Fusion Of Folds Research Articles

The Biology of the Development of the Genital Organs. A Multimedia Teaching Program

In my presentation, I review the sexual differentiation from the genetic sex until the appearance of the external genitalia and the developmental anomalies to use an animated cartoon. The first critical stage of sexual differentiation occurs at the moment of fertilization, when the genetic sex of the zygote is determined by the nature of the sex chromosome contributed by the sperm. Although an XY zygote is destined to become a male, no distinctive differences between the early development of male and female embryos have been noted. This is accomplished after migration of the primordial germ cell into the early gonad. Because of the early commonality of genital structures, anomalies are the result of abnormal retention or loss of appropriate genital structures. Therefore, most genital anomalies are some form of intersex. During the early differentiation of the gonads, while the mesonephros is still the dominant excretory organ, the gonads arise as ridge like thickenings (gonadal ridge) on its ventromedial face. Differentiation of the indifferent gonads into ovaries or testes occurs after the arrival of the primordial germ cells. The primordial germ cells arise from the endodermal cells of the yolk. The principal function of the Y chromosome is to direct the differentiation of the presented indifferent gonad into a testis from the sixth week, while two X chromosome are presented the ovaries start to develop, from the 12th week. The next and most obvious phase in sexual differentiation of the embryo is the differentiation of the somatic sex. The early embryo develops a dual set of potential genital ducts, one is the original mesonephric (Wolff ) ducts, which persists after degeneration of the mesonephros as an excretory organ, and the another is newly formed pair of ducts called the paramesonephric (Müllerian) ducts. Under the influence of testosterone secreted by the testes, the mesonephric ducts develop into the duct system through which the spermatozoa are conveyed from the testes to the urethra. The potentially female paramesonephric ducts regress under the influence of another product of the embryonic testes, the Müllerian inhibitory factor, a glycoprotein secreted by the Sertoli cells. In genetically female embryos, neither testosterone nor Müllerian inhibitory factor are secreted by the gonads. In the absence of testosterone the mesonephric ducts regress and lack of Müllerian inhibitory factor permits the paramesonephric ducts to develop into oviducts, the uterus and part of the vagina. The next stage is the development of the external genitalia. In very young embryos, a vaguely outlined elevation known as the genital eminence can be seen in the midline, just cephalic to the proctodeal depression. This is soon differentiated into a central prominence (genital tubercle) closely flanked by a pair of folds (genital folds) extending toward the proctodeum. Somewhat farther to either side are rounded elevation known as the genital swellings. From this common starting point the external genitalia of both sex differentiate. If the individual is to develop into a male the genital tubercle, under the influence of dihydrotestosterone, becomes greatly elongated to form the penis and the genital swellings become enlarged to form the scrotal pouches. During the growth of the penis a groove develops along the entire length of its caudal face and is continuous with the slit‐like opening of the urogenital sinus. This groove later becomes closed over by a ventral fusion of the genital folds, establishing the penile portion of the urethra. The portion of the urogenital sinus between the neck of the bladder and the original opening of the urogenital sinus becomes the prostetic urethra. In the female, the genital tubercle becomes the clitoris, the genital folds become the labia minora, and the genital swellings become the labia majora. The urethra in the female is derived from the urogenital sinus, being homologous with the prostatic portion of the male urethra.

Neurulation in the cranial region – normal and abnormal

Cranial neurulation is the embryonic process responsible for formation of the brain primordium. In the mouse embryo, cranial neurulation is a piecemeal process with several initiation sites and two neuropores. Variation in the pattern of cranial neurulation occurs in different mouse strains, and a simpler version of this morphogenetic scheme has been described in human embryos. Exencephaly is more common in females than in males, an unexplained phenomenon seen in both mice and humans. As the cranial neural tube closes, a critical morphogenetic event is the formation of dorsolateral bending points near the neural fold tips, which enables subsequent midline fusion of the neural folds. Many mutant and gene-targeted mouse strains develop cranial neural tube defects, and analysis of the underlying molecular defects identifies several requirements for normal dorsolateral bending. These include a functional actin cytoskeleton, emigration of the cranial neural crest, spatio-temporally regulated apoptosis, and a balance between cell proliferation and the onset of neuronal differentiation. A small number of mouse mutants exhibit craniorachischisis, a combined brain and spine neurulation defect. Recent studies show that disturbance of a single molecular signalling cascade, the planar cell polarity pathway, is implicated in mutants with this defect.

Maternal and Paternal Risk Factors for Hypospadias

Hypospadias is a common congenital anomaly, characterized by incomplete fusion of the urethral folds during fetal development, which results in the urethra opening on the ventral surface of the penis or on the scrotum. In their article, Pierik et al. (2004) proposed multiple possible maternal and paternal risk factors related to the development of isolated hypospadias, including genetic, endocrine, and environmental factors. Concerns and alarms have been raised about the potential effects of endocrine disruptors, which include derivatives of poly-aromatic hydrocarbons and pesticides, on the developing male reproductive tract. We have recently published two case–control studies on risk factors and hypospadias, one on a possible association with maternal age (Bianca et al. 2005) and the other on the role of endocrine disruptors (Bianca et al. 2003). In the first study (Bianca et al. 2005), we evaluated 415 newborns with isolated hypospadias and 812 controls. Our results suggest that an increased risk for hypospadias exists in women at the extremes of the age distribution ( 40 years; p = 0.000 and 0.026, respectively) relative to women in the middle of the distribution, with a mechanism probably related to hormonal disruption. It has been postulated that changes in concentrations of sex hormones during the fetal critical period of genital development (weeks 8–14), caused by endogenous or exogenous factors, may play a role in the development of hypospadias and that hypospadias could be associated with early malfunction of the placenta, resulting in decreased secretion of placental and fetal hormones that could in turn disturb fetal development (Akre et al. 1999). Mothers at the extremes of the age distribution may be more susceptible to this hormonal disruption. The association between hypospadias and maternal age, both for younger and older women, might be explained as a “defect in nature’s quality control” caused by a reduction of defensive maternal mechanisms that would be less efficient in the elimination of malformed fetuses in the mothers of hypospadias cases. This hypothesis may also apply to other birth defects where a relationship with maternal age has been demonstrated. In the second study of 68 cases and 211 controls (Bianca et al. 2003), we identified a high incidence of hypospadias in two towns in southeastern Sicily, which have intense industrial (Augusta) and agricultural (Vittoria) activities. Our results showed an incidence that was 3.8 [95% confidence interval (CI), 2.16–6.14] and 2.3 times (95% CI, 1.48–3.43) higher, respectively, than expected (3.2 per 1,000 male live births in southeastern Sicily). The odds ratios for fathers’ job exposure alone were 5.5 (95% CI, 1.22–24.7) for working in an oil refinery in Augusta and 2.9 (95% CI, 1.01–8.55) for working in hothouses in Vittoria. Pollutants in both areas include compounds with proven estrogenic activity and other chemicals: pesticides and herbicides and their metabolites; dieldrin, chlordane, and endosulfan; polychlorinated biphenyls and dioxins; bisphenols used in epoxy resins; and some phthalates used as plasticizers in a wide variety of applications, including polyvinyl compounds and alkyphenol ethoxylates. The direct cause–effect relationship between environmental pollutants, which act as endocrine disruptors, and the increased incidence of hypospadias in these areas is difficult to establish and demonstrate. Our study (Bianca et al. 2003), suggested that exposure to large amounts of industrial and agricultural pollutants is sufficient to increase the risk of hypospadias. Other risk factors, such as reproductive history, may be involved in the etiology of hypospadias. Disturbances in sexual differentiation occur when endogenous and/or exogenous factors act to disrupt the metabolism of gonadal hormones during development. Further epidemiologic and biologic studies are needed to explain, in a multi-factorial model, which factors (genetic and/or environmental) interact and influence the etiology of this congenital abnormality.

IN UTERO PREDNISONE EXPOSURE AFFECTS GENITAL DEVELOPMENT

The etiology of the worldwide incidence of hypospadias remains unexplained. Data from the Center for Disease Control suggest that maternal asthma exposure may be a risk factor. The development of the mouse and human urethra shows significant similarities, such as fusion of 2 epithelial edges, closure of a midline epithelial seam and subsequent cellular remodeling, to justify the use of the mouse as an experimental animal model. Prednisone may act as a weak androgen directly or alternatively lowering testosterone by hypothalamic suppression of gonadotropins. We describe the effects of prednisone on urethral formation in the mouse. The model comprised 10, 20 and 20 timed pregnant C57/6 mice exposed to 1,000, 200 and 100 mg/kg prednisone, respectively, on gestational days 12 through 18. The morphology of the genital tubercles of both sexes were examined on gestational day 19 using histological techniques and 3-dimensional computer reconstruction. Specific attention was focused on the developing urethral seam. Microscopic serial analysis confirmed the presence of an arrest in seam formation in approximately 25% of male fetuses given supraphysiological doses of prednisone (1,000 mg/kg). In contrast, acceleration of urethral fold fusion and a longer urethral tube were observed in those males treated with 100 and 200 mg/kg prednisone. The female fetuses treated with the same dosages of prednisone did not show any change at the level of the urethral seam area or in the remainder of the genital tubercles. Supraphysiological doses of prednisone treatment has an inhibitory effect on urethral fold fusion leading to hypospadias whereas low doses accentuate urethra formation in male mouse fetuses. In mice lower doses do not cause hypospadias in the males or affect urethral formation in females.

The development of the male genitourinary system: III. The formation of the spongiose and glandar urethra

It is generally agreed that the urethral plate disintegrates, resulting in the urethral groove. This is subsequently transformed into the urethra by fusion of the urethral folds, which flank its sides. Recently, the existence of such a groove and folds has been denied and this challenge to the long accepted existence of such folds is significant since hypospadias is considered to result from failure of their fusion. The present studies indicate that mesodermal fold formation and its subsequent subepithelial fusion across the midline plays an essential role in urethral tube formation. Disruption of this process readily explains common congenital abnormalities of the urethra.

Occipital extra- and intracranial lipoencephalocele associated with tectocerebellar dysraphia: case for discussion

Y. Yamanouchi ()) Department of Neurosurgery, Kansai Medical University, 10–15 Fuimizono-cho, 570–8507 Moriguchi-city, Osaka, Japan e-mail: yamauchy@takii.kmu.ac.jp Tel.: +81-6-69921001 Fax: +81-6-69916207 The author presented a rare and interesting case in which embryological consideration and pathogenesis of tectocerebellar dysraphia with lipoma was discussed. I felt a strong similarity between this case and lipomyelomeningoceles in the spine after a first reading. I agreed with almost everything the authors wrote. However, it does not seem to me that the co-existence of the lipoencephalocele and tectocerebellar dysraphia suggests multiple sites of neural closure. Intracranial lipomas can occur in various locations: in the corpus callosum, quadrigeminal area, brainstem, and cerebellum, mainly in the midline. Many of them occur without skull defect and other cerebral malformations. Regarding lipomas, we should consider ordinary lipoencephaloceles or lipomeningoceles totally separately from lipomas, such as those located in the corpus callosum or spinal lipomas without dysraphism. Truwit and Barkovich, cited by the author, stated that intracranial lipomas are congenital malformations that result from abnormal persistence and maldifferentiation of meninx primitiva during the development of the subarachnoid cisterns [3]. But in this case, it is possible that abnormal development occurred earlier than the stage of development of the subarachnoid cisterns. In the dysraphic state, I don’t think there is the same pathogenesis in skin-covered lesions and lesions with the neural tissue exposed. Tarara et al. [2] induced central nervous system malformations in primates by using triamcinolone acetonide. They suggested encephaloceles may result from a combination of mesenchymal and neural tube abnormalities. In this case, considering the coexistence of lipomatous tissue, we may apply the theory of embryogenesis of spinal lipomyelomeningoceles resulting from premature dysjunction, which is that lipomas arise when the cutaneous ectoderm prematurely separates from the neuroepithelium prior to neural fold fusion. Under these circumstances, the surrounding mesenchyme ingresses between the neural tube and overlying cutaneous ectoderm [1].

Loss of AP-2α impacts multiple aspects of ventral body wall development and closure

Human birth defects involving the ventral body wall are common, yet little is known about the mechanism of body wall closure in mammals. The AP-2α transcription factor knock-out mouse provides an exceptional tool to understand this particular pathology, since it has one of the most severe ventral body wall closure defects, thoracoabdominoschisis. To gain insight into the complex morphological events responsible for body wall closure, we have studied this developmental process in AP-2α knock-out mice. Several tissues involved in normal ventral body wall closure are defective in the absence of AP-2α, including those associated with the primary body wall, the umbilical ring, and the mesoderm of the secondary body wall. These defects, coupled with the expression pattern of AP-2α, suggest that AP-2α is involved in multiple developmental mechanisms directing the morphogenesis of the ventral body wall, including cell migration, differentiation, and death. There is a failure of migration and fusion of the body folds at the umbilical ring, as well as in the formation and migration of the abdominal bands and ventral musculature. Furthermore, the mechanism of cell deposition at the umbilical ring is disturbed. Consequently, the mesodermal compartment of the body wall is underdeveloped. We also suggest that AP-2α is required for signaling from the surface ectoderm to the underlying mesoderm for proper development and closure of the ventral body wall. These findings provide a fundamental understanding of how AP-2α functions in the closure of the ventral body wall, as well as offer insight into related human birth defects.

Development of the penile urethra.

In humans hypospadias consists of an abnormal ventral urethral meatus, incomplete formation of the prepuce and penile curvature (Baskin et al., 1998). The cause of hypospadias is thought to be failure of formation or fusion of the urethral folds bounding the urethral groove. While failure of urethral fold fusion may be the ultimate cause of hypospadias, there are many other possible causes of this malformation. In this review, the development of the penile urethra will be described for both the human and the mouse. The purpose of this paper is to review the developmental anatomy of the external genitalia to provide a framework for subsequent chapters that will focus on mechanisms of penile development. The second goal of this review is to discuss the development of the external genitalia of the human and the mouse so as to evaluate the mouse as a relevant animal model for development of the external genitalia.

The effect of oestrogen and testosterone on the urethral seam of the developing male mouse genital tubercle.

To describe the effects of exogenous oestrogens and androgens on urethral formation in the mouse, as the development of the mouse and human urethra have significant similarities, and understanding normal male urethral development may help to identify the causes of abnormal development, e.g. hypospadias. Timed-pregnant C57/6 mice were exposed to synthetic oestrogens and androgens. The morphology of the genital tubercles was examined histologically and with three-dimensional computer reconstruction. Specific attention was focused on the developing urethral seam. Microscopic serial analysis confirmed the presence of an arrest in seam formation in about half of oestrogen-treated male fetuses. In contrast, there was acceleration of urethral fold fusion and a longer urethral tube in those treated with androgens. Oestrogen-treated fetuses had a thin periurethral spongiosa, in contrast to androgen-treated fetuses which developed a thicker periurethral spongiosa. The effect of oestrogens on seam area formation did not depend on the dose, but in contrast, in the androgen-treated fetuses it was. Oestrogens and androgens have a direct effect on the fusion of the urethral fold that leads to seam formation. Normal urethral development depends on the delicate balance of these complementary hormones.

The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures.

During vertebrate development, an organizing signaling center, the isthmic organizer, forms at the boundary between the midbrain and hindbrain. This organizer locally controls growth and patterning along the anteroposterior axis of the neural tube. On the basis of transplantation and ablation experiments in avian embryos, we show here that, in the caudal midbrain, a restricted dorsal domain of the isthmic organizer, that we call the isthmic node, is both necessary and sufficient for the formation and positioning of the roof plate, a signaling structure that marks the dorsal midline of the neural tube and that is involved in its dorsoventral patterning. This is unexpected because in other regions of the neural tube, the roof plate has been shown to form at the site of neural fold fusion, which is under the influence of epidermal ectoderm derived signals. In addition, the isthmic node contributes cells to both the midbrain and hindbrain roof plates, which are separated by a boundary that limits cell movements. We also provide evidence that mid/hindbrain roof plate formation involves homeogenetic mechanisms. Our observations indicate that the isthmic organizer orchestrates patterning along the anteroposterior and the dorsoventral axis.

The absence of lateral fusion in cloacal partition

Background/purpose: The mechanism by which the cloaca becomes partitioned into a dorsal rectal part and a ventral genitourinary sinus has been the subject of speculation for more than a century. Despite repeated suggestions that partitioning of the cloaca by fusion of lateral folds does not occur, the concept continues to hold sway in many student and surgical texts. The authors reviewed the histologic and 3-dimensional appearance of the urorectal septum in human and rat embryos to see if there was any evidence of lateral fusion in its formation. Methods: Sprague-Dawley rat embryos (n = 143) were examined between 11 and 21 days’ gestation and compared with human embryo sections (57 embryos) held in historical collections in Europe. Rat embryos were examined by microscopy, dissection, and serial histologic section. In addition, some specimens were sectioned in wax until the lumen of the cloaca was reached, after which they were dewaxed and the internal cavities imaged with scanning electronmicroscopy (n = 18 of 143). Results: Cloacal “partitioning” resulted from a combination of growth of the mesenchyme of the hindgut and genitourinary sinus, an alteration in the position of the cloaca in relation to surrounding structures secondary to growth in the ventral, infraumbilical abdominal wall and changes in the curvature of the developing spine, and apoptosis in the dorsal wall of the cloaca with shortening of the dorsal cloacal wall. There was no septum, as it is usually defined, between the developing bladder and hindgut. There was no evidence on either histologic section or scanning electronmicroscopy of any process of fusion occurring between the 2 lateral folds within the lumen of the cloaca. Conclusions: Lateral fusion of the side walls of the cloaca does not play a role in cloacal “partition.” Development of the bladder and hindgut occurs by a process that involves growth, differentiation, and remodeling.

Spatial and temporal expression of folate-binding protein 1 (Fbp1) is closely associated with anterior neural tube closure in mice.

Periconceptional folate supplementation is widely believed to have significant preventive effects on the production of neural tube defects. Folate-binding protein 1 (FBP1) is one of the membrane proteins that mediate cellular uptake of folate. Although recent studies suggest that Fbp1 is essential for neural tube closure, the pattern of Fbp1 expression in embryonic tissues has not been examined in detail. To elucidate how Fbp1 contributes to neural tube closure, we examined the spatial and temporal expression patterns of Fbp1 in the developing neural folds and tube of mouse embryos by in situ hybridization. Fbp1 showed a distinct expression pattern in the neural folds, which preceded initiation of neural tube closure at the cervical region and the prosencephalic/mesencephalic boundary. Fbp1 expression was mainly localized to the most dorsal regions of the neural folds where fusion was to occur. With proceeding of neural fold fusion, Fbp1 expression extended to the adjacent unfused neural folds. In the rhombencephalon, robust expression of Fbp1 was observed in rhombomere2 (r2) and r6, suggesting its roles in development of neural crest cells. Fbp1 also showed intense expression in the yolk sac, indicating that FBP1 may mediate transferring maternal folate to embryos during neurulation. These findings indicate close association between Fbp1 and anterior neural tube closure.

Embryogenesis of the glowworm Rhagophthalmus ohbai Wittmer (Insecta: Coleoptera, Rhagophthalmidae), with emphasis on the germ rudiment formation

The early embryonic development and features of the developing embryo of the glowworm Rhagophthalmus ohbai are described chiefly by light microscopy, with emphasis on the germ rudiment formation and its phylogenetic implication. The egg period is 30-34 days at about 23 degrees C. The newly laid egg is a short ellipsoid, 1.09 by 0.78 mm in size, and the size increases to 1.15 by 0.95 mm by 17 days after oviposition. Cleavage is of the typical superficial type. The germ disk is formed by cell aggregation of the embryonic area at the anterior end of the egg. The central part of the germ disk then sinks into the yolk and the spherical germ rudiment is formed by fusion of the amnioserosal folds extended from all margins of the germ disk. The inner region of the germ rudiment soon becomes slender and develops into the short embryo, whereas the outer region facing the anterior end is extended to form the thin amnion. The embryo then rapidly elongates, the elongation being accompanied by embryo segmentation and formation of appendages. The submerged condition of the embryo persists until about 17 days after oviposition (about 1 day before embryonic revolution) and thereafter the embryo becomes superficial in position. The presence of the following embryonic characters in R. ohbai supports the molecular data placing it within the Lampyridae: 1) formation of a spherical germ rudiment near the anterior end of the egg, and 2) the submerged condition of the developing embryo persists until shortly before revolution.

2001 Warkany lecture: to die or not to die, the role of apoptosis in normal and abnormal mammalian development.

Cell death is a common and reproducible feature of the development of many mammalian tissues/organs. Two well-known examples of programmed cell death (PCD) are the cell deaths associated with fusion of the neural folds and removal of interdigital mesenchymal cells during digit formation. Like normal development, abnormal development is also associated with increased cell death in tissues/organs that develop abnormally after exposure to a wide variety of teratogens. At least in some instances, teratogens induce cell death in areas of normal PCD, suggesting that there is a link between programmed and teratogen-induced cell death. Although researchers recognized early on that cell death is an integral part of both normal and abnormal development, little was known about the mechanisms of cell death. In 1972, Kerr et al. ('72) showed conclusively that cell deaths, induced in a variety of contexts, followed a reproducible pattern, which they termed apoptosis. The next breakthrough came in the 1980s when Horvitz and his colleagues identified specific cell death genes (ced) that controlled PCD in the roundworm, Caenorhabditis elegans (C. elegans). Identification of ced genes in the roundworm quickly led to the isolation of their mammalian homologues. Subsequent research in the 1990s led to the identification of a cadre of proteins controlling cell death in mammals, i.e., receptors/ligands, caspases, cytochrome c, Apaf-1, Bcl-2 family proteins, and IAPs. Two major pathways of apoptosis have now been elucidated, the receptor-mediated and the mitochondrial apoptotic pathways. The latter pathway, induced by a wide variety of toxic agents, is activated by the release of cytochrome c from mitochondria. Cytochrome c then facilitates the activation of a caspase cascade involving caspase-9 and -3. Activation of these caspases results in the cleavage of a variety of cellular proteins leading to the orderly demise of the cell. Work from my laboratory in the last 5 years has shown that teratogens, such as hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine, induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway, i.e., mitochondrial release of cytochrome c, activation of caspase-9 and -3, inactivation of poly (ADP-ribose) polymerase (PARP), and systematic degradation of DNA. Our work, as well as the work of others, has also shown that different tissues within the early post implantation mammalian embryo are differentially sensitive to the cell death inducing potential of teratogens, from exquisite sensitivity of cells in the developing central nervous system to complete resistance of cells in the developing heart. More importantly, we have shown that the resistance of heart cells is directly related to the failure to activate the mitochondrial apoptotic pathway in these cells. Thus, whether a cell dies in response to a teratogen and therefore contributes to the pathogenesis culminating in birth defects, depends, at least in part, by the cell's ability to regulate the mitochondrial apoptotic pathway. Future research aimed at understanding this regulation should provide insight not only into the mechanism of teratogen-induced cell death but also the role of cell death in the genesis of birth defects.

The two sites of fusion of the neural folds and the two neuropores in the human embryo.

Since reports on a pattern of multiple sites of fusion of the neural folds in the mouse appeared, it has been widely assumed that a similar pattern must be valid for the human. In the absence of embryological evidence, claims have been made that such a pattern can be discerned by classifying neural tube defects. The neural folds and tube, as well as the neuropores, were reassessed in 98 human embryos of Stages 8-13; 61 were controlled by precise graphic reconstructions. Careful study of an extensive series of staged human embryos shows that two de novo sites of fusion of the neural folds appear in succession: alpha in the rhombencephalic region and beta in the prosencephalic region, adjacent to the chiasmatic plate. Fusion from Site alpha proceeds bidirectionally (rostrad and caudad), whereas that from beta is unidirectional (caudad only). The fusions terminate in neuropores, of which there are two: rostral and caudal. Highly variable accessory loci of fusion, without positional stability and of unknown frequency, may be encountered in Stage 10 but seemingly not later, and their existence has been known for more than half a century. Two sites of fusion (a term preferred to closure) of the neural folds and two neuropores are found in the human embryo. No convincing embryological evidence of a pattern of multiple sites of fusion, such as has been described in the mouse, is available for the human. The construction of embryological details from information derived from other species or from the examination of later anomalies is liable to error. Neural tube defects are reviewed and although they have been considered on the basis of five, four, or three sites of fusion, interpretations based on two sites can as readily be envisaged.

Pigmented epidermal cysts.

There are few reports documenting the presence of melanin or melanocytes in epidermal cysts. One hundred and twenty five epidermal cysts from Indian patients were analyzed for the presence of melanin pigment and their sites were noted. A Masson's Fontana stain and immunohistochemistry for S-100 protein and HMB 45 was performed for localization of melanin and melanocytes within the epidermal cysts. Seventy-nine (63%) of the epidermal cysts showed presence of melanin pigment or melanocytes to a variable extent. Melanin was not present in epidermal cysts occurring along lines of fusion of skin folds during embryonogenesis (e.g., ventral midline, inner canthus, nose, upper lip and in the distal most parts like leg and foot and also scrotum). Ten of the 79 epidermal cysts showed extensive accumulation of melanin pigment and infiltration with melanophages in the cyst wall. Four of these patients gave history of trauma and the follow-up was uneventful in two of them. Pigmentation of epidermal cysts thus follows a definite anatomic pattern and is dependent on the natural skin color. Large amount of pigment accumulation within epidermal cysts occurs after cyst rupture and is not associated with hemochromatosis as was believed in the past.

Salpingographic demonstration of “cobblestone” mucosa of the distal tubes is indicative of irreversible mucosal damage

Objective: To establish the predictive value of the observation of a “cobblestone” pattern of the distal tube mucosa shown on the selective salpingogram as an indicator of significant and probably irreparable damage to the ciliated epithelium.Design: Clinical study.Setting: Fertility and Laser Center and Academic Radiology Department.Patient(s): Patients with primary or secondary infertility and obstruction of the fallopian tubes documented on hysterosalpingogram.Intervention(s): Transcervical recanalization of obstructed tubes followed by reassessment by selective salpingogram.Main Outcome Measure(s): Progression of mucosal disease with resultant low likelihood of intrauterine pregnancies and high probability of ectopic pregnancy.Result(s): In 32 asymptomatic patients, there was progression to coalescence and agglutination of mucosal folds in 13, to a thick wall pyosalpinx in 4. Five thin wall and 8 thick wall hydrosalpinges in asymptomatic patients showed no significant progression of mucosal disease but increasing peritubular fibrosis. In 12 symptomatic patients, progressive fusion of mucosal folds with polypoid hyperplasia was observed in 11, a thin wall hydrosalpinx in 2.Conclusion(s): Cobblestone appearance of the distal tubes heralds significant mucosal damage, prone to progressive disease and hence, a poor chance for conception.

Abnormal neural crest cell migration after the in vivo knockdown of tenascin-C expression with morpholino antisense oligonucleotides.

A key feature of vertebrate development is the formation of the neural crest. In the trunk, neural crest cells delaminate from the neural tube shortly after the fusion of the neural folds and migrate ventrally along specific pathways to form the neurons and glia of the peripheral nervous system. As neural crest cells leave the neural tube during the initial stages of their migration, they express the extracellular matrix glycoprotein tenascin-C, which is also found in the stroma of many tumors. We have studied the possible role for tenascin-C during neural crest morphogenesis in vivo by microinjecting tenascin-C morpholino antisense oligonucleotides into the lumen of the avian neural tube in ovo and electroporating the morpholino antisense oligonucleotides into the precursors of the neural crest. After 24 hr, tenascin-C immunostaining is reduced around the dorsal neural tube in the experimental microinjected embryos (12 of 13) but not in embryos microinjected with control morpholino antisense oligonucleotides (n = 3) or subjected to electroporation only (n = 2). In each of the 12 tenascin-C knockdown embryos neural crest cells are seen ectopically in the lumen of the neural tube and in the neuroepithelium; cells that do leave the neural tube after the microinjection fail to disperse laterally from the surface of the neural tube into the somites. The observation that neural crest cells must express tenascin-C to migrate normally is consistent with a role for this glycoprotein in contributing to the invasive behavior of neural crest cells.

Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters

The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.

Intrauterine Growth Retardation in Experimental Diabetes: Possible Role of the Placenta

Fetal growth disorders are common in pregnancy complicated by diabetes. Whereas macrosomia often occurs in infants of diabetic women, growth retardation is almost a rule in spontaneous and experimental diabetes in animals. However, it is not clear when during development growth inhibition starts and how placental pathology might affect fetal growth in maternal diabetes. In this study pregnant Wistar rats were injected (ip) with a single dose of 50 mg/kg of streptozotocin (STZ) on gestation day (GD) 2 and a blood glucose level of 200 mg/dl or more determined 24 hrs later indicated diabetes. The controls were non-treated, buffer treated or, following confirmation of diabetes, injected with a single dose of 2–6 IU of insulin (Novo Ultralente) once daily. Fetuses and placentae were collected from GD 14–20. Intrauterine growth retardation (IUGR) in STZ group was significant as early as GD 15 and persisted to GD 20. Insulin produced a significant recovery in fetal weight gain. The placentas of STZ-treated group were significantly heavier than those of the control groups. The reduction in cord length of the STZ group became apparent on GD 16 and remained so to term. The placenta of GD 14 STZ group had a thicker decidua basalis and dilated maternal sinusoids. By GD 16, the decidua basalis contained glycogen-containing decidual cells and scattered glycogen cells confirmed by Best' carmine with or without diastase. The glycogen cells of the basal zone were more abundant, and had degenerated in some sites leaving behind cysts with eosinophilic mass. The giant cells had proliferated enormously. The labyrinthine zone appeared spongy with persistent fetal mesenchyme, peri-vascular fibrosis, and enhanced placental barrier. The trophoblasts of the labyrinths also contained traces of glycogen unlike the controls. By GD 18, the decidua basalis of the STZ group was thinner than that of the controls and contained necrotic giant cells and lymphocytic aggregations. In the basal zone, the giant cells had proliferated further; more glycogen cells had degenerated. Perivascular fibrosis was still extensive in the labyrinthine zone. Bloodless maternal sinusoids, extensive vacuolization, degeneration of glycogen islands and formation of cysts characterized the labyrinthine zone. These changes varied in intensity from one area to another in the same placenta and between placentas of the same and of different litters. The development of the upper and lower jaws, elevation and fusion of palatal shelves, reduction of physiological umbilical hernia, descent of the testes, fusion of the urethral folds and separation of digits of the paws were significantly delayed in the STZ group. The consistent association of placental pathology with fetal growth retardation is suggestive of an alteration in placental function possibly contributing to IUGR in STZ-induced diabetes in rats.