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Female Reproductive Tract |
Departments of Obstetrics and Gynecology,3
Dairy Science,4
Animal Sciences,5
Pediatrics,6 University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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injections (7.5 mg i.m. 4 h apart). At CIDR removal, Experimental Day 0, zero (n = 9), 500 IU (n = 8), or 1000 IU (n = 7) eCG was injected i.m.; UBF was monitored continuously for 55–75 h. Jugular blood was sampled every 8 h to evaluate levels of P4, estradiol-17ß (E2ß) and luteinizing hormone (LH). The inhibitor of nitric oxide synthase, L- nitro-arginine methyl ester (L-NAME) was infused in a stepwise fashion unilaterally into one uterine artery at 48–50 h after 500 IU eCG and the effects on UBF were examined (n = 7). The zero-eCG group gradually increased UBF from a baseline of 17.4 ± 3.9 to 80.5 ± 1.1 ml/min. The 500-IU-eCG group increased UBF between 10 and 15 h from a baseline of 11 ± 3.3 to 83.3 ± 1.0 ml/min, whereas UBF for the 1000-IU-eCG group was higher (100.1 ± 1.7 ml/min) than that seen in either of the other groups. Plasma P4 fell to baseline within 8 h of CIDR removal, while E2ß rose gradually in association with elevations in UBF. LH surges occurred between 32 and 56 h after CIDR removal and the LH surge occurred earlier in the 1000-IU-eCG group than the other two groups (P < 0.01). L-NAME infusion dose dependently reduced maximum levels of UBF ipsilaterally by 54.6% ± 6.2%, but contralaterally only by 27.4% ± 8.5%. Regardless of season, either dose of eCG will result in analogous UBF responses. During the follicular phase, elevations in UBF are in part locally controlled by the de novo production of nitric oxide.
menstrual cycle, nitric oxide, seasonal reproduction, steroid hormones, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast with studies in ovx sheep, UBF during the normal estrous cycle has not been as extensively studied and no studies are currently available that directly examine whether the vasodilatory response to cyclical changes in endogenous follicular-derived estrogen is fully analogous to the response to exogenous estrogen. Markee [11] first described the changes in uterine hyperemia (blushing) in association with the development of follicular structures on the ovaries and with injections of crude ovarian estrogen extracts. Using intact ewes, Greiss and Anderson [12] reported that UBF is elevated during the periovulatory period in association with behavioral receptivity to a ram. Ford et al. [13–16] and Roman-Ponce et al. [17] reported that the higher the estrogen/progesterone (P4) ratio in intact animals, the greater the magnitude of UBF. However, in studies using cycling cows, ewes, and sows, the timing and magnitude of the pattern of UBF of individual animals was quite variable [16, 17] compared with very low, nearly nonexistent variability in the ovx model. While this made the ovx model a vastly predictable and easier model to study than the intact sheep, it may not, however, represent what is actually occurring in the uterus during the estrous cycle. Moreover, in nearly all studies to date using the ovx animal model, pharmacologic, not physiologic, doses of estrogen have been administered to study UBF responses.
Another major limitation of the intact cycling sheep model for evaluating UBF changes is the fact that sheep are photoreceptive breeders, making it quite difficult, if not impossible, to perform these studies throughout the year. The change from the follicular to the luteal phase during the estrous cycle is predominantly due to the sequential production of estrogen and P4 by the ovary and their negative and, to a lesser extent, positive influence on the hypothalamic GnRH pulse generator [18, 19]. Ewes in the Northern Hemisphere generally enter seasonal anestrus and lose cyclicity in the spring and remain anovulatory until early fall; the reverse is true in the Southern Hemisphere [20, 21].
Nitric oxide is a potent vasodilator, produced and released from endothelial cells during the enzymatic conversion of L-arginine to L-citrulline [22–28]. The endothelial NO synthase (eNOS) enzyme is inhibited by various analogues of L-arginine, e.g., L-nitro-arginine methyl ester (L- NAME). In ovx estradiol-17ß (E2ß)-treated sheep, local infusion of L-NAME at the time of maximum UBF reduces UBF and cyclic guanosine monophosphate, the endogenous second messenger of NO production, in a dose-dependent manner [9, 10], demonstrating a role for NO in regulating the pharmacological estrogen-mediated elevations in UBF.
The objective of the current study was to develop a synchronized animal model using CIDR's PGF2 and eCG that will allow for the consistent study of the magnitude, pattern, and mechanisms by which UBF is regulated during ovarian cycles in sheep regardless of season. We then tested the utility of using this model by evaluating the hypothesis that L-NAME will decrease the elevated UBF noted in the follicular-phase cycling animal, thus demonstrating a role for local de novo production of NO in mediating the endogenous E2ß-induced rise in UBF.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research and Animal Care and Use Committees of both the Medical School and the College of Agriculture and Life Sciences. Nonpregnant, intact multiparous ewes (50–65 kg) of mixed western breeds were exposed to a 16L: 8D cycle daily beginning 3–4 days before surgery. This light schedule mimics the daylight timing during the normal breeding season (September–March) in the Northern Hemisphere. There was no specific designated day of the ovarian cycle for surgical instrumentation. Food and water was withheld 24 h before surgery that was performed as previously described [7, 8, 29, 30]. At surgery, the ewes received i.m. ketamine (16 mg/kg), atropine (12 µg/kg), and antibiotics (400 000 U Penicillin, 200 mg Gentamicin). A percutaneous jugular venous catheter was then inserted for i.v. administration of ketamine (10 mg/ml) in 0.9% saline and 5% dextrose with supplemental pentobarbital sodium (Nembutal; 50 mg/ml) as needed for additional anesthesia. The abdominal, inguinal, and cervical regions were shaved and aseptically scrubbed. Under sterile conditions, the uterus was exposed following a midventral laparotomy. Transonic flow probes (3 or 4 mm; Transonic Systems Inc., Ithaca, NY) were implanted around the middle uterine artery of each uterine horn. Polyvinyl catheters (Tygon, Cleveland, OH) containing heparinized saline (25 IU/ml) were implanted retrograde into the right and left distal branches of the uterine artery (0.23- mm inner diameter [ID], 0.47-mm outer diameter [OD]). After closure of the midline incision, a catheter (0.40-mm ID, 0.70-mm OD) was inserted into both superficial saphenous femoral arteries through an inguinal incision and it was advanced through the femoral circulation and into the abdominal aorta (20 cm). All catheters were filled with sterile, heparinized saline (25 IU/ml), sealed, and exteriorized with the flow probe leads to a pouch on the ewe's left flank. The ewes were given immediate postsurgical analgesia (Flunixin Meglumine; 75 mg i.m.) and allowed free access to food and water ad libitum throughout the remainder of the studies.
Experimental Protocol for the Study of Uterine Blood Flow
The 500- and 1000-IU-eCG (Sioux Biochemical, Inc., Sioux Center, IA) protocols were used throughout the entire year. By necessity, the control (zero-eCG) group, however, could only be used during the normal breeding season. Sheep were allowed a 5- to 7-day postsurgical recovery period. At this time, they were then implanted with a vaginal P4-controlled internal drug-releasing device (CIDR; 0.3 g) (Latinagro de Mexico, Monterrey, Mexico), for 7 days [31]. After 6 days of P4 treatment, ewes received two 7.5-mg i.m. injections of PGF2 (Dinoprost Tromethamine- Lutalyse; Upjohn, Kalamazoo, MI) 4 h apart to lyse any existing corpora lutea. The following day, designated as Experimental Day 0, the CIDR was removed and each animal received zero eCG (n = 9), 500 IU eCG (n = 8), or 1000 IU eCG (n = 7) i.m. Animals from each group were monitored continuously for 55–75 h or until UBF returned approximately to baseline levels. Animals were then given a 1 µg/kg i.v. bolus doses of E2ß (stock solution 100 µg/ml in ethanol) once UBF levels had returned to baseline, and the UBF response was monitored for 2–3 h as previously described [3, 6–8].
A group of six sheep also were ovariectomized at the time of chronic instrumentation [7, 8] as described above for the ovary-intact groups. This ovx group was studied to show that, in the absence of ovarian/follicular- derived estrogen, eCG cannot increase UBF. To verify a normal uterine vasodilatory responsiveness to exogenous estrogen treatment, ovx sheep were treated with i.v. 1 µg/kg E2ß at 66 h after the time of CIDR removal and a 500-IU-eCG i.m. injection and were monitored for 2–3 h [6–8].
Circulating Levels of Progesterone, Estrogen, and LH
Blood was collected every 8 h via the jugular vein catheter starting at Time 0, when the CIDR was removed and eCG was injected. Plasma and serum were obtained and stored frozen (–20°C) until plasma P4 or serum E2ß and luteinizing hormone (LH) were analyzed. Serum LH (200 µl) was analyzed by RIA using methods [32] that were previously validated in our laboratory [33]. Plasma P4 was analyzed using the commercial Coat-A-Count RIA kit from Diagnostic Products Corporation (DPC, Los Angeles, CA). To analyze E2ß, serum (500 µl) was extracted twice with diethyl ether and concentrations of E2ß were evaluated by RIA as previously reported [34]. All samples were analyzed in duplicate and all samples were analyzed in a single assay (intraassay coefficients of variation = 3.6% for LH, 8.4% for P4, and 7.1% for E2ß).
L-NAME Infusion Experiments
L-NAME (Sigma Chemical Co., St. Louis, MO) infusion experiments were performed to determine the role of NO in the follicular phase rise in UBF and to test the utility of using this newly developed animal model. Based on results from the first group of animals (Figs. 1 and 2), only the 500-IU-eCG dose of eCG was used. All animals were monitored during a control ovulation cycle before undergoing the L-NAME experiment during the following ovarian cycle. UBF was allowed to rise to maximum levels (48–50 h) before unilateral administration of L-NAME infused in a stepwise fashion to observe the local dose-dependent effects of NOS blockade at peak UBF. The range of doses of L-NAME were based on our previous experiments [10] using ovx sheep and infusing L-NAME at maximum UBF after E2ß was given. Estimated concentrations of L-NAME in the uterine arterial blood ranged from 0.05 to 1 mg/ml. The first two doses were infused for 25 min; all other doses were infused for 10 min each. Therefore, concentrations of L-NAME in uterine artery blood were slightly different for each individual animal due to the difference in maximum UBF levels. The opposite uterine horn UBF for which final drug concentrations cannot be calculated served as a contralateral internal control within each animal.
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Statistical Analysis
Differences in treatment groups for UBF patterns and steroid hormone levels were analyzed using split plot analysis of variance (ANOVA). The data describing the time of the LH surge was analyzed by one-way ANOVA. Data are reported as means ± SEM. Correlation coefficients and least squares best-fit lines between the temporal relationships of hormone profiles and UBF were also analyzed. A P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Baseline levels of UBF were equal (P > 0.05) in the three ovary-intact groups, averaging 14.8 ± 1.7 ml/min. Regardless of which season (cycling or anestrous) this treatment regimen was used, a rise in UBF was observed in the 500- and 1000-IU-eCG groups (Fig. 1). The zero-eCG treatment was only used during the breeding season due to the absence of ovulation during the anestrous period. The three treatment groups (zero eCG, 500 IU, and 1000 IU eCG) revealed remarkably similar timing of the overall pattern of rise in UBF, with the initial rise beginning about 5– 10 h and reaching maximum flows at approximately 45–55 h. Naturally-cycling control ewes gradually increased UBF from baseline levels of 17.4 ± 3.9 ml/min to a plateau of 80.5 ± 1.1 ml/min. The 500-IU-eCG group increased UBF between 10–15 h, with a gradual rise from baseline UBF of 11 ± 3.3 ml/min to a plateau in UBF of 83.3 ± 1.0 ml/ min. The plateau in UBF for the 1000-IU-eCG-treated sheep occurred earlier and thus was higher at these times than those seen in either the zero-eCG or 500-IU-eCG groups, increasing from a baseline UBF of 11.9 ± 1.9 ml/ min and reaching 100.1 ± 1.7 ml/min (P < 0.01). In Figure 1, with results from all experiments shown (mean ± SEM), it appears that the zero-eCG control group maintained elevated levels of UBF longer than both the 500- and 1000- IU-eCG groups. We also observed spikes or acute fluctuations of UBF in each individual animal that appeared to occur in a random fashion before and during the overall rise in UBF. However, Figure 1, which illustrates the total mean UBF in all animals, does not illustrate these fluctuations due to summing the flows from both sides and then averaging of individual values at specific time points. Figure 2 is a profile of individual representative animals from each group. This figure illustrates the acute spikes in UBF that cannot be seen in the combined data for Figure 1. It is noteworthy that, when the spikes in UBF occurred, for the most part, they were observed bilaterally and were correlated. An additional interesting observation that was seen in some animals of all three groups was an initial transient rise in UBF (35–50 ml/min) within the first 10–12 h, maximizing at 15–20 h, and returning to baseline levels until approximately 30–35 h. This event did not occur in all animals and there was no relationship between the eCG dose and season with the incidence of this rise. As observed with the isolated spikes in UBF, the observed initial transient rise in UBF is not accurately represented in the combined data for Figure 1 but can be seen in some of the individual UBF patterns in Figure 2. In the ovx model, because there was no observable rise in UBF at any time due to eCG, these studies demonstrate the need for the presence of the ovaries during the follicular phase to induce both this initial transient rise in UBF and also the sustained vasodilatory response described for all three intact groups (Fig. 3, top). Moreover, because we have recently shown that the periovulatory elevations in UBF are reduced by local uterine arterial infusion of the pure estrogen receptor antagonist ICI 182 780, the current data clearly demonstrate that ovarian follicular-derived estrogen indeed controls this rise in UBF [35].
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The classic pattern of vasodilatory response in UBF to 1 µg/kg exogenous E2ß was observed in each animal, regardless of treatment group, i.e., a rise in UBF was observed 30 min after administration and maximum UBF levels were reached at 90–120 min (Fig. 3, bottom). Additionally, when all intact animal E2ß responses were combined (eCG has no effect at this time), the magnitude of response in UBF of the intact animals to exogenous E2ß was significantly higher than the response of ovx animals to exogenous E2ß (P < 0.05). The response in maximum UBF to rising endogenous ovarian-derived estrogen levels was significantly lower (P < 0.05) and substantially more protracted in all ovary-intact cycling groups than either the maximum or timing of the UBF responses observed to exogenous (1 µg/kg) E2ß systemic administration in either intact or ovx ewes (Fig. 4).
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Progesterone Concentrations During the Periovulatory Period
Zero-eCG (n = 8), 500-IU-eCG (n = 5), and 1000-IU- eCG (n = 7) groups exhibited similar (P > 0.05) concentrations of P4 at Time 0 (5.21 ± 1.02 ng/ml) (Fig. 5) and throughout the 55–75-h experiment. At 8 h, plasma P4 levels decreased (P > 0.001) to 1.50 ± 0.39 ng/ml, reaching baseline levels (<0.5 ng/ml) at 
16 h.
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Estradiol-17ß Concentrations During the Periovulatory Period
Zero-eCG (n = 6), 500-IU-eCG (n = 6) and 1000-IU- eCG (n = 5) groups exhibited similarly (P > 0.05) low concentrations of E2ß at Time 0, averaging 5.0 ± 0.5 pg/ ml (Fig. 6). E2ß levels began to rise in a similar time frame as UBF during the periovulatory period and peak E2ß levels were not different between the three groups. In the 1000-IU-eCG group, however, the time of peak E2ß levels (24 h) was considerably earlier than that observed in the 500-IU-eCG group, which reached peak E2ß levels at 40 h. It is noteworthy that the zero-eCG group maintained elevated E2ß concentrations from 24 to 40 h, i.e., there was a longer E2ß plateau in this versus the other two groups, which showed a peak followed by a more rapid fall (P < 0.05). There was no significant difference in E2ß concentrations between the three groups until 40 h after CIDR removal and eCG injection. At this time, the 1000-IU-eCG group exhibited decreasing levels of E2ß while the 500-IU- eCG group maintained peak E2ß levels at 40–48 h (P < 0.01 500 IU eCG versus 1000 IU eCG). At 48 h, the 1000- IU-eCG group had reached baseline levels (4.5 ± 0.8 pg/ ml), whereas the other two groups reached baseline levels at 56 h (4.5 ± 0.8 pg/ml; zero eCG) and 64 h (3.7 ± 1.1 pg/ml; 500 IU eCG).
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LH Levels During the Periovulatory Period
The time of the LH surge was not different between the zero-eCG and the 500-IU-eCG groups (49.6 ± 3 h and 49.3 ± 1.6 h, respectively). The time of the LH surge in the 1000-IU-eCG group was earlier than the other two groups (35.2 ± 2.0 h) (P < 0.01) (Fig. 7). This earlier LH surge in the 1000-IU-eCG group corresponds with the earlier peak and fall E2ß seen in this group.
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Correlations Between Ovarian Hormones and UBF
Table 1 shows correlation coefficients between circulating levels of E2ß, P4, and the E2ß:P4 ratios with changes in UBF during the periovulatory period. We found both hormones and the ratio to be strongly correlated (P < 0.05; E2ß positively, P4 negatively) with alterations in UBF in the zero-eCG and 500-IU-eCG groups. In the 1000-IU-eCG group, E2ß and UBF were not significant; however, both P4 and the E2ß:P4 were found to be significant (P < 0.05). All relationships were best described by a cubic polynomial relationship except E2ß in the 1000-IU-eCG group. The individual points and best-fit lines describing changes in UBF with circulating levels of E2ß, P4, and E2ß:P4 are shown in Figure 8, A, B, and C, respectively.
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UBF Responses to NOS Inhibition in Follicular Phase Sheep
When unilaterally infused in a stepwise fashion, L- NAME dramatically decreased ipsilateral UBF in a dose- dependent manner (Fig. 9). With the highest dose of L- NAME infused, ipsilateral UBF was decreased by 54.6% ± 6.2%, while the contralateral UBF was decreased by only 27.4% ± 8.5%. An ipsilateral effect was obtained by the second dose of L-NAME infused (0.005–0.036 mg L- NAME/ml ipsilateral UBF), while a significant contralateral UBF response did not occur until the fifth dose used (0.185–0.471 mg L-NAME/ml ipsilateral ml UBF). Ipsilateral became significantly different from contralateral (P < 0.05) by the third dose of L-NAME and remained different throughout the rest of the experiment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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and eCG, indeed allows for study of UBF during synchronized ovarian cycles in sheep, regardless of season. We also observed that either dose of eCG (500 or 1000 IU) produces results that are analogous but not identical with the natural cycling animals, i.e., eCG reduces the variability of time during the ovarian cycle when the rise and decline in UBF occurs without altering the decline in systemic P4 levels on CIDR removal. The 1000-IU-eCG dose also caused the LH surge to occur earlier in association with an earlier observed rise and fall in E2ß and UBF during the periovulatory period. Additionally, we present the first report directly comparing UBF responses in both ovary-intact and ovx sheep to endogenous versus exogenous E2ß. We then demonstrated that, as seen in the ovx E2ß-treated model [9, 10], L-NAME locally and dose-dependently decreased (55–60%) the elevated UBF of the follicular phase, demonstrating that elevated de novo uterine vascular synthesis of NO is partly responsible for the periovulatory rise in UBF. Only a few studies in the ovary-intact sheep [12, 14, 16, 17, 35–37], cow [13, 16, 38–40], horse [41], donkey [42], and pig [15, 16, 43] described the UBF profiles during the estrous cycle. Indeed, the ovine studies described the tremendous variability of the changes in UBF that occurred. Moreover, the ovine reports had the added difficulties associated with housing a ram in the laboratory and performing behavioral estrous detection 2–3 times per day. Using the animal model described herein alleviates the major difficulty of these very practical issues. It is possible that similar synchronization methods can also simplify the study of endogenous follicular-derived E2ß in other species [13, 15, 16, 38–40, 43].
Use of P4-releasing devices and exogenous gonadotropin allows for successful synchronization during the cycling or anestrous season and eCG after P4 is a well-used tool for increasing conception and pregnancy rates [44–49]. We chose this synchronization model because it results in approximately 60–70% out-of-season pregnancy rates (unpublished data). To our knowledge, this is the first use of this synchronization procedure for the study of the physiologic alterations in UBF during the periovulatory period in ewes.
A major objective of the current experiment was to completely eliminate ovarian-derived P4 from the corpora lutea as a variable. Luteolytic injections of PGF2 were given 6 days after CIDR insertion (i.e., 1 day before P4 withdrawal). P4 profiles did not show any significant differences between the three groups studied. Thus, eCG did not affect absolute rates of P4 decline after CIDR removal. We also tested the dose of eCG, i.e., 500 versus 1000 IU, in animals under CIDR/P4 control to determine what dose results in similar responses to that seen in sheep synchronized during the breeding season (i.e., without eCG injection). The eCG at either dose produced results which were analogous to normal ovarian cycle changes (zero-eCG group). However, the patterns of UBF were not identical with the natural cycling animals because the use of eCG had the added benefit of reducing the variability during the ovarian cycle when the rise and decline in UBF occurred. When shown as means ± SEM within the three groups (Fig. 1), 1000 IU eCG elicited an earlier reduction in UBF than that seen in the other groups. These UBF changes are supported by the earlier reduction in the levels of E2ß in this group as well as the earlier occurrence of the LH surge, which were expected based on previous reports [46–49].
In a previous study, Roman-Ponce et al. [17] evaluated UBF changes during the estrous cycle in four sheep and related this to E2ß and P4 in peripheral blood sampled twice daily during proestrus. In agreement with this report [17], all three groups in the current study demonstrated that P4 and 
UBF were negatively correlated and that the E2ß:P4 ratio and UBF were positively correlated (Table 1). In contrast, they did not find any significant correlation between E2ß concentration alone and UBF, whereas our correlation was significant in both the 500-IU- and zero-eCG groups (Table 1). Although we were unable to find significant correlations between E2ß and UBF in the 1000-IU- eCG group, we attribute this to the earlier decreases in E2ß and the occurrence of the LH surge. Correlations between UBF and circulating P4 concentrations are also consistent with early experiments in which exogenous estrogen and P4 were administered [5, 17]. Our data support previous suggestions [12–17, 36, 50] that there are indeed biologically relevant changes in UBF due to the changes in the E2ß:P4 ratio, but that neither E2ß alone nor P4 can completely define this relationship.
Regardless of whether ewes are ovx or intact, we observed the classic flow response with regard to the timing of the rise in UBF to 1 µg/kg exogenous E2ß treatment, i.e., the 30-min delay followed by the attainment of maximum UBF levels between 90 and 120 min (Fig. 3, bottom). Huckabee et al. [2] showed this response in intact nonpregnant ewes in 1970 using estradiol benzoate and estrone. Most other studies, including those from our laboratory, have treated ovx sheep with E2ß [1, 3–10, 28–31, 35]. To our knowledge, this is the first report directly comparing the differences in UBF response of intact cycling ewes versus ovx ewes to exogenous E2ß. Because the maximum rise in UBF to exogenous E2ß was greater in intact versus ovx ewes, these data suggest either that the uterus of the ovx sheep may be somewhat smaller without cyclical ovarian steroids or that the intact animals were primed with estrogen from the periovulatory rise in follicular-derived estrogen. The intact animal responses to endogenous versus exogenous E2ß also were substantially different. Intact ewes responded with a consistently higher maximum UBF in response to the maximum exogenous dose of E2ß than during the follicular phase. This is likely due to the substantially higher blood levels of estrogen achieved with exogenous 1 µg/kg E2ß treatment (6511 pg/ml at 15 min) [51] as compared with the periovulatory rise in E2ß concentrations (
10–12 pg/ml; Fig. 6). It is probable that a lower dose of exogenously administered E2ß will achieve a blood estrogen level and a final rise of UBF similar to that seen in the ovary-intact cycling group; however, the classic acute pattern of vasodilatation described over a 2-h period is only seen with exogenous E2ß administration regardless of whether the sheep are intact or ovx. Therefore, both the magnitude and temporal pattern of these responses clearly demonstrate that there truly are some differences in pharmacological responses of the uterus to E2ß as compared with naturally physiologically occurring ovarian-derived estrogen, although both animal models are useful. Furthermore, we have also demonstrated the additional vasodilatory reserve by the uterine vascular bed above the follicular phase.
To demonstrate the utility of using the proposed synchronization model, we evaluated the hypothesis that elevated UBF during the follicular phase is dependent on the local uterine NO production. L-NAME was infused into one uterine artery at the time of maximum periovulatory UBF. 500 IU of eCG was chosen instead of 1000 IU eCG because the temporal pattern in UBF and timing of the LH surge was more analogous to that of the zero-eCG control group. Previously, Van Buren et al. [9] and Rosenfeld et al. [10] used L-NAME and decreased UBF in a dose-dependent manner in response to exogenous E2ß in ovx sheep. They reported up to 60–70% inhibition when administering L- NAME after a maximum steady-state UBF level had been reached. Herein, we used a similar dose-response protocol in intact follicular phase sheep to study the UBF response with endogenous ovarian-derived E2ß. L-NAME did indeed dose dependently reduce UBF during the follicular phase by up to 67% (average maximum decrease of 54.6% ± 6.2%). Thus, 55–70% inhibition of both endogenous and exogenous E2ß UBF responses are similarly mediated by NO production. It is unclear what accounts for the other non-L-NAME-sensitive 30–45% of the rise in UBF. Previously, we reported that the follicular phase of the ovarian cycle was characterized by significant elevations in uterine artery endothelial expression of eNOS protein and mRNA [27, 30, 52]. Moreover, exogenous E2ß treatment of ovx ewes increases endothelial eNOS protein and mRNA levels [28, 30, 31, 52, 53]. Data from the current studies demonstrate that these elevated eNOS protein levels are also activated in vivo and show for the first time that the rise in UBF during the ovarian cycle is increased to a great extent (55%) due to the de novo production of NO. Moreover, the use of this synchronization model in this manner illustrates that this is an effective animal model to provide further insights into the natural ovarian E2ß-mediated changes in UBF. For example, determination of what accounts for the 30–45% of the non-L-NAME-sensitive UBF rise during the follicular phase responses to antagonists of other proposed pathways needs to be explored, e.g., estrogen receptors [35], voltage-gated K+ channels [54], or other vasodilators.
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FOOTNOTES |
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2 Correspondence: Ronald R. Magness, Department of Obstetrics and Gynecology, University of Wisconsin, Perinatal Research Laboratories, 7E Meriter Hospital, 202 South Park Street, Madison, WI 53715. FAX: 608 257 1304; rmagness{at}wisc.edu
Received: 2 June 2003.
First decision: 1 July 2003.
Accepted: 11 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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