Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatmentinduced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulsechase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/ medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.

The cytochemical distribution of two different acid phosphatases, nicotinamide adenine dinucleotide phosphatase (NADPase) and cytidine monophosphatase (CMPase), was analyzed within the Golgi apparatus, acrosomic system, and chromatoid body of early spermatids. Within the saccules composing the Golgi cortex, the phosphatases were localized in specific and nonoverlapping compartments: the cis-tubular network, or cis-element, and the first underlying saccule were unreactive for both enzymes, the following four to five saccules were reactive for NADPase, and the last three saccules were reactive for CMPase. Vesicles of various sizes and short membranous tubules, present near the edges of saccules and in the medulla of the Golgi apparatus, were NADPase- and/or CMPase-positive. The acrosomic system showed a weak but significant NADPase reactivity and a strong CMPase reactivity. The vesicles associated with the chromatoid body were reactive for NADPase and/or CMPase. The combined observations indicate that the two cytochemically distinct compartments of the Golgi cortex, the NADPase- and CMPase-positive saccules, independently contribute to the formation of the acrosomic system and the vesicular component of the chromatoid body.

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Summary -The glucose-6-phosphatase (G6Pase) activity of cytoplasmic components of spermatocytes and spermatids of the rat was examined by electron microscope cytochemistry using cerium chloride as a capture agent. G6Pase activity, a recognized ERresident enzyme, was present in all ER cisternae of spermatocytes. In spermatids, while some ER cisternae were G6Pase-rcactive, others were negative or only slightly reactive, indicating an unequal distribution of the enzymatic activity throughout the network of ER cisternae in these cells. In spermatocytes, the c/s-and trans-elements of the stacks of Golgi saccules were slightly but significantly reactive for G6Pase. In the Golgi apparatus of spermatids, the c/s-element, 4 or 5 underlying saccules, as well as one or two thick trans Golgi elements were G6Pase reactive. The G6Pase activity of the various Golgi elements, fike that of the ER cisternae was not affected by the pH of the medium and was completely inhibited by Na-vanadate, a known G6Pase inhibitor. Sertoli and Leydig cells, submitted to the same cytochemical conditions, showed complete G6Pase reactivity of their ER; however in Sertoli cells, all Golgi components were consistently negative while in Leydig cells the c/s-and trans-elements of the Golgi stacks were slightly reactive, as in spermatocytes. Thus, the G6Pase reactivity of Golgi elements, appeared variable from one cell type to another. The compact juxtanuclear Golgi apparatuses of spermatocyte, and spermatids were both associated with numerous G6Pase reactive ER cisternae; some were present at their Curface, others crossed their cortices between Golgi stacks and formed elaborate networks in their cores. Other ER cisternae, in tur:,, were closely apposed to or intertwined with trans-elements of the Golgi stacks, a relationship raising the possibility of molecular ex~,,langes at this pole of the stacks in germinal cells.lllueose-6-phosphatme I endopluml¢ retleulum I Golgi apparatus I spermulocytes I spermatids

In hepatocytes from inflamed rats an apparent switch had occurred in the type of secretory material processed by the Golgi apparatus. Furthermore, the inflammation-induced increase in the size of individual Golgi stacks apparently was not due to a parallel increase in size of all Golgi saccules. Rather, saccules within given Golgi compartments responded in a characteristic and specific manner to the increase in glycoprotein processing that occurs during inflammation.