0013-7227/79/1056-1330$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society

Vol. 105, No. 6 Printed in U.S.A.

A New Perspective on the Mechanism of Corpus Luteum Regression* M. M. BUHR, J. C. CARLSON,! AND J. E. THOMPSON Biology Department, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

regression. This was accompanied by a parallel rise in the lipid phase transition temperature. In addition, the proportion of lipid in the gel phase increased with time after prostaglandin F2(1 treatment. These results indicate that the mechanism of corpus luteum regression may involve phase changes in the phospholipid bilayer of cellular membranes. The resulting presence of gel phase lipid in the membrane matrices could contribute to the loss of tissue function. (Endocrinology 105: 1330, 1979)

ABSTRACT. Wide angle x-ray diffraction has been used to examine the phase behavior of microsomal membranes from regressing corpora lutea of prepubertal pseudopregnant rats. During periods of optimal progesterone secretion, all of the membrane lipid was in the liquid-crystalline phase at physiological temperature and, therefore, was fluid. However, mixtures of liquid-crystalline and gel phase lipid were observed under identical conditions in microsomal membrane preparations from animals undergoing spontaneous or prostaglandin F2u-induced

T

REATMENT of prepubertal rats with gonadotropic hormones results in extensive luteinization of the ovary. The corpus luteum (CL) remains functional for 14-15 days after hCG treatment (1). Prostaglandin F2a (PGF2«) is believed to be responsible for luteal regression in several species (2). The administration of PGF2« results in a rapid drop in plasma progesterone concentration and involution of luteal tissue and increased uterine levels of PGF near the end of pseudopregnancy. Despite a number of experiments on the effects of PG on CL function, we still do not clearly understand its basic mechanism of action in inducing regression. Recently, it has been established that there are marked changes in the phase properties of membranes during functional regression of senescing plant tissue. For example, microsomal membranes from senescing bean cotyledons acquire increasing proportions of gel phase lipid as regression intensifies; this is paralleled by a corresponding rise in the lipid phase transition temperature of the membranes (3, 4). Thus, at physiological temperature, there is a mixture of liquid-crystalline and gel phase lipid in the older membranes. In addition, experiments with liposomes of pure phospholipids have demonstrated that this mixture of lipid phases renders the membranes leaky (5, 6). Consequently, the presence of both lipid phases in natural membranes could result in the loss of ionic gradients and intracellular compart-

mentation. In the present study, microsomal membranes from regressing rat CL were examined by wide angle xray diffraction to determine if this tissue undergoes similar phase changes during spontaneous and PGF2,,-induced luteolysis. Materials and Methods Animal treatments Female Wistar rats, between 23-33 days of age, were injected sc with 50 IU PMS (Gestyl, Organon Pharmaceuticals, West Orange, NJ) and, 72 h later (day 0), with 25 IU hCG (Pregnyl, Organon). The animals were assigned at random to two groups. Group 1 consisted of control rats sacrificed 5, 8, 9, 10, 11, or 13 days after hCG administration on day 0. Group 2 animals were injected sc with 500 jug PGF2o in two doses (125 and 375 /ig) 7 h apart. They were sacrificed either 24 h after treatment on days 4, 8 or 12 or 48 or 72 h after treatment on day 8. At sacrifice, the ovaries from two or three animals were collected and pooled for the membrane preparation. This collection constituted a single trial, and a minimum of three trials were performed for each time interval in the three groups. Immediately before sacrifice, the animals were placed under light ether anesthesia and a 1-ml blood sample was removed by cardiac puncture. The isolated plasma samples were stored at —20 C and subsequently assayed for progesterone concentration (7). Membrane isolation

Received February 21,1979. * This work was supported by the Medical and National Research Councils of Canada. f To whom requests for reprints should be addressed.

Smooth microsomal membranes were isolated essentially according to a procedure described previously for animal tissues (8). The excised ovaries were cleared of surrounding tissue, weighed, and homogenized at 4 C in 0.3 M sucrose-50 mM NaHC0 3 (pH 7.0) for 15 sec with a Polytron homogenizer

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CORPUS LUTEUM REGRESSION (Brinkmann Instruments, Inc., Westbury, NY). After a 10-min centrifugation (500 X g), the supernatant was spun for 20 min at 10,000 X g. The resultant supernatant was brought to a final CsCl concentration of 15 mM and layered on 3.5 ml of a 1.3 M sucrose-15 mM CsCl solution in a polycarbonate centrifuge tube. This was spun at 165,000 X g for 3 h; the layer of smooth microsomes which formed at the interface was removed, mixed 1:3 (vol/vol) with 50 mM NaHCOa and pelleted by centrifuging at 165,000 X g for an additional hour.

X-Ray diffraction Samples of the microsomal fraction were prepared for x-ray diffraction as previously described (3, 4). Wide angle diffraction patterns were recorded using CuK a-radiation from a pointfocused x-ray tube (type PW 2103/01) on a Philips (type 1030, Philips Electronic Instruments, Inc., Mount Vernon, NY) camera under conditions in which the samples retain 50-75% moisture with respect to final dried weight (4). For each sample, diffraction patterns were recorded at physiological temperature for the rat (39 C), and the lipid phase transition temperature, the highest temperature at which gel phase lipid could be detected, was determined to within 1 C. A densitometer (model 345, Clifford) was used to compare the intensities of the diffraction reflections on the x-ray film.

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The x-ray diffraction patterns recorded at 39 C from smooth microsomal membranes prepared from rat CL (group 1) featured two broad diffuse bands centered at Bragg spacings of 4.6 A and ~10 A for days 5, 8, 9, 10, and 11 after hCG administration (Fig. 1, A-E). These wide angle patterns are typical of those obtained for biological membranes (3, 4,13). The inner 10-A reflection is not well characterized and presumably derives at least in part from protein (14). The outer broad ring at 4.6 A derived from membrane phospholipid which was in a liquid-crystalline state and was essentially disordered (3, 4, 13). Thus, through day 11, the lipid of smooth micro-

Assays The activities of several marker enzymes were determined as an index of membrane origin. (Na+-K+)-ATPase (EC 3.6.1.3) was assayed in the presence of 10 mM fluoride ion, according to a procedure described previously for CL tissue (9). Rotenoneinsensitive NADH-cytochrome c reductase (EC 1.6.99.3) and succinate dehydrogenase (EC 1.3.99.1) were assayed by the methods of Sottocasa et al. (10) and Pennington (11), respectively. In addition, protein content was measured by the method of Lowry et al. using bovine serum albumin as a standard (12).

Statistics Student's paired t test was used to determine the level of significance of the observations.

Results Phase properties of microsomal membranes The phase properties of membrane lipids are temperature dependent. The transition temperature can be defined as the highest temperature at which gel phase lipid can be detected. Below the transition temperature, there is a mixture of liquid-crystalline and gel phase lipid in the membrane matrix, and as the temperature is lowered, the proportion of lipid in the gel phase increases. Above the transition temperature, all of the membrane lipid is in the liquid-crystalline phase. Normally, the transition temperature for membrane lipid is below physiological temperature, which means that at physiological temperature the membrane lipids are exclusively liquid-crystalline.

FIG. 1. Wide angle x-ray diffraction of microsomal membranes from CL removed at various days during pseudopregnancy. Wide angle xray diffraction patterns were recorded at 39 C for smooth microsomal membranes isolated from CL after hCG treatment (day 0). A-E, Patterns for membranes isolated 5, 8, 9, 10, and 11 days after treatment, respectively, showing (from outside to inside) diffuse bands at Bragg spacings of 4.6 and -10 A. F, Pattern for membranes isolated 13 days after treatment showing (from outside to inside) a sharp band at 4.15 A and diffuse bands at 4.6 and ~10 A.

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Endo • 1979 Voll05 • No 6

BUHR, CARLSON, AND THOMPSON

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TABLE 1. Ovarian weight, transition temperature (TT), and plasma progesterone (P) concentration in untreated and PGF2n-treated pseudopregnant rats PGF2«

Control Day 5 8 9 10 11 13

Paired ovarian wt (mg)

TT(C)

P (ng/ml)

180 ± 27 150 ± 7 160 ± 18 170 ± 15 170 ± 22 170 ± 0

39 ± 0.6 40 ± 1.3 41 ± 1.7 42 ± 0.7 40 ± 0.9 43 ± 2.7

363.9 ± 33.4 281.5 ± 24.3 249.2 ± 42.8 284.8 ± 82.0 150.7 ± 21.4* 70.5 ± 24.4e

Paired ovarian wt (mg)

TT(C)

P (ng/ml)

180 ± 14"

47 ± 0.8*

66.7 ± 10.2c

180 ± 11° 180 ± 13" 210 ± 2 ^ 210 ± 17"

47 ± 0.5* 48 ± 0.5* 49 ± 1.2* 47 ± 0.7

11.4 ± 1.6C 17.1 ± 7.2C 7.7 ± l.l c 9.1 ± 1.3"

Values represent the mean ± SE. The spontaneous changes (Control) associated with aging are arranged vertically and the corresponding treated changes appear horizontally. " Killed 24 h after PGF2n. * Significantly greater (P < 0.005) than corresponding control value. c Significantly less (P < 0.005) than corresponding control value. d Killed 48 h after PGF2. ' Significantly less (P < 0.005) than progesterone concentration on days 5, 8, 9, and 10. 'Killed 72 h after PGF2a. * Significantly less (P < 0.05) than corresponding control value.

somal membranes remained exclusively liquid-crystalline after hCG administration. Diffraction patterns recorded at 39 C from membranes isolated 13 days after hCG treatment were essentially identical to those obtained for the earlier stages, except that it was possible to discern a sharp reflection peripheral to the outer diffuse lipid band and centered at a Bragg spacing of 4.15 A (Fig. IF). The appearance of this sharp ring coincides with the transition of a portion of the membrane lipid from the fluid liquid-crystalline state, depicted by the broad 4.6-A reflection, to a gel state in which the fatty acid side chains are packed hexagonally (3, 4, 13). Thus, 13 days after hCG treatment there is a mixture of liquid-crystalline and gel phase lipids in these membranes at physiological temperature. This is paralleled by a rise in the lipid phase transition temperature and a decline in plasma progesterone concentration between days 11-13 (Table 1). Treatment of rats with PGF2« (group 2) resulted in an earlier appearance of lipid phase changes in the luteal cell membranes. Microsomal preparations from luteal tissue of animals sacrificed on days 5,9, and 13, 24 h after PGF2o administration, revealed the sharp 4.15-A band as well as the broad 4.6-A reflection upon x-ray diffraction at 39 C, again indicating a mixture of lipid phases (Fig. 2). In addition, the transition temperatures of the membrane lipid were significantly higher, (except on day 13) and the plasma progesterone levels were significantly lower than the corresponding control values (Table 1). Despite these changes, however, there was no apparent loss in CL weight. In the rats sacrificed at various intervals (0-72 h) after PG treatment on day 8, we examined the time dependence of the PGF2a-induced lipid phase changes. X-ray

diffraction patterns of the smooth microsomal membranes from these CL all featured the 4.15-A band at 39 C, indicating that gel phase lipid was present at each sampling interval after PG treatment (Fig. 3). The transition temperature rose from a low of 40.3 ± 1.3 C before initiation of luteolysis to a high of 49.0 ± 1.2 C 72 h after PGF2« administration (Table 1), and the greatest drop in plasma progesterone concentration occurred in the first 24 h of PGF2«-induced luteolysis. Densitometer scans of the x-ray diffraction patterns revealed a progressive increase in the proportion of gel phase lipid with time after exposure to PGF2« (Fig. 4). Enzymatic properties of the smooth microsomal fraction A smooth microsomal fraction is by nature heterogeneous, comprising small vesicles of membrane derived from almost all organelles of the cell. To gauge this heterogeneity for the microsomal fraction from rat CL, enzymes serving as markers for specific types of membrane were assayed. The fraction used in this study contained plasma membrane and endoplasmic reticulum, as indicated by the presence of (Na+-K+)ATPase and rotenone-insensitive NADH-cytochrome c reductase, although their relative proportions were variable among experiments. Mitochondrial membrane was not present, as indicated by the absence of succinate dehydrogenase (Table 2).

Discussion It is clear from the diffraction patterns recorded at physiological temperature that before luteolysis, the lipid of microsomal membranes from rat CL is exclusively liquid-crystalline. Moreover, the periods during which

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CORPUS LUTEUM REGRESSION

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FIG. 3. Wide angle x-ray diffraction of microsomal membranes from CL removed 0-72 h after PGF2,, treatment. Wide angle x-ray diffraction patterns were recorded at 39 C for smooth microsomes isolated from CL after various periods of PGF2,, treatment. Animals were treated with PGF2U 8 days after hCG treatment. A, Control (0 h) showing (from outside to inside) diffuse bands at Bragg spacings of 4.6 and ~10 A. B, C, and D, Twenty-four, 48 and 72 h after PGF2« treatment, respectively, showing (from outside to inside) a sharp band at 4.15 A and diffuse bands at 4.6 and -10 A.

FIG. 2. Wide angle x-ray diffraction of microsomal membranes from CL removed from control and PGF2u-treated rats. Wide angle x-ray diffraction patterns were recorded at 39 C for smooth microsomes isolated from CL after hCG treatment. The left half of each panel represents samples removed from control rats, and the right half represents samples from rats treated with PGF2n 24 h before sacrifice. Both control and treated rats were killed on days 5, 9, or 13 (A-C, respectively) after hCG administration on day 0. The left halfoi A and B show (from outside to inside) diffuse bands at Bragg spacings of 4.6 and —10 A; the left half of C and the right halves of A-C show (from outside to inside) a sharp band at 4.15 A and diffuse bands at 4.6 and -10 A.

these membranes retained this exclusively liquid-crystalline character correlated temporally with periods of optimal progesterone secretion. By contrast, once spontaneous or PGF2tt-induced luteolysis had been initiated, the sharp 4.15-A Bragg reflection depicting gel phase lipid appeared in diffraction patterns recorded at physiological temperature. This indicates a mixture of liquid-crystalline and gel phase lipids in the membranes. The appearance of gel phase lipid also coincided temporally with the loss of CL function, as gauged by the fall in plasma

progesterone levels. The simultaneous change in membrane phase behavior and sharp decline in blood progesterone occurred in animals undergoing spontaneous as well as PGF2«-induced luteolysis. In addition, as involution of the tissue intensified, the proportion of membrane lipid in the gel phase increased and there was a corresponding rise in transition temperature. This increase in transition temperature indicates that additional types of phospholipid join the gel phase as involution of the tissue progresses. The close correlation between morphological and functional changes is consistent with the fact that formation of gel phase lipid appears to be related to membrane deterioration. For example, regions of the membrane in which the lipid is in the gel phase are likely to be devoid of enzyme activity, since it is known that during formation of the gel phase, protein is displaced into adjacent more fluid regions (15). The mixture of liquid-crystalline and gel phase lipids may also render the membranes leaky, for it has been previously demonstrated that the permeability of liposomes to various ions increases at the phase transition temperature (5). This in turn would disrupt essential ionic balances and lead to breakdown of

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BUHR, CARLSON, AND THOMPSON

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4

5 BRAGG

6 SPACING

o

10

(A)

FIG. 4. Densitometer tracings of wide angle x-ray diffraction patterns of microsomal membranes from CL removed 0-72 h after PGF2,, treatment. Densitometer tracings of wide angle x-ray diffraction patterns recorded at 39 C from the preparations illustrated in Fig. 3. Each tracing represents half of the diffraction pattern. Reading from top to bottom, the lines represent densitometer tracings of the diffraction pattern of samples removed 72, 48, 24, and 0 h after PGF2(I. TABLE 2. Enzyme activities of two different microsomal preparations from CL of prepubertal pseudopregnant rats

Microsomal preparation

(Na+-K+)ATPase

Cytochrome c reductase

Succinate dehydrogenase

SA"

Enrichment*

SAC

Enrichment*

SAd

320.7 428.1

12.0 3.9

82.2 37.7

12.5 11.8

NDe ND

Enrichment*

" Micrograms of PO4 per mg protein/h. * Specific activity of microsomal preparation/specific activity of homogenate. c Micromoles of cytochrome c reduced per mg protein/h. '' Moles of p-iodonitrotetrazolium violet reduced per mg protein/h. '" Not detectable.

intracellular compartmentation and, ultimately, to the rapid loss of tissue function. Mixtures of both lipid phases have been observed previously in membranes isolated from plant and animal

Endo • 1979 Voll05 • No6

tissues during periods of cell deterioration caused by natural aging or treatment with infectious agents. In plants, bean cotyledons function as temporary storage organs, which atrophy shortly after germination. During cotyledon senescence, gel phase lipid becomes detectable in the membranes at physiological temperature; these changes are progressive, and they occur in both rough and smooth microsomes (3, 4). At the same time, membrane-bound enzymatic activity in these cells decreases substantially (16). Similar degenerative changes have also been observed in microsomal and chloroplast membranes of senescing leaves and algae (17, 18) and in plasma membranes of chick intestinal cells infected with Coccidia (Thompson, J. E., A. Fernando, and J. Pasternak, unpublished observations). Accordingly, the present data for rat CL add to a growing body of evidence which suggests that the formation of gel phase lipid in membranes may be a common feature of cell deterioration across both plant and animal systems regardless of whether the deterioration is attributable to natural aging, spontaneous involution, or infectious agents. In the present study, the diffraction analyses were carried out on smooth microsomal fractions, but such fractions invariably contain membranes derived from several organelles of the cell, including the plasmalemma. Moreover, the proportion of plasma membrane in the fraction increases as the severity of homogenization used to disrupt the tissue is increased (19). The method of homogenization used in the present study (Polytron) can certainly be regarded as severe, inasmuch as it deploys two forces of disruption, shearing and sonication. It is not surprising, therefore, to find that smooth microsomal fractions isolated from these homogenates contain significant proportions of plasma membrane, as evidenced by measurements of the surface membrane marker (Na+K+)ATPase. Thus, it is likely that the gel phase lipid detected in the smooth microsomal fractions derives, at least in part, from the plasma membrane present in the fraction. A particularly intriguing aspect of this study is the extent to which this prospective change in plasma membrane phase behavior in the CL may influence the gonadotropin receptors on the cell surface. As proteins are squeezed out of the gel phase regions, the whole pattern of protein organization in the plasmalemma, including the innate distribution of hormone receptor sites, could be altered. Recently, Behrman et al. (20) reported that PGF2« treatment inhibits gonadotropin binding in rat CL tissue. In light of the present study, it is conceivable that this decreased binding capability is related to phase changes in the lipid component of the plasma membrane which have affected the LH receptors. Without tropic hormone support, progesterone synthesis drops. In addition, gel phase lipid in cytoplasmic membranes, partic-

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CORPUS LUTEUM REGRESSION ularly endoplasmic reticulum, would also be expected to curtail the steroid synthetic capability of this tissue. At present, our findings do not pinpoint the initial membrane changes associated with CL regression. However, they do suggest that the mechanism of luteal regression may involve physical alteration in the lipid component of one or more of the cellular membrane systems which leads to loss of tissue function.

References 1. Lee, C. Y., K. Tateishi, R. J. Ryan, and N. S. Jiang, Binding of human chorionic gonadotropin by rat ovarian slices: Dependence on the functional state of the ovary, Proc Soc Exp Biol Med 148: 505, 1975. 2. Pharris, B. B., S. A. Tillson, and R. R. Erickson, Prostaglandins in luteal function, Recent Prog Horm Res 28: 51, 1972. 3. McKersie, B. D., J. E. Thompson, and J. K. Brandon, X-Ray diffraction evidence for decreased lipid fluidity in senescent membranes from cotyledons, CanJBot 54: 1074, 1976. 4. McKersie, B. D., and J. E. Thompson, Lipid crystallization in senescent membranes from cotyledons, Plant Physiol 59: 803,1977. 5. Papahadjopoulos, D., K. Jacobsen, S. Nir, and T. Isac, Phase transitions in phospholipid vesicles. Florescence polarization and permeability measurements concerning the effect of temperature and cholesterol, Biochim Biophys Acta 311: 330, 1973. 6. Van Dijck, F. W. M., P. H. J. T. Ververgaert, A. J. Verkleij, L. L. M. Van Deenen, and J. De Gier, Influence of Ca2+ and Mg2+ on the thermotropic behaviour and permeability properties of liposomes prepared from dimyristoyl phosphatidylglycerol and mixtures of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine, Biochim Biophys Acta 406: 465,1975. 7. Carlson, J. C, and J. W. D. Gole, CL regression in the pseudopregnant rabbit and the effects of treatment with prostaglandin F2 and arachidonic acid, J Reprod Fertil 55: 381, 1978. 8. Dallner, G., P. Siekevitz, and G. E. Palade, Biogenesis of endoplasmic reticulum membranes. I. Structural and chemical differentiation in developing rat hepatocyte, J Cell Biol 30: 73, 1966.

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9. Bramley, T. A., and R. J. Ryan, Interaction of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary, Endocrinology 103: 778, 1978. 10. Sottocasa, G. L., B. Kuylenstierna, L. Ernster, and A. Bergstrand, An electron transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study, J Cell Biol 32: 415, 1967. 11. Pennington, R. J., Biochemistry of dystrophic muscle, Biochem J 80: 649, 1961. 12. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. 13. Esfahani, M., A. R. Limbrick, S. Knutton, T. Oka, and S. J. Wakil, The molecular organization of lipids in the membrane of E. coli: phase transitions, Proc Natl Acad Sci USA 68: 3180, 1971. 14. Finean, J. B., R. Coleman, S. Knutton, A. R. Limbrick, and J. E. Thompson, Structural studies of cell membrane preparations, J Gen Physiol 51: 19S, 1968. 15. Shechter, E., L. Letellier, and T. Gulik-Kuzywiki, Relations between structure and function in cytoplasmic membrane vesicles isolated from an E. coli fatty acid auxotroph, Eur J Biochem 49: 61, 1974. 16. Thompson, J. E., The behaviour of cytoplasmic membranes in Phaseolus vulgar is cotyledons during germination, Can J Bot 52: 535, 1974. 17. McKersie, B. D., and J. E. Thompson, Phase behaviour of chloroplast and microsomal membranes during leaf senescence, Plant Physiol 61: 639, 1978. 18. Thompson, J. E., C. I. Mayfield, W. E. Inniss, D. E. Butler, and J. Kruuv, Senescence-related changes in the lipid transition temperature of microsomal membranes from algae, Physiol Plantarum 43: 114, 1978. 19. Coleman, R., R. H. Michell, J. B. Fihean, and J. N. Hawthorne, A purified plasma membrane fraction isolated from rat liver under isotonic conditions, Biochim Biophys Acta 135: 573, 1967. 20. Behrman, H. R., D. L. Grinwich, M. Hichens, and G. J. MacDonald, Effect of hypophysectomy, prolactin, and prostaglandin F?,, on gonadotropin binding in vivo and in vitro in the corpus luteum, Endocrinology 103: 349, 1978.

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A new perspective on the mechanism of corpus luteum regression.

0013-7227/79/1056-1330$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society Vol. 105, No. 6 Printed in U.S.A. A New Perspective on the Me...
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