Proliferative Effects of Estradiol, Progesterone, and Two CB Congeners and Their Metabolites on Gray Seal (Halichoerus grypus) Uterine Myocytes in Vitro

Britt-Marie Bäcklin*, Carolina Bredhult{dagger} and Matts Olovsson{dagger},1

* Swedish Museum of Natural History, Contamination Research Group, Box 50007, SE-104 05 Stockholm, Sweden, and {dagger} Department of Women’s and Children’s Health and The Centre for Reproductive Biology in Uppsala, Uppsala University, SE-751 85 Uppsala, Sweden

Received March 10, 2003; accepted May 27, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gray seal females living in the Baltic Sea have been found to exhibit a high prevalence of uterine leiomyomas. These animals are also known to accumulate lipid-soluble PCBs in their blubber. PCBs have documented endocrine-disrupting effects; to investigate whether the PCBs could be part of the genesis of uterine smooth muscle tumors in this species, gray seal myometrial cell cultures were exposed to two CBs and their metabolites, as well as to estradiol and progesterone, after which the effects were analyzed in terms of proliferative activity by measurements of BrdU absorbance and protein content. Progesterone was found to have an inhibitory effect, whereas one CB acted as a stimulant on the myometrial cell proliferation. One of the CB metabolites also seemed to have an inhibitory effect, although this could not be statistically verified. These results suggest that some CBs have effects on uterine myometrial cell proliferation in gray seals and, thus, may also take part in the growth regulation of uterine leiomyomas.

Key Words: grey seal; CB; myometrium; proliferation; in vitro.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Earlier studies on Baltic gray seals (Halichoerus grypus) have revealed a high incidence of lesions in various organs occurring in combination as a disease complex (Bergman and Olsson, 1985Go, 1989Go). The lesions indicated hormonal imbalance, metabolic disorder, and immunosuppression. These findings have been correlated to organochlorine contaminants, and especially to high levels of {Sigma}DDT (DDT: 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane + DDD: 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane + DDE: 2-bis(4-chlorophenyl)-1,1-dichloroethene and polychlorinated biphenyls (PCBs), in the seals (Bergman and Olsson, 1985Go; Helle et al., 1976Go). It is also well known that some fish consumed by the Baltic gray seals contain these organochlorines (Bignert et al., 1998Go; Strandberg et al., 1998Go).

Some PCBs and their metabolites have been documented to have estrogenic or anti-estrogenic effects (Hansen et al., 1992Go; Kester et al., 2000Go; Korach et al., 1988Go; Kramer et al., 1997Go; Moore et al., 1997Go; Safe et al., 1991Go). The hormonal effects might be due to estrogen receptor binding activity (Korach et al., 1988Go) or inhibition of the estrogen inactivating enzyme estrogen sulfotransferase, resulting in increased estrogen bioavailability in target tissues (Kester et al., 2000Go). Other studies have also presented evidence that PCBs can exert their hormonal effects through nonreceptor-mediated mechanisms (Kramer et al., 1997Go; Moore et al., 1997Go).

The endocrine effects of organochlorines are probably important when taking into account that studies on Baltic seals have revealed that organochlorines, and especially PCBs, seem to be responsible for significant reduction in reproduction (Bergman, 1999Go; Bergman and Olsson, 1989Go). Among autopsied gray seals older than 15 years, one type of lesion in particular, uterine leiomyomas, showed an incidence of about 50% (Bergman, 1999Go). Uterine leiomyomas are benign neoplasms that are thought to arise from smooth muscle cells and depend on ovarian steroids for proliferation (Englund, 2001Go; Kawaguchi et al., 1985Go; Townsend et al., 1970Go). No prevalence of uterine leiomyomas has been reported in gray seal populations outside the Baltic Sea. In another Baltic seal species (ringed seal, Phoca hispida botnica) in the Bothnian bay, leiomyomas were only found in 2 seals out of 18 autopsied females (unpublished results). The different seal species have been found to vary in concentrations of certain CBs in their blubber, possibly due to differences in metabolism (Haraguchi et al., 1992Go; Olsson et al., 1992Go). These facts together suggest that organochlorines, and the way in which they are metabolized, may be important factors in the process of developing uterine leiomyomas in Baltic gray seals, but this has to be elucidated further.

The effects of seven organochlorine pesticides on uterine leiomyoma cells have been investigated in vitro in the Eker rat (Hodges et al., 2000Go). The tested compounds (2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane, toxaphene, kepone, methoxychlor, dieldrin, endosulfan isomers {alpha} and ß) all acted as estrogen receptor agonists and induced transcription of an estrogen-inducible reporter gene, as well as of the endogenous estrogen-responsive gene encoding the progesterone receptor. Some of the tested organochlorines also stimulated uterine leiomyoma cell growth through the estrogen receptor. These results indicate that exposure to organochlorine pesticides may have a role in the pathogenesis of uterine leiomyomas in the Eker rat.

At present, little is known about what causes normal myometrium to change growth pattern and start developing into leiomyomas. This investigation was designed to explore whether progesterone, estradiol, two CB congeners, and three of their metabolites previously found in Baltic gray seals (Bergman et al., 1994aGo,bGo; Haraguchi et al., 1992Go), exert any effects on gray seal uterine myometrial cell proliferation in vitro.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sacrificed Baltic gray seal females were sampled on the ice in the Bothnian Bay in May 2000. Within 30 min, myometrium from uteri of three seals (referred to as A, B, and C) was collected from the middle part of the uterine horns. The samples were immediately placed in tubes containing sterile phosphate-buffered saline (PBS) without Mg2+ and Ca2+, cooled in ice water, and transported to the laboratory within 3–4 days. Recent research has shown that human myometrial cells are quite resistant to hypoxia and are viable even after culturing for several days under anaerobic conditions (Olovsson et al., 2000Go), and there were no difficulties in obtaining viable cells from the grey seal uteri. The ovaries were cut into 3-mm slices and examined for corpora lutea. The ages of the seals were 21 (A), 5 (B), and 6 (C) years as determined by examining the annual growth pattern in cementum zones in undecalcified tooth sections (Johnston and Watt, 1980Go).

Establishment of cell cultures.
Using a sterile technique, the tissues were prepared by mincing into about 1-mm3 pieces followed by enzymatic digestion (collagenase: Gibco, 17101-015, 2.5 mg/ml + DNase: Sigma, D-4138, 50 µg/ml + hyaluronidase: Sigma, H-4272, 200 µg/ml in Dulbecco’s modified Eagle’s medium: DMEM, Gibco) for 2–3 h at 37°C.

Cell suspensions were centrifuged at 5 x g for 2 min in order to pellet undigested fragments. The supernatants containing dissociated cells were centrifuged at 400 x g for 10 min. The pellets were resuspended in the culture medium, DMEM supplemented with Glutamax I 0.29 mg/ml (Life, Gibco), 10% fetal bovine serum (Gibco), penicillin G 100 IU/ml (Gibco), and streptomycin sulfate 100 µg/ml (Gibco). The centrifugation was repeated and the resulting cell-containing pellets were resuspended in culture medium.

Cultures were carried out in chamber slides (Super Cell SlideTM, Histolab, Sweden) for immunostaining, 96-well plates (Falcon) for studies of proliferative activity and 25-cm2 bottles (Falcon) for protein measurements, in an atmosphere of 5% CO2 in humidified air at 37°C (Forma Scientific CO2 incubator).

The cells were repeatedly checked for growth using an inverted Nikon Diaphot phase contrast microscope.

Subcultivation of cells was done by trypsinization according to standard procedures, giving rise to three different subconfluent preparations, corresponding to each of the three seals, for further experiments.

Immunostaining for verification of cell culture purity.
Immunostaining was performed for the smooth muscle cell-specific cytoskeletal protein {alpha}-actin after 6–8 days in primary culture. The primary antibody used was mouse anti-{alpha}-actin (Sigma, A-2547) diluted 1:200 in PBS. The PBS used for the immunostaining contained calcium and magnesium.

The cells were rinsed with PBS and then fixed with 3.7% buffered formalin for 10 min, rinsed with PBS, fixed in cold (-20°C) 70% methanol in the -20°C freezer for 5 min, rinsed with cold (+4°C) PBS and blocked for unspecific binding with 2% bovine serum albumin for 30 min. The cultures were then incubated with primary antibody for 30 min at room temperature, rinsed with PBS and incubated for another 30 min with the secondary antibody, goat anti-mouse IgGAM:FITC (Kirkegaard & Perry Lab. Inc., cat. no. 02-18-07), followed by rinsing with PBS.

Examination was conducted using an inverted Nikon Diaphot microscope equipped with a reflected light fluorescence system for FITC application. The cells were accepted for further culturing only if almost all (95–100%) cells stained positively for {alpha}-actin.

Cell Culture Experiments
Preparation of myometrial cells for further experiments.
Twenty-four h prior to the exposure of the myometrial cells to steroids and PCB, the fetal bovine serum content of the culture medium was decreased to 1% in order to depress the cellular activity. This medium was used throughout the conducted experiments.

Exposure to estradiol and progesterone.
Cells from each of the three seals (A, B, and C) were exposed to fresh culture medium containing different concentrations (ranging from 0–100 nmol/ml culture medium) and combinations of estradiol (ß-estradiol, Sigma E-8875) and progesterone (Sigma P-0130) for 24 h. Cells in six wells per seal and hormone combination were exposed. In 12 wells per seal, fresh culture medium without any addition of estradiol or progesterone served as control.

In order to investigate whether priming the myometrial cells with estradiol before addition of progesterone would have any effect on the results, cell cultures were exposed to estradiol (100 nmol/ml) for 24 h, after which the medium was removed and replaced with fresh 100 nmol/ml estradiol-solution combined with 0, 1, 10, or 100 nmol/ml progesterone. Cells in 24 wells per seal and hormone combination were exposed. The exposure lasted for an additional 24 h. In 32 wells per seal, fresh culture medium without any addition of estradiol or progesterone was used as control.

For each seal, the myometrial cells in each well originated from the same batch of cultured cells.

Exposure to CB congeners and metabolites.
Myometrial cells from seal C were exposed to 2,2',4,5,5'-pentachlorobiphenyl (CB 101) and two of its metabolites, 3'-methylsulphonyl-2,5,2',4',5'-pentachlorbiphenyl (3'-MeSO2-CB 101) and 4'-methylsulphonyl-2,2',4,5,5'-pentachlorobiphenyl (4'-MeSO2-CB 101) as well as 2,3',4,4',5-pentachlorobiphenyl (CB 118) and one of its metabolites, 2,3,3',4',5-pentachloro-4-biphenylol (4-OH-CB 107).

The organochlorines were dissolved in 1 ml DMSO (dimethyl sulfoxide, Labassco) and diluted in fresh culture medium to 20 µM. Six wells were used for each tested substance and exposure lasted for 24 h. The myometrial cells in each well originated from the same batch of cultured cells.

The DMSO-content (v/v) of the final solutions ranged between 0.11–0.77%. In 12 wells, culture medium with an equivalent amount of DMSO served as control. Due to the environmental toxicity and health risks, the organochlorines and DMSO were handled in a fumehood and all waste was collected and disposed according to applicable directions.

Evaluation of effects.
Cell proliferation was estimated by measuring BrdU incorporation. After addition of the 5-bromo-2'-deoxyuridine (BrdU) labeling reagent (Cell proliferation ELISA, BrdU [colorimetric], Roche, Mannheim, Germany) and exposure to estradiol and/or progesterone, the cells were analyzed for proliferation by measuring BrdU incorporation during DNA synthesis in terms of absorbance at 405 nm, according to the supplier’s manual. As stated in the manual, the absorbance values directly correlate to the amount of DNA synthesis and thereby also to the number of proliferating cells.

BrdU-measurements were performed in cells from all three seals after exposure to each concentration of estradiol and/or progesterone, and in the priming experiment after priming with estradiol and the subsequent addition of progesterone.

After the myometrial cells from seal C had been exposed to the CB congeners and metabolites, the proliferative activity was evaluated with measurements of BrdU-absorbance.

Cell protein content was also analyzed, using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) based on the method of Bradford (Bradford, 1976Go) in order to quantify the cellular activity of the myometrial cells. A standard curve was prepared using human serum albumin in serial dilutions. The analysis was performed according to the manufacturer’s instructions in order to measure the total amount of protein in the cells in terms of absorbance at 595 nm. The analysis was performed in one series, which explains why mean and SDs could not be calculated.

For the experiments with estradiol and progesterone, a general linear model (GLM) adjusting for repeated measurements on the subjects was used as method of statistical analysis. For the experiment with PCBs, an ANOVA using Dunnett’s technique for comparing treatment groups vs. a control group was used as method of statistical analysis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The occurrence of corpora lutea implied that ovulation had occurred in all three seals.

Cell purity was considered acceptable for further trials only when 95–100% of the cells stained positively for the smooth muscle-specific protein {alpha}-actin, confirming that the cultures mainly consisted of myometrial smooth muscle cells.

The effects of estradiol and progesterone differed among the seals. When studying the mean values of the three gray seal myometrial cell cultures, those exposed to progesterone exhibited significantly (p = 0.0003) inhibited cellular proliferation, as measured in terms of BrdU-absorbance, in comparison with the control (Fig. 1Go). However, a clear pattern with stronger inhibition in response to higher doses did not appear to exist, which could be due to a saturation of the effect. The results for the myometrial cell cultures exposed to estradiol were not statistically significant.



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FIG. 1. Myometrial cell proliferation in response to different concentrations of estradiol and/or progesterone (nmol/ml), determined by measurements of BrdU-absorbance at 405 nm (mean and SDs of all three seals).

 
When the myometrial cells were primed with 100 nmol/ml estradiol for 24 h before a combination of estradiol and progesterone was added, the proliferation was not significantly different from those of unprimed equivalents from the previous experiment.

BrdU-measurements on myometrial cell cultures from seal C exposed to the CB congeners and metabolites (Fig. 2Go) revealed that CB 101 gave rise to an increased cell proliferation (p = 0.0009) compared with the control. 4'-MeSO2-CB 101 seemed to have an inhibitory effect on cellular proliferation, but this could however not be statistically proven (p = 0.0537). The other tested PCBs did not differ from control.



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FIG. 2. Myometrial cell proliferation in response to the addition of CB congeners and metabolites, determined by measurements of BrdU-absorbance at 405 nm (mean and SDs of results from seal C). *Significantly different from control (p = 0.0009).

 
Protein measurements of cell cultures exposed to CB congeners and metabolites (Fig. 3Go) exhibited similar patterns as observed with the BrdU measurements. The most pronounced increase in protein production was evoked by CB 101 while the increase was rather limited after exposure to 3'-MeSO2-CB 101 and CB 118. Exposure to 4'-MeSO2-CB 101 and 4-OH-CB 107 gave results similar to those for the control.



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FIG. 3. Amount of protein (mg/ml) in myometrial cells in response to the addition of CB congeners and metabolites (seal C), determined by measurements of absorbance at 595 nm.

 
Among the CB congeners and their metabolites, the largest effect was thus exerted by CB 101, which stimulated both cell proliferation and protein synthesis. Two of the tested substances, 3'-MeSO2-CB 101 and CB 118, did not evoke any myometrial cell response in terms of proliferation in comparison with the control, yet they stimulated protein synthesis but to a lesser extent than CB 101. 4-OH-CB 107 influenced neither the cellular proliferation nor the protein synthesis. 4'-MeSO2-CB 101 seemed to have an inhibitory effect, similar to progesterone, and also had little effect on protein synthesis.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, progesterone was found to inhibit the gray seal myometrial cell proliferation. The occurrence of corpora lutea implied that ovulation had occurred in all three seals and that they hence were in a progesterone dominated phase when the myometrial cells were collected. An earlier study on human myometrial cell cultures has shown that the addition of progesterone gave no significant mitogenic effect (Shimomura et al., 1998Go). These authors, however, did not distinguish between cells collected in different phases of the menstrual cycle. Neither did Kawaguchi et al. (1985)Go, who reported that human myometrial and leiomyoma cells treated with progesterone for 14 days exhibited poor proliferation. Our results with progesterone are thus consistent with the findings for human myometrial cells in these previous studies.

For leiomyomas, it has been noticed that treatment of leiomyoma-bearing women with the anti-progestin RU 486 results in reduced leiomyoma sizes (Murphy et al., 1993Go). And conversely, progesterone treatment has been reported to result in higher mitotic activity in leiomyomas than in an untreated control (Tiltman, 1985Go). Moreover, in concordance with those findings, leiomyomas have been found to proliferate most in the secretory, progesterone dominated phase (Shimomura et al., 1998Go).

Our results with in vitro exposure of uterine myometrial cells with progesterone are thus quite the opposite of results for leiomyomas, strengthening the implications that the control of proliferation is differentially regulated in myometrium and leiomyomas.

Exposure to estradiol was predicted to influence the gray seal myometrial cells more than the present experiments revealed, and the estradiol was expected to have a stimulatory—not inhibitory—effect on proliferation in concordance with previous findings for humans (Shimomura et al., 1998Go). Again, however, these authors had not distinguished between myometrial cells from different phases of the menstrual cycle. The same is true for Kawaguchi et al. (1985)Go who reported no deviations in proliferation of estradiol-treated myometrial and leiomyoma cells from controls. Furthermore, another study on myometrial cells from women who were menstruating or in the secretory phase, has revealed a transcriptional response after two days of culturing with ethinyl estradiol that was three times greater than for the nonhormone treated control (Andersen et al., 1995Go).

Why the estradiol did not seem to have a stimulatory effect on proliferation in the present study needs to be further elucidated. Perhaps it is of importance that estrogens have been demonstrated to be able to act not only through the estrogen receptor, but also through nonreceptor mediated mechanisms (Brann et al., 1995Go; Fausett et al., 1999Go; Paroo et al., 2002Go). That, in combination with the possibility that incubating the myometrial cells with the steroid for a longer period of time than 24 h might be needed for any stimulatory effects to be detected, might be a possible explanation. Another possible explanation might be that after the cultures had been established, the fetal bovine serum content in the culture medium was decreased to 1%. This might be too little to allow the myometrial cells to proliferate, and it may therefore have contributed to the overall low proliferation observed in the conducted experiments.

The results obtained in the present study might partly also be due to the seals all being under the influence of progesterone when the uterine tissues were collected. The breeding season of gray seals in the Baltic starts in the middle of February. Usually these seals give birth to a single pup. Gestation lasts about 350 days, which includes a period of delayed implantation of about 150 days (Hewer and Backhouse, 1968Go). Estrus and mating occur 14–18 days after parturition (Boness and James, 1979Go). After ovulation, the plasma progesterone concentration increases and remains elevated for most of gestation, including the period of delayed implantation. During the final month of gestation, plasma progesterone increases further and then declines sharply at parturition (Boyd, I. L., 1983Go; Boyd, L., 1984Go). Seals included in this study were in the middle of the period of delayed implantation. Such hormonal conditions suggest that estrogen and progesterone receptors reasonably should exhibit quite a low level of expression in the collected myometrium, at least in its inner subendometrial layers, as other investigators previously have reported for humans and canines (Englund, 2001Go; Englund et al., 1998Go; Mertens et al., 2001Go; Noe et al., 1999Go; Vermeirsch et al., 1999Go, 2000Go). Although low, the progesterone receptor expression in the secretory phase should still somewhat exceed that of the estrogen receptor. An assumption that there was a relative overweight of progesterone receptors in the gray seal myometrium might partly explain why progesterone tended to affect the cells more than the estradiol did in the present experiments. The actual receptor situation in gray seals has not been investigated since no method to quantify estrogen and progesterone receptors in this species currently exists. However, by using antibodies against human estrogen and progesterone receptors, we were able to stain gray seal myometrial cells grown in vitro for both receptors at the time of the exposure experiments (unpublished data). If the observations regarding steroid receptor expression in other species are valid for gray seals as well, they do not, however, hold any explanation of why estradiol did not seem to effect myometrial cell proliferation. However, given the expected low estrogen receptor content, a presumed stimulatory response might have been difficult to detect.

The reason for wanting to prime the cells with estradiol was to resemble the natural course in females as much as possible, when high levels of estrogen precede progesterone and correlate to increased amounts of both estrogen and progesterone receptors in uterine tissues (Englund, 2001Go; Englund et al., 1998Go; Mertens et al., 2001Go; Noe et al., 1999Go; Vermeirsch et al., 1999Go, 2000Go). Therefore, when priming the cells with estradiol, one would expect the response to the subsequently added estradiol and progesterone to be greater due to the increased levels of receptors. However, since priming could not be shown to influence the myometrial cells, the question of whether or not the 24-h incubation period would be sufficient to alter the expression of steroid receptors to any detectable degree remains to be answered.

The selection of organochlorines to be tested was based on the fact that CB 101, 3'-MeSO2-CB 101, 4'-MeSO2-CB 101, CB 118, and 4-OH-CB 107 have been detected in Baltic gray seals (Bergman et al., 1994aGo,bGo; Haraguchi et al., 1992Go; Letcher et al., 2000Go). One of the tested CB-metabolites (4'-MeSO2-CB 101) gave rise to a response in gray seal myometrial cell proliferation similar to that evoked by progesterone, whereas another CB congener (CB 101) had the opposite effect. The stimulatory effects on both proliferation and protein synthesis suggest that CB 101 could be a supporter of myometrial proliferation in the seals. Whether or not these effects were exerted through the estrogen or progesterone receptors, either in an agonistic or antagonistic fashion, as have been reported for some PCB (Korach et al., 1988Go; Kramer et al., 1997Go), is not known. Growing evidence for nonreceptor mediated mechanisms of action for estradiol and progesterone has been presented (Brann et al., 1995Go), and the same is true for some PCBs (Kester et al., 2000Go; Kramer et al., 1997Go; Moore et al., 1997Go). The situation regarding myometrial cells, however, does not seem to have been investigated.

When Hodges et al. (2000)Go investigated the effects of some organochlorine pesticides on Eker rat leiomyoma cells it was found that all the tested compounds had estrogenic actions. Furthermore, some of the substances also stimulated leiomyoma cell growth. These results strongly indicate that exposure to organochlorine pesticides could contribute to the growth of uterine leiomyomas in the Eker rat. Whether or not the observed effects also are valid for gray seal myometrial cells is not known. However, the observed effects of CB 101 on gray seal uterine myocytes seen in this study support the hypothesis that certain organochlorines could influence the growth of uterine leiomyomas in gray seals.


    ACKNOWLEDGMENTS
 
This study was financially supported by the European Chemical Industry Council (CEFIC) and the Swedish Environmental Protection Agency, Project no. I-59-02. We acknowledge Professor Åke Bergman at the Department for Environmental Chemistry, Stockholm University, who kindly provided the CB congeners and some of their metabolites, and Mrs. Margareta Nordling for excellent technical assistance.


    NOTES
 
1 To whom correspondence should be addressed at Department of Women’s and Children’s Health, Kvinnokliniken, Entrance 95/96, Uppsala University Hospital, SE-751 85 Uppsala, Sweden. Fax: +46 18 559775. E-mail: matts.olovsson{at}kbh.uu.se. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Andersen, J., DyReyes, V. M., Barbieri, R. L., Coachman, D. M., and Miksicek, R. J. (1995). Leiomyoma primary cultures have elevated transcriptional response to estrogen compared with autologous myometrial cultures. J. Soc. Gynecol. Investig. 2, 542–551.[CrossRef][ISI][Medline]

Bergman, A. (1999). Health condition of the Baltic gray seal (Halichoerus grypus) during two decades: Gynaecological health improvement but increased prevalence of colonic ulcers. APMIS 107, 270–282.[ISI][Medline]

Bergman, Å., Klasson-Wehler, E., and Kuroki, H. (1994a). Selective retention of hydroxylated PCB metabolites in blood. Environ. Health. Perspect. 102, 464–469.[ISI][Medline]

Bergman, Å., Norstrom, R. J., Haraguchi, K., Kuroki, H., and Béland, P. (1994b). PCB and methyl sulfones in mammals from Canada and Sweden. Environ. Toxicol. Chem. 13, 121–128.[ISI]

Bergman, A., and Olsson, M. (1985). Pathology of Baltic gray seal and ringed seal females with special reference to adrenocortical hyperplasia: Is environmental pollution the cause of a widely distributed disease syndrome? Finn. Game Res. 44, 47–62.

Bergman, A., and Olsson, M. (1989). Pathology of Baltic grey seal and ringed seal males. Report regarding animals sampled 1977–1985. In Influence of Human Activities on the Baltic Ecosystem. Proceeding of the Soviet-Swedish Symposium "Effects of Toxic Substances on Dynamics of Seal Populations," Moscow, USSR, April 1418. (A. V. Yablokov and M. Olsson, Eds.), pp. 74–86. Leningrad Gidrometeoizdat.

Bignert, A., Olsson, M., Persson, W., Jensen, S., Zakrisson, S., Litzen, K., Eriksson, U., Häggberg, L., and Alsberg, T. (1998). Temporal trends of organochlorines in Northern Europe, 1967–1995. Relation to global fractionation, leakage from sediments and international measures. Environ. Pollut. 99, 177–198.[CrossRef][ISI][Medline]

Boness, D. J., and James, H. (1979). Reproductive behaviour of the gray seal (Halichoerus grypus) on Sable Island, Nova Scotia. J. Zool. Lond. 188, 477–500.

Boyd, I. L. (1983). Luteal regression, follicle growth and the concentration of some plasma steroids during lactation in grey seals (Halichoerus grypus). J. Reprod. Fertil. 69, 157–164.[Abstract]

Boyd, L. (1984) Development and regression of the corpus luteum in gray seal (Halichoerus grypus) ovaries and its use in determining fertility rates. Can. J. Zool. 62, 1095–1100.[ISI]

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.[CrossRef][ISI][Medline]

Brann, D. W., Hendry, L. B., and Mahesh, V. B. (1995). Emerging diversities in the mechanism of action of steroid hormones. J. Steroid. Biochem. Mol. Biol. 52, 113–133 (Review).[CrossRef][ISI][Medline]

Englund, K. (2001). Hormonal regulation of sex steroid receptors and growth related genes in human myometrium and leiomyomas. Ph.D. dissertation. Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden.

Englund, K., Blanck, A., Gustavsson, I., Lundkvist, U., Sjöblom, P., Norgren, A., and Lindblom, B. (1998). Sex steroid receptors in human myometrium and fibroids: Changes during the menstrual cycle and gonadotropin-releasing hormone treatment. J. Clin. Endocrinol. Metab. 83, 4092–4096.[Abstract/Free Full Text]

Fausett, M. B., Belfort, M. A., Nanda, R., Saade, G. R., and Vedernikov, Y. (1999). The effects of sex steroids on human umbilical artery and vein. J. Soc. Gynecol. Investig. 6, 27–31.[CrossRef][ISI][Medline]

Hansen, L. G., Jansen, H. T., Cooke, P. S., and Porcelli, J. (1992). Estrogenic and anti-estrogenic actions of polychlorinated biphenyls (PCBs) on uterine tissue in the immature rat. Biol. Reprod. 46, 87.

Haraguchi, K., Athanasiadou, M., Bergman, Å., Hovander, L., and Jensen, S. (1992). PCB and PCB methylsulphones in selected groups of seals from Swedish waters. AMBIO 21, 546–549.[ISI]

Helle, E., Olsson, M., and Jensen, S. (1976). PCB levels correlated with pathological changes in seal uteri. AMBIO 5, 261–263.

Hewer, H. R., and Backhouse, K. (1968) Embryology and foetal growth rate in the gray seal (Halichoerus grypus). J. Zool. Lond. 155, 507–533.

Hodges, L. C., Bergerson, J. S., Hunter, D. S., and Walker, C. L. (2000). Estrogenic effects of organochlorine pesticides on uterine leiomyoma cells in vitro. Toxicol. Sci. 54, 355–364.[Abstract/Free Full Text]

Johnston, D. H., and Watt, I. D. (1980) A rapid method for sectioning undecalcified carnivore teeth for aging. Worldwide Furbearer Conference Proceedings, August 3–11, vol. 1, pp. 407–422. Frostburg, MD.

Kawaguchi, K., Fujii, S., Konishi, I., Okamura, H., and Mori, T. (1985). Ultrastructural study of cultured smooth muscle cells from uterine leiomyomas and myometrium under the influence of sex steroids. Gynecol. Oncol. 21, 32–41.[ISI][Medline]

Kester, M. H., Bulduk, S., Tibboel, D., Meinl, W., Glatt, H., Falany, C. N., Coughtrie, M. W., Bergman, Å., Safe, S. H., Kuiper, G. G., et al. (2000). Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: A novel pathway explaining the estrogenic activity of PCBs. Endocrinology 141, 1897–1900.[Abstract/Free Full Text]

Korach, K. S., Sarver, P., Chae, K., Mclachlan, J. A., and Mckinney, J. D. (1988). Estrogen receptor-binding activity of polychlorinated hydroxybiphenyls: Conformationally restricted structural probes. Mol. Pharmacol. 33, 120–126.[Abstract]

Kramer, V. J., Helferich, W. G., Bergman, Å., Klasson-Wehler, E., and Giesy, J. P. (1997). Hydroxylated polychlorinated biphenyl metabolites are anti-estrogenic in a stably transfected human breast adenocarcinoma (MCF7) cell line. Toxicol. Appl. Pharmacol. 144, 363–376.[CrossRef][ISI][Medline]

Letcher, R. J., Klasson-Wehler, E., and Bergman, Å. (2000). Methyl sulfone and hydroxylated metabolites of polychlorinated biphenyls. In The Handbook of Environmental Chemistry (J. Paasivirta, Ed.), Vol. 3, pp. 315–359. Springer-Verlag, Berlin, Heidelberg.

Mertens, H. J., Heineman, M. J., Theunissen, P. H., de Jong, F. H., and Evers, J. L. (2001). Androgen, estrogen and progesterone receptor expression in the human uterus during the menstrual cycle. Eur. J. Obstet. Gynecol. Reprod. Biol. 98, 58–65.[CrossRef][ISI][Medline]

Moore, M., Mustain, M., Daniel, K., Chen, I., Safe, S., Zacharewski, T., Gillesby, B., Joyeux, A., and Balaguer, P. (1997). Antiestrogenic activity of hydroxylated polychlorinated biphenyl congeners identified in human serum. Toxicol. Appl. Pharmacol. 142, 160–168.[CrossRef][ISI][Medline]

Murphy, A. A., Kettel, L. M., Morales, A. J., Roberts, V. J., and Yen, S. S. (1993). Regression of uterine leiomyomata in response to the antiprogesterone RU 486. J. Clin. Endocrinol. Metab. 76, 513–517.[Abstract]

Noe, M., Kunz, G., Herbertz, M., Mall, G., and Leyendecker, G. (1999). The cyclic pattern of the immunocytochemical expression of oestrogen and progesterone receptors in human myometrial and endometrial layers: Characterization of the endometrial-subendometrial unit. Hum. Reprod. 14, 190–197.[Abstract/Free Full Text]

Olovsson, M., Nordling, M., Ulmsten, U., Lindblom, B., Waldenström, A., and Ronquist, G. (2000). Human uterine myocytes retain their energy charge with no gross alterations in morphology for at least 8 days when cultured under anaerobic conditions. Gynecol. Obstet. Invest. 49, 165–169.[CrossRef][ISI][Medline]

Olsson, M., Andersson, Ö., Bergman, Å., Blomqvist, G., Frank, A., and Rappe, C. (1992). Contaminants and diseases in seals from Swedish waters. AMBIO 21, 561–562.[ISI]

Paroo, Z., Haist, J. V., Karmazyn, M., and Noble, E. G. (2002). Exercise improves postischemic cardiac function in males but not females: Consequences of a novel sex-specific heat shock protein 70 response. Circ. Res. 90, 911–917.[Abstract/Free Full Text]

Safe, S., Astroff, B., Harris, M., Zacharewski, T., Dickerson, R., Romkes, M., and Biegel, L. (1991). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds as antioestrogens: Characterization and mechanism of action. Pharmacol. Toxicol. 69, 400–409.[ISI][Medline]

Shimomura, Y., Matsuo, H., Samoto, T., and Maruo, T. (1998). Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma. J. Clin. Endocrinol. Metab. 83, 2192–2198.[Abstract/Free Full Text]

Strandberg, B., Strandberg, L., van Bavel, B., Bergqvist, P. A., Broman, D., Falandysz, J., Naf, C., Papakosta, O., Rolff, C., and Rappe, C. (1998). Concentrations and spatial variations of cyclodienes and other organochlorines in herring and perch from the Baltic Sea. Sci. Total. Environ. 215, 69–83.[CrossRef][ISI][Medline]

Tiltman, A. J. (1985). The effect of progestins on the mitotic activity of uterine fibromyomas. Int. J. Gynecol. Pathol. 4, 89–96.[ISI][Medline]

Townsend, D. E., Sparkes, R. S., Baluda, M. C., and McClelland, G. (1970). Unicellular histogenesis of uterine leiomyomas as determined by electrophoresis of glucose-6-phosphate dehydrogenase. Am. J. Obstet. Gynecol. 107, 1168–1173.[ISI][Medline]

Vermeirsch, H., Simoens, P., Hellemans, A., Coryn, M., and Lauwers, H. (2000). Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 53, 773–788.[CrossRef][ISI][Medline]

Vermeirsch, H., Simoens, P., Lauwers, H., and Coryn, M. (1999). Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 51, 729–743.[CrossRef][ISI][Medline]





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