Co-localization of neuron-specific enolase-like and kallikrein-like immunoreactivity in ductal and tubular epithelium of sheep salivary gland and kidney

Neuron-specific enolase-like and kallikrein-like immunoreactivity was found to be co-localized in the ductal elements of the submandibular gland and in the more distal portions of the nephron in the kidney of newborn lambs. Some glomerular peripolar cells in the kidney were immunopositive for neuron-specific enolase without detectable kallikrein-like immunoreactivity.     

Morphogenesis of the renal juxtaglomerular apparatus and peripolar cells in the sheep

Summary The morphogenesis of the juxtaglomerular apparatus and peripolar cells was studied in the metanephros of fetal sheep (from 24 to 147 days of gestation) using light and electron microscopy. The first juxtaglomerular apparatus was detected at 45 days of gestation, following constriction of the edges of Bowman's capsule and formation of the vascular pole of the renal corpuscle. Mesenchymal cells gave rise to lacis cells and to smooth muscle and epithelioid cells of the juxtaglomerular arterioles. Epithelioid cells developed only sparse cytoplasmic granulation, first detectable at 92 days. The macula densa developed from tubular cells at the junction of the middle and upper limbs of the S-shaped body of the developing nephron. Peripolar cells arose from epithelial cells in the lower limb of the S-shaped body, at the constricting edges of Bowman's capsule, and formed a cuff around the origin of the glomerular tuft. Cytoplasmic granules were first detected in peripolar cells at 53 days, and remained more prominent than epithelioid cell granulation throughout gestation.     

Non-granulated peripolar cells exist in the rat glomerulus

Summary The peripolar cell is a unique cell type in the mammalian glomerulus. Peripolar cells are said to be identifiable during light microscopy by their cytoplasmic granules and by their position at the vascular pole; and during scanning electron microscopy by their distinctive surface morphology. We used both techniques to count peripolar cells in 6 normal rat kidneys. Scanning microscopy revealed that 55(±5)% of glomeruli contained at least one peripolar cell whereas light microscopy revealed granulated peripolar cells in only 4(±2)% of glomeruli. Vascular poles which contained peripolar cells previously identified by scanning were then examined by light and by transmission electron microscopy. Serial sections through these peripolar cells demonstrated the absence of cytoplasmic granules. Our observations suggest that the majority of peripolar cells in the rat contain no granules.     

Scanning and transmission electron-microscopic study of peripolar cells in the newborn lamb kidney

Scanning and transmission electron microscopy were used to study the ultrastructural characteristics and positions of granulated peripolar cells in newborn lamb kidney. Following tissue fixation by vascular perfusion in situ, the vascular pole region of the glomerulus was exposed for examination by scanning electron micoscopy following removal of the glomerular tuft. Peripolar cells were recognized by their surface morphology enabling their quantification and an assessment of the relationship of their position in the renal cortex. The prominent expression of peripolar cells in this species was confirmed. Almost every vascular pole examined revealed peripolar cells (405 out of 407; 99.5%) and thus, throughout the cortex, the distribution of peripolar cells was the same as the distribution of renal corpuscles. Larger, more protruding peripolar cells were observed in the outer cortical renal corpuscles. The numbers of peripolar cells encircling each vascular pole ranged from 1 to 10. There was no correlation between number of granulated peripolar cells at the vascular pole and the position of the renal corpuscle within the renal cortex. As viewed by transmission electron microscopy, organelles of protein synthesis were abundant in the cytoplasm of peripolar cells. Exocytosis of cytoplasmic granules was observed by both scanning and transmission electron microscopy implying that a process of regulative secretion occurs from these cells. The use of ultrastrural techniques has provided evidence supporting the concept that peripolar cells are prominent in the cuff region of each renal corpuscle of the newborn lamb and further-more that peripolar cells in this species most likely have a secretory function.     

Immunohistochemical localization of transthyretin in glomerular peripolar cells of newborn sheep

Summary Purified transthyretin has been isolated from sheep serum. Antiserum raised against this protein has been used with an indirect immunoperoxidase histochemical technique to identify transthyretin in newborn lamb kidney tissue. Transthyretin was found in proximal tubule cells and in glomerular peripolar cells. Preabsorption studies using purified transthyretin protein indicate that the immunoreactivity of the antiserum is specific to transthyretin.     

A comparative study of the glomerular peripolar cell and the renin-secreting cell in twelve mammalian species

The peripolar cell is a glomerular epithelial cell situated within Bowman's capsule at its vascular pole. It is believed to be a secretory cell which forms part of the juxtaglomerular apparatus. Scanning electron microscopy was used to perform a comparative study of the morphology and number of peripolar cells in twelve mammalian species. The number of renin-secreting cells in kidney sections stained by renin antibodies and immunocytochemistry was counted. There was a marked inter-species variation in the number, size and appearance of peripolar cells. They were largest and most abundant in sheep and goat and fewest in dog, cow and human. There was no correlation between the numbers of peripolar cells and renin-secreting cells. This does not support the view that the peripolar cell is part of the juxtaglomerular apparatus.     

Distribution of glomerular peripolar cells in different mammalian species

Summary Peripolar cells are granulated glomerular epithelial cells that form a cuff around the vascular pole of the glomerulus. Quantitation of these cells in 17 species of mammals (including man, several laboratory animals and a variety of other species) indicated that they were detectable by light microscopy in all but one of the mammals that were examined (the Australian hopping mouse). In adult mammals with detectable peripolar cells, the peripolar cell index (the percentage of randomly sectioned glomeruli that displayed peripolar cells in histological sections of kidney) ranged from 0.15 (for echidna) to 11.86 (for sheep). Newborn lambs and rats showed strikingly high values (23.30 and 10.76, respectively) compared with their adult counterparts. Using electron microscopy, peripolar cells were observed in all species that were examined, including the Australian hopping mouse. Morphologically, peripolar cells were similar in all species although their size and granule population varied. They showed a predominantly outer cortical glomerular distribution and a close anatomical relationship with the renin-containing myoepithelioid cells. These findings indicate that peripolar cells are present in a wide variety of species and support the view that such cells may play a significant role in the regulation of normal renal function.     

Cholinergic reactivity of cerebral arteries in the developing fetal and newborn lamb.

Cerebral arteries of newborn pigs and baboons constrict to acetylcholine, suggesting that endothelium-dependent dilator mechanisms may be lacking in immature cerebral arteries. The present study tested this possibility in the immature sheep by examining the response of cerebral arterioles in fetal and newborn sheep to endothelium-dependent dilator, acetylcholine. Pial arteriolar diameter was measured in 9 anaesthetized foetuses in utero (4 preterm, 90-111 days gestation and 5 term, 128-143 days gestation) and in 5 anaesthetized, newborn lambs (14 days) using a closed cranial window with intravital microscopy. Application of acetylcholine to the pial surface induced dose-dependent increase in pial arteriolar diameter in all age groups; EC50 for acetylcholine was 0.10 +/- 0.03, 0.28 +/- 0.08 and 0.26 +/- 0.17 microM for preterm fetal, term fetal, and newborn lambs, respectively. The data demonstrate a sensitive dilator response to acetylcholine in immature fetuses as well as newborn lambs suggesting that cholinergic-mediated release of endothelium-dependent relaxing factor is functional early in gestation. The contractile response to acetylcholine observed in newborn pigs and premature baboons may reflect a species difference rather than maturational lack of endothelium-dependent dilator mechanisms.     

IL-10 secreting CD21+ B cells are present in jejunal Peyer’s patches of sheep during fetal development

We recently reported a novel interleukin-10 (IL-10)-secreting CD21⁺ B cell population in jejunal Peyer’s patches (JPP) of sheep with a regulatory function (B_regs) suppressing Toll-like receptor 9 (TLR9)-induced cytokine responses. However, little is known about the development of these cells. Therefore, we investigate their existence in JPP cells from fetal and newborn lambs. CD21⁺ B cells were purified from JPP cells by magnetic cell sorting and subsequently stimulated with the TLR9 agonist, CpG ODN (CpG oligodeoxynucleotide). Lymphocyte proliferative responses, cytokine production (IL-10, IL-12 and interferon-γ [INF-γ]) and antibody secretion were assayed. We found that fetal and neonatal CD21⁺ B cells spontaneously secreted high levels of IL-10 regardless of CpG stimulation but that these cells did not produce any IL-12 or INF-γ upon stimulation with CpG. The observed responses are consistent with those previously reported for B_regs characterized in JPP of older lambs. Surprisingly, unlike in older lambs, fetal and neonatal JPP CD21⁺ B cells proliferated in response to CpG stimulation. Our investigations of fetal and neonatal lambs provide evidence for the development of IL-10-secreting CD21⁺ B cells in PPs prior to antigen exposure.     

The influence of plasma lipoproteins on steroidogenesis of cultured ovine fetal and neonatal adrenal cells

The present study examined the effects of serum and lipoproteins on the function of cultured adrenal cells from 115-127-day-old ovine fetuses and from newborn lambs. On day 1 of culture, corticosteroid output was similar in medium containing 2% horse serum or in serum-free medium, both for fetal and neonatal cells. However, on day 5, cells cultured in the absence of serum produced smaller amounts of these steroids than cells maintained in medium containing serum; the difference was more marked under ACTH1-24 stimulation. Conversely, cAMP production was never lower in the absence than in the presence of serum. When stimulated by ACTH1-24 on day 2 of culture, fetal or neonatal adrenal cells incubated in the presence of a saturating concentration of ovine LDL produced more corticosteroids than cells incubated in serum-free medium; HDL also enhanced ACTH1-24-induced steroidogenesis, but to a lesser extent. VLDL was effective only with neonatal cells. In fetal and neonatal cells cultured for 6 days in ACTH-free medium, VLDL and LDL increased ACTH-induced steroidogenesis, but HDL did not. On the other hand, when cells were cultured in the presence of ACTH1-24, LDL and HDL were equipotent in supporting ACTH1-24-induced steroid output. Three major lipoprotein fractions were observed in serum of fetal and newborn lambs. The concentration of cholesterol was very low in the VLDL fraction of fetuses, but it was similar to that of newborns in LDL. Conversely, 4 times more cholesterol was present in HDL of newborns than in HDL of fetuses. These results suggest that: (i) after several days of cell culture, cholesterol availability is an important limiting factor for the steroidogenesis of cells maintained under serum-free conditions; (ii) both an "LDL pathway" and an "HDL pathway" are operating in adrenal cells from fetal as well as newborn sheep; (iii) LDL and HDL are important physiological sources of cholesterol to support steroidogenesis by fetal and neonatal adrenal cells.     

Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish

Summary Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinaced), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphryna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowman's capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish.     

Experimental Modelling of the Consequences of Brief Late Gestation Asphyxia on Newborn Lamb Behaviour and Brain Structure

Brief but severe asphyxia in late gestation or at the time of birth may lead to neonatal hypoxic ischemic encephalopathy and is associated with long-term neurodevelopmental impairment. We undertook this study to examine the consequences of transient in utero asphyxia in late gestation fetal sheep, on the newborn lamb after birth. Surgery was undertaken at 125 days gestation for implantation of fetal catheters and placement of a silastic cuff around the umbilical cord. At 132 days gestation (0.89 term), the cuff was inflated to induce umbilical cord occlusion (UCO), or sham (control). Fetal arterial blood samples were collected for assessment of fetal wellbeing and the pregnancy continued until birth. At birth, behavioral milestones for newborn lambs were recorded over 24 h, after which the lambs were euthanased for brain collection and histopathology assessments. After birth, UCO lambs displayed significant latencies to (i) use all four legs, (ii) attain a standing position, (iii) find the udder, and (iv) successfully suckle - compared to control lambs. Brains of UCO lambs showed widespread pathologies including cell death, white matter disruption, intra-parenchymal hemorrhage and inflammation, which were not observed in full term control brains. UCO resulted in some preterm births, but comparison with age-matched preterm non-UCO control lambs showed that prematurity per se was not responsible for the behavioral delays and brain structural abnormalities resulting from the in utero asphyxia. These results demonstrate that a single, brief fetal asphyxic episode in late gestation results in significant grey and white matter disruption in the developing brain, and causes significant behavioral delay in newborn lambs. These data are consistent with clinical observations that antenatal asphyxia is causal in the development of neonatal encephalopathy and provide an experimental model to advance our understanding of neuroprotective therapies.     

Plasma and urinary clearance rates of atrial natriuretic factor during ontogeny in sheep

The present study was designed to determine the plasma clearance rate of atrial natriuretic factor (ANF) during development in chronically-instrumented fetal, newborn and adult non-pregnant sheep. To determine the contribution of the kidney in the metabolism of ANF, urinary clearance of ANF was also measured. Intravenous infusion of ANF (0.025 and 0.1 microgram.min-1.kg-1) produced a significant decrease in mean arterial blood pressure in newborn lambs and in adult non-pregnant sheep. Estimated plasma ANF clearance rate for the 0.025 and 0.1 microgram.min-1.kg-1 ANF infusion rate were respectively 177 +/- 55 and 155 +/- 34 ml.min-1.kg-1 in fetuses, 138 +/- 26 and 97 +/- 13 ml.min-1.kg-1 in newborn lambs and, 148 +/- 33 and 103 +/- 25 ml.min-1.kg-1 in adult nonpregnant ewes. Fetal, newborn and adult ANF plasma clearance rates during high ANF infusion rate (0.1 microgram.min-1.kg-1) were not significantly different. Low or high ANF infusion rate was not associated with significant changes in urinary ANF concentration or urinary ANF excretion rate. Taken together, the present study demonstrates that ANF plasma clearance rate is similar in fetal, newborn and adult non-pregnant sheep and that the excretory function of the kidney contributes only minimally to ANF plasma clearance rate.     

Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells.

Bone marrow transplantation to reconstitute defective hematopoietic cell lines in children with congenital defects is limited by donor availability, graft rejection, and graft-versus-host disease (GVHD). These problems can be eliminated by transplanting normal preimmune fetal hematopoietic stem cells (HSC) into an unrelated preimmune fetal recipient. We show here that injections of allogeneic fetal stem cells into preimmune fetal lambs and monkeys result in long-term stable hematopoietic chimerism. HSCs harvested from the livers of preimmune fetal sheep and monkeys when injected into the peritoneal cavity of young unrelated fetal sheep and monkey recipients results in stable, long-term postnatal hematopoietic chimerism involving lymphoid, erythroid, and myeloid cells of donor origin. Donor cell engraftment was achieved without the use of cytoablative procedures and without the development of GVHD.     

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.     

妊娠晚期胎羊宫内外科手术的实验研究

目的　以妊娠晚期孕羊为研究对象 ,探讨宫内外科手术的可行性 ,为开展胎儿外科研究建立动物实验模型。方法　通过对妊娠 12 0d左右的 4只双胎孕羊进行宫腔手术 ,切除双胎中 1只胎羊的脚趾或唇部皮肤 ,观察妊娠结果。并取分娩后羔羊脑组织及手术切除部位皮肤作组织形态学观察和评价。结果　妊娠羊经阴道足月分娩后羔羊均成活 ,脚趾及唇部切除处未见疤痕形成 ,皮肤组织形态学观察对照组与实验组未见明显差异 ;脑组织形态学检查仅有 1例实验组羔羊有轻度病理学变化 ,与对照羔羊有差异 ,其余 3例实验羔羊与对照羔羊脑组织结构均正常 ,未见有明显的病理学改变。结论　妊娠羊经宫内胎羊外科手术后能够持续妊娠状态并经阴道自然分娩 ,产出羔羊的精神状态及病理组织学观察结果表明 ,利用妊娠晚期孕羊进行胎儿外科动物实验研究是可行的 ,为进一步开展人类先天性畸形胎儿的早期治疗奠定了动物实验基础     

Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus specifically associated with a contagious lung tumor of sheep, sheep pulmonary adenomatosis (SPA). JSRV replicates actively in the transformed epithelial cells of the lung, and JSRV DNA and RNA have been detected in lymphoid tissues of naturally affected animals. To determine the lymphoid target cells of JSRV, CD4(+) T cells, CD8(+) T cells, B lymphocytes, and adherent cell (macrophage/monocyte) populations were isolated from the mediastinal lymph nodes of naturally affected sheep and lambs inoculated with JSRV. Cells were enriched to high purity and then analyzed for JSRV proviral DNA by heminested PCR, and the proviral burden was quantitated by limiting dilution analysis. JSRV proviral DNA was found in all subsets examined but not in appropriate negative controls. In sheep naturally affected with SPA, JSRV proviral burden was greatest in the adherent cell population. In the nonadherent lymphocyte population, surface immunoglobulin-positive B cells contained the greatest proviral burden, while CD4(+) and CD8(+) T cells contained the lowest levels of JSRV proviral DNA. In most of the cases (5 of 8), provirus also could be detected in the peripheral blood mononuclear cell (PBMC) population. A kinetic study of JSRV infection in the mediastinal lymphocyte population of newborn lambs inoculated with JSRV found that JSRV proviral DNA could be detected as early as 7 days postinoculation before the onset of pulmonary adenomatosis, although the proviral burden was greatly reduced compared to adult natural cases. This was reflected in the levels found in PBMC since proviral DNA was detected in 2 of 13 animals. At the early time points studied (7 to 28 days postinoculation) no one subset was preferentially infected. These data indicate that JSRV can infect lymphoid and phagocytic mononuclear cells of sheep and that dissemination precedes tumor formation. Infection of lymphoid tissue, therefore, may play an important role in the pathogenesis of SPA.     

Sister-chromatid exchange in cultured lymphocytes of ewes and their newborn lambs

The incidence of sister-chromatid exchange (SCE) in cultured lymphocytes of ewes and their newborn lambs was determined using the BrdU-Giemsa technique. In all ewe-lamb pairs, the SCE rate in the lambs was less than that of the ewes. The mean SCE frequencies per chromosome of the ewes after lambing and of the newborn lambs were 0.1909 and 0.1581, respectively. The statistical analysis shows that a significant difference exists between SCE in the adult female sheep and their lambs. At the same time, a negative correlation was observed between SCE rate and cell proliferation. The results of this study are compared with those of previous reports on age-dependency of SCE.     

Immunogold co-localization of ovine placental lactogen and the antigen recognized by the SBU-3 monoclonal antibody in sheep placental granules

Ovine placental lactogen and the SBU-3 antigen (derived from a trophoblast membrane preparation), two proteins of widely different structure, function and destination, were shown by ultrastructural immunogold techniques to localize in identical structures in the sheep placentome throughout most of pregnancy. Both were restricted to the ultrastructurally similar membrane-bounded granules in the chorionic fetal binucleate cell and the syncytium at the fetomaternal interface. The Golgi body from which the granules form was also doubly labelled but only in the binucleate cell, never the syncytium. This provides further evidence that the binucleate cells migrate and fuse to form the syncytium. The two proteins were homogeneously distributed in the granules and would be released together by exocytosis. Only the lactogen reaches the fetal and maternal circulations so the SBU-3 may have some more local function. In early pregnancy the SBU-3 antigen is found by itself in the granules, indicating that the association with the lactogenic hormone is not obligatory. Neither antigen was found consistently in the otherwise ultrastructurally similar interplacentomal binucleate cell granules, corroborating the presence of two functional populations of binucleate cells.     

Regulation of brain water during acute glucose-induced hyperosmolality in ovine fetuses, lambs, and adults.

We tested the hypothesis that, during acute glucose-induced hyperosmolality, the brain shrinks less than predicted on the basis of an ideal osmometer and that brain volume regulation is present in fetuses, premature and newborn lambs. Brain water responses to glucose-induced hyperosmolality were measured in the cerebral cortex, cerebellum, and medulla of fetuses at 60% of gestation, premature ventilated lambs at 90% of gestation, newborn lambs, and adult sheep. After exposure of the sheep to increases in osmolality with glucose plus NaCl, brain water and electrolytes were measured. The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress. In the absence of volume regulation, brain solute remains constant as osmolality changes. The osmotically active solute demonstrated direct linear correlations with plasma osmolality in the cerebral cortex of the fetuses at 60% of gestation (r = 0.72, n = 24, P = 0.0001), premature lambs (r = 0.58, n = 22, P = 0.005), newborn lambs (r = 0.57, n = 24, P = 0.004), and adult sheep (r = 0.70, n = 18, P = 0.001). Similar findings were observed in the cerebellum and medulla. Increases in the quantity of osmotically active solute over the range of plasma osmolalities indicate that volume regulation was present in the brain regions of the fetuses, premature lambs, newborn lambs, and adult sheep during glucose-induced hyperosmolality. We conclude that, during glucose-induced hyperosmolality, the brain shrinks less than predicted on the basis of an ideal osmometer and exhibits volume regulation in fetuses at 60% of gestation, premature lambs, newborn lambs, and adult sheep.