J. Anat. (1979), 129, 4, pp. 707-718 With 19 figures Printed in Great Britain
707
Uptake of marker proteins by glycoprotein-containing cells of the pregnant rat uterus and placenta R. SHARMA AND S. PEEL
Human Morphology, Faculty of Medicine, University of Southampton, Southampton, U.K.
(Accepted 20 November 1978) INTRODUCTION
There are several regions of the pregnant rat uterus and placenta where glycoprotein inclusions have been demonstrated in cells with the periodic acid-Schiff technique after digestion with diastase: these include the placental giant cells, labyrinth, visceral yolk sac and the granulated cells of the metrial gland (Wislocki, Weiss, Burgos & Ellis, 1957; Bulmer & Dickson, 1960; Padykula, Deren & Wilson, 1966). The yolk sac shows extensive endocytotic activity and here the glycoprotein inclusions may be related to lysosomal bodies associated with transport and digestion by the yolk sac placenta (Beck, Lloyd & Griffiths, 1967; Seibel, 1974). It has long been accepted that the placental giant cells ingest decidual debris and blood cells (Jenkinson, 1913) and their endocytotic activity has been demonstrated by Bridgman (1948) and Beck et al. (1967). The glycoprotein inclusions in giant cells were described by Bulmer & Dickson (1960) and Deane et al. (1962), but it was not established whether they represented ingested material or a secretory product. It is also not known whether the glycoprotein content of the metrial gland cell granules is synthesised in situ or arises by endocytosis although, in an ultrastructural study, Larkin & Flickinger (1969) described a series of changes which involved differentiation of the granules in situ. The precise nature of the glycoprotein inclusions in these various cell types is not known. Endocytosis of serum proteins would involve the uptake of numerous glycoproteins, including immunoglobulins. These have recently been demonstrated in giant cells, yolk sac, labyrinth and metrial gland cells (Bernard, Ripoche & Bennett, 1977; Bulmer & Peel, 1977a, b). The present study records an attempt to characterise the inclusions of the giant cells, labyrinth, visceral endoderm and granulated metrial gland cells in relation to any endocytosis of marker proteins injected into the maternal blood stream. MATERIALS AND METHODS
Pregnant Wistar rats (38) were used and the day on which spermatozoa were found in vaginal smears was recorded as day 0 of pregnancy. Horseradish peroxidase (Sigma, type II) 12-14 mg/100 g of body weight (dissolved in 1 0 ml of 0-9 % saline), and rat serum proteins (65 mg) conjugated to fluorescein isothiocyanate (FITC serum) by the method of Rinderknecht (1960) were used as tracers. Single intravenous injections of the tracers were given at stages of pregnancy between days 10 and 18 and tissues were obtained 2 or 24 hours after injection. The animals were anaesthetised with ether and conceptuses removed, immersed in cold 4 % paraformaldehyde or 0021-8782/79/2828-6810 $02.00 © 1979 Anat. Soc. G.B. & I. 45-2
708 R. SHARMA AND S. PEEL 95 % ethanol and allowed to fix overnight. Tissues obtained from the animals injected with peroxidase were fixed in paraformaldehyde and were trimmed to small blocks after the first 2 hours of fixation. After 16 hours fixation they were washed in 0-1 M phosphate buffer pH 7-2-7-4 and treated for 1 hour in a solution of 3,3'diaminobenzidine tetrahydrochloride in 0 5 M Tris HCl buffer (pH 7 6) containing 0 1 % hydrogen peroxide (DAB solution). After four rinses in distilled water tissues were dehydrated in ethanol and embedded in glycol methacrylate. 1,um sections were cut and stained with the periodic acid-Schiff (PAS) technique after diastase digestion, or with haematoxylin and eosin. Tissues from the animals injected with FITC serum, fixed either in paraformaldehyde or ethanol, were subjected to the cold processing technique of Sainte-Marie (1962) and embedded in paraffin wax. Sections (5 /%m) were de-waxed and mounted in Uvinert (G.T. Gurr, High Wycombe, England) and examined with a Zeiss Universal microscope. Some paraffin sections from the animals injected with FITC serum were treated with DAB solution to demonstrate endogenous peroxidase. Control tissues from animals not injected with marker proteins were obtained and processed in similar manner. Some sections were subjected to the PAS and DAB techniques and others were stained for haemoglobin by the method of Dunn & Thompson (1945). RESULTS
Yolk sac and Reichert's membrane At day 10 the tall columnar endoderm cells of the visceral layer of the yolk sac are separated from the maternal decidua capsularis by the yolk sac cavity, the flattened cells of the parietal layer of the yolk sac, Reichert's membrane, and the giant cells of the central zone. As pregnancy proceeds there is progressive degeneration of the decidua capsularis and attenuation of the giant cell layer. Figure 1 is a diagrammatic representation of the relations of the various uterine and fetal tissues at day 12. It is generally accepted that by about day 16 the visceral layer of the yolk sac is exposed to the uterine lumen following the rupture of Reichert's membrane. At about this time endodermal invaginations into the labyrinth first become apparent. Glycoprotein inclusions up to 2 ,um in diameter were found in all cells of the visceral endoderm up to day 12 (Fig. 2). The glycoprotein inclusions were smaller and less densely stained in the later stages (Fig. 3) and were restricted to that part of the yolk sac adjacent to the chorio-allantoic placenta. Inclusions were not seen in the endodermal invaginations in the labyrinth, nor in parietal yolk sac cells, although Reichert's membrane itself showed a high glycoprotein content. Endogenous peroxidase and haemoglobin inclusions were not detected in the yolk sac. At all stages of pregnancy examined, 2 hours after injection of peroxidase or FITC serum these tracers were detected as supranuclear inclusions in the visceral endodermal cells, and they were still present after 24 hours. Up to day 13 the inclusions were relatively large (Fig. 4) and were present in all visceral endodermal cells. After day 13, however, some visceral endodermal cells at the antimesometrial aspect of the implantation sites did not appear to have taken up the tracer molecules. At day 18, inclusions showing tracers were relatively small (Fig. 5), and were restricted to visceral endodermal cells adjacent to the chorio-allantoic placenta. No uptake of tracer molecules was detected in cells of the endodermal invaginations, nor in the yolk sac mesoderm. There was no significant uptake of the fluorescent marker by the cells of the parietal layer of the yolk sac, but Reichert's membrane
Protein uptake in rat placenta
709
Fig. 1. A diagrammatic representation of a transverse section through a pregnant uterus on day 12 of pregnancy. Yolk sac cavity, YS; visceral endoderm, V; parietal endoderm, P; labyrinth, L; junctional zone, J; decidua basalis, DB; decidua capsularis, DC; metrial gland, MG; junctional zone giant cells, JG; marginal giant cells, MZG; central giant cells, CG; uterine lumen, UL.
showed fluorescence in areas corresponding to the uptake by the different areas of the visceral endoderm. After the peroxidase injections, no peroxidase activity was detected in either the parietal yolk sac or Reichert's membrane.
Giant cells At 10 days the trophoblastic giant cells form an almost complete investment for the conceptus. The giant cells of the central zone are in contact with the decidua capsularis around the lateral and antimesometrial surfaces of the conceptus, while the giant cells of the ectoplacental cone are in contact with the decidua basalis. By 13 days the central zone giant cells form a much attenuated layer, particularly antimesometrially, and they apparently disappear with the rupture of Reichert's membrane. With the development of the junctional zone, and the horizontal flattening of the ectoplacental cone, giant cells appear as a layer two or three cells
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707
Uptake of marker proteins by glycoprotein-containing cells of the pregnant rat uterus and placenta R. SHARMA AND S. PEEL
Human Morphology, Faculty of Medicine, University of Southampton, Southampton, U.K.
(Accepted 20 November 1978) INTRODUCTION
There are several regions of the pregnant rat uterus and placenta where glycoprotein inclusions have been demonstrated in cells with the periodic acid-Schiff technique after digestion with diastase: these include the placental giant cells, labyrinth, visceral yolk sac and the granulated cells of the metrial gland (Wislocki, Weiss, Burgos & Ellis, 1957; Bulmer & Dickson, 1960; Padykula, Deren & Wilson, 1966). The yolk sac shows extensive endocytotic activity and here the glycoprotein inclusions may be related to lysosomal bodies associated with transport and digestion by the yolk sac placenta (Beck, Lloyd & Griffiths, 1967; Seibel, 1974). It has long been accepted that the placental giant cells ingest decidual debris and blood cells (Jenkinson, 1913) and their endocytotic activity has been demonstrated by Bridgman (1948) and Beck et al. (1967). The glycoprotein inclusions in giant cells were described by Bulmer & Dickson (1960) and Deane et al. (1962), but it was not established whether they represented ingested material or a secretory product. It is also not known whether the glycoprotein content of the metrial gland cell granules is synthesised in situ or arises by endocytosis although, in an ultrastructural study, Larkin & Flickinger (1969) described a series of changes which involved differentiation of the granules in situ. The precise nature of the glycoprotein inclusions in these various cell types is not known. Endocytosis of serum proteins would involve the uptake of numerous glycoproteins, including immunoglobulins. These have recently been demonstrated in giant cells, yolk sac, labyrinth and metrial gland cells (Bernard, Ripoche & Bennett, 1977; Bulmer & Peel, 1977a, b). The present study records an attempt to characterise the inclusions of the giant cells, labyrinth, visceral endoderm and granulated metrial gland cells in relation to any endocytosis of marker proteins injected into the maternal blood stream. MATERIALS AND METHODS
Pregnant Wistar rats (38) were used and the day on which spermatozoa were found in vaginal smears was recorded as day 0 of pregnancy. Horseradish peroxidase (Sigma, type II) 12-14 mg/100 g of body weight (dissolved in 1 0 ml of 0-9 % saline), and rat serum proteins (65 mg) conjugated to fluorescein isothiocyanate (FITC serum) by the method of Rinderknecht (1960) were used as tracers. Single intravenous injections of the tracers were given at stages of pregnancy between days 10 and 18 and tissues were obtained 2 or 24 hours after injection. The animals were anaesthetised with ether and conceptuses removed, immersed in cold 4 % paraformaldehyde or 0021-8782/79/2828-6810 $02.00 © 1979 Anat. Soc. G.B. & I. 45-2
708 R. SHARMA AND S. PEEL 95 % ethanol and allowed to fix overnight. Tissues obtained from the animals injected with peroxidase were fixed in paraformaldehyde and were trimmed to small blocks after the first 2 hours of fixation. After 16 hours fixation they were washed in 0-1 M phosphate buffer pH 7-2-7-4 and treated for 1 hour in a solution of 3,3'diaminobenzidine tetrahydrochloride in 0 5 M Tris HCl buffer (pH 7 6) containing 0 1 % hydrogen peroxide (DAB solution). After four rinses in distilled water tissues were dehydrated in ethanol and embedded in glycol methacrylate. 1,um sections were cut and stained with the periodic acid-Schiff (PAS) technique after diastase digestion, or with haematoxylin and eosin. Tissues from the animals injected with FITC serum, fixed either in paraformaldehyde or ethanol, were subjected to the cold processing technique of Sainte-Marie (1962) and embedded in paraffin wax. Sections (5 /%m) were de-waxed and mounted in Uvinert (G.T. Gurr, High Wycombe, England) and examined with a Zeiss Universal microscope. Some paraffin sections from the animals injected with FITC serum were treated with DAB solution to demonstrate endogenous peroxidase. Control tissues from animals not injected with marker proteins were obtained and processed in similar manner. Some sections were subjected to the PAS and DAB techniques and others were stained for haemoglobin by the method of Dunn & Thompson (1945). RESULTS
Yolk sac and Reichert's membrane At day 10 the tall columnar endoderm cells of the visceral layer of the yolk sac are separated from the maternal decidua capsularis by the yolk sac cavity, the flattened cells of the parietal layer of the yolk sac, Reichert's membrane, and the giant cells of the central zone. As pregnancy proceeds there is progressive degeneration of the decidua capsularis and attenuation of the giant cell layer. Figure 1 is a diagrammatic representation of the relations of the various uterine and fetal tissues at day 12. It is generally accepted that by about day 16 the visceral layer of the yolk sac is exposed to the uterine lumen following the rupture of Reichert's membrane. At about this time endodermal invaginations into the labyrinth first become apparent. Glycoprotein inclusions up to 2 ,um in diameter were found in all cells of the visceral endoderm up to day 12 (Fig. 2). The glycoprotein inclusions were smaller and less densely stained in the later stages (Fig. 3) and were restricted to that part of the yolk sac adjacent to the chorio-allantoic placenta. Inclusions were not seen in the endodermal invaginations in the labyrinth, nor in parietal yolk sac cells, although Reichert's membrane itself showed a high glycoprotein content. Endogenous peroxidase and haemoglobin inclusions were not detected in the yolk sac. At all stages of pregnancy examined, 2 hours after injection of peroxidase or FITC serum these tracers were detected as supranuclear inclusions in the visceral endodermal cells, and they were still present after 24 hours. Up to day 13 the inclusions were relatively large (Fig. 4) and were present in all visceral endodermal cells. After day 13, however, some visceral endodermal cells at the antimesometrial aspect of the implantation sites did not appear to have taken up the tracer molecules. At day 18, inclusions showing tracers were relatively small (Fig. 5), and were restricted to visceral endodermal cells adjacent to the chorio-allantoic placenta. No uptake of tracer molecules was detected in cells of the endodermal invaginations, nor in the yolk sac mesoderm. There was no significant uptake of the fluorescent marker by the cells of the parietal layer of the yolk sac, but Reichert's membrane
Protein uptake in rat placenta
709
Fig. 1. A diagrammatic representation of a transverse section through a pregnant uterus on day 12 of pregnancy. Yolk sac cavity, YS; visceral endoderm, V; parietal endoderm, P; labyrinth, L; junctional zone, J; decidua basalis, DB; decidua capsularis, DC; metrial gland, MG; junctional zone giant cells, JG; marginal giant cells, MZG; central giant cells, CG; uterine lumen, UL.
showed fluorescence in areas corresponding to the uptake by the different areas of the visceral endoderm. After the peroxidase injections, no peroxidase activity was detected in either the parietal yolk sac or Reichert's membrane.
Giant cells At 10 days the trophoblastic giant cells form an almost complete investment for the conceptus. The giant cells of the central zone are in contact with the decidua capsularis around the lateral and antimesometrial surfaces of the conceptus, while the giant cells of the ectoplacental cone are in contact with the decidua basalis. By 13 days the central zone giant cells form a much attenuated layer, particularly antimesometrially, and they apparently disappear with the rupture of Reichert's membrane. With the development of the junctional zone, and the horizontal flattening of the ectoplacental cone, giant cells appear as a layer two or three cells
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