J. Anat. (1979), 128, 2, pp. 239-251 With 11 figures Printed in Great Britain
239
The small luteal cell of the sheep J. D. O'SHEA*, D. G. CRANt AND MARY F. HAY
Agricultural Research Council Institute of Animal Physiology, Animal Research Station, 307 Huntingdon Road, Cambridge CB3 OJQ
(Accepted 14 February 1978) INTRODUCTION
Sheep are widely used to study female reproductive endocrinology; in particular the physiology of the ovine corpus luteum has been intensively investigated in recent years, and it was in sheep that the role of prostaglandin F2a as a luteolytic hormone was first clearly demonstrated (McCracken et al. 1972). Surprisingly, no comprehensive account of the ultrastructural cytology of the corpus luteum in the sheep has been published, although the large luteal cells, believed to be of granulosa-cell origin, have been described in some detail by Deane et al. (1966), Bjersing et al. (1970) and Gemmell, Stacy & Thorburn (1974, 1976). For many years the existence of a second, smaller type of luteal cell, possibly of thecal or stromal origin, has been recognized as a feature of the corpora lutea of artiodactyls, including the sheep (Mossman & Duke, 1973). It is the main purpose of this paper to describe the distribution, ultrastructure and relationships of this smaller type of luteal cell in the sheep. In addition, some aspects of the ultrastructure of other cell types in the corpus luteum are considered, particularly where these relate to the identification and possible functions of the small luteal cells. MATERIALS AND METHODS
Ovaries containing corpora lutea were obtained from adult ewes under pentobarbitone and fluothane anaesthesia at the following stages of the oestrous cycle or pregnancy-oestrous cycle day 10 (3 corpora lutea); pregnancy days 15 (2), 25 (1), 50 (2), 100 (4), 125 (4) and 140 (4). Thin slices of luteal tissue from all corpora lutea up to day 50 of pregnancy were rapidly fixed in 10 % phosphate-buffered formalin (Pease, 1964) at 0-4 'C, postfixed in Dalton's chrome-osmium fixative (Glauert, 1965) at 0-4 °C, and embedded in Araldite. Tissues from corpora lutea removed at or after day 100 of pregnancy were collected at a separate time, using a different method of fixation which gave generally superior results, but which did not affect their interpretation. These tissues were fixed in 6-25 % glutaraldehyde in 0-2 M collidine buffer, pH 7-2, at room temperature, and post-fixed in 1 % osmium tetroxide in the same buffer. Blocks were stained for 1 hour in 2 % uranyl acetate in maleate buffer, pH 5*15, and embedded in Epon. All sections were cut on an LKB Ultratome III ultra* Permanent address: Department of Veterinary Preclinical Sciences, University of Melbourne,
Parkville, Victoria 3052, Australia.
t Reprint requests should be addressed to: Dr D. G. Cran.
240
J. D. O'SHEA, D. G. CRAN AND MARY F. HAY
241 Small luteal cell of sheep microtome, stained with 2 0 aqueous uranyl acetate and lead citrate, and examined in an AEI EM801 electron microscope. Thick sections (1 ,um) for light microscopy were stained in a 1 % toluidine blue in 1 % borax solution. An additional eight mature corpora lutea showing no signs of structural regression were obtained from non-pregnant ewes and studied for the distribution of A5-3flhydroxysteroid dehydrogenase, using Wattenburg's method (Hay & Moor, 1975). For determining the proportions of different cell types in the corpora lutea, low magnification (x 1000 plate magnification) electron micrographs were taken of randomly selected grid squares from single sections of corpora lutea at day 10 of the oestrous cycle and days 50 and 100 of pregnancy. All cells in which the nucleus was included in the section were counted. RESULTS
General features of luteal tissue Within the lobules of all corpora lutea studied, two major populations of cells were recognizable by light microscopy in addition to numerous small blood vessels. The first population consisted of large luteal cells (Fig. 1) which were polyhedral, somewhat angular but relatively uniform in shape, and 25-50 Sm in diameter. These cells possessed central, rounded nuclei approximately 10 um in diameter, and abundant cytoplasm. The second population was composed of smaller, more angular or elongated cells (Fig. 1) whose outlines were less easily traced. Because of their irregular shape, meaningful measurements were difficult to obtain, but their narrower dimensions seldom exceeded 15 dam. Their nuclei were less rounded, and generally slightly smaller than those of the large luteal cells. With electron microscopy, the large luteal cells comprised a discrete population, and no cells of intermediate structure were observed to link them with any other cell type. However, the smaller cells were of two distinct types: fibroblast-like cells, and cells with which this report is primarily concerned, referred to hereafter as small luteal cells. The remaining cells of the luteal tissue were endothelial cells and pericytes of capillaries, and occasional leucocytes, particularly lymphocytes and
eosinophils (Fig. 10). The cellular structure of the corpora lutea examined was relatively constant, irrespective of luteal age, up to day 100 of pregnancy. Certain marked and progressive changes were, however, observed in the later stages of pregnany. Fig. 1. Luteal tissue at day 100 of pregnancy. Large (L) and small (S) luteal cells are present, together with capillaries (C). Epon/toluidine blue. x 640. Fig. 2. Luteal tissue at day 125 of pregnancy. Both large (L) and small (S) luteal cells contain lipid droplets, which appear pale. Epon/toluidine blue. x 640. Fig. 3. Luteal tissue at day 10 of the oestrous cycle, stained to demonstrate A5-3,8-hydroxysteroid dehydrogenase activity. Sites of enzyme activity appear dark, and unstained nuclei of large (L) and small (S) luteal cells appear pale. x 740. Fig. 4. Electron micrograph showing a capillary (C) partially surrounded by two small luteal cells (S) which are interposed between the capillary and neighbouring large luteal cells (L). A few lipid droplets (arrows) are present in both the small luteal cells. Pregnancy day 125. x 4800.
ANA
128
242
J. D. O SHEA, D. G. CRAN AND MARY F. HAY
Table 1. Numbers and proportions of different cell types in corpora lutea of ewes during the oestrous cycle or pregnancy Stage of oestrous cycle or pregnancy
Type of cell
Large luteal Small luteal Fibroblast Endothelium and pericyte Miscellaneous or unidentified Total
Oestrous cycle (Day 10) 11 23 11 58 0 103
Pregnancy A
Day 50
Day 100
23 56 21 110 9 219
10 33 13 48 7 111
Total (%) 44 (10) 112 (26) 45 (10) 216 (50) 16 (4) 433 (100)
Quantitative aspects of luteal cytology The numbers and proportions of each of the major cell types in a count of 433 cells are shown in Table 1. Ninety six per cent of all cells were classified under four main headings, with similar findings at each of the three stages studied. Overall it was observed that half of all nuclei present belonged either to endothelial cells or pericytes of capillaries, while small luteal cells accounted for about a quarter of the total. A further 20 % was divided evenly between large luteal cells and fibroblasts. The remaining 4 % of cells consisted of leucocytes or of cells which were not identified with confidence.
Ultrastructure of luteal cells Small luteal cells These cells (Figs. 4, 5) were irregular in outline, with elongated, tapering cytoplasmic processes extending for considerable distances from the region of the nucleus. Each cell possessed a single nucleus situated within the main mass of cytoplasm. Nuclei were generally oval in shape, with an irregular outline (Figs. 4, 5), and contained one or more nucleoli. In corpora lutea of all stages studied a small proportion of these cells (less than 10 %) contained an area of modified or unmodified cytoplasm bounded by a complete, inverted nuclear envelope (Fig. 6). No continuity with the surrounding cytoplasm was observed, but serial sectioning would be necessary to establish this. The cytoplasm of the small luteal cells contained a large amount of endoplasmic reticulum of predominantly tubular type, and associated with scattered clusters of attached ribosomes (Fig. 7). Thus, although this endoplasmic reticulum was predominantly smooth many profiles had at least some ribosomes. Small numbers of free ribosomes were also present. These cells contained a moderate number of mitochondria of variable size and with profiles which were round, oval, elongated or branching (Fig. 6). Both tubular and lamellar cristae were present, in variable proportions. Many mitochondria contained small, electron-dense matrix granules (Fig. 6). A small number of cells at all stages which, in other respects, conformed to the structure of small luteal cells, contained larger than usual numbers of mitochondria. One or more small Golgi complexes associated with numerous small coated or uncoated vesicles were present (Fig. 7). There were also small numbers of dense,
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239
The small luteal cell of the sheep J. D. O'SHEA*, D. G. CRANt AND MARY F. HAY
Agricultural Research Council Institute of Animal Physiology, Animal Research Station, 307 Huntingdon Road, Cambridge CB3 OJQ
(Accepted 14 February 1978) INTRODUCTION
Sheep are widely used to study female reproductive endocrinology; in particular the physiology of the ovine corpus luteum has been intensively investigated in recent years, and it was in sheep that the role of prostaglandin F2a as a luteolytic hormone was first clearly demonstrated (McCracken et al. 1972). Surprisingly, no comprehensive account of the ultrastructural cytology of the corpus luteum in the sheep has been published, although the large luteal cells, believed to be of granulosa-cell origin, have been described in some detail by Deane et al. (1966), Bjersing et al. (1970) and Gemmell, Stacy & Thorburn (1974, 1976). For many years the existence of a second, smaller type of luteal cell, possibly of thecal or stromal origin, has been recognized as a feature of the corpora lutea of artiodactyls, including the sheep (Mossman & Duke, 1973). It is the main purpose of this paper to describe the distribution, ultrastructure and relationships of this smaller type of luteal cell in the sheep. In addition, some aspects of the ultrastructure of other cell types in the corpus luteum are considered, particularly where these relate to the identification and possible functions of the small luteal cells. MATERIALS AND METHODS
Ovaries containing corpora lutea were obtained from adult ewes under pentobarbitone and fluothane anaesthesia at the following stages of the oestrous cycle or pregnancy-oestrous cycle day 10 (3 corpora lutea); pregnancy days 15 (2), 25 (1), 50 (2), 100 (4), 125 (4) and 140 (4). Thin slices of luteal tissue from all corpora lutea up to day 50 of pregnancy were rapidly fixed in 10 % phosphate-buffered formalin (Pease, 1964) at 0-4 'C, postfixed in Dalton's chrome-osmium fixative (Glauert, 1965) at 0-4 °C, and embedded in Araldite. Tissues from corpora lutea removed at or after day 100 of pregnancy were collected at a separate time, using a different method of fixation which gave generally superior results, but which did not affect their interpretation. These tissues were fixed in 6-25 % glutaraldehyde in 0-2 M collidine buffer, pH 7-2, at room temperature, and post-fixed in 1 % osmium tetroxide in the same buffer. Blocks were stained for 1 hour in 2 % uranyl acetate in maleate buffer, pH 5*15, and embedded in Epon. All sections were cut on an LKB Ultratome III ultra* Permanent address: Department of Veterinary Preclinical Sciences, University of Melbourne,
Parkville, Victoria 3052, Australia.
t Reprint requests should be addressed to: Dr D. G. Cran.
240
J. D. O'SHEA, D. G. CRAN AND MARY F. HAY
241 Small luteal cell of sheep microtome, stained with 2 0 aqueous uranyl acetate and lead citrate, and examined in an AEI EM801 electron microscope. Thick sections (1 ,um) for light microscopy were stained in a 1 % toluidine blue in 1 % borax solution. An additional eight mature corpora lutea showing no signs of structural regression were obtained from non-pregnant ewes and studied for the distribution of A5-3flhydroxysteroid dehydrogenase, using Wattenburg's method (Hay & Moor, 1975). For determining the proportions of different cell types in the corpora lutea, low magnification (x 1000 plate magnification) electron micrographs were taken of randomly selected grid squares from single sections of corpora lutea at day 10 of the oestrous cycle and days 50 and 100 of pregnancy. All cells in which the nucleus was included in the section were counted. RESULTS
General features of luteal tissue Within the lobules of all corpora lutea studied, two major populations of cells were recognizable by light microscopy in addition to numerous small blood vessels. The first population consisted of large luteal cells (Fig. 1) which were polyhedral, somewhat angular but relatively uniform in shape, and 25-50 Sm in diameter. These cells possessed central, rounded nuclei approximately 10 um in diameter, and abundant cytoplasm. The second population was composed of smaller, more angular or elongated cells (Fig. 1) whose outlines were less easily traced. Because of their irregular shape, meaningful measurements were difficult to obtain, but their narrower dimensions seldom exceeded 15 dam. Their nuclei were less rounded, and generally slightly smaller than those of the large luteal cells. With electron microscopy, the large luteal cells comprised a discrete population, and no cells of intermediate structure were observed to link them with any other cell type. However, the smaller cells were of two distinct types: fibroblast-like cells, and cells with which this report is primarily concerned, referred to hereafter as small luteal cells. The remaining cells of the luteal tissue were endothelial cells and pericytes of capillaries, and occasional leucocytes, particularly lymphocytes and
eosinophils (Fig. 10). The cellular structure of the corpora lutea examined was relatively constant, irrespective of luteal age, up to day 100 of pregnancy. Certain marked and progressive changes were, however, observed in the later stages of pregnany. Fig. 1. Luteal tissue at day 100 of pregnancy. Large (L) and small (S) luteal cells are present, together with capillaries (C). Epon/toluidine blue. x 640. Fig. 2. Luteal tissue at day 125 of pregnancy. Both large (L) and small (S) luteal cells contain lipid droplets, which appear pale. Epon/toluidine blue. x 640. Fig. 3. Luteal tissue at day 10 of the oestrous cycle, stained to demonstrate A5-3,8-hydroxysteroid dehydrogenase activity. Sites of enzyme activity appear dark, and unstained nuclei of large (L) and small (S) luteal cells appear pale. x 740. Fig. 4. Electron micrograph showing a capillary (C) partially surrounded by two small luteal cells (S) which are interposed between the capillary and neighbouring large luteal cells (L). A few lipid droplets (arrows) are present in both the small luteal cells. Pregnancy day 125. x 4800.
ANA
128
242
J. D. O SHEA, D. G. CRAN AND MARY F. HAY
Table 1. Numbers and proportions of different cell types in corpora lutea of ewes during the oestrous cycle or pregnancy Stage of oestrous cycle or pregnancy
Type of cell
Large luteal Small luteal Fibroblast Endothelium and pericyte Miscellaneous or unidentified Total
Oestrous cycle (Day 10) 11 23 11 58 0 103
Pregnancy A
Day 50
Day 100
23 56 21 110 9 219
10 33 13 48 7 111
Total (%) 44 (10) 112 (26) 45 (10) 216 (50) 16 (4) 433 (100)
Quantitative aspects of luteal cytology The numbers and proportions of each of the major cell types in a count of 433 cells are shown in Table 1. Ninety six per cent of all cells were classified under four main headings, with similar findings at each of the three stages studied. Overall it was observed that half of all nuclei present belonged either to endothelial cells or pericytes of capillaries, while small luteal cells accounted for about a quarter of the total. A further 20 % was divided evenly between large luteal cells and fibroblasts. The remaining 4 % of cells consisted of leucocytes or of cells which were not identified with confidence.
Ultrastructure of luteal cells Small luteal cells These cells (Figs. 4, 5) were irregular in outline, with elongated, tapering cytoplasmic processes extending for considerable distances from the region of the nucleus. Each cell possessed a single nucleus situated within the main mass of cytoplasm. Nuclei were generally oval in shape, with an irregular outline (Figs. 4, 5), and contained one or more nucleoli. In corpora lutea of all stages studied a small proportion of these cells (less than 10 %) contained an area of modified or unmodified cytoplasm bounded by a complete, inverted nuclear envelope (Fig. 6). No continuity with the surrounding cytoplasm was observed, but serial sectioning would be necessary to establish this. The cytoplasm of the small luteal cells contained a large amount of endoplasmic reticulum of predominantly tubular type, and associated with scattered clusters of attached ribosomes (Fig. 7). Thus, although this endoplasmic reticulum was predominantly smooth many profiles had at least some ribosomes. Small numbers of free ribosomes were also present. These cells contained a moderate number of mitochondria of variable size and with profiles which were round, oval, elongated or branching (Fig. 6). Both tubular and lamellar cristae were present, in variable proportions. Many mitochondria contained small, electron-dense matrix granules (Fig. 6). A small number of cells at all stages which, in other respects, conformed to the structure of small luteal cells, contained larger than usual numbers of mitochondria. One or more small Golgi complexes associated with numerous small coated or uncoated vesicles were present (Fig. 7). There were also small numbers of dense,
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