Cell Tiss. Res. 164, 179- 192 (1975) - 9 by Springer-Verlag 1975
Ultrastructural Changes in Granulosa Lutein Cells and Progesterone Levels during Preimplantation, Implantation, and Early Placentation in the Western Spotted Skunk* Akhouri A. Sinha and Rodney A. Mead Section of Metabolism and Endocrinology,Veterans Administration Hospital and Department of Zoology, Universityof Minnesota, Minneapolis, USA, Department of BiologicalSciences,Universityof Idaho, Moscow, USA
Summary. The ultrastructure of corpora lutea obtained during the preimplantation, implantation and early post-implantation periods has been studied in 20 western spotted skunks. Fine structure of granulosa lutein cells was correlated with progesterone levels. The corpus luteum of the prolonged (7 month) preimplantation period contained undifferentiated small granulosa cells and differentiated large granulosa lutein cells. The former ranged in size between 12 and 20 It and the latter between 20 and 45 It. The ratio of small and large cells was about equal in an animal 2 days prior to nidation whereas only few small cells and numerous large cells were observed in an animal estimated to be 8 to 12 hours from nidation. Occasionally small cells were observed amidst large ones during the 24 hour nidation period, i.e. adhesion of trophoblast with the luminal uterine epithelium, but small cells were absent in animals after this period. Small cells had some smooth and rough endoplasmic reticulum, rod-shaped mitochondria with plate-llike cristae, small Golgi complex, and relatively smooth plasma membranes. Large lutein cells had abundant smooth endoplasmic reticulum, membranous whorls of smooth endoplasmic reticulum, usually round mitochondria with tubular and lamellar cristae, a well developed Golgi complex, variable amounts of lipid droplets, and highly plicated and ruffled plasma membranes. Peripheral plasma progesterone levels during the prolonged preimplantation period ranged between 1.1 and 7.9 ng/ml, but during implantation it was between 8 and 16.6 ng/ml. It is suggested that plasma progesterone levels fluctuate during the time of implantation and should not be regarded as a basis to predict actual nidation in the western spotted skunk. words: Granulosa lutein cells - Western spotted skunk - Progesterone levels - Implantation.
Key
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Ultrastructure
Dr. Akhouri A. Sinha, Bldg. 49, Rm. 207, Veterans Administration Hospital, 54th Street and 48th Avenue South, Minneapolis, Minnesota 55417, USA. * This research was supported in part by Grant Number HD06556 from the National Institute of Child Health and Human Development. Send offprint requests to:
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The western spotted skunk (SpilogaIe putorius latifrons) breeds in late September and early embryonic development proceeds as in most mammals. However, by the time the embryos enter the uterus, the corpora lutea become involuted, plasma progesterone levels remain relatively low, and embryonic development becomes arrested at the blastocyst stage for approximately 7 months (Mead, 1968; Mead and Eik-Nes, 1969). Light-microscopic studies of corpora lutea indicated that lutein cells and their nuclei double in size in implanted animals from those of the delay period. Plasma progesterone levels increase in implanted animals (Mead and Eik-Nes, 1969; Mead, 1975; Foresman and Mead, 1974). Ultrastructural changes in pre-and-postimplantation corpora lutea of species exhibiting obligate delay of implantation such as the mink Mustela vison (Enders and Enders, 1963; Moller, 1973), European badger Meles meles (Canivenc et al., 1967) and a few spotted skunks (Sinha, 1974) have been described. This paper extends the above observations by critically examining ultrastructural changes in lutein cells in a series of spotted skunks killed during the final days of the preimplantation period, during implantation, and early placentation and correlates such changes with plasma progesterone levels in the same skunks.
Table 1. Pertinent data regarding reproductive state of each skunk, cell measurements and peripheral plasma progesterone values Animal number
Date
Mean of granulosa lutein cells (in micra)
Plasma progesterone (ng/ml)
Remarks stage of implantation
1214 1219 1153" 1188" 1218 a 1184 1234 1238 1200" 1197 1247 1202 1218 (right) 1240 1245 1224 1222 1217 1271 1273
3/8/71 3/3/71 4/22/70 4/7/71 4/7/71 5/7/71 4/26/72 4/23/72 5/5/71 4/11/71 5/13/72 4/13/72 4/11/71 4/22/72 4/21/72 4/2/71 5/12/71 4/1/71 5/2/73 5/3/73
13 18 23 23 31 32 33 34 35 34 38 38 39 41 28 40 40
1.1 3.7 5.0 7.9 6.3 9.6 16.6 8.3 12.5 8.0 11.8 14.0 8.3 9.5
U U U+ U U I I Ib Ib I I I I I Ib PL PL PL PL PL
24
-10.3 12.0 32.0 8.7
a Tissues obtained by laparotomy of uterus ; U = Unimptanted; I = Implanted ; + Birth of one young on 5/23/71 ; P L = Early Placenta. b Morphological features of uterus, blastocyst and corpus luteum suggested that implantation had occurred, but actual adhesion of trophoblast with uterine epithelium was not seen.
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Materials and Methods Corpora lutea were obtained from five western spotted skunks (Spilogaleputorius latifrons) during the prolonged preimplantation period, from ten skunks killed during various stages of implantation, and from five skunks with early post-implantation embryos. Tissues were obtained by laparotomy or by sacrificing the animals (Table 1). Appropriate portions of corpora were fixed for 2 to 4 hours in 3% gluteraldehyde in 0.1 M phosphate buffer at pH 7.3, washed four times in phosphate buffer, and post-fixed in 2% buffered osmiumtetroxide for 1.5 hours. Specimens were dehydrated in a graded series of ethyl alcohol, embedded in Epon 812 (Luft, 1961)following two changes in propylene oxide, and sectioned with an LKB 111 ultratome. Thin sections were stained with a combination uranyl acetate and lead citrate and examined with an RCA-EMU-4 electron microscope. Unstained thin sections and thick sections stained with methylene blue were also examined. In addition, several corpora were fixed in 3% glutaraldehyde or a mixture of alcohol, formalin and acetic acid, embedded in paraffin, cut at 6-8 p., and stained with Groat-s tetrachrome stain. From each corpus luteum, the greatest diameter of ten large granulosa lutein cells was measured with an ocular micrometer. In addition, blood from these animals was obtained by cardiac puncture, and the plasma assayed for progesterone by a previously validated radio-immunoassay (Mead, 1975). In this study, nidation was considered to have occurred when histological examination revealed adhesion of trophoblastic cells with luminal epithelia of the uterus (Sinha and Mead, 1975).
Results
Granulosa Lute& Cells from the Delay Period. C o r p o r a lutea obtained f r o m five skunks during the preimplantation period contained r a n d o m l y distributed small granulosa cells, large granulosa lutein cells and connective tissues (Figs. 1-3, 5). The small cells ranged f r o m 12 and 20 la in size, and the large ones between 20 and 45 Ix, with an average o f 35 Ix. C o r p o r a lutea f r o m animals 1214 and 1219 (estimated to be m o r e than 4 weeks away f r o m implantation) contained n u m e r o u s small cells, and some large cells. However, in skunk 1153 (about 2 days f r o m implantation) the n u m b e r o f small and large cells appeared equal. C o r p o r a lutea f r o m female 1218 (estimated to be 8 to 12 hours before nidation) contained a few small granulosa cells and n u m e r o u s large granulosa lutein cells (Fig. 5). The small cells had small a m o u n t s o f s m o o t h and r o u g h endoplasmic reticulum, rod-shaped m i t o c h o n d r i a with plate-like and some tubular cristae and relatively s m o o t h plasma m e m b r a n e s (Figs. 2, 9). The small cells differentiated into large lutein cells. T h e large lutein cells possessed large a m o u n t s o f s m o o t h endoplasmic reticulum, n u m e r o u s r o u n d - s h a p e d m i t o c h o n d r i a with tubular cristae, sometimes m i t o c h o n d r i a contained crystalloids, well-developed Golgi complex with cisternae a n d vesicles, variable a m o u n t s o f lipid droplets, and plicated and ruffled plasma m e m b r a n e s (Figs. 9, 17, 18). Each cell had a nearly r o u n d nucleus with a p r o m i n e n t nucleolus which was occasionally associated with electron o p a q u e bodies (Fig. 12). Plasma progesterone levels in these animals ranged between 1.1 and 7.0 ng]ml (Table 1). Theca lutein cells were not observed in any o f the c o r p o r a lutea examined in this study. Granulosa Lutein Cells from the Implantation Period. C o r p o r a lutea f r o m ten animals representing stages o f nidation were studied. Each corpus contained p r e d o m i n a n t l y large granulosa lutein cells. Occasional small granulosa cells were observed in animals within the first 24 hours o f nidation (as in animals 1184, 1234, 1197), but no small cells were observed in animals after this period (as in
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animals 1218, 1202, 1247, 1240, Table 1) (Figs. 4, 6). The corpora lutea were highly vascular and had attenuated capillaries. Ultrastructural features of large lutein cells from these implanted animals were comparable to the large lutein cells described previously (Figs. 9, 10). However, some variations in their fine structural features were also observed. For example, the cells enclosed variable amounts intercellular fluid and macromolecules between the ruffled and microvillous plasma membranes. In favorable sections some intercellular material appeared absorbed by the microspinocytotic vesicles which sometimes became associated with the subjacent smooth endoplasmic reticulum (Figs. 15.16). Morphologically the absorbed material appeared comparable to the lipid droplets as shown in other cells (Figs. 10, 13). However, histochemical studies should be undertaken to determine the nature of absorbed intercellular material. The large lutein cells of this period frequently showed apposing smooth endoplasmic reticulum and mitochondria (Figs. 11, 14, 17, 18). Occasionally granular endoplasmic reticulum was associated with mitochondria (Figs. 11, 18). In addition, close association of Golgi complex cisternae with mitochondria was also observed (Fig. 13). In some cells, several mitochondria contained crystalloids (Figs. 17, 18). The large lutein cells of this period also contained many membranous whorls of smooth and/or fenestrated endoplasmic reticulum (Figs. 7, 8, 9, 11, 14, 15) which sometimes showed electron opaque and less electron opaque layers between the membranes of the whorls (Fig. 11). Plasma progesterone levels of these skunks ranged between 8 and 16.6 ng/ml (Table 1, Fig. 19). Granulosa Lutein Cells during Early Placentation. Corpora lutea obtained from five animals exhibiting early stages ofplacentation showed only large granulosa lutein cells, connective tissues and blood vessels. Small granulosa cells were not observed. Most of the large lutein cells ranged in size from 35 to 45 g and exhibited ultrastructural features of large lutein cells described earlier. However, some lutein cells from this period contained more lipid droplets than those ob-
Fig. 1. Portion of a corpus luteum illustrating the ratio of granulosa cells (GC), granulosa lutein cells (arrow) in the corpus from female 1214 which was estimated to be 4 or more weeks away from implantation. Note the blood vessel (B). x 567 Fig. 2. A very low power electron micrograph from the above animal illustrating granulosa cells (GC) and granulosa lutein cells (GLC) with a m e m b r a n o u s whorl (MW). The corpus luteum was poorly fixed for electron microscopy, hence the fine structure of cells is not identified, x3,160 Fig. 3. A portion of a corpus luteum illustrating large granulosa lutein cells and occasional small granulosa cells (arrow) in skunk 1153. This tissue was obtained by laparotomy approximately 2 days prior to nidation, x 567 Fig. 4. A portion of a corpus luteum from female 1184 shows many large granulosa lutein cells
(GLC) containing variable a m o u n t s of lipid droplets (L). This animal possessed blastocysts in early stages ofnidation, x 576 Fig. 5. A portion of a corpus luteum obtained by laparotomy from an animal with unimplanted blastocysts (1218, left ovary) approximately 8 to 12 hours prior to implantation. Note the numerous large granulosa lutein cells (GLC), occasional small granulosa cells (GC), and intercellular space contains unidentified intercellular material (arrow). x 559
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Fig. 6. A portion of right ovary obtained 4 days later from skunk 1218 (Table 1, Fig. 5), Note the large granulosa lntein cells with variable amounts of lipid droplets. The ovary did not contain small granulosa cel{s, x 567
Fig. 7, A Portion of an electron micrograph from female 1218 (right ovary) showing several granulosa lutein cells (GLC), some cells are more electron opaque than others. The cells show many membranous whorls (MW), smooth endoplasmic reticulum, lipid droplets (L) and spherically shaped mitochondria. Note the intercellular spaces with intercellular fluid and materials, and perivascular cells (PV). • 5,980 Fig. 8. Detail of a portion of a lutein cell from skunk 1200 showing a m e m b r a n o u s whorl (MW) which illustrates longitudinal, oblique and transverse, and surface grazing of the smooth endoplasmic reticulum (SER). Note arrangement of r i b o s o m e s x 18,200
Fig. 9. Electron micrograph from female 1200 illustrating organelles of small granulosa cells (GC) and large granulosa lutein cells (GLC). Note that the mitochondria in the granulosa cells are usually rod-shaped with plate-like cristae but that there are some mitochondria (M) with tubular cristae. Also note the microvilli, intercellular spaces (IS) and portions of smooth endoplasmic reticulum (arrows) with lipid-like material, x 9,250 Fig. 10. Electron micrograph illustrating a portion of a granulosa lutein cell with fenestrated endoplasmic reticulum (ER), lipid droplets (L), smooth endoplasmic reticulum, round-shaped mitochondria with tubular cristae, microvilli (MV), and lysosome (arrow). • 8,100
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Fig. 11. Detail of a granulosa lutein cell from animal 1245 shows the nucleus (N), a well developed Golgi complex (G), membranous whorl (MW) with layers of electron opaque bands of SER alternating with less electron opaque bands of SER, tysosomes (small arrow), polyribosomes (large arrow), and mostly spherically shaped mitochondria with some tubular cristae, x 15,400
Granulosa Lutein Cells in the Western Spotted Skunk
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served during the implantation period. Plasma progesterone levels ranged from 8.7 to 32 ng/ml at this time.
Discussion
Although our data (Table 1) were obtained from a limited number of animals which do not represent a closely graded series of stages of the preimplantation, implantation, and early post-implantation period, they demonstrate that morphological changes in the granulosa lutein cells are correlated with changes in plasma progesterone levels (Fig. 19). Mead and Eik-Nes (1969) reported that plasmaprogesterone levels during the late preimplantation period were 6 to 12 ng/ml but attained levels in excess of 20 ng/ml after nidation. We have shown that plasma progesterone levels are variable late in the preimplantation period and during nidation (Table 1) and appear to be correlated with the extent of differentiation of the lutein cells. Our analysis of fine structure and cytology of granulosa cells and granulosa lutein cells from a few days prior to implantation through nidation has revealed that changes in cell size during luetinization are mostly due to increased synthesis of smooth endoplasmic reticulum, differentiation of mitochondria, plications of plasma membranes, and accumulation of lipid material. It also appears that differentiation of mitochondria may be a limiting factor during the luteinization of cells. This limitation is probably reflected by fluctuations in the levels of plasma progesterone during the time of implantation. In fact, mitochondria have been considered as a rate limiting step in the biosynthesis of steroids (Garren et al., 1971 ; Koritz, 1962; Savard, 1973). The process ofluteinization is essentially completed within 72 hours of nidation in the skunk. The processes of luteinization have also been reported for other species (Enders, 1973; Bjersing, 1967; Blanchette, 1966; Koering et al., 1973). Membranous whorls of smooth endoplasmic reticulum are considered indicative of highly active lutein cells in sows (Bjersing, 1967) and other species (Enders, 1973; Christensen and Gillim, 1969; Sinha, 1974). Since such whorls first appear at a time when plasma progesterone levels are increasing in the western spotted skunks, we have likewise assumed their presence to be associated with increased steroidogenesis. However, highly active lutein cells of some species do not contain membranous whorls (Gillim et al., 1969, Crisp and Browning, 1968). Fig. 12. Details of a nucleus of a lutein cell of female 1245 shows the nucleolus (NU) and several electron-opaque bodies (arrows) closely associated with the nucleolonema. • 24,500 Fig. 13. Details of a lutein cell from skunk 1245 showing close association of Golgi complex (G) with a mitochondrium (M). Note that the mitochondria membranes are contiguous (arrow) with cisternae of the Golgi complex. Also note lysosome (LY) and lipid droplet (L). • 21,100 Fig. 14. A portion of lutein cells illustrating contiguous membranes of membranous whorl (MW), and a mitochondrial membrane (small arrows) from female 1202 with implanted blastocysts. Note the plasma membranes, microvilli, micropinocytotic vesicles (large arrows), and a perivascular cell (PV). • 12,900
Fig. 15. Details of an intercellular space (IS)and subjacent portion of a lutein cell illustrating membranous whorl (M W) and tubules of SER containing lipid/cholesterol-like material (large arrows). Note that the tubules of SER are often continguous with the membranes of the whorl (MW). Note the microvilli (MV), micropinocytotic vesicles (small arrows). • 19,500 Fig. 16. Details ofa granulosa lutein cell female 1197 showing portions of SER containing lipid/cholesterol-like material (small arrows), while other portions of SER (large arrow) are without the lipid material. Note the mitochondria (M), Golgi complex (G), folds and microvilli, intercellular space (IS) and subjacent micropinocytotic vesicles (V). • 34,600
Fig. 17. Details of a granulosa lutein cell from animal 1245 show large mitochondria (M) containing nearly rectangular intramitochondrial crystalloids (CR). Note the close association of mitochondrial membranes and smooth endoplasmic reticulum (SER) and plasma membranes and associated vesicles (V). Also note the lipid droplet (L), granular endoplasmic reticulum (GER), and intercellular spaces (IS). Crystalloids have a lattice-like arrangement, x 42,000 Fig. 18. Details of a granulosa lutein cell from the above animal showing close association of Golgi complex cisternae (G) with mitochondrial membranes. Note the mitochondria (M) and a mitochondrion with a crystalloid (CR). x 26,200
190
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Fig. 19. The graph illustrates relationshipof granulosa lutein cell size and progesterone levels during delay and implantationin the western spotted skunk. (I implanted; U unimplanted)
Plasma membranes of skunk lutein cells have plications, ruffles, and microvilli much as in lutein cells in other species (Bjersing et al., 1970; Enders, 1973) and other steroid secreting cells (Christensen and Gillim, 1969; Fawcett et al., 1969; Priedkalns and Weber, 1968; Enders and Lyons, 1964). Apparently, these specializations have developed to compensate for increased surface activities such as absorption of lipid/cholesterol and other macromolecules from the perivascular spaces (Adams and Hartig, 1969 a, b; Enders, 1962, 1973), and for release of hormones and other materials into the intercellular spaces (Green and Maqueo, 1965; Tokida, 1965; Gillim et al., 1969). Our study suggests that once lipid accumulation has begun, it continues until the lutein cells are ladened with large numbers of lipid droplets much as in the raccoon (Procyon lotor), deer (Odocoileus virginianus), and crabeater seal (Lobodon carcinophagus), and leopard seal (Hydrurga leptonyx) (Sinha et al., 1971 a, b; Sinha, 1974). These cells essentially become lipid storage cells and have sparse cytoplasmic organelles. On the other hand active lutein cells are reported to have abundant smooth endoplasmic reticulum and mitochondria but small numbers of lipid droplets (Enders, 1973 ; Christensen and Gillim, 1969; Sinha et al., 1971 a, b; Fawcett et al., 1969). Based on morphological analysis of lutein cells in the skunk and other carnivores (Sinha, 1974) it is suggested that most of the transfer of cholesterol and other lipids occurs at the smooth endoplasmic reticulum and mitochondrial membrane apposition sites or where tubular smooth endoplasmic reticulum is contiguous with mitochondrial cristae (Sinha et al., 1971 a). Similar organelle to organelle apposition sites have been reported in other steroid secreting tissues. Likewise the functional significance of the association of lipid droplets, mitochondria and smooth endoplasmic reticulum and pathways for stereoidogenesis have been discussed (Deane, 1958; Long and Jones, 1967; Hirschfield and Koritz, 1966; Jackanicz and Armstrong, 1968; Flint and Armstrong, 1972; Salhanick et al., 1973). Williamson (1964) considers caveolae to be involved in lipid transport in other cells.
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References Adams, E.C., Hertig, A.T.: Studies on the corpus luteum. I. Observations on the ultrastructure of development and regression of the luteal cells during the menstrual cycle. J. Cell Biol. 41, 696 715 (1969a) Adams, E.C., Hertig, A.T. : Studies on the human corpus luteum. II. Observations on the ultrastructure of luteal cells during pregnancy. J. Cell Biol. 41, 716-735 (1969b) Bjersing, L. : On the ultrastructure of granulosa lutein cells in porcine corpus luteum. Z. Zellforsch. 82, 187-211 (1967) Bjersing, L., Hay, M.F., Moor, R.M., Short, R.V. : Endocrine activity, histochemistry and ultrastructure of ovine corpora lutea. Further observations on regression at the end of the oestrous cycle. Z. Zellforsch. l l l , 437~157 (1970) Blanchette, E.J. : Ovarian steroid cells. II. The luteal cell. J. Cell Biol. 31,517-542 (1966) Canivenc, R., Cohere, G., Brechenmacher, C. : Quelques aspects ultrastructuraux de la cellule lut6ale chez le Blaireau (Meles meles L.). C.R. Acad. Sci. (Paris) 264, 1187-1189 (1967) Christensen, A.K., Gillim, S.W. : The correlation of fine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In : The gonads (K.W. McKerns, ed.), p. 415-488.New York : Appleton-Century-Crofts 1969 Crips, T.M., Browning, H.C.: The fine structure of corpora lutea in ovarian transplants of mice following luteotrophin stimulation. Amer. J. Anat. 122, 169-192 (1968) Deane, H.W.: Intracellular lipids: Their detection and significance. I: Frontiers in cytology (S.L. Palay, ed.), p. 227-263. New Haven: Yale University Press 1958 Enders, A.C.: Observations on the fine structure of lutein cells. J. Cell Biol. 12, 101-113 (1962) Enders, A.C. : Cytology of the corpus luteum. Biol. Reprod. 8, 158 182 (1973) Enders, A.C., Lyons, W.R.: Observations on the fine structure of lutein cells. II. The effects of hypophysectomy and mammotrophic hormone in the rat. J. Cell Biol. 22, 127-141 (1964) Enders, R.K., Enders, A.C. : Morphology of the female reproductive tract during delayed implantation in the mink. In: Delayed implantation, p. 129-139 (ed. A.C. Enders). Chicago: Chicago Univ. Press 1963 Fawcett, D.T., Long, J.A., Jones, A.U : The ultrastructure of endocrine glands. Recent Prog. Hormone Res. 25, 315-380 (1969) Flint, A.P.F., Armstrong, D.T.: Dynamic aspects of ovarian cholesterol metabolism: regulation by gonadotropins. In: Gonadotropins (B.B. Saxena, C.G. Beling, and H.M. Gandy, eds.), p. 269-286. New York: Wiley-Interscience 1972 Foresman, K.R., Mead, R.A. : Pattern of luteinizing hormone secretion during delayed implantation in the spotted skunk (Spilogaleputorius latifrons). Biol. Reprod. 11, 475-480 (1974) Garren, L.D., Gill, G.N., Masui, H., Walton, G.M. : On the mechanism of action of ACTH. Recent Progr. Hormone Res. 27, 443-478 (1971) Gillim, S.W., Christensen, A.K., McLennan, C.E. : Fine structure of the human menstrual corpus luteum at its stage of maximum secretory activity. Amer. J. Anat. 126, 409-428 (1969) Green, J.A., Maqueo, M. : Ultrastructure of the human ovary. I. The luteal cell during the menstrual cycle. Amer. J. Obstet. Gynec. 92, 946~57 (1965) Hirschfeld, I.H., Kortiz, S.B. : Pregnenolone synthesis stimulation in the large particles from bovine adrenal cortex and bovine corpus luteum. Endocrinology 78, 165 168 (1966) Jackanicz, T.M., Armstrong, D.T.: Progesterone biosynthesis in rabbit ovarian interstitial tissue mitocbondria. Endocrinology 83, 789-776 (1968) Koering, M.J., Wolf, R.C., Meyer, R.K.: Morphological changes in the corpus luteum correlated with progestin levels in the rhesus monkey during early pregnancy. Biol. Reprod. 9, 254-271 (1973) Koritz, S.B.: The effect of calcium ions and freezing on the in vitro synthesis of pregnenolone by rat adrenal preparations. Biochim. biophys. Acta (Amst.) 56, 63-75 (1962) Long, J.A., Jones, A.L.: The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum. Amer. J. Anat. 120, 463488 (1967) Luft, J.H. : Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409-414 (1961) Mead, R.A. : Reproduction in western forms of the skunk (genus Spilogale). J. Mammal. 49, 373-390 (1968)
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Mead, R.A. : Effect of luteinizing hormone-releasing hormone on luteal recrudescence during obligate delay of implantation in the western spotted skunk. Submitted for publication 1975 Mead, R.A. : Effects of hypophysectomy on blastocyst survival, progesterone secretion and nidation in the spotted skunk. Biol. Reprod. 12, 526 533 (1975) Mead, R.A., Eik-Nes, K.B.: Seasonal variation in plasma levels of progesterone in western forms of the spotted skunk. J. Reprod. Fertil., Suppl. 6, 397403 (1969) Moller, O.M. : The fine structure of the lutein cells in the mink (Mustela vision) with special reference to the secretory activity during pregnancy. Z, Zellforsch. 138, 523-544 (1973) Priedkalns, J., Weber, A.F. : Ultrastructural studies on the bovine Graafian follicle and corpus luteum. Z. Zellforsch. 91, 554-573 (1968) Salhanick, H.A., McIntosh, E.N., Uzgiris, V.I., Whipple, C.A., Mitani, F.: Mitochondrial systems of pregnenolone synthesis and its inhibition. In: The regulation of mammalian reproduction, p. 520-534. Illinois: Charles C. Thomas 1973 Savard, K. : The biochemistry of the corpus luteum. Biol. Reprod. 8, 183~02 (1973) Sinha, A.A.: Comparative ultrastructure of the corpus luteum of implantation and pregnancy in carnivora. In : Electron microscopic concepts of secretion ultrastructure of endocrinal and reproductive organs, p. 53-69 (ed. Melvin Hess). New York: John Wiley and Sons, Inc., eds. 1974 Sinha, A.A., Mead, R.A.: Morphological changes in the trophoblast, uterus and corpus luteum during delayed implantation and implantation in the western spotted skunk. Submitted for publication 1975 Sinha, A.A., Seal, U.S., Doe, R.P.: Fine structure of the corpus luteum of the raccoon during pregnancy. Z. Zellforsch. 117, 3545 (1971 a) Sinha, A.A., Seal, U.S., Doe, R.P.: Ultrastructure of the corpus luteum of the white-tailed deer during pregnancy. Amer. J. Anat. 132, 189-206 (1971 b) Tokida, A. : Electron microscopic studies on the corpora lutea obtained from normal human ovaries. Mie med. J. 15, 27-76 (1965) Williamson, J.R. : Adipose tissue. Morphological changes associated with lipid mobilization. J. Cell Biol. 20, 57 74 (1964)
Received July 9, 1975 / &final form August 14, 1975
Ultrastructural Changes in Granulosa Lutein Cells and Progesterone Levels during Preimplantation, Implantation, and Early Placentation in the Western Spotted Skunk* Akhouri A. Sinha and Rodney A. Mead Section of Metabolism and Endocrinology,Veterans Administration Hospital and Department of Zoology, Universityof Minnesota, Minneapolis, USA, Department of BiologicalSciences,Universityof Idaho, Moscow, USA
Summary. The ultrastructure of corpora lutea obtained during the preimplantation, implantation and early post-implantation periods has been studied in 20 western spotted skunks. Fine structure of granulosa lutein cells was correlated with progesterone levels. The corpus luteum of the prolonged (7 month) preimplantation period contained undifferentiated small granulosa cells and differentiated large granulosa lutein cells. The former ranged in size between 12 and 20 It and the latter between 20 and 45 It. The ratio of small and large cells was about equal in an animal 2 days prior to nidation whereas only few small cells and numerous large cells were observed in an animal estimated to be 8 to 12 hours from nidation. Occasionally small cells were observed amidst large ones during the 24 hour nidation period, i.e. adhesion of trophoblast with the luminal uterine epithelium, but small cells were absent in animals after this period. Small cells had some smooth and rough endoplasmic reticulum, rod-shaped mitochondria with plate-llike cristae, small Golgi complex, and relatively smooth plasma membranes. Large lutein cells had abundant smooth endoplasmic reticulum, membranous whorls of smooth endoplasmic reticulum, usually round mitochondria with tubular and lamellar cristae, a well developed Golgi complex, variable amounts of lipid droplets, and highly plicated and ruffled plasma membranes. Peripheral plasma progesterone levels during the prolonged preimplantation period ranged between 1.1 and 7.9 ng/ml, but during implantation it was between 8 and 16.6 ng/ml. It is suggested that plasma progesterone levels fluctuate during the time of implantation and should not be regarded as a basis to predict actual nidation in the western spotted skunk. words: Granulosa lutein cells - Western spotted skunk - Progesterone levels - Implantation.
Key
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Ultrastructure
Dr. Akhouri A. Sinha, Bldg. 49, Rm. 207, Veterans Administration Hospital, 54th Street and 48th Avenue South, Minneapolis, Minnesota 55417, USA. * This research was supported in part by Grant Number HD06556 from the National Institute of Child Health and Human Development. Send offprint requests to:
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The western spotted skunk (SpilogaIe putorius latifrons) breeds in late September and early embryonic development proceeds as in most mammals. However, by the time the embryos enter the uterus, the corpora lutea become involuted, plasma progesterone levels remain relatively low, and embryonic development becomes arrested at the blastocyst stage for approximately 7 months (Mead, 1968; Mead and Eik-Nes, 1969). Light-microscopic studies of corpora lutea indicated that lutein cells and their nuclei double in size in implanted animals from those of the delay period. Plasma progesterone levels increase in implanted animals (Mead and Eik-Nes, 1969; Mead, 1975; Foresman and Mead, 1974). Ultrastructural changes in pre-and-postimplantation corpora lutea of species exhibiting obligate delay of implantation such as the mink Mustela vison (Enders and Enders, 1963; Moller, 1973), European badger Meles meles (Canivenc et al., 1967) and a few spotted skunks (Sinha, 1974) have been described. This paper extends the above observations by critically examining ultrastructural changes in lutein cells in a series of spotted skunks killed during the final days of the preimplantation period, during implantation, and early placentation and correlates such changes with plasma progesterone levels in the same skunks.
Table 1. Pertinent data regarding reproductive state of each skunk, cell measurements and peripheral plasma progesterone values Animal number
Date
Mean of granulosa lutein cells (in micra)
Plasma progesterone (ng/ml)
Remarks stage of implantation
1214 1219 1153" 1188" 1218 a 1184 1234 1238 1200" 1197 1247 1202 1218 (right) 1240 1245 1224 1222 1217 1271 1273
3/8/71 3/3/71 4/22/70 4/7/71 4/7/71 5/7/71 4/26/72 4/23/72 5/5/71 4/11/71 5/13/72 4/13/72 4/11/71 4/22/72 4/21/72 4/2/71 5/12/71 4/1/71 5/2/73 5/3/73
13 18 23 23 31 32 33 34 35 34 38 38 39 41 28 40 40
1.1 3.7 5.0 7.9 6.3 9.6 16.6 8.3 12.5 8.0 11.8 14.0 8.3 9.5
U U U+ U U I I Ib Ib I I I I I Ib PL PL PL PL PL
24
-10.3 12.0 32.0 8.7
a Tissues obtained by laparotomy of uterus ; U = Unimptanted; I = Implanted ; + Birth of one young on 5/23/71 ; P L = Early Placenta. b Morphological features of uterus, blastocyst and corpus luteum suggested that implantation had occurred, but actual adhesion of trophoblast with uterine epithelium was not seen.
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Materials and Methods Corpora lutea were obtained from five western spotted skunks (Spilogaleputorius latifrons) during the prolonged preimplantation period, from ten skunks killed during various stages of implantation, and from five skunks with early post-implantation embryos. Tissues were obtained by laparotomy or by sacrificing the animals (Table 1). Appropriate portions of corpora were fixed for 2 to 4 hours in 3% gluteraldehyde in 0.1 M phosphate buffer at pH 7.3, washed four times in phosphate buffer, and post-fixed in 2% buffered osmiumtetroxide for 1.5 hours. Specimens were dehydrated in a graded series of ethyl alcohol, embedded in Epon 812 (Luft, 1961)following two changes in propylene oxide, and sectioned with an LKB 111 ultratome. Thin sections were stained with a combination uranyl acetate and lead citrate and examined with an RCA-EMU-4 electron microscope. Unstained thin sections and thick sections stained with methylene blue were also examined. In addition, several corpora were fixed in 3% glutaraldehyde or a mixture of alcohol, formalin and acetic acid, embedded in paraffin, cut at 6-8 p., and stained with Groat-s tetrachrome stain. From each corpus luteum, the greatest diameter of ten large granulosa lutein cells was measured with an ocular micrometer. In addition, blood from these animals was obtained by cardiac puncture, and the plasma assayed for progesterone by a previously validated radio-immunoassay (Mead, 1975). In this study, nidation was considered to have occurred when histological examination revealed adhesion of trophoblastic cells with luminal epithelia of the uterus (Sinha and Mead, 1975).
Results
Granulosa Lute& Cells from the Delay Period. C o r p o r a lutea obtained f r o m five skunks during the preimplantation period contained r a n d o m l y distributed small granulosa cells, large granulosa lutein cells and connective tissues (Figs. 1-3, 5). The small cells ranged f r o m 12 and 20 la in size, and the large ones between 20 and 45 Ix, with an average o f 35 Ix. C o r p o r a lutea f r o m animals 1214 and 1219 (estimated to be m o r e than 4 weeks away f r o m implantation) contained n u m e r o u s small cells, and some large cells. However, in skunk 1153 (about 2 days f r o m implantation) the n u m b e r o f small and large cells appeared equal. C o r p o r a lutea f r o m female 1218 (estimated to be 8 to 12 hours before nidation) contained a few small granulosa cells and n u m e r o u s large granulosa lutein cells (Fig. 5). The small cells had small a m o u n t s o f s m o o t h and r o u g h endoplasmic reticulum, rod-shaped m i t o c h o n d r i a with plate-like and some tubular cristae and relatively s m o o t h plasma m e m b r a n e s (Figs. 2, 9). The small cells differentiated into large lutein cells. T h e large lutein cells possessed large a m o u n t s o f s m o o t h endoplasmic reticulum, n u m e r o u s r o u n d - s h a p e d m i t o c h o n d r i a with tubular cristae, sometimes m i t o c h o n d r i a contained crystalloids, well-developed Golgi complex with cisternae a n d vesicles, variable a m o u n t s o f lipid droplets, and plicated and ruffled plasma m e m b r a n e s (Figs. 9, 17, 18). Each cell had a nearly r o u n d nucleus with a p r o m i n e n t nucleolus which was occasionally associated with electron o p a q u e bodies (Fig. 12). Plasma progesterone levels in these animals ranged between 1.1 and 7.0 ng]ml (Table 1). Theca lutein cells were not observed in any o f the c o r p o r a lutea examined in this study. Granulosa Lutein Cells from the Implantation Period. C o r p o r a lutea f r o m ten animals representing stages o f nidation were studied. Each corpus contained p r e d o m i n a n t l y large granulosa lutein cells. Occasional small granulosa cells were observed in animals within the first 24 hours o f nidation (as in animals 1184, 1234, 1197), but no small cells were observed in animals after this period (as in
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animals 1218, 1202, 1247, 1240, Table 1) (Figs. 4, 6). The corpora lutea were highly vascular and had attenuated capillaries. Ultrastructural features of large lutein cells from these implanted animals were comparable to the large lutein cells described previously (Figs. 9, 10). However, some variations in their fine structural features were also observed. For example, the cells enclosed variable amounts intercellular fluid and macromolecules between the ruffled and microvillous plasma membranes. In favorable sections some intercellular material appeared absorbed by the microspinocytotic vesicles which sometimes became associated with the subjacent smooth endoplasmic reticulum (Figs. 15.16). Morphologically the absorbed material appeared comparable to the lipid droplets as shown in other cells (Figs. 10, 13). However, histochemical studies should be undertaken to determine the nature of absorbed intercellular material. The large lutein cells of this period frequently showed apposing smooth endoplasmic reticulum and mitochondria (Figs. 11, 14, 17, 18). Occasionally granular endoplasmic reticulum was associated with mitochondria (Figs. 11, 18). In addition, close association of Golgi complex cisternae with mitochondria was also observed (Fig. 13). In some cells, several mitochondria contained crystalloids (Figs. 17, 18). The large lutein cells of this period also contained many membranous whorls of smooth and/or fenestrated endoplasmic reticulum (Figs. 7, 8, 9, 11, 14, 15) which sometimes showed electron opaque and less electron opaque layers between the membranes of the whorls (Fig. 11). Plasma progesterone levels of these skunks ranged between 8 and 16.6 ng/ml (Table 1, Fig. 19). Granulosa Lutein Cells during Early Placentation. Corpora lutea obtained from five animals exhibiting early stages ofplacentation showed only large granulosa lutein cells, connective tissues and blood vessels. Small granulosa cells were not observed. Most of the large lutein cells ranged in size from 35 to 45 g and exhibited ultrastructural features of large lutein cells described earlier. However, some lutein cells from this period contained more lipid droplets than those ob-
Fig. 1. Portion of a corpus luteum illustrating the ratio of granulosa cells (GC), granulosa lutein cells (arrow) in the corpus from female 1214 which was estimated to be 4 or more weeks away from implantation. Note the blood vessel (B). x 567 Fig. 2. A very low power electron micrograph from the above animal illustrating granulosa cells (GC) and granulosa lutein cells (GLC) with a m e m b r a n o u s whorl (MW). The corpus luteum was poorly fixed for electron microscopy, hence the fine structure of cells is not identified, x3,160 Fig. 3. A portion of a corpus luteum illustrating large granulosa lutein cells and occasional small granulosa cells (arrow) in skunk 1153. This tissue was obtained by laparotomy approximately 2 days prior to nidation, x 567 Fig. 4. A portion of a corpus luteum from female 1184 shows many large granulosa lutein cells
(GLC) containing variable a m o u n t s of lipid droplets (L). This animal possessed blastocysts in early stages ofnidation, x 576 Fig. 5. A portion of a corpus luteum obtained by laparotomy from an animal with unimplanted blastocysts (1218, left ovary) approximately 8 to 12 hours prior to implantation. Note the numerous large granulosa lutein cells (GLC), occasional small granulosa cells (GC), and intercellular space contains unidentified intercellular material (arrow). x 559
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183
Fig. 6. A portion of right ovary obtained 4 days later from skunk 1218 (Table 1, Fig. 5), Note the large granulosa lntein cells with variable amounts of lipid droplets. The ovary did not contain small granulosa cel{s, x 567
Fig. 7, A Portion of an electron micrograph from female 1218 (right ovary) showing several granulosa lutein cells (GLC), some cells are more electron opaque than others. The cells show many membranous whorls (MW), smooth endoplasmic reticulum, lipid droplets (L) and spherically shaped mitochondria. Note the intercellular spaces with intercellular fluid and materials, and perivascular cells (PV). • 5,980 Fig. 8. Detail of a portion of a lutein cell from skunk 1200 showing a m e m b r a n o u s whorl (MW) which illustrates longitudinal, oblique and transverse, and surface grazing of the smooth endoplasmic reticulum (SER). Note arrangement of r i b o s o m e s x 18,200
Fig. 9. Electron micrograph from female 1200 illustrating organelles of small granulosa cells (GC) and large granulosa lutein cells (GLC). Note that the mitochondria in the granulosa cells are usually rod-shaped with plate-like cristae but that there are some mitochondria (M) with tubular cristae. Also note the microvilli, intercellular spaces (IS) and portions of smooth endoplasmic reticulum (arrows) with lipid-like material, x 9,250 Fig. 10. Electron micrograph illustrating a portion of a granulosa lutein cell with fenestrated endoplasmic reticulum (ER), lipid droplets (L), smooth endoplasmic reticulum, round-shaped mitochondria with tubular cristae, microvilli (MV), and lysosome (arrow). • 8,100
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Fig. 11. Detail of a granulosa lutein cell from animal 1245 shows the nucleus (N), a well developed Golgi complex (G), membranous whorl (MW) with layers of electron opaque bands of SER alternating with less electron opaque bands of SER, tysosomes (small arrow), polyribosomes (large arrow), and mostly spherically shaped mitochondria with some tubular cristae, x 15,400
Granulosa Lutein Cells in the Western Spotted Skunk
187
served during the implantation period. Plasma progesterone levels ranged from 8.7 to 32 ng/ml at this time.
Discussion
Although our data (Table 1) were obtained from a limited number of animals which do not represent a closely graded series of stages of the preimplantation, implantation, and early post-implantation period, they demonstrate that morphological changes in the granulosa lutein cells are correlated with changes in plasma progesterone levels (Fig. 19). Mead and Eik-Nes (1969) reported that plasmaprogesterone levels during the late preimplantation period were 6 to 12 ng/ml but attained levels in excess of 20 ng/ml after nidation. We have shown that plasma progesterone levels are variable late in the preimplantation period and during nidation (Table 1) and appear to be correlated with the extent of differentiation of the lutein cells. Our analysis of fine structure and cytology of granulosa cells and granulosa lutein cells from a few days prior to implantation through nidation has revealed that changes in cell size during luetinization are mostly due to increased synthesis of smooth endoplasmic reticulum, differentiation of mitochondria, plications of plasma membranes, and accumulation of lipid material. It also appears that differentiation of mitochondria may be a limiting factor during the luteinization of cells. This limitation is probably reflected by fluctuations in the levels of plasma progesterone during the time of implantation. In fact, mitochondria have been considered as a rate limiting step in the biosynthesis of steroids (Garren et al., 1971 ; Koritz, 1962; Savard, 1973). The process ofluteinization is essentially completed within 72 hours of nidation in the skunk. The processes of luteinization have also been reported for other species (Enders, 1973; Bjersing, 1967; Blanchette, 1966; Koering et al., 1973). Membranous whorls of smooth endoplasmic reticulum are considered indicative of highly active lutein cells in sows (Bjersing, 1967) and other species (Enders, 1973; Christensen and Gillim, 1969; Sinha, 1974). Since such whorls first appear at a time when plasma progesterone levels are increasing in the western spotted skunks, we have likewise assumed their presence to be associated with increased steroidogenesis. However, highly active lutein cells of some species do not contain membranous whorls (Gillim et al., 1969, Crisp and Browning, 1968). Fig. 12. Details of a nucleus of a lutein cell of female 1245 shows the nucleolus (NU) and several electron-opaque bodies (arrows) closely associated with the nucleolonema. • 24,500 Fig. 13. Details of a lutein cell from skunk 1245 showing close association of Golgi complex (G) with a mitochondrium (M). Note that the mitochondria membranes are contiguous (arrow) with cisternae of the Golgi complex. Also note lysosome (LY) and lipid droplet (L). • 21,100 Fig. 14. A portion of lutein cells illustrating contiguous membranes of membranous whorl (MW), and a mitochondrial membrane (small arrows) from female 1202 with implanted blastocysts. Note the plasma membranes, microvilli, micropinocytotic vesicles (large arrows), and a perivascular cell (PV). • 12,900
Fig. 15. Details of an intercellular space (IS)and subjacent portion of a lutein cell illustrating membranous whorl (M W) and tubules of SER containing lipid/cholesterol-like material (large arrows). Note that the tubules of SER are often continguous with the membranes of the whorl (MW). Note the microvilli (MV), micropinocytotic vesicles (small arrows). • 19,500 Fig. 16. Details ofa granulosa lutein cell female 1197 showing portions of SER containing lipid/cholesterol-like material (small arrows), while other portions of SER (large arrow) are without the lipid material. Note the mitochondria (M), Golgi complex (G), folds and microvilli, intercellular space (IS) and subjacent micropinocytotic vesicles (V). • 34,600
Fig. 17. Details of a granulosa lutein cell from animal 1245 show large mitochondria (M) containing nearly rectangular intramitochondrial crystalloids (CR). Note the close association of mitochondrial membranes and smooth endoplasmic reticulum (SER) and plasma membranes and associated vesicles (V). Also note the lipid droplet (L), granular endoplasmic reticulum (GER), and intercellular spaces (IS). Crystalloids have a lattice-like arrangement, x 42,000 Fig. 18. Details of a granulosa lutein cell from the above animal showing close association of Golgi complex cisternae (G) with mitochondrial membranes. Note the mitochondria (M) and a mitochondrion with a crystalloid (CR). x 26,200
190
A.A. Sinhaand R.A. Mead 123'$
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Fig. 19. The graph illustrates relationshipof granulosa lutein cell size and progesterone levels during delay and implantationin the western spotted skunk. (I implanted; U unimplanted)
Plasma membranes of skunk lutein cells have plications, ruffles, and microvilli much as in lutein cells in other species (Bjersing et al., 1970; Enders, 1973) and other steroid secreting cells (Christensen and Gillim, 1969; Fawcett et al., 1969; Priedkalns and Weber, 1968; Enders and Lyons, 1964). Apparently, these specializations have developed to compensate for increased surface activities such as absorption of lipid/cholesterol and other macromolecules from the perivascular spaces (Adams and Hartig, 1969 a, b; Enders, 1962, 1973), and for release of hormones and other materials into the intercellular spaces (Green and Maqueo, 1965; Tokida, 1965; Gillim et al., 1969). Our study suggests that once lipid accumulation has begun, it continues until the lutein cells are ladened with large numbers of lipid droplets much as in the raccoon (Procyon lotor), deer (Odocoileus virginianus), and crabeater seal (Lobodon carcinophagus), and leopard seal (Hydrurga leptonyx) (Sinha et al., 1971 a, b; Sinha, 1974). These cells essentially become lipid storage cells and have sparse cytoplasmic organelles. On the other hand active lutein cells are reported to have abundant smooth endoplasmic reticulum and mitochondria but small numbers of lipid droplets (Enders, 1973 ; Christensen and Gillim, 1969; Sinha et al., 1971 a, b; Fawcett et al., 1969). Based on morphological analysis of lutein cells in the skunk and other carnivores (Sinha, 1974) it is suggested that most of the transfer of cholesterol and other lipids occurs at the smooth endoplasmic reticulum and mitochondrial membrane apposition sites or where tubular smooth endoplasmic reticulum is contiguous with mitochondrial cristae (Sinha et al., 1971 a). Similar organelle to organelle apposition sites have been reported in other steroid secreting tissues. Likewise the functional significance of the association of lipid droplets, mitochondria and smooth endoplasmic reticulum and pathways for stereoidogenesis have been discussed (Deane, 1958; Long and Jones, 1967; Hirschfield and Koritz, 1966; Jackanicz and Armstrong, 1968; Flint and Armstrong, 1972; Salhanick et al., 1973). Williamson (1964) considers caveolae to be involved in lipid transport in other cells.
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References Adams, E.C., Hertig, A.T.: Studies on the corpus luteum. I. Observations on the ultrastructure of development and regression of the luteal cells during the menstrual cycle. J. Cell Biol. 41, 696 715 (1969a) Adams, E.C., Hertig, A.T. : Studies on the human corpus luteum. II. Observations on the ultrastructure of luteal cells during pregnancy. J. Cell Biol. 41, 716-735 (1969b) Bjersing, L. : On the ultrastructure of granulosa lutein cells in porcine corpus luteum. Z. Zellforsch. 82, 187-211 (1967) Bjersing, L., Hay, M.F., Moor, R.M., Short, R.V. : Endocrine activity, histochemistry and ultrastructure of ovine corpora lutea. Further observations on regression at the end of the oestrous cycle. Z. Zellforsch. l l l , 437~157 (1970) Blanchette, E.J. : Ovarian steroid cells. II. The luteal cell. J. Cell Biol. 31,517-542 (1966) Canivenc, R., Cohere, G., Brechenmacher, C. : Quelques aspects ultrastructuraux de la cellule lut6ale chez le Blaireau (Meles meles L.). C.R. Acad. Sci. (Paris) 264, 1187-1189 (1967) Christensen, A.K., Gillim, S.W. : The correlation of fine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In : The gonads (K.W. McKerns, ed.), p. 415-488.New York : Appleton-Century-Crofts 1969 Crips, T.M., Browning, H.C.: The fine structure of corpora lutea in ovarian transplants of mice following luteotrophin stimulation. Amer. J. Anat. 122, 169-192 (1968) Deane, H.W.: Intracellular lipids: Their detection and significance. I: Frontiers in cytology (S.L. Palay, ed.), p. 227-263. New Haven: Yale University Press 1958 Enders, A.C.: Observations on the fine structure of lutein cells. J. Cell Biol. 12, 101-113 (1962) Enders, A.C. : Cytology of the corpus luteum. Biol. Reprod. 8, 158 182 (1973) Enders, A.C., Lyons, W.R.: Observations on the fine structure of lutein cells. II. The effects of hypophysectomy and mammotrophic hormone in the rat. J. Cell Biol. 22, 127-141 (1964) Enders, R.K., Enders, A.C. : Morphology of the female reproductive tract during delayed implantation in the mink. In: Delayed implantation, p. 129-139 (ed. A.C. Enders). Chicago: Chicago Univ. Press 1963 Fawcett, D.T., Long, J.A., Jones, A.U : The ultrastructure of endocrine glands. Recent Prog. Hormone Res. 25, 315-380 (1969) Flint, A.P.F., Armstrong, D.T.: Dynamic aspects of ovarian cholesterol metabolism: regulation by gonadotropins. In: Gonadotropins (B.B. Saxena, C.G. Beling, and H.M. Gandy, eds.), p. 269-286. New York: Wiley-Interscience 1972 Foresman, K.R., Mead, R.A. : Pattern of luteinizing hormone secretion during delayed implantation in the spotted skunk (Spilogaleputorius latifrons). Biol. Reprod. 11, 475-480 (1974) Garren, L.D., Gill, G.N., Masui, H., Walton, G.M. : On the mechanism of action of ACTH. Recent Progr. Hormone Res. 27, 443-478 (1971) Gillim, S.W., Christensen, A.K., McLennan, C.E. : Fine structure of the human menstrual corpus luteum at its stage of maximum secretory activity. Amer. J. Anat. 126, 409-428 (1969) Green, J.A., Maqueo, M. : Ultrastructure of the human ovary. I. The luteal cell during the menstrual cycle. Amer. J. Obstet. Gynec. 92, 946~57 (1965) Hirschfeld, I.H., Kortiz, S.B. : Pregnenolone synthesis stimulation in the large particles from bovine adrenal cortex and bovine corpus luteum. Endocrinology 78, 165 168 (1966) Jackanicz, T.M., Armstrong, D.T.: Progesterone biosynthesis in rabbit ovarian interstitial tissue mitocbondria. Endocrinology 83, 789-776 (1968) Koering, M.J., Wolf, R.C., Meyer, R.K.: Morphological changes in the corpus luteum correlated with progestin levels in the rhesus monkey during early pregnancy. Biol. Reprod. 9, 254-271 (1973) Koritz, S.B.: The effect of calcium ions and freezing on the in vitro synthesis of pregnenolone by rat adrenal preparations. Biochim. biophys. Acta (Amst.) 56, 63-75 (1962) Long, J.A., Jones, A.L.: The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum. Amer. J. Anat. 120, 463488 (1967) Luft, J.H. : Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409-414 (1961) Mead, R.A. : Reproduction in western forms of the skunk (genus Spilogale). J. Mammal. 49, 373-390 (1968)
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Mead, R.A. : Effect of luteinizing hormone-releasing hormone on luteal recrudescence during obligate delay of implantation in the western spotted skunk. Submitted for publication 1975 Mead, R.A. : Effects of hypophysectomy on blastocyst survival, progesterone secretion and nidation in the spotted skunk. Biol. Reprod. 12, 526 533 (1975) Mead, R.A., Eik-Nes, K.B.: Seasonal variation in plasma levels of progesterone in western forms of the spotted skunk. J. Reprod. Fertil., Suppl. 6, 397403 (1969) Moller, O.M. : The fine structure of the lutein cells in the mink (Mustela vision) with special reference to the secretory activity during pregnancy. Z, Zellforsch. 138, 523-544 (1973) Priedkalns, J., Weber, A.F. : Ultrastructural studies on the bovine Graafian follicle and corpus luteum. Z. Zellforsch. 91, 554-573 (1968) Salhanick, H.A., McIntosh, E.N., Uzgiris, V.I., Whipple, C.A., Mitani, F.: Mitochondrial systems of pregnenolone synthesis and its inhibition. In: The regulation of mammalian reproduction, p. 520-534. Illinois: Charles C. Thomas 1973 Savard, K. : The biochemistry of the corpus luteum. Biol. Reprod. 8, 183~02 (1973) Sinha, A.A.: Comparative ultrastructure of the corpus luteum of implantation and pregnancy in carnivora. In : Electron microscopic concepts of secretion ultrastructure of endocrinal and reproductive organs, p. 53-69 (ed. Melvin Hess). New York: John Wiley and Sons, Inc., eds. 1974 Sinha, A.A., Mead, R.A.: Morphological changes in the trophoblast, uterus and corpus luteum during delayed implantation and implantation in the western spotted skunk. Submitted for publication 1975 Sinha, A.A., Seal, U.S., Doe, R.P.: Fine structure of the corpus luteum of the raccoon during pregnancy. Z. Zellforsch. 117, 3545 (1971 a) Sinha, A.A., Seal, U.S., Doe, R.P.: Ultrastructure of the corpus luteum of the white-tailed deer during pregnancy. Amer. J. Anat. 132, 189-206 (1971 b) Tokida, A. : Electron microscopic studies on the corpora lutea obtained from normal human ovaries. Mie med. J. 15, 27-76 (1965) Williamson, J.R. : Adipose tissue. Morphological changes associated with lipid mobilization. J. Cell Biol. 20, 57 74 (1964)
Received July 9, 1975 / &final form August 14, 1975
