Studies on Synthetic and Natural Melanin and Its Affinity for Fe(III) Ion.

In this work, we measured the metal-binding sites of natural and synthetic dihydroxyindole (DHI) melanins and their respective interactions with Fe(III) ions. Besides the two acid groups detected for the DHI system: catechol (Cat) and quinone-imine (QI), acetate groups were detected in the natural oligomer by potentiometric titrations. At acidic pH values, Fe(III) complexation with synthetic melanin was detected in an Fe(OH)(CatH(2)Cat) interaction. With an increase of pH, three new interactions occurred: dihydroxide diprotonated catechol, Fe(OH)(2)(CatH(2)Cat)(-), dihydroxide monoprotonated catechol, [Fe(OH)(2)(CatHCat)](2-), and an interaction resulting from the association of one quinone-imine and a catechol group, [Fe(OH)(2)(Qi(-))(CatHCat)](3-). In the natural melanin system, we detected the same interactions involving catechol and quinone-imine groups but also the metal interacts with acetate group at pH values lower than 4.0. Furthermore, interactions in the synthetic system were also characterized by infrared spectroscopy by using the characteristic vibrations of catechol and quinone-imine groups. Finally, scanning electronic microscopy (SEM) and energy-dispersive X-ray (EDS) analysis were used to examine the differences in morphology of these two systems in the absence and presence of Fe(III) ions. The mole ratio of metal and donor atoms was obtained by the EDS analysis.

Development of the gubernaculum during testicular descent in the rat.

Gubernacular elongation during inguinoscrotal testicular descent and cremaster muscle development remains poorly described in mammals. The role of the genitofemoral nerve (GFN) remains elusive. We performed detailed histological analysis of testicular descent in normal rats to provide a comprehensive anatomical description for molecular studies. Fetuses and neonatal male offspring (5-10 per group) from time-mated Sprague-Dawley dams (embryonic days 15, 16, and 19; postnatal days 0, 2, and 8) were prepared for histology. Immunohistochemistry was performed for nerves (Class III tubulin, Tuj1) and muscle (desmin). At embryonic days 15 and 16, the gubernaculum and breast bud are adjacent and both supplied by the GFN. By embryonic day 19, the breast bud has regressed and the gubernacular swelling reaction is completed. Postnatally, the gubernacular core regresses, except for a cranial proliferative zone. The cremaster is continuous with internal oblique and transversus abdominis. By postnatal day 2 (P2), the gubernaculum has everted, locating the proliferative zone caudally and the residual mesenchymal core externally. Eversion creates the processus vaginalis, with the everted gubernaculum loose in subcutaneous tissue but still remote from the scrotum. By P8, the gubernaculum has nearly reached the scrotum with fibrous connections attaching the gubernaculum to the scrotal skin. A direct link between GFN, gubernaculum, and breast bud suggests that the latter may be involved in gubernacular development. Second, the cremaster muscle is continuous with abdominal wall muscles, but most of its growth occurs in the distal gubernacular tip. Finally, gubernacular eversion at birth brings the cranial proliferative zone to the external distal tip, enabling gubernacular elongation similar to a limb bud.

Substance P and vasoactive intestinal peptide are reduced in right transverse colon in pediatric slow-transit constipation.

BACKGROUND: Slow-transit constipation (STC) is recognized in children but the etiology is unknown. Abnormalities in substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide (NO) have been implicated. The density of nerve fibers in circular muscle containing these transmitters was examined in colon from children with STC and compared to other pediatric and adult samples. METHODS: Fluorescence immunohistochemistry using antibodies to NO synthase (NOS), VIP and SP was performed on colonic biopsies (transverse and sigmoid colon) from 33 adults with colorectal cancer, 11 children with normal colonic transit and anorectal retention (NAR) and 51 with chronic constipation and slow motility in the proximal colon (STC). The percentage area of nerve fibers in circular muscle containing each transmitter was quantified in confocal images. KEY RESULTS: In colon circular muscle, the percentage area of nerve fibers containing NOS > VIP > SP (6 : 2 : 1). Pediatric groups had a higher density of nerve fibers than adults. In pediatric samples, there were no regional differences in NOS and VIP, while SP nerve fiber density was higher in sigmoid than proximal colon. STC children had lower SP and VIP nerve fiber density in the proximal colon than NAR children. Twenty-three percent of STC children had low SP nerve fiber density. CONCLUSIONS & INFERENCES: There are age-related reductions in nerve fiber density in human colon circular muscle. NOS and VIP do not show regional variations, while SP nerve fiber density is higher in distal colon. 1/3 of pediatric STC patients have low SP or VIP nerve fiber density in proximal colon.

Decrease in nerve fibre density in human sigmoid colon circular muscle occurs with growth but not aging.

BACKGROUND: Studies in animals suggest that enteric neurons decrease in density or number with increasing age. Neurons containing nitric oxide (NO), vasoactive intestinal peptide (VIP) and Substance P (SP) have been implicated. In human large intestine, NO-utilizing neurons decrease during childhood or early adulthood but it is not known if the innervation of the muscle changes. This study examined the density of nerve fibres containing these transmitters in sigmoid colon circular muscle from children and adults. METHODS: Fluorescence immunohistochemistry using antibodies to neuronal NO synthase (nNOS), VIP and SP was performed on sigmoid colon from 18 adults with colorectal cancer, two children with familial adenomatous polyposis, and normal colon from nine children with Hirschsprung's disease. The percentage area of immunoreactive (IR) nerve fibres containing each transmitter in circular muscle was quantified in confocal images. KEY RESULTS: In the adult sigmoid colon circular muscle, the percentage area of nerve fibres containing nNOS>VIP>SP (6 : 2 : 1). Paediatric groups had significantly higher percentage area of nerve fibres containing nNOS, VIP or SP-IR than adults, with the decrease in nerve fibre density occurring from birth to 30 years. Circular muscle thickness increased between 12 and 30 years. Total nerve fibre area remained constant, while the muscle increased in thickness. CONCLUSIONS & INFERENCES: In human sigmoid colon circular muscle, there are reductions in nNOS-, VIP- and SP-IR nerve fibre density with growth from newborn to late adolescence but little further change with aging. The reduction in nerve density is due to an increase in circular muscle thickness rather than a loss of nerve fibres.

O atom transfer from nitric oxide catalyzed by Fe(TPP).

The reaction of NO-Fe(TPP) with low pressures of NO gas proceeds through three distinct transformations, the first of which we suggest is the formation of an N--N-coupled, (NO)(2) adduct intermediate. The subsequent formation of NO(NO(2))Fe(TPP), which under these conditions readily loses NO, suggests that it is formed by addition of free NO(2) to the starting nitrosyl. A mechanism is proposed which implies that the addition of a competitive O atom acceptor would lead to catalytic production of N(2)O. In agreement with the proposed mechanism, the formation of N(2)O is decoupled from the formation of the nitrite by using PPh(3) as the competitive acceptor. The mechanism of O atom transfer was examined by cross-labeling experiments, which show that both O atoms in the intermediate are equivalent, even under catalytic conditions. The formation of an intermediate was confirmed by IR spectroscopy of the heterogeneous reaction of an NO-Fe(TPP) film with gaseous NO, in which transient, isotope-sensitive nu(NO) bands are seen prior to NO(NO(2))Fe(TPP) formation. Mixed (14)N/(15)N label experiments demonstrate coupling between the two bound nitrosyls in the transient species.

Enlargement of the processus vaginalis during testicular descent in rats.

The role of the processus vaginalis (PV) during inguinoscrotal testicular descent remains controversial. Some authors propose passive dragging of the PV by the migrating testis, while others suggest active elongation. In addition, the exact site of growth is unknown. Our aim was to determine whether the PV actively proliferates at its tip or stretches passively during the inguinoscrotal phase of descent in the rat. Gubernacula were removed from Sprague-Dawley (SD) rats and congenitally-cryptorchid TS mutants. Animals (at days 3, 7, 10, and 11) were treated with bromo-uridine deoxyribose (BUdR) 2 h before death. BUdR incorporation into newly-synthesised DNA served as a marker for cell division. The gubernacula were processed for haematoxylin and eosin and immunoperoxidase staining. Three sites were examined: (1) the tip of the PV on either side of the gubernacular bulb; (2) the proximal gubernacular cord; and (3) the proximal parietal PV. At each site, 50 adjacent cells were counted and the number of positive cells recorded. The highest BUdR labelling in SD rats was at the tip (site 1) on day 3 (17/50) compared with sites 2 (11/50) and 3 (9/50) (P < 0.05). Labelling decreased by 7 and 11 days to similar levels in all three sites. In TS rats, labelling rates were lower at day 3 and were highest at the tip at day 11. These results suggest active growth of the caudal tip of the PV during testicular descent. In normal rats, the growth rate slows as the testis approaches the scrotum. By contrast, in TS rats growth continued longer. We propose that the PV elongates actively from the tip to allow the intraperitoneal testis to leave the abdomen in a special peritoneal diverticulum.

Leydig insulin-like hormone, gubernacular development and testicular descent.

PURPOSE: Testicular descent is controlled by 2 morphological and hormonal steps. Transabdominal testicular descent is mediated by gubernacular swelling and regression of the cranial suspensory ligament. Müllerian inhibiting substance (MIS) has been proposed to stimulate the swelling but this remains controversial. Recently, a mouse mutant for Leydig insulin-like hormone (Insl3) was found to have undescended testis and deficient gubernaculum. We examine the testicular position of Insl3 mutant mice and the development of gubernacula. MATERIALS AND METHODS: Mice with Insl3 homozygotes (-/-), heterozygotes (+/-) and wild-types (+/+) were examined at embryonic day 16.5 and birth. Macroscopic dissections and measurements of the testicular position, as well as microscopic analysis (hematoxylin and eosin, and Masson's trichrome) were performed. RESULTS: Of the mice 11 Insl3 homozygote males had significantly impaired testicular descent at embryonic day 16.5 and birth (p <0.01), and the cord was thin and elongated, while 14 heterozygotes and 7 wild-types had normal testicular descent. Microscopically, the gubernaculum of Insl3 homozygotes was small with some muscle development but no central core of mesenchyme at embryonic day 16.5. On the other hand, heterozygotes and wild-types had normal gubernacular development with a swelling reaction. CONCLUSIONS: Insl3 mutants show feminized gubernaculum with deficient mesenchymal core. Insl3 appears to have some role in the gubernacular swelling reaction in mice.

Permeability of endothelial monolayers to albumin is increased by bradykinin and inhibited by prostaglandins.

Using monolayers of bovine aortic endothelial cells (BAEC) in modified Boyden chambers, we examined the role of prostaglandins (PGs) in the bradykinin (BK)-induced increase of albumin permeability. BK induced a concentration-dependent increase of the permeability of BAEC, which reached 49.9 +/- 1% at the concentration of 10(-8) M. Two inhibitors of the prostaglandin G/H synthase, indomethacin (2.88 microM) and ibuprofen (10 microM), potentiated BK-induced permeability 1.8- and 3.9-fold, respectively. Exogenously administered PGE2 and iloprost, a stable analog of prostacyclin, attenuated the effect of BK in a concentration-dependent manner. Butaprost equally reduced the effect of BK, suggesting the participation of the EP2 receptor in this phenomenon. However, the EP4-selective antagonist AH-23848 did not significantly inhibit the protective effect of PGE2. The inhibitory effect of PGE2 was reversed by the adenylate cyclase inhibitor MDL-12330A (10 microM). These results suggest that BK-induced increase of permeability of BAEC monolayer to (125)I-labeled albumin is negatively regulated by PGs. This postulated autocrine activity of PGs may involve an increase in the intracellular level of cAMP.

Undescended testis is accompanied by calcitonin gene related peptide accumulation within the sensory nucleus of the genitofemoral nerve in trans-scrotal rats.

PURPOSE: Recent data suggest that calcitonin gene related peptide (CGRP) released from the sensory branch of the genitofemoral nerve may regulate testicular descent. We studied the number of CGRP immunoreactive cells in the sensory nucleus of the genitofemoral nerve (L1 to L2 dorsal root ganglia) in cryptorchid trans-scrotal rats. Four-week-old trans-scrotal rats with unilateral undescended testis underwent bilateral genitofemoral nerve dissection and retrograde nerve labeling with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI). Animals were sacrificed 48 hours later and the L1 to L2 dorsal root ganglia were removed. Serial sections were obtained and double fluorescent labeled with antibody to CGRP. Retrograde labeled and CGRP immunoreactive cells were counted using an epi-fluorescent microscope. In the 6 male trans-scrotal rats evaluated we noted a mean plus or minus standard deviation of 1,272 +/- 98 retrograde labeled dorsal root ganglion cells ipsilateral to a fully descended testicle, including 98 +/- 34 that were also CGRP immunoreactive. On the side of the undescended testis there was a mean of 1,600 +/- 304 DAPI positive cells and 160 +/- 51 CGRP immunoreactive, DAPI labeled cells. The difference was significant (p <0.02). This study shows that in trans-scrotal rats the sensory nucleus of the genitofemoral nerve contains more CGRP immunoreactive cells ipsilateral to an undescended testis than on the contralateral side, highlighting the significance of CGRP supply through the sensory branch of the genitofemoral nerve for testicular descent.

Apoptotic cell death and fertility in three unilateral cryptorchid rat models.

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.

Unusual voltammetry of manganese-substituted myoglobin in surfactant film: evidence for two redox pathways.

The surfactant film methodology is used to examine the electrochemistry of manganese-substituted myoglobin. Cyclic voltammograms at different scan rates depict a dynamic exchange between two redox couples, E1 (-0.25 V vs. SCE) and E2 (-0.41 V). Similar behavior is seen for Mn-substituted cytochrome c peroxidase, but the free cofactor, Mn(protoporphyrin IX) yields a single couple (-0.32 V) under the same conditions. A square scheme is proposed which describes equilibration between two different redox pathways associated with different forms of the protein. Overlapping oxidative currents from these two couples can be deconvoluted, and a pseudo first-order rate constant of 2.3 s(-1) is obtained for the reaction following reduction of Mn(III)Mb. Experiments have been performed to probe possible mechanisms for this equilibrium, such as ligand dissociation or reversible adsorption at the electrode surface. A cofactor-induced reorganization of the protein structure is suggested as the basis of the behavior.

Growth factor and somatic cell regulation of mouse gonocyte-derived colony formation in vitro.

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.

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.

Does the sensory nucleus of the genitofemoral nerve have a role in testicular descent?

BACKGROUND/PURPOSE: A role for the genitofemoral nerve (GFN) and its neurotransmitter, CGRP, in testicular descent has been well established. The exact mechanism, however, by which circulating androgens act on the GFN is not yet known. The authors studied the sensory nucleus of the GFN (L1-L2 dorsal root ganglia [DRG]) to determine whether it is sexually dimorphic and able to be influenced by intrauterine antiandrogen treatment. METHODS: Sprague-Dawley rats were injected daily with 100 mg/kg/d of the antiandrogen flutamide on day 16 to 19 of pregnancy. Control animals were treated with vehicle only. At the age of 2 to 3 days the newborn rats underwent unilateral dissection of the GFN. The proximal end was labelled with fluorescent dye, diamidinophenyl indole. The rats were killed 48 hours later, and the relevant ganglia (L1,L2) were removed. Cryostat frozen serial sections were cut, and retrogradely labelled fluorescent cells were counted under an epifluorescence microscope. In 32 animals, the cells were double fluorescent labelled with antibody to CGRP and FITC. RESULTS: Of 75 rats evaluated, the mean number of the DAPI-positive, retrogradely labelled cells in the control groups was 266 +/- 55 in the male, and 230 +/- 67 in the female as opposed to 186 +/- 45 and 161 +/- 35 in the flutamide-treated male and female groups, respectively. In 32 animals the DRG sections were double labelled for CGRP. The number of CGRP plus DAPI-positive cells were as follows: control males, 60 +/-12; control females, 50 +/- 9; flutamide males, 36 +/- 8; flutamide females, 40 +/- 10. CONCLUSIONS: These findings show a sexual dimorphism in the number of GFN cell bodies in the DRG. Flutamide decreases the number of GFN cell bodies in the DRG of both males and females. Our results are consistent with a role for circulating androgens acting on the sensory nucleus of the GFN (DRG) instead of the motor nucleus as previously thought. The release of CGRP from the nerve endings may occur via the sensory branch of the GFN.

Catalytic two-electron reductions of N2O and N3- by myoglobin in surfactant films.

Myoglobin (Mb), in films of dimethyldidodecylammonium bromide (ddab) on graphite electrodes, is used as a catalyst to mediate the electrochemical reduction of nitrous oxide (N2O) as well as the isoelectronic ion azide (N3-) in aqueous solutions. The electrocatalytic reductions are characterized by a rate-dependent irreversibility in cyclic voltammograms of Mb/ddab in the presence of the substrates. Bulk electrolysis shows that the reduction of 15N15NO by Mb/ddab yields 15N15N as shown by GC/MS. The catalytic reduction of azide results in almost quantitative formation of ammonia. These electrocatalytic processes are rationalized as two-electron reductions, with the catalyst cycling between the Fe(I) and Fe(III) states of Mb. To our knowledge, this is the first characterization of N2O reduction by an Fe porphyrin or heme protein.

Localization of calcitonin gene-related peptide within the genitofemoral nerve in immature rats.

BACKGROUND/PURPOSE: Calcitonin gene-related peptide (CGRP) has been proposed to influence migration and testicular descent by release from the genitofemoral nerve. The site of CGRP within the nerve has been controversial, with conflicting views on whether CGRP is synthesised and released from the motor nerves. METHODS: The genitofemoral nerve (GFN) was retrogradely labelled by fluorescent dye (DAPI) in 25 Sprague-Dawley rats (days 5, 16, and 31, n = 8 in each group; day 35, n = 1). Spinal cords and dorsal root ganglia (DRG) were removed two to three days later and sectioned for immunofluorescence. Substance P and CGRP-containing cells were labelled with fluorescein-linked antibodies. Specimens were examined by double fluorescence to identify cells with both markers. RESULTS: The motor nucleus of the GFN contained 119 cells on day 7 and 284 cells by days 19 through 34. A prominent band of CGRP-containing fibers, arising from the dorsal horn, synapsed with the GFN motor nucleus itself. CGRP-labelled GFN cells were found in the DRG by double labelling. CONCLUSIONS: CGRP from the GFN may affect gubernacular migration by release from the sensory nerves, rather than motor nerves as previously thought. The GFN motor nucleus receives CGRP-containing innervation from the dorsal horn, which may form part of the cremasteric reflex.

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.

Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method.

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.

Temperature sensitivity of primary spermatocyte DNA synthesis in immature mice confirmed by bromodeoxyuridine labelling in vitro.

OBJECTIVE: To investigate the relationship between temperature and DNA synthesis of immature germ cells and to determine whether the early primary spermatocytes proliferate at a 'scrotal' temperature of 32 degrees C in vitro. MATERIALS AND METHODS: Day-7 mouse testes (n = 16) were cultured with 10% fetal calf serum (FCS) for 3 days at 32 degrees C or 37 degrees C and labelled by bromodeoxyuridine (BrDU) for a further hour. The BrDU-labelled cells were detected by immunohistochemical staining using a monoclonal anti-BrDU antibody. The numbers of primary spermatocytes with BrDU-labelling or unlabelled in each tubule were determined as an index of spermatocyte DNA synthesis. In addition, the cultured media at different temperatures were collected and the testosterone levels measured by radioimmunoassay. RESULTS: The numbers of primary spermatocytes (P < 0.01) and the BrDU-labelling index in primary spermatocytes per tubule at 32 degrees C in vitro were significantly higher (P < 0.001) than in the culture at 37 degrees C. The testosterone levels in the culture media at 32 degrees C were also markedly higher than in the culture at 37 degrees C (P < 0.01). CONCLUSION: These observations indicate that DNA synthesis of early primary spermatocytes and testosterone production can be stimulated by lower testicular temperature, even in immature mouse testes that are naturally located in the intra-abdominal cavity.

Congenital undescended testes in neonatal pigs and the effect of exogenous calcitonin gene-related peptide.

PURPOSE: We investigated the neonatal piglet as a possible animal model for cryptorchidism and to determine whether calcitonin gene-related peptide (CGRP), which has been proposed to regulate inguinoscrotal testicular descent, could induce testicular descent in piglets with congenital cryptorchidism. MATERIALS AND METHODS: We examined 38 cryptorchid piglets to document the anatomy in 8 and to investigate the role of CGRP in 30. The 2-week-old piglets were allocated randomly to receive a mini-osmotic pump containing CGRP at various concentrations or phosphate buffered saline. The pump was inserted surgically into the ipsilateral scrotum, with the contents blinded to the surgeon. The positions of the testes, pump and anatomical landmarks were measured and photographed. The pigs were sacrificed and dissected 2 weeks later, and the positions were remeasured and photographed. The testes were examined histologically. RESULTS: The 3 variants of cryptorchidism observed were intra-abdominal in 20 cases, inguinal in 9 and lateral inguinal ectopic in 9. CGRP had no effect on intra-abdominal or ectopic testes. In contrast, for inguinal testes exogenous CGRP caused a slight but significant 10 +/- 7.9 mm. descent towards the pump in 5 cases compared to -2.9 +/- 5.8 mm. in 4 controls. CONCLUSIONS: Exogenous CGRP stimulated migration of inguinal testes that had been arrested in the line of descent while ectopic testes did not respond. These results support a role for CGRP in testicular descent and suggest that a slow release depot preparation might be useful as a possible treatment in some forms of cryptorchidism.