FUNCTIONAL LUTEOLYSIS IN THE PSEUDOPREGNANT RAT: EFFECTS OF PROSTAGLANDIN
PROSTAGLANDIN
F2\g=a\AND
F2\g=a\IN
16-ARYLOXY
VITRO
A. K. HALL AND J. ROBINSON Department of Zoology, The University of Hull, Hull, HU6 7RX
(Revised manuscript received 26 October 1978) SUMMARY
The present work concerns the luteolytic effects of prostaglandin (PG) F2\g=a\and its analogue, 16-aryloxy PGF2\g=a\,upon isolated luteal cells. Varying doses of these two prostaglandins were incubated with cells in the presence or absence of an optimum stimulatory dose of LH (1 \g=m\g/ml).The total contents of progesterone and 20\g=a\-dihydroprogesterone in flasks were determined after the incubation periods by radioimmunoassay. Both prostaglandins inhibited basal synthesis of progesterone and 20\g=a\-dihydroprogesterone, maximum inhibition occurring at concentrations of either PG of between 250 and 500 ng/ml. In this dose range both prostaglandins were found to abolish LH-stimulated progestogen synthesis completely. These effects were discernible within 5 min of incubation. The studies demonstrated that the onset of PG-induced luteolysis in vitro is characterized by an inhibition of the biosynthesis of both progesterone and its weakly progestogenic metabolite, 20\g=a\-dihydroprogesterone; induction of 20\g=a\-hydroxysteroid dehydrogenase activity by either PG was not found in incubations extending up to 60 min. In contrast to their relative potencies in vivo, PGF2\g=a\and 16-aryloxy PGF2\g=a\were essentially equipotent in this in-vitro system.
INTRODUCTION
Functional
luteolysis comprises
the cessation of progesterone-secreting activity by the well established that administration of prostaglandin F^ (PGF2a) can induce functional luteolysis in the rat (Pharriss & Wyngarden, 1969) and in many other non-primate mammalian species (review, Horton & Poyser, 1976). However, attempts to demonstrate inhibitory effects of PGF^ on luteal progesterone biosynthesis in vitro have produced inconsistent results. Paradoxically, steroidogenesis is often stimulated in such situations (Pharriss, Wyngarden & Gutknecht, 1968; Sellner & Wickersham, 1970; Hansel, Concannon & Lukaszewska, 1973). However, O'Grady, Kohorn, Glass, Caldwell, Brock & Speroff (1972) were able to demonstrate inhibitory effects of PGF^ on steroidogenesis by rabbit corpora lutea in organ culture. More recently, McNatty, Henderson 8c Sawers (1975) showed that PGF^ diminishes progesterone secretion in long-term cultures of human granulosa cells. Related studies by the same workers have since confirmed and extended these findings to porcine and bovine cultured granulosa cells (Henderson & McNatty, 1977). In addition, Henderson (1976) showed that 16-aryloxy PGF2a induces functional luteolysis in 8-day cultures of porcine granulosa cells. Watson & Maule-Walker (1977), using superfused porcine corpora lutea, showed that progesterone secretion could be impaired by PGF^ or (and to a greater extent) by uterine extracts. These studies in vitro have demonstrated unequivocally that the process of PGF^-induced functional luteolysis requires only the participation of the prostaglandins and intact granulosa cells : previous corpus luteum. It is
now
theories postulating actions involving ovarian haemodynamics (Pharriss, Cornette & Gutknecht, 1970) or the hypothalamo-pituitary-ovarian axis (Labhsetwar, 1970, 1974, 1976) now seem untenable. Hall & Robinson (1978) recently demonstrated that the hormonally induced pseudopregnant rat is potentially suitable as a model for the study of luteolysis. The aim of the present study was to establish an isolated cell system derived from these rats which would allow investigation of the luteolytic process in vitro. Current work in vivo indicates that the onset of functional luteolysis is rapid ; plasma progesterone levels fall within 30 min of administration of PGF^ (Behrman & Hichens, 1976). It was thought desirable, therefore, that an in-vitro system should be capable of a similar short response time. In addition, it was intended to obtain comparative data relating to PGF2a and 16-aryloxy PGF^ (ICI 80 996), the latter being an analogue possessing greatly enhanced luteolytic potency in several species, including the rat (Dukes, Russell & Walpole, 1974). A preliminary report of this study has already been presented (Hall, Merry & Robinson, 1978). MATERIALS AND METHODS
Animal
treatment
(24-day-old) female rats (Sprague-Dawley, CFY strain) received 50 i.u. pregnant gonadotrophin (Gestyl ; Organon Laboratories Ltd, Surrey), s.c. in 0-2 ml 72 h later by 25 i.u. human chorionic gonadotrophin (HCG; Pregnyl; followed saline, 0-9% Organon Laboratories Ltd), s.c. in 0-2 ml 0-9% saline. Rats were killed 6 or 7 days after the injection of HCG. Various characteristics of this animal preparation have been described previously (Tan & Robinson, 1977; Hall & Robinson, 1978). Immature
mare serum
Preparation of ovarian cell suspensions Isolated cells were prepared by a modification of procedures described elsewhere (Clark, Azhar & Menon, 1976). Excised ovaries (usually 3 g wet wt) were trimmed of fat, weighed, quartered and placed in an isolation mixture comprising collagenase (Worthington Bio¬ chemical Corporation, Freehold, New Jersey, U.S.A.; 1500units/g tissue), deoxyribonuclease (Sigma Chemical Co., Poole, Dorset; 0-005%, w/v), fatty acid free bovine serum albumin (BSA ; Sigma Chemical Co. ; 4%, w/v) and 5-5 mM-glucose, all were made up in a modified Krebs bicarbonate buffer (KRB; Umbreit, Burris & Stouffer, 1972) which contained no calcium component. The KRB lacking calcium ions was used throughout all the following procedures. The complete disaggregation mixture and quartered ovaries were shaken at 37 °C under an atmosphere of 02 and C02 (95 : 5, v/v) for 2 h. After 1 h, dis¬ persion was achieved by repeated aspiration of the suspension through a 1 ml disposable tuberculin syringe without a needle. The digestion was then continued for a further 1 h. After filtration through nylon gauze (60 µ mesh), the products of the disaggregation were centrifuged at 170 g for 10 min at 4 °C. The 170 g pellet was resuspended in fresh KRB containing 1% (w/v) fatty acid free BSA, with a Pasteur pipette, and re-centrifuged at 90 g for 5 min at 4 °C. After this, the 90 g pellet was resuspended and re-centrifuged at 25 g for 5 min at 4 °C. Cells were finally suspended in KRB containing 0-5% (w/v) fatty acid free BSA and 5-55 mM-glucose; before use they were aspirated once, using a 1 ml tuberculin syringe fitted with a 23-gauge needle (30 mm long), and transferred to a flask which was magnetically stirred continuously while portions of cells were dispensed. Examination of cell suspensions by phase-contrast microscopy revealed that 85% of the cells were typical luteal cells; the principal contaminant being red blood cells. Cells
give a
Incubation procedure divided into 12 75 mm plastic tubes (Sterilin Ltd, Teddington, Middlesex) to population of approximately 3-2 107 cells/ml, as determined by total DNA content
were
(Burton, 1956). Incubations and no Ca2+).
were
carried out in 1 ml KRB
(containing
5-5
mM-glucose,
Cells were incubated under these experimental conditions for 1 h at 37 °C under an atmosphere of 95% 02 and 5% C02. Incubations were terminated by immersion in solid C02/methanol at -80 °C, followed by storage at -25 °C. Stock PGF^ was dissolved in redistilled ethanol (1 mg/ml); after serial dilution the PGF^ was added to incubations in a delivery volume of 5 µ ethanol. Both luteinizing hormone (LH; NIH-LH-B9) and 16-aryloxy PGF2a were added in 50 µ distilled water. The viability of the cells after incubation under the various treatments was not significantly different from control cells, as judged by Trypan blue exclusion tests (Tennant, 1964). Steroid radioimmunoassays Frozen samples were thawed and centrifuged at 200 g for 15 min. Portions of supernatant fluid were taken for direct radioimmunoassay; the specificity of the two radioimmunoassay antisera was such that chromatography of the samples was shown to be unnecessary.
20
PROSTAGLANDIN
F2\g=a\AND
F2\g=a\IN
16-ARYLOXY
VITRO
A. K. HALL AND J. ROBINSON Department of Zoology, The University of Hull, Hull, HU6 7RX
(Revised manuscript received 26 October 1978) SUMMARY
The present work concerns the luteolytic effects of prostaglandin (PG) F2\g=a\and its analogue, 16-aryloxy PGF2\g=a\,upon isolated luteal cells. Varying doses of these two prostaglandins were incubated with cells in the presence or absence of an optimum stimulatory dose of LH (1 \g=m\g/ml).The total contents of progesterone and 20\g=a\-dihydroprogesterone in flasks were determined after the incubation periods by radioimmunoassay. Both prostaglandins inhibited basal synthesis of progesterone and 20\g=a\-dihydroprogesterone, maximum inhibition occurring at concentrations of either PG of between 250 and 500 ng/ml. In this dose range both prostaglandins were found to abolish LH-stimulated progestogen synthesis completely. These effects were discernible within 5 min of incubation. The studies demonstrated that the onset of PG-induced luteolysis in vitro is characterized by an inhibition of the biosynthesis of both progesterone and its weakly progestogenic metabolite, 20\g=a\-dihydroprogesterone; induction of 20\g=a\-hydroxysteroid dehydrogenase activity by either PG was not found in incubations extending up to 60 min. In contrast to their relative potencies in vivo, PGF2\g=a\and 16-aryloxy PGF2\g=a\were essentially equipotent in this in-vitro system.
INTRODUCTION
Functional
luteolysis comprises
the cessation of progesterone-secreting activity by the well established that administration of prostaglandin F^ (PGF2a) can induce functional luteolysis in the rat (Pharriss & Wyngarden, 1969) and in many other non-primate mammalian species (review, Horton & Poyser, 1976). However, attempts to demonstrate inhibitory effects of PGF^ on luteal progesterone biosynthesis in vitro have produced inconsistent results. Paradoxically, steroidogenesis is often stimulated in such situations (Pharriss, Wyngarden & Gutknecht, 1968; Sellner & Wickersham, 1970; Hansel, Concannon & Lukaszewska, 1973). However, O'Grady, Kohorn, Glass, Caldwell, Brock & Speroff (1972) were able to demonstrate inhibitory effects of PGF^ on steroidogenesis by rabbit corpora lutea in organ culture. More recently, McNatty, Henderson 8c Sawers (1975) showed that PGF^ diminishes progesterone secretion in long-term cultures of human granulosa cells. Related studies by the same workers have since confirmed and extended these findings to porcine and bovine cultured granulosa cells (Henderson & McNatty, 1977). In addition, Henderson (1976) showed that 16-aryloxy PGF2a induces functional luteolysis in 8-day cultures of porcine granulosa cells. Watson & Maule-Walker (1977), using superfused porcine corpora lutea, showed that progesterone secretion could be impaired by PGF^ or (and to a greater extent) by uterine extracts. These studies in vitro have demonstrated unequivocally that the process of PGF^-induced functional luteolysis requires only the participation of the prostaglandins and intact granulosa cells : previous corpus luteum. It is
now
theories postulating actions involving ovarian haemodynamics (Pharriss, Cornette & Gutknecht, 1970) or the hypothalamo-pituitary-ovarian axis (Labhsetwar, 1970, 1974, 1976) now seem untenable. Hall & Robinson (1978) recently demonstrated that the hormonally induced pseudopregnant rat is potentially suitable as a model for the study of luteolysis. The aim of the present study was to establish an isolated cell system derived from these rats which would allow investigation of the luteolytic process in vitro. Current work in vivo indicates that the onset of functional luteolysis is rapid ; plasma progesterone levels fall within 30 min of administration of PGF^ (Behrman & Hichens, 1976). It was thought desirable, therefore, that an in-vitro system should be capable of a similar short response time. In addition, it was intended to obtain comparative data relating to PGF2a and 16-aryloxy PGF^ (ICI 80 996), the latter being an analogue possessing greatly enhanced luteolytic potency in several species, including the rat (Dukes, Russell & Walpole, 1974). A preliminary report of this study has already been presented (Hall, Merry & Robinson, 1978). MATERIALS AND METHODS
Animal
treatment
(24-day-old) female rats (Sprague-Dawley, CFY strain) received 50 i.u. pregnant gonadotrophin (Gestyl ; Organon Laboratories Ltd, Surrey), s.c. in 0-2 ml 72 h later by 25 i.u. human chorionic gonadotrophin (HCG; Pregnyl; followed saline, 0-9% Organon Laboratories Ltd), s.c. in 0-2 ml 0-9% saline. Rats were killed 6 or 7 days after the injection of HCG. Various characteristics of this animal preparation have been described previously (Tan & Robinson, 1977; Hall & Robinson, 1978). Immature
mare serum
Preparation of ovarian cell suspensions Isolated cells were prepared by a modification of procedures described elsewhere (Clark, Azhar & Menon, 1976). Excised ovaries (usually 3 g wet wt) were trimmed of fat, weighed, quartered and placed in an isolation mixture comprising collagenase (Worthington Bio¬ chemical Corporation, Freehold, New Jersey, U.S.A.; 1500units/g tissue), deoxyribonuclease (Sigma Chemical Co., Poole, Dorset; 0-005%, w/v), fatty acid free bovine serum albumin (BSA ; Sigma Chemical Co. ; 4%, w/v) and 5-5 mM-glucose, all were made up in a modified Krebs bicarbonate buffer (KRB; Umbreit, Burris & Stouffer, 1972) which contained no calcium component. The KRB lacking calcium ions was used throughout all the following procedures. The complete disaggregation mixture and quartered ovaries were shaken at 37 °C under an atmosphere of 02 and C02 (95 : 5, v/v) for 2 h. After 1 h, dis¬ persion was achieved by repeated aspiration of the suspension through a 1 ml disposable tuberculin syringe without a needle. The digestion was then continued for a further 1 h. After filtration through nylon gauze (60 µ mesh), the products of the disaggregation were centrifuged at 170 g for 10 min at 4 °C. The 170 g pellet was resuspended in fresh KRB containing 1% (w/v) fatty acid free BSA, with a Pasteur pipette, and re-centrifuged at 90 g for 5 min at 4 °C. After this, the 90 g pellet was resuspended and re-centrifuged at 25 g for 5 min at 4 °C. Cells were finally suspended in KRB containing 0-5% (w/v) fatty acid free BSA and 5-55 mM-glucose; before use they were aspirated once, using a 1 ml tuberculin syringe fitted with a 23-gauge needle (30 mm long), and transferred to a flask which was magnetically stirred continuously while portions of cells were dispensed. Examination of cell suspensions by phase-contrast microscopy revealed that 85% of the cells were typical luteal cells; the principal contaminant being red blood cells. Cells
give a
Incubation procedure divided into 12 75 mm plastic tubes (Sterilin Ltd, Teddington, Middlesex) to population of approximately 3-2 107 cells/ml, as determined by total DNA content
were
(Burton, 1956). Incubations and no Ca2+).
were
carried out in 1 ml KRB
(containing
5-5
mM-glucose,
Cells were incubated under these experimental conditions for 1 h at 37 °C under an atmosphere of 95% 02 and 5% C02. Incubations were terminated by immersion in solid C02/methanol at -80 °C, followed by storage at -25 °C. Stock PGF^ was dissolved in redistilled ethanol (1 mg/ml); after serial dilution the PGF^ was added to incubations in a delivery volume of 5 µ ethanol. Both luteinizing hormone (LH; NIH-LH-B9) and 16-aryloxy PGF2a were added in 50 µ distilled water. The viability of the cells after incubation under the various treatments was not significantly different from control cells, as judged by Trypan blue exclusion tests (Tennant, 1964). Steroid radioimmunoassays Frozen samples were thawed and centrifuged at 200 g for 15 min. Portions of supernatant fluid were taken for direct radioimmunoassay; the specificity of the two radioimmunoassay antisera was such that chromatography of the samples was shown to be unnecessary.
20
