Crucial Roles of the Mesenchymal Androgen Receptor in Wolffian Duct Development.

Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2, and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice.

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Mesonephric-like Differentiation of Ovarian Endometrioid and High-grade Serous Carcinomas: Clinicopathological and Molecular Characteristics Distinct from Those of Mesonephric-like Adenocarcinoma.

BACKGROUND/AIM: Ovarian endometrioid carcinoma (EC) and high-grade serous carcinoma (HGSC) may exhibit various growth patterns and mimic mesonephric-like adenocarcinoma (MLA). We investigated the clinicopathological and molecular features of ovarian carcinomas with mesonephric-like differentiation (MLD). PATIENTS AND METHODS: We analyzed the electronic medical records and pathology slides of two EC-MLD and three HGSC-MLD patients, and conducted immunostaining and targeted sequencing of their samples. RESULTS: All cases showed architectural diversity, compactly aggregated small tubules and ducts, and eosinophilic intraluminal secretions, indicating the possibility of an ovarian MLA. However, the following histological and immunophenotypical features confirmed the diagnoses of EC-MLD and HGSC-MLD: squamous, tubal, and sertoliform differentiation; serous tubal intraepithelial carcinoma; solid, endometrioid, transitional (SET) feature; solid, transitional, endometrioid, mucinous-like (STEM) feature; diffuse expression of hormone receptors and Wilms tumor 1; mutant p53 immunostaining pattern; and wild-type v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog gene. CONCLUSION: A subset of ovarian ECs and HGSCs can display MLD and mimic an MLA. A thorough histological examination combined with ancillary tests is crucial to differentiate between these ovarian neoplastic entities.

Increased rates of vesicoureteral reflux in mice from deletion of Dicer in the peri-Wolffian duct stroma.

BACKGROUND: Vesicoureteral reflux (VUR), backflow of urine into the kidney, is associated with urinary tract infections and chronic kidney disease. Integrity of the vesicoureteral junction (VUJ), where reflux occurs, is determined largely by proper induction of the ureteric bud from the Wolffian duct. Induction is modulated by signals from the surrounding peri-Wolffian duct stroma. We evaluated whether miRNAs in the peri-Wolffian duct stroma are necessary for proper ureteric induction, VUJ formation, and suppression of VUR. METHODS: We generated a mouse with loss of miRNAs in the peri-Wolffian duct stroma. We evaluated embryos for ureteric bud induction defects and expression of genes that regulate induction. We performed cystograms to assess for reflux and assessed VUJs in postnatal mice. RESULTS: Mutant embryos had cranially displaced ureteric bud induction sites vs. controls. We observed no changes in expression of genes known to regulate induction. While mutants were early postnatal lethal, they had high rates of VUR vs. controls. Mutant VUJs that refluxed had low inserting ureters and shortened intravesicular tunnels vs. non-refluxing mice. CONCLUSIONS: We found that miRNAs in the peri-Wolffian duct stroma are required for normal ureteric bud induction, VUJ formation, and prevention of VUR.

Sprouty1 Controls Genitourinary Development via its N-Terminal Tyrosine.

BACKGROUND: Studies in mice suggest that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT). Mutations in Sprouty1, an intracellular Ret inhibitor, results in supernumerary kidneys, megaureters, and hydronephrosis in mice. But the underlying molecular mechanisms involved and which structural domains are essential for Sprouty1 function are a matter of controversy, partly because studies have so far relied on ectopic overexpression of the gene in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for the function of Sprouty1 in vitro. METHODS: We generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We characterized the development of the genitourinary systems in these mice via different methods, including the use of reporter mice expressing EGFP from the Ret locus, and whole-mount cytokeratin staining. RESULTS: Mice lacking this tyrosine grow ectopic ureteric buds that will ultimately form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. CONCLUSIONS: Tyrosine 53 is absolutely necessary for Sprouty1 function during genitourinary development in mice.

Pkd2 deletion during embryo development does not alter mesonephric programmed cell senescence.

Programmed cell senescence during embryo development is a recently described process that opens a new perspective to understand the senescence response and that adds a new player whose contribution to development needs to be addressed. Identifying developmental syndromes with a root in deregulated programmed cell senescence will undoubtedly reinforce our view of senescence and could provide a new angle to confront disease. One of the structures that was initially reported to undergo cellular senescence is the mesonephros. During E12.5-E14.5, before regression, mesonephric tubules are positive for the most widely used marker of cell senescence, SAßG, and negative for proliferation marker, Ki67, in a p21Cip1-dependent manner. PKD2 is one of the genes defective in autosomal dominant polycystic kidney disease (ADPKD). Inherited mutations in this gene result in cyst formation in adults after a secondary hit. Polycystin-2 (PC2) protein, the product of PKD2 gene expression, inhibits cell cycle progression by inducing p21Cip1, whereas mutated PKD2 results in increased proliferation and defective differentiation of kidney epithelial cells. Here, we addressed the possibility of defective programmed cell senescence as a consequence of Pkd2 deletion in mice. We analyzed embryos for the expression of the senescence marker SAßG, for the proliferative status of mesonephric tubule cells, and for the expression of p21Cip1, without identifying any noticeable deregulation of cell senescence. Our results exclude defective programmed cell senescence upon Pkd2 ablation as an initial event in ADPKD.

Reciprocal Spatiotemporally Controlled Apoptosis Regulates Wolffian Duct Cloaca Fusion.

The epithelial Wolffian duct (WD) inserts into the cloaca (primitive bladder) before metanephric kidney development, thereby establishing the initial plumbing for eventual joining of the ureters and bladder. Defects in this process cause common anomalies in the spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). However, developmental, cellular, and molecular mechanisms of WD-cloaca fusion are poorly understood. Through systematic analysis of early WD tip development in mice, we discovered that a novel process of spatiotemporally regulated apoptosis in WD and cloaca was necessary for WD-cloaca fusion. Aberrant RET tyrosine kinase signaling through tyrosine (Y) 1062, to which PI3K- or ERK-activating proteins dock, or Y1015, to which PLCγ docks, has been shown to cause CAKUT-like defects. Cloacal apoptosis did not occur in RetY1062F mutants, in which WDs did not reach the cloaca, or in RetY1015F mutants, in which WD tips reached the cloaca but did not fuse. Moreover, inhibition of ERK or apoptosis prevented WD-cloaca fusion in cultures, and WD-specific genetic deletion of YAP attenuated cloacal apoptosis and WD-cloacal fusion in vivo Thus, cloacal apoptosis requires direct contact and signals from the WD tip and is necessary for WD-cloacal fusion. These findings may explain the mechanisms of many CAKUT.

Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

BackgroundFibroblast growth factor receptor 2 (Fgfr2) deletion from murine peri-Wolffian duct stroma (ST) results in aberrant ureteric bud induction, abnormal ureteral insertion into the bladder, and high rates of vesicoureteral reflux (VUR). It is unclear which receptor docking protein(s) is/are responsible for Fgfr2 actions in these tissues. We investigated whether the docking protein, fibroblast receptor substrate 2α (Frs2α), had a role in peri-Wolffian duct ST similar to Fgfr2.MethodsWe conditionally deleted Frs2α in peri-Wolffian duct ST with a Tbx18cre mouse line (Frs2αST-/-). We assessed for ureteric induction defects and alterations in downstream targets mediating defects. We performed euthanized cystograms and assessed ureter-bladder junctions by three-dimensional (3D) reconstructions.ResultsEmbryonic day (E) 11.5 Frs2αST-/- embryos had many displaced ureteric bud induction sites when compared with controls. E11.0 Frs2αST-/- embryos had decreased Bmp4 expression and signaling, which can cause abnormal ureteric bud induction. Postnatal day 1 (P1) and P30 Frs2αST-/- mice had higher VUR rates and grades vs. CONTROLS: Mutant refluxing ureters that inserted improperly into the bladder had shortened intravesicular tunnels (IVTs) when compared with controlsConclusionFrs2αST-/- embryos have aberrant ureteric induction sites, improper ureteral insertion, shortened intravesicular lengths, and VUR. Induction site defects appear secondary to reduced Bmp4 expression, similar to Fgfr2 mutants.

Novel androgen-induced activity of an antimicrobial ß-defensin: Regulation of Wolffian duct morphogenesis.

The Wolffian duct (WD) undergoes morphological changes induced by androgens to form the epididymis, which is an organ essential for sperm maturation. Androgen action in WD epithelium involves paracrine factors of mesenchymal origin that function by still poorly understood mechanisms. Here we studied the antimicrobial ß-defensin SPAG11C as a new player in duct morphogenesis, localized prenatally in the WD mesenchyme. Organotypic culture of rat WDs and tissues from Androgen Receptor (AR) knockout mice (ARKO) were used. Our results show that androgen/AR signaling differentially regulated SPAG11C expression at mRNA and protein levels in the developing WD. WDs incubated with recombinant human SPAG11C were shorter and less coiled as a result of reduced epithelial cell proliferation, but not increased apoptosis. Our results suggested ß-defensin SPAG11C as an androgen-target required for WD morphogenesis. This highlights the multifunctional repertoire of the ß-defensin protein family and their potential contribution to the in utero environment that determines male reproductive success.

Role of Ca2+ in the IVM of spermatozoa from the sterlet Acipenser ruthenus.

The role of Ca2+ in sturgeon sperm maturation and motility was investigated. Sperm from mature male sterlets (Acipenser ruthenus) were collected from the Wolffian duct and testis 24h after hormone induction. Testicular spermatozoa (TS) were incubated in Wolffian duct seminal fluid (WDSF) for 5min at 20°C and were designated 'TS after IVM' (TSM). Sperm motility was activated in media with different ion compositions, with motility parameters analysed from standard video microscopy records. To investigate the role of calcium transport in the IVM process, IVM was performed (5min at 20°C) in the presence of 2mM EGTA, 100µM Verapamil or 100µM Tetracaine. No motility was observed in the case of TS (10mM Tris, 25mM NaCl, 50mM Sucr with or without the addition of 2mM EGTA). Both incubation of TS in WDSF and supplementation of the activation medium with Ca2+ led to sperm motility. The minimal Ca2+ concentration required for motility activation of Wolffian duct spermatozoa, TS and TSM was determined (1-2nM for Wolffian duct spermatozoa and TSM; approximately 0.6mM for TS). Motility was obtained after the addition of verapamil to the incubation medium during IVM, whereas the addition of EGTA completely suppressed motility, implying Ca2+ involvement in sturgeon sperm maturation. Further studies into the roles of Ca2+ transport in sturgeon sperm maturation and motility are required.

Early formation of the Müllerian duct is regulated by sequential actions of BMP/Pax2 and FGF/Lim1 signaling.

The Müllerian duct (MD) and Wolffian duct (WD) are embryonic tubular tissues giving rise to female and male reproductive tracts, respectively. In amniote embryos, both MD and WD emerge in both sexes, but subsequently degenerate in the males and females, respectively. Here, by using MD-specific gene manipulations in chicken embryos, we identify the molecular and cellular mechanisms that link early MD specification to tubular invagination. Early (pre-)specification of MD precursors in the coelomic epithelium requires BMP signaling and its downstream target Pax2 in a WD-independent process. Subsequently, the BMP/Pax2 axis induces Lim1 expression, a hallmark of MD specification, for which FGF/ERK and WD-derived signals are also required. Finally, the sequential actions of the BMP/Pax2 and FGF/Lim1 axes culminate in epithelial invagination to form a tubular structure driven by an apical constriction, where apical accumulation of phospho-myosin light chain is positively regulated by FGF/ERK signaling. Our study delineates mechanisms governing the early formation of the MD, and also serves as a model of how an epithelial cell sheet is transformed to a tubular structure, a process seen in a variety of developmental contexts.

Novel function of LHFPL2 in female and male distal reproductive tract development.

Congenital reproductive tract anomalies could impair fertility. Female and male reproductive tracts are developed from Müllerian ducts and Wolffian ducts, respectively, involving initiation, elongation and differentiation. Genetic basis solely for distal reproductive tract development is largely unknown. Lhfpl2 (lipoma HMGIC fusion partner-like 2) encodes a tetra-transmembrane protein with unknown functions. It is expressed in follicle cells of ovary and epithelial cells of reproductive tracts. A spontaneous point mutation of Lhfpl2 (LHFPL2(G102E)) leads to infertility in 100% female mice, which have normal ovarian development, ovulation, uterine development, and uterine response to exogenous estrogen stimulation, but abnormal upper longitudinal vaginal septum and lower vaginal agenesis. Infertility is also observed in ~70% mutant males, which have normal mating behavior and sperm counts, but abnormal distal vas deferens convolution resulting in complete and incomplete blockage of reproductive tract in infertile and fertile males, respectively. On embryonic day 15.5, mutant Müllerian ducts and Wolffian ducts have elongated but their duct tips are enlarged and fail to merge with the urogenital sinus. These findings provide a novel function of LHFPL2 and a novel genetic basis for distal reproductive tract development; they also emphasize the importance of an additional merging phase for proper reproductive tract development.

Epithelial Wnt/ßcatenin signalling is essential for epididymal coiling.

Organ shape and size are important determinants of their physiological functions. Epithelial tubes are anlagen of many complex organs. How these tubes acquire their complex shape and size is a fundamental question in biology. In male mice, the Wolffian duct (WD; postnatally known as epididymis) undergoes an astonishing transformation, where a straight tube only a few millimetres long elongates to over 1000 times its original length and fits into a very small space, due to extensive coiling of epithelium, to perform the highly specialized function of sperm maturation. Defective coiling disrupts sperm maturation and leads to male infertility. Recent work has shown that epithelial cell proliferation is a major driver of WD coiling. Still, very little is known about the molecular signals involved in this process. Testicular androgens are known regulators of WD development. However, epithelial androgen receptor signalling is dispensable for WD coiling. In this study, we have shown that Wnt signalling is highly active in the entire WD epithelium during its coiling, and is limited to only a few segments of the epididymis in later life. Pharmacological and genetic suppression of Wnt signalling inhibited WD coiling by decreasing cell proliferation and promoting apoptosis. Comparative gene expression analysis identified Fibroblast growth factor 7 (Fgf7) as a prime Wnt target gene involved in WD coiling and in vitro treatment with Fgf7 protein increased coiling of WDs. In summary, our work has established that epithelial canonical Wnt signalling is a critical regulator of WD coiling and its precise regulation is essential for WD/epididymal differentiation.

Protein tyrosine kinase 7 is essential for tubular morphogenesis of the Wolffian duct.

The Wolffian duct, the proximal end of the mesonephric duct, undergoes non-branching morphogenesis to achieve an optimal length and size for sperm maturation. It is important to examine the mechanisms by which the developing mouse Wolffian duct elongates and coils for without proper morphogenesis, male infertility will result. Here we show that highly proliferative epithelial cells divide in a random orientation relative to the elongation axis in the developing Wolffian duct. Convergent extension (CE)-like of cell rearrangements is required for elongating the duct while maintaining a relatively unchanged duct diameter. The Wolffian duct epithelium is planar polarized, which is characterized by oriented cell elongation, oriented cell rearrangements, and polarized activity of regulatory light chain of myosin II. Conditional deletion of protein tyrosine kinase 7 (PTK7), a regulator of planar cell polarity (PCP), from mesoderm results in loss of the PCP characteristics in the Wolffian duct epithelium. Although loss of Ptk7 does not alter cell proliferation or division orientation, it affects CE and leads to the duct with significantly shortened length, increased diameter, and reduced coiling, which eventually results in loss of sperm motility, a key component of sperm maturation. In vitro experiments utilizing inhibitors of myosin II results in reduced elongation and coiling, similar to the phenotype of Ptk7 knockout. This data suggest that PTK7 signaling through myosin II regulates PCP, which in turn ensures CE-like of cell rearrangements to drive elongation and coiling of the Wolffian duct. Therefore, PTK7 is essential for Wolffian duct morphogenesis and male fertility.

Fibroblast growth factor receptor signaling in kidney and lower urinary tract development.

Fibroblast growth factor receptors (FGFRs) and FGF ligands are highly expressed in the developing kidney and lower urinary tract. Several classic studies showed many effects of exogenous FGF ligands on embryonic renal tissues in vitro and in vivo. Another older landmark publication showed that mice with a dominant negative Fgfr fragment had severe renal dysplasia. Together, these studies revealed the importance of FGFR signaling in kidney and lower urinary tract development. With the advent of modern gene targeting techniques, including conditional knockout approaches, several publications have revealed critical roles for FGFR signaling in many lineages of the kidney and lower urinary tract at different stages of development. FGFR signaling has been shown to be critical for early metanephric mesenchymal patterning, Wolffian duct patterning including induction of the ureteric bud, ureteric bud branching morphogenesis, nephron progenitor survival and nephrogenesis, and bladder mesenchyme patterning. FGFRs pattern these tissues by interacting with many other growth factor signaling pathways. Moreover, the many genetic Fgfr and Fgf animal models have structural defects mimicking numerous congenital anomalies of the kidney and urinary tract seen in humans. Finally, many studies have shown how FGFR signaling is critical for kidney and lower urinary tract patterning in humans.

GATA3 Is a Sensitive and Specific Marker of Benign and Malignant Mesonephric Lesions in the Lower Female Genital Tract.

GATA3 is a transcription factor critical for embryogenesis, development, and cell differentiation. Recent studies have suggested that GATA3 is a sensitive and relatively specific biomarker for urothelial and breast carcinomas, with most Müllerian carcinomas being negative. We investigated GATA3 expression in mesonephric/Wolffian remnants and tumors in the female genital tract. A western blot was performed to assess specificity for the GATA3 antibody. GATA3 immunohistochemistry was performed on 59 formalin-fixed paraffin-embedded mesonephric samples, including 17 mesonephric remnants (MR; 11 cervical and 6 fallopian tube), 15 mesonephric hyperplasias, 21 mesonephric carcinomas, and 6 female adnexal tumors of probable Wolffian origin. Thirty conventional endocervical adenocarcinomas (ENDO-CA), 9 gastric-type cervical adenocarcinomas, and 165 endometrial adenocarcinomas (EM-CA) were also evaluated. GATA3 nuclear intensity and extent of staining was evaluated. The western blot revealed GATA3 expression in seminal vesicle and cell lines derived from breast and urothelial carcinomas, but not in other cell lines including ovarian, cervical, and endometrial cancers. All cervical MRs and mesonephric hyperplasias, 5/6 (83%) fallopian tube MRs, and 20/21 (95%) mesonephric carcinomas were GATA3 positive, although with great variability in both intensity (weak to strong) and extent (1+ to 3+) of staining. Only 1/6 (17%) female adnexal tumors of probable Wolffian origin showed weak multifocal staining. One of 30 (3%) usual-type ENDO-CAs and 3/165 EM-CAs exhibited weak-moderate GATA3 immunoreactivity; all gastric-type cervical adenocarcinomas were negative. GATA3 is a highly sensitive and specific marker for mesonephric lesions in the lower genital tract; however, its utility in the upper genital tract may be more limited. In addition, GATA3 can aid in distinguishing lower genital mesonephric lesions from usual-type and gastric-type ENDO-CAs and uterine EM-CAs.

FGF8 coordinates tissue elongation and cell epithelialization during early kidney tubulogenesis.

When a tubular structure forms during early embryogenesis, tubular elongation and lumen formation (epithelialization) proceed simultaneously in a spatiotemporally coordinated manner. We here demonstrate, using the Wolffian duct (WD) of early chicken embryos, that this coordination is regulated by the expression of FGF8, which shifts posteriorly during body axis elongation. FGF8 acts as a chemoattractant on the leader cells of the elongating WD and prevents them from epithelialization, whereas static ('rear') cells that receive progressively less FGF8 undergo epithelialization to form a lumen. Thus, FGF8 acts as a binary switch that distinguishes tubular elongation from lumen formation. The posteriorly shifting FGF8 is also known to regulate somite segmentation, suggesting that multiple types of tissue morphogenesis are coordinately regulated by macroscopic changes in body growth.

Dynamic changes in the spatio-temporal expression of the ß-defensin SPAG11C in the developing rat epididymis and its regulation by androgens.

Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the ß-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of ß-defensins in the epididymis.

Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations.

The antioxidant system of sterlet seminal fluid in testes and Wolffian ducts.

Oxidative stress is a possible source of spermatozoa function deterioration. Seminal fluid (SF) protects spermatozoa against reactive oxygen species (ROS) attack during development in testes and transit through the reproductive tract. Spermatozoa curvilinear velocity and percent of motile cells as well as changes in thiobarbituric acid-reactive substance (TBARS) content, superoxide dismutase, and catalase activity, and uric acid concentration in SF were evaluated in sterlet sperm collected from testes 24 h after hormone induction of spermiation and from Wolffian ducts at 12, 24, 36, and 60 h after hormone injection (HI). While testicular spermatozoa motility was not initiated in activating medium, Wolffian duct sperm showed low motility at 12 h, significant increase at 24 and 36 h, and decrease at 60 h. Testicular SF was characterized by the highest level of TBARS and activity of studied enzymes compared with SF from Wolffian duct sperm at 24 h post-HI. In fluid from Wolffian duct sperm, a significant increase in TBARS content was shown at 36-60 h post-HI. In contrast to testicular SF, in SF from Wolffian duct sperm, this increase was not counterbalanced by changes in the studied variables of antioxidant system. This may be the source of the observed decrease in spermatozoa motility parameters 60 h post-HI. The results may confirm a dual role of ROS in fish sperm physiology. The data with respect to decrease in sturgeon spermatozoa motility parameters at 60 h post-HI should be taken into account in artificial sturgeon propagation.