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Biology of Reproduction 63, 1179-1184 (2000)
© 2000 Society for the Study of Reproduction, Inc.

ARTICLES

Repeated Exposure to Prolactin Is Required to Induce Luteal Regression in the Hypophysectomized Rat1

Jennifer M. Bowen2,a, and P. Landis Keyesa

a Department of Physiology and Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109-0622


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated whether prolactin (PRL) treatments resembling the intermittent PRL surges of estrous cycles could induce luteal regression in hypophysectomized rats. Immature female rats were stimulated to ovulate and form corpora lutea with exogenous gonadotropins, and were hypophysectomized following ovulation. A single s.c. injection of either vehicle (VEH) or PRL was administered to each rat on post-hypophysectomy Day 8 and again on Day 11. The four resulting treatment groups consisted of rats that received two injections of VEH, VEH followed by PRL, PRL followed by VEH, or two injections of PRL. Rats were killed 24 or 72 h following the second injection. Plasma 20{alpha}-dihydroprogesterone, luteal weight, and total luteal protein were determined. One ovary was sectioned for immunohistochemistry for monocytes/macrophages, apoptotic nuclei, and major histocompatibility class II (MHC II) molecules. No effect of time (following injection) was observed on any endpoint, indicating that PRL does not have an ongoing regressive action. Time groups from within each treatment group were therefore pooled for analysis. Significant declines (P < 0.05) in plasma concentrations of 20{alpha}-dihydroprogesterone, luteal weight, and protein per corpus luteum occurred only after two injections of PRL. Numbers of luteal monocytes/macrophages, apoptotic nuclei, and MHC II-positive cells were low in all groups; numbers of luteal monocytes/macrophages increased following two injections of PRL (P < 0.05). We conclude that PRL has a cumulative regressive effect on the corpus luteum of the hypophysectomized rat. Drawing a parallel with the estrous cycle, we suggest that continued exposure to PRL, over several cycles, is necessary to induce full luteal regression.

corpus luteum, corpus luteum function, prolactin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the estrous cycle of the rat, secretion of prolactin (PRL) is largely restricted to a single surge on proestrus, prior to ovulation [1]. This predictable elevation in plasma PRL concentrations is presumed to initiate regression of corpora lutea of the preceding cycle (i.e., those that were formed at the previous ovulation). Evidence for this proposed luteolytic role of PRL has been obtained from studies using a chemical blockade of PRL secretion, which, over several cycles, results in accumulation of corpora lutea on the ovaries and an increase in ovarian weight [2, 3]. Furthermore, chemical blockade of the proestrous PRL surge diminishes apoptosis and accumulation of immune cells in regressing corpora lutea [4–6].

Additional evidence for a role for PRL in initiation of luteal regression has come from studies that have been carried out in hypophysectomized rats. After hypophysectomy, corpora lutea formed prior to surgery are maintained for more than a month in the absence of pituitary hormones; these corpora lutea remain structurally intact and continue to produce steroids [7–9], predominantly 20{alpha}-dihydroprogesterone [9, 10], a metabolite of progesterone. Rapid regression of corpora lutea of hypophysectomized rats can be induced by injections of PRL, provided at least 3 days have passed since hypophysectomy [7–10]. To date, PRL treatments administered to hypophysectomized rats to induce luteolysis have consisted of multiple injections administered either every 12 h [9, 10] or every day [7, 8, 11] for periods of 3–13 days. This pattern of PRL replacement more closely resembles the pattern of PRL secretion induced by mating and observed during pregnancy or pseudopregnancy than it resembles that of the estrous cycle [1]. We have administered PRL in a manner that approximates PRL secretion during the estrous cycle to determine whether this pattern of PRL exposure is an effective stimulus to induce regression of the corpora lutea of hypophysectomized rats. We have used immature rats that were stimulated to undergo their first ovulation prior to hypophysectomy. This provides a single population of corpora lutea of one age that can be studied after one or two injections of PRL, thereby mimicking the proestrous PRL surges of consecutive cycles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Immature female Sprague-Dawley rats were obtained from the Portage, MI facility of Charles River (Wilmington, MA). Rats received a s.c. injection of 5 IU eCG at 29 days of age to stimulate follicular development, followed by an s.c. injection of 5 IU hCG 56 h later to induce ovulation of follicles and formation of corpora lutea. The rats were hypophysectomized following ovulation (Day 32) by the vendor. All rats were provided with rat chow, 5% glucose in water ad libitum, and sliced oranges. Animal procedures were approved by the University Committee on the Use and Care of Animals at the University of Michigan.

Hormones

Ovine PRL (lot AFP10677C) was obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). The PRL was diluted in 0.15 M NaCl, 0.03 M NaHCO3, and 0.1% BSA to a final concentration of 1.25 mg/ml and pH of 8.2–8.6. Diluted PRL was stored at 4°C and used within 1 wk.

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 has been reported previously [12]. The monoclonal antibody to MHC II molecules (clone MRC OX-6) was obtained from Cedarlane Laboratories, Ltd. via Accurate Chemical & Scientific Corp. (Westbury, NY). A characterization of this antibody has been published [13]. Biotinylated secondary antibodies to mouse and rabbit immunoglobulin were obtained from Vector Laboratories (Burlingame, CA).

Immunohistochemistry

Ovaries were frozen in ornithine carbamyl transferase compound (OCT; Miles Laboratories, Inc., Elkhart, IN) for sectioning, for immunohistochemistry and in situ detection of apoptosis. Frozen sections of corpora lutea (7 µm) were air-dried and fixed in 95% ethanol (10 min), and then placed in 0.3% H2O2 in methanol (4°C) for 15 min to quench endogenous peroxidase activity. The tissue sections were then rinsed three times (5 min each) in PBS containing 1% BSA (PBS-1% BSA; Fraction V) 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) or MHC II molecules (OX-6 antibody; 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 Scotts tap water (0.04 M MgSO4·7H2O, 0.025 M NaHCO3 in 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 using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Frozen sections 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. Following postfixing, sections were rinsed again in two changes of PBS and then quenched in 3% H2O2 in PBS for 5 min at room temperature. Sections were rinsed in PBS, equilibration buffer from the kit was applied to cover each section, and 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, then rinsed with PBS and incubated with antidigoxigenin antibody conjugated with peroxidase for 30 min at room temperature. After rinsing in PBS, sections were incubated for 6 min with diaminobenzidine substrate, washed in three changes of distilled water for 1 min each, and then in distilled water for 5 min. The sections were counterstained with methyl green for 10 min, followed by three washes in distilled water, three washes in 100% butanol, and three washes in xylene before mounting. Negative controls consisted of replacement of the TdT enzyme solution with PBS; this replacement completely eliminated staining. Atretic follicles in the sections served as an internal positive control.

Radioimmunoassays

Plasma concentration of 20{alpha}-dihydroprogesterone was determined by radioimmunoassay. After decapitation of the rats, trunk blood was collected into heparinized tubes and then centrifuged at 1740 x g for 20 min. Plasma was stored at -20°C until extracted and assayed for 20{alpha}-dihydroprogesterone according to the method previously described by Bender et al. [14].

Quantitation of Monocytes/Macrophages, MHC II-Positive Cells, and Apoptotic Nuclei

Numbers of macrophages and MHC II-positive cells per high power field were determined for coded slides by visual observation of immunodetectable cells. An immunodetectable cell was defined as a nucleus surrounded by immunostained material. Immunostained material that did not contain a nucleus was not counted as a cell. A light microscope with a 45x objective was used. Cells were counted only within corpora lutea and within as many nonoverlapping high power fields as possible for each corpus luteum. Two ovarian sections were examined for each rat and an average number of monocytes/macrophages or MHC II-positive cells per high power field (450x; 0.06 mm2) was obtained for each rat; this number was used as n = 1 for statistical analysis. For quantitation of apoptotic nuclei, one ovarian section was examined as just described, using coded slides. Again, as many fields as possible were counted for each corpus luteum and the average number of apoptotic nuclei per high power field for each animal was used as n = 1 for statistical analysis.

Assay of Total Luteal Protein

Corpora lutea were homogenized in 300 µl modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA) containing detergents (1% NP-40 and 0.015% Na deoxycholate) using a tissue homogenizer. The homogenate was diluted (1:3) for assay of total protein using the Bio-Rad Protein Microassay (Bio-Rad Laboratories, Hercules, CA) with BSA as the standard. All standards and samples were assayed in triplicate.

Experimental Design

The experimental design is depicted in Figure 1. Hypophysectomized rats (n = 44) were divided into seven groups. Rats received s.c. injections of VEH (0.15 M NaCl, 0.03 M NaHCO3, 0.1% BSA; 0.2 ml) or ovine PRL (250 µg in 0.2 ml VEH) at 0800 h on Days 40 and 43 of age (Days 8 and 11 post-hypophysectomy), and were killed by decapitation between 0800 and 1100 h on Days 44 or 46 of age (24 or 72 h following the second injection). Groups consisted of rats that received two VEH injections (VEH/VEH; n = 6 killed at 24 h and n = 6 killed at 72 h), rats that received one injection of VEH followed by one of PRL (VEH/PRL; n = 6 killed at 24 h and n = 7 killed at 72 h), and rats that received two injections of PRL (PRL/PRL; n = 6 killed at 24 h and n = 7 killed at 72 h). Additional rats were given one injection of PRL followed by one of VEH (PRL/VEH; n = 6) and were killed at 72 h following the second injection (144 h following prolactin). Trunk blood was collected from all rats, and plasma was obtained for later radioimmunoassay of 20{alpha}-dihydroprogesterone. The ovaries from each rat were immediately removed and placed on ice. One ovary was frozen in OCT compound for sectioning. Corpora lutea from the other ovary were dissected free, weighed as a group, and frozen in liquid nitrogen. These corpora lutea were stored at -80°C for later determination of total protein content. The sella turcica was visually inspected in all rats for the presence of pituitary fragments; rats with incomplete hypophysectomies were excluded from experiments and are not reflected in the numbers given in this paragraph.



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  		FIG. 1. Experimental design. Immature rats were stimulated to ovulate and form corpora lutea by treatment with 5 IU eCG on Day 29 of age and with 5 IU hCG 56 h later, then hypophysectomized on Day 32 of age. Injections of PRL or VEH were carried out on Days 40 and 43 of age (Days 8 and 11 post-hypophysectomy) and rats were then killed on Days 44 or 46 (24 or 72 h following the second injection)

Statistical Analysis

Data were transformed to normalize variance, prior to statistical analysis, when this was necessary. Plasma 20{alpha}-dihydroprogesterone concentrations and the number of apoptotic nuclei were natural log-transformed, numbers of monocytes/macrophages and MHC II-positive cells were square root-transformed, and luteal weight and protein were analyzed without transformation.

Two-way ANOVA was conducted on the data for rats in the VEH/VEH, VEH/PRL, and PRL/PRL groups that were killed at 24 and 72 h following the second injection (6 of the 7 groups). In the absence of interaction between time of tissue collection and treatment, one-way ANOVA was carried out for time and for treatment, followed by Tukeys multiple comparisons test, when applicable. When there was interaction between treatment and time, one-way ANOVA was carried out on all seven groups, with each group representing a different time and treatment combination. This was followed by Tukey's multiple comparisons test.

To test for an effect of time of tissue collection for rats that received one injection of PRL, an additional one-way ANOVA followed by Tukeys multiple comparisons test when applicable was carried out on data from rats that had been killed 24, 72, or 144 h after a single PRL injection (VEH/PRL and PRL/VEH groups).

P values of less than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 1 contains mean (± SEM) results for the individual groups of rats, for each of the measurements.


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  		TABLE 1. Individual group means (mean ± SEM) for all measurements

Plasma 20{alpha}-Dihydroprogesterone

There was no significant interaction between treatment and time of tissue collection (P = 0.821) for plasma 20{alpha}-dihydroprogesterone concentrations of rats killed at 24 and 72 h; however, a significant effect of treatment was observed (P = 0.002). Plasma concentrations of 20{alpha}-dihydroprogesterone were 16.3 ± 1.8 ng/ml for rats that received two VEH injections (VEH/VEH; n = 12), 13.3 ± 1.4 ng/ml for rats that received one PRL injection (VEH/PRL; n = 13), and 8.8 ± 0.7 ng/ml for rats that received two PRL injections (PRL/PRL; n = 13; Fig. 2). There was a significant decrease in plasma 20{alpha}-dihydroprogesterone following two PRL injections compared with groups that received one PRL injection or VEH alone (P = 0.031 vs. one injection; P = 0.001 vs. VEH alone). There was no significant effect of time (P = 0.336) for rats killed at 24 and 72 h, or for rats killed at 24, 72, and 144 h after one PRL injection (P > 0.9).



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  		FIG. 2. Mean ± SEM plasma 20{alpha}-dihydroprogesterone concentrations (ng/ml) for hypophysectomized rats receiving two VEH injections (VEH/VEH; n = 12), one PRL injection (VEH/PRL; n = 13), or two PRL injections (PRL/PRL; n = 13). Results are for combined 24-h and 72-h groups. Different letters indicate significant differences at P < 0.05

Luteal Weight

There was no significant interaction between treatment and time of tissue collection (P = 0.431) for luteal weights (mg/corpus luteum) of rats killed at 24 and 72 h; however, there was a significant effect of treatment (P = 0.00005). Average weight per corpus luteum was 0.8 ± 0.03 mg for rats that received two VEH injections (VEH/VEH; n = 12), 0.73 ± 0.04 mg for rats that received one PRL injection (VEH/PRL; n = 13), and 0.58 ± 0.02 mg for rats that received two PRL injections (PRL/PRL; n = 13; Figure 3). There was a significant decrease in weight per corpus luteum following two PRL injections compared with groups that received one PRL injection or VEH alone (P < 0.008 vs. one injection; P = 0.00005 vs. vehicle alone). There was no significant effect of time (P = 0.689) on luteal weights for rats killed at 24 and 72 h, or for rats killed at 24, 72, and 144 h after one PRL injection (P = 0.165).



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  		FIG. 3. Mean ± SEM luteal weight (mg) for hypophysectomized rats receiving two VEH injections (VEH/VEH; n = 12), one PRL injection (VEH/PRL; n = 13), or two PRL injections (PRL/PRL; n = 13). Results are for combined 24-h and 72-h groups. Different letters indicate significant differences at P < 0.01

Luteal Protein

Total luteal protein was analyzed both as µg protein/mg luteal tissue and as µg protein/corpus luteum. When considered as µg protein/mg luteal tissue, there was no effect of either treatment (P = 0.621) or time (P = 0.056) for rats killed at 24 and 72 h, and no interaction between these terms (P = 0.481). When protein was considered as µg protein/corpus luteum, there was also no effect of time (P = 0.309) and no interaction between time and treatment (P = 0.292) for rats killed at 24 and 72 h, but there was a significant effect of treatment (P = 0.011). This consisted of a decline in protein/corpus luteum for PRL/PRL groups (37 ± 2.7 µg; n = 13), compared with VEH/VEH groups (52.4 ± 3.1; n = 12; P = 0.007). The mean value for µg protein/corpus luteum for VEH/PRL groups was not different from either PRL/PRL or VEH/VEH groups (45 ± 4 µg; P = 0.209 and P = 0.271, respectively).There was no effect of time after one PRL injection (24–144 h) when considered either as µg protein/mg luteal tissue (P = 0.992) or as µg protein/corpus luteum (P = 0.497).

Monocytes/Macrophages

There was no significant interaction between treatment and time of tissue collection (P = 0.092), and no significant effect of time of tissue collection (P = 0.321) for luteal monocytes/macrophages from rats killed at 24 and 72 h. There was, however, a significant effect of treatment (P = 0.011). There was a significant increase in monocytes/macrophages per high power field in corpora lutea from PRL/PRL groups (3.1 ± 0.6; n = 13) compared with VEH/VEH groups (1.5 ± 0.7; n = 12; P = 0.021). Numbers of monocytes/macrophages per high power field were 2.6 ± 0.4 (n = 13) for VEH/PRL groups, and this was not significantly different from either PRL/PRL (P = 0.865) or VEH/VEH groups (P = 0.067). There was no effect of time following one PRL injection (24–144 h) on the number of luteal monocytes/macrophages per high power field (P = 0.101).

MHC II-Positive Cells

Two-way ANOVA revealed no effect of treatment (P = 0.99) or time (P = 0.713) on the number of MHC II-positive cells, and no interaction between the terms (P = 0.439). The average number of MHC II-positive cells per high power field for rats in all treatment groups was 1.8 ± 0.3.

Apoptotic Nuclei

There was no significant effect of treatment (P = 0.076) on the number of luteal apoptotic nuclei from rats killed at 24 and 72 h; however, there was a significant effect of time of tissue collection (P = 0.003) and a significant interaction between the two terms (P = 0.015). The number of luteal apoptotic nuclei per high power field was greater for the VEH/PRL group at 24 h than for the VEH/PRL group at 72 h (P = 0.001), the PRL/VEH group at 72 h (144 h after PRL; P = 0.008), the VEH/VEH group at 24 h (P = 0.009) or 72 h (P = 0.012), or the PRL/PRL group at 72 h (P = 0.046). There were no other significant differences among groups (P > 0.05). Comparison of numbers of luteal apopototic nuclei from rats killed at 24, 72, and 144 h after one PRL injection showed a significantly greater number of apoptotic nuclei in corpora lutea of rats killed at 24 h compared with those killed at 72 h (P = 0.001) or 144 h (P = 0.007).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Significant declines in luteal weight and protein and in plasma concentrations of 20{alpha}-dihydroprogesterone, indicating luteal regression, were observed only in hypophysectomized rats that received two PRL injections. This result contrasts with the effect of PRL in the cycling rat in which one proestrous PRL surge or injection of PRL induces a significant decline in luteal weight [15, 16]. The absence of a time effect in this study indicates that PRL initiates a rapid permanent change rather than an ongoing process of luteal regression. Furthermore, comparison of these results with prior studies suggests that the total number of PRL injections, rather than the timing of those injections, determines the amount of luteal regression. Consistent with this hypothesis, the amount of luteal wet weight loss (27.5%) observed after treatment with two injections of PRL is similar to that observed in hypophysectomized rats given two injections of PRL over a 24-h time period [17], while a considerably greater decline in luteal weight (50%–60%) was observed after six injections of PRL given within a 72-h time period [17, 18]. It is interesting that the decline in plasma 20{alpha}-dihydroprogesterone observed in this and a previous [17] study parallels the decline in luteal weight. It is possible that the decline in plasma progestin levels is at least partially due to the loss of steroidogenic tissue.

Luteal regression in rats is a two-part process. The first phase, referred to as "functional luteolysis," is defined as termination of secretion of appreciable quantities of progesterone [19] and occurs during the 4- to 5-day estrous cycle. Corpora lutea that have passed through this first phase of regression do not lose the ability to secrete steroid and can produce significant amounts of 20{alpha}-dihydroprogesterone, as in this study. However, the functional ability of the corpus luteum to sustain pregnancy is lost. The second phase of luteal regression, "structural luteolysis," is defined as the complete morphological regression of the corpus luteum [19] and is addressed in this study. Structural luteolysis is a slower process than functional luteolysis, and corpora lutea may remain on the ovary throughout several estrous cycles before their complete dissolution [20]. As we have observed in this study, the remaining ability of the corpora lutea to produce steroid is lost in parallel with morphological degeneration.

An increase in luteal monocytes/macrophages, which has been associated with structural luteolysis in rats [10, 15, 21], was observed following two injections of PRL, although total numbers of these cells were still low. The numbers of monocytes/macrophages observed after two injections of PRL in this study were similar to those observed in corpora lutea of hypophysectomized rats that received two injections of PRL in 24 h (3.1 per high power field) but lower than numbers in corpora lutea of hypophysectomized rats that received six injections in 72 h (49.3 per high power field) [10] or numbers in regressing corpora lutea following a proestrous PRL surge (9.4 ± 0.9 per high power field) [15]. Monocytes/macrophages may be recruited into corpora lutea by the chemokine monocyte-chemoattractant protein-1 (MCP-1), which is chemotactic for monocytes [22, 23]. Immunohistochemical staining for this chemokine is intense and widely distributed in corpora lutea of hypophysectomized rats following two PRL injections in 24 h, and remains high when PRL injections are administered every 12 h [10]. In the absence of frequent PRL exposure, however, expression of MCP-1 may be transient, resulting in the recruitment of fewer immune cells. The number of luteal monocytes/macrophages was low in all groups in this study, and it is difficult to say whether these cells remained in the corpora lutea after recruitment. A significant reduction in the number of monocytes/macrophages was not observed over 144 h following PRL treatment; however, there did appear to be a nonsignificant trend toward a decline, which may have reached significance if the groups had been larger. The number of MHC II-positive cells did not increase following treatments which elevated monocyte/macrophage numbers, which suggests that these immune cells are not acting primarily as antigen-presenting cells in these corpora lutea.

Prolactin failed to consistently induce apoptosis in the corpora lutea of hypophysectomized rats in this experiment. The increase in apoptotic nuclei observed 24 h after one injection of PRL was minimal in comparison to the numbers previously observed in regressing corpora lutea of the cycle [15] and is due to higher values in two rats. It is possible that detection of apoptosis at this single time point may have been fortuitous, reflecting the tail end of a rapid and transient increase in cell death induced by PRL. Whereas increases in apoptotic nuclei have been observed 24 h after the proestrous PRL surge in cycling rats [15], apoptosis in this reproductive state may be influenced by other factors in addition to PRL. Expression of apoptosis-related genes increases in cycling rats during the latter half of the cycle, prior to the proestrous PRL surge [24]. In addition, Gaytán et al. [6] have reported that progesterone is necessary to allow initiation of apoptosis by PRL in the cycling rat. This may at least partially explain the difference in numbers of apoptotic cells that we observed between hypophysectomized rats, which have little circulating progesterone, and the cycling rat [15].

Although only limited numbers of apoptotic nuclei and monocytes/macrophages were observed in corpora lutea of rats receiving two injections of PRL, the corpora lutea decreased in weight and total protein content. The loss of cells via apoptosis may not account for all of the observed weight and protein loss by the corpora lutea. PRL can affect sodium and water transport across the plasma membrane, leading to changes in cell volume [25–27]. PRL also alters calcium transport across the plasma membrane [28, 29] and has been shown to affect protein synthesis via a calcium-dependent system in the corpus luteum [30]. Additional destruction of tissue, in this case extracellular matrix, may be carried out via matrix metalloproteinases, which have been reported to increase in the corpus luteum following PRL treatment to induce luteal regression [31, 32], and to be associated with a depletion of protein from the tissue [31].

The most significant result of this study is the requirement for repeated exposures to PRL to initiate significant luteal regression in hypophysectomized rats. These data and data from recent reports [17, 18] indicate that the number of exposures to PRL determines the level of luteal regression that occurs. Drawing a parallel between the hypophysectomized rat and the cycling rat, we suggest that continued exposure to PRL is necessary to induce full regression, which explains the persistence of corpora lutea of the rat through several estrous cycles [20]. Furthermore, the greater number of apoptotic nuclei and of monocytes/macrophages in corpora lutea that enter regression during the estrous cycle [15] compared with corpora lutea of rats in this study suggests that regression of corpora lutea during the estrous cycle is enhanced by the presence of factors other than PRL.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge R.K. Brabec and R.W. Crawford of the P30 Center Morphology Core Facility for sectioning of tissues, and S. Kitzsteiner for performing radioimmunoassays. We thank Dr. M. Brown of the P30 Center Biostatistic Core and A. Phillips of the Center for Statistical Consultation and Research for their statistical advice.


   FOOTNOTES
 
First decision: 20 October 1999.

1 This work was supported by grant HD-33478 from the National Institutes of Health (NIH). Support was also provided by the Morphology Core Facility and the Assays and Reagents Core Facility of the P30 Centre for the Study of Reproduction (through NIH HD-18258). Back

2 Correspondence and current address: J.M. Bowen, Department of Pharmacology and Clinical Pharmacology, University of Auckland, Private Bag 92019, 85 Park Road, Grafton, Auckland, New Zealand. FAX: 64 9 373 7556; j.bowen{at}auckland.ac.nz Back

Accepted: May 31, 2000.

Received: September 22, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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