Sperm antioxidant system in ocellate river stingray Potamotrygon motoro at transition from seminal vesicle to cloaca.

The importance of reactive oxygen species and the antioxidant system in sperm biology has been recognized for different bony fishes but nothing is known in this regard for chondrichthyans. For the first time for cartilaginous fishes, the enzymatic antioxidant system was shown herein to be present in both fractions of sperm (spermatozoa and seminal fluid) collected from two different places (seminal vesicle and cloaca). In internally fertilizing freshwater ocellate river stingray, Potamotrygon motoro, the activity of superoxide dismutase and glutathione peroxidase was not changed upon sperm transition from the seminal vesicle to the cloaca. The activity of catalase was significantly increased for both sperm fractions at transition from the seminal vesicle to the cloaca (1.6 times for spermatozoa and 1.9 times for seminal fluid). The role of the sperm antioxidant system for different aspects of internal fertilization is discussed. The presented results are the initiatory step in uncovering the biochemical events of internal reproduction in Chondrichthyes.

Bmp7 functions via a polarity mechanism to promote cloacal septation.

BACKGROUND: During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse. CONCLUSION/SIGNIFICANCE: Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.

The cloacal plate: the missing link in anorectal and urogenital development.

OBJECTIVE: To determine whether differences in the embryology of the anorectal and urogenital area, previously examined in sheep, pigs, rats, rabbits and guinea pigs, producing varying conclusions, may be secondary to differences in species development. MATERIALS AND METHODS: Rat and human embryos were studied at the time of genital tubercle development and cloacal partition by standard serial-section histology, and by immunohistochemistry, dissection and scanning electron microscopy. The images obtained were compared with those previously reported for pig and sheep embryos. RESULTS: The cloacal plate, a vertically orientated midline plate of epithelial cells in the caudal half of the genital tubercle, was the key structure that varied between the different species. In rats the plate maintained a vertical height along its length, while in humans and pigs it reverted to a two-layer membrane dorsally, shortly before it degenerated to expose both the anorectal and urogenital tracts. In sheep the plate was taller ventrally than in the other species but also reverted to a short membrane dorsally that exposed the hindgut when it degenerated. The anterior part of the cloacal plate persisted in all embryos as the urethral plate, which then participated in the formation of the urethra in the male and the vestibule in the female. The animal that most closely resembled humans in anogenital development was the pig. CONCLUSIONS: The cloacal plate is the key to understanding early anorectal and urogenital development and yet it is barely recognized in published reports. An appreciation of its relevance helps to explain the variations in the described embryology of the region, and is mandatory when interpreting embryological findings in animal models of human anomalies. Given the similarities between porcine and human development, the pig may be the most legitimate animal model for the study of anorectal and urogenital anomalies in humans.

Pre- and post-translational regulation of aromatase by steroidal and non-steroidal aromatase inhibitors.

Treatment of castrated quail with testosterone (T) reliably activates male copulatory behavior and, at the same time, increases the aromatase activity (AA), the number of aromatase-immunoreactive (ARO-ir) cells and the concentration of aromatase mRNA as measured by RT-PCR in the brain. All these effects can be mimicked by estrogens. The behavioral effects of T can be blocked by a variety of aromatase inhibitors and, in parallel, the AA is strongly inhibited in the preoptic area (POA). We showed recently that the steroidal inhibitor, 4-OH-androstenedione (OHA) markedly decreases the immunostaining density of brain ARO-ir cells while the non-steroidal inhibitor, R76713 (racemic Vorozole; VOR) unexpectedly increased the density of this staining, despite the fact that the enzyme activity was completely inhibited. To generalize these findings and try to identify the underlying mechanism, we compared here the effects of two steroidal (OHA and androstatrienedione [ATD]) and two non-steroidal (VOR and Fadrozole [FAD]) aromatase inhibitors on the aromatase immunostaining and aromatase mRNA concentration in the brain of castrated quail concurrently treated with T. The 4 inhibitors significantly blocked the activation by T of male copulation. The two steroidal inhibitors decreased the immunostaining of brain ARO-ir cells but both VOR and FAD markedly enhanced the density of this staining. In parallel, OHA and ATD completely blocked the T-induced increase in aromatase mRNA concentration, while VOR and FAD had no effect on these RNA concentrations in the POA-anterior hypothalamus and they decreased them only slightly in the posterior hypothalamus. Taken together these results suggest that the inhibition of AA by ATD or OHA and the subsequent removal of locally produced estrogens blocks the synthesis of aromatase presumably at the transcriptional level. By contrast, the two non-steroidal inhibitors tested here block AA but in parallel increase the aromatase immunostaining. This effect does not result from an enhanced transcription and it is therefore speculated that these compounds increase either the translation of the aromatase mRNA or the half-life of the protein itself.

The integrative segment of the quail Coturnix coturnix japonica. Occurrence and distribution of carbonic anhydrase and complex carbohydrates.

As part of a more extensive study into the involvement of carbonic anhydrase in avian excretory function, the occurrence and distribution of this enzyme was investigated in the quail integrative segment. The integrative segment represents, in birds, that part of the intestinal tract where ureteral urine undergoes postrenal modification to form definitive urine. To define the structural peculiarities within the intestinal epithelium, the constituent parts, namely cloaca, rectum and caecum, as well as the posterior ileum, were examined histochemically to visualise complex carbohydrates. The histochemical findings for carbonic anhydrase activity were compared with the results from a correlative immunohistochemical approach performed with a specific antiserum to avian CA II. Most of the enzyme activity unhomogeneously distributed in the intestinal enterocytes within the mucosal epithelium, was shown to be due to the cytosolic isoenzyme CA II. Additional carbonic anhydrase isoenzymes, distinct from CA II, seem to occur both at the enterocyte brush border and at the smooth muscle layer of the muscularis externa.

Distribution of esterase activity at the level of the epithelium of the diffusely infiltrated area (DIA) and of the cloaca in the Gallus domesticus: an ultrastructural study.

Esterase activity studies on the areas of lymphoid infiltration in the bursa of Fabricius (diffusely infiltrated area) and the dorsal wall of the cloaca showed that the epithelial cells exhibit varying degrees of diffuse esterase activity; it was possible to demonstrate the presence of spot-like esterase positivity in star-shaped cells in the epithelium itself. These cells can be found at various levels from the proximal to the distal region and have also been observed in the connective tissue of the tunica propria; as a result, the hypothesis has been advanced that they are connective tissue.

Ecdysone 20-mono-oxygenase in the desert locust, Schistocerca gregaria.

The enzyme catalysing the hydroxylation of ecdysone to 20-hydroxyecdysone, ecdysone 20-mono-oxygenase (EC 1.14.99.22), was investigated in the Malpighian tubules of fifth-instar locusts, Schistocerca gregaria. Enzyme activity was optimal at 35 degrees C and pH 6.8-8.0. Under these conditions the mono-oxygenase exhibited an apparent Km for ecdysone of 7.1 X 10(-7) M, a maximal specific activity of 1.1 nmol/h per mg of protein and was competitively inhibited by 20-hydroxyecdysone with an apparent Ki of 6.3 X 10(-7) M. Enzyme activity was decreased in the presence of Ca2+, Mg2+, EDTA and non-ionic detergents. The Malpighian tubule ecdysone 20-mono-oxygenase was localized primarily in the subcellular fraction sedimenting at 7500 g and, on the basis of marker enzyme profiles, was assigned mainly to the mitochondria. NADPH was required for activity, although addition of NADH together with NADPH had a synergistic effect. NADP+-dependent isocitrate dehydrogenase (EC 1.1.1.42) and an energy-dependent NAD(P) transhydrogenase (EC 1.6.1.1.) appeared to be the major sources of reducing equivalents, with the contribution from the 'malic enzyme' (EC 1.1.1.40) being less important. The monooxygenase was characterized as a cytochrome P-450-containing mixed-function oxidase from the inhibition patterns with metyrapone, CO and cyanide; CO inhibition was reversible with monochromatic light at 450 nm. However, the ecdysone 20-mono-oxygenase shows much lower sensitivity to CO inhibition and to photodissociation of the CO-inhibited complex than do vertebrate cytochrome P-450-dependent hydroxylation systems. The concentration of cytochrome P-450 in the Malpighian tubule mitochondria was 30 pmol/mg of protein. The properties of the mono-oxygenase are discussed in relation to hydroxylation enzymes from other sources.

5 Beta-reductase activity in the brain and cloacal gland of male and female embryos in the Japanese quail (Coturnix coturnix japonica).

Testosterone metabolism was studied by an in-vitro technique in the brain and cloacal gland of young male and female quail at different ages ranging from 7 days of incubation to 2 days after hatching. Very active metabolism, leading almost exclusively to the production of 5 beta-reduced compounds, was observed. 5 beta-Reductase activity remained high throughout the incubation period in the hypothalamus, decreased around the time of hatching in the cerebellum and decreased progressively between days 7 and 15 of incubation in the cloacal gland. These changes could be involved in the control of sexual differentiation: the high 5 beta-reductase in the brain possibly protects males from being behaviourally demasculinized by their endogenous testosterone while the decreasing 5 beta-reductase in the cloacal gland would progressively permit the masculinization of that structure.