Figure 1. Diagram of cannulation to test for secretion of vitamin C in response to treatment with PGF2.
B.K. Petroff*, R.E. Ciereszko*, K. Dabrowski+,
A.C. Ottobre*, W.F. Pope*, and J.S. Ottobre*
*Department of Animal Sciences and
+Department of Natural Resources
The corpus luteum (CL) is an ovarian tissue that produces progesterone (P4) necessary for successful pregnancy. If pregnancy does not occur, the CL is destroyed following release of prostaglandin F2-alpha (PGF2-alpha) from the uterus. The luteolytic effects of PGF2-alpha are thought to be mediated in part by the promotion of an increasingly oxidative cellular environment. Loss of antioxidants is one mechanism by which PGF2-alpha might induce oxidative damage within the corpus luteum. This study was performed to establish whether vitamin C depletion is an acute effect of PGF2-alpha on the porcine CL and to gain insight into the mechanism of luteal vitamin C loss at luteolysis. Gilts (n = 4) were anesthetized, and both utero-ovarian veins (UOVs) and an ear vein were catheterized. Each CL on the treated ovary received an intraluteal injection of PGF2-alpha (1 g), followed by a sustained release implant containing 100 g of the prostaglandin. The other ovary served as control, with each CL receiving corresponding volumes of injection vehicle and blank implant. Blood was collected from the ear vein and both UOVs every 15 minutes beginning 15 minutes prior to the onset of treatment. Blood collection was ended when animals were ovariectomized and corpora lutea collected at 2 hours post-treatment. Progesterone (P4) and vitamin C concentrations were measured in tissue and plasma samples. PGF2-alpha-treated luteal tissue had similar P4 but significantly lower vitamin C concentrations when compared with control CL. PGF2-alpha treatment resulted in a rapid and sustained increase in plasma vitamin C within the treatment-side UOV, while the control UOV and ear vein showed little change in plasma vitamin C during the experimental period. No effect of PGF2-alpha on plasma P4 concentrations was evident. Thus, PGF2-alpha action depletes the porcine corpus luteum of vitamin C by inducing secretion of the vitamin into the bloodstream. These results are consistent with the hypothesis that vitamin C depletion contributes to the demise of the porcine CL.
The porcine corpus luteum is destroyed near the end of a nonfertile estrous cycle in response to prostaglandin F2-alpha released from the uterus. This results in a waning of the production of progesterone, a steroid required for successful implantation of the embryo and maintenance of pregnancy. The mechanisms by which PGF2-alpha destroys the CL are only partially understood.
Recent studies have established a role for reactive oxygen species (i.e., superoxides and peroxides) in the destruction of the CL at the end of the nonfertile estrous cycle. This process of luteolysis is associated with an accumulation of superoxide and peroxide radicals within the CL (Sawada and Carlson, 1989; Riley and Behrman, 1991). Prostaglandin F2-alpha is known to induce accumulation of such reactive oxygen species within the CL (Riley and Behrman, 1991). Exposure of luteal cells to these oxidants results in oxidation and loss of fluidity in cellular membranes (Sawada and Carlson, 1991) and interruption of progesterone production (Behrman and Preston, 1989; Gatzuli et al., 1991). These and other studies support an oxidant-mediated mechanism of PGF2-alpha action on the CL.
Vitamin C (ascorbate) is a multifunctional antioxidant that is synthesized by the liver of the pig and sequestered within the corpus luteum via an ATP-dependent process (Stansfield and Flint, 1967; Musicki et al., 1996). The concentrations of vitamin C found in the functional porcine CL are as much as 100-fold greater than systemic plasma concentrations (Petroff et al., 1995) and rival the ascorbate concentrations found in the adrenal gland, the richest source of vitamin C in the body (Levine and Morita, 1985). In a recent study, we found that concentrations of vitamin C in regressing porcine corpora lutea were greatly reduced in comparison to corpora lutea collected during the mid-luteal phase and early pregnancy (Petroff et al., 1995). Furthermore, exogenous PGF2-alpha rapidly causes such depletion of luteal ascorbate in intact and hypophysectomized rats (Musicki et al., 1996; Aten et al., 1992; Sato et al., 1974).
Several mechanisms might be responsible for depletion of luteal ascorbate stores following treatment with PGF2-alpha. The vitamin may be irreversibly oxidized while neutralizing the reactive oxygen species that accumulate in luteal tissue following exposure to PGF2-alpha (Sawada and Carlson, 1989). Periods of luteal ascorbate depletion are associated with an accumulation of lipid peroxides, suggesting that the vitamin does play a significant role as a luteal antioxidant (Aten et al., 1992). PGF2-alpha may also induce secretion of luteal stores of vitamin C by impairing the mechanisms of ascorbate sequestration within the CL. Treatment of cultured rat luteal cells that have been preloaded with radiolabelled ascorbate causes a rapid loss of the vitamin which is thought to be due to interruption of ascorbate transport (Musicki et al., 1996). In this experiment, we wanted to determine whether PGF2-alpha causes a loss of luteal ascorbate from the porcine CL in vivo and gain insight into the mechanism of any loss of vitamin C following treatment with PGF2-alpha.
Experimental Model (Figure 1). Landrace x Duroc gilts (n = 4), weighing from 260 to 300 pounds, were used in this study. The onset of estrus was determined by daily observation (0830) in the presence of intact boars. Surgical procedures were performed on day 13 following the onset of estrus. This protocol was approved by the Agricultural Animal Care and Use Committee at The Ohio State University.
Figure 1. Diagram of cannulation to test for
secretion of vitamin C in response to treatment with PGF2.
A surgical plane of anesthesia was maintained throughout the experimental procedures using halothane inhalation to effect. Following exposure of the reproductive tract via a midventral incision, both utero-ovarian veins (UOVs) were catheterized with polyethylene catheters (I.D. 0.35 mm, O.D. 0.58 mm; Clay Adams, Parasippany, N.J.). An ear vein was also catheterized using an 18 ga x 1.25" over-the-needle IV catheter (Terumo Medical Corp., Elkton, MD) for the collection of systemic blood samples. All catheters were flushed with heparinized saline (100 IU/ml; Elkins-Sinn, Inc., Cherry Hill, NJ) following emplacement and each blood sample collection. Proper catheter place-ment was tested by palpation and by comparison of UOV plasma P4 concentrations with those of systemic plasma within each animal. Because utero-ovarian veins normally contain substantially greater concentrations of P4 than systemic vessels, UOVs that yielded blood samples containing concentrations of P4 similar to systemic (ear vein) samples at all time points were considered to have been improperly cannulated. Blood samples were collected from both UOVs and the ear vein at -15, 0, 15, 30, 45, 60, 75, 90, 105, and 120 minutes relative to the onset of treatment.
Ovaries were randomly designated as receiving PGF2-alpha or control treatment. To achieve acute and prolonged increases in PGF2-alpha within treated CL, PGF2-alpha treatment entailed intraluteal injections of 1 g PGF2-alpha (Lutalyse; Upjohn Co., Kalamazoo, MI) delivered in 250 l normal saline to all corpora lutea on that ovary followed by implantation of each CL with approximately 100 g PGF2-alpha (tris salt, Sigma Chemical Co., St. Louis, MO) in the form of a 50 l sustained-release silastic implant (Ford and Christenson, 1991). Such intraluteal PGF2-alpha implants have been shown previously to induce luteal regression and loss of tissue progesterone in the porcine CL (Hehnke et al., 1994). Corpora lutea on the remaining (control) ovary received an intraluteal injection of normal saline and a blank silastic implant. The order of treatment was randomly determined. Intraluteal injections were completed by 5 minutes and implants delivered by 15 minutes from the onset of treatment.
Immediately following collection of each set of blood samples, plasma from each blood sample was divided into two aliquots. One aliquot was immediately placed on dry ice and maintained at -70oC until assay of progesterone. The second aliquot was acidified, transferred to dry ice, and maintained at -70oC until assay of vitamin C. Two hours after the onset of treatment both ovaries were removed. Corpora lutea were immediately dissected, examined for the presence of implants, and stored at -70oC until assay for tissue concentrations of progesterone and vitamin C.
Progesterone RIA. The concentrations of P4 in plasma and tissue samples were measured by radioimmunoassay using a proven and specific antibody (GDN #377; donated by Dr. Gordon Niswender, Colorado State University) as described previously (Petroff et al., 1995).
Measurement of Vitamin C. Vitamin C (ascorbate + dehydroascorbate) was measured using a colorimetric assay as described previously (Petroff et al., 1995).
Tissue Progesterone Concentrations. Concentrations of P4 were similar in control and PGF2-alpha-treated corpora lutea (Figure 2). These high concentrations of progesterone were similar to those seen in fully functional corpora lutea from our previous work (Petroff et al., 1995). Moreover, the PGF2-alpha implants used in this study required 12 hours to elicit changes in luteal progesterone in a previous study (Hehnke et al., 1994). Thus, luteal function was probably uncompromised by 2 hours post-treatment in the present study.
Tissue Vitamin C Concentrations. Mean concentrations of vitamin C within PGF2-alpha-treated corpora lutea were significantly decreased in comparison to controls at 2 hours post-treatment (Figure 3). Vitamin C concentrations in control CL were similar to those seen previously in mid-cycle corpora lutea (Petroff et al., 1995). Thus, PGF2-alpha appeared to rapidly deplete luteal vitamin C stores by a local mechanism.
Figure 2. Concentrations of progesterone in luteal
tissue following treatment with PGF2
vs vehicle. Each bar represents the mean SE of the progesterone
concentrations present in luteal tissue from four gilts at the end of
the experimental procedure.
Figure 3. Concentrations of vitamin C present in luteal
tissue following treatment with PGF2
vs vehicle. Each bar represents the mean SE of the progesterone
concentrations present in luteal tissue from four gilts at the end of
the experimental procedure.
Plasma Progesterone Concentrations. Figure 4A depicts plasma P4 concentrations in the utero-ovarian veins draining control and PGF2-alpha-treated ovaries. Data from the control UOV of one pig was excluded, as P4 concentrations did not differ from those of systemic plasma, indicating suspect cannula placement. Concentrations of P4 in UOV plasma from both treated and control sides varied greatly from one time point to the next. Control side utero-ovarian plasma appeared to contain greater concentrations of P4 throughout the experiment. However, this was not attribut-able to any treatment effect, as progesterone con-centrations did not change significantly from zero hour controls for either of the UOVs. Similarly, systemic concentrations of progesterone did not change significantly following PGF2-alpha-treatment (Figure 4B). These data further support that luteal function was uncompromised by PGF2-alpha to 2 hours post-treatment when the experiment was ended.
Figure 4A and B. Concentrations of progesterone present
in ear vein plasma and plasma from utero-ovarian veins
draining control and PGF2-treated ovaries collected prior
to and during intraluteal treatment with PGF2 and vehicle.
Data from one control UOV were excluded due to poor
cannulation. Values are least squared means for data from
four gilts
Plasma Vitamin C Concentrations. Figure 5 depicts the concentrations of vitamin C present in plasma samples collected from the ear vein and both utero-ovarian veins prior to and following local ovarian treatment with prostaglandin F2-alpha. Data from the control UOV of one pig were excluded due to suspect cannulation. Ascorbate concentrations in plasma from the excluded vessel did not differ from systemic plasma values. Vitamin C concentrations were quite similar in plasma collected from the ear vein and both UOV prior to treatment with PGF2-alpha. Vitamin C concentrations in the UOV plasma from the PGF2-alpha-treated side rose significantly from pretreatment values by 15 minutes and remained elevated to 75 minutes post-treatment. These concentrations were greater than those present in both the ear vein plasma and plasma collected from the control UOV. Maximal concentrations of vitamin C in plasma from the treated-side UOV were more than twice pretreatment values. Although ascorbate concentrations appeared to rise slightly with time in both systemic plasma and plasma from the control side UOV, this effect was not statistically significant. Thus, PGF2-alpha treatment specifically induced a significant release of vitamin C into the venous bloodstream.
Figure 5. Concentrations of vitamin C present in
plasma from an ear vein and utero-ovarian veins draining control and
PGF2 treated ovaries prior to and
throughout intraluteal treatment with PGF2 and vehicle. Data from one control UOV
were excluded due to poor cannulation. Values are least squared means
for data from four gilts. Asterisks denote values significantly
different from their respective zero hour controls.
The high concentrations of vitamin C found in the fully functional corpus luteum have led many investigators to postulate as to the role of the vitamin in luteal function. Ascorbate is known to serve as a cofactor in collagen synthesis, an important process in the rapidly growing CL (Luck et al., 1995). Vitamin C also is involved in steroid (e.g., P4) and peptide hormone production (Luck et al., 1995) and may thereby directly promote luteal function (Byrd et al., 1993; Luck and Jungclas, 1987a,b; Biskind and Glick, 1936; Biswas and Deb, 1970). Vitamin C further serves as a cellular antioxidant, functioning especially in combination with vitamin E (Packer et al., 1979) to neutralize reactive oxygen species produced by such processes as cellular respiration and steroidogenesis. Interruption of these functions of vitamin C would result in the reversal of tissue growth, waning progesterone production, and increasingly oxidative cellular environment seen during regression of the corpus luteum.
Previous studies conducted in our and other laboratories have established that concentrations of vitamin C are diminished in regressing corpora lutea but remain elevated should luteal function continue due to rescue of the CL in the event of pregnancy (Petroff et al., 1995; Biskind and Glick, 1936; Luck and Jungclas, 1987a). In the present study, treatment of porcine corpora lutea with prostaglandin F2-alpha, the agent that destroys the CL, resulted in a rapid loss of luteal ascorbate. This is in agreement with previous studies in which rats were treated systemically with PGF2-alpha (Aten et al., 1992; Sato et al., 1974), although a portion of this effect may have been attributable to indirect effects of PGF2-alpha mediated through other hormones. The within-animal control and local nature of PGF2-alpha administration used in the present study enabled us to demonstrate significant ascorbate depletion within the CL that was attributable solely to a direct action of PGF2-alpha. This is in agreement with a recent study performed using cultured rat luteal cells (Musicki et al., 1996). In the present study, the loss of luteal vitamin C preceded any change in luteal function (i.e., P4 production). Thus, depletion of luteal ascorbate stores appeared to be an early change of PGF2-alpha-induced luteal regression in the porcine CL.
Prostaglandin F2-alpha appeared to deplete the porcine CL of vitamin C by inducing secretion of the vitamin into the bloodstream. This is indicated by the rapid and profound increase in plasma ascorbate seen in the venous blood from PGF2-alpha-treated ovaries in the present study. Using the utero-ovarian concentrations of ascorbate in our study combined with data documenting the ovarian blood flow on day 13 of the porcine estrous cycle (15 ml/min; Magness et al., 1983), we estimated that approximately 8 mg of vitamin C was secreted into the bloodstream by the PGF2-alpha-treated ovary in each animal. This loss via secretion would be sufficient to deplete vitamin C from nearly all functional luteal tissue present.
This study documents a rapid depletion of vitamin C from the porcine corpus luteum following administration of PGF2-alpha. This loss of luteal ascorbate preceded any change in luteal function as assessed by tissue and plasma progesterone concentrations. The depletion of luteal vitamin C stores was associated with rapid and sustained release of the vitamin into the venous blood draining from the treated ovary. It appeared, therefore, that PGF2-alpha action depleted the porcine corpus luteum of vitamin C by inducing secretion of the vitamin into the bloodstream. This vitamin C depletion may contribute to the destruction of the porcine CL.
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