Suzanne Hasthorpe

Abnormalities of testicular descent

Testicular descent occurs in two stages. The transabdominal phase (8–15 weeks) is controlled by enlargement of the caudal genito-inguinal ligament (gubernaculum) and regression of the cranial ligament. Insulin-like 3 from the Leydig cell appears to be the prime stimulator of gubernacular growth, augmented by Müllerian inhibiting substance/anti-Müllerian hormone. Testosterone causes regression of the cranial ligament. The inguinoscrotal phase (25–35 weeks) requires the migration of the gubernaculum from the groin to the scrotum; this migration is guided by the genito-femoral nerve releasing calcitonin gene-related peptide under the influence of androgen. The neonatal gonocyte transforms into a type A spermatogonium at 3–12 months of age, a step that is now known to be crucial for subsequent fertility, as the stem cells for spermatogenesis are created in this structure. This step is blocked in undescended testis and, hence, orchidopexy is currently recommended at 6–12 months of age. Congenital cryptorchidism is caused by the failure of gubernacular migration to the scrotum (1%–2%) but we now recognise that another 1%–2% of boys have acquired cryptorchidism, secondary to the failure of spermatic cord elongation with growth of the boy. These latter cases come to operation at 5–10 years of age. Surgery remains the mainstay of treatment, as hormonal therapy has not been proven to be effective, presumably because testicular descent is a complex anatomical mechanism.

The vagus and recurrent laryngeal nerves in the rodent experimental model of esophageal atresia

BACKGROUND After surgical correction of their esophageal atresia and tracheoesophageal fistula (EA-TEF), many patients exhibit evidence of esophageal dysmotility. Controversy exists as to whether the esophageal motility disorders result from denervation caused by surgery or from an inherent abnormal innervation of the esophagus. METHODS The present study used an Adriamycin-induced EA-TEF fetal rat model to trace the course and branching of both the vagus and recurrent laryngeal nerves. Abnormalities observed in EA-TEF rat fetuses include: (1) fewer branches from both recurrent laryngeal nerves; (2) deviation of the left vagus from its normal course below the aorta, passing behind the fistula to approach and join with the right vagus to form a single nerve trunk on the right side of the esophagus; (3) relatively few branches from the single vagal nerve trunk (composed of fibers of the left and the right vagus) on the surface of the lower esophagus. CONCLUSIONS Fetuses affected by EA-TEF have inherent abnormalities in the course and branching pattern of the vagus nerves as they descend through the thorax, culminating in a deficient extrinsic nerve fiber plexus in the lower esophagus. These observations may account for the esophageal motility disorders seen in patients who have EA-TEF even before surgical intervention.

Histopathologic study of esophageal atresia and tracheoesophageal fistula in an animal model

A histopathologic study of tracheoesophageal anomalies was conducted on an Adriamycin-treated animal model to determine how closely it resembles the human pattern. Adriamycin was administered (2 mg/kg body weight) to timed-pregnant rats on days 6 through 9 of gestation. The fetuses were recovered at term, dissected and prepared for histological studies. Dissection showed a similar range of variants of tracheoesophageal anomalies as seen in humans. Esophageal atresia with distal tracheoesophageal fistula was by far the most common type. Other varieties were seen such as esophageal atresia without a fistula, tracheal atresia and hypoplastic esophagus with atrophic mucosa, and muscle coat. Serial sectioning of the distal segment showed tracheobronchial elements extending to a variable distance from the origin of the fistula.

Relationship between esophageal atresia with tracheoesophageal fistula and vertebral anomalies in mammalian embryos

BACKGROUND/PURPOSE The association of esophageal atresia with tracheoesophageal fistula and vertebral anomalies is well known, although the embryology of the combined defects has not yet been analysed. The present study describes the origin and development of esophageal atresia with tracheoesophageal fistula and vertebral anomalies in embryos using a rat model of VATER association produced by Adriamycin administration. RESULTS The lung buds were seen to develop from the laryngotracheal groove but the trachea failed to grow normally and the foregut overgrowth compensated for this failure. The trachea developed by trachealization of the foregut, which continued as a fistula to the lower esophageal segment. The notochord did not separate from the foregut at the correct time (before day 11 in rat embryos, Carnegie stage 11 in human embryos). Overgrowth of the foregut ventrally and caudally carried with it the attached notochord in the same direction leading to abnormal bending of this structure. CONCLUSION This irregularity of the notochord may be responsible for abnormal development of the vertebrae.

In vitro fusion of human inguinal hernia with associated epithelial transformation.

The processus vaginalis (PV) is a peritoneal diverticulum which forms to allow descent of the fetal testis to the scrotum. During human development fusion and obliteration of the PV often fails to occur with the result that inguinal hernias are the most prevalent congenital abnormality requiring surgery in childhood. Androgen is proposed to regulate testicular descent via the genitofemoral nerve which releases the neuropeptide calcitonin gene-related peptide (CGRP). It is possible that subsequent fusion of the PV and tissue remodelling following descent is indirectly controlled by androgen via CGRP action. An organ culture assay was developed to assess fusion of the PV taken from inguinal herniotomy in infants. Fusion was induced in vitro by CGRP but not by CGRP 8–37, CGRP 27–37 or dihydrotestosterone in equimolar concentrations. Fusion was accompanied by transformation of the epithelium, as shown by staining of intermediate filament proteins, cytokeratin and vimentin. Localization studies for CGRP receptors on 25 specimens indicated CGRP acts on mesenchymal fibroblasts but not directly on PV epithelium suggesting an indirect pathway. Hepatocyte growth factor/scatter factor was found to induce fusion of PV and may be involved as an intermediate molecule in the fusion cascade. This study represents the first approach to understanding the humoral control and underlying mechanism by which the PV fuses.

Clonogenic Culture of Normal Spermatogonia: In Vitro Regulation of Postnatal Germ Cell Proliferation

Abstract The stem cell properties of neonatal germ cells have recently been demonstrated by in vivo transplantation. Regulation of proliferation of these cells, however, is not yet understood, and an in vitro system is needed for directly testing the action of differentiation and proliferation-related factors for germ cells. We developed an in vitro model involving micromanipulation and a single-cell clonogenic assay in which results from independent experiments on spermatogonia and gonocytes have been analyzed and compared. Neonatal germ cells can be distinguished by their large size both in vivo and in vitro in a single-cell suspension. These cells are picked up singly using a micropipette and deposited into a 96-well plate precoated with an extracellular matrix component, e.g., collagen IV. The effect of growth factors or cocultured somatic cells was assayed by counting the percentage of wells containing a colony and comparing this percentage with that of control cultures. Addition of platelet-derived growth factor significantly shifted the modal colony size for gonocytes from >16–64 to >64–128 cells/colony (P < 0.001, χ2) but had no effect on spermatogonia-derived colony size and number. For testis somatic cell underlays, there was a profound inhibition of all colony types, and immunohistochemical staining of testis cell underlays showed inhibin/activinβA subunit expression. This finding suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant recovery (P = 0.046) of gonocyte-derived colony numbers but not spermatogonia-derived colonies, which may reflect the functional regulation by these factors observed in vivo. This proliferation assay also highlights many similarities in the regulation of gonocyte and spermatogonia proliferation in vitro, suggesting that proliferation potential is not noticeably affected by the transition of gonocytes to spermatogonia. For example, the average colony cloning efficiency was 80% for gonocytes and 76% for spermatogonia. This technology forms a basis for optimizing growth of neonatal germ cells for applications such as introduction of genetic material into the germ line to produce transgenic mice and to explore gene therapy.

Apoptosis in tracheoesophageal embryogenesis in rat embryos with or without adriamycin treatment.

PURPOSE The aim of this study was to determine whether apoptosis participates in separation of the foregut into trachea and esophagus and to evaluate the potential role of apoptosis in the development of esophageal atresia and tracheoesophageal fistula (EA + TEF) induced by Adriamycin. METHODS Timed-pregnant rats were injected daily with either saline or Adriamycin (2 mg/kg) intraperitoneally on days 6 to 9 of gestation. Paraffin sections were prepared from 31 experimental and 31 control embryos at days 12 and 13 of gestation. Condensed nuclei were identified on the paraffin sections using the TUNEL method. Apoptosis was quantified by counting the positively stained cell nuclei in transverse sections of embryos. RESULTS In day 12 control embryos the number of apoptotic nuclei in both lateral ridges of the foregut was high (15.67 +/- 1.38) but relatively low (4.17 +/- 0.80) in Adriamycin-treated embryos (P< .0001). In day 13 Adriamycin-treated embryos, the number of apoptotic nuclei in the region of the upper esophageal pouch was extremely high (23.78.5 +/- 2.20) compared with no detectable apoptotic nuclei in the control embryos. CONCLUSIONS Apoptosis is required for normal tracheoesophageal embryogenesis and may be an important mechanism to be involved in the embryological development of esophageal atresia and tracheoesophageal fistula.

Visceral anomalies in prenatally adriamycin-exposed rat fetuses : a model for the VATER association

Abstract Adriamycin is teratogenic if given to pregnant rats. A wide range of anomalies involving the gastrointestinal, renal, and cardiovascular systems has been described, similar to the VATER association, yet it is not known if they are identical to the human pattern. The aim of this study was to document the visceral anomalies in rat fetuses exposed to adriamycin and to determine their similarities with the congenital defects in humans with the VATER association. The results revealed a spectrum of very similar anomalies. Furthermore, the characteristics of the tracheo-oesophageal anomalies had a lot of features in common with the human pattern. We conclude that the adriamycin-treated fetal rat is an excellent model for studying the VATER association.

Timing and embryology of esophageal atresia and tracheo-esophageal fistula.

The embryology of tracheo‐esophageal anomalies is controversial. The development of an adriamycin‐treated animal model has enabled improved understanding of the embryogenesis of these anomalies. Using this model, we aimed to describe the events leading to esophageal atresia and tracheo‐esophageal fistula.

Cardiovascular malformations in rat fetuses with oesophageal atresia and tracheo-oesophageal fistula induced by adriamycin

Associated congenital anomalies have emerged as the most significant prognostic factor in babies born with oesophageal atresia and/or tracheo-oesophageal fistula (OA-TOF). The most frequently encountered groups of anomalies are cardiovascular (CV) and gastrointestinal, the former being more significant from a prognostic point of view. Some, such as a right-sided aortic arch (RAA), vascular ring, or major heart defects, may alter the timing and surgical approach for the repair of OA-TOF or adversely affect the prognosis. The rat fetal OA model induced by adriamycin (Adr) has been described previously. In the present experiments, information was sought regarding the incidence and type of CV abnormalities in fetal rats obtained from Adr-treated dams. OA-TOF was induced in 24 of 36 fetal rats from Adr-treated dams. DV abnormalities were found in 18 (75%) OA-TOF fetuses and 10 (83%) Adr-treated fetuses without OA-TOF. The difference was not significant (P > 0.05). The most frequently found anomalies were ventricular and atrial septal defects. A RAA was present in 8/36 fetuses and a double aortic arch in 2/36. A patent ductus arteriosus was present in all treated fetuses compared with two-thirds of controls. The findings in the present study emphasise the importance of CV anomalies in association with OA, and reinforce the value of the Adr-induced rat fetal OA model by adding to our knowledge of the basic embryogenesis of both OA and CV anomalies.