Premature rupture of fetal membranes can harm infant and mother. It is unclear whether structural changes predispose these membranes to breaking. We thus assessed rat visceral yolk sac placenta (VYSP) and amnion by light and by transmission electron microscopy on Days 18-21 of gestation. Light microscope sections were stained for connective tissue (extracellular matrix) components: collagen, glycoprotein, and glycosaminoglycans/proteoglycans. Some tissue was incubated with chondroitinase ABC. We observed that fetal membranes became increasingly fragile, rupturing readily on Day 21. On Days 18-20, the two epithelial layers of the capsular VYSP were separated by a well-developed, well-vascularized connective tissue layer that stained intensely for all matrix components studied; on Day 21, the connective tissue layer was thinner, moderately stained, and less vascularized. On Days 18-20, the two cellular layers of the amnion were separated by a narrow, compact connective tissue layer that stained modestly for all matrix components; on Day 21, this area was widened and stained faintly. Transmission electron microscopy showed that collagen fibrils of the amnion were abundant, closely packed, and well organized on Days 18-20, whereas on Day 21 they were few in number, widely spaced, and disorganized. Similar changes were present after incubation with chondroitinase ABC. In addition, amniotic epithelial cells were moribund and delaminating, basal laminae were deteriorating or absent, and few cells were at the outer surface of the amnion. All changes preceded parturition. We conclude that the structural integrity of rat fetal membranes is impaired before birth through the loss of connective tissue components and cells, changes that presumably underlie membrane rupture. Lastly, the similarity of structural changes in rat and human fetal membranes point to the potential usefulness of the rat model.

We report the cloning of a complementary DNA for the mouse homolog of the very low density lipoprotein (VLDL)/apolipoprotein-E receptor (VLDLR), the deduced amino acid sequence of the protein, and the mapping of the gene encoding the receptor to mouse chromosome 19. Northern hybridization revealed that the VLDLR messenger RNA (mRNA) is most abundant in skeletal muscle, heart, kidney, and brain. It was also detected in lung and in low levels in liver, but it was not found in spleen or testes. Levels of VLDLR mRNA in mouse placenta increased from days 8-18 of gestation. The VLDLR mRNA was induced in 3T3-L1 cells undergoing differentiation into adipocytes. The increase in VLDLR mRNA paralleled the rise in lipoprotein lipase and hormone-sensitive lipase mRNAs. However, VLDLR and low density lipoprotein receptor-related protein were increased in the presence of retinoic acid, whereas the induction of lipoprotein lipase and hormone-sensitive lipase mRNAs was inhibited. Our observations demonstrate regulated expression of the VLDLR gene in placenta and adipocytes, where the receptor protein may play roles in the uptake of triglyceride-rich particles for storage of lipid (adipocytes) or for lipid transport to the fetus (placenta). The availability of a murine complementary DNA probe and the knowledge of the map position of the VLDLR gene in the mouse genome will facilitate studies on the function and regulation of this protein.

The origin of thylakoid membranes was studied in Chlamydomonas reinhardtii y-1 cells during greening at 38-degrees-C. Previous studies showed that, when dark-grown cells are exposed to light under these conditions, the initial rates of accumulation of chlorophyll and the chlorophyll a/b-binding proteins in membranes are maximal (MA Maloney JK Hoober, DB Marks [1989] Plant Physiol 91: 1100-1106; JK Hoober MA Maloney, LR Asbury, DB Marks [1990] Plant Physiol 92: 419-426). As shown in this paper, photosystem II activity, which was nearly absent in dark-grown cells, also increased at a linear rate in parallel with chlorophyll. As compared with those made at 25-degrees-C, photosystem II units assembled during greening at 38-degrees-C were photochemically more efficient, as judged by saturation at a lower fluence of light and a negligible loss of excitation energy as fluorescence. Electron microscopy of cells in light for 5 or 15 minutes at 38-degrees-C showed that these initial, functional thylakoid membranes developed in association with the chloroplast envelope.

We used electron microscopy, acid hydrolase cytochemistry, and biochemistry to analyze the uptake and metabolism of colloidal gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein (LDL) by cultured rat granulosa cells. The initial interaction of gold-LDL conjugates with granulosa cells occurred at binding sites diffusely distributed over the plasma membrane. After incubation with ligand in the cold, 99.9% of the conjugates were at the cell surface but less than 4% lay over coated pits. Uptake was specific since it was decreased 93-95% by excess unconjugated LDL and heparin, but only 34-38% by excess unconjugated human high density lipoprotein. LDL uptake was related to granulosa cell differentiation; well-luteinized cells bound 2-3 times as much gold-LDL as did poorly luteinized cells. Ligand internalization was initiated by warming and involved coated pits, coated vesicles, pale multivesicular bodies (MVBs), dense MVBs, and lysosomes. A key event in this process was the translocation of gold-LDL conjugates from the cell periphery to the Golgi zone. This step was carried out by the pale MVB, a prelysosomal compartment that behaves like an endosome. Granulosa cells exposed to LDL labeled with gold and [3H]cholesteryl linoleate converted [3H]sterol to [3H]progestin in a time-dependent manner. This conversion was paralleled by increased gold-labeling of lysosomes and blocked by chloroquine, an inhibitor of lysosomal activity. In brief, granulosa cells deliver LDL to lysosomes by a receptor-mediated mechanism for the hydrolysis of cholesteryl esters. The resulting cholesterol is, in turn, transferred to other cellular compartments, where conversion to steroid occurs. These events comprise the pathway used by steroid-secreting cells to obtain the LDL-cholesterol vital for steroidogenesis.

Secretory granules, which are released by exocytosis and are speculated to contain progesterone, have been described in luteal cells of sheep and other large domestic animals. These granules are small and densely staining. Gemmell and Stacy ('79) suggested that luteal cells of guinea pigs also contain secretory granules, although they could not document exocytosis of granule content at the fine structural level. For the present study, quantitative methods were used to reexamine the possibility that luteal cells of guinea pigs possess secretory granules. Ovaries of guinea pigs were fixed in situ by vascular perfusion at the time of maximum progesterone secretion, when such granules would be most abundant, as well as other stages. Two types of granules that might be confused with secretory granules are microperoxisomes and lysosomes. Therefore, slices of perfusion-fixed corpora lutea were incubated for the fine structural localization of a peroxisomal enzyme, catalase, or for the lysosomal enzymes, acid phosphatase (ACPase) and arylsulfatase. Other tissue was prepared for conventional electron microscopy. Granule types were classified on the basis of size, morphology, and enzyme content. Quantitation of granule types was carried out on both cytochemically reacted and conventionally prepared luteal tissue. More than 5500 microperoxisomes, 2800 lysosomes, and 1100 multivesicular bodies (MVBs) were tabulated. The results indicate that luteal cells of guinea pigs have three main types of granules: 1) Microperoxisomes, about 0.2 micrometer in diameter and containing catalase; 2) lysosomes, about 0.5 micrometer in diameter and positive for ACPase and arylsulfatase; and 3) MVBs, about 0.4 micrometer in diameter and containing small vesicles. At the time of peak steroid secretion during pregnancy and the estrous cycle, the granule population in luteal cells of guinea pigs consists of 73-80% microperoxisomes, 13-17% lysosomes, and 7-9% MVBs. These proportions are similar in tissue reacted for cytochemistry and tissue prepared by conventional means. Greater than 99% of the small 0.2-0.3 micrometer diameter granules in guinea pig luteal cells are catalase reactive. This finding eliminates from further consideration most of the prime candidates for secretory granules in these cells. Finally, neither a sequential appearance of granules nor exocytosis of secretory product was detected. Our data thus argue against the suggestion that luteal cells of guinea pig have secretory granules of the type observed in corpora lutea of large domestic animals.

Activated macrophages phagocytize moribund luteal cells and thus play a central role in the postpartum regression of corpora lutea in guinea pigs (Paavola, '79). When viewed by transmission electron microscopy (TEM), these luteal macrophages exhibit many surface protrusions. To characterize more fully the nature and extent of these evaginations, as well as to gain further understanding of phagocytes in their natural surroundings, luteal macrophages were studied in situ by scanning electron microscopy of regressing corpora lutea. Correlated TEM was carried out to confirm the identity of the various cell types. Even in low power scanning electron micrographs, macrophages are conspicuous, and can be readily distinguished from luteal cells by their surface topography. Luteal cell surfaces bear low ridge‐like folds and sparse microvilli. In contrast, macrophages characteristically exhibit highly developed surface projections, the most common of which are knob‐like or clubbed processes of varying size and shape. Other distinctive surface modifications displayed by luteal macrophages include long, slender filopodia, and well developed pseudopodia. These processes generally have an uneven distribution over the cell; thus, luteal macrophages may appear polarized with regard to surface activity. Both filopodia and pseudopodia occur in close contact with luteal cell surfaces. In addition, occasional luteal macrophages have surfaces that are covered with large, crater‐like depressions. The phagocytosis ofcells and cellular debris by macrophages was also observed. In summary, the highly pleomorphic surface activity of luteal macrophages appears to be correlated with their role in the removal of senescent luteal cells.