Exposure of male Albino Swiss rats to the nonsteroidal antiandrogen flutamide during the period from gestational day (d) 10 to birth resulted in feminisation of the external genitalia and the suppression of growth of the male reproductive tract. In adulthood, testes were found to be located in diverse positions. True cryptorchidism occurred in 10% of cases, whereas 50% of testes descended to the scrotum and 40% were located in a suprainguinal ectopic region. Varying degrees of tubule abnormality were seen in the testes of flutamide-treated animals, ranging from completely normal tubules with full spermatogenesis (and the expected frequency of the stages of spermatogenesis) to severely abnormal tubules lined with Sertoli cells only. For each individual testis, the overall severity of tubule damage was strongly correlated with its adult location, with intra-abdominal testes worst affected and scrotally-located testes least; only the latter contained normal tubules. Similarly, intra-abdominal testes were the smallest in weight and contained the least testosterone. By contrast, postnatal treatment of male rats with flutamide from birth to postnatal d 14 did not impair development of the external genitalia, the process of testicular descent or adult spermatogenesis. These findings confirm that androgen blockade during embryonic development interferes with testicular descent but also demonstrate that (1) prenatal flutamide treatment per se has a detrimental effect on adult testis morphology but (2) the degree of abnormality of the testes is strongly influenced by location.

During the 20th century, the human vomeronasal organ (VNO) has been controversial regarding its structure, function, and even identity. Despite reports that provide evidence for its presence throughout prenatal and postnatal ontogeny, some studies and numerous textbooks declare its absence in late fetal and postnatal humans. To that end, the present study was designed to establish firmly whether the human VNO is homologous with that of other mammals and whether it degenerates (partially or completely) or persists throughout prenatal development. Fifty human embryos and fetuses (33 d to 32 wk fertilisation age) and 2 neonates were examined by light microscopy. Four embryonic primates (mouse lemurs) were examined for a comparison of VNO embryogenesis. The presence or absence and structural characteristics of the VNO and supporting tissues are described. The first appearance of the VNO was in the form of bilateral epithelial thickenings of the nasal septum, the vomeronasal primordium. The primordia invaginated between 37 and 43 d of age and formed the tubular VNO. The tubular VNO was located dorsally at a variable distance from, but was always spatially separated from the paraseptal cartilages. The mouse lemurs examined in this study and other reports from the literature indicate that the human VNO resembles that of primates having functional VNOs until just after a tubular VNO is formed. Examination of the VNO and adjacent tissues suggested that the VNO may lose receptor cells and corresponding vomeronasal nerves and become a ciliated, pseudostratified epithelium between ∼ 12 and 14 wk of age. Our findings indicate the prenatal human VNO goes through 3 successive stages: early morphogenesis, transformation (of the epithelium), and growth. These observations indicated that (1) all embryonic humans develop a vomeronasal organ which is homologous with the VNOs of other mammals, but which has become displaced and highly variable in relative location during embryogenesis; (2) the human vomeronasal organ does not degenerate prenatally, but very likely loses the functional components of the vomeronasal complex of other mammals; and (3) the remnant of the human VNO persists until birth and beyond.

Mutations in the gene for peripheral myelin protein 22 (PMP22) are associated with peripheral neuropathy in mice and humans. Although PMP22 is strongly expressed in peripheral nerves and is localised largely to the myelin sheath, a dual role has been suggested as 2 differentially expressed promoters have been found. In this study we compared the initial stages of postnatal development in transgenic mouse models which have, in addition to the murine pmp22 gene, 7 (C22) and 4 (C61) copies of the human PMP22 gene and in homozygous and heterozygous Trembler-J (TrJ) mice, which have a point mutation in the pmp22 gene. The number of axons that were singly ensheathed by Schwann cells was the same in all groups indicating that PMP22 does not function in the initial ensheathment and separation of axons. At both P4 and P12 all mutants had an increased proportion of fibres that were incompletely surrounded by Schwann cell cytoplasm indicating that this step is disrupted in PMP22 mutants. C22 and homozygous TrJ animals could be distinguished by differences in the Schwann cell morphology at the initiation of myelination. In homozygous TrJ animals the Schwann cell cytoplasm had failed to make a full turn around the axon whereas in the C22 strain most fibres had formed a mesaxon. It is concluded that PMP22 functions in the initiation of myelination and probably involves the ensheathment of the axon by the Schwann cell, and the extension of this cell along the axon. Abnormalities may result from a failure of differentiation but more probably from defective interactions between the axon and the Schwann cell.

Vertebrate somitogenesis has been shown to be associated with a molecular oscillator, the segmentation clock, whose periodicity matches that of the process of somitogenesis. The existence of such a clock in presomitic mesoderm (PSM) cells was originally proposed in theoretical models such as the ‘clock and wavefront’. Molecular evidence for the existence of this clock in vertebrates has been obtained on the basis of the periodic expression of several genes, most of which are related to the Notch signalling pathway. These genes are expressed in a dynamic sequence which appears as a wave sweeping caudo-rostrally along the whole PSM once during each somite formation. Notch-pathway mouse and fish mutants lose the dynamic expression of the cycling genes, indicating that Notch signalling is required for their periodic expression, or is required to coordinate the oscillations between PSM cells. Therefore Notch signalling is either part of the mechanism of the oscillator itself or acts as a cofactor required for cycling gene expression. A further potentially important role for the segmentation clock is to periodically activate Notch signalling in the rostral presomitic mesoderm, thereby generating the periodic formation of somite boundaries.

Facet capsule injury has been hypothesised as a mechanism for neck pain. While qualitative studies have demonstrated the proximity of neck muscles to the cervical facet capsule, the magnitude of their forces remains unknown owing to a lack of quantitative muscle geometry. In this study, histological techniques were employed to quantify muscle insertions on the human cervical facet capsule. Computerised image analysis of slides stained with Masson's trichrome was performed to characterise the geometry of the cervical facet capsule and determine the total insertion area of muscle fibres into the facet capsule for the C4–C5 and C5–C6 joints. Muscle insertions were found to cover 22·4±9·6 % of the capsule area for these cervical levels, corresponding to a mean muscle insertion area of 47·6±21·8 mm2. The magnitude of loading to the cervical facet capsule due to eccentric muscle contraction is estimated to be as high as 51 N. When taken in conjunction with the forces acting on the capsular ligament due to vertebral motions, these forces can be as high as 66 N. In that regard, these anatomical data provide quantitative evidence of substantial muscle insertions into the cervical facet capsular ligament and provide a possible mechanism for injury to this ligament and the facet joint as a whole.

Development of the neural crest involves a remarkable feat of coordinated cell migration in which cells detach from the neural tube, take varying routes of migration through the embryonic tissues and then differentiate at the end of their journey to participate in the formation of a number of organ systems. In general, neural crest cells appear to migrate without the guidance of long-range physical or chemical cues, but rather they respond to heterogeneity in the extracellular matrix that forms their migration substrate. Molecules such as fibronectin and laminin act as permissive substrate components, encouraging neural crest cell attachment and spreading, whereas chondroitin sulphate proteoglycans are nonpermissive for migration. A balance between permissive and nonpermissive substrate components seems to be necessary to ensure successful migration, as indicated by a number of studies in mouse mutant systems where nonpermissive molecules are over-expressed, leading to inhibition of neural crest migration. The neural crest expresses cell surface receptors that permit interaction with the extracellular matrix and may also modify the matrix by secretion of proteases. Thus the principles that govern the complex migration of neural crest cells are beginning to emerge.

The amount/distribution of liver melanin in 3 amphibian species (Rana esculenta, Triturus a. apuanus, Triturus carnifex) was studied during 2 periods of the annual cycle (summer activity–winter hibernation) by light and electron microscopy, image analysis and microspectrofluorometry. The increase in liver pigmentation (melanin content) during winter appeared to be correlated with morphological and functional modifications in the hepatocytes, which at this period were characterised by a decrease in metabolic activity. These findings were interpreted according to the functional role (e.g. phagocytosis, cytotoxic substance inactivation) played by the pigment cell component in the general physiology of the heterothermic vertebrate liver and, in particular, in relation to a compensatory engagement of these cells against hepatocellular hypoactivity during the winter period.

The treefrog Eleutherodactylus coqui is a direct developer — it has no tadpole stage. The limb buds develop earlier than in metamorphosing species (indirect developers, such as Xenopus laevis). Previous molecular studies suggest that at least some mechanisms of limb development in E. coqui are similar to those of other vertebrates and we wished to see how limb morphogenesis in this species compares with that in other vertebrates. We found that the hind limb buds are larger and more advanced than the forelimbs at all stages examined, thus differing from the typical amniote pattern. The limb buds were also small compared to those in the chick. Scanning and transmission electron microscopy showed that although the apical ectoderm is thickened, there was no apical ectodermal ridge (AER). In addition, the limb buds lacked the dorsoventral flattening seen in many amniotes. These findings could suggest a mechanical function for the AER in maintaining dorsoventral flattening, although not all data are consistent with this view. Removal of distal ectoderm from E. coqui hindlimb buds does not stop outgrowth, although it does produce anterior defects in the skeletal pattern. The defects are less severe when the excisions are performed earlier. These results contrast with the chick, in which AER excision leads to loss of distal structures. We suggest that an AER was present in the common ancestor of anurans and amniotes and has been lost in at least some direct developers including E. coqui.

The pectoral girdle articulates the forelimb with the axial skeleton in all vertebrates with paired anterior appendages. The structure of the pectoral girdle and its position along the axial skeleton has changed significantly during vertebrate evolution. These morphological changes have been well described, but there is little comparative embryology to indicate how these changes may have occurred. It is equally obscure how the muscles that connect the head with the pectoral girdle have maintained appropriate attachments even though these 2 structures have become separated. Here I review the changes in the pectoral girdle across different vertebrate taxa, indicating, where known, the developmental mechanisms underlying these changes. I also suggest how the muscular connections between the head and pectoral girdle have been maintained between these once adjacent bones, displaced during vertebrate evolution.

The anatomy of the lymphoid organs was studied during the course of detailed dissections of 50 beach-stranded bottlenose dolphins, Tursiops truncatus. Constant lymph nodes occur in 4 groups, based on their location and structure. These groups are somatic, including nodes of the cervical region and pelvic recess; lung-associated, included marginal, diaphragmatic and hilar nodes; visceral, including the mesenteric, pancreatic, pericolic and porta hepatis nodes; and aortic arch nodes. Lymphatic drainage of the lung is primarily to the marginal and diaphragmatic nodes. The mesenteric node mass is well-endowed with capsular and trabecular smooth muscle, and a network of muscle fascicles within the organ implies an important contractile function in the circulation of lymph. In addition to constant nodes, occasionally nodes are found in relation to the thoracic aorta, the kidney, and under the scapula. Gut-associated structures include dorsal and ventral oropharyngeal tonsils, mucosal aggregates in the straight segment of the intestine (colon) and anal tonsils; this gut-associated lymphoid tissue tends to involute with age, being greatly reduced by puberty. Formed lymphoid organs include the thymus and the spleen, the latter being relatively small in relation to body size. None of these structures is unique among cetaceans, but the anal tonsils are particularly well developed in T. truncatus. The lymphoid aggregates in the colon resemble the arrangement in the vermiform appendix, which is lacking in most cetaceans, and may have functions analogous to that organ.

The variations which occur in the medial and lateral ligament complexes of the elbow were investigated. These occurred frequently with the standard appearances occurring in no more than half the specimens on the medial side and one quarter of those on the lateral side. Surgeons who regularly perform elbow arthroplasty must be aware of these considerations, especially with the introduction of unconstrained prostheses which rely upon the ligament complex for their postoperative stability.

It is widely accepted that Meckel's cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckel's cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckel's cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckel's cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckel's cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral-type mechanism of calcification.

Double-label immunocytochemistry was used to investigate the colocalisation of various neuropeptides and the enzymes nitric oxide synthase (NOS) and tyrosine hydroxylase (TH) in intramural ganglia of the human male urinary bladder neck and trigone. Postmortem specimens were obtained from 7 male infants and children ranging in age from 2 mo to 3 y who had died as a result of cot death or accidental trauma. On average 60% of the intramural neurons were non-TH-immunoreactive (-IR) (i.e. presumptive cholinergic) and 40% were TH- and DbβH-IR (i.e. noradrenergic). Within the non-TH-IR population, calcitonin gene-related peptide (CGRP) was found in 65% of cells, neuropeptide Y (NPY) in 90%, nitric oxide synthase (NOS) in 45%, somatostatin (SOM) in 90%, and vasoactive intestinal polypeptide (VIP) in 40%. The corresponding values for the TH-IR neurons were CGRP (54%), NPY (70%), NOS (58%), SOM (73%) and VIP (40%). All the observed bombesin (BOM)-immunoreactivity was colocalised with TH while 90% of VIP and almost all the CGRP was colocalised with NPY. Less than 5% of neurons were immunoreactive for substance P (SP) or met-enkephalin (m-ENK) and some of these also contained TH. Varicose nerve fibres were seen in close proximity to some of the intramural neurons, the majority of such varicosities showing immunoreactivity to CGRP, VIP or TH. Less common were pericellular varicosities immunoreactive to NPY, SOM or SP. These results demonstrate the neurochemical heterogeneity of intramural neurons in the human bladder neck and provide indirect evidence for the complexity of the peripheral innervation of the human urinary bladder.

Na+/H+ exchange has been implicated in models of ion transport across the branchial epithelium of marine and freshwater fishes. In this preliminary study, we present immunohistochemical data using a polyclonal antibody raised against NHE3 which show NHE3-like immunoreactivity (IR) in the gills from a freshwater and a marine teleost species. In both species, branchial epithelial cells demonstrating NHE3-like IR were localised predominantly to the junction between the filament and the secondary lamellae. However, there was a marked difference in the morphology of the NHE3-like immunoreactive epithelial cells between the species. This morphological difference between the species suggests functional differences in the exchanger, which may be related to marine versus freshwater environments.

We have re-evaluated, using the anterograde tracer biocytin, supraspinal efferent projections from the parabrachial complex (PBN) to gain new information about the nature of its connections and nerve terminal patterns. We selectively injected biocytin into the 3 main regions of the nucleus (lateral PBN, medial PBN and Kölliker-Fuse nucleus). We observed distinct groups of ascending and descending fibres of different calibre from the PBN running throughout the brain and reaching many brain areas involved in the regulation of autonomic function. Here we detected labelled bouton-like terminals and fibres with en-passage varicosities. The ascending efferents from the lateral PBN mainly reached the reticular, raphe and thalamic nuclei, the zona incerta (ZI), central nucleus of the amygdala (CeA) and lateral area of the periaqueductal grey (PAG). Thin descending efferents reached the ventral region of the solitary tract nucleus (STN). The ascending efferents from the medial PBN were seen in the raphe nuclei, reticular nuclei, ventral and lateral areas of the PAG, thalamic nuclei, and in the medial and lateral nuclei of the amygdala. Descending efferents were seen in the STN and in some reticular nuclei. The ascending projections from the Kölliker-Fuse targeted the ventral area of PAG, CeA, ZI, lateral hypothalamic area, ventromedial thalamic nucleus and, with only a few terminals, the ipsi and contralateral reticular area. A large number of descending efferents reached STN, caudal and paragigantocellular reticular nuclei. The higher sensitivity of biocytin compared with other types of markers allowed us to determine more effectively the distribution, nature and extent of the supraspinal PBN connections. This suggested that in several nerve circuits the PBN probably plays a more important role than previously thought.

Tibial torsion, twisting of the tibia about its longitudinal axis, varies during development and early childhood. Knowledge of the normal range of tibial torsion at various ages and its accurate clinical measurement is important in the assessment of the extent of a torsional deformity. To evaluate tibial torsion a reliable technique for its measurement in vivo is therefore required. The aim of this study was to determine which of 4 existing in vivo methods of measuring tibial torsion was the most accurate and had the highest repeatability, by comparing them with direct measurement of the tibia. A wide range of mean values for tibial torsion was observed, using the various techniques, with none of the indirect techniques employed having a strong correlation with direct measurement of tibial torsion. The repeatability of the indirect techniques was observed to be low both in cadavers (n=4) and the living (n=3). Since none of the in vivo techniques appear to measure true tibial torsion or be of a reasonable repeatability, alternative easy to use and inexpensive methods need to be developed. Accurate clinical measurement of tibial torsion is important in the assessment of the extent of a torsional deformity. It is recommended that data gained using the methods reviewed here are interpreted with caution.

Connexin 43 (Cx43) has been reported to be expressed in vascular smooth muscle cells and endothelial cells. Evidence for possible variations in Cx43 distribution within different parts of the vascular system is limited. We have therefore investigated the expression of Cx43 in the endothelia and media of 11 vessels of different size and function in the rat, using immunofluorescence and confocal laser scanning microscopy. The results showed that punctate Cx43 staining was abundant in the endothelia and media of all of the 5 elastic arteries examined. In the media, the amount of Cx43 staining decreased as the size of the elastic arteries became smaller. In the 6 muscular arteries examined, 2 different patterns of Cx43 staining were observed. In the first type, Cx43 expression was high in the endothelium but virtually absent from the media. Mesenteric resistance, hepatic and tail arteries were examples. In the second type, Cx43 staining was absent from both the media and the endothelia. The coronary, basilar, and middle cerebral arteries showed this appearance. The results suggest that expression of Cx43 is largely restricted to elastic arteries in the arterial system of the rat. The lack of immunodetectable Cx43 from the media of all muscular arteries examined, and from the endothelia of some of these arteries, raises the possibility of significant differences in the form of expression of Cx43 in these vessels or the presence of other connexins.

The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI-1 and PAI-2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI-1 and PAI-2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohr's and Nitabuch's striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI-2 was noted in villous trophoblast whereas tPA and PAI-1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI-1 and PAI-2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI-1 and PAI-2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI-1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI-2 appears mainly to play a role in degradation of trophoblast cell-associated extracellular matrix, and thus may be of greatest importance during early stages of placentation.

Traumatic avulsions of ligament or tendon insertions rarely occur at the actual interface with bone, which suggests that this attachment is strong or otherwise protected from injury by the structure of the insertion complex. In this study we describe the terminal extent of quadriceps tendon fibres where they insert into the patellae of adult rabbits, humans, dogs and sheep. Specimens were examined by scanning electron microscopy (SEM) and light microscopy (LM). To facilitate tracing of tendon fibres the specimens were decalcified for SEM, and polarised light microscopy (PLM) was used in the LM segment of the study. By SEM it was possible to identify mature bone by the presence of osteocytes and a lamellar organisation. PLM and SEM showed that, unlike tendon fibres elsewhere, those in the calcified fibrocartilage were not crimped. No specific cement line was identified by SEM. Tendon fibres interdigitated among separate bone lamellar systems, (osteons or marrow spaces), but did not merge with the collagen systems of individual lamellae. The interdigitation was more extensive and the margin between tendon and bone was less distinct in the anterior third of the insertion. The segment of calcified tendon which interdigitated with bone stained less intensely blue and was less cellular than the more proximal calcified fibrocartilage zone adjacent to the tidemark. Lamellar collagen fibres of the bony trabeculae in the anterior patella were unusually parallel and longitudinal in orientation, making distinction of interposed tendon fibres difficult on LM and PLM sections. LM, SEM and transmission electron microscopy of rabbit patellae at birth revealed that anterior quadriceps tendon fibres extended over the patella in a fibrous cellular layer. By 2 wk of age, this layer had acquired chondroid features (i.e. cell lacunae and metachromasia) and contained vessels extending from patellar marrow. At 6 wk of age, part of this fibrocartilaginous layer was replaced by mature bone and osteoid. In the young adult animal, the quadriceps tension interdigitates extensively with the patellar bone. This segment of the insertion is perhaps the remnant of calcified fibrocartilage which has been remodelled by bone formation.

Following the discovery that the proto-oncogene protein kinase B (PKB) functions as a downstream element in signalling from phosphoinositide 3′-kinase (PI3-kinase) (Burgering & Coffer, 1995), PKB has been shown to mediate a diverse array of PI3-kinase dependent cellular responses. Most recently PKB-dependent phosphorylation of 3 members of the family of Forkhead transcription factors has been demonstrated to play a role in PI3-kinase dependent effects on transcription. This review focuses on this newly discovered function of PKB in conveying the diversity of PI3-kinase dependent cellular responses.