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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.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The age distribution for presentation of 64 inguinal hernias in children had a biphasic distribution with cases initially occurring from birth to 4 months, then a lag and thereafter the incidence rose after 1 year of age. Males constituted 51/64 of the cases. A neuropeptide, calcitonin gene related peptide (CGRP), that is present in the genitofemoral nerve (GFN) which innervates the processus vaginalis was found to induce fusion of the hernial sac tissue. In vitro organ cultures of human processus vaginalis were used to assay for hernial fusion and to study the morphological changes associated with this process. The processus vaginalis was folded so that the surfaces lining the sac were lying next to each other. The length of fused surface along this fold line was assayed by microscopy and quantitated for each specimen that fulfilled the criteria of healthy intact tissue. The average length of fusion for children ≤ 4 months of age was 67.7% for CGRP with a background of 49.4% fusion when tissue was incubated with phosphate buffered saline (PBS) addition to the medium. In children over 4 months the degree of fusion was less with 31.7% and 23.3% for CGRP and PBS, respectively. The mean difference between CGRP and PBS groups was 17.5% for children ≤ 4 months and 4.5% for older children with the difference being significantly different (p=0.0018) for the younger group only. The morphological changes of the fused processus vaginalis tissue resembled transformation from an epithelial to mesenchymal cell type. The cells lining the processus vaginalis expressed cytokeratin and appeared to be a differentiated epithelial cell type. Transformation of this type is a common mechanism of tissue remodelling in embryological development. A growth factor often associated with such embryological changes is hepatocyte growth factor/scatter factor (HGF/SF) and this factor was also found to induce fusion of the processus vaginalis. In hernial tissue from children of ≤ 4 months of age HGF/SF induced a significant degree of fusion (p=0.038) as it did in older cases (p=0.015). This in vitro model approach to the understanding of inguinal hernia closure could lead to a nonsurgical treatment of this condition. [ABSTRACT FROM AUTHOR]

Cell cycle progression is prevented by signal transduction pathways known as checkpoints which are activated in response to replication interference and DNA damage. We cloned a G2/M cell cycle phase-related checkpoint gene from a neonatal mouse testis cDNA library which was identified as mouse claspin, a proposed adaptor protein for Chk1. As part of a study on germ cell differentiation we examined the expression of the checkpoint gene, Chk1, and claspin at 12.5 and 14.5 days post coitum (dpc) and in the post-natal phase. Chk1 mRNA expression increased from 12.5 to 14.5 dpc in female gonads and was strong in males at both time points. Claspin however, was not detected until 14.5 dpc. This suggests there may be some dissociation of claspin expression from Chk1 in fetal germ cell development. Chk1 and claspin expression was also studied in testis over the first 3 days following birth, when apoptosis regulates germ stem cell number. We modulated checkpoint-related gene expression in testis using the anti-metabolite, 5-fluorouracil, resulting in increased apoptosis and upregulation of Chk1 (P<0.0001) and Cdc2 (P<0.02) mRNA. Although we do not fully understand the role checkpoint gene expression has during mammalian germ cell development this report is the first to show the expression of checkpoint-related genes in early mammalian germ cells., (Copyright (c) 2007 Wiley-Liss, Inc.)

Background: Calcitonin gene-related peptide (CGRP) is proposed to indirectly cause inguinal hernia closure via hepatocyte growth factor (HGF). Studies have shown that CGRP and HGF cause processus vaginalis (PV) fusion in vitro. We localized the HGF receptor in the PV and tested whether CGRP was responsible for HGF release., Method: Hernial sacs collected from 20 children (15 males, 4 females, 1 XY female) undergoing inguinal hernia repair were immunohistochemically stained for HGF receptor (c-met). Parietal peritoneum was stained for comparison. Hernial sacs from another 16 children (12 males, 4 females), with each sac divided into 4, were cultured, with and without CGRP, for 24 and 48 hours. Hepatocyte growth factor content was then assayed in the culture medium (4/16 children) and tissue extracts (12/16 children), using enzyme-linked immunosorbent assay. Children were aged 1 month to 10 years. Data were analyzed using paired Student t tests., Results: C-met localized to the PV epithelial surface in 17 of 20 hernial sacs and in the parietal peritoneum. Hepatocyte growth factor levels increased over time in 4 of 4 culture medium assays, with a significant difference in 1 of 4. Seven of 12 tissue extract assays had significant differences; however, 3 of 7 had decreased HGF levels., Conclusion: The presence of HGF receptors in the PV is consistent with a role for HGF in triggering epithelial-mesenchymal transformation during inguinal hernia closure. The presence of HGF receptors in the parietal peritoneum suggests that regulation of this process is complex. Enzyme-linked immunosorbent assay results indicate that, in a subset of patients, exogenous CGRP may be responsible for HGF elevation and potentially implicates deficient endogenous CGRP as one cause for inguinal hernia patency.

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 understanding of testicular descent has changed much in the 20 years since the authors' laboratory began studying the mechanism. The process is now known to occur in 2 steps with different anatomy and hormonal regulation but with many still unresolved controversies. Recent advances include the recognition of acquired cryptorchidism of critical early postnatal germ cell development and the recommendation for surgery at 6 months of age. The authors still await long-term outcome studies.

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, chi2) 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/activinbetaA 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.

Background/purpose: Spermatogenesis in postnatal testes is controlled by the hypothalamic-pituitary-gonadal axis. To determine if pituitary hormones can induce precocious spermatogenesis once primary spermatocytes (PS) have formed, prepubertal mice were treated with human chorionic gonadotrophin (hCG)., Methods: Day 12 immature mice (n = 10) were injected every third day with hCG (3 or 6 IU) dissolved in 100 microL phosphate-buffered saline (PBS). Control mice (n = 10) were either uninjected or injected with 100 microL PBS alone. On day 20 to 22 the excised testes were examined histologically with tubule counts., Results: HCG-treated mice had fewer tubules at stage I (P <.001) and more at stage III than the PBS-treated group (P <.001). Mean thickness of the round spermatid layer per tubular cross section in the hCG-treated group was significantly increased compared with the PBS-treated group (P <.01). Similarly, the percentage of the tubules at stage III (containing round spermatids) in the hCG-treated group was significantly increased, from 25% to 71%, compared with the PBS-treated group (P <.01). With increasing doses of hCG the testosterone levels were significantly higher than in controls (P <.01), but hCG did not alter testis weight or position., Conclusions: These results show that hCG stimulates the transformation of PS to round spermatids even in immature mouse testes. These findings suggest that hCG treatment of prepubertal cryptorchid boys may initiate premature spermatogenesis., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

We have cloned and characterized a novel murine DNA-binding protein Desrt, with a motif characteristic of the ARID (A-T rich interaction domain) family of transcription factors. The Desrt gene encodes an 83-kD protein that is shown to bind DNA and is widely expressed in adult tissues. To examine the in vivo function of Desrt, we have generated mice with a targeted mutation in the ARID domain of Desrt. Homozygous mutants have reduced viability, pronounced growth retardation, and a high incidence of abnormalities of the female and male reproductive organs including cryptorchidism. This may thus serve as a model to dissect the mechanisms involved in the development of the reproductive tract including testicular descent. Gene-targeted mice also display a reduction in the thickness of the zona reticularis of the adrenal gland and transient aberrations of the T and B cell compartments of primary lymphoid organs. These data show that this novel DNA-binding protein, Desrt, has a nonredundant function during growth and in the development of the reproductive system.

Three rat strains have been studied, using a sensitive apoptotic detection method for germ-cell degeneration, to resolve the controversy regarding the effect of cryptorchidism on the contralateral descended testis (CDT). Sprague Dawley and Buffalo rats were made cryptorchid by operation at 20-22 days of age, while trans-scrotal (T-S) rats were a congenitally unilateral cryptorchid strain. Sham operated rats or normal T-S littermates were used as controls. Experiments were performed over a period ranging from 2 weeks to 18 months. Testis weight was assayed, as was the detection of apoptosis by agarose gel laddering and immunohistochemistry by using the TUNEL method. Labeled cells in 150 cross-sectioned testis tubules were counted on the TUNEL stained slides and the mean number of labeled cells per tubule was calculated. Paternity studies on Sprague Dawley and T-S rats were carried out at 12 and 24 weeks of age to assess fertility by the resultant number of pregnancies and litter sizes. Both Sprague Dawley and T-S rat models showed a biphasic distribution of apoptosis levels. This biphasic distribution was not observed in Buffalo rats as they were only studied at later time points (12-20 weeks). A significant effect on either testis weight or apoptosis in the CDT compared with the control descended testis (P > or = 0.1) has not been found in these three cryptorchid models, and the present results are discussed with reference to observations of other researchers in rodents and humans. While the cryptorchid testis showed a high level of labeled apoptotic cells per tubule in all rat strains, fertility was not affected and remained the same as controls at 12 and 24 weeks. There was, however, a marked strain difference in fertility in T-S as compared with Sprague Dawley rats. After 24 weeks of cryptorchidism, both control and cryptorchid T-S rats had a 44% pregnancy incidence compared with a 90% pregnancy incidence in Sprague Dawley rats. In addition, litter size in T-S control and cryptorchid rats were small compared with those of Sprague Dawley rats at 12 and 24 weeks.

At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture. Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro. A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003). Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies. The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01). Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes. Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone. However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation. Evidence for the expression of the Müllerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive. This finding was in agreement with the lack of a direct action of Müllerian inhibiting substance on the formation of gonocyte-derived colonies in vitro. Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation. An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.

Background/purpose: Recent evidence has suggested that calcitonin gene-related peptide (CGRP), which is released from the genitofemoral nerve, may trigger fusion of the patent processus vaginalis in children with inguinal hernia. The purpose of this study was to determine whether CGRP triggers the release of mesenchymal factors leading to subsequent fusion of the processus vaginalis., Methods: The response of cultured epithelial cells derived from the patent processus vaginalis was analysed by a novel in vitro culture system. Epithelial cells lining fresh hernial sacs (removed at inguinal herniotomy) were detached enzymatically and cultured for 72 hours on Micropore filters, in the presence of either 100 ng/mL hepatocyte growth factor (HGF), 7.4 x 10(-6) mol/L CGRP (amino acids 1 to 37), 7.4 x 10(-6) mol/L CGRP (8 to 37) antagonist, 10% fetal calf serum (FCS), or serum-free medium (SFM) alone. Transformation from an epithelial cell morphology to motile mesenchymal fibroblast-like cells was assessed by an average migration score (AMS), ranging from 0 with no sign of migration, to 3 with greater than 75% of cells migrating. Confocal microscopy was used to record changes in expression of epithelial (cytokeratin) and mesenchymal (vimentin) markers, as well as actin. Beta-catenin also was examined because it is part of the molecular complex that links cadherins to actin resulting in cell-cell adhesion., Results: Epithelial and mesenchymal markers underwent either down-regulation or up-regulation as epithelial cell sheets broke apart and individual cells commenced migration. The AMS after 72 hours of culture was 0.22 with SFM (control); with FCS the score was 1.4 (P < .01). The AMS score with CGRP (1 to 37) was 0.55 (P = .165) and with its analogue, CGRP (8 to 37), which is a competitive inhibitor, 0.67 (P = .309). Neither was significant. HGF caused a significant increase in the AMS to 1.56 (P = .01)., Conclusion: Both HGF and FCS (which contains various undefined peptides and growth factors) produced transformation of hernial sac epithelial cells, whereas CGRP and its inactive analogue did not. CGRP receptors are localised to mesenchymal fibroblasts within the processus vaginalis connective tissue, suggesting that CGRP could act indirectly via HGF, which, in turn, promotes fusion of the processus vaginalis. In the future, a nonsurgical treatment of inguinal herniae in children might be possible by the local administration of agents which promote fusion.

Survival and proliferation of mouse gonocytes was studied using a single cell clonogenic assay in vitro. The effect of growth factors and extracellular matrix on clonogenic development was quantitated. Fundamental requirements for growth of neonatal gonocytes included addition of fetal calf serum and coating culture wells with collagen IV alone or with added fibronectin. After 4-5 days, colonies ranged in size from four to > 128 cells, and some contained very elongated cells indicating migratory behaviour. Soluble stem cell factor did not have any effect on clonogenicity, although STO (subline of SIM mouse fibroblasts) cells, which produce membrane-bound stem cell factor, reduced colony formation from 79 +/- 5.9% to 20 +/- 3.3% without added growth factor. The majority of gonocytes and type A spermatogonia express the c-kit receptor according to in situ hybridization studies. However, the results indicate that the receptor may not be functional in neonatal gonocytes and their immediate progeny. The current assay for gonocytes can be extended to test new growth factors or proliferation-inducing agents directly, as well as to study cell-cell interactions. This assay and long-term propagation of neonatal germ cells will provide the much needed resources to enable greater understanding of the early development of germ cells.

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.

Ets-1 is a transcription factor with restricted expression in lymphocytes, and it has been implicated in the regulation of T cell genes such as TCR alpha, TCR beta, CD4, IL-2, and TNF-alpha. We show in this study that Ets-1 is also expressed in some mast cells constitutively and can be induced in primary mast cells with stimuli that activate mast cells. We also show that Ets-1 plays a role in the regulation of granulocyte-macrophage CSF (GM-CSF), a cytokine expressed by activated mast cells. We have characterized a murine growth factor-independent mast cell line, FMP6-, derived from a factor-dependent cell line, FMP1.6. FMP6- has acquired a distinct connective tissue mast cell-like phenotype, as characterized by the expression of mast cell proteases MMCP-4 and MMCP-6, expression of IL-12, and the down-regulation of IL-4. The parental FMP1.6 cell line displays a mucosal mast cell-like phenotype. FMP6- cells have increased Ets-1 expression and achieve growth-factor independence by the autocrine production of GM-CSF and IL-3. Transient transfection of an Ets-1 expression construct in FMP6- cells results in transactivation of a GM-CSF reporter, while a point mutation in the consensus Ets binding site in the conserved lymphokine element, CLE0, abolishes Ets-1 transactivation. Importantly, antisense Ets-1 demonstrates an ability to repress the activity of the GM-CSF reporter. These data suggest a role for Ets-1 in mast cell growth regulation and activation, and because of the central role of mast cells in inflammatory processes, such as asthma and rheumatoid arthritis, they identify Ets-1 as potentially contributing to the pathophysiology of such diseases.

Objectives: To determine whether cryptorchidism is a congenital malformation and whether the impaired spermatogenesis in immature testes can be reversed by early orchidopexy, using the mutant trans-scrotal (T-S) rat which is normally masculinized but has cryptorchidism in 85% of males., Materials and Methods: First, T-S rats (six per group) with ectopic testes destined to be undescended were investigated histologically at 4, 7 and 14 days after birth. Secondly, 12-day-old T-S rats were divided into four groups which underwent different procedures, i.e. 1, with normally descending testes (normal control, 10 rats); 2, with undescended testes (UDT) treated by orchidopexy (treated UDT, eight rats); 3, with UDT treated by a sham operation (sham-operated UDT, six rats); and 4, with UDT left untreated (untreated UDT, six rats). Thirty days after operation the testicular anatomy was recorded; excised testes were examined histologically and different types of germ cells were counted per tubule cross-section microscopically., Results: There were no quantitative or morphological differences in the numbers of gonocytes, type-A spermatogonia or Leydig cells in the seminiferous tubules between normal and ectopic testes in the first 14 days after birth. However, by 21 days of age spermatogenesis in the UDT had declined with transformation from primary leptotene spermatocytes to spermatids. There were significantly more Leydig cells in the untreated UDT at 30 days than in normal control testes. The impaired spermatogenesis in UDT was restored by early orchidopexy and there were significantly more seminiferous tubules at stage 3 (pachytene spermatocytes) and stage 4 (spermatids) than in the untreated or sham-operated groups (P < 0.001)., Conclusions: These results show that in the T-S rat with cryptorchidism, the testicular damage is not a congenital malformation and can be reversed with early surgical correction.

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.

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.

Background: Many patients who have esophageal atresia and tracheoesophageal fistula (EA-TEF) have associated tracheomalacia, which is thought to be one of the reasons for respiratory complications after surgical correction of the abnormality., Methods: In this study, tracheas from Adriamycin-induced EA-TEF fetal rats were examined histologically and relevant cross-sectional parameters of the tracheas were measured., Results: The tracheal lumen in tracheomalacia was small and irregular, losing its normal "D" shape. In most rats, the cartilaginous ring was broken into two to four segments, making the trachea lose its rigid support. The submucosa was thickened with prominent bulging of its membranous part into the tracheal lumen. The ratio of the inner luminal cross-sectional area to the outer tracheal cross-sectional area in EA-TEF rats was 15.7%, compared with a control ratio of 47.2%. In EA-TEF rats, the length of the cartilaginous ring was significantly shortened (P < .001), but not the length of membranous trachea, thus resulting in a cartilaginous/membranous (C/M) ratio of 1.55:1, markedly lower than that of normal rats (4.34:1, P < .001). The reduction of anterior-posterior diameter of the tracheal lumen was more marked than that of the transverse diameter., Conclusions: These observations suggest that the trachea in EA-TEF rats has a smaller lumen and is more flaccid than normal, making it prone to airway obstruction. The fact that tracheomalacia developed only in fetuses who had EA-TEF indicates that the factors that result in EA-TEF also cause tracheomalacia.

Interferons (IFNs) are naturally occurring cytokines which have pleiotropic actions including regulation of cell growth and differentiation, important for control of tumour growth and development. In this study we investigated the association between the presence of IFN genes in NSCLC cell lines, receptor expression and function, and sensitivity to IFNs. Some of the NSCLC lines had partial or complete IFN gene deletion but others contained all genes. However, all NSCLC lines were resistant to the antiproliferative effects of IFN alpha 2 and IFN beta ser. While the lack of sensitivity to IFNs did not appear to be associated with the presence of IFN genes, numbers of receptors or with low binding affinities it did correlate with abnormal regulation of receptor expression. When analyzed by Northern blotting it was notable that IFNA receptor expression on a sensitive cell line, Daudi, was upregulated following IFN exposure however, in the insensitive NSCLC lines IFN mediated upregulation of receptors did not occur. This defect in the regulation of receptor expression in NSCLC lines could contribute to the insensitivity of the antiproliferative effects of IFN's and thus potentiate tumour development or progression.

Purpose: Recently evidence has been accumulating that some undescended testes present later in childhood after apparent normalcy in infancy. These ascending testes appear to explain why a significant number of orchiopexies are performed later in childhood despite the recommendation that surgery for cryptorchidism be performed in infancy. We aimed to determine whether the cause of these ascending testes was persistence of the processus vaginalis., Materials and Methods: A total of 25 boys 4 to 13 years old with no history of testicular maldescent at birth underwent transscrotal orchiopexy in a 2-year period. A total of 33 orchiopexies was performed (8 bilateral). The spermatic cord was carefully dissected and operative findings of the nature of the spermatic cord were noted., Results: In all cases dissection within the spermatic cord revealed a similar finding, namely a fibrous string situated deep to the cremasteric muscle and spermatic fasciae. Transection of this string allowed adequate elongation of the vas deferens and gonadal vessels to permit scrotal placement of the testis. Histological examination revealed characteristic processus vaginalis tissue in which the peritoneal derived mesothelial lining cells were present within a partially obliterated processus vaginalis., Conclusions: Cryptorchidism presenting later in childhood may be an acquired abnormality caused by failure of natural growth of the spermatic cord when the processus vaginalis leaves a fibrous remnant, which prevents normal elongation. These observations suggest that the ascending testis is acquired postnatally and the cause may be related to inguinal hernia.

Calcitonin gene-related peptide (CGRP) receptors are located in the cremaster muscle of the gubernaculum of rats, and causes gubernacular contraction in vitro, suggesting that CGRP plays an important role in testicular descent. It has been postulated that CGRP released from the genitofemoral nerve acts as a "chemoattractant" for gubernacular migration to the scrotum. The aim of this study is to investigate whether the gubernacular cells of rats exhibit a chemotactic response to CGRP in vitro. Chemotaxis of gubernacular cells from Sprague-Dawley rats (1 to 6 days old) was measured using blind-well chambers. The migration of cells, which passed from the upper to the lower compartment through a polycarbonate filter, were counted using microscopy. The lower compartment included 10(-11), 10(-10), 10(-9), and 10(-8) M CGRP or phosphate buffered saline (PBS) as control. The chemotactic index (CI) was defined as the ratio of migration toward CGRP versus PBS as control. There was no significant migration even at varying CGRP concentrations. Isolated rat gubernacular cells therefore did not exhibit a chemotactic response to CGRP and the role CGRP plays in testicular descent still remains unclear. This result does not exclude the possibility that the gubernaculum responds to CGRP as a whole organ rather than as individual cells.

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.

Early spermatogenesis from gonocytes to A-type spermatogonia is inhibited in boys with cryptorchidism. Histological lesions in the undescended testes (UDT) are presumably secondary to the extrascrotal position. The purpose of this study is to determine the abnormalities in number and maturation of germ cells in pigs with undescended testes and to examine the effect of UDT location on early germ cell differentiation. Testicular biopsies from 4-week-old pigs with natural unilateral cryptorchidism (n = 20) were divided into three groups: (1) intraabdominal testes, (2) superficial inguinal pouch testes, and (3) contralateral descended testes (CDT). The testis weight (P < .01) and the area of circular cross-section of the seminiferous tubules (P < .05) in intraabdominal testes was lower than in CDT. The number of germ cells (per tubular cross-section) (P < .05) and A-type spermatogonia (P < .05) in intraabdominal and inguinal testes was significantly lower than in CDT. In contrast, the number of gonocytes in intraabdominal testes was similar to that in CDT. The number of Sertoli cells was similar in the three groups. These results demonstrate that defective transformation of gonocytes to A-type spermatogonia in pigs is similar to that observed in boys with unilateral cryptorchidism. The different locations of UDT in the pig are correlated with the degree of impaired spermatogenesis.

The cell line CIPL38 was derived from the pleural effusion of a patient with small cell lung cancer. The karyotype was hyperdiploid and complex with a variable number of marker chromosomes. Two of the markers had large homogeneously staining regions (hsr), which were shown to consist of amplified MYCN by in situ hybridization. One hsr bearing a marker chromosome could not be identified with G-banding, but the other was situated on a der(14). This was elucidated further with FISH analysis, which enabled the identification of sequences of chromosome i involved in a complex rearrangement with chromosome 14 and the hsr.

A cytogenetic study of pleural effusions (PE) containing metastatic or invasive tumor cells from 11 patients with non-small cell lung cancer (NSCLC) (3 squamous cell carcinomas [SQC] and 8 adenocarcinomas [ADC] including 1 giant cell variant) was performed to identify non-random chromosome abnormalities. Numerical abnormalities seen in > or = 30% of cases included gain of chromosomes 7 and 20, and loss of chromosomes 4, 9, 10, 13, 15, 16, 18, 19, 21, and 22. The most frequent structural abnormality involved rearrangement in 1p with breakpoints clustering at 1p10-p13. Other recurrent breakpoint regions, seen in > or = 30% of cases, occurred in chromosome region 3p10-p21, 3q11-q25, 6p11-p25, 6q13-q23, 7q11-q36, 9q32-q34, 11p11-p13, 11q13-q24, 13p/14p and/or 15p, 17p and 19p, with, in particular, apparent loss of 6q21-q27, 3p21-p26, 7q21-q22, 9p22-p24 (shortest regions of common overlap) and 17p. There was also recurrent gain of 1q23-q44, 8q13-q24, and 11q13-q23. These abnormalities were not restricted to a particular histological subtype, with the exception of +8 and a breakpoint in 9q32-q34, which were seen only in ADC. The 9q32-q34 breakpoint observed in 4 ADC PE (including 1 giant cell variant) represents a new observation in NSCLC. These findings, when compared to those reported for primary NSCLC indicate cytogenetic differences between the two which may be associated with pleural invasion of NSCLC.

Establishing the presence of various cell types in long-term marrow culture (LTMC) has an important bearing on understanding the regulation of stromal cell-related hemopoiesis. Controversy has surrounded the identity of the very large cells in murine LTMC; they have been given a variety of designations, including blanket cells. Using dual immunogold labeling with a recently derived monoclonal antibody, H513E3, and anti-human factor VIII antibodies, we have conclusively located endothelial cells in LTMC. Endothelial cells are relatively large, with thinly spread cytoplasm, and they overlie macrophages, granulocytes, and other less differentiated developing hemopoietic cells. Previously, demonstration of endothelial cells in LTMC had been difficult due to lack of specific markers in the mouse. We have demonstrated that LTMC endothelial cells have the exact location and ultrastructural characteristics as the previously described blanket cells. We propose that previous designations should not be continued and that these cells should be referred to as endothelial cells. The known functions of endothelial cells now become relevant to the understanding of stromal regulation of hemopoiesis.

Evidence has been sought for megakaryocyte maturation in long-term cultures of mouse bone marrow. Cultures up to 14 weeks of age were examined for the presence of megakaryocytes with processes, that is, resembling the morphological appearance seen in vivo prior to platelet liberation. Such cells were found floating just above the adherent stromal layer using low magnification phase contrast microscopy. It was rare to observe as many as 20 of these cells per 25-cm2 flask. At higher magnification, processes were seen to be attenuated with constrictions at intervals along their length. Time-lapse photography was used to follow the development and behavior of the processes. Direct evidence of rupture was very rare; generally the megakaryocytes retracted their processes within 48 h. Careful searching of cultures occasionally revealed the presence of several process fragments, and sometimes individual platelets were found. Ultrastructurally, the processes were seen to contain organelles that are usually associated with platelets. The observations applied to both Dexter and Whitlock-Witte cultures. It is concluded that maturation of megakaryocytes occurs in long-term marrow culture to the point where platelet release appears imminent. Final rupture is rare and may require shearing forces, which in vivo would be provided by blood flow.

The binding of a marrow cell-related monoclonal antibody (H513E3 MAb) has been investigated in long-term marrow cultures (LTMC) and in vivo. Immunogold labeling and electron microscopy revealed that this antibody labeled an endothelial-like cell. Cross-reaction of anti-human Factor VIII confirmed endothelial specificity of the H513E3 MAb. In addition, vessel endothelium (vena cava, aorta, and marrow) exhibited binding of the antibody. This antibody provides a unique tool to study the cellular architecture of LTMC and implicates endothelium as an important component of LTMC. The function of the endothelial cell-specific surface antigen is unknown, although preferential labeling of the upper surface of endothelial cells suggests that it may play a role in cell communication, particularly with the floating population. The H513E3 MAb reacts with an external membrane antigen, a property that makes this antibody particularly useful for fluorescences-activated sorting of endothelial cells.

Cytogenetic analysis of ten primary non-small cell lung carcinomas (NSCLC), including five adenocarcinomas (ADC), three squamous cell (SQC), and two large cell (LCC) carcinomas has been carried out in an attempt to determine karyotype changes involved in the early stage of disease. The tumors were all aneuploid and exhibited complex karyotypes with multiple structural and numerical abnormalities. Clonal structural rearrangements were identified and in particular loss of material from the short arm of chromosome 9 had a 90% incidence. This loss was due to non-reciprocal translocation, deletion, or chromosome loss. Breakpoints were in the region 9q13 to p22. Other chromosome regions that were non-randomly involved are as follows: I cen to p13, 3p, 5q11 to q13, 6p, 6q15 to q27, 7p, 8p, 11q12 to q23, 13p, 14p, 15p, 17p, and 19p. While a primary cytogenetic change in NSCLC has not been identified conclusively, our findings implicate loss of material from 9p as a potentially important event.

The technique of somatic cell hybridization has been applied to obtain new sources of immunogen for monoclonal antibody production. A marrow population enriched for rare and undifferentiated cells was hybridized with A9 cells. Mature cells were depleted by using mice at 8 days following 5-fluorouracil treatment and by using a sorting procedure. Selection of hybrids was in medium containing hypoxanthine, aminopterin, and thymidine (HAT). Chromosome analysis was used to verify that clones contained hybrid cells. An example of an antibody (H513E3) obtained by immunizing with a hybrid cell line (H5Scl1.4.4) is presented. The H513E3 antibody showed low reactivity with normal marrow (0%-3%), and hemopoietic cell lines of various types exhibited no cross-reaction. However, about 10% of cells from the adherent stromal layer of long-term marrow cultures reacted with the H513E3 antibody. A specific cell type was labeled that appeared to have endothelial-like morphology when observed with immunogold labeling and electron microscopy. The hybridization technique allows a cell of a particular differentiation lineage to be conserved, wholly or partially, in a cloned form, thus overcoming the heterogeneity of a normal marrow population.

Haemopoietic regeneration was studied following x-irradiation and transplantation of bone marrow from either normal or hydroxyurea-treated donor mice, to ascertain the contribution of proliferating progenitor cells to regeneration. With transplantation of equivalent numbers of CFU-S, total DNA and 3HTdR uptake into DNA in spleen and femoral bone marrow and the erythroid, granulocytic and mononuclear cell populations were not significantly different between normal (NBM) and hydroxyurea-treated (HUBM) marrow. The response of hypertransfused x-irradiated mice to erythropoietin (EPO) administration was also not significantly different in spleens of mice receiving normal or hydroxyurea-treated marrow.