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Corpora lutea (CL) from cows on day 12 of the oestrous cycle were studied by electron microscopy to investigate whether, and how, different subpopulations of luteal cells can be identified in tissue sections. Tissues from 6 CL were examined, and nucleated profiles of luteal cells were classified as large, medium or small on the basis of their areas in electron micrographs. Cut-off points for area categories for large, medium and small-sized cells were based on diameters of greater than 25, 20-25 and less than 20 microns, respectively, if the measured areas were converted to a circular shape after correction for shrinkage. The only qualitative features which distinguished cells of large size from those of small size were the presence of clusters of secretory granules, and of exocytosis of these granules, in large cells only. However, these features were observed in only 59% of large cells, probably primarily due to sampling limitations in single sections. Other qualitative features which have been regarded as diagnostic of large or small luteal cells were observed in cells in all size categories. It was concluded that large and small luteal cells in the cyclic CL of the cow are distinguishable by their ultrastructural features. However, these data do not support the recent suggestion that the mid-cycle CL of the cow contain two subpopulations of large luteal cells in approximately equal numbers.

Corpora lutea from cyclic ewes were dissociated by collagenase digestion and trypsin/EGTA treatment. Enriched fractions of endothelial cells, small luteal cells and large luteal cells were prepared on a stepped gradient of Ficoll 400. Progesterone was measured by radioimmunoassay and the results corrected so that progesterone production by each cell type could be determined. Endothelial cells did not produce significant amounts of progesterone, with or without LH stimulation, and endothelial cell contamination of small and large luteal cell fractions did not influence progesterone production by these fractions. Mean +/- s.e.m. basal progesterone production (n = 10) by large luteal cells was greater (P less than 0.001) on a per cell basis than that by small luteal cells (1.16 +/- 0.16 compared with 0.25 +/- 0.06 pg/h/cell). However LH, which stimulated a maximal 3-4-fold increase in progesterone production by small luteal cells (LH ED50 = 0.14 ng/ml), had no significant effect on production by large luteal cells, when contamination by small luteal cells was taken into account. The response of small luteal cells was specific to LH, other hormones having had no significant effect. Basal progesterone production by small luteal cells (0.12 +/- 0.03 fg/h/micron3) calculated per unit volume of cell was not significantly different from that of large luteal cells (0.17 +/- 0.02 fg/h/micron3). After LH stimulation, small luteal cells produced more progesterone than did large luteal cells (0.40 +/- 0.09 compared with 0.18 +/- 0.03 fg/h/micron3) (P less than 0.05). When the amounts of progesterone produced per cell were multiplied by the absolute numbers of large luteal (1 X 10(7] and small luteal (5 X 10(7] cells in the intact corpus luteum, basal progesterone production by large luteal cells (11.6 +/- 1.6 micrograms/h) was similar to that by small luteal cells (12.3 +/- 3.0 micrograms/h). However, under LH stimulation, progesterone production by the small luteal cell type (39.9 +/- 9.5 micrograms/h) was approximately 3 times greater than that by the large luteal cell type (12.3 +/- 1.6 micrograms/h) (P less than 0.05). We therefore conclude that small luteal cells may be the principal source of luteal progesterone production in the sheep.

Changes in luteal weight from about Day 20 to near term, and in quantitative histology as assessed by ultrastructural morphometry and light microscopic counts of mitosis and cell death on Days 30, 60, 100 and 142, were studied in 168 pregnant ewes. Luteal weight (mean +/- s.d.) remained constant at 0.56 +/- 0.11 g until Day 120, and fell thereafter to reach 0.31 +/- 0.11 g after Day 140 (P less than 0.01). Up to Day 100, quantitative aspects of the composition of the luteal tissue showed no significant change, and values for volume density, cytoplasmic:nuclear ratio, cell number/mm3 and cell volume were comparable to values previously obtained for corpora lutea (CL) of the cycle. By Day 142 structural evidence of luteal regression was present, but regressive changes were much more marked in some CL than others. Mitosis was seen in a few cells (0.02-0.04%) on all of the days studied, but never in large luteal cells. Cell death was rarely seen up to Day 100, but had increased in incidence by Day 142 (P less than 0.01). Luteal progesterone content, 55.2 +/- 15.9 nmol/g on Day 30, was not significantly changed on Days 60, 100 or 142. It is concluded that (1) structural regression of the CL of pregnancy does not begin until much later than the time (about Day 50) when pregnancy ceases to depend on the CL; (2) structural luteal regression begins before parturition, but its time of onset and/or rate of progression vary widely between animals; and (3) large and small luteal cells remain as distinctive populations throughout pregnancy, and their numbers at all stages can be accounted for by survival of the cells which differentiate during the genesis of the CL.

An electron microscopical, morphometric study has been made of the cellular and non-cellular composition of the ovine corpus luteum fixed by perfusion, together with an estimate of total cell numbers in the corpus luteum based on measurement of cell and tissue DNA content. Normal cyclical corpora lutea from six ewes were studied by each method. Total cell numbers per corpus luteum were estimated at 258 X 10(6) by morphometry and 238 X 10(6) by DNA measurement. The existence of four structurally distinct major cell populations in ovine luteal tissue was confirmed. Percentages of the total cell number were as follows: large luteal cells - 4.2%; small luteal cells - 18.5%; fibrocytes - 16.8%; endothelial cells and pericytes - 52.6%. The remaining 7.9% comprised other miscellaneous cell types. Of the four major cell types, cell volume (X 10(3) micron 3 +/- standard error of the mean) was greatest for large luteal cells (13.08 +/- 2.10) and least for endothelial cells and pericytes (0.52 +/- 0.05), with small luteal cells (2.08 +/- 0.19) and fibrocytes (1.61 +/- 0.27) intermediate in size. Large luteal cells occupied 25.4% of luteal volume density, small luteal cells 17.5%, fibrocytes 11.2% and endothelial cells and pericytes 12.7%. Other cell types occupied 5.4%, with the remaining volume occupied by vascular lumina (10.8%) and intercellular space (17.1%). The data on cell volume support morphological findings suggesting that large and small luteal cells of the ovine corpus luteum are distinct populations rather than the upper and lower limits of a single population whose sizes are normally distributed. Of these two types of luteal cell, small luteal cells outnumbered large luteal cells by more than 4:1, while the individual large luteal cells were more than six times the mean volume of the small luteal cells. These facts need to be considered when assessing the relative contributions of the two types of luteal cell to overall luteal steroidogenic function.

The effects of a synthetic prostaglandin analogue, cloprostenol, on luteal function in the guinea pig were studied. At a dose of 250 micrograms, cloprostenol administered I-P on day 9 of the cestrous cycle caused a reduction in the length of the oestrous cycle from 17.4 +/- s.d.0.9 to 14.5 +/- 1.1 days (p less than 0.01). Lower doses were ineffective, and post-treatment cycles were not different in length from pre-treatment cycles. Cloprostenol also caused a dose-dependent reduction in luteal weight, which fell from 3.52 +/- 0.82 to 1.82 +/- 0.41mg (p less than 0.01) 48 h after administration of a 250 micrograms dose on day 9. Plasma progesterone, measured by radioimmunoassay, was reduced from 4.67 +/- 0.59 to 2.69 +/- 0.66 ng ml-1 (p less than 0.01) 48 h after administration of 250 micrograms cloprostenol on day 9. 250 micrograms cloprostenol also reduced blood flow per corpus luteum, measured by 85Sr-labelled 15 microns microspheres, both at 3 h (20.20 +/- 10.36 to 9.40 +/- 4.20 microliters min-1; p less than 0.05) and at 48 h (18.47 +/- 8.27 to 5.23 +/- 1.90 microliters min-1; p less than 0.01) after administration on day 9. No adverse side-effects were observed at any dose level of cloprostenol used. It was concluded that cloprostenol is a useful experimental luteolysin in the guinea pig.

Ovarian and luteal blood flow rates were studied using radioactive microspheres in guinea-pigs between Day 6 of the oestrous cycle and Day 1 of the following cycle. Peripheral plasma progesterone levels were measured by radioimmunoassay on the same days of the oestrous cycle. Ovarian blood flow was greatest between Days 9 and 12 and had fallen by Day 16 both in absolute (ml . min-1) and relative (ml.min-1.g-1) terms. Luteal weight and blood flow were also greatest between Days 9 and 12 and had fallen sharply by Day 16. The highest mean (+/- s.d.) luteal flows measured were 0.10 +/- 0.04 ml.min-1 per corpus luteum, and 24.26 +/- 9.3 ml.min-1.g-1 luteal tissue on Day 10 of the cycle. Mean peripheral plasma progesterone levels reached a maximum of 3.66 +/- 1.1 ng/ml at Day 12 of the cycle and fell thereafter, reaching 0.74 +/- 0.5 ng/ml by Day 1 of the following cycle. Plasma progesterone levels declined significantly between Days 12 and 14 of the cycle, whereas no significant drop in luteal blood flow was demonstrable until after Day 14. These data do not support the idea that declining luteal blood flow is an initiating mechanism in luteal regression in the guinea-pig.

Quantitative and qualitative changes in the microvasculature of the corpus luteum of the guinea pig during cyclical luteal regression were studied by light and electron microscopy. During luteal regression, between day 12 of the oestrous cycle and day 1 of the next cycle, there was a reduction in the extent of the luteal capillary bed as measured both by 'hit' counts and by differential counts of all nucleated cells. Luteal cells made up a constant proportion of congruent to 60% of the luteal tissue area up to day 1 of the next cycle, but had increased as a proportion of all nucleated cells by that stage. Both fibroblasts and macrophages increased in number and in area occupied during luteal regression. Ultrastructural changes in capillaries during luteal regression included an increased incidence and degree of protrusion of endothelial cells into the lumina of small blood vessels, and degeneration and death of many endothelial cells. In spite of the widespread presence of endothelial cell degeneration, most small vessels retained a continuous lining of apparently viable endothelium. It is concluded that in this species there is a rapid and substantial reduction in the luteal capillary bed, which commences during the early stages of luteal regression, and a mechanism is proposed by which the capillary bed may be reduced while retaining its integrity.

A method is presented for the isolation and purification of three cell types, endothelial cells, small luteal cells and large luteal cells, from the ovine corpus luteum. The method involves enzymatic dispersion of luteal tissue followed by centrifugation of separated cells on a Ficoll gradient. The three purified cell types and others, particularly fibrocytes and smooth muscle cells, that were removed during purification, were identified by their morphology. The cell yield, the cellular composition and cellular progesterone content of each fraction from the Ficoll gradient were measured. The endothelial cell fractions were relatively free of contamination by other cell types and had negligible progesterone. Fractions of small luteal cells and those of large luteal cells contained endothelial cells but were relatively free of other cell types. Large luteal cells contained significantly more progesterone, produced more progesterone when incubated in culture, but were less responsive to luteinizing hormone than small luteal cells.

To determine the cellular origin of oxytocin produced by the cyclical corpus luteum (CL) of the sheep, enriched fractions of enzymatically dispersed small and large luteal cells from 12 CL were prepared on a Ficoll 400 gradient. Oxytocin was measured by RIA. Large luteal cells contained 1.08 +/- (SD) 0.70 fg/cell oxytocin, which was congruent to 30 X the content of small luteal cells. Endothelial cells contained little if any oxytocin. During a 12-h incubation, large luteal cells produced 0.28 fg/cell.h oxytocin: small luteal cells did not produce measurable amounts of oxytocin. It is concluded that the large luteal cells are the source of the oxytocin produced by the CL of the sheep.

Corpora lutea from cyclic ewes were dissociated by collagenase and trypsin/EGTA treatments, and enriched fractions of small and large luteal cells were prepared on gradients of Ficoll. These fractions were incubated separately or remixed before incubation. Colchicine, cytochalasin B and the calcium channel-blocker verapamil significantly reduced progesterone production by both small and large luteal cell fractions, while isoprenaline stimulated an increase in progesterone production by large luteal cell fractions only. When fractions of small and large luteal cells were remixed, no more and no less progesterone was produced than would have been predicted from equivalent fractions incubated separately. There was therefore no evidence of synergism between small and large luteal cells in the production of progesterone. Prostaglandin F-2 alpha, which can inhibit LH-stimulated progesterone production by ovine luteal tissue in vitro, had no effect on LH-stimulated progesterone production by small luteal cell fractions, but significantly inhibited that by enriched fractions of large luteal cells. Since large luteal cell fractions were contaminated with small luteal cells, which are probably responsible for the progesterone-secretory response of these fractions to LH, it was concluded that the inhibition of LH-stimulated progesterone production by small luteal cells is dependent on the presence of large luteal cells. Oxytocin added to large and small luteal cell fractions did not affect progesterone production by either fraction. It was therefore concluded that the inhibitory action of PGF-2 alpha on LH-stimulated progesterone production may require the interaction of large and small luteal cells, but that oxytocin is not likely to be an intermediary in this interaction.

Ovulation was induced by 3 X 30 micrograms LHRH i.v. at 90-min intervals in anoestrous Corriedale ewes. Plasma LH surges occurred in all of 31 ewes given LHRH, but ovulation occurred in only 16 of these ewes. Luteal weight and plasma progesterone concentration were lower in ewes in which ovulation was induced during anoestrus than in cyclic control ewes in the breeding season, and when data from induced and control ewes were pooled luteal weight was strongly correlated with plasma progesterone concentration (r = +0.612, P less than 0.01). Five mature corpora lutea (CL) resulting from ovulation induced during anoestrus were compared by morphometric methods with 5 CL from cyclic control ewes. When data from induced and control CL were pooled, luteal volume was positively correlated with total number of cells per CL (r = +0.869, P less than 0.01) but negatively correlated with number of cells per mm3 luteal tissue (r = -0.676, P less than 0.05), i.e. smaller CL contained fewer cells, but more cells per unit volume. Relative numbers of large to small luteal cells, at approximately equal to 1:6, were similar in LHRH-induced and cyclic control CL. Large and small luteal cells were smaller in induced CL than in control CL, but cytoplasmic:nuclear ratio did not differ between induced and control CL. Basal and LH-stimulated progesterone production by dispersed luteal cells in vitro were lower for CL from LHRH-treated ewes than from controls. However, percentage increase in progesterone production in response to LH was not different between LHRH-treated and control ewes at any dose rate of LH used. It is concluded that the small size of CL induced by LHRH is due primarily to the low numbers and small volumes of the luteal cells in these CL, and that subnormal luteal weight and subnormal progesterone production per luteal cell contribute to the low plasma progesterone concentrations in ewes treated with LHRH during seasonal anoestrus.

Groups of ewes received either saline or prostaglandin F2alpha (PGF2alpha) as an injection directly into the corpus luteum. Changes in circulating progesterone levels were measured as well as subsequent histological examination of the corpora lutea. Saline, or PGF2alpha given at the two lower doses (60 and 120 mug respectively), failed to suppress progesterone levels permanently, or to induce degenerative changes in the corpora lutea. Treatment with a higher dose of PGF2alpha (240 mug) was followed by a marked elevation in progesterone levels. These results are discussed in relation to reported effects of PGF2alpha arriving at the ovary via the arterial circulation.

Corpora lutea (CL) from naturally cycling Corriedale ewes were obtained in the mid- and late luteal phases of the oestrous cycle (Days 9 and 13; 5 ewes per group). The cellular composition of these CL was compared by ultrastructural morphometry to determine whether there were changes in numbers of large and small luteal cells consistent with differentiation of some small luteal cells to large luteal cells during the last part of the luteal phase. No differences between Days 9 and 13 were detected in luteal volume, plasma progesterone concentration, or volume density of any component of the luteal tissue. Large luteal cell numbers (mean +/- s.e.m.) were lower per unit volume of luteal tissue on Day 13 than on Day 9 (14.1 +/- 0.5 vs 18.4 +/- 1.3 X 10(3)/mm3, P less than 0.05). Mean volume of the individual large luteal cells was greater on Day 13 than on Day 9 (19.65 +/- 0.72 vs' 15.60 +/- 1.34 micrograms 3 X 10(3), P less than 0.05). However, there were no significant differences in numbers or volumes of small luteal cells between Days 9 and 13, and total numbers of large luteal cells per CL were not different between these two days. These results provide no support for the hypothesis that small luteal cells differentiate into large luteal cells during the oestrous cycle of the sheep.

Corpora lutea of sheep were examined by electron microscopy at day 10 of the oestrous cycle and at days 15, 25, 50, 100, 125 and 140 of pregnancy. Small luteal cells were present in all corpora lutea, and were two to three times as numerous as large luteal cells. The former were irregular in shape, with tapering cytoplasmic processes. Their major cytoplasmic organelles were a predominantly smooth endoplasmic reticulum, mitochondria with tubular and lamellar cristae, and one or more Golgi complexes. The enzyme delta 5-3 beta-hydroxysteroid dehydrogenase was present in their cytoplasm. Small luteal cells were often interposed between large luteal cells and capillaries, and formed close, complex surface relationships with large luteal cells. Small and large luteal cells differed in many ways, including the restriction of numerous approximately 0.2 micron cytoplasmic granules to the large cells, and no cells of intermediate structure were observed. These features of small luteal cells suggest a steroid hormone synthetic function, and direct interaction with large luteal cells.

The cellular composition of CL from 6 cows on approximately Day 12 of the oestrous cycle, after synchronization with cloprostenol, was studied by ultrastructural morphometry. Point-count measurements of volume density (mean +/- s.d.) showed that large luteal cells occupied 40.2 +/- 7.0% of the luteal tissue, and small luteal cells 27.7 +/- 6.3%. Of the total of 393.4 +/- 52.0 x 10(3) cells per mm3 of luteal tissue, large luteal cells made up only 3.5% and small luteal cells 26.7%, a ratio of 1:7.6. Endothelial cells/pericytes, at 52.3%, were the most numerous cell type. The mean volume per large luteal cell was 29.6 +/- 6.3 x 10(3) microns 3, while that of small luteal cells was 2.7 +/- 0.4 x 10(3) microns 3. In spherical form, these volumes would represent mean diameters of 38.4 microns and 17.2 microns respectively, and are consistent with published measurements on dispersed luteal cells. However, the values for cell numbers are much higher than published values based on luteal tissue dispersion, suggesting that dispersion may result in substantial and possibly selective losses of luteal cells.

Data on the structure, quantitation, origins and functions of the large luteal (LL) and small luteal (SL) cells of sheep, goats and cattle are reviewed. Both LL and SL cells show ultrastructural features consistent with a steroidogenic function. However, in addition to differences in size and shape, LL cells differ from SL cells primarily in possessing large numbers of secretory granules, suggesting an additional protein/polypeptide synthetic and secretory function. In sheep, morphometric estimates show that the corpus luteum (CL) contains approximately equal to 10 X 10(6) LL cells and approximately equal to 50-60 X 10(6) SL cells: individual LL cells are approximately equal to X 6 greater in volume than SL cells. During formation of the CL, granulosa and theca cells are incorporated, and evidence suggests that granulosa cells give rise to LL cells and theca cells to SL cells. However, SL cells, or cells of thecal origin, may also give rise to some LL cells. Both LL and SL cells produce progesterone in vitro. On a per cell basis, LL cells produce more progesterone than do SL cells, but SL cells show a much greater progesterone-secretory response to LH. Oxytocin is synthesized, and secreted in granule form, only by the LL cells, and relaxin, whose presence has been demonstrated convincingly only in cattle, also appears to be produced only by LL cells. The two types of luteal cell in ruminants therefore show major differences in function: the occurrence of any significant functional interaction remains to be established.