartment.

Ultrastructure of the kidney of a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona). II. Distal tubule, connecting tubule, collecting duct and Wolffian duct.

The ultrastructure of the distal nephron, the collecting duct and the Wolffian duct was studied in a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) by transmission and scanning electron microscopy (TEM, SEM). The distal tubule (DT) is made up of one type of cell that has a well-developed membrane labyrinth established both by interdigitating processes and by interlocking ramifications. The processes contain large mitochondria, the ramifications do not. The tight junction is shallow and elongated by a meandering course. The connecting tubule (CNT) is composed of CNT cells proper and intercalated cells, both of which are cuboidal in shape. The CNT cells are characterized by many lateral interlocking folds. The intercalated cells have a dark cytoplasm densely filled with mitochondria. Their apical cell membrane is typically amplified by microplicae beneath which a layer of globular particles (studs) is found. The collecting duct (CD) is composed of principal cells and intercalated cells, again both cuboidal in shape. The CD epithelium is characterized by dilated intercellular spaces, which are often filled with lateral microfolds projecting from adjacent principal cells. The apical membrane is covered by a prominent glycocalyx. The intercalated cells in the CD are similar to those in the CNT. The Wolffian duct (WD) has a tall pseudostratified epithelium established by WD cells proper, intercalated cells and basal cells. The WD cells contain irregular-shaped dense granules located beneath the apical cell membrane. The intercalated cells of the WD have a dark cytoplasm with many mitochondria; their nuclei display a dense chromatin pattern.

The pig mesonephros. III. Distal tubule, collecting tubule, and Wolffian duct: SEM- and TEM-studies.

The ultrastructure of the distal and collecting tubules of mature pig mesonephroi (41st gestational day) was studied in perfusion-fixed embryos. In the distal tubule, the three subsegments postulated on the basis of enzyme histochemistry show only minor differences of their luminal surfaces, mostly of cell size. TEM photographs reveal a single cell type with interdigitating basolateral processes, frequently flattened to 30-120 nm lamellae devoid of organelles. Larger interdigitating processes harbor vertically oriented mitochondria in the form of indented plates. The macula densa cells are small, do not interdigitate, and have distended intercellular spaces. The collecting tubule starts with a dorsal convolution, in which intercalated cells (with apical microfolds and numerous mitochondria) occur in addition to interdigitating cells. Further down this segment, the interdigitating cells are gradually replaced by principal cells characterized by interlocking lateral microvilli, basal infoldings, and relatively few organelles. Intercalated cells extend into the Wolffian duct. Although the pig mesonephros has the most differentiated nephron of the mammals studied so far, with metanephros-like cells, its intrinsic urinary concentrating capacity appears to be low in view of its vascularization pattern and nephron architecture.

Epithelio-mesenchymal interface and fibronectin in the differentiation of the rat mesonephric and paramesonephric ducts.

The distribution of fibronectin and the morphological differentiation of the genital ducts was studied in rat fetuses at ages from 15 to 21 days. Fibronectin was localized with the peroxidase-antiperoxidase and avidin-biotin method at the electron- and light-microscope level. In 15-day-old male and female fetuses, fibronectin was localized as a continuous lamella around the mesonephric duct and as a discontinuous lamella around the paramesonephric duct. During the differentiation of the female paramesonephric duct, the fibronectin layer became continuous and remained so after the age of 16 days. The fibronectin layer of the male mesonephric duct remained continuous at all ages. The accumulation of mesenchymal cells on the outer surface of the female mesonephric duct and the concomitant detachment of the fibronectin layer around the duct suggests that mesenchymal regulation plays a role in the regression of the mesonephric duct. In the regressing male paramesonephric duct fibronectin was simultaneously lost in the condensed periductal mesenchyme, the places of epithelio-mesenchymal contact, and the epithelial cytoplasmic protrusions towards the mesenchyme. Ultrastructurally, fibronectin was localized in the basal laminae, on the cell membrane in contact with the extracellular material, and on the surface of the fibrillar and flocculent extracellular material. In addition to auto- and heterophagy, epithelio-mesenchymal interactions seem to play an important role in the regression of the genital ducts, although in different ways in males and females. The present results give additional support to the theory of the possible migration of epithelial cells into the surrounding mesenchyme during the regression of the paramesonephric duct.

Contacts between Wolffian and Müllerian cells at the tip of the outgrowing Müllerian duct in rat embryos.

The developing Müllerian duct was studied at the light microscopic as well as the electron microscopic level in rat embryos, especially in the section of the terminal bud and its tip, where Wolffian and Müllerian duct are enclosed by a common basal membrane. In this zone desmosomes can be found among Wolffian cells and also among Müllerian cells. In addition, we found cell contacts between Müllerian and Wolffian cells, namely short electron-dense segments on adjacent surfaces or disc-shaped thickenings within opposite plasma membranes, as well as fusions of the plasmalemmata over short distances. Until now, these cell contacts have not been described in rat embryos.

Ultrastructural study of a female adnexal tumor of probable wolffian origin.

A broad-ligament tumor, of probable wolffian origin, is presented. The ultrastructural features of this histologically benign tumor have not been previously described. The fine structure of this tumor is compared with normal structures and tumors derived from wolffian and müllerian anlage. The findings are consistent with derivation from wolffian duct.

The mature mesonephric nephron of the rabbit embryo. I. SEM-studies.

The luminal surface ultrastructure of the mature mesonephric nephron in 18 day rabbit embryos was studied in order to classify the nephron segments and to compare them with their metanephric counterparts. The proximal tubule has two slightly different segments. Its brush-bordered cells, with lateral ridges and basal microvilli (revealed in disjoined cells) exhibit structural principles similar to those of metanephric cells. The short distal tubule, starting with an abrupt border, cannot be subdivided. Its surface differs from one specimen to the next; the various cellular patterns are regarded as different functional states rather than evidence of a true cellular heterogeneity. Cells with leaf-like meandering borders correspond to similar metanephric cells favoring a paracellular transport mechanism. The collecting tubule shares common features with the metanephric collecting duct in spite of its different origin. Among principal cells, clearly demarcated by marginal microvillous rows and studded with sparse apical microvilli, non-ciliated and strongly bulging intercalated cells occur in small numbers. The latter have exaggerated, sometimes branched microvilli, and occasional microplicae. In the Wolffian duct, which has no metanephric counterpart, the single cilia dominate the picture of a homogeneous cell population. Apical globular protrusions of the tubular epithelia, which have been depicted in almost every paper on the mesonephros, are all fixation artefacts that can only be avoided by properly perfusing the living embryo.

A comparative study of the differentiation and involution of the Mullerian duct and Wolffian duct in the male and female fetal mouse.

The present investigation has examined the ultrastructural differentiation of the genital ducts of both sexes of fetal mice. The emphasis of observations was placed on the phenomenon of morphogenetic cytolysis, particularly during the critical periods of Wolffian duct stabilization and Mullerian duct involution. Both developing and regressing genital ducts evidence extensive cytolysis. Autophagy appears to be the mechanism of morphogenetic changes in the developing male Wolffian duct. Autophagy, heterophagy, and degeneration in situ are all prominent cytolytic activities in female Wollfian duct involution. The developing female Mullerian duct undergoes extensive morphogenetic remodeling by the mechanisms of autophagy, heterophagy, and degeneration in situ. In the male Mullerian duct, autophagy, heterophagy, and degeneration in situ are also prominent. In addition, whole degenerated epithelial cells are extruded from the duct early in regression which may be realted to the transformation of periductal mesenchymal cells into an "epithelioid cell cuff" which does not form around the regressing Wolffian duct. The formation of this mesenchymal condensation surrounding the duct is also accompanied by the protrusion of Mullerian epithelial cell cytoplasm into the mesenchymal cells. These observations may evidence a complex epithelial-mesenchymal interaction occurring during male Mullerian duct involution.

Contribution to the study of the development of the terminal portion of the Wolffian duct and the ureter.

Ten human embryos ranging from 10 to 20 mm in vertex-coccyx length (Horizons XVI and XVII Streeter) aged between 33 +/- 1 and 39 +/- 1 days, were studied in order to interpret the mechanism which determines the displacement of the ureter from the dorsal part of the Wolffian duct toward the lateral wall of the bladder. We demonstrated that this movement occurs due to the fact that the duct common to the ureter and the Wolffian duct undergoes a process of internal rotation before it is absorbed by the urogenital sinus. This determines that the ureteral orifice enter the urogenital sinus laterally with respect to the Wolffian duct. This absorption indicates that the mucosa of the trigone is of mesoblastic origin and the remainder of the besical of endoblastic origin. The origin of the muscular apparatus of the detrusor is entirely mesoblastic.