This report describes the fine structure of guinea pig luteal cells during the period of maximum progesterone secretion and throughout involution, in ovaries that have been fixed by perfusion, a technique that provides optimal preservation of steroid-secreting tissues. Smooth endoplasmic reticulum (smooth ER), a prominent organelle in these cells, is particularly well preserved by this method of fixation. During the time of maximal progesterone secretion, luteal cells contain abundant tubules and cisternae of smooth ER, many mitochondria, a well-developed Golgi complex and some lipid droplets. This fine structural picture is consistent with active steroidogenesis.Autophagy plays an important role in the regression of luteal cells in the corpus luteum. The onset of luteolysis is marked by the appearance of structurally complex autophagic vacuoles, one of which has not been described previously in luteal cells. This autophagic vacuole seems to originate from GERL (Golgi-endoplasmic reticulum-lysosomes) as a cup-shaped structure, which subsequently increases in size and complexity. Regressing luteal cells also contain increased numbers of both dense bodies (lysosomes) and lipid droplets, and exhibit changes in nuclear and mitochondria1 morphology. In contrast to previous reports in the literature, changes in the morphology of smooth ER were not observed as a characteristic feature of involution in corpora lutea of guinea pigs in the present study. Advanced regression of luteal cells is characterized by multiple fusion of lipid droplets and a decrease in the amount of smooth ER.Another mechanism active in the breakdown of the corpus luteum is the phagocytosis of luteal cells by macrophages. Although present a t all stages, macrophages are most abundant in older corpora lutea, where they often surround dead or dying luteal cells.The corpus luteum of the ovary is a tissue ideally suited for the study of cellular regression because, following their appearance, mature luteal cells synthesize and secrete steroid hormones for a limited period, then degenerate in response to hormones produced by the uterus and eventually disappear. The involution of cells in response to physiological signals is of general interest. Although the degeneration of luteal cells has been described for a number of mammalian species, the mechanisms underlying this normal involution are still imperfectly understood. Major fine structural studies have dealt with human (Adams and Hertig, '69; van Lennep and Madden, '65; Green and Maqueo, ,651, rabbit '66). hamster (Leavitt et al., '73) sheep (Deane et al., '66; Bjersing et al., '70a,b; Gemmell et al., '761, cow (Priedkalns and Webeq '68) and sow (Bjersing, '67; Cavazos et al., '69). Some observations have also been published on regression of guinea pig corpus luteum (Crombie et al., '71; Bourneva, '73), although the process has not been studied in depth in this species.Cellular self-destruction (autophagy) and Thus, it is evident that the involution of the corpus luteum deserves further...

The fetal membranes undergo striking changes in structure before delivery that involve catabolism of the extracellular matrix. To investigate the role of specific enzymes in this process, we examined gelatinase activities in rat amnion, visceral yolk sac placenta, and placenta and amniotic fluid between Days 18-21 of pregnancy. Matrix metalloproteinase (MMP)-2 was present in amnion on all days, and its activity increased slightly on Day 21. The 92-kDa gelatinase, MMP-9, was not detected on Days 18-20 but appeared by the morning of Day 21. There was a marked increase in MMP-9 mRNA in the amnion on Day 20, preceding the appearance of MMP-9 activity. Western blotting confirmed an increase in MMP-9 protein in amnion on Day 21. MMP-2 and MMP-9 activities were detected in extracts of whole yolk sac placenta, placenta, and amniotic fluid, but there were no striking changes in these gelatinases between Days 18 and 21. However, the capsular regions of the visceral yolk sac placentae, which thin and rupture during labor, did show higher MMP-9 activity on Day 21 than on Days 18 and 20. We suggest that the striking increase in MMP-9 expression in amnion and possibly the capsular region of the visceral yolk sac placenta approximately 12 h prior to delivery is responsible, in part, for the alterations in the structure of these fetal membranes before parturition.

Little information is available on the ultrastructure of macrophages in the corpus luteum or their importance in the regression of luteal tissue. In the present study, the fine structure of activated luteal macrophages during pregnancy and the postpartum period was examined by electron microscopy of guinea pig ovaries fixed by vascular perfusion. In these corpora lutea, macrophages can readily be distinguished from luteal cells. Activated macrophages typically display three prominent inclusions in their cytoplasm: (1) heterophagic vacuoles, (2) distinctive large dense inclusions, and (3) large and small electron-lucent vacuoles. In addition, they contain numerous smaller lysosome-like dense bodies. Activated macrophages in corpora lutea also characteristically show many surface protrusions, such as processes, folds or pseudopodia, which often occur in close contact with nearby luteal cells. Generally, nuclei of macrophages are irregular in shape and display a dense border of heterochromatin, thus differing from those of luteal cells. Macrophages seem to be most abundant in regressing corpora lutea, where they commonly display heterophagic vacuoles containing recognizable luteal cell fragments, evidence that these phagocytes ingest senescent luteal cells. The digestion of luteal cell components in heterophagic vacuoles presumably gives rise to the distinctive large dense inclusions typically seen in macrophages. The findings of this study indicate that macrophages play a central role in luteolysis by phagocytizing luteal cells or their remnants. They therefore appear to bring about the reduction in volume of the corpus luteum that occurs as this tissue regresses. These results taken together with those previously published (Paavola, '78) further indicate that breakdown of the corpus luteum during postpartum luteolysis in guinea pigs involves both autophagy and heterophagy.

The postpartum involution of corpora lutea was examined by electron microscope cytochemistry of guinea pig ovaries previously fixed by vascular perfusion, a method which produces optimal preservation of steroid-secreting cells and yet maintains enzyme activity. The intracellular digestive apparatus was identified through the localization of two acid hydrolases, acid phosphatase (ACPase) and arylsulfatase. Other marker enzymes localized were thiamine pyrophosphatase (in Golgi cisternae) and alkaline phosphatase (along plasma membranes). Prolonged osmication was used to mark the outer Golgi cisterna. The results demonstrate that luteal cell regression is characterized by a striking increase in the number of lysosomes and the appearance of numerous, double-walled autophagic vacuoles. Both lysosomes and the space between the double walls of autophagic vacuoles exhibit ACPase and arylsulfatase activity. In contrast to earlier periods, just before and during regression, Golgi complex-endoplasmic reticulum-lysosomes (GERL) is markedly hypertrophied, displaying intense acid hydrolase activity. On the basis of various criteria, GERL is proposed to function in the formation of lysosomes and autophagic vacuoles. Lysosomes seem to develop from GERL as focal protuberances of varying size and shape, which detach from the parent structure. Double-walled autophagic vacuoles, often large and complex in structure, initially are produced as GERL cisternae envelop small areas of cytoplasm. Lytic enzymes, perhaps furnished by the engulfing membranes and trapped lysosomes, presumably bring about digestion of the contents of these vacuoles, producing first aggregate-type inclusions, then, as the contents are further degraded, myelin figure-filled residual bodies. ACPase activity occasionally appears within smooth endoplasmic reticulum tubules and cisternae in advanced regression, possibly suggesting that lytic enzymes utilize this membrane system as an access route to GERL. These data indicate that cellular autophagy is a prominent mechanism underlying luteal cell involution during normal postpartum J. CELL BIOLOGY t~ The Rockefeller University Press

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.