Ultrastructure of the radula protractor of Busycon canaliculatum

Summary The distribution of the sarcoplasmic reticulum and sarcolemmic tubules in the radula protractor muscle of the whelk, Busycon canaliculatum, has been investigated. The sarcoplasmic reticulum consists of an interconnected system of cisternae and tubular channels. The cisternae are closely associated with the sarcolemma. The tubular channels project from the cisternae into the interior of the cell and run parallel to the long axis of the myofilaments. Parallel tubular channels are interconnected with one another by short branches. This finding of an elaborate sarcoplasmic reticulum supports previous physiological work on this smooth muscle which indicated the presence of an intracellular compartmentalization of calcium ions. There is also an extensive system of tubular invaginations of the sarcolemma which we have termed sarcolemmic tubules. These tubules are 600 Å in diameter and about 0.5 microns in length. There is a substructure associated with the leaflet of the tubular membrane bordering the extracellular space. The sarcolemmic tubules penetrate only half a micron from the surface of the cell and interdigitate with the sarcoplasmic reticulum associated with the sarcolemma. Calculations have shown that the surface area of this smooth muscle cell is more than doubled by the presence of sarcolemmic tubules.     

Phosphate transport after acute changes in total NAD content in renal proximal tubules

Suspensions of proximal tubules were obtained by collagenase digestion of rat renal cortex followed by centrifugation on a percoll gradient. NAD content in tubules incubated at 37 degrees C was decreased by 40-60% compared with tubules incubated at 4 degrees C. This change occurred within 30 min and was maintained for up to 2 hr. Inhibitors of NAD hydrolysing enzymes prevented the depletion of cellular NAD at 37 degrees C. Acute changes in proximal tubule NAD content at 37 degrees C were not accompanied by changes in phosphate uptake by brush border membrane vesicles subsequently prepared from the same tubules. In contrast, incubation of tubules with parathyroid hormone (10(-6) M) produced the expected inhibition (20%) of brush border membrane transport of phosphate. One implication of these findings is that acute changes in total NAD content of proximal tubules at 37 degrees C may not influence the phosphate transport system in the renal brush border membrane. Other interpretations are discussed.     

Hormonal stimulation of adenosine 3',5'-cyclic monophosphate formation in cell-free particles and intact cells of smooth muscle in the aorta and femoral artery of rats during immubilization stress]

The ability of adrenaline and histamine to stimulate the formation of cyclic AMP was investigated in broken cell preparation and intact cells of smooth muscle of the aorta and femoral artery of rats which had been subjected to daily intermittent immobilization for 1, 3, and 17 weeks. It was found that this type of stress led to an instability of the blood pressure which was associated with an increase in the sensitivity of adenylate cyclase in the broken cell preparations from the arteries to adrenaline and histamine and with a heightened cyclic AMP response to the two hormones in the intact arterial smooth muscle cells. The sensitivity of cardiac adenylate cyclase for adrenaline remained unchanged.     

Possible role of calcium mediators in parathyroid hormone action on phosphate transport in rabbit renal brush border membrane

The possibility of the involvement of intracellular calcium in the action of parathyroid hormone on phosphate transport in renal brush border membrane was examined. Preincubation of rabbit renal proximal tubules with parathyroid hormone or 8-bromo-cAMP induced a significant inhibition on phosphate uptake by the brush border membrane vesicles isolated therefrom. The addition of intracellular Ca antagonists, trifluoperazine or W-7, to the preincubation medium, alone was without effect on phosphate uptake by the brush border membrane vesicles, but abolished the inhibitory effects of parathyroid hormone and 8-bromo-cAMP.     

Thiobarbituric acid-reactive substance formation of rat kidney brush border membrane vesicles induced by ferric nitrilotriacetate

An iron chelate, ferric nitrilotriacetate (Fe3+-NTA), is nephrotoxic and also carcinogenic to the kidney in experimental animals. Iron-promoted lipid peroxidation in the proximal tubules is thought to be responsible for the pathologic process. In the present study, iron-promoted lipid peroxidation, with thiobarbituric acid (TBA) formation as an indication, in the tubular surface was simulated in vitro using rat kidney brush border membrane vesicles and the results were compared with those using linoleate micelles and rat liver microsomal lipid liposomes. Addition of ascorbate, cysteine, or dithiothreitol to the Fe3+-NTA solution resulted in consumption of dissolved oxygen and promoted the lipid peroxidation in the micelles and in the liposomes. In contrast, addition of glutathione to the Fe3+-NTA solution caused only sluggish oxygen consumption and far less peroxidation in these lipid systems. When the brush border membrane vesicles were used for the peroxidation substrate, Fe3+-NTA and glutathione could promote TBA formation at a rate comparable to that elicited by Fe3+-NTA with cysteine or dithiothreitol. Acivicin, a gamma-glutamyl transpeptidase inhibitor, suppressed the peroxidation of the brush border membrane vesicles promoted by Fe3+-NTA and glutathione. These results suggest the following mechanism of proximal tubular cell lipid peroxidation promoted by Fe-NTA: Fe3+-NTA filtered through glomeruli is rapidly reduced by cysteine and Fe2+-NTA starts lipid peroxidation at the site, leading to proximal tubular necrosis. Cysteine is amply supplied by the decomposition of glutathione within the lumen by the action of gamma-glutamyl transpeptidase and dipeptidase situated at the proximal tubular brush border membrane.     

Effect of phosphate deprivation on enzymes of the cyclic adenosine monophosphate metabolism in rat proximal convoluted and proximal straight tubules.

Phosphate deprivation causes a resistance to the phosphaturic effect of parathyroid hormone (PTH). The present study determined whether acute phosphate deprivation alters basal or stimulated activities of key enzymes of the cyclic adenosine monophosphate (cAMP) metabolism in microdissected proximal convoluted and proximal straight tubules, since blunted cAMP levels in these proximal subsegments might account for refractoriness to the effect of PTH on phosphate reabsorption in the proximal convoluted and proximal straight tubule segments. In the proximal convoluted tubules of rats fed a normal-phosphate diet (NPD), PTH increased the adenylate cyclase activity by tenfold. In the proximal convoluted tubule of rats fed a low-phosphate diet (LPD), PTH also increased the adenylate cyclase activity by tenfold. In addition, forskolin increased the adenylate cyclase activity to levels similar to PTH in the proximal convoluted tubule of rats fed NPD or LPD. In the proximal straight tubule of rats fed NPD, PTH resulted in an approximately fivefold increase in adenylate cyclase activity. In the proximal straight tubule of rats fed LPD, PTH resulted in a fourfold increase in adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was markedly decreased (46%) in the proximal straight tubule of phosphate-deprived rats. The cAMP-phosphodiesterase activity in the proximal convoluted tubule was significantly increased by 26% in phosphate-deprived rats. The cAMP-phosphodiesterase activities in the proximal straight tubules from rats fed NPD or LPD were similar. We conclude that distinct differences in key enzymes of cAMP metabolism exist in the proximal convoluted and proximal straight tubule subsegments. Further, phosphate deprivation affects the cAMP-phosphodiesterase and adenylate cyclase activities differently in these nephron subsegments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of parathyroid hormone-stimulated adenylate cyclase in plasma membranes of cells of the kidney cortex.

Free flow electrophoresis was employed to separate renal cortical plasma membranes into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. During the separation adenylate cyclase activity was found to parallel the activity of Na+-K+-activated ATPase, an enzyme which is present in contraluminal but not in luminal membranes. In the basal-lateral membrane fraction the specific activities of adenylate cyclase and Na+-K+-activated ATPase were 4.4 and 4.6 times greater, respectively, than in the brush border fraction. The adenylate cyclase of the basal-lateral membrane fraction was specifically stimulated by parathyroid hormone which maximally increased enzyme activity eightfold. The biologically active (1-34) peptide fragment of paratyhroid hormone produced a 350% increase in adenylate cyclase activity. In contrast, calcitonin, epinephrine and vasopressin maximally stimulated the enzyme by only 55, 35 and 30%, respectively. These results indicate that adenylate cyclase, specifically stimulated by parathyroid hormone, is distributed preferentially in the contraluminal region of the plasma membrane of renal cortical epithelial cells.     

Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia

Rabbit nephron segments of proximal convoluted tubules (PCT); proximal straight tubules (PST); cortical and medullary thick ascending limbs of Henle's loop (CAL, MAL); and cortical, outer medullary, and inner medullary collecting tubules (CCT, OMCT, IMCT) were individually microdissected and grown in monolayer culture in hormone supplemented, defined media. Factors favoring a rapid onset of proliferation included young donor age, distal tubule origin, and the addition of 3% fetal calf serum to the medium. All primary cultures had polarized morphology with apical microvilli facing the medium and basement membrane-like material adjacent to the dish. Differentiated properties characteristic of the tubular epithelium of origin retained in cultures included ultrastructural characteristics and cytochemically demonstrable marker enzyme proportions. PCT and PST were rich in alkaline phosphatase; CAL stained strongly for NaK-ATPase; CCT contained two cell populations with regard to cytochrome oxidase reaction. A CCT-specific anti-keratin antibody (aLEA) was immunolocalized in CCT cultures, and a PST cytokeratin antibody stained PST cultures. The biochemical response of adenylate cyclase to putative stimulating agents was the same in primary cultures as in freshly isolated tubules. In PCT and PST adenylate cyclase activity was stimulated by parathyroid hormone (PTH) but not by arginine vasopressin (AVP); CAL and MAL adenylate cyclase was stimulated by neither PTH nor AVP; CCT, OMCT, and IMCT adenylate cyclase was stimulated by AVP but not by PTH. NaF stimulated adenylate cyclase activity in every cultured segment. It is concluded that primary cultures of individually microdissected rabbit PCT, PST, CAL, MAL, CCT, OMCT, and IMCT retain differentiated characteristics with regard to ultrastructure, marker enzymes, cytoskeletal proteins, and hormone response of adenylate cyclase and provide a new system for studying normal and abnormal functions of the heterogeneous tubular epithelia in the kidney.     

Distribution of prostaglandin E2-sensitive adenylate cyclase along the rat nephron

To define sites of prostaglandin action of renal tubules, the distribution of adenylate cyclase sensitive to prostaglandin E₂ (PGE₂) was examined in single nephron segments dissected from rat kidney. Further, the interaction between PGE₂ and vasopressin on adenylate cyclase activity in nephron segments sensitive to vasopressin was evaluated. Procedures involved in isolating nephron segments were without effects on adenylate cyclase stimulation by PGE₂. PGE₂ stimulated adenylate cyclase activity of the thin descending limb of Henle (tDL), cortical collecting tubules (CCT), and medullary collecting tubules (MCT) at concentrations of 1.4 × 10^?5 to 2.8 × 10^?5 M. PGE₂ was without effects in other nephron segments tested including proximal convoluated tubules, proximal pars recta, the thin and thick ascending limb of Henle's loop, and distal and connecting tubules. PGE₂, at both high (2.8 × 10^?5 M) and low (2.8 × 10^?8 M) concentrations, did not inhibit adenylate cyclase activity stimulated by submaximal doses of vasopressin in medullary thick ascending limb of Henle (MTAL), CCT, and MCT. These data define the distribution of PGE₂-sensitive adenylate cyclase in the rat nephron, i.e., tDL, CCT, and MCT, and show the lack of direct inhibitory actions of PGE₂ on vasopressin sensitive adenylate cyclase in MTAL, CCT, and MCT.     

Metabolism of cyclic AMP in isolated renal tubules: effects of prostaglandins and parathyroid hormone.

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) with maximal effects of PGE1 being 6-8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2-3 fold greater in tubules exposed to PTH than to PGE1. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates. The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Cellular differentiation in the kidneys of newborn mice studies with the electron microscope

The structure of the kidney of the Swiss albino mouse changes progressively during the first 2 weeks after birth. Cells proliferate to form new nephrons, cells differentiate by acquiring specialized membranous components, and certain cytological features which are present at birth diminish in abundance or disappear. The differentiation of the cells of the cortical tubules has been studied using the light and electron microscopes. The tubules are partially and variably differentiated at birth. During the first 2 weeks after birth the brush border develops in the proximal tubules by the accumulation of numerous microvilli on the apical cell margins. Basal striations develop in proximal and distal tubules as an alignment of mitochondria, the result of what appears to be progressive interlocking of adjacent fluted cells. The mitochondria increase in number and size, accumulate homogeneous matrix, and acquire small, very dense granules. The collecting ducts develop tight pleating of the basal cell membranes, and dark cells containing numerous small cytoplasmic vesicles and microvilli appear. At birth there are dense irregular cytoplasmic inclusions presumed to be lipide in renal cells, the cytoplasmic granules of Palade are abundant, and there are large round bodies in the cells of the proximal tubules. The lipide inclusions disappear a few days after birth, and the cytoplasmic granules of Palade diminish in abundance as the cells differentiate. The large round bodies in the proximal tubules consist of an amorphous material and contain concentrically lamellar structures and mitochondria. They resemble the cytoplasmic droplets produced in the proximal tubules of adult rats and mice by the administration of proteins. The large round bodies disappear from the proximal tubules of infant mice during the first week after birth, but the concentric lamellar structures may be found in adult mice.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

Studies on vertebrate kidney. V. PAS-positive lipids in kidneys of fishes from different habitats.

Five fish species living in different habitats, i.e. fresh water, estuarine and marine, were studied for the distribution of PAS-positive materia in various regions of the kidney, 10 minutes' oxidation with 0-5 per cent. HIO4 being employed prior to treatment with Schiff's reagent. PAS-positive material was detected in different sites of the kidney, i.e. brush border of proximal tubules, proximal tubule cells' cytoplasm, distal tubule cells' cytoplasm, glomerulus, basal cell border of proximal tubules and the interstitial cells. Of these sites, the brush border of the proximal tubule of Scoliodon sorrakowah showed the presence of PAS-positive lipids. Elsewhere the PAS-positive reaction was due to carbohydrates. Free aldehyde groups were absent. In Tilapia mossambica and Labeo rohita, PAS staining was enhanced after chloroform-methanol extraction, particularly in the brush border of the proximal tubule. The significance of these findings is discussed.     

Evaluation of the effects of adenosine on hepatic and adipocyte adenylate cyclase under conditions where adenosine is not generated endogenously.

Direct effects of adenosine on adipocyte and hepatic adenylate cyclase have been demonstrated in an assay system where adenosine is not generated. The substrate used, 2'-deoxy ATP may, on metabolism, only give rise to 2'-deoxyadenosine, which does not act at adenosine receptors. With a slight modification of existing assay techniques this assay system has been used to detect a hitherto undiscovered adenosine receptor on liver plasma membranes, which is antagonised by methylxanthines and which stimulates adenylate cyclase activity in a GTP-dependent manner. The potent inhibitory effects of purine-modified adenosine analogs on fat cell adenylate cyclase are reproduced by adenosine in this assay system. An application of this approach to the study of adenylate cyclase not only simplifies detection of the role of adenosine, but also yields insights into the interaction between guanine nucleotides and hormones.     

Cholera toxin facilitates calcium transport in jejunal brush border vesicles

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1. This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.     

Metabolism of cyclic AMP in isolated renal tubules: Effects of prostaglandins and parathyroid hormone

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E₁ (PGE₁) with maximal effects of PGE₁ being 6–8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2–3 fold greater in tubules exposed to PTH than to PGE₁. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates.The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Presence of “Ra” and “P”-site receptors for adenosine coupled to adenylate cyclase in cultured vascular smooth muscle cells

The existence of adenosine receptors coupled to adenylate cyclase in cultured vascular smooth muscle cells from rat aorta is demonstrated in these studies. Adenosine, N⁶-phenylisopropyladenosine, adenosine N′-oxide and 2-chloroadenosine stimulated adenylate cyclase in a concentration dependent manner. The stimulation was dependent on the presence of guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine. In contrast, 2′ deoxyadenosine inhibited adenylate cyclase activity. Adenosine and 2-chloroadenosine showed a biphasic effect on adenylate cyclase, stimulation occurred at low concentrations. The activation of adenylate cyclase by N⁶-phenylisopropyladenosine was also dependent on the Mg²⁺ concentration. The data suggest that vascular smooth muscle cells have both “Ra” and “P” receptors for adenosine, and it can be postulated that the relaxant effect of adenosine on vascular smooth muscle may be mediated by its interaction with “Ra” receptors associated with adenylate cyclase.     

Mn2+-sensitive, soluble adenylate cyclase in rat testis. Differentiation from other testicular nucleotide cyclases.

In subcellular fractions prepared from homogenate of adult rat testis adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity was found in the particulate, primarily 600 X g for 10 min, fractions, as well as in the cytosol. The properties of the adenylate cyclase in the cytosol differs substantially from the adenylate cyclase system associated with the 600 X g for 10 min particulate fraction. The cytosol enzyme, in contrast to the particulate adenylate cyclase, was found to be fluoride- and gonadotropin hormone-insensitive. The cytosol adenylate cyclase appears to be located in the germ cell while the particulate enzyme system in the non-germ cell component of the seminiferous tubules, The cytosol adenylate cyclase was found to be distinct also from the guanylate cyclase present in the rat testis cytosol. The adenylate cyclase appears to be located in the germ cell component while the guanylate cyclase, in the non-germ cell tubular component. Furthermore, it was found that the cytosol guanylate cyclase develops at an earlier stage of spermatogenesis, and precedes the development of the cytosol adenylate cyclase.     

CELLULAR DIFFERENTIATION IN THE KIDNEYS OF NEWBORN MICE STUDIED WITH THE ELECTRON MICROSCOPE

The structure of the kidney of the Swiss albino mouse changes progressively during the first 2 weeks after birth. Cells proliferate to form new nephrons, cells differentiate by acquiring specialized membranous components, and certain cytological features which are present at birth diminish in abundance or disappear. The differentiation of the cells of the cortical tubules has been studied using the light and electron microscopes. The tubules are partially and variably differentiated at birth. During the first 2 weeks after birth the brush border develops in the proximal tubules by the accumulation of numerous microvilli on the apical cell margins. Basal striations develop in proximal and distal tubules as an alignment of mitochondria, the result of what appears to be progressive interlocking of adjacent fluted cells. The mitochondria increase in number and size, accumulate homogeneous matrix, and acquire small, very dense granules. The collecting ducts develop tight pleating of the basal cell membranes, and dark cells containing numerous small cytoplasmic vesicles and microvilli appear. At birth there are dense irregular cytoplasmic inclusions presumed to be lipide in renal cells, the cytoplasmic granules of Palade are abundant, and there are large round bodies in the cells of the proximal tubules. The lipide inclusions disappear a few days after birth, and the cytoplasmic granules of Palade diminish in abundance as the cells differentiate. The large round bodies in the proximal tubules consist of an amorphous material and contain concentrically lamellar structures and mitochondria. They resemble the cytoplasmic droplets produced in the proximal tubules of adult rats and mice by the administration of proteins. The large round bodies disappear from the proximal tubules of infant mice during the first week after birth, but the concentric lamellar structures may be found in adult mice.     

Morphology of the pronephros of the juvenile brown trout, Salmo trutta

The pronephros in juvenile brown trout (Salmo trutta) consists of a large ovoid renal corpuscle and a pair of tubules. The corpuscle is retained for 11 months, after which the glomerulus regresses. The glomerular arteries come directly from the dorsal aorta. The interstitium is permeated with venous blood vessels that arise from the anterior cardinal veins and are closely apposed to the tubules. Two distinct segments of the pronephric tubular system are distinguished by the histological and ultrastructural features of their component cells: 1) a short, transitional neck in which cells change from capsular epithelium to columnar epithelium, typical of tubules; 2) the convoluted segment composed of cells similar to first proximal tubular cells of the opisthonephros with well-formed brush borders, apical vesicles that vary in size and number along this segment, and lysosomes. Pinocytosis and exocytosis are also evident in this segment. The tubular system increases in length and in its convolutions until about week 9, when the opisthonephros develops. Distally each tubule connects with a Wolffian duct, with cells marked by the absence of apical inclusions and the presence of a uniform brush border, numerous mitochondria, and elaborate infolding of the basalar membrane. Nephrostomes, which are often characteristic of pronephroi, are not present. Cells with long cilia are found throughout the tubular system but are most characteristic of the neck and Wolffian-duct segments.