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An electron microscopic study of the chorioallantoic placenta and the subplacenta of the cane rat (Thryonomys swinderianus temminck)
Placenta(I984), 5, 433-442
An Electron Microscopic Study of the Chorioallantoic Placenta and the Subplacenta of the Cane Rat (Thryonomys swinderianus Temminck) DOMINIC ODUORoOKELO Department of VeterinaryAnatomy, Universityof Nairobi, PO Box 3or97, Nairobi, Kenya
INTRODUCTION The cane rat belongs to the African pbiomnrph rnrlen* gro,!p u,b;eh togothor x~,;th the. q.,~!eh American caviomorphs constitute the rodent subfamily commonly known as the Hystricomorpha. The close phylogenetic relationship between the phiomorphs and the caviomorphs is supported by several findings. Lavocat (i 967, 1974) observed that the middle ear ofDiamantomys, a Miocene rodent in Kenya, was very similar to those of the present day cane rat (Thryonomys swinderianus) in Africa and the plains viscacha (Lagostomus maximus) in South America. The cane rat also shares a number of reproductive characteristics with the caviomorphs of S. America; these include a relatively long gestation period of about i55 days, a mean litter size of four (Asibey, x974), the presence of accessory corpora lutea and a well-developed interstitial gland tissue in the ovaries (Weir and Rowlands I974; Oduor-Okelo, I978) and the presence of a vaginal closure membrane (Weir, I974; Oduor-Okelo, I979)- In a recent study, Oduor-Okelo and Gombe (1982) have shown that the histological structure of the fetal membranes and chorioallantoic placenta of the cane rat is very similar to that of the guinea pig and other caviomorphs. Such similarities include the division of the definitive placenta into labyrinthine and spongy zones; the presence of a specialized zone of the chorion, the subplacenta, situated between the placental disc and the basal decidua, and having PAS-positive, diastase-resistant contents (Amoroso, I952; Tibbitts and Hillemann, I959; Davies, Dempsey & Amoroso, i96Ib; Hillemann and Gaynor, I96I; Roberts and Perry, I974); the presence of a nonvascular collar (Perrotta, I959) that attaches the chorio-allantoic membrane to the placenta, and numerous white avascular membrane-pustules similar to those found in the plains viscacha (Roberts and Perry, I974). In this paper the ultrastructure of the cane rat placenta and subplacenta is presented for the first time, and the similarities with those of the guinea pig (Davies, Dempsey & Amoroso, I96Ia; Enders, I965; Kaufmann and Davidoff, I977) and the chinchilla (King and Tibbitts, i976) are discussed. MATERIALS AND M E T H O D S Six pregnant cane rats were used in this study, and in all of them the definitive chorio-allantoic placenta was already formed, with one near term. In the most advanced pregnancy, the average 433
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weight of the three fetuses was i24. 5 g, and the mean crown-rump length (CR-L) was I6.2 cm. In the least advanced pregnancy the average weight of the four fetuses was 1.8 g, and the mean CR-L was 2. 4 cm. (Asibey, 1974, has recorded a mean birthweight of I28.5 g.) Relatively large pieces of placental tissue were fixed in Bouin's solution and processed for conventional histological examination. Small pieces of placental and subplacental tissues were fixed in glutaraldehyde-formaldehyde trinitrocresol (Ito and Karnovsky, 1968), and postfixed in osmium tetroxide overnight. The tissues were then washed in physiological saline, dehydrated in ascending grades of alcohol and cleared with propyline oxide. They were then embedded in Epon-Araldite (R. P. Cargille Laboratories Inc., New Jersey, USA). Thin sections (6o nm thick) cut with a diamond knife were stained successively with uranyl acetate and lead citrate for examination with a Philips 2Ol electron microscope, while thick sections cut with glass knife were stained with toluidine blue and examined with the light microscope.
RESULTS Observations with the light microscope revealed the division of the cane rat placenta into labyrinthine and spongy zones and a distinct subplacental region (Figure I). In the labyrinthine zone the syncytiotrophoblast formed the immediate wall of the maternal blood spaces and stained darkly with toluidine blue due to the presence of numerous basophilic granules. The
Photomicrographof the definitiveplacenta of the cane rat showingthe labyrinthine (L) and spongy(S) zones and the well-developedsubplacenta (arrow). It also shows the non-vascularcollar of Perrotta (X). H & E, x 6.8.
Figure I.
Figure 2. A section of the labyrinthine zone of the chorio-aUantoic placenta showing mitotic figures in the cytotrophoblastic layer (arrow). Embedded in Epon-Araldite and stained with toluidine blue. x 415.
Figure3. The interhaemal membrane of the cane rat placenta consisting of a single layer ofsyncytiotrophoblast (S) a basal lamina and the fetal capillary endothelium (fc). T h e syncytial microvilli protrude into the maternal blood spaces (rob). Note the short strands of granular endoplasmic reticulum and also the infoldings of the basal plasmallema (arrows). Epon section cut at 6o nm and stained with uranyl acetate and lead citrate. • 656O,
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Figure 4. The interhaemal membrane of the chorio-allantoic placenta showing numerous coated micropinocytotic
vesicles(arrows)and a pair ofCentrioles(C) in the syncytialcytoplasm,mb = maternal bloodspaces;fc = fetalcapillary endothelium. Epon section cut at 6onto and stained with uranyl acetate and lead citrate, x 816o. cytotrophoblast, on the other hand, stained very lightly, had distinct cell bqundaries and showed mitotic figures (Figure z). The spongy zone consisted of a maze of darkly staining syncytiotrophoblast enclosing large maternal blood spaces. In this zone there was no cytotrophoblast and no fetal vessels penetrated into it. Ultrastructural observations on the labyrinthine zone showed that fetal and maternal blood streams were closely approximated, especially in those areas where the interhaemal membranes consisted of a thin layer of syncytial trophoblast, a basal lamina and the fetal capillary endothelium (Figure 3)- The surface of the syncytiotrophoblast facing the maternal blood space had numerous microvilli, while the basal surface had recesses which contained cytoplasmic infoldings. The cytoplasm was rich in short strands of granular endoplasmic reticulum (Figure 3). Centrioles were sometimes observed in sections of the syncytiotrophoblast (Figure 4). Large mitochondria and well-developed Golgi complexes were conspicuous features of the syncytial cytoplasm (Figure 5). Some coated vesicles appeared to be in open communication with the maternal blood space (Figure 4). In the spongy zone, syncytiotrophoblast lined the vascular channels which drained maternal blood from the labyrinth. The trophoblastic surface was marked by numerous microvilli, and the cytoplasm had a well developed rER with dilated cisternae containing an amorphous finely granular precipitate. In some areas of the syncytium there were deep recesses into which microvilli projected (Figure 6). Whether these 'intracellular canaliculi' were real or artefacts was difficult to ascertain. The Golgi apparatus was well developed and scattered within the
437
Placental Ultrastructure of the Cane Rat
Figure5. Highpowermagnificationof the syncytiotrophoblast.Notethe welldevelopedGolgicomplexes(G), granular endoplasmicreticulum,largemitochondria,and numerouscoatedvesicles,someof whichare associatedwith the Golgi complexes.Epon section cut at 6ohm and stained with uranyl acetateand lead citrate, x 95oo.
cytoplasm. Other cytoplasmic organelles included rounded mitochondria, coated vesicles and numerous polyribosomes. Lipid droplets and glycogen deposits were frequent findings. The subplacenta consisted of folded lamellae ofcytotrophoblast and syncytiotrophoblast. The lamellae were separated from each other by fetal mesenchyme. The cytotrophoblastic cells had an undifferentiated appearance with free ribosomes, few rounded mitochondria and small Golgi apparatus. Cytotrophoblastic cells were attached to each other by desmosomes and large vacuoles appeared in the cytoplasm (Figure 7). Desmosomal connections between the syncytioand cyto-trophoblast were frequently observed, and the intercellular spaces were dilated to form lacunae occupied by short microvilli mainly from the syncytiotrophoblast. The lacunae contained an electron-dense precipitate. Scattered throughout the syncytial cytoplasm were numerous spherical granules (Figure 8). Such granules seemed to be peculiar to the subplacental syncytium as they were not observed in the syncytium of the labyrinthine and spongy zones. The cytoplasmic zone immediately below the microvillous border was free of organelles but was occupied by a finely granular, electron-dense substance similar to that in the lacunae. There were strands ofrER with dilated cisterns (Figure 8) and widely scattered Golgi membranes (Figure 9). DISCUSSION The suggestion by Roberts and Perry (x974) that a haemoendothelial condition may develop in late pregnancy in hystricomorph rodents has not been supported by ultrastructural finding. In
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Figure 6. Syncytiotrophoblast of the spongy zone. Granular endoplasmic reticulum and coated vesicles are demonstrated. Note also the intracytoplasmie canaliculi (Ic) studded with microvilli. Numerous microvilli also project into the maternal blood space (nab). Epon section cut at 6ohm and stained with uranyl acetate and lead citrate. • 43oo.
the chinchilla (King and Tibbitts, I976) as well as in the present study of the cane rat, the minimal placental barrier, up to the end of pregnancy, consists of a single layer of syncytiotrophoblast, a common basal lamina and fetal capillary endothelium. The interhaemal membrane in the cane rat placenta is of the haemomonochorial type according to the classification of Enders (I965). The fine structure of the trophospongium is also similar to that of the guinea pig and chinchilla. It is therefore probable that the cane rat trophospongium carries out biosynthetic activities similar to those described in the guinea pig (see Tam and Burgess, I977; Burgess and Tam, x978; Bambra et al, I98o). A unique feature of hystricognathous hystricomorph placentation is the presence of the subplacenta. It contains PAS-positive diastase-resistant material occupying what appears to be lacunae or spaces (see Davies, Dempsey and Amoroso, I96Ib; Oduor-Okelo and Gombe, I98z ). Ultrastructurally, the subplacenta consists of both cyto- and syncytiotrophoblast. The cytotrophoblast is very much undifferentiated while the syncytiotrophoblast has a well developed granular endoplasmic reticulum, scattered Golgi complexes, and numerous electrondense granules of uniform size. King and Tibbitts (i 976) have suggested that these granules may be related to secretory activities, although the nature of the secretion is not yet known. A finely granular precipitate is present in the intercellular spaces and also in the syncytial cytoplasm
Placental Ultrastructure of the Cane Rat
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Figure 7. The subplacenta of the cane rat chorio-allantoicplacenta. The cytotrophoblast(ct) and syncytialtrophoblast (st) are held togetherby desmosomes.The intercellular spacesbetweenthese two typesoftrophoblast are studded with microvilli. Vacuoles (arrows) seem to be an important transport route from the mesenchyme (m) through the cytotrophoblast. Epon sections cut at 6o nm and stained with uranyl acetate and lead citrate, x 495o.
immediately below the microvillous border. It is suggested that this precipitate is the PASpositive material seen in histological sections and is therefore both extra- and intra--cellular. Several physiological functions have been suggested for the subplacenta (see Perrotta, I959; Davies, Dempsey and Amoroso, 1961 b; Roberts and Perry, 1974; Luckett and Mossman, 198 i). Heap and Illingworth (i974) have isolated a progesterone-binding globulin (PBG) from the plasma of several hystricomorph rodents during pregnancy and have suggested that such a substance may be produced in the subplacenta. This, however, is also speculative and further experimental work is needed to determine whether the subplacenta is the site of PBG synthesis and, if so, how PBG gets into maternal circulation.
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Figure 8. The subplacentaof the cane rat chofio-allantoicplacenta.The syncytiotrophoblast(st) has a well developed granular endoplasmicreticulum (er) and contains rounded electron-densegranules (g) of uniform size. Numerous microvilli protrude into the intercellular spaces. Note the precipitate in the intercellular space (arrow). The cytotrophoblasticcells(ct) are poor in organellesand have fewermicrovilli.Epon section cut at 6o nm and stained with uranyl acetate and lead citrate, x 396o.
SUMMARY The ultrastructure of the cane rat chorio-allantoic placenta has been studied. In its thinnest regions the interhaemal membrane is composed of a single layer of syncytiotrophoblast, a common basal lamina, and the fetal capillary endothelium. Hence this is a haemomonochorial placenta. The cytotrophoblastic cells show many mitotic figures but they are never seen directly bounding the maternal blood spaces. T h e syncytiotrophoblast of the spongy zone is rich in dilated cisterns of granular endoplasmic reticulum and is also characterized by many 'intrasyncytial canaliculi'. The subplacenta consists of both cytotrophoblast and syncytiotrophoblast. The syncytiotrophoblast has numerous short microvilli projecting into the intercellular spaces, granular endoplasmic reticulum, a well-developed Golgi complex, and numerous electron--dense granules of uniform size. T h e intercellular spaces or lacunae contain a precipitate of moderate electron density which is similar in texture to the intracellular precipitate in the 'brush border' region of the syncytiotrophoblast. The similarities between the ultrastructure of the cane rat placenta and those of the guinea pig and chinchilla are discussed.
Placental Uhrastructure of the Cane Rat
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Figure 9. The subplacenta of the cane rat chorio-allantoic placenta. The syncytiotrophoblast (st) has well developed Golgi complexes (arrows), granular endoplasmic reticulum and electron-dense granules (g). Note the organelle-free brush border (*) which contains an electron-dense homogenous precipitate. The cytotrophoblast (ct) is poor in organelles. The intercellular space (Is) is occupied by short microvilli mainly from the syncytiotrophoblast. Epon section cut at 6onm and stained with uranyl acetate and lead citrate, x ~26oo.
ACKNOWLEDGEMENTS I wish to thank Mr L. Brown and his family for organizing the trapping of cane rats at his Lukenya farm. I am grateful to Prof. T. R. Odhiambo, the Director of the International Centre for Insect Physiology & Ecology (ICIPE) for permission to use the electron microscope facilities at ICIPE. My appreciation also goes to the WHO Special Programme of Research Development and Training in Human Reproduction, and the Department of Cell Biology, University of Texas Health Science Center at Dallas where part of this work was carried out. I also wish to thank Mr J. Gichiri and Mrs R. Katema for their technical assistance, and Mrs G. Hinga for typing the script. REFERENCES Amoroso, E. C. (1952) Placentation. In Marshall's Physiology of Reproduction (Ed.) Parkes, A. S. Volume z, pp. I27-31t. London: Longmans. Asibey, E. O. A. (x974) Reproduction in the grass cutter (Thryonomys swmderianusTemminck) in Ghana. Symposia of the ZoologicalSociety of London, 34, 251-263. Bambra, C. S., Robinson, G., Foxcroft, G. R. & Amoroso, E. C. (t98o) Immunohistochemical localization of a chorionic gunadotrophin-like material in the guinea pig placenta. In Abstracts of the Winter Meeting of the Societyfor the study of Fertility, London, pp. 20. Burgess, S. M. & Tam, W. H. (1978) Ultrastructural changes in the guinea pig placenta with special reference to organdies associated with steroidogenesis. Journal of Anatomy, i26, 319-327 . Davies, J., Dempsey, E. W. & Amoroso, E. C. (t961a) The subplacenta of the guinea pig: an electron microscopic study. Journal of Anatomy, 95, 311-324.
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Davies, J., Dempsey, E. W. & Arnoroso, E. C. (I 961b) The subplacenta of the guinea pig: Development, histology and histochemistry. Journal of Anatomy, 95, 457-473. Enders, A. C. 0965) A comparative study of the fine structure of the trophoblast in several haemochorial placentas. American Journal of Anatomy, H6, 29-68. Heap, R. B. & Illingworth, D. V. (x974) The maintenance of gestation in the guinea pig and other hystricomorph rodents: Changes in the dynamic of progesterone metabolism and the occurrence of progesterone-binding globulin (PBG). Sympozia of the Zoological Society, 34, 385-415 . Hillemann, H. H. & Gaynor, A. I. (I961) The definitive architecture of the placenta of nutria, Myocastor coypu (Monila). AmericanJournal of Anatomy, lO9, 229-318. Ito, S. & Karnovsky, M. J. (t968) Formaldehyde~lutaraidehyde fixatives containing trinitro compounds. Journal of Cell Biology, 39, 168a-169a. Kaufmann, P. & Davidoff, M. (I977) The guinea pig placenta. Advances in Anatomy, Embryologyand Cell Biology, 53, I-9. King, B. F. & Tibbitts, F. D. (t976) The fine structure of the chinchilla placenta. AmericanJournal of Anatoray, i45 , 33-56. Lavocat, R. (x967) Observations sur la region auditive des Rongeurs Theridomorphes. In Problems actueles de paleontologie. ColloquesInternationaux centre National de la RechercheScientifulue, x63, 49I-5oi. Lavocat, R. (I974) What is an hystricomorph? Symposia of the Zoological Society of London, 34, 7-20. Luckett, E. P. & Mossman, H. W. (i98i) Development and phylogenetic significance of the foetal membranes and placenta of the African hystricognathous rodents Bathyergusand Hystrix. AmericanJournalofAnatomy, 162, 265-285. Oduor-Okelo, D. (1978) A histological study on the ovary of the African cane rat (Thryonomys swinderianus). East African WildlifeJournal, I6, 257-264. Oduor-Okelo, D. (I979) A study of thefoetal membranesand placensa of the African cane rat ( Thryonomysswinderianus) with some observations on the placentation in the elephant shrews-familyMacroscelididae. Ph.D. Thesis, University of Nairobi. Oduor-Okelo, D. & Gombe, S. (I98Z) Placentation in the cane rat (Thryonomys swinderianus). African Journal of
Ecology, 20, 49-'66. Perrotta, C. A. (1959) Fetal membranes of the Canadian porcupine (Erethizondorsatum). AmericanJournal of Anatomy, Io4, 35-59. Roberts, C. M. & Perry, J. S. (I974) Hystricomorph embryology. Symposia of the ZoologicalSociety of London, 34, 334-36o. Tam, W. H. & Burgess, S. M. (1977) The developmental changes in the guinea pig.Journal of Anatomy, 123, 6oi-6i 4. Tibbitts, F. D. & Hillemann, H. H. (1959) The development and histology of the chinchilla placenta. Journal of Morphology. io5, 317-365. Weir, B. J. 0974) Reproductive characteristics of hystricomorph rodents. Symposia of the ZoologicalSociety of London, 34, 265-3oL Weir, B. J. & Rowlands, I. W. 0974) Functional anatomy of the hystricomorph ovary. Symposia of the Zoological Society of London, 34, 303-332.
An Electron Microscopic Study of the Chorioallantoic Placenta and the Subplacenta of the Cane Rat (Thryonomys swinderianus Temminck) DOMINIC ODUORoOKELO Department of VeterinaryAnatomy, Universityof Nairobi, PO Box 3or97, Nairobi, Kenya
INTRODUCTION The cane rat belongs to the African pbiomnrph rnrlen* gro,!p u,b;eh togothor x~,;th the. q.,~!eh American caviomorphs constitute the rodent subfamily commonly known as the Hystricomorpha. The close phylogenetic relationship between the phiomorphs and the caviomorphs is supported by several findings. Lavocat (i 967, 1974) observed that the middle ear ofDiamantomys, a Miocene rodent in Kenya, was very similar to those of the present day cane rat (Thryonomys swinderianus) in Africa and the plains viscacha (Lagostomus maximus) in South America. The cane rat also shares a number of reproductive characteristics with the caviomorphs of S. America; these include a relatively long gestation period of about i55 days, a mean litter size of four (Asibey, x974), the presence of accessory corpora lutea and a well-developed interstitial gland tissue in the ovaries (Weir and Rowlands I974; Oduor-Okelo, I978) and the presence of a vaginal closure membrane (Weir, I974; Oduor-Okelo, I979)- In a recent study, Oduor-Okelo and Gombe (1982) have shown that the histological structure of the fetal membranes and chorioallantoic placenta of the cane rat is very similar to that of the guinea pig and other caviomorphs. Such similarities include the division of the definitive placenta into labyrinthine and spongy zones; the presence of a specialized zone of the chorion, the subplacenta, situated between the placental disc and the basal decidua, and having PAS-positive, diastase-resistant contents (Amoroso, I952; Tibbitts and Hillemann, I959; Davies, Dempsey & Amoroso, i96Ib; Hillemann and Gaynor, I96I; Roberts and Perry, I974); the presence of a nonvascular collar (Perrotta, I959) that attaches the chorio-allantoic membrane to the placenta, and numerous white avascular membrane-pustules similar to those found in the plains viscacha (Roberts and Perry, I974). In this paper the ultrastructure of the cane rat placenta and subplacenta is presented for the first time, and the similarities with those of the guinea pig (Davies, Dempsey & Amoroso, I96Ia; Enders, I965; Kaufmann and Davidoff, I977) and the chinchilla (King and Tibbitts, i976) are discussed. MATERIALS AND M E T H O D S Six pregnant cane rats were used in this study, and in all of them the definitive chorio-allantoic placenta was already formed, with one near term. In the most advanced pregnancy, the average 433
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weight of the three fetuses was i24. 5 g, and the mean crown-rump length (CR-L) was I6.2 cm. In the least advanced pregnancy the average weight of the four fetuses was 1.8 g, and the mean CR-L was 2. 4 cm. (Asibey, 1974, has recorded a mean birthweight of I28.5 g.) Relatively large pieces of placental tissue were fixed in Bouin's solution and processed for conventional histological examination. Small pieces of placental and subplacental tissues were fixed in glutaraldehyde-formaldehyde trinitrocresol (Ito and Karnovsky, 1968), and postfixed in osmium tetroxide overnight. The tissues were then washed in physiological saline, dehydrated in ascending grades of alcohol and cleared with propyline oxide. They were then embedded in Epon-Araldite (R. P. Cargille Laboratories Inc., New Jersey, USA). Thin sections (6o nm thick) cut with a diamond knife were stained successively with uranyl acetate and lead citrate for examination with a Philips 2Ol electron microscope, while thick sections cut with glass knife were stained with toluidine blue and examined with the light microscope.
RESULTS Observations with the light microscope revealed the division of the cane rat placenta into labyrinthine and spongy zones and a distinct subplacental region (Figure I). In the labyrinthine zone the syncytiotrophoblast formed the immediate wall of the maternal blood spaces and stained darkly with toluidine blue due to the presence of numerous basophilic granules. The
Photomicrographof the definitiveplacenta of the cane rat showingthe labyrinthine (L) and spongy(S) zones and the well-developedsubplacenta (arrow). It also shows the non-vascularcollar of Perrotta (X). H & E, x 6.8.
Figure I.
Figure 2. A section of the labyrinthine zone of the chorio-aUantoic placenta showing mitotic figures in the cytotrophoblastic layer (arrow). Embedded in Epon-Araldite and stained with toluidine blue. x 415.
Figure3. The interhaemal membrane of the cane rat placenta consisting of a single layer ofsyncytiotrophoblast (S) a basal lamina and the fetal capillary endothelium (fc). T h e syncytial microvilli protrude into the maternal blood spaces (rob). Note the short strands of granular endoplasmic reticulum and also the infoldings of the basal plasmallema (arrows). Epon section cut at 6o nm and stained with uranyl acetate and lead citrate. • 656O,
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Figure 4. The interhaemal membrane of the chorio-allantoic placenta showing numerous coated micropinocytotic
vesicles(arrows)and a pair ofCentrioles(C) in the syncytialcytoplasm,mb = maternal bloodspaces;fc = fetalcapillary endothelium. Epon section cut at 6onto and stained with uranyl acetate and lead citrate, x 816o. cytotrophoblast, on the other hand, stained very lightly, had distinct cell bqundaries and showed mitotic figures (Figure z). The spongy zone consisted of a maze of darkly staining syncytiotrophoblast enclosing large maternal blood spaces. In this zone there was no cytotrophoblast and no fetal vessels penetrated into it. Ultrastructural observations on the labyrinthine zone showed that fetal and maternal blood streams were closely approximated, especially in those areas where the interhaemal membranes consisted of a thin layer of syncytial trophoblast, a basal lamina and the fetal capillary endothelium (Figure 3)- The surface of the syncytiotrophoblast facing the maternal blood space had numerous microvilli, while the basal surface had recesses which contained cytoplasmic infoldings. The cytoplasm was rich in short strands of granular endoplasmic reticulum (Figure 3). Centrioles were sometimes observed in sections of the syncytiotrophoblast (Figure 4). Large mitochondria and well-developed Golgi complexes were conspicuous features of the syncytial cytoplasm (Figure 5). Some coated vesicles appeared to be in open communication with the maternal blood space (Figure 4). In the spongy zone, syncytiotrophoblast lined the vascular channels which drained maternal blood from the labyrinth. The trophoblastic surface was marked by numerous microvilli, and the cytoplasm had a well developed rER with dilated cisternae containing an amorphous finely granular precipitate. In some areas of the syncytium there were deep recesses into which microvilli projected (Figure 6). Whether these 'intracellular canaliculi' were real or artefacts was difficult to ascertain. The Golgi apparatus was well developed and scattered within the
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Figure5. Highpowermagnificationof the syncytiotrophoblast.Notethe welldevelopedGolgicomplexes(G), granular endoplasmicreticulum,largemitochondria,and numerouscoatedvesicles,someof whichare associatedwith the Golgi complexes.Epon section cut at 6ohm and stained with uranyl acetateand lead citrate, x 95oo.
cytoplasm. Other cytoplasmic organelles included rounded mitochondria, coated vesicles and numerous polyribosomes. Lipid droplets and glycogen deposits were frequent findings. The subplacenta consisted of folded lamellae ofcytotrophoblast and syncytiotrophoblast. The lamellae were separated from each other by fetal mesenchyme. The cytotrophoblastic cells had an undifferentiated appearance with free ribosomes, few rounded mitochondria and small Golgi apparatus. Cytotrophoblastic cells were attached to each other by desmosomes and large vacuoles appeared in the cytoplasm (Figure 7). Desmosomal connections between the syncytioand cyto-trophoblast were frequently observed, and the intercellular spaces were dilated to form lacunae occupied by short microvilli mainly from the syncytiotrophoblast. The lacunae contained an electron-dense precipitate. Scattered throughout the syncytial cytoplasm were numerous spherical granules (Figure 8). Such granules seemed to be peculiar to the subplacental syncytium as they were not observed in the syncytium of the labyrinthine and spongy zones. The cytoplasmic zone immediately below the microvillous border was free of organelles but was occupied by a finely granular, electron-dense substance similar to that in the lacunae. There were strands ofrER with dilated cisterns (Figure 8) and widely scattered Golgi membranes (Figure 9). DISCUSSION The suggestion by Roberts and Perry (x974) that a haemoendothelial condition may develop in late pregnancy in hystricomorph rodents has not been supported by ultrastructural finding. In
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Figure 6. Syncytiotrophoblast of the spongy zone. Granular endoplasmic reticulum and coated vesicles are demonstrated. Note also the intracytoplasmie canaliculi (Ic) studded with microvilli. Numerous microvilli also project into the maternal blood space (nab). Epon section cut at 6ohm and stained with uranyl acetate and lead citrate. • 43oo.
the chinchilla (King and Tibbitts, I976) as well as in the present study of the cane rat, the minimal placental barrier, up to the end of pregnancy, consists of a single layer of syncytiotrophoblast, a common basal lamina and fetal capillary endothelium. The interhaemal membrane in the cane rat placenta is of the haemomonochorial type according to the classification of Enders (I965). The fine structure of the trophospongium is also similar to that of the guinea pig and chinchilla. It is therefore probable that the cane rat trophospongium carries out biosynthetic activities similar to those described in the guinea pig (see Tam and Burgess, I977; Burgess and Tam, x978; Bambra et al, I98o). A unique feature of hystricognathous hystricomorph placentation is the presence of the subplacenta. It contains PAS-positive diastase-resistant material occupying what appears to be lacunae or spaces (see Davies, Dempsey and Amoroso, I96Ib; Oduor-Okelo and Gombe, I98z ). Ultrastructurally, the subplacenta consists of both cyto- and syncytiotrophoblast. The cytotrophoblast is very much undifferentiated while the syncytiotrophoblast has a well developed granular endoplasmic reticulum, scattered Golgi complexes, and numerous electrondense granules of uniform size. King and Tibbitts (i 976) have suggested that these granules may be related to secretory activities, although the nature of the secretion is not yet known. A finely granular precipitate is present in the intercellular spaces and also in the syncytial cytoplasm
Placental Ultrastructure of the Cane Rat
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Figure 7. The subplacenta of the cane rat chorio-allantoicplacenta. The cytotrophoblast(ct) and syncytialtrophoblast (st) are held togetherby desmosomes.The intercellular spacesbetweenthese two typesoftrophoblast are studded with microvilli. Vacuoles (arrows) seem to be an important transport route from the mesenchyme (m) through the cytotrophoblast. Epon sections cut at 6o nm and stained with uranyl acetate and lead citrate, x 495o.
immediately below the microvillous border. It is suggested that this precipitate is the PASpositive material seen in histological sections and is therefore both extra- and intra--cellular. Several physiological functions have been suggested for the subplacenta (see Perrotta, I959; Davies, Dempsey and Amoroso, 1961 b; Roberts and Perry, 1974; Luckett and Mossman, 198 i). Heap and Illingworth (i974) have isolated a progesterone-binding globulin (PBG) from the plasma of several hystricomorph rodents during pregnancy and have suggested that such a substance may be produced in the subplacenta. This, however, is also speculative and further experimental work is needed to determine whether the subplacenta is the site of PBG synthesis and, if so, how PBG gets into maternal circulation.
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Figure 8. The subplacentaof the cane rat chofio-allantoicplacenta.The syncytiotrophoblast(st) has a well developed granular endoplasmicreticulum (er) and contains rounded electron-densegranules (g) of uniform size. Numerous microvilli protrude into the intercellular spaces. Note the precipitate in the intercellular space (arrow). The cytotrophoblasticcells(ct) are poor in organellesand have fewermicrovilli.Epon section cut at 6o nm and stained with uranyl acetate and lead citrate, x 396o.
SUMMARY The ultrastructure of the cane rat chorio-allantoic placenta has been studied. In its thinnest regions the interhaemal membrane is composed of a single layer of syncytiotrophoblast, a common basal lamina, and the fetal capillary endothelium. Hence this is a haemomonochorial placenta. The cytotrophoblastic cells show many mitotic figures but they are never seen directly bounding the maternal blood spaces. T h e syncytiotrophoblast of the spongy zone is rich in dilated cisterns of granular endoplasmic reticulum and is also characterized by many 'intrasyncytial canaliculi'. The subplacenta consists of both cytotrophoblast and syncytiotrophoblast. The syncytiotrophoblast has numerous short microvilli projecting into the intercellular spaces, granular endoplasmic reticulum, a well-developed Golgi complex, and numerous electron--dense granules of uniform size. T h e intercellular spaces or lacunae contain a precipitate of moderate electron density which is similar in texture to the intracellular precipitate in the 'brush border' region of the syncytiotrophoblast. The similarities between the ultrastructure of the cane rat placenta and those of the guinea pig and chinchilla are discussed.
Placental Uhrastructure of the Cane Rat
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Figure 9. The subplacenta of the cane rat chorio-allantoic placenta. The syncytiotrophoblast (st) has well developed Golgi complexes (arrows), granular endoplasmic reticulum and electron-dense granules (g). Note the organelle-free brush border (*) which contains an electron-dense homogenous precipitate. The cytotrophoblast (ct) is poor in organelles. The intercellular space (Is) is occupied by short microvilli mainly from the syncytiotrophoblast. Epon section cut at 6onm and stained with uranyl acetate and lead citrate, x ~26oo.
ACKNOWLEDGEMENTS I wish to thank Mr L. Brown and his family for organizing the trapping of cane rats at his Lukenya farm. I am grateful to Prof. T. R. Odhiambo, the Director of the International Centre for Insect Physiology & Ecology (ICIPE) for permission to use the electron microscope facilities at ICIPE. My appreciation also goes to the WHO Special Programme of Research Development and Training in Human Reproduction, and the Department of Cell Biology, University of Texas Health Science Center at Dallas where part of this work was carried out. I also wish to thank Mr J. Gichiri and Mrs R. Katema for their technical assistance, and Mrs G. Hinga for typing the script. REFERENCES Amoroso, E. C. (1952) Placentation. In Marshall's Physiology of Reproduction (Ed.) Parkes, A. S. Volume z, pp. I27-31t. London: Longmans. Asibey, E. O. A. (x974) Reproduction in the grass cutter (Thryonomys swmderianusTemminck) in Ghana. Symposia of the ZoologicalSociety of London, 34, 251-263. Bambra, C. S., Robinson, G., Foxcroft, G. R. & Amoroso, E. C. (t98o) Immunohistochemical localization of a chorionic gunadotrophin-like material in the guinea pig placenta. In Abstracts of the Winter Meeting of the Societyfor the study of Fertility, London, pp. 20. Burgess, S. M. & Tam, W. H. (1978) Ultrastructural changes in the guinea pig placenta with special reference to organdies associated with steroidogenesis. Journal of Anatomy, i26, 319-327 . Davies, J., Dempsey, E. W. & Amoroso, E. C. (t961a) The subplacenta of the guinea pig: an electron microscopic study. Journal of Anatomy, 95, 311-324.
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