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a Department of Physiology and Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female Sprague-Dawley rats were obtained from Charles River (Portage, MI) at 10 wk of age, after the animals had shown two consecutive 4-day estrous cycles. Examination of vaginal epithelium was carried out daily, and only those rats showing consecutive, regular 4-day cycles after arrival were used. These rats were killed after a minimum of two additional consecutive, 4-day cycles, at 12–14 wk of age. Animals were housed under a controlled lighting schedule of 12L:12D (lights-on 0500–1700 h). Animal procedures were approved by the University Committee on the Use and Care of Animals at the University of Michigan.
Hormones
Ovine prolactin (NIDDK-oPRL, lot #AFP10677C) was obtained from the National Institute of Diabetes and Digestive and Kidney Diseases of the NIH (Bethesda, MD). The prolactin was diluted in buffered saline (0.15 M NaCl, 0.03 M NaHCO3, and 0.1% BSA fraction V) to a final concentration of 1.25 mg/ml and pH of 8.2–8.6.
Experimental Design
Experiment 1 Rats were killed either on the morning (0900–1000 h) of proestrus (Proestrus group, n = 7) or on the afternoon (1500–1700 h) of estrus (n = 25). Rats killed on estrus were given either an injection of bromocryptine (BRC; Sigma Chemical Co., St. Louis, MO; 0.2 mg in 0.2 ml 70% ethyl alcohol; n = 18) or vehicle (0.2 ml ethyl alcohol; n = 7) s.c. at 1100 h on proestrus and an injection of either prolactin (PRL; 250 µg in 0.2 ml buffered saline; n = 10) or buffered saline (0.2 ml; Estrus+BRC group; n = 15) s.c. at 1600 h on proestrus. The resulting treatment groups consisted of rats receiving bromocryptine alone (n = 8), bromocryptine followed by prolactin (Estrus+BRC+PRL group; n = 10), or vehicle alone (Estrus group; n = 7). The 0.2-mg dose of bromocryptine was selected based on a publication by Matsuyama et al. [9] showing that this dose suppresses the proestrous prolactin surge of rats. Rats were killed by decapitation following methoxyflurane (Metofane; Mallinckrodt Veterinary Inc., Mundelein, IL) anesthesia, and the ovaries were immediately removed and placed on ice. Specific sets of corpora lutea were dissected from both ovaries, consisting of those from the current cycle on proestrus or those from the preceding cycle on estrus. Typical ovaries from proestrus and estrus are shown in Figure 1. The populations of corpora lutea used in this study were readily visible. These corpora lutea were large, distinct, opaque/white structures, frequently marked by pronounced vascularity in the apical region. The corpora lutea from one ovary (n = 5–10) were frozen as a group in OCT compound (Miles Laboratories, Inc., Elkhart, IN) for sectioning for immunohistochemistry and in situ detection of apoptosis. Rats that had no fluid accumulation in the uterus on proestrus and those that had no visible ovulations on estrus were excluded from the experiment.
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Experiment 2 The results of experiment 1 showed that blockade of the proestrous prolactin surge did not completely block regressive changes in the corpus luteum. To confirm that this was not due to incomplete suppression of prolactin secretion, we repeated the experiment using a higher dose of bromocryptine. This dose was equivalent to that used by Gaytán et al. [8]. Rats were killed on either the morning (0900–1000 h) of proestrus (Proestrus group; n = 12) or the afternoon (1500–1700 h) of estrus (n = 36). Rats killed on estrus had been treated on proestrus with an injection of either bromocryptine (1 mg in 0.2 ml 70% ethyl alcohol; n = 24) or vehicle (0.2 ml ethyl alcohol; n = 12) s.c. at 1100 h and with an injection of either prolactin (250 µg in 0.2 ml buffered saline; n = 12) or buffered saline (0.2 ml; n = 24) s.c. at 1600 h. The resulting treatment groups consisted of rats receiving bromocryptine alone (Estrus+BRC group; n = 12), bromocryptine followed by prolactin (Estrus+BRC+PRL group; n = 12), or vehicle alone (Estrus group; n = 12). Rats were killed, and specific sets of corpora lutea were dissected from both ovaries as described for experiment 1. For 5 rats in each group, 6 corpora lutea from each rat were incubated for 10 h in Medium 199 with one complete change of medium (at 5 h) for assays of progestin production/release. The remaining corpora lutea (n = 3–10; all rats) were frozen for sectioning for immunohistochemistry and in situ detection of apoptosis. Rats that had no fluid accumulation in the uterus on proestrus and those that had no visible ovulations on estrus were excluded from the experiment.
Experiment 3 To confirm that the higher bromocryptine treatment used in experiment 2 was effective in blocking prolactin secretion but did not interfere with ovulation, rats were killed on proestrus at the estimated time of the prolactin surge (1500–1600 h), after treatment at 1100 h with either bromocryptine (1 mg in 0.2 ml 70% ethyl alcohol; n = 7) or vehicle (0.2 ml 70% ethyl alcohol; n = 7) s.c. An additional group of rats were killed on estrus, after treatment with bromocryptine on proestrus (n = 7), and were examined for the presence of new ovulations. Rats were quickly decapitated without anesthesia to avoid any effect of stress or anesthetic on prolactin secretion. Trunk blood was taken at the time animals were killed, and the plasma was assayed for prolactin. Rats that had no fluid accumulation in the uterus on proestrus were excluded from the experiment.
Experiment 4 To eliminate the possibility of rebound secretion of prolactin occurring after clearance of the injected bromocryptine, repeated injections of bromocryptine were administered. Rats were killed on either the morning (0900–1000 h) of proestrus (Proestrus group; n = 6) or the afternoon (1400–1700 h) of estrus (n = 21). Rats killed on estrus had been treated with 4 consecutive injections of either bromocryptine (1 mg in 0.2 ml 70% ethyl alcohol; n = 14) or vehicle (0.2 ml ethyl alcohol; n = 7) every 8 h beginning at 1200 h on proestrus and also with an injection of either prolactin (250 µg in 0.2 ml buffered saline; n = 7) or saline (0.2 ml; n = 14) at 1600 h on proestrus. The resulting treatment groups consisted of rats receiving bromocryptine alone (Estrus+BRC group; n = 7), bromocryptine followed by prolactin (Estrus+BRC+PRL group; n = 7), or vehicle alone (Estrus group; n = 7). Rats were decapitated following metofane anesthesia, and specific sets of corpora lutea were dissected from both ovaries as described for experiment 1. Six corpora lutea from each rat were incubated for 10 h in Medium 199 with one complete change of medium (at 5 h) for assays of progestin production/release. The remaining corpora lutea (n = 4–11) were frozen for sectioning for immunohistochemistry and in situ detection of apoptosis. Rats that had no fluid accumulation in the uterus on proestrus and those that had no visible ovulations on estrus were excluded from the experiment.
Experiment 5 During this study, a report was published [10] describing much higher numbers of apoptotic nuclei in regressing corpora lutea than we had observed here or previously [11]. The authors of that study used sections of paraffin-preserved ovaries to examine apoptosis, whereas we used cryopreserved sections. Therefore, the following experiment was carried out to directly compare the numbers of apoptotic nuclei in frozen vs. paraffin sections of corpora lutea from the same rats. Rats were killed on either the morning (0900–1030 h) of proestrus (n = 9) or the afternoon (1400–1630 h) of estrus (n = 8). Specific sets of corpora lutea were dissected from both ovaries as described for experiment 1. The corpora lutea from each rat were divided into two groups (n = 5–9 each): one group was frozen in OCT for sectioning, while the other was fixed in 4% paraformaldehyde in Sorenson's buffer (0.1 M NaH2PO4, 0.1 M Na2HPO4, pH 7.3) for 24 h before embedding in paraffin. Rats that had no fluid accumulation in the uterus on proestrus and those that had no visible ovulations on estrus were excluded from the experiment.
Antibodies
The monoclonal antibody against rat monocytes/macrophages (clone ED1) used for immunohistochemical staining was obtained from Chemicon International Inc. (Temecula, CA). The specificity of this antibody for rat monocytes/macrophages has been reported previously [12]. The monoclonal antibody against rat differentiated macrophages (clone ED2) was obtained from Serotec via Accurate Chemical & Scientific Corp. (Westbury, NY) and has been shown to specifically recognize differentiated, resident tissue macrophages [13]. The monoclonal antibody to major histocompatibility molecules class II (clone MRC OX-6) was obtained from Cedarlane Laboratories Ltd. via Accurate Chemical & Scientific Corp. A characterization of this antibody has been published [14]. Biotinylated secondary antibody to mouse immunoglobulin was obtained from Vector Laboratories (Burlingame, CA).
Immunohistochemistry
Frozen sections of corpora lutea were air dried and fixed in 95% ethanol (10 min); they were then placed in 0.3% H2O2 in methanol (4°C) for 15 min to quench endogenous peroxidase activity. The tissue sections were next rinsed 3 times (5 min each) in PBS containing 1% BSA (fraction V; PBS-1% BSA) and then incubated with 10% normal goat serum for 15 min. After this blocking procedure, the sections were rinsed again in PBS-1% BSA and then incubated with monoclonal antibody against either rat monocytes/macrophages (ED1; 1:200 dilution), differentiated macrophages (ED2; 1:200 dilution), or major histocompatibility complex (MHC) II molecules (OX-6; 1:500 dilution; 30 min at 37°C). After the incubation with primary antibody, the sections were rinsed in PBS-0.1% BSA and then exposed to biotinylated goat anti-mouse immunoglobulin (1:200 dilution; 30 min at 37°C). Detection of the antigen-antibody complex was achieved by using a Vector avidin-biotin-peroxidase kit and 3-amino-9 ethylcarbazole as the substrate. The tissue sections were counterstained with hematoxylin, rinsed in distilled water, and dipped in Scott's tap water (a mordant) before being mounted with aqueous mounting medium. Nonspecific staining was assessed by replacing primary or secondary antibodies with the appropriate serum, and was undetectable in all instances.
In Situ Detection of Apoptosis
Nuclei exhibiting DNA fragmentation were detected by in situ hybridization using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Frozen sections (7 µm) were air dried and fixed in 10% neutral buffered formalin for 10 min. They were then rinsed in two changes of PBS for 5 min each before being postfixed in ethanol:acetic acid (2:1) for 5 min at -20°C. After postfixing, sections were rinsed again in two changes of PBS and then quenched in 3% H2O2 in PBS for 5 min at room temperature. Paraffin sections (5 µm) were deparaffinized in 3 changes of xylene (5 min each) and then washed in 100% ethanol (2 times, 5 min each), 95% ethanol (3 min), and 70% ethanol (3 min). The sections were then rinsed in PBS and placed in proteinase K solution (20 µg/ml in PBS; Boehringer-Mannheim, Indianapolis, IN) for 5 min at room temperature. After enzymatic digestion, the sections were rinsed twice in distilled water (2 min each) and quenched in 3% H2O2 in PBS for 5 min at room temperature. From this point, treatment of the two types of sections was essentially identical. Sections were rinsed in PBS, and equilibration buffer from the kit was applied to cover each section; the buffer was blotted and replaced with a solution of terminal deoxynucleotide transferase (TdT). The sections were incubated with this enzyme for 1 h at 37°C to allow for tagging of 3' DNA ends with digoxigenin residues. The sections were next placed in stop buffer for 10 min; they were then rinsed with PBS and incubated with anti-digoxigenin antibody conjugated with peroxidase for 30 min at room temperature. After rinsing in PBS, sections were incubated with diaminobenzidine substrate for 6 min, washed in 3 changes of distilled water for 1 min each, and then washed in distilled water for 5 min. The sections were counterstained with methyl green (10 min for frozen sections, 1 min for paraffin sections), followed by 3 washes in distilled water, 3 washes in 100% butanol, and 3 washes in xylene before mounting. Negative controls consisted of replacement of the TdT enzyme solution with PBS; this replacement completely eliminated staining. Positive control slides consisted of ovarian sections containing large numbers of atretic follicles.
Quantitation of Macrophages and Apoptotic Nuclei
Numbers of macrophages per high-power field were determined for coded slides by visual observation of immunodetectable cells. A light microscope with a x45 objective was used. As many fields as possible (1–3) were counted for each corpus luteum, and two sections of the frozen group of corpora lutea (processed in different staining runs) were examined for each rat. An average number of macrophages per high-power field (x450; 0.06 mm2) was obtained for each rat, and this number was used as n = 1 for statistical analysis.
For quantitation of apoptotic nuclei, two sections from each group of corpora lutea were examined as described above, using coded slides, and an average number of apoptotic nuclei per high-power field was obtained. Again, as many fields as possible (1–3) were counted for each corpus luteum in these sections. For experiment 5, only one section from each group of corpora lutea was examined.
In Vitro Steroid Production
Six corpora lutea from each rat were dissected out at the time animals were killed, and three intact corpora lutea were placed in each of two 16 x 125-mm stoppered glass culture tubes containing 1.0 ml of filter-sterilized Medium 199 (with Earle's salts and L-glutamine; Gibco, Grand Island, NY) containing 0.1% BSA and 50 mg/L gentamicin sulfate (Sigma). The tubes were filled with an atmosphere of 95% O2 and 5% CO2, stoppered, and then placed in a shaking water bath at 37°C for incubation. After 5 h of incubation, the conditioned medium was removed and frozen; new medium, 1.0 ml/tube, was added to each tube, and the incubation was continued for an additional 5 h. At the end of the second incubation period, the medium was removed and frozen, and the incubated corpora lutea were blotted on filter paper and weighed. This incubation procedure has been previously published for rabbit corpora lutea [15]. Two consecutive incubation periods are carried out to determine ongoing progestin production by corpora lutea during in vitro incubation; progestin released into the medium during the first period of incubation may consist in part of progestin synthesized prior to the onset of incubation.
Conditioned, unextracted medium from both incubation periods was assayed for progesterone and 20
-dihydroprogesterone using assays previously described [16]. Medium that had not been incubated with corpora lutea was assayed as a negative control for both steroids. Samples for each experiment were assayed in one assay for progesterone and in one assay for 20-dihydroprogesterone. The sensitivity of the progesterone assays was 0.13 ng/ml, and the sensitivity of the 20-dihydroprogesterone assay was 0.31 ng/ml. Intraassay coefficients of variation were 1.6% and 2.6% for the progesterone assays and 4.6% and 4.0% for the 20-dihydroprogesterone assays, for samples from experiment 2 and experiment 4, respectively.
RIA for Prolactin
Serum prolactin concentration was determined in duplicate measurement by RIA with NIDDK-rat (r)PRL-RP-3 as the standard, NIDDK-anti-rPRL-S-9 as the primary antibody, and NIDDK-rPRL-I-6 as the iodinated trace. All samples were run in one assay, having a sensitivity of 0.02 ng/ml and an intraassay coefficient of variation of 2.9%. The assay was performed in the laboratory of Dr. N. Schwartz at Northwestern University and was supported by a grant at that institution (P01-HD21921).
Statistics
For experiments 1, 2, and 4, the distribution of the data was examined by box plot to identify outliers and to determine whether transformation was required. Square root transformation was performed on all variables with the exception of luteal weight, which was not transformed, and progestin data, which were log transformed. A two-way ANOVA was performed with group and experiment as the two factors for all variables except in vitro progestin production. Since there was no significant interaction between group and experiment for any variable, the ANOVA was repeated without the interaction. Tukey's multiple range test was performed to identify differences among experiments and groups. One outlying value was deleted from the luteal weight data set, as it had an inordinate influence on the analysis; however, its exclusion did not affect the conclusions.
Data on in vitro progestin production were analyzed using a two-way ANOVA with time point and group as factors. There was no significant interaction. Therefore the analysis was repeated without the interaction. Tukey's multiple range test was performed to determine differences between time points and groups.
Plasma prolactin concentrations for experiment 3 were compared using Student's t-test. Numbers of apoptotic nuclei per high-power field in paraffin and frozen sections (experiment 5) were compared using a paired t-test. Results for all experiments are described as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inhibition of the Proestrous Prolactin Surge by Bromocryptine (Experiment 3)
Plasma prolactin concentration in rats killed on proestrus afternoon averaged 122.6 ± 12.0 ng/ml, while in rats killed at the same time point but after bromocryptine treatment, the plasma prolactin concentration averaged 17.9 ± 6.3 ng/ml (P < 0.001). This indicates that 1 mg of bromocryptine was effective in blocking the proestrous prolactin surge. Of the 7 animals treated with bromocryptine on proestrus and killed on estrus, 5 had ovulated, as determined by the presence of ovulation points on the ovarian surface. The remaining 2 did not ovulate, suggesting that the bromocryptine dose was sufficiently high to interfere with normal LH secretion in some individuals, as has been previously described [17]. Rats so affected were readily identifiable, however, by the lack of visible ovulation points. As expected, prolactin was low on estrus and was not different between animals that had undergone ovulation (5.9 ± 1.8 ng/ml) and those that had not (4.4 ± 1.0; P > 0.05).
Monocytes/Macrophages and Differentiated Macrophages (Experiments 1, 2, 4)
Numbers of luteal monocytes/macrophages (ED1+ cells) and differentiated macrophages (ED2+ cells) per high-power field are shown in Table 1. One value is presented for each group, as there was no interaction between group and experiment for either analysis. For monocytes/macrophages, there was a significant effect of experiment (P < 0.001), with the values from experiment 1 being lower than those for the other two experiments. There was no significant effect of experiment for differentiated macrophages (P > 0.05). For both monocytes/macrophages and differentiated macrophages, the number per high-power field was not different for rats killed on estrus (Estrus) and those killed on estrus after treatment with bromocryptine and prolactin (Estrus+BRC+PRL; P > 0.05). However, corpora lutea in both of these groups contained greater numbers of immunostained cells than did corpora lutea of rats killed on estrus after treatment with bromocryptine alone (Estrus+BRC; P < 0.05). All rats killed on estrus, regardless of treatment, had significantly higher numbers of luteal monocytes/macrophages and differentiated macrophages than did rats killed on proestrus (P < 0.05).
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Apoptotic Nuclei (Experiments 1, 2, 4)
Numbers of luteal apoptotic nuclei per high-power field, as distinguished by the ApopTag in situ hybridization kit, are shown in Table 1. One value is presented for each group, as there was no interaction between group and experiment. In addition, there was no significant effect of experiment for this analysis (P > 0.05). There was no difference in number of apoptotic nuclei per high-power field between the Estrus and Estrus+BRC+PRL groups (P > 0.05). The number of apoptotic nuclei was significantly decreased in Estrus+BRC-treated rats (P < 0.05) compared to the Estrus and Estrus+BRC+PRL groups; however, the number of apoptotic nuclei was higher for all groups killed on estrus than for those killed on proestrus (P > 0.05).
Apoptotic Nuclei: Comparison of Frozen and Paraffin Sections (Experiment 5)
Corpora lutea from each rat were divided into two groups, one of which was frozen for sectioning and the other of which was fixed in 4% paraformaldehyde and embedded in paraffin. The number of apoptotic nuclei per high-power field on proestrus was 1.4 ± 0.3 for frozen sections and 1.8 ± 0.5 for paraffin sections (n = 9). The number of apoptotic nuclei per high-power field on estrus was 5.5 ± 1.2 for frozen sections and 8.0 ± 0.7 for paraffin sections (n = 8). The method of tissue preservation had no effect on the number of apoptotic nuclei detected (P > 0.05). Background staining, consisting of a pale brown tint to all nuclei, was observed around the edges of corpora lutea in paraffin sections, and in some cases throughout the section. Higher concentrations of proteinase K, and longer periods of incubation in this enzyme, increased this background staining greatly and also caused damage to the tissue sections. No background staining was observed in cryopreserved sections.
MHC II Molecules (Experiments 1, 2, 4)
Numbers of MHC II-positive cells (OX6+ cells) per high-power field are shown in Table 1. One value is presented for each group, as there was no interaction between group and experiment. There was no significant effect of experiment (P > 0.05) or group (P > 0.05) for this analysis.
Luteal Weight (Experiments 1, 2, 4)
Weight per corpus luteum (mg) for experiments 1, 2, and 4 is shown in Table 1. One value is presented for each group, as there was no interaction between group and experiment. There was a significant effect of experiment (P < 0.001), however, with values from experiment 1 being lower than those for the other two experiments. One outlying value was deleted from the Estrus+BRC group for experiment 4, as it had an extreme effect on the analysis. Weight per corpus luteum was not different between Proestrus and Estrus+BRC groups (P > 0.05) or between Estrus and Estrus+BRC+PRL groups (P > 0.05). Luteal weight was significantly greater in the first two groups, Proestrus and Estrus+BRC, than in the latter two (P < 0.05).
In Vitro Steroid Production
Experiment 2
Production of 20-dihydroprogesterone and progesterone by incubated corpora lutea is expressed in ng/mg luteal tissue. The two values were combined into total progestin produced per milligram luteal tissue. After 5 h of incubation, progestin production was 163.4 ± 30.0, 467.0 ± 84.5, 419.6 ± 77.4, and 515.6 ± 100.8 ng/mg tissue, respectively, for corpora lutea obtained from Proestrus (n = 5), Estrus (n = 5), Estrus+BRC (n = 5), and Estrus+BRC+PRL (n = 5) groups. After the second 5 h of incubation, total progestin production was 127.8 ± 24.4, 437.3 ± 31.2, 355.5 ± 58.5, and 451.2 ± 70.2 ng/mg tissue, respectively, for those groups. There was no significant difference between the two time points for each group (P > 0.05). Progestin production/mg luteal tissue was significantly greater for all groups killed on estrus than for the Proestrus group (P < 0.05). There were no differences among groups killed on estrus (P > 0.05). Values are shown graphically in Figure 2.
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Experiment 4
As for experiment 2, production of 20-dihydroprogesterone and progesterone by incubated corpora lutea was combined and expressed as nanograms of total progestin per milligram of luteal tissue. After 5 h of incubation, progestin production was 74.6 ± 7.0, 209.8 ± 18.3, 151.7 ± 20.6, and 241.0 ± 29.5 ng/mg tissue, respectively, for corpora lutea obtained from Proestrus (n = 6), Estrus (n = 7), Estrus+BRC (n = 7), and Estrus+BRC+PRL (n = 7) groups. After the second 5 h of incubation, production of progestin was 86.4 ± 8.7, 287.9 ± 39.8, 225.0 ± 40.5, and 332.8 ± 44.8 ng/mg tissue, respectively, for those groups. There was a significant effect of time point (P < 0.01), with the amount of progestin produced being greater for the second 5 h than for the first 5 h (P < 0.05). At both time points, the amount of progestin produced per milligram of luteal tissue was significantly greater for all rats killed on the afternoon of estrus, regardless of treatment, than for rats killed on proestrus (P < 0.05). In addition, progestin values for Estrus+BRC+PRL were higher than those for Estrus+BRC (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prolactin does seem to be responsible for the drop in luteal wet weight detected during this time period. This observation is consistent with earlier reports that chemical blockade of the proestrous prolactin surge results in accumulation of corpora lutea in the rat ovary [6, 7]. Multiple mechanisms may underlie the loss of weight by corpora lutea first exposed to the proestrous prolactin surge. The proestrous prolactin surge appears to promote increased incidence of apoptosis in the corpora lutea [9, 10, 11]. However, in the current study we observed that much of the increase in apoptotic nuclei between proestrus and estrus appears to be independent of the prolactin surge at proestrus, suggesting that prolactin may act, at least in part, by other pathways to reduce luteal mass. One possibility is a direct action of prolactin on macrophages, to increase their phagocytic activity [18, 19]. Another possibility is that some of the change in wet weight observed over this time period is due to loss of tissue water. Prolactin is known to affect Na+ transport in a variety of ways [20], including via the Na+-K+ ATPase, which has a direct role in regulating cell volume. Prolactin stimulates Na+ transport by the Na+-K+ ATPase in the mammary gland [21], for example, but inhibits this transport in erythrocytes [22]. A distinct possibility, therefore, is that prolactin has a role in regulating cell volume in the corpus luteum.
Despite the increase in markers of luteal regression from proestrus to estrus, the steroidogenic capacity of the luteal tissue was not impaired. In fact, the amount of steroid produced per milligram of luteal tissue was greater for corpora lutea obtained on estrus than for those obtained on proestrus. This observed increase in steroid production on estrus may be the result of stimulation of luteal cells by the LH surge of the preceding day. Overall, therefore, it appears that regressing corpora lutea on estrus have lost neither the ability to produce steroids nor the ability to respond to stimulatory signals.
Detection of MHC II molecules in this study was intended to quantify the presence of antigen-presenting cells, thus serving as a measurement of immune system activity in the corpus luteum. The number of cells that were positively immunostained for MHC II molecules did not change between proestrus and estrus, despite an increase in monocyte/macrophage and macrophage numbers. This suggests that the role of these cells as antigen-presenting cells may be less critical than their role as phagocytes during regression of the corpus luteum. A further consideration is that the average number of MHC II-positive cells present on proestrus is greater than the average number of monocytes/macrophages in the tissue at that time. This suggests another possibility, that luteal cells themselves are expressing MHC II molecules. The presence of MHC II molecules on luteal cells has been previously reported for bovine luteal cells, with the percentage of luteal cells expressing MHC II molecules increasing near the end of the cycle and after administration of prostaglandin F2 to cause luteolysis [23]. Further, bovine luteal cells expressing MHC II were able to function as antigen-presenting cells, and to cause proliferation of T cells [24]. It is possible that the corpus luteum of the cycle gains antigenicity as it ages and that an increase in MHC II molecules occurs earlier than proestrus. Alternatively, an increase in MHC II molecules, whether on luteal steroidogenic cells or luteal macrophages, may occur later in the process of regression. It has been previously reported that MHC II-positive cells are more predominant in rat corpora lutea that have regressed through a cycle than in those first entering regression [25].
As previously mentioned, our results concur with the majority of previous reports. In concordance with the observations of Wüttke and Meites and Billeter and Flückiger [6, 7], we observe a prolactin-mediated decline in luteal weight. In addition, we are able to confirm the results of Matsuyama et al. [9], who reported that the proestrous prolactin surge was responsible for a significant increase in apoptosis. Only by directly comparing markers of regression in corpora lutea on proestrus and those on estrus were we are able to determine that the proestrous prolactin surge is not solely responsible for induction of regressive changes between proestrus and estrus. The absence of the proestrus-estrus comparison of the most recent corpora lutea in prior studies could explain why prolactin has been previously perceived as the signal for the onset of luteolysis during the cycle. Our results are at variance with those of Gaytán et al. [10], who report that infiltration of monocytes/macrophages and induction of apoptosis in corpora lutea can be completely prevented by blockade of the proestrous prolactin surge. This discrepancy cannot be readily explained. One possible source of the discrepancy is that we dissected out and examined a specific set of corpora lutea on proestrus and estrus, while the work of Gaytán et al. [10] was performed on whole ovarian sections containing several populations of corpora lutea. Preferential luteolysis of older generations of corpora lutea by prolactin has been reported for the hypophysectomized rat [3], and increased numbers of MHC II-positive cells are also present in older corpora lutea [25], suggesting that these populations may respond differently to luteolytic signals than those first entering regression. Gaytán et al. [10] also reported that the presence of progesterone on proestrus was required for the increase in apoptosis induced by prolactin, which supports the concept that other factors are involved in regression of corpora lutea of the cycle, whether permissively or as active signals for the onset of luteolysis. Additional support for this idea comes from the work of Guo et al. [26], who report an increase in apoptosis-associated gene expression in corpora lutea on the latter days of the cycle, coinciding with the decrease in progesterone production by the corpora lutea, but prior to the proestrous prolactin surge.
The importance of factors other than prolactin in luteal regression is in apparent conflict with observations in the hypophysectomized rat treated with prolactin. In the hypophysectomized rat, prolactin clearly induces a full panel of regressive changes in the corpora lutea, including expression of monocyte chemoattractant protein-1 and infiltration of monocytes/macrophages [5], decreases in ovarian [2, 3] and luteal [27, 28] weight, increases in apoptotic nuclei (unpublished results), and decreased 3ß-hydroxysteroid dehydrogenase mRNA, protein, and activity [29]. Further, none of these regressive events occurs in the continued absence of prolactin. A critical difference between observations of prolactin action in the estrous cycle and in the hypophysectomized rat is that in the latter, multiple injections of prolactin were always used, either every 12 h for several days [4, 5], or daily for multiple days [2, 3, 29]. This type of prolactin replacement is closer to the pattern of secretion observed after mating, during pregnancy or pseudopregnancy, than to that of the cycle [30]. To our knowledge, replacement of prolactin in a manner mimicking the proestrous prolactin surge of the estrous cycle, or successive cycles (i.e., a single prolactin surge every 4–5 days), has not been carried out in hypophysectomized rats. We are currently investigating whether this type of replacement will result in luteal regression in the hypophysectomized rat or will reveal the necessity for other luteolytic factors.
We conclude from our results that prolactin alone is not responsible for the initiation of many of the elements of regression in the cycling rat. Although our results are unexpected, in light of current concepts it is not surprising that a process as critical as luteal regression is not dependent on one signal alone. Scientific experience, particularly the results from studies of transgenic mice, has taught us that redundancy is the hallmark of biological systems.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J.M. Bowen, Department of Physiology, University of Michigan, 7627 Medical Sciences II Building, Ann Arbor, MI 48109-0622. FAX: 734 936 8813; bowenjm{at}umich.edu
Accepted: June 4, 1999.
Received: March 10, 1999.
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REFERENCES |
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-ergokryptine (CB 154). Experientia 1971; 27:464–465.[CrossRef][Medline]
A, Presl J, 
idovsk J, Man
al P. The localization of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and fallopian tube. Immunology 1983; 48:587–596.[Medline]
5-4 isomerase expression and activity in the hypophysectomized rat ovary: interactions between the stimulatory effect of human chorionic gonadotropin and the luteolytic effect of prolactin. Endocrinology 1990; 127:2726–2737.[Abstract]This article has been cited by other articles:
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F. Gaytan, C. Bellido, C. Morales, and J.E. Sanchez-Criado Luteolytic Effect of Prolactin Is Dependent on the Degree of Differentiation of Luteal Cells in the Rat Biol Reprod, August 1, 2001; 65(2): 433 - 441. [Abstract] [Full Text] [PDF]
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J. M. Bowen and P. L. Keyes Repeated Exposure to Prolactin Is Required to Induce Luteal Regression in the Hypophysectomized Rat Biol Reprod, April 1, 2000; 63(4): 1179 - 1184. [Abstract] [Full Text]
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