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Progesterone receptor immunoreactivity differs in the uterus of pseudopregnant and medroxyprogesterone acetate-treated rabbits
Animal Reproduction Science 120 (2010) 173–178
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Progesterone receptor immunoreactivity differs in the uterus of pseudopregnant and medroxyprogesterone acetate-treated rabbits Santiago R. Anzaldúa Arce a , C. Adriana Mendoza-Rodríguez b , Ignacio Camacho-Arroyo b , Marco Cerbón b,∗ , Mario Pérez-Martínez a a b
Facultad de Medicina Veterinaria y Zootecnia, Departamento de Morfología, Universidad Nacional Autónoma de México, D.F., Mexico Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México, D.F., Mexico
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Article history: Received 22 April 2009 Received in revised form 10 March 2010 Accepted 15 March 2010 Available online 24 March 2010 Keywords: Immunohistochemistry Medroxyprogesterone acetate Progesterone receptor Pseudopregnancy Rabbit Uterus
a b s t r a c t Progesterone receptor (PR) plays an important role in mammals pregnancy which is characterized by greater progesterone plasma concentrations. We assessed PR protein distribution in the rabbit uterus by immunohistochemistry in two progestational conditions: pseudopregnancy (intact adult animals treated with hCG) and after application of a synthetic progestin, medroxyprogesterone acetate (MPA), to ovariectomized animals (OVX). PR immunoreactivity in uterine epithelium of pseudopregnant rabbits was increased in relation to non-pseudopregnant (NP) rabbits. Amounts were similar on Days 1, 3, and 5 of treatment, and was greater on Day 7 (P < 0.001). In contrast, a significant diminution in PR immunoreactivity was observed in stroma cells from Days 1 to 7 (P < 0.001). In OVX rabbits treated with MPA, an increase in PR immunoreactivity was observed in the uterine epithelium on Days 1 to 5 of treatment, reaching a maximum on Day 3 (P < 0.001). In contrast, in stromal cells a diminution in PR immunoreactivity was observed when compared to the OVX group on Days 1, 3 and 7 of MPA treatment (P < 0.001), and there was a slight increase on Day 5. Results suggest a differential time course and tissue specific immunoreactivity for PR in the uterus of the rabbit in two progestational conditions. The present study indicated synthetic progestins have different mechanisms of receptor regulation than those of natural hormones and it should be taken into account in reproductive applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The structure and function of the rabbit uterus is regulated by estradiol (E2 ) and progesterone (P4 ). It has been reported that estrogenic hormonal environment induces cellular proliferation (Conti et al., 1981) and P4 participates in the maturation of endometrial glands, implantation of blastocyst and maintenance of pregnancy (Spencer and
∗ Corresponding author at: Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán 04510, México, D.F., Mexico. Tel.: +52 55 5622 3820; fax: +52 55 5622 3820. E-mail address: [email protected] (M. Cerbón). 0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.03.005
Bazer, 2002). Moreover, P4 participates in the secretion of proteins such as uteroglobin (Beier, 2000). Most P4 actions are mediated by intracellular progesterone receptors (PR) which belong to the nuclear receptor superfamily, that are ligand dependent transcription factors. In the rabbit uterus and other tissues of many species, PRs are up-regulated by E2 and down-regulated by P4 (Zaino et al., 1989; HegeleHartung et al., 1992; Camacho-Arroyo et al., 1996, 2003; Kraus and Katzenellenbogen, 1993), and amount of PR in tissues are hormonally controlled (Blauer et al., 2005). PR immunolocalization has been described in the uterus of prepuberal rabbits, pregnant adult animals (Perrot-Applanat et al., 1985, 1988), estrous rabbits (Hegele-Hartung et al., 1992), and during human chorionic
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gonadotropin (hCG) induced pseudopregnancy (HegeleHartung et al., 1992; Iwai et al., 1991; Karbowski et al., 1992). Endogenous P4 induces down-regulation of PR (Gutiérrez-Sagal et al., 1993) because maximal endometrial content of PR mRNA and PR immunoreactive cells (epithelium and stromal cells) (Anzaldúa et al., 2007) was observed when plasma P4 concentrations were less (Challis et al., 1973), and was markedly diminished when plasma P4 concentrations were greater (on days 3–5 after mating) (Anzaldúa et al., 2007; Gutiérrez-Sagal et al., 1993). Medroxyprogesterone acetate (MPA) is a potent synthetic progestin which is employed as regulator of the estrous cycle in ruminants (Iida et al., 2004; Ross et al., 2004), and it is considered as an antitumoral agent (Actis et al., 1998). MPA has a high affinity to PR in immature, pregnant and the adult rabbit uterus (Feil and Bardin, 1979; Zhang et al., 1989). Moreover, it is able to maintain the placental function in absence of P4 (Gadsby et al., 1983). However, MPA effects on PR regulation and distribution in the rabbit uterus are unknown. There are no comparative studies between the two induced progestational conditions: pseudopregnancy induced by hCG treatment, which is under endogenous P4 control, and the exogenous administration of progestins in rabbits. It is also unknown whether changes in PR distribution and amounts in the rabbit uterus during pseudopregnancy are similar to those observed in early pregnancy. The objective of the present study was to assess PR distribution and amounts in the epithelium and stroma of rabbit uterus in two different induced progestational conditions: pseudopregnancy by treatment with hCG and exogenous application of a synthetic progestin (MPA). 2. Materials and methods 2.1. Animals Adult virgin female New Zealand white rabbits (n = 30; 3.5–4.5 kg; Harlan Mexico) were used throughout the study. Animals were housed in individual cages with food and water available ad libitum. A group of 12 animals was ovariectomized under anesthesia with xylazine (3–5 mg/kg) and ketamine (35 mg/kg). Three weeks after surgery, the rabbits were divided into two groups: a group formed by ovariectomized animals (OVX) treated with vehicle (corn oil) was assigned control group (n = 3), and another group that received a subcutaneous injection of 5 mg of MPA on Days 0 and 5 of treatment (Lee and Fields, 1991). This dose maintains placental function in the absence of luteal P4 (Gadsby et al., 1983). Another group (n = 12) of intact female rabbits was treated with a single 100 IU of human chorionic gonadotropin (hCG) dose by IM injection (Chorulon, Intervet). The day of the first injection of MPA, as well as that of hCG, was designated as Day 0. Rabbits were anesthetized with ketamine and were sacrificed by an overdose (90 mg/kg) of sodium pentobarbital applied by IM injection; (Rhóne, Mérieux, Qro. México) on Days 1, 3, 5, and 7 after MPA or hCG treatment (n = 3). Nonpregnant intact animals (NP) (n = 3) were used as control for hCG treated animals and other group of ovariectomized
animals treated with vehicle (OVX + veh) (n = 3) were used as control of the MPA-treated OVX rabbits. Experiments in this study were performed according to the Law for the Protection of Animals (México), and NOM-062-200-1999 (Aluja, 2002). 2.2. Immunohistochemistry The rabbit uterus was removed and immersion-fixed in 4% paraformaldehyde for 8 h and stored in PBS solution (pH 7.4) at 4 ◦ C and thereafter dehydrated and embedded in paraffin. Serial sections from the middle portion of uterine horns were cut at 5 m thickness and mounted on slides coated with poly-l-lysine (Sigma). Slides were deparaffinized, rehydrated through graded concentrations of alcohol to distilled water, transferred to sodium citrate buffer (pH 6.0), and heated two times for 10 min in a microwave oven (Mabe, Hot Point) set at 800 W. Slides were cooled between microwave irradiations for 5 min. After this procedure, slides were washed twice with 10 mM PBS, pH 7.4, and incubated successively in: 3% hydrogen peroxide in PBS for 30 min (at room temperature), 1% normal goat serum plus 1% hydrogen peroxide in PBS for 30 min; 0.5% Triton X-100 in PBS for 30 min; PR mouse monoclonal antibody (Mi 60-10 antibody was a kind gift from Dr. Edwin Milgrom and Dr. Perrot-Applanat, Perrot-Applanat et al., 1985) 10 g/ml prepared in PBS containing 0.3% Triton X-100 for 48 h at 4 ◦ C in a humid chamber. Slides were incubated with a biotinylated secondary antibody for 2 h at room temperature and later with conjugated streptavidin-peroxidase for 1 h. Sections were washed twice with PBS among incubations. Peroxidase activity was evidenced by using 3,3 -diaminobenzidine chromogen solution in the presence of hydrogen peroxide for 10 min. After washing, sections were dehydrated and a cover slip was applied with mounting medium (Permount). Some sections were counterstained with Mayerˇıs hematoxylin. Control sections were incubated with preimmune goat serum (1:2000) in place of the primary antibody. Immunostaining was visualized by using a digital camera (Leica DMLS). The number of immunopositive nuclei and staining intensity in the epithelium and stroma were determined using Metamorph Imaging System (Westchester, PA, USA). All the nuclei from the epithelium and stroma were quantified in each section. Three sections per animal (3 animals/day) were analyzed for PR immunoreactivity. Staining intensity of cell nuclei was assigned by the following scores: 0, absent; 1, weak; 2, moderate and 3, intense. The histological score (HSCORE) was calculated as follows: HSCORE = Pi (i + 1), where i = 0, 1, 2 or 3, and Pi is the percentage of cells with each intensity from 0 to 100% (Lessey et al., 1988). 2.3. Data analysis Data were analyzed by using a one-way analysis of variance (ANOVA) followed by a multiple comparison of Tukey. Prism 2.01 program (Graph Pad, CA, USA) was used for calculating probability values.
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Fig. 1. Immunohistochemical localization of PR in the uterus of pseudopregnant and MPA-treated rabbits. PR protein was observed by immunohistochemistry in NP rabbit (A), pseudopregnant rabbit on Day 1 (B), 3 (C), 5 (D) and 7 (E). PR protein in OVX + vehicle treated animals (G) and OVX + MPA rabbits on Days 1 (H), 3 (I), 5 (J) and 7 (K). Arrows indicate specific nuclear immunostaining. In (F) there was a negative control section of a NP rabbit uterus, and in (L) a negative control section of an OVX + vehicle rabbit. In both negative controls, the primary antibody was omitted. Cells were counterstained with Mayer’s hematoxylin. Barr = 100 m.
3. Results There was similar glandular morphology in the uterus of hCG- and MPA-treated rabbits. Small endometrial glands were present the first 3 days both in pseudopregnant and OVX animals treated with MPA (Fig. 1B, C and H, I, respectively). Great arborizations formed by endometrial glands
were observed on Day 7 of pseudopregnancy, and there was less arborization in OVX + MPA-treated rabbits (Fig. 1 E vs. K, respectively). PR immunostaining was observed in the nucleus of several uterine cells. In NP and OVX + vehicle rabbits, the number of stromal PR immunopositive cells and the intensity of the immunostaining were greater as compared
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Fig. 2. PR protein in epithelial and stromal cells in the rabbit uterus. The HSCORE of PR was calculated, as described in Section 2, in uterine epithelial (A) and stromal cells (B): black bars, pseudopregnant rabbits (Days 1 to 7); open bars, OVX rabbits treated with MPA (Days 1 to 7). Control animals (0) are non-pregnant rabbits (black bar) or OVX + vehicle rabbits (open bars). Results are expressed as mean ± SEM. Different letters indicate a difference between groups (P < 0.001).
with epithelial cells (P < 0.001) (Fig. 1A and G). No staining was observed in negative control of NP rabbit uterus and OVX + vehicle uterus where the primary antibody was omitted (Fig. 1F and L, respectively). In the epithelium of NP and OVX + vehicle animals PR HSCORE was 131.0 ± 2.0 and 168.9 ± 4.72, respectively, whereas in the lamina propia (stromal cells) the score was greater (P < 0.001) (253.0 ± 6.67 and 206.66 ± 8.95, respectively) (Fig. 2). There were changes in PR immunoreactivity in pseudopregnant and OVX + MPA-treated rabbits (P < 0.001) in relation to NP and OVX + vehicle group, respectively, with exception of epithelial cells on Day 7 OVX + MPA and stromal cells on Day 5 OVX + MPA, where the HSCORE was similar to OVX animals (Fig. 2A and B). In uterine epithelial cells of pseudopregnant rabbits PR HSCORE was increased (P < 0.001) in relation to that of NP rabbits (Fig. 2A). The amount of immunoreactivity was similar from Day 1 to 5, and was robust on day 7 (Fig. 2A). In contrast, there was a decrease (P < 0.001) in the HSCORE in stromal cells from Days 1 to 7 of pseudopregnancy as compared with the NP rabbits (Fig. 2B). A gradual increase (P < 0.001) was observed in PR HSCORE of the uterine epithelium of OVX + MPA from Day 1 to day 5, reaching a maximum on Day 3 (Fig. 2A). In stromal cells, there was a decrease in PR immunostaining during treatment with MPA (P < 0.001) (Fig. 2B), although an increase (P < 0.001) was observed in PR HSCORE on Day 5, in relation to the previous days (Fig. 2B).
4. Discussion In the present study PR immunoreactivity and distribution of receptors were evaluated in the uterus of the female rabbit in two progestational conditions: pseudopregnancy and after MPA treatment. In the rabbit endometrium hCG stimulates formation of glands by epithelial cell proliferation (Conti et al., 1981) between Days 6 and 8 after treatment (Hegele-Hartung et al., 1992); this observation is in agreement with results of the present study. On Day 7 of pseudopregnancy induced by hCG, significant changes in endometrium, including great arborizations formed by endometrial glands were observed in the present study.
Estrogens are potent mitogenic compounds that induce cellular proliferation in the female reproductive tract. In the present study, pseudopregnant rabbits had greater arborizations formed by endometrial glands. During pseudopregnancy in rabbits a significant increase on E2 concentrations is observed (Browning et al., 1980; Challis et al., 1973). This increase in E2 may be related with the increase in arborization observed in the present study. In contrast, in OVX + MPA-treated rabbits on Day 7 there was a lesser glandular arborization pattern. This may be due to the fact that these animals are OVX, and E2 concentrations were less than in pseudopregnant rabbits. In addition, MPA has an antiestrogenic effect in the rabbit uterus (Misao et al., 1995) where it exerts antiproliferative effects on estrogen-stimulated uterine growth. In the present study, there was an increase in PR HSCORE in uterine epithelial cells during the first 7 days of pseudopregnancy. This could be due to the well known priming effect of E2 on PR because E2 is required for the induction of PR synthesis in female reproductive tracts and there is an increase of E2 serum concentrations during early pseudopregnancy (Browning et al., 1980; Challis et al., 1973). In contrast with the present results, Hegele-Hartung et al. (1992) found a gradual diminution of PR in epithelial cells after Day 4. Iwai et al. (1991) reported a diminution in number of PR immunoreactive cells in the uterine glandular epithelium by 48 h after hCG injection and disappearance of PR 3 days after hCG administration. These differences may be explained by the method of quantification because in those reports number of glandular epithelial immunoreactive cells were only analyzed, while in the present study PR HSCORE in luminal and glandular epithelial cells after treatment with hCG was assessed. There is a diminution of PR gene expression in the rabbit endometrium (epithelial and stromal cells) on Day 3 and 4 of pregnancy (Anzaldúa et al., 2007; GutiérrezSagal et al., 1993), this could be due to an increase of progesterone plasma concentrations (Camacho-Arroyo et al., 1996). Although progesterone and estrogen plasma concentrations are similar in pregnancy and pseudopregnancy conditions (Browning et al., 1980; Challis et al., 1973), a different PR HSCORE pattern was observed in stromal and epithelial cells. This suggests regulation of PR gene expression not only depends on progesterone and
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estrogen plasma concentrations, but also on other local or paracrine factors such as hCG/LH receptors (Jensen and Odell, 1992) and the fertilized egg which is present in the uterus between 60 and 72 h post-coitus (Osada et al., 1999). Presence of hCG/LH receptors in the rabbit endometrium (Jensen and Odell, 1992; Sawitzke and Odell, 1991) can mediate hCG effects in the uterus (Ziecik et al., 1992). MPA induced an initial increase in PR HSCORE of uterine epithelial cells on Days 1 and 3, followed by a gradual diminution on Days 5 and 7 (Fig. 2 A); whereas in stromal cells of the lamina propia a PR diminution occurred from the first to third day of treatment as compared with the OVX rabbits (Fig. 2B). On Day 5 post-MPA treatment an increase in PR HSCORE was observed, however, this increase was similar to OVX control group. Changes on Day 5 are not related to the fact that animals were treated with MPA every 5 days, because animals on Day 5 had not been treated on that day yet. Thus, a diminution of amounts of MPA could be related to the increase in PR HSCORE observed on this day. In the present study, results indicate an opposite regulation of PR HSCORE between epithelium and stromal uterine cells of pseudopregnant rabbits and OVX + MPA treatments, because PR increases in the uterine epithelial cells of both progestin-treated groups and there were decreases in the stromal cells (Fig. 2A and B). These results suggest a differential regulation of PR in two different cell types in the uterus. The reason of this particular pattern of regulation is unknown. Interestingly, MPA increases PR binding in the ewe and the rabbit (Zhang et al., 1989) and it has a potent progestational effect on the rabbit uterus (Spilman et al., 1986), but it also functions as an antiestrogenic drug by reducing the number of estrogen receptors in some target tissues of rabbit (Di Carlo et al., 1984) (which, on the contrary, are increased by E2 ), besides, in the rabbit uterus MPA causes a very evident reduction in weight (Di Carlo et al., 1984). These results could be related with the PR down-regulation in epithelial cells observed from Day 5 to 7 post-MPA treatment in the present study. Moreover, MPA down-regulates PR gene expression in the mouse uterus (Actis et al., 1998). In the present study, there was PR down-regulation in the stromal cells of OVX + MPA rabbits. Comparable results were obtained for immunohistochemistry detection of two isoforms of PR (A and B) in stromal cells of the human endometrium after intermittent treatment with oral MPA for 3 months, in patients with endometrial hyperplasia. This reduction was also observed in PR isoforms in the epithelial cells (Vereide et al., 2006). Although there is a progestational hormonal environment both in pseudopregnant and OVX + MPA rabbits in the present study, the mechanisms of signaling pathways of paracrine interactions between epithelial and stromal cells are not similar. In other reports, PR gene expression via ER␣ was completely opposite between the uterine and vaginal epithelium in mice; estrogen up-regulates PR in the vaginal epithelium and down-regulates PR in the uterine epithelium (Kurita et al., 2000). In summary, a specific gene expression pattern of PR was observed between pseudopregnant and OVX + MPA-
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treated rabbits in uterine epithelial and stromal cells. Further studies are needed to understand the mechanisms by which progestins mediate stromal–epithelial interactions in female reproductive tissues. Acknowledgements The authors express their gratitude to Francisco López López for technical support. We thank Dr. Edwin Milgrom and Dr. Perrot-Applanat for the kind gift of Mi 60-10 antibody. This work was supported in part by grant from DGAPA-UNAM (PAPIIT-ES212101 and IN219710-2), and CONACyT (AC-89544 and 80338). References Actis, A.M., Dorfman, V.B., Caruso, S.P., Levin, E., 1998. Effects on mouse uterus of three antitumoral drugs acting upon estrogen and progesterone receptors directly and through other transduction pathways. Biochem. Pharmacol. 55, 273–278. Aluja, A.S., 2002. Animales de Laboratorio y la Norma Oficial Mexicana (NOM-062-ZOO-1999). Gaceta Médica de México 138, 295–298. Anzaldúa, A.S., Camacho-Arroyo, I., Reyna-Neyra, A., Pérez-Martínez, M., Cerbón, M., 2007. Regional differences in expression of progesterone receptor in oviduct and uterus of rabbit during early pregnancy. Comp. Biochem. Physiol. 147 A, 685–690. Beier, H.M., 2000. The discovery of uteroglobin and its significance for reproductive biology and endocrinology. Ann. N.Y. Acad. Sci. 923, 9–24. Blauer, M., Heinonen, P.K., Martikainen, P.M., Tomas, E., Ylikomi, T., 2005. A novel organotypic culture model for normal human endometrium: regulation of epithelial cell proliferation by estradiol and medroxyprogesterone acetate. Hum. Reprod. 20, 864–871. Browning, J.Y., Keyes, P.L., Woli, R.C., 1980. Comparison of serum progesterone, 20␣ dihydroprogesterone and 17 estradiol in pregnant and pseudopregnant rabbits: evidence for postimplantation recognition of pregnancy. Biol. Reprod. 23, 1014–1019. Camacho-Arroyo, I., Pasapera, A.M., Cerbón, M.A., 1996. Regulation of progesterone receptor gene expression by sex steroid hormones in the hypothalamus and the cerebral cortex of the rabbit. Neurosci. Lett. 214, 25–28. Camacho-Arroyo, I., González-Arenas, A., González-Agüero, G., GuerraAraiza, C., González-Morán, G., 2003. Changes in content of progesterone receptor isoforms and estrogen receptor alpha in the chick brain during embryonic development. Comp. Biochem. Physiol. 136 A, 447–452. Challis, J.R., Davies, I.J., Ryan, K.J., 1973. The concentrations of progesterone, estrone and estradiol 17 in the plasma of pregnant rabbits. Endocrinology 93, 971–976. Conti, C.J., Giménez-Conti, I.B., Zerbe, G.O., Gerschenson, L.E., 1981. Differential effects of estradiol-17 beta and progesterone on the proliferation of glandular and luminal cells of rabbit uterine epithelium. Biol. Reprod. 24, 643–648. Di Carlo, F., Racca, S., Conti, G., Gallo, E., Muccioli, G., Sapino, A., Bussolati, G., 1984. Effect of long-term administration of high doses of medroxyprogesterone acetate on hormone receptors and target organs in the female rat. J. Endocrinol. 103, 287–293. Feil, P.D., Bardin, C.W., 1979. The use of medroxyprogesterone acetate to study progestin receptors in immature, pregnant, and adult rabbit uterus. Adv. Exp. Med. Biol. 117, 241–254. Gadsby, J.E., Keyes, L., Bill, C.H.2nd., 1983. Control of corpus luteum function in the pregnant rabbit: role of estrogen and lack of a direct luteolytic role of the placenta. Endocrinology 113, 2255–2262. Gutiérrez-Sagal, R., Pérez-Palacios, G., Langley, E., Pasapera, A.M., Castro, I., Cerbón, M.A., 1993. Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit. Mol. Reprod. Dev. 34, 244–249. Hegele-Hartung, C., Chwalisz, K., Beier, H.M., 1992. Distribution of estrogen and progesterone receptors in the uterus and immunohistochemical study in the immature and adult pseudopregnant rabbit. Histochemistry (Germany) 97, 39–50. Iida, K., Kobayashi, N., Cono, H., Miyamoto, A., Fukui, Y., 2004. A comparative study of induction of estrous and ovulation by three different intravaginal devices in ewes during the non-breeding season. J. Reprod. Dev. 50, 63–69.
178
S.R.A. Arce et al. / Animal Reproduction Science 120 (2010) 173–178
Iwai, T., Fujii, S., Nanbu, Y., Nonogaki, H., Konishi, I., Mori, T., Okamura, H., 1991. Effect of chorionic gonadotropin on the expression of progesterone receptor and estrogen receptors in rabbit ovarian granulose cells and the uterus. Endocrinology 129, 1840–1848. Jensen, J.D., Odell, W.D., 1992. Identification of LH/hCG receptors in rabbit uterus. Proc. Soc. Exp. Biol. Med. 189, 28–30. Karbowski, B., Vollmer, E., Schneider, P.G., 1992. Steroid receptors in the fallopian tube–morphological and functional investigation in rabbits. Prog. Histochem. Cytochem. 26, 140–144. Kraus, W.L., Katzenellenbogen, B.S., 1993. Regulation of progesterone expression and growth in the rat uterus: modulation of estrogen actions by progesterone and sex steroid hormone antagonists. Endocrinology 132, 2371–2379. Kurita, T., Lee, K.J., Cooke, P.S., Taylor, J.A., Lubahn, D.B., Cunha, G.R., 2000. Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract. Biol. Reprod. 62, 821–830. Lee, V.H., Fields, P.A., 1991. Rabbit relaxin: the influence of pregnancy and ovariectomy during pregnancy on the plasma profile. Biol. Reprod. 45, 209–214. Lessey, B.A., Killman, A.P., Metzger, D.A., Haney, A.F., Greene, G.L., McCarty Jr., K.S., 1988. Immunohistochemical analysis of the human uterine estrogen and progesterone receptors throughout the menstrual cycle. J. Clin. Endocrinol. Metab. 67, 334–340. Misao, R., Nishigaki, M., Hori, M., Ichigo, S., Fujimoto, J., Tamaya, T., 1995. Effects of danazol and medroxyprogesterone acetate on estrogen(estradiol and estriol) specific binding sites in rabbit uterus. Gynecol. Endocrinol. 9, 29–35. Osada, H., Tsumoda, I., Matsuura, M., Satoh, K., Kanayama, K., Nakayama, Y., 1999. Investigation of ovum transport in the oviduct: the dynamics of the oviductal fluids in domestic rabbit. J. Int. Med. Res. 27, 176–180. Perrot-Applanat, M., Logeat, F., Groyer-Picard, M.T., Milgrom, E., 1985. Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies. Endocrinology 116, 1473–1484.
Perrot-Applanat, M., Groyer-Picard, M.T., García, E., Lorenzo, F., Milgrom, E., 1988. Immunocytochemical demonstration of estrogen and progesterone receptors in muscle cells of uterine arteries in rabbits and humans. Endocrinology 123, 1511–1519. Ross, P.J., Adler, J.F., Cellejas, S.S., Butler, H., Alberio, R.H., 2004. Estradiol benzoate given 0 or 24 h after the end of a progestagen treatment in postpartum suckled beef cows. Theriogenology 62, 265–273. Sawitzke, A.L., Odell, W.D., 1991. Uterine binding sites for LH/hCG can be modulate by hormonal status in rabbits and rats. Acta Endocrinol. (Copenh.) 124, 322–330. Spencer, T.E., Bazer, F.W., 2002. Biology of progesterone action during pregnancy recognition and maintenance of pregnancy. Front. Biosci. 7, 1879–1898. Spilman, C.H., Gibson, R.E., Beuving, D.C., Campbell, J.A., 1986. Progestin and antiprogestin effects on progesterone receptor transformation. J. Steroid. Biochem. 24, 383–389. Vereide, A.B., Kaino, T., Sager, G., Arnes, M., Ørbo, A., 2006. Effect of levonorgestrel IUD and oral medroxyprogesterone acetate on glandular and stromal progesterone receptors (PRA and PRB), and estrogen receptors (ER-␣ and ER-) in human endometrial hyperplasia. Gynecol. Oncol. 101, 214–223. Zaino, R.J., Clarke, C.L., Feil, P.D., Satyaswaroop, P.G., 1989. Differential distribution of estrogen and progesterone receptors in rabbit uterus detected by dual immunofluorescence. Endocrinology 125, 2728–2734. Zhang, X., Stone, G.M., Miller, B.G., 1989. In vitro binding of progesterone, cronolone and medroxyprogesterone acetate to uterine progesterone receptors of the sheep, rabbit and mouse. Reprod. Fertil. Dev. 1, 223–230. Ziecik, A.J., Jedlinska, M., Rzucidllo, S.J., 1992. Effect of estradiol and progesterone on myometrial LH/hCG receptors in pigs. Acta Endocrinol. (Copenh.) 127, 185–188.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Progesterone receptor immunoreactivity differs in the uterus of pseudopregnant and medroxyprogesterone acetate-treated rabbits Santiago R. Anzaldúa Arce a , C. Adriana Mendoza-Rodríguez b , Ignacio Camacho-Arroyo b , Marco Cerbón b,∗ , Mario Pérez-Martínez a a b
Facultad de Medicina Veterinaria y Zootecnia, Departamento de Morfología, Universidad Nacional Autónoma de México, D.F., Mexico Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México, D.F., Mexico
a r t i c l e
i n f o
Article history: Received 22 April 2009 Received in revised form 10 March 2010 Accepted 15 March 2010 Available online 24 March 2010 Keywords: Immunohistochemistry Medroxyprogesterone acetate Progesterone receptor Pseudopregnancy Rabbit Uterus
a b s t r a c t Progesterone receptor (PR) plays an important role in mammals pregnancy which is characterized by greater progesterone plasma concentrations. We assessed PR protein distribution in the rabbit uterus by immunohistochemistry in two progestational conditions: pseudopregnancy (intact adult animals treated with hCG) and after application of a synthetic progestin, medroxyprogesterone acetate (MPA), to ovariectomized animals (OVX). PR immunoreactivity in uterine epithelium of pseudopregnant rabbits was increased in relation to non-pseudopregnant (NP) rabbits. Amounts were similar on Days 1, 3, and 5 of treatment, and was greater on Day 7 (P < 0.001). In contrast, a significant diminution in PR immunoreactivity was observed in stroma cells from Days 1 to 7 (P < 0.001). In OVX rabbits treated with MPA, an increase in PR immunoreactivity was observed in the uterine epithelium on Days 1 to 5 of treatment, reaching a maximum on Day 3 (P < 0.001). In contrast, in stromal cells a diminution in PR immunoreactivity was observed when compared to the OVX group on Days 1, 3 and 7 of MPA treatment (P < 0.001), and there was a slight increase on Day 5. Results suggest a differential time course and tissue specific immunoreactivity for PR in the uterus of the rabbit in two progestational conditions. The present study indicated synthetic progestins have different mechanisms of receptor regulation than those of natural hormones and it should be taken into account in reproductive applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The structure and function of the rabbit uterus is regulated by estradiol (E2 ) and progesterone (P4 ). It has been reported that estrogenic hormonal environment induces cellular proliferation (Conti et al., 1981) and P4 participates in the maturation of endometrial glands, implantation of blastocyst and maintenance of pregnancy (Spencer and
∗ Corresponding author at: Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán 04510, México, D.F., Mexico. Tel.: +52 55 5622 3820; fax: +52 55 5622 3820. E-mail address: [email protected] (M. Cerbón). 0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.03.005
Bazer, 2002). Moreover, P4 participates in the secretion of proteins such as uteroglobin (Beier, 2000). Most P4 actions are mediated by intracellular progesterone receptors (PR) which belong to the nuclear receptor superfamily, that are ligand dependent transcription factors. In the rabbit uterus and other tissues of many species, PRs are up-regulated by E2 and down-regulated by P4 (Zaino et al., 1989; HegeleHartung et al., 1992; Camacho-Arroyo et al., 1996, 2003; Kraus and Katzenellenbogen, 1993), and amount of PR in tissues are hormonally controlled (Blauer et al., 2005). PR immunolocalization has been described in the uterus of prepuberal rabbits, pregnant adult animals (Perrot-Applanat et al., 1985, 1988), estrous rabbits (Hegele-Hartung et al., 1992), and during human chorionic
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gonadotropin (hCG) induced pseudopregnancy (HegeleHartung et al., 1992; Iwai et al., 1991; Karbowski et al., 1992). Endogenous P4 induces down-regulation of PR (Gutiérrez-Sagal et al., 1993) because maximal endometrial content of PR mRNA and PR immunoreactive cells (epithelium and stromal cells) (Anzaldúa et al., 2007) was observed when plasma P4 concentrations were less (Challis et al., 1973), and was markedly diminished when plasma P4 concentrations were greater (on days 3–5 after mating) (Anzaldúa et al., 2007; Gutiérrez-Sagal et al., 1993). Medroxyprogesterone acetate (MPA) is a potent synthetic progestin which is employed as regulator of the estrous cycle in ruminants (Iida et al., 2004; Ross et al., 2004), and it is considered as an antitumoral agent (Actis et al., 1998). MPA has a high affinity to PR in immature, pregnant and the adult rabbit uterus (Feil and Bardin, 1979; Zhang et al., 1989). Moreover, it is able to maintain the placental function in absence of P4 (Gadsby et al., 1983). However, MPA effects on PR regulation and distribution in the rabbit uterus are unknown. There are no comparative studies between the two induced progestational conditions: pseudopregnancy induced by hCG treatment, which is under endogenous P4 control, and the exogenous administration of progestins in rabbits. It is also unknown whether changes in PR distribution and amounts in the rabbit uterus during pseudopregnancy are similar to those observed in early pregnancy. The objective of the present study was to assess PR distribution and amounts in the epithelium and stroma of rabbit uterus in two different induced progestational conditions: pseudopregnancy by treatment with hCG and exogenous application of a synthetic progestin (MPA). 2. Materials and methods 2.1. Animals Adult virgin female New Zealand white rabbits (n = 30; 3.5–4.5 kg; Harlan Mexico) were used throughout the study. Animals were housed in individual cages with food and water available ad libitum. A group of 12 animals was ovariectomized under anesthesia with xylazine (3–5 mg/kg) and ketamine (35 mg/kg). Three weeks after surgery, the rabbits were divided into two groups: a group formed by ovariectomized animals (OVX) treated with vehicle (corn oil) was assigned control group (n = 3), and another group that received a subcutaneous injection of 5 mg of MPA on Days 0 and 5 of treatment (Lee and Fields, 1991). This dose maintains placental function in the absence of luteal P4 (Gadsby et al., 1983). Another group (n = 12) of intact female rabbits was treated with a single 100 IU of human chorionic gonadotropin (hCG) dose by IM injection (Chorulon, Intervet). The day of the first injection of MPA, as well as that of hCG, was designated as Day 0. Rabbits were anesthetized with ketamine and were sacrificed by an overdose (90 mg/kg) of sodium pentobarbital applied by IM injection; (Rhóne, Mérieux, Qro. México) on Days 1, 3, 5, and 7 after MPA or hCG treatment (n = 3). Nonpregnant intact animals (NP) (n = 3) were used as control for hCG treated animals and other group of ovariectomized
animals treated with vehicle (OVX + veh) (n = 3) were used as control of the MPA-treated OVX rabbits. Experiments in this study were performed according to the Law for the Protection of Animals (México), and NOM-062-200-1999 (Aluja, 2002). 2.2. Immunohistochemistry The rabbit uterus was removed and immersion-fixed in 4% paraformaldehyde for 8 h and stored in PBS solution (pH 7.4) at 4 ◦ C and thereafter dehydrated and embedded in paraffin. Serial sections from the middle portion of uterine horns were cut at 5 m thickness and mounted on slides coated with poly-l-lysine (Sigma). Slides were deparaffinized, rehydrated through graded concentrations of alcohol to distilled water, transferred to sodium citrate buffer (pH 6.0), and heated two times for 10 min in a microwave oven (Mabe, Hot Point) set at 800 W. Slides were cooled between microwave irradiations for 5 min. After this procedure, slides were washed twice with 10 mM PBS, pH 7.4, and incubated successively in: 3% hydrogen peroxide in PBS for 30 min (at room temperature), 1% normal goat serum plus 1% hydrogen peroxide in PBS for 30 min; 0.5% Triton X-100 in PBS for 30 min; PR mouse monoclonal antibody (Mi 60-10 antibody was a kind gift from Dr. Edwin Milgrom and Dr. Perrot-Applanat, Perrot-Applanat et al., 1985) 10 g/ml prepared in PBS containing 0.3% Triton X-100 for 48 h at 4 ◦ C in a humid chamber. Slides were incubated with a biotinylated secondary antibody for 2 h at room temperature and later with conjugated streptavidin-peroxidase for 1 h. Sections were washed twice with PBS among incubations. Peroxidase activity was evidenced by using 3,3 -diaminobenzidine chromogen solution in the presence of hydrogen peroxide for 10 min. After washing, sections were dehydrated and a cover slip was applied with mounting medium (Permount). Some sections were counterstained with Mayerˇıs hematoxylin. Control sections were incubated with preimmune goat serum (1:2000) in place of the primary antibody. Immunostaining was visualized by using a digital camera (Leica DMLS). The number of immunopositive nuclei and staining intensity in the epithelium and stroma were determined using Metamorph Imaging System (Westchester, PA, USA). All the nuclei from the epithelium and stroma were quantified in each section. Three sections per animal (3 animals/day) were analyzed for PR immunoreactivity. Staining intensity of cell nuclei was assigned by the following scores: 0, absent; 1, weak; 2, moderate and 3, intense. The histological score (HSCORE) was calculated as follows: HSCORE = Pi (i + 1), where i = 0, 1, 2 or 3, and Pi is the percentage of cells with each intensity from 0 to 100% (Lessey et al., 1988). 2.3. Data analysis Data were analyzed by using a one-way analysis of variance (ANOVA) followed by a multiple comparison of Tukey. Prism 2.01 program (Graph Pad, CA, USA) was used for calculating probability values.
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Fig. 1. Immunohistochemical localization of PR in the uterus of pseudopregnant and MPA-treated rabbits. PR protein was observed by immunohistochemistry in NP rabbit (A), pseudopregnant rabbit on Day 1 (B), 3 (C), 5 (D) and 7 (E). PR protein in OVX + vehicle treated animals (G) and OVX + MPA rabbits on Days 1 (H), 3 (I), 5 (J) and 7 (K). Arrows indicate specific nuclear immunostaining. In (F) there was a negative control section of a NP rabbit uterus, and in (L) a negative control section of an OVX + vehicle rabbit. In both negative controls, the primary antibody was omitted. Cells were counterstained with Mayer’s hematoxylin. Barr = 100 m.
3. Results There was similar glandular morphology in the uterus of hCG- and MPA-treated rabbits. Small endometrial glands were present the first 3 days both in pseudopregnant and OVX animals treated with MPA (Fig. 1B, C and H, I, respectively). Great arborizations formed by endometrial glands
were observed on Day 7 of pseudopregnancy, and there was less arborization in OVX + MPA-treated rabbits (Fig. 1 E vs. K, respectively). PR immunostaining was observed in the nucleus of several uterine cells. In NP and OVX + vehicle rabbits, the number of stromal PR immunopositive cells and the intensity of the immunostaining were greater as compared
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Fig. 2. PR protein in epithelial and stromal cells in the rabbit uterus. The HSCORE of PR was calculated, as described in Section 2, in uterine epithelial (A) and stromal cells (B): black bars, pseudopregnant rabbits (Days 1 to 7); open bars, OVX rabbits treated with MPA (Days 1 to 7). Control animals (0) are non-pregnant rabbits (black bar) or OVX + vehicle rabbits (open bars). Results are expressed as mean ± SEM. Different letters indicate a difference between groups (P < 0.001).
with epithelial cells (P < 0.001) (Fig. 1A and G). No staining was observed in negative control of NP rabbit uterus and OVX + vehicle uterus where the primary antibody was omitted (Fig. 1F and L, respectively). In the epithelium of NP and OVX + vehicle animals PR HSCORE was 131.0 ± 2.0 and 168.9 ± 4.72, respectively, whereas in the lamina propia (stromal cells) the score was greater (P < 0.001) (253.0 ± 6.67 and 206.66 ± 8.95, respectively) (Fig. 2). There were changes in PR immunoreactivity in pseudopregnant and OVX + MPA-treated rabbits (P < 0.001) in relation to NP and OVX + vehicle group, respectively, with exception of epithelial cells on Day 7 OVX + MPA and stromal cells on Day 5 OVX + MPA, where the HSCORE was similar to OVX animals (Fig. 2A and B). In uterine epithelial cells of pseudopregnant rabbits PR HSCORE was increased (P < 0.001) in relation to that of NP rabbits (Fig. 2A). The amount of immunoreactivity was similar from Day 1 to 5, and was robust on day 7 (Fig. 2A). In contrast, there was a decrease (P < 0.001) in the HSCORE in stromal cells from Days 1 to 7 of pseudopregnancy as compared with the NP rabbits (Fig. 2B). A gradual increase (P < 0.001) was observed in PR HSCORE of the uterine epithelium of OVX + MPA from Day 1 to day 5, reaching a maximum on Day 3 (Fig. 2A). In stromal cells, there was a decrease in PR immunostaining during treatment with MPA (P < 0.001) (Fig. 2B), although an increase (P < 0.001) was observed in PR HSCORE on Day 5, in relation to the previous days (Fig. 2B).
4. Discussion In the present study PR immunoreactivity and distribution of receptors were evaluated in the uterus of the female rabbit in two progestational conditions: pseudopregnancy and after MPA treatment. In the rabbit endometrium hCG stimulates formation of glands by epithelial cell proliferation (Conti et al., 1981) between Days 6 and 8 after treatment (Hegele-Hartung et al., 1992); this observation is in agreement with results of the present study. On Day 7 of pseudopregnancy induced by hCG, significant changes in endometrium, including great arborizations formed by endometrial glands were observed in the present study.
Estrogens are potent mitogenic compounds that induce cellular proliferation in the female reproductive tract. In the present study, pseudopregnant rabbits had greater arborizations formed by endometrial glands. During pseudopregnancy in rabbits a significant increase on E2 concentrations is observed (Browning et al., 1980; Challis et al., 1973). This increase in E2 may be related with the increase in arborization observed in the present study. In contrast, in OVX + MPA-treated rabbits on Day 7 there was a lesser glandular arborization pattern. This may be due to the fact that these animals are OVX, and E2 concentrations were less than in pseudopregnant rabbits. In addition, MPA has an antiestrogenic effect in the rabbit uterus (Misao et al., 1995) where it exerts antiproliferative effects on estrogen-stimulated uterine growth. In the present study, there was an increase in PR HSCORE in uterine epithelial cells during the first 7 days of pseudopregnancy. This could be due to the well known priming effect of E2 on PR because E2 is required for the induction of PR synthesis in female reproductive tracts and there is an increase of E2 serum concentrations during early pseudopregnancy (Browning et al., 1980; Challis et al., 1973). In contrast with the present results, Hegele-Hartung et al. (1992) found a gradual diminution of PR in epithelial cells after Day 4. Iwai et al. (1991) reported a diminution in number of PR immunoreactive cells in the uterine glandular epithelium by 48 h after hCG injection and disappearance of PR 3 days after hCG administration. These differences may be explained by the method of quantification because in those reports number of glandular epithelial immunoreactive cells were only analyzed, while in the present study PR HSCORE in luminal and glandular epithelial cells after treatment with hCG was assessed. There is a diminution of PR gene expression in the rabbit endometrium (epithelial and stromal cells) on Day 3 and 4 of pregnancy (Anzaldúa et al., 2007; GutiérrezSagal et al., 1993), this could be due to an increase of progesterone plasma concentrations (Camacho-Arroyo et al., 1996). Although progesterone and estrogen plasma concentrations are similar in pregnancy and pseudopregnancy conditions (Browning et al., 1980; Challis et al., 1973), a different PR HSCORE pattern was observed in stromal and epithelial cells. This suggests regulation of PR gene expression not only depends on progesterone and
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estrogen plasma concentrations, but also on other local or paracrine factors such as hCG/LH receptors (Jensen and Odell, 1992) and the fertilized egg which is present in the uterus between 60 and 72 h post-coitus (Osada et al., 1999). Presence of hCG/LH receptors in the rabbit endometrium (Jensen and Odell, 1992; Sawitzke and Odell, 1991) can mediate hCG effects in the uterus (Ziecik et al., 1992). MPA induced an initial increase in PR HSCORE of uterine epithelial cells on Days 1 and 3, followed by a gradual diminution on Days 5 and 7 (Fig. 2 A); whereas in stromal cells of the lamina propia a PR diminution occurred from the first to third day of treatment as compared with the OVX rabbits (Fig. 2B). On Day 5 post-MPA treatment an increase in PR HSCORE was observed, however, this increase was similar to OVX control group. Changes on Day 5 are not related to the fact that animals were treated with MPA every 5 days, because animals on Day 5 had not been treated on that day yet. Thus, a diminution of amounts of MPA could be related to the increase in PR HSCORE observed on this day. In the present study, results indicate an opposite regulation of PR HSCORE between epithelium and stromal uterine cells of pseudopregnant rabbits and OVX + MPA treatments, because PR increases in the uterine epithelial cells of both progestin-treated groups and there were decreases in the stromal cells (Fig. 2A and B). These results suggest a differential regulation of PR in two different cell types in the uterus. The reason of this particular pattern of regulation is unknown. Interestingly, MPA increases PR binding in the ewe and the rabbit (Zhang et al., 1989) and it has a potent progestational effect on the rabbit uterus (Spilman et al., 1986), but it also functions as an antiestrogenic drug by reducing the number of estrogen receptors in some target tissues of rabbit (Di Carlo et al., 1984) (which, on the contrary, are increased by E2 ), besides, in the rabbit uterus MPA causes a very evident reduction in weight (Di Carlo et al., 1984). These results could be related with the PR down-regulation in epithelial cells observed from Day 5 to 7 post-MPA treatment in the present study. Moreover, MPA down-regulates PR gene expression in the mouse uterus (Actis et al., 1998). In the present study, there was PR down-regulation in the stromal cells of OVX + MPA rabbits. Comparable results were obtained for immunohistochemistry detection of two isoforms of PR (A and B) in stromal cells of the human endometrium after intermittent treatment with oral MPA for 3 months, in patients with endometrial hyperplasia. This reduction was also observed in PR isoforms in the epithelial cells (Vereide et al., 2006). Although there is a progestational hormonal environment both in pseudopregnant and OVX + MPA rabbits in the present study, the mechanisms of signaling pathways of paracrine interactions between epithelial and stromal cells are not similar. In other reports, PR gene expression via ER␣ was completely opposite between the uterine and vaginal epithelium in mice; estrogen up-regulates PR in the vaginal epithelium and down-regulates PR in the uterine epithelium (Kurita et al., 2000). In summary, a specific gene expression pattern of PR was observed between pseudopregnant and OVX + MPA-
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treated rabbits in uterine epithelial and stromal cells. Further studies are needed to understand the mechanisms by which progestins mediate stromal–epithelial interactions in female reproductive tissues. Acknowledgements The authors express their gratitude to Francisco López López for technical support. We thank Dr. Edwin Milgrom and Dr. Perrot-Applanat for the kind gift of Mi 60-10 antibody. This work was supported in part by grant from DGAPA-UNAM (PAPIIT-ES212101 and IN219710-2), and CONACyT (AC-89544 and 80338). References Actis, A.M., Dorfman, V.B., Caruso, S.P., Levin, E., 1998. Effects on mouse uterus of three antitumoral drugs acting upon estrogen and progesterone receptors directly and through other transduction pathways. Biochem. Pharmacol. 55, 273–278. Aluja, A.S., 2002. Animales de Laboratorio y la Norma Oficial Mexicana (NOM-062-ZOO-1999). Gaceta Médica de México 138, 295–298. Anzaldúa, A.S., Camacho-Arroyo, I., Reyna-Neyra, A., Pérez-Martínez, M., Cerbón, M., 2007. Regional differences in expression of progesterone receptor in oviduct and uterus of rabbit during early pregnancy. Comp. Biochem. Physiol. 147 A, 685–690. Beier, H.M., 2000. The discovery of uteroglobin and its significance for reproductive biology and endocrinology. Ann. N.Y. Acad. Sci. 923, 9–24. Blauer, M., Heinonen, P.K., Martikainen, P.M., Tomas, E., Ylikomi, T., 2005. A novel organotypic culture model for normal human endometrium: regulation of epithelial cell proliferation by estradiol and medroxyprogesterone acetate. Hum. Reprod. 20, 864–871. Browning, J.Y., Keyes, P.L., Woli, R.C., 1980. Comparison of serum progesterone, 20␣ dihydroprogesterone and 17 estradiol in pregnant and pseudopregnant rabbits: evidence for postimplantation recognition of pregnancy. Biol. Reprod. 23, 1014–1019. Camacho-Arroyo, I., Pasapera, A.M., Cerbón, M.A., 1996. Regulation of progesterone receptor gene expression by sex steroid hormones in the hypothalamus and the cerebral cortex of the rabbit. Neurosci. Lett. 214, 25–28. Camacho-Arroyo, I., González-Arenas, A., González-Agüero, G., GuerraAraiza, C., González-Morán, G., 2003. Changes in content of progesterone receptor isoforms and estrogen receptor alpha in the chick brain during embryonic development. Comp. Biochem. Physiol. 136 A, 447–452. Challis, J.R., Davies, I.J., Ryan, K.J., 1973. The concentrations of progesterone, estrone and estradiol 17 in the plasma of pregnant rabbits. Endocrinology 93, 971–976. Conti, C.J., Giménez-Conti, I.B., Zerbe, G.O., Gerschenson, L.E., 1981. Differential effects of estradiol-17 beta and progesterone on the proliferation of glandular and luminal cells of rabbit uterine epithelium. Biol. Reprod. 24, 643–648. Di Carlo, F., Racca, S., Conti, G., Gallo, E., Muccioli, G., Sapino, A., Bussolati, G., 1984. Effect of long-term administration of high doses of medroxyprogesterone acetate on hormone receptors and target organs in the female rat. J. Endocrinol. 103, 287–293. Feil, P.D., Bardin, C.W., 1979. The use of medroxyprogesterone acetate to study progestin receptors in immature, pregnant, and adult rabbit uterus. Adv. Exp. Med. Biol. 117, 241–254. Gadsby, J.E., Keyes, L., Bill, C.H.2nd., 1983. Control of corpus luteum function in the pregnant rabbit: role of estrogen and lack of a direct luteolytic role of the placenta. Endocrinology 113, 2255–2262. Gutiérrez-Sagal, R., Pérez-Palacios, G., Langley, E., Pasapera, A.M., Castro, I., Cerbón, M.A., 1993. Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit. Mol. Reprod. Dev. 34, 244–249. Hegele-Hartung, C., Chwalisz, K., Beier, H.M., 1992. Distribution of estrogen and progesterone receptors in the uterus and immunohistochemical study in the immature and adult pseudopregnant rabbit. Histochemistry (Germany) 97, 39–50. Iida, K., Kobayashi, N., Cono, H., Miyamoto, A., Fukui, Y., 2004. A comparative study of induction of estrous and ovulation by three different intravaginal devices in ewes during the non-breeding season. J. Reprod. Dev. 50, 63–69.
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S.R.A. Arce et al. / Animal Reproduction Science 120 (2010) 173–178
Iwai, T., Fujii, S., Nanbu, Y., Nonogaki, H., Konishi, I., Mori, T., Okamura, H., 1991. Effect of chorionic gonadotropin on the expression of progesterone receptor and estrogen receptors in rabbit ovarian granulose cells and the uterus. Endocrinology 129, 1840–1848. Jensen, J.D., Odell, W.D., 1992. Identification of LH/hCG receptors in rabbit uterus. Proc. Soc. Exp. Biol. Med. 189, 28–30. Karbowski, B., Vollmer, E., Schneider, P.G., 1992. Steroid receptors in the fallopian tube–morphological and functional investigation in rabbits. Prog. Histochem. Cytochem. 26, 140–144. Kraus, W.L., Katzenellenbogen, B.S., 1993. Regulation of progesterone expression and growth in the rat uterus: modulation of estrogen actions by progesterone and sex steroid hormone antagonists. Endocrinology 132, 2371–2379. Kurita, T., Lee, K.J., Cooke, P.S., Taylor, J.A., Lubahn, D.B., Cunha, G.R., 2000. Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract. Biol. Reprod. 62, 821–830. Lee, V.H., Fields, P.A., 1991. Rabbit relaxin: the influence of pregnancy and ovariectomy during pregnancy on the plasma profile. Biol. Reprod. 45, 209–214. Lessey, B.A., Killman, A.P., Metzger, D.A., Haney, A.F., Greene, G.L., McCarty Jr., K.S., 1988. Immunohistochemical analysis of the human uterine estrogen and progesterone receptors throughout the menstrual cycle. J. Clin. Endocrinol. Metab. 67, 334–340. Misao, R., Nishigaki, M., Hori, M., Ichigo, S., Fujimoto, J., Tamaya, T., 1995. Effects of danazol and medroxyprogesterone acetate on estrogen(estradiol and estriol) specific binding sites in rabbit uterus. Gynecol. Endocrinol. 9, 29–35. Osada, H., Tsumoda, I., Matsuura, M., Satoh, K., Kanayama, K., Nakayama, Y., 1999. Investigation of ovum transport in the oviduct: the dynamics of the oviductal fluids in domestic rabbit. J. Int. Med. Res. 27, 176–180. Perrot-Applanat, M., Logeat, F., Groyer-Picard, M.T., Milgrom, E., 1985. Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies. Endocrinology 116, 1473–1484.
Perrot-Applanat, M., Groyer-Picard, M.T., García, E., Lorenzo, F., Milgrom, E., 1988. Immunocytochemical demonstration of estrogen and progesterone receptors in muscle cells of uterine arteries in rabbits and humans. Endocrinology 123, 1511–1519. Ross, P.J., Adler, J.F., Cellejas, S.S., Butler, H., Alberio, R.H., 2004. Estradiol benzoate given 0 or 24 h after the end of a progestagen treatment in postpartum suckled beef cows. Theriogenology 62, 265–273. Sawitzke, A.L., Odell, W.D., 1991. Uterine binding sites for LH/hCG can be modulate by hormonal status in rabbits and rats. Acta Endocrinol. (Copenh.) 124, 322–330. Spencer, T.E., Bazer, F.W., 2002. Biology of progesterone action during pregnancy recognition and maintenance of pregnancy. Front. Biosci. 7, 1879–1898. Spilman, C.H., Gibson, R.E., Beuving, D.C., Campbell, J.A., 1986. Progestin and antiprogestin effects on progesterone receptor transformation. J. Steroid. Biochem. 24, 383–389. Vereide, A.B., Kaino, T., Sager, G., Arnes, M., Ørbo, A., 2006. Effect of levonorgestrel IUD and oral medroxyprogesterone acetate on glandular and stromal progesterone receptors (PRA and PRB), and estrogen receptors (ER-␣ and ER-) in human endometrial hyperplasia. Gynecol. Oncol. 101, 214–223. Zaino, R.J., Clarke, C.L., Feil, P.D., Satyaswaroop, P.G., 1989. Differential distribution of estrogen and progesterone receptors in rabbit uterus detected by dual immunofluorescence. Endocrinology 125, 2728–2734. Zhang, X., Stone, G.M., Miller, B.G., 1989. In vitro binding of progesterone, cronolone and medroxyprogesterone acetate to uterine progesterone receptors of the sheep, rabbit and mouse. Reprod. Fertil. Dev. 1, 223–230. Ziecik, A.J., Jedlinska, M., Rzucidllo, S.J., 1992. Effect of estradiol and progesterone on myometrial LH/hCG receptors in pigs. Acta Endocrinol. (Copenh.) 127, 185–188.