B.K. Petroff, R.E. Ciereszko, and J.S. Ottobre
Department of Animal Sciences
Prostaglandin F2 (PGF2-alpha) is a luteolytic agent in the pig. Treatment of porcine luteal tissue with PGF2-alpha in vitro, however, often has little effect on, or even stimulates, progesterone synthesis. In this study, we investigated the impact of gonadotropic support, lipoprotein substrate, intercellular contact, and duration of exposure on the effect of PGF2-alpha in short-term incubations of porcine luteal tissue. Porcine corpora lutea (CL) of pregnancy were minced or dissociated and incubated with increasing concentrations of PGF2-alpha in the presence and absence of porcine low-density lipoproteins (LDLs) and/or human chorionic gonadotropin (hCG). Progesterone (P4) accumulation in the medium during 8-hour incubations of dissociated luteal cells was slightly stimulated by PGF2-alpha. Low-density lipoproteins and hCG both increased P4 accumulation as well. Prostaglandin F2, gonadotropin, and LDL did not interact in their effects on steroidogenesis during incubation of dispersed cells. The stimulatory effect of PGF2-alpha on steroidogenesis by dissociated cells was most evident during hours 2 to 4 of incubation, but PGF2-alpha was without effect for the remainder of the incubation. When minced luteal tissue was used, P4 accumulation in the medium during an 8-hour incubation was inhibited by PGF2-alpha, while pLDL and hCG exerted only a slight stimulatory effect. Progeserone (P4) production by minced CL substantially was diminished during the first 4 hours of incubation, but the inhibitory effects of PGF2-alpha were lost thereafter. We concluded that the effects of PGF2-alpha on steroidogenesis by porcine CL studied in vitro were affected by tissue preparation and length of exposure. Furthermore, short-term incubations of minced porcine CL may be a promising model for investigating the antisteroidogenic effects of PGF2-alpha.
Prostaglandin F2 (PGF2-alpha) inhibits progesterone (P4) production by the corpus luteum (CL) of many species, including the pig. Release of PGF2-alpha from the uterus destroys the CL at the end of the nonfertile estrous cycle, thus removing the supply of progesterone that would have been required in the event of pregnancy. This luteolytic action of PGF2-alpha can be used therapeutically to "short-cycle" animals (especially cattle) that fail to conceive. Despite the physiological and therapeutic importance of PGF2-alpha, the means by which the prostaglandin acts are not clearly understood.
Progress in determining the mechanism of PGF2-alpha action within the porcine CL has been hindered by the ambiguity of experimental models. Experiments focusing on the entire animal (in vivo models), while consistently yielding physiologically relevant data, often are of limited value when focusing upon phenomenae occurring at the cellular or subcellular level. Experiments using tissue removed from the body (in vitro models) and incubated in physiological solutions allow unimpeded observation of the function of a single tissue or cell type. However, investigators must be sure that these in vitro models reflect what actually is occurring in the body.
Short-term incubations of dissociated luteal cells have proven to be a useful and convenient in vitro model to investigate PGF2-alpha action in CL of several species, particularly when incubations are supplemented with gonadotropins [i.e., luteinizing hormone (LH) or prolactin] or lipoprotein substrate. The use of such models to investigate luteal function in the pig, however, has yielded variable results. Treatment of dissociated porcine luteal cells with PGF2-alpha often results in lack of effect or even stimulation of progesterone production (Yuan et al., 1993; Mattioli et al., 1985; Wiesak et al., 1992). This disparity between the effect of PGF2-alpha on the porcine CL in vivo vs in vitro has limited the use of short-term incubations for investigating the action of PGF2-alpha.
Many explanations have been offered for the inability to replicate PGF2-alpha-mediated inhibition of luteal steroidogenesis in vitro using short-term incubations of dispersed porcine luteal cells. Some possibilities include 1) inadequate concentration of substrate for progesterone production; 2) the lack of gonadotropic (e.g., LH) support for luteal function; 3) the need for intracellular communication during the action of PGF2-alpha; 4) inadequate time or pattern of PGF2-alpha exposure; and 5) loss of PGF2-alpha action on vascular and/or immune components found in the intact CL. Furthermore, the exposure of luteal cells to light during tissue processing and incubation is fraught with possibility for nonphysiological effects. In this study, we investigated the impact of ambient light, substrate availability, gonadotropic support, intracellular contact, and time of exposure on the action of PGF2-alpha on steroidogenesis in short-term incubations of luteal cells derived from the porcine CL.
Chemicals. Prostaglandin F2 (tris salt) was purchased from Sigma Chemical Co., St. Louis, Missouri. The human chorionic gonadotropin (hCG) used was CR 127 (14,900 U/mg, NIADDK) and the luteinizing hormone was USDA P-LH-B2. Human chorionic gonadotropin (hCG) is nearly identical to LH in structure and acts through binding to the LH receptor. Medium 199 (M199) was purchased from Gibco Co., Grand Island, NewYork. Collagenase (Cls I) was purchased from Worthington Biochemicals, Freehold, New Jersey. Specific and proven antibody (GDN #377) against progesterone was donated by Dr. Gordon Niswender, Colorado State University, Fort Collins, Colorado. 3H-progesterone was obtained from New England Nuclear, Boston, Massachusetts. Porcine low density lipoproteins (pLDLs) were prepared as described previously (Spies et al., 1967). All other chemicals were purchased from Sigma Chemical Co., St. Louis, Missouri.
Tissue Preparation. Ovaries were collected from sows during mid-gestation (days 35 to 85) at slaughter and transported to the laboratory on ice in M199 containing Earles salts and supplemented with 25 mM HEPES (M199, pH = 7.35). Porcine corpora lutea of pregnancy are sensitive to the effects of both LH (or hCG) and PGF2-alpha in vivo (Diehl and Day, 1974; Johnson et al., 1988) and thus represent a readily available model for the study of porcine luteolysis. Corpora lutea were dissected from the ovarian stroma and decapsulated. Dissociated luteal cells were prepared by mincing luteal tissue with fine scissors followed by gentle mechanical agitation and enzymatic digestion for 1 hour at 37oC using a collagenase (600 U/ml) and DNase (0.02%) solution. Following dissociation, dispersed cells were thoroughly washed with fresh M199 and counted by hemocytometry. Viability, as determined by trypan blue exclusion, was 70 to 90%. Minced luteal tissue was prepared by mincing corpora lutea with two no. 10 scalpel blades. Minced tissue sections were approximately 1.0 mm3.
Experimental Design. Experiment 1: Effects of ambient light and PGF2-alpha on progesterone accumulation during short-term incubation of dissociated porcine luteal cells (n = 4).
Ovaries were collected at slaughter as previously described. Upon collection, one ovary immediately was placed in a plastic collection vial impermeable to visible light. This ovary was transported to the laboratory, processed, and dissociated in an atmosphere of near total darkness. The minimal illumination needed to dissect and mince corpora lutea was provided by indirect lighting using a shielded 5 watt red light. Enzymatic dispersion of these corpora lutea and incubation of tissue samples were carried out in complete darkness. The remaining ovary was processed and incubated normally under fluorescent lighting. Luteal cells processed in a light or dark atmosphere were treated factorially with PGF2-alpha (0 and 500 ng/ml) and LH (0 and 100 ng/ml). These cells (100,000 cells/ml M199) then were incubated for 6 hours at 37oC under an atmosphere of 95% O2 / 5% CO2 in the presence and absence of ambient light. Following the incubation period, conditioned medium was collected and stored at -20oC until assay of progesterone by RIA. Viability of luteal cells was determined following the incubation period using trypan blue exclusion.
Experiment 2: Effects of increasing concentrations of PGF2-alpha on progesterone accumulation during short-term incubation of dissociated porcine luteal cells and luteal minces in the presence and absence of hCG (n = 5).
Dissociated luteal cells (100,000 viable cells/ml) and minced luteal tissue (100 mg/ml) were prepared from CL of pregnancy and incubated for 8 hours at 37oC under an atmosphere of 95% O2 / 5% CO2. Both tissue preparations were derived from corpora lutea of each animal. Treatments were factorially arranged for each tissue preparation, performed in duplicate, and included PGF2-alpha (0, 0.005, 0.05, 0.10, 0.20, 0.50, 1.0, and 1.5 g/ml) and hCG (0 and 100 ng/ml). Following incubation, medium was collected and stored at -20oC. Progesterone was measured by RIA.
Experiment 3: Effects of increasing concentrations of PGF2-alpha on progesterone accumulation during incubation of dissociated porcine luteal cells and minced porcine CL in the presence and absence of hCG and low-density lipoproteins (n = 4).
Dissociated luteal cells (100,000 cells/ml) and minced CL (100 mg/ml) were prepared from CL of pregnancy. The incubation conditions were as described for Experiment 2. Both tissue preparations were derived from each animal. Treatments were performed in duplicate for each tissue preparation and included PGF2-alpha (0, 5, 50, 500 ng/ml); hCG (0 and 100 ng/ml) and pLDL (0 and 140 g LDL cholesterol). Following incubation, medium was collected and stored at -20¡C. Progesterone within the medium samples was determined by RIA.
Experiment 4: Progesterone production by dissociated luteal cells over time in the presence and absence of PGF2-alpha (n = 3).
Dispersed luteal cells prepared from CL collected from sows during gestation (100,000/ml) were incubated in M199 + gentamicin (1 g/ml) for 12 hours with PGF2-alpha (0 and 500 ng/ml) under the incubation conditions used for previous experiments. Treatments and controls were performed in duplicate. Cells were pelleted by brief centrifugation every 2 hours, and conditioned medium was collected and replaced with fresh medium and treatment. A zero-hour control was included for each animal to determine progesterone concentrations at the start of the incubation. Medium samples were stored at -20oC until RIA of progesterone.
Experiment 5: Progesterone production by minced luteal tissue over time in the presence and absence of PGF2-alpha (n = 3).
Minced luteal tissue was prepared from corpora lutea of pregnant sows. These minces were incubated in M199 + gentamicin (1 g/ml) for 12 hours with PGF2-alpha (0 and 500 ng/ml) under the incubation conditions used for previous experiments. Treatments and controls were performed in duplicate. Tissue was pelleted by brief centrifugation every 2 hours, and conditioned medium was collected and replaced with fresh medium and treatment. A zero-hour control was included for each animal to determine P4 concentrations at the start of the incubation. Medium samples were stored at -20oC until radioimmunoassay of progesterone.
Radioimmunoassay of Progesterone. Radioimmunoassay was used to measure concentrations of progesterone in samples of M199 incubated with dispersed luteal cells and minced CL as previously described. The validation of this assay for use with luteal-conditioned medium previously has been reported (Watson and Maule-Walker, 1977).
Experiment 1: No differences were found in either the viability or basal and LH-stimulated progestin accumulation (Figure 1) between luteal cells prepared and incubated in the dark and those prepared and incubated normally under ambient illumination. Prostaglandin F2 stimulated basal progesterone production significantly in the absence of light, but this tendency was present in the illuminated cells as well. The presence of gonadotropin was associated with an attenuation of the stimulatory effects of PGF2-alpha during dark incubations but was otherwise without effect.
Figure 1. Progesterone accumulation following
treatment of luteal cells prepared and incubated in complete darkness vs
normal ambient light with luteinizing hormone and prostaglandin
F2 (n = 4). Bars bearing common superscripts denote
treatments resulting in similar (P > 0.05) concentrations of
progesterone.
Experiment 2: Analysis confirmed a slight but significant stimulatory effect of hCG on progesterone accumulation in short-term (8 hours) incubations of dissociated porcine luteal cells (Figure 2A; mean P4 accumulation hCG was 201.22 vs 176.30 ng/ml, respectively).
Prostaglandin F2 slightly increased progesterone accumulation, although a dose-dependent effect was not consistently evident.
In contrast to dissociated cell incubations, minced CL of the same animals failed to respond to hCG (Figure 2B). Furthermore, PGF2-alpha signifi-cantly inhibited progesterone accumulation during short term incubation of minced luteal tissue at several concentrations. Prostaglandin F2 and hCG did not interact significantly in their effects.
Figure 2A and B. Progeserone accumulation during
incubation of dissociated (Panel A) or minced (Panel B)
porcine luteal tissue with increasing concentrations of
prostaglandin F2 in the presence and absence of human
chorionic gonadotropin (n = 5). Asterisks and double
asterisks denote treatment means which differ significantly
(P 0.05 and P 0.10, respectively) from corresponding
controls.
Experiment 3: Human chorionic gonado-tropin and pLDL both modestly increased the accumulation of progesterone during an 8-hour incubation of dissociated luteal cells (Figure 3A). The mean concentrations of progesterone that accumulated in the presence and absence of hCG were 269.92 vs 231.38 ng P4/ml, while incubation of cells in the presence and absence of pLDL resulted in mean P4 accumulation of 264.44 vs 236.85 ng P/ml, respectively. Prostaglandin F2 failed to significantly alter progesterone accumulation by dissociated luteal cells regardless of other treatments.
There was a significant interaction among the effects of hCG, pLDL, and PGF2-alpha on progesterone accumulation during incubations of minced luteal tissue prepared from the same animals. Steroidogenesis appeared unaffected by hCG (Figure 3B), while pLDL exerted a stimulatory effect in the presence of hCG. In the absence of hCG and pLDL, PGF2-alpha exerted a significant inhibitory effect at the highest dose used but was without effect under other incubation conditions.
Figure 3A and B. Progesterone accumulation during
incubation of dissociated (Panel A) or minced (Panel B) luteal tissue
for 8 hours (n = 4). Treatments were factorially arranged and included
increasing concentrations of prostaglandin F2, as well as
human chorionic gonadotropin and low-density lipoproteins. Asterisks
denote treatment means which differ significantly (P 0.05)
from corresponding controls.
Experiment 4: Progesterone production by dispersed luteal cells was moderate initially,reached a zenith during hours 2 to 4, and declined thereafter (Figure 4). Treatment with PGF2-alpha significantly increased P4 production vs controls during hours 2 to 4. This stimulatory effect was not evident during the remainder of the incubation.
Figure 4A and B. Progesterone production by
dispersed luteal cells and luteal minces during 2-hour intervals
throughout a 12-hour incubation in the presence and absence of
prostaglandin F2 (n = 3 each). Asterisks denote treatments
with means differing significantly (P 0.05) from controls for
that time point.
Experiment 5: The effects of PGF2-alpha on progesterone production throughout a 12-hour incubation of minced porcine CL of pregnancy are depicted in Figure 4. Progesterone production by control tissues was greatest during the first 4 hours of incubation, decreased from 4 to 6 hours, and remained steady for the duration of the incubation. Basal progesterone production substantially was attenuated by PGF2-alpha during hours 0 to 2 and 2 to 4 of incubation. After 4 hours of incubation, control and treated tissues produced similar amounts of P4. Thus, there was a change in the impact of PGF2-alpha on steroidogenesis over time.
Demonstrating an inhibitory effect of PGF2-alpha on the porcine CL using an in vitro model often has proven to be a difficult task. In previous work, Watson and Maule-Walker (1978) and Uggla (1988) showed that PGF2-alpha was capable of acutely inhibiting progesterone production by superfused porcine luteal slices prepared from CL collected during the mid-luteal phase and pregnancy. Subsequent attempts to repeat this effect in preparations of dispersed luteal cells generally resulted in no effect or stimulation of luteal steroidogenesis (Yuan et al., 1993; Mattioli et al., 1985; Wiesak et al., 1992). The results of the current study demonstrated that differences in tissue preparation and the time of incubation probably contributed to the disparate effects of PGF2-alpha observed to date in studies performed using luteal tissue incubated outside of the body.
This study was the first to investigate the impact of light on steroidogenesis and viability of luteal cells. Visible light induced DNA damage and prevented cell division in Chinese hamster ovary cells (Boder et al., 1983; Girsh et al., 1995). Furthermore, light is known to catalyze many reactions which are unnatural for the luteal cell, including production of destructive molecules such as peroxide, superoxide, and hydroxyl radicals. Nonetheless, the intensity of ambient light used in our dissociations and incubations did not appear to affect cell viability, basal steroidogenesis, or responsiveness to PGF2-alpha or LH during short-term incubations of porcine luteal cells.
The observation that minced luteal tissue and dissociated luteal cells seemed to respond differently to PGF2-alpha treatment in vitro was a major finding of this study. In general, preparations of dispersed luteal cells responded to PGF2-alpha with slight increase in progesterone synthesis. In contrast, progesterone production by luteal slices significantly was inhibited in all experiments, although this inhibition was modest and not seen at all doses in incubations lasting more than 4 hours. These data are in agreement with similar studies performed using other ovarian tissues (Gadsby et al., 1993). The observed lack of inhibitory effects of PGF2-alpha in dispersed luteal preparations may have been due to a loss of necessary intracellular contact. The majority of high affinity binding sites for PGF2-alpha in the pig CL are found on a single type of cell - the large luteal cell (Gadsby et al., 1991; Gregoraszczuk, 1983). Overall, these results suggested that minced luteal tissue may represent a more favorable laboratory model for investigation of the inhibitory action of PGF2-alpha in the porcine CL.
In the current study, hCG and pLDL were more effective in stimulating P4 production in dispersed luteal cell preparations. Similar stimulation of luteal P4 production by LDL and gonadotropins was observed in previous studies of dispersed luteal cells (Rajkumar et al., 1985; Buhr, 1987; Gleeson and Thorburn, 1973). In our study, hCG was without effect on steroidogenesis by minced luteal tissue, while pLDL only slightly stimulated P4 production. This may have been due to difficulties in these relatively (vs PGF2-alpha) larger molecules gaining access to luteal cells within the interior of the tissue pieces.
An unexpected observation within this study was the loss of inhibitory effect of PGF2-alpha on P4 synthesis by luteal minces after the first 2 to 4 hours of incubation. PGF2-alpha profoundly decreased progesterone production during the first 2 hours, but this inhibition was gone by hours 4 to 6 of incubation. The stimulatory effect of PGF2-alpha seen in the dispersed cell preparations also was lost over time. Because of this loss of PGF2-alpha effect over time in the current study, progesterone accumulation within our treated and control samples approached equality with increasing length of incubation. The mechanism responsible for the observed loss of PGF2-alpha action may involve receptor down-regulation due to chronic exposure to high concentrations of the prostaglandin. During normal luteal regression, CL are exposed to PGF2-alpha episodically (Dusza et al., 1988) and may therefore avoid any such loss of PGF2-alpha inhibition due to receptor down regulation.
In conclusion, the effects of PGF2-alpha on steroidogenesis by porcine CL studied in vitro were affected by tissue preparation and length of exposure. Ambient light did not appear to affect luteal progesterone accumulation under any conditions studied. Short-term incubations of minced luteal tissue may be a promising model for investigating the inhibitory effects of PGF2-alpha on progesterone production.
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