[Organogenesis of the mesonephros in birds. Realization in vitro of quail-chicken chimera mesonephros].
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Effects of the mesonephros and insulin-like growth factor I on chondrogenesis of limb explants.
The mesonephros has been shown to have a growth-promoting influence in vivo on limb outgrowth. This influence has been studied in detail using an organ culture system. The results show that in the presence of the mesonephros limb explants formed larger cartilages than cultures without mesonephros. Furthermore, with mesonephros, morphology of the cartilages is comparable to that of skeletal elements in vivo while cartilages formed in cultures lacking mesonephros were amorphous. The mesonephric influence also promoted the formation of a well-organized extracellular matrix in the cartilage while cartilage in cultures without mesonephros formed an abnormal appearing matrix. Cartilage matrices in cultures with or without mesonephros were immunoreactive to type IX and type II collagens, cartilage proteoglycan PGH, and link protein although cultures lacking mesonephros had a very restricted distribution of type IX collagen immunoreactivity. Despite the different distribution of type IX collagen, long-form-type IX collagen transcripts appeared similar in both types of culture based on in situ hybridization. The mesonephric effect on limb explants could be partially duplicated by the addition of insulin-like growth factor I (IGF-I) to cultures without mesonephros. Furthermore, the mesonephric influence on cartilage growth and morphological differentiation could be blocked by the addition of a blocking antibody to IGF-I to cultures with mesonephros. The results support the hypothesis that IGF-I is one of the growth factors produced by the mesonephros which may play a role in early limb development and chondrogenesis.
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beta-Catenin expression during vascular development and degeneration of avian mesonephros.
beta-Catenin is a structural component of adherens junctions, a regulator of the Wnt signalling pathway and a transcriptional co-activator with a key role in vascular patterning. The avian mesonephros is a transitory embryonic kidney that is used in the study of vascular development and degeneration. Here we examine beta-catenin expression in this model during vascular development and degeneration. Quail embryos with developing or degenerating mesonephros were studied, on day 6 (30HH) or day 11 of incubation (40HH), respectively. QH1 whole mounts of developing mesonephros revealed numerous angioblast-like cells situated in the paramesonephric duct that seem to invade the mesonephros. Although these cells did not express beta-catenin, the surrounding periductal mesenchymal cells translocated high levels of beta-catenin into the nucleus. In contrast, degenerating mesonephros were devoid of angioblast-like cells and beta-catenin was lower than in the developing mesonephros. beta-Catenin was significantly reduced in the glomerular capillary tuffs, indicating that it was particularly down-regulated in the vascular system. No sex-related differences in beta-catenin expression were observed in degenerating mesonephros. Furthermore, two special populations of glomerular and peritubular endothelial cells were observed in degenerating mesonephros: one translocating beta-catenin into the nucleus and the other in apoptosis that did not translocate it. In conclusion, our results indicate that the paramesonephric duct is a potential new vasculogenetic pathway, and suggest that beta-catenin plays a role in the fate of mesonephric endothelial cells.
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Influence of mesonephros on foetal and neonatal rabbit gonads. II. Sex-steroid release by the ovary in vitro.
To test whether the mesonephros influences the steroid hormone release by the foetal and neonatal ovary, rabbit ovaries were cultured with and without the mesonephros for a period of 5 days. The media were harvested once every day and analyzed by RIA for the content of testosterone, oestradiol and progesterone. Mesonephros produced progesterone only at day 20 pc. At day 21 and 28 pc significant amounts of all three hormones were produced. The media of the co-cultures of ovaries with the mesonephros contained on day 23 and 28 pc less testosterone. On day 23 pc less les and on day 3 pp more oestradiol was found in the media of the co-cultures. At day 21 pc progesterone release was increased by the mesonephros. It could be concluded that the mesonephros is able to synthetize steroids and possesses the capacity both to lower and to increase ovarian steroid release in culture. The enhancement of ovarian steroid synthesis in cocultures with the mesonephros at certain ages might be due either to increased substrate production and/or increased enzymatic activity. The lowering of steroids seen at different ages might be due to mesonephric steroid binding. The mesonephros appeared to influence the male and female rabbits differently at the same ages.
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Influence of the mesonephros on the development of fetal mouse ovaries following transplantation into adult male and female mice.
We previously reported that fetal mouse ovaries frequently develop testicular structure following transplantation into adult male mice. The mechanism involved in gonadal sex reversal of ovarian grafts is not known. In the present study, we examined the influence of the adjacent mesonephros on development of the ovarian grafts. The results show that (1) when fetal ovaries were transplanted with the attached mesonephros, the frequency of ovotestis development was higher in male hosts than in female hosts, (2) the fetal ovaries that had been separated from mesonephros developed testicular structures more frequently than those with the mesonephros, and the incidence of ovotestis development was comparable in male and female hosts, (3) removal of the cranial or caudal half of the mesonephros resulted in a similar frequency of ovotestis development, and (4) when fetal ovaries were separated and reattached to the mesonephros, they developed testicular structures at a frequency similar to that of ovaries left attached to the mesonephros, and the sex of mesonephroi reattached to ovarian grafts did not influence the incidence of ovotestis development. These findings suggest that fetal ovaries can develop testicular structures after transplantation regardless of the sex of host, and that the adjacent mesonephros protects ovarian grafts from masculinizing stimuli more efficiently in female host than male hosts.
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Mesonephros has a role in limb development and is related to thalidomide embryopathy.
Recent studies have demonstrated a link between limb reduction defects and mesonephros removal [Geduspan and Solursh, 1992) Dev. Biol., 151:242-250]. However, there is some question as to whether the limb-reduction defects seen in that study resulted from the removal of mesonephros or from the formation of scar tissue medial to the limb territory. The current study was conducted to test the hypothesis that elimination of the mesonephros without producing scar tissue adjacent to the limb will adversely affect limb morphogenesis. The hypothesis was tested by the insertion of tantalum foil barriers into various levels of the intermediate mesoderm of developing chick embryos to prevent the caudal elongation of the mesonephros. Limb reduction defects were obtained when the mesonephros was prevented from forming caudal to somite 14. No limb defects were seen when a foil barrier was placed into the intermediate mesoderm at the level of somite 21 or 25. Our results support the notion that a signal from the mesonephros is necessary for normal limb development. In addition, it appears that a craniocaudal factor emanating from the mesonephros plays a role in limb development. The limb reduction defects obtained in this study were also compared to the pattern of thalidomide embryopathy in humans. There is a close correspondence between the types of limb reduction anomalies seen with thalidomide and mesonephric blocks and between the severity of defects vs. the timing of thalidomide intake or mesonephric blockage. A model for possible thalidomide embryopathy is presented.
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A growth-promoting influence from the mesonephros during limb outgrowth.
It has been suggested that the mesonephros has a role in normal limb development. This hypothesis was directly tested by removing the mesonephros adjacent to the presumptive limb region of stage 12-18 chick embryos using microsurgery or laser ablation. The experimental manipulation resulted in reduced limb outgrowth on the operated side. The poor limb outgrowth was correlated with either the lack of or the presence of a rudimentary mesonephros on the operated side. Furthermore, the presence of nephric tissue in limb bud organ culture enhanced growth and morphological differentiation of cartilage formed in culture. In vivo, the influence of the mesonephros resulted in significantly higher cell proliferation in the adjoining medial half of the limb mesoderm compared with the lateral half. The removal of the mesonephros adjoining the prospective limb region reduced the number of dividing cells in the medial mesoderm. The higher proliferation in the medial limb mesoderm is significant to limb outgrowth since grafting experiments showed that most of the cells that form the limb are derived from the medial mesoderm. The results suggest that the influence from the mesonephros may provide some signal for limb outgrowth.
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3D reconstruction of the mouse's mesonephros.
The present work reports on the three-dimensional reconstruction of the segmented mesonephros during the embryonic development of the mouse. With a light microscope and an automatic reconstruction of surfaces, aspects of the mesonephros are described. These surfaces are obtained by using digitized contour lines. A new interpolation method called DSI (Discrete Smooth Interpolation) enables correction of the distortion induced by microtomy in paraffin sections. After a triangulation step, this method uses a smoothing algorithm, which implies a spatial redistribution of the vertices of the triangles to correct the rotational and translational misalignment. The use of this 3D program improves the understanding of the development patterns and helps us to appreciate changes in the rebuilt mesonephros. By 10.5 embryonic days, tubules emerge from the Wolffian body and begin their formation, then between 11.5 and 13.5 embryonic days, tortuous mesonephric tubules bound to the Wolffian duct form small curls, which grow and finally unwind. At the same time, mesonephric tubules unbound to the Wolffian duct appear, and on 13.5 embryonic days, the Müllerian duct is visible. After 14.5 embryonic days, the segmented mesonephros keeps its general aspect but decreases in size. At this time, each gonad is provided with both Wolffian and Müllerian ducts. Later, the Wolffian duct differentiates into the definitive male duct system, whilst the Müllerian duct regresses. Conversely, the paramesonephric duct differentiates into the definitive female duct system, whilst the mesonephric duct in turn degenerates. By this time degeneration has begun in the cranial portion of the mesonephros and this process progresses caudally. The spatial organization of the mesonephric tubules and the precise organization of all connections between these elements and the ducts may be well defined. Such approach can allow for a high definition of the normal pattern of mesonephros differentiation.
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Limb initiation and development is normal in the absence of the mesonephros.
With rapid progress in understanding the genes that control limb development and patterning interest is becoming focused on the factors that permit the emergence of the limb bud. The current hypothesis is that FGF-8 from the mesonephros induces limb initiation. To test this, the inductive interaction between the Wolffian duct and intermediate mesoderm was blocked rostral to the limb field, preventing mesonephric differentiation while maintaining the integrity of the limb field. The experimental outcome was monitored by following expression of cSim1 and Lmx1, molecular markers for the duct and the mesonephros, respectively. Evidence is presented that the intermediate mesoderm undergoes apoptosis when the inductive interaction with the Wolffian duct is blocked. fgf-8 expression was undetectable in the mesonephric area of embryos with confirmed absence of mesonephros; nevertheless, limb buds formed and limb development was normal. The mesonephros in general, and specifically its fgf-8 expression, was shown to be unnecessary for limb initiation and development; the hypothesis linking the mesonephros and limb development is not supported. Further studies of axial influences on limb initiation should now concentrate on medial structures such as Hensen's node and paraxial mesoderm; the alternative that no axial influences are required should also be examined.
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The chicken embryonic mesonephros synthesizes link protein, an extracellular matrix molecule usually found in cartilage.
Link protein is a macromolecule that is relatively abundant in the extracellular matrix of cartilage, where it acts as a stabilizing component in aggregates of the large chondroitin sulfate proteoglycan and hyaluronic acid. In the present study, link protein transcripts were demonstrated in the chicken embryonic mesonephros by RNA in situ hybridization using a cartilage link protein cDNA probe. The link protein transcripts of the mesonephros are of the same size as those seen in cartilage. In addition, mesonephroi contain a protein that is immunologically reactive with a link protein polyclonal antiserum and this protein is identical in size to link protein isolated from cartilage. No transcripts for cartilage proteoglycan core protein were detected in the mesonephros. Type II collagen and cartilage matrix protein transcripts were also not detectable in the mesonephros. From previous data on chondrogenesis in the developing limb bud, the transcription of link protein and the proteoglycan core protein genes appeared to be spatially and temporally regulated in a coordinated fashion. However, the presence of link protein transcripts in the mesonephros, independent of cartilage proteoglycan core protein gene expression, indicates that these genes can be regulated independently of each other.
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Steroid contents of and steroidogenesis in vitro by the developing gonad and mesonephros around sexual differentiation in fetal sheep.
The aim of the present study was to establish whether the steroids, progesterone, androstenedione, testosterone and oestradiol, were present in the mesonephric-gonadal complex of female and male sheep fetuses around sexual differentiation (that is, from day 28 to day 45 of gestation, with sexual differentiation occurring at approximately day 32). A second aim was to test whether the mesonephric-gonadal complex, mesonephros (days 35-45 only) and gonad (days 35-45 only) were capable of steroid synthesis in vitro. The steroid contents in the mesonephric-gonadal complex were not detectable before sexual differentiation. However, from day 35 of gestation onwards, the mesonephric-ovarian complex contained mainly oestradiol and the mesonephric-testicular complex contained mainly testosterone: from day 35 until day 45 the increase in content of these two steroids exceeded the increase in the mass of tissue by more than fivefold. From day 40 to day 45 of gestation, the contents of the other steroids in the pathways to oestradiol increased progressively in both sexes but more in parallel with the increase in tissue mass. In contrast to the steroid contents in the tissue at recovery, the mesonephric-gonadal tissue from both sexes in tissue culture was able to synthesize most steroids before and after sexual differentiation and also to metabolise supplementary androstenedione to oestradiol. These findings suggest that many, if not all, of the steroidogenic enzymes in the pathway from cholesterol to oestradiol are present before sexual differentiation. Most of the aforementioned steroids were present in detectable amounts in isolated mesonephros and gonad of both sexes after sexual differentiation. Moreover, for both the isolated mesonephros and gonad, there were increases in the mean contents of most steroids after culture relative to the contents in the tissues at recovery. These data suggest that the mesonephros, as well as the gonad, in both sexes is capable of synthesizing steroid. It is concluded that, in the sheep fetus, the female and male gonads are steroidogenically active after sexual differentiation, that the steroidogenic enzymes develop before sexual differentiation, and that the mesonephros is a site of steroid synthesis.
Androstenedione↗
Endogenous galectins and effect of galectin hapten inhibitors on the differentiation of the chick mesonephros.
Galectins are galactoside-binding lectins. In the mesonephros of the chick embryo, the 16-kDa galectin is abundant in the glomerular and tubular basement membranes where it colocalizes with fibronectin and laminin. To test whether galectin-glycoprotein interactions could play a role in mesonephric development, the effects of the galectin hapten inhibitors thiodigalactoside (TDG) and lactose on the differentiation of the cultured mesonephros were investigated. When compared to control saccharide-free or maltose-treated cultures, mesonephroi cultured in the presence of TDG and lactose exhibited defects in tissue organization. These included a distorted tubule shape, pseudo-stratification of the tubular epithelium, and detachment of glomerular podocytes from the basement membrane. The presence of molecular differentiation markers in the developing mesonephros was investigated. In vivo, expression of the epithelial-specific cell adhesion molecule E-cadherin is restricted to differentiated tubular epithelial cells, whereas the intermediate filament protein vimentin is present in mesonephrogenic mesenchyme and is undetectable in tubular epithelial cells. In mesonephroi cultured in the absence of sugars or in the presence of maltose, the expression pattern of these two marker molecules resembles that found in the mesonephros in vivo. In contrast, in the mesonephroi cultured in the presence of TDG and lactose, the epithelial tubular cells expressing E-cadherin also express vimentin. Re-expression of vimentin in the tubular epithelial cells could indicate a partial reversal to a mesenchymal phenotype. Results suggest that galectin-glycoprotein interactions in the basement membrane are important in the maintenance of the renal epithelial phenotype. Dev Dyn 1999;215:248-263.
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Microvascular assembly and cell invasion in chick mesonephros grafted onto chorioallantoic membrane.
Embryonic tissues, in common with other tissues, including tumours, tend to develop a substantial vasculature when transplanted onto the chorioallantoic membrane (CAM). Studies conducted to date have not examined in any detail the identity of vessels that supply these grafts, although it is known that the survival of transplanted tissues depends on their ability to connect with CAM vessels supplying oxygen and nutrients. We grafted the mesonephros, a challenging model for studies in vascular development, when it was fully developed (HH35). We used reciprocal chick-quail transplantations in order to study the arterial and venous connections and to analyse the cell invasion from the CAM to the organ, whose degeneration in normal conditions is rapid. The revascularization of the grafted mesonephros was produced by the formation of peripheral anastomoses between the graft and previous host vasculatures. The assembly of graft and CAM blood vessels occurred between relatively large arteries or veins, resulting in chimeric vessels of varying morphology depending on their arterial or venous status. Grafts showed an increased angiogenesis from their original vasculature, suggesting that the normal vascular degeneration of the mesonephros was partially inhibited. Three types of isolated host haemangioblast were identified in the mesonephros: migrating angioblast-like cells, indicating vasculogenesis, undifferentiated haematopoietic cells and macrophages, which might have been involved in the angiogenesis. Tomato lectin was found to bind activated macrophages in avian embryos.
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[The blood vessels of the mesonephros of domestic cattle (Bos taurus), a corrosion cast study].
The blood vessels of the bovine mesonephros - a corrosion cast study Using scanning electron microscopy plastoid corrosion cast specimens of the mesonephros of 30-45-day-old bovine foetuses were investigated. The studies showed that the blood vessels of the mesonephros are organized in two separate networks similar to these of the teleosts and amphibians. The glomerula mesonephrica originate from the aorta implying that they belong to the high pressure system. The tubuli mesonephrici are surrounded by a filamentous independent capillary network which is shown for the first time in these studies. This capillary network originates from the caudal cardinal veins and represents a mesonephric portal system with a relatively low blood pressure. The blood from the two capillary networks diverts into terminal veins and into the subcardinal veins. The questions of haemodynamics in the mesonephros are discussed.
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The quail mesonephros: a new model for renal senescence?
BACKGROUND/AIMS: Renal senescence during normal aging is associated with specific vascular alterations and tissue degeneration. Although the degenerative program executed during embryonic kidney development is known to include vascular alterations, studies yet have to examine whether it involves replicative senescence. In this study, we assessed the potential of the quail mesonephros, a transitory embryonic kidney, as a model of human renal senescence. METHODS: Quail embryos with developing or degenerating mesonephros were studied on day 6 or day 11 of incubation, respectively. Senescence-associated beta-galactosidase activity, a marker of replicative senescence, was examined on whole mounts and sections. Senescent vascular characterization was performed by the scanning electron-microscopic analysis of vascular corrosion casts. RESULTS: Senescence-associated beta-galactosidase activity was found only in old mesonephros. Moreover, at 11 days of incubation glomerular capillaries showed discontinuities and were thinner and more tortuous than those observed at 6 days, characteristics also reported for the aging human kidney. CONCLUSION: The degenerating quail mesonephros is a potential model of renal senescence, showing biochemical and morphological characteristics of the aging human kidney.
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[Hematopoietic growth factors expressed in human aorta-gonad-mesonephros (AGM)-derived stromal cells in vitro].
This study was aimed to investigate the hematopoietic growth factors expressed in human aorta-gonad-mesonephros (AGM)-derived stromal cells in vitro in order to provide the basic data for elucidating the role of AGM -derived-stromal cells in embryo-hematopoiesis and its hematopoietic suppoitive effect. RT-PCR was used to analyze the expression of IL-6, SCF, Flt3-L, oncostatin M (OSM), IL-3, TPO, M-CSF and LIF in human aorta-gonad-mesonephros-derived stromal cells (hAGMS1-S5) at mRNA level. IL-6, SCF and Flt3-L levels were detected in the supernatant of hAGMS1-S5 stromal cells by ELISA assay. Umbilical cord blood CD34(+) cells were cocultured with hAGMS1-S5 feeder cells, and hematopoietic cells were collected at day 14 for colony analysis in methylcellulose semisolid medium. The results showed that human aorta-gonad-mesonephros-derived stromal cells S1-S5 expressed IL-6, SCF, Flt3-L and OSM mRNA, but did not express IL-3, TPO, M-CSF and LIF mRNA. In the supernatant of hAGMS1-S5 cells, IL-6, SCF and Flt3-L could be detected by ELISA assay at different levels, while there was no significant difference between groups of hAGMS1-S5 (P > 0.05). When cocultured with umbilical cord blood CD34(+) cells, hAGMS1-S5 could support the expansion of CFU-GM, BFU-E, and CFU-Mix. The supportive effects of hAGM S1-S5 were significantly different (P < 0.05), hAGM S3 and S4 were better than hAGM S1, S2, and S5. It is concluded that detection of hematopoietic growth factors expressed in human aorta-gonad-mesonephros-derived stromal cells provided a solid foundation to elucidate the mechanism of hematopoiesis and the hematopoietic supportive effect of these stromal cells.
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Changes in the protein spectrum of the chick embryo mesonephros and metanephros in the course of development.
The protein spectra of two fractions (the soluble and the membrane fraction) of chick embryo kidney homogenates were isolated by electrophoresis on polyacrylamide gels with the aim of detecting the kidney differentiation process at the molecular level and, at the same time, of evaluating similarities in the construction of the mesonephros and metanephros at this level. Corresponding stages of the above two types of kidney were chosen for studying changes in protein structure during differentiation--i.e. the outset of differentiation (the 6-day mesonephros, the 11-day metanephros) and the stage of full maturity (the 14-day mesonephros, the 20-day metanephros). A total of 36 proteins was distinguished. The analysis of the protein spectra showed that the number of proteins changes but slightly during differentiation; the protein composition of the two types of kidney during differentiation altered by 20-35% of the total number of proteins; the similarity of the protein composition of the corresponding stages of mesonephros and metanephros, expressed as the proportion of the number of identical proteins, was greater than the mutual similarity of different developmental stages of the same type of kidney. The percentage of different proteins at corresponding stages of the kidneys varied from 5% to 23% of the total number of proteins detected.
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Gonadal development of the chick embryo following microsurgically caused agenesis of the mesonephros and using interspecific quail-chick chimaeras.
Mesonephric agenesis was achieved by microsurgical excision of the left Wolffian duct and the underlying intermediate mesoderm of different regions between somites 16 and 23 in chickens after 50-52 h of incubation (stage 14 HH). Quail-chick chimaeras were produced by transplantation of corresponding quail tissue in the region of somites 18-21. A morphometrical analysis of the mesonephric and gonadal area in cross sections shows that the intermediate mesoderm from somites 16 to 23 develops into the mesonephros. A partial agenesis of the mesonephros brought about by removal of the intermediate mesoderm at the level of somites 18 to 21 at stage 14 leads to a mean reduction of the gonadal volume of 37.8% compared to the volume of the untreated side at stage 30. Transplantation of quail intermediate mesoderm in this region of the excision results in development of a hybrid mesonephros. Consequently, the gonads are invaded and colonized by quail cells mobilized from mesonephric corpuscles examined at stage 30, 35 and 36. These results are discussed in terms of the origin of the gonadal stroma during this developmental period; they show that in the region from the third to the sixth segment the ventromedial part of the differentiating mesonephros participates in the contribution of stromal cells to the gonad.
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