118 TRANSFER OF IRON FROM MOTHER TO FETUS IN WATER BUFFALO: ERYTROPHAGOCYTOSIS AND UTEROFERRIN

The transplacental transport of iron by uteroferrin (Uf), and the hemophagous areas in the water buffalo placenta were analyzed to clarify the mechanism of blood extravasation in the materno-fetal interface with consequent transfer of iron to the fetus through the trophoblastic erythrophagocytosis (Murai and Yamauchi 1986 Nippon Juigaki Zasshi 48, 75–88) and in the endometrial glands (Bazer et al. 1991 Exp. Hematol. 19 910–19 915). In the water buffalo placenta this mechanism remains unclear; uteroferrin is very important in this process because it is both an iron transporter and a progesterone-induced hematopoietic growth factor. Our objective was to characterize these hemophagous areas and the endometrium of the water buffalo, focusing in the materno-fetal transfer of iron. Small pieces of the placentomes and interplacentomal region of water buffaloes (Bubalus bubalis bubalis, n = 51) in all of the gestation periods were fixed in 4% paraformoldehyde, and 2.5% glutaraldehyde in PBS, processed, and stained for light and transmission electron microscopy to characterize the hemophagous areas and endometrial glands morphologically, histochemically (Perls, acid phosphatase reaction), and immunohistochemically with rabbit anti-pig uteroferrin antibody to confirm the iron transfer. The hemophagous areas were present in the placentome from 4–10-months-pregnant placentae. The Perls reaction for ferric iron staining was negative in the placentome and positive in the endometrial glands, and the acid phosphatase reaction to detect phagocytic activity was positive in the placentome as well as in the interplacentomal region. The uteroferrin immunohistochemical reaction was positive in the trophoblast, mainly in determined regions of the materno-fetal interface and in other points deep in the placentome, and the endometrial glands showed a strong reaction in the epithelium and in the lumina. The ultrastructure of the hemophagous areas revealed ingested erythrocytes inside the epithelial cells of trophoblasts, endocytic vesicles, and caveolae. The endometrial gland epithelium is of the columnar type with microvilli and basal nuclei. The results obtained mainly by histochemistry and immunohistochemistry indicated that the hemophagous areas and endometrial glands are very important sites for iron transfer in water buffalo, and are thus involved in the regulation of fetal hematopoiesis. This work was funded by FAPESP.

Arrested apoptosis without nuclear fragmentation produced by efferent duct ligation in round spermatids and multinucleated giant cells of rat testis

The apoptotic process evoked by efferent duct ligation in the testes of adult rats was followed for 10 days by differential staining for haematoxylin-eosin, periodic acid-Schiff and a modified trichrome technique in optical microscopy and by ultrastructural localization of acid phosphatase. Round spermatids showed the first effects of efferent duct ligation. At day 3 after ligation, annular clumps of chromatin with typical apoptotic characteristics appeared against the nuclear membrane of these cells. Afterwards, membranous structures and a wide separation between the two layers of the nuclear membrane were observed but nuclear fragmentation did not occur and apoptotic granules were not seen. Cytoplasmic components were also altered, and severely damaged organoids and empty vacuoles lacking acid phosphatase reaction were frequently seen. On day 2 after efferent duct ligation, multinucleated giant cells appeared, which displayed similar characteristics as spermatids and showed no acid phosphatase reaction. Although abnormal spermatids and multinucleated giant cells were surrounded by the cytoplasm of Sertoli cells, neither lysosomal acid phosphatase nor phagocytic activity was detected. It is concluded that efferent duct ligation specifically affects round immature spermatids eliciting a partial nuclear apoptotic response that is not accompanied by autophagic or heterophagic activity and without lysosomal participation in Sertoli cells.

Structural responses of amoebae to the injection of heterologous cytoplasm

Responses to the introduction of heterologous cytoplasm and the fate of foreign organelles were investigated in amoebae. Heterologous cytoplasm was transferred by microinjection from Pelomyxa carolinensis to Amoeba discoides. In control experiments, homologous cytoplasm was transferred from one A. discoides to another. Recipient cells were observed by light microscopy, and samples were prepared for ultrastructural study at intervals between 15 min nad 3 days after operation. Recipients of heterologous cytoplasm showed two main responses. First, about 40% recipients expelled small amounts of cytoplasm by a blebbing process within 30 min after injection. Second, organelles were segregated and broken down in membrane-bounded cytoplasmic vacuoles between 6 h and 2 days after operation. Acid phosphatase reaction product was observed in these vacuoles along with altered organelles. Use of electron-dense thoria particles to mark donor cells demonstrated the presence of injected cytoplasm in the vacuoles. In contrast, when amoebae were injected with homologous cytoplasm, none was expelled and vacuoles containing degenerating organelles were uncommon. The survival rate and general appearance of recipients of heterologous cytoplasm were much poorer than those of homologous recipients, and most of the former died by I week after operation. It is postulated that amoeba are capable of recognizing heterologous organelles introduced into the cytoplasm and that they respond by expulsion and/or destruction of the foreign cellular components. The previously described lethal effect of heterologous cytoplasm was confirmed.

Elimination of excess smooth endoplasmic reticulum after phenobarbital administration.

Livers from Charles River rats during and after treatment with phenobarbital were studied in order to investigate possible mechanisms involved in the elimination of excess smooth endoplasmic reticulum. The most pronounced structural change during compound administration was proliferation of smooth endoplasmic reticulum; depletion of glycogen and an increase in lipid deposits were also observed. After termination of treatment, these changes were reversed. The appearance of an increased number of autophage vacuoles and lysosomes plus the localization of acid phosphatase reaction product in these bodies suggests autophagy as one possible mechanism for the elimination of excess smooth endoplasmic reticulum. Cytoplasmic blebs and fragments replete with smooth endoplasmic reticulum were observed within the sinusoids. The presence of Kupffer cell cytoplasmic extensions surrounding these fragments and acid phosphatase reaction product within Kupffer cell inclusions suggests heterophagy as another process participating in the removal of excess smooth endoplasmic reticulum.