Nuclear estrogen and progesterone receptors in the ...

Animal Reproduction Science 101 (2007) 28–37

Nuclear estrogen and progesterone receptors in the oviduct of heifers under natural and superovulated estrous cycles Guilherme Ribeiro Valle a,∗ , Geovanni Dantas Cassali b , José Carlos Nogueira b , Antônio Carlos Santana Castro b , Adelina Martha Reis b , Fábio Maur´ıcio Cardoso a , Cláudia Beatriz Oliveira Figueiredo b , Ernane Fagundes Nascimento c a

Departamento de Medicina Veterinária, Pontif´ıcia Universidade Católica de Minas Gerais, Brazil b Instituto de Ciˆ encias Biológicas, Universidade Federal de Minas Gerais, Brazil c Escola de Veterin´ aria, Universidade Federal de Minas Gerais, Brazil Received 7 November 2005; accepted 21 August 2006 Available online 30 August 2006

Abstract Oviducts from 22 crossbred heifers were examined for the presence of nuclear estrogen (ER␣) and progesterone (PR) receptors at different phases (estrus, metaestrus and diestrus) of naturally occurring estrous cycles and estrous cycles during which superovulation was induced. Receptors were detected by immunohistochemistry in the epithelial cells, connective tissue and muscular layer of oviductal infundibulum, ampulla, ampullary/isthmic transition and isthmus. Epithelial ER␣ was found along the entire oviduct regardless of the cycle phase and of the circulating concentrations of 17␤-estradiol (E2 ) and progesterone (P4 ). Epithelial PR was found mainly at the ampullary-isthmic transition and isthmus and more intensely at the estrus phase but their amount was not correlated with P4 concentrations. ER␣ in the connective tissue was more abundant at the infundibulum and ampulla, regardless of the phase of the estrous cycle and of E2 and P4 circulating concentrations. PR in the connective tissue was found mostly at the ampulla, regardless of the estrous cycle phase but no correlations were found between amount and P4 concentrations. ER␣ staining intensity in the muscular layer was similar at all phases of the estrous cycle and at all anatomical segments of the oviducts. However, PR staining was more intense at the isthmus during the metaestrus phase and it was negatively correlated with P4 concentrations. In general, data from the present research suggest that P4 exerts

∗ Corresponding author at: Rua do Ros´ ario 1081, Bairro Angola, Betim, CEP 32630-000 Minas Gerais, Brazil. Fax: +55 31 3539 6908. E-mail address: [email protected] (G.R. Valle).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.08.026

G.R. Valle et al. / Animal Reproduction Science 101 (2007) 28–37

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an inhibitory role upon ER␣ and PR. Also, no differences were found between animals subjected or not to superovulation. © 2006 Elsevier B.V. All rights reserved. Keywords: Bovine; Oviduct; Estrogen receptor; Progesterone receptor; Estrous cycle

1. Introduction Steroid hormones act on target cells by binding to specific receptors, which are located in the cytosol and/or nucleus and activate the genome (King and Greene, 1984; Perrot-Applanat et al., 1985; Leake and Habib, 1987). There are two types of estrogen receptors (ER), the alpha (ER␣) and the beta (ER␤), the former being preferentially found in the oviduct (Pelletier et al., 2000; Wang et al., 2000). ER occur predominantly at the estrogenic phase of the human oviduct (Press et al., 1986; Coppens et al., 1993; Amso et al., 1994) whereas in the rat Pelletier et al. (2000) and Wang et al. (2000) have found ER␣ along the mucosa and muscular layers at all phases of the estrous cycle. Under natural estrous cycles, 17␤-estradiol (E2 ) stimulates the production of both ER and progesterone receptors (PR) whereas progesterone (P4 ) exerts an inhibitory effect on these receptors in several species (Wathes and Lamming, 1995; Wu et al., 1996; Meikle et al., 2001). In the human oviduct, Devoto and Pino (1991) have identified a down regulation mechanism whereby E2 stimulates both ER and PR production while P4 reduces PR. According to Ulbrich et al. (2003), the amount of mRNA for ER␣ and PR within the bovine oviduct is more abundant during the follicular phase of the estrous cycle but the intensity of these receptors is greater at the beginning of the luteal phase. In heifers, superovulation did not have any effect upon the presence of both ER and PR within the oviduct (van de Leemput et al., 2001). The objective of the present study was to investigate possible relationships among circulating concentrations of E2 and P4 concentrations and ER␣ and PR within the oviduct of crossbred heifers at different phases of natural (physiological E2 and P4 concentrations) and superovulated (high E2 and P4 concentrations) estrous cycles. 2. Materials and methods 2.1. Animals and treatments Crossbred heifers (Bos taurus taurus × Bos taurus indicus; n = 22) with ages ranging from 2 to 4 years were kept at pasture and checked for signs of behavioral estrus twice a day. As the animals displayed the first signs of estrus, they were randomly assigned to each one of the following groups: (a) slaughtered 17 h after estrus (Est group); (b) slaughtered 4 days after estrus (Met group); (c) slaughtered 11 days after estrus (Die group); (d) submitted to superovulatory treatment and slaughtered 17 h after estrus (Estsup group); (e) submitted to superovulatory treatment and slaughtered 4 days after estrus (Metsup group). Superovulation was obtained by i.m. injections of follicle stimulating hormone (FSH) (Pluset, Calier, Brazil) in decreasing doses (total dosage of 350 IU) at intervals of 12 h from the 10th day following estrus. At the 12th day after estrus, 25 mg of dinoprost trometamina (Lutalyse, Rhodia, Brazil) were also i.m. injected. Immediately before slaughter, 3 mL of venous blood were collected into assay tubes containing ethylenediaminetetraacetic acid (Vacutainer EDTA K3 , B.D., Brazil) and centrifuged for 10 min

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at 400 × g. Blood plasma obtained was frozen at −20 ◦ C until analysis by radioimmunoassay (Estradiol Maia Kit and Progesterone Maia Kit, BioChem ImmunoSystems, Italy). All samples were analyzed in the same assay. The intra-assay variation coefficients for E2 and P4 were 7.0 and 8.1%, respectively. Minimum detection concentrations for P4 and for E2 were 0.022 ng/mL and 1.0 pg/mL, respectively. 2.2. Tissue preparation and immunohistochemical analysis Tissue samples from the infundibulum, ampulla, ampullary/isthmic transition and isthmus of both oviducts were fixed in 10% neutral buffer formalin, embedded in paraffin wax and sectioned at 5 ␮m. Immunohistochemistry was performed by using the streptavidin–biotin–peroxidase complex method (Ultra Vision Large Volume Detection System antipolyvalent, HRP ready to use, Lab Vision, USA) with a monoclonal antibody against PR (clone hPRa2, dilution 1:20, Neomarkers, USA) and with a monoclonal antibody against ER␣ (clone Ab-17, dilution 1:200, Neomarkers, USA), both displaying reactivity to bovine. Heat-induced antigen retrieval using the Dako Antigen Retrieval Solution (Dako, Denmark) was previously obtained in a wet bath for 20 min. The reagents were applied according to a protocol described elsewhere (Cassali et al., 2001). Diaminobenzidine (Large volume DAB substrate system, Lab Vision, USA) was used as a chromogen and followed by counterstaining. Positive controls were assessed by using samples of endometrium of estrus heifers, and negative controls were assessed by using normal serum as the primary antibody. The intensity of nuclear staining for the primary antibodies in the oviductal epithelium, connective tissue and muscular layer was blindly analyzed (no previous knowledge of the tissue samples) by two observers, according to the following staining score criteria: 0 = less than 10% as lack; 1 = between 10 and 40% as weak; 2 = between 40 and 80% as moderate; 3 = more than 80% as intense. Although steroid receptors may also be located in the cytosol, only nuclear expression was considered as specific and the mean score of the two observers was considered. 2.3. Statistical analysis The Wilcoxon test was used for comparing right and left oviducts, as well as ipsi and contralateral oviduct in relation to the ovary containing either a corpus luteum in formation (Met group), formed (Die group) or a preovulatory follicle (Est group), in those groups of heifers under natural estrous cycle. Comparisons among experimental groups, including data of hormonal concentrations, were performed by using Kruskal–Wallis and Mann–Whitney tests; Friedman method was employed for comparisons among different regions of the oviducts. The relationships among variables were identified by Spearman correlation coefficient. The level of significance considered for the statistical analysis was 5% (P < 0.05) and results were expressed as means ± standard deviation (age, body weight and hormonal concentrations) or medians (receptors staining scores). 3. Results 3.1. Hormone and receptor concentrations Data are provided in Table 1 for circulating concentrations of E2 and P4 . In all segments of the oviducts, the staining intensity for both ER␣ and PR was similar in right and left oviducts, and in ipsi and contralateral oviducts in relation to the ovary with a corpus luteum or a pre-ovulatory follicle, regardless of the estrous cycle phase.


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Table 1 Age, body weight and hormonal concentrations (mean ± S.D.) of heifers Groupa

Age (years)

Body weight (kg)

Hormonal concentrationsb E2 (pg/mL)

Est Estsup Met Metsup Die

3.0 3.3 2.7 3.5 3.3

± ± ± ± ±

0.0 0.5 0.6 0.6 0.5

398.0 374.0 366.3 397.5 368.3

± ± ± ± ±

30.2 17.9 18.6 14.9 37.4

10.27 27.40 12.23 10.80 16.55

± ± ± ± ±

1.75 AB 11.58 B 0.65 AB 1.24 A 10.55 AB

P4 (ng/mL) 0.18 0.65 0.73 21.58 11.55

± ± ± ± ±

0.13 A 0.42 AC 0.12 AB 18.60 BC 7.07 B

Letters (A–C) within the same column indicate significant differences (P < 0.05). a Est, estrus; Estsup, superovulated estrus; Met, metaestrus; Metsup, superovulated metaestrus; Die, diestrus. b E , 17␤-estradiol plasma concentration; P , progesterone plasma concentration. 2 4

3.2. ERα nuclear staining Fig. 1(a–c) shows ER␣ staining and Table 2 presents the mean scores for nuclear ER␣ staining at the different regions of the oviducts according to the experimental groups and to the estrus (Est + Estsup) and metaestrus (Met + Metsup) phases. Differences in the ER␣ staining intensity were not observed among the segments of the oviducts, regardless of the estrous cycle phase. Regarding the nuclear staining in connective tissue, the staining intensity was more intense at the infundibulum and ampulla at the estrus phase; and more intense at the infundibulum, intermediate at the isthmus and less intense at the ampulla and ampullary/isthmic transition at the metaestrus phase (Table 2). 3.3. PR nuclear staining Results for PR nuclear staining (Fig. 1h–j) in the epithelium indicate the staining was more intense in the isthmus, intermediate at the ampullary/isthmic transition and less intense at the ampulla and infundibulum. This pattern of reduction of staining intensity from the isthmus towards the infundibulum was also seen when the animals were considered as grouped into estrus and metaestrus phases. Along the oviduct, the greatest staining intensity was always found in animals at the estrus phase (Table 3). PR staining in the connective tissue was more intense in the ampulla at the estrus phase of the estrous cycle; and less intense staining was seen in Est group as compared to the Estsup group at the isthmus (Table 3). However, the nuclei in the muscular layer were more intensely stained for PR at the ampullary/isthmic transition during the estrus phase of the estrous cycle (Table 3). 3.4. Correlations between steroid levels and ERα and PR staining intensity Significant correlations between circulating concentrations of E2 and P4 and ER␣ staining intensity were not found. However, epithelial PR staining intensity at the ampulla and at ampulla/isthmus was negatively correlated with P4 circulating concentrations. This pattern followed that observed for the connective tissue of the entire oviduct, and for the ampullary/isthmic transition and isthmic muscle layer (Table 4). Significant correlations between ER␣ and PR within the same oviduct segment were also found. The following positive correlations were also observed: between epithelial ER␣ and con-

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Fig. 1. Nuclear immunostaining of alpha estrogen (a–c) and progesterone (h–j) receptors at different cells of heifers oviduct. Immunostaining nuclei are brown and non-immunostaining nuclei are blue. (a) Epithelial (e), connective tissue (ct) and muscular (m) cells of the ampullary/isthmic transition (Die group). (b) Intense immunostaining of epithelial (e) and connective tissue (ct) cells of the ampulla (Estsup group). (c) Intense immunostaining of epithelial cells (e) and moderate of connective tissue cells (ct) of the infundibulum (Die group). (d–g) Estrus heifer endometrium (Est group) as nuclear immunostaining control: alpha estrogen receptors negative (d) and positive (e) control and progesterone receptors negative (f) and positive (g) control. (h) Lack of immunostaining of epithelial (e) and connective tissue (ct) cells of the infundibulum (Met group). (i) Intense immunostaining of epithelial cells (e) and weak of connective tissue cells (ct) of the infundibulum (Met group). (j) Isthmic immunostaining (Met group): intense in epithelial cells (e), lack in connective tissue cells (ct) and intense in smooth muscle cells (m). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)


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Table 2 Scores for staining intensitya (median) of alpha estrogen receptors in the oviducts of heifers at different phases of natural and superovulated estrous cycles Groupb

Epithelium Est Estsup Met Metsup Die Estrus phase Metaestrus phase Connective tissue Est Estsup Met Metsup Die Estrus phase Metaestrus phase Muscular layer Est Estsup Met Metsup Die Estrus phase Metaestrus phase

Region Infundibulum

Ampulla

Ampulla/isthmus

Isthmus

2.9 3.0 3.0 2.8 2.6

3.0 3.0 3.0 3.0 3.0

3.0 3.0 3.0 3.0 3.0

3.0 3.0 3.0 3.0 3.0

3.0 3.0

3.0 3.0

3.0 3.0

3.0 3.0

2.4 2.4 2.5 2.3 1.6

2.1 1.6 1.5 1.6 1.9

1.6 1.5 1.3 1.5 1.0

1.8 1.5 1.3 2.0 1.6

2.4 b 2.5 b

1.9 b 1.5 a

1.5 a 1.5 a

1.5 a 1.8 ab

– – – – –

– – – – –

3.0 3.0 3.0 3.0 2.8

3.0 3.0 3.0 3.0 3.0

– –

– –

3.0 3.0

3.0 3.0

Letters (a and b) within the same line indicate significant differences (P < 0.05). a 0, none; 1, weak; 2, moderate; 3, intense. b Est, estrus; Estsup, superovulated estrus; Met, metaestrus; Metsup, superovulated metaestrus; Die, diestrus; Estrus phase, Est + Estsup; Metaestrus phase, Met + Metsup.

nective tissue PR at the infundibulum (r = 0.50); between connective tissue ER␣ and epithelial PR (r = 0.51); between connective tissue ER␣ and connective tissue PR at the ampulla (r = 0.70); and between muscular ER␣ and muscular PR at the isthmus (r = 0.51). At the ampulla/isthmus segment, positive correlation between muscular ER␣ and muscular PR approached significance (r = 0.47; P = 0.056). 4. Discussion The use of superovulation provided a great amplitude of E2 and P4 circulating concentrations, greater than those normally found in estrous cycle, allowing the establishment of correlations between these hormone concentrations and the receptors, despite the estrous cycle phase. Studies in primates (McClellan et al., 1984; Press et al., 1986; Devoto and Pino, 1991; Amso et al., 1994; Brenner and Slayden, 1994) and rats (Pelletier et al., 2000; Wang et al., 2000) have shown

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Table 3 Scores for staining intensitya (median) of progesterone receptors in the oviducts of heifers at different phases of natural and superovulated estrous cycles Groupb

Epithelium Est Estsup Met Metsup Die Estrus phase Metaestrus phase Connective tissue Est Estsup Met Metsup Die Estrus phase Metaestrus phase Muscular layer Est Estsup Met Metsup Die Estrus phase Metaestrus phase

Region Infundibulum

Ampulla

Ampulla/isthmus

Isthmus

0.9 1.5 0.0 0.0 0.5

1.5 1.3 0.3 0.5 0.5

2.5 2.9 3.0 2.8 2.7

3.0 3.0 3.0 2.8 2.7

1.1 B,a 0.0 A,a

1.4 B,a 0.3 A,ab

3.0 B,b 1.8 A,bc

3.0 B,b 2.8 A,c

0.6 1.6 1.8 0.0 0.0

1.9 2.0 0.8 0.4 1.3

1.5 1.4 0.0 0.4 0.0

1.3** 1.8** 0.0 0.1 0.3

1.8 B,a 0.0 A

2.0 B,b 0.5 A

1.5 B,a 0.3 A

1.4 B,a 0.0 A

– – – – –

– – – – –

3.0 2.9 1.0 0.5 0.0

3.0 3.0 3.0 1.8 2.0

– –

– –

3.0 B 1.0 A

3.0 2.3

Letters (a–c) within the same line and (A and B) within the same region indicate significant differences (P < 0.05). a 0, none; 1, weak; 2, moderate; 3, intense. b Est, estrus; Estsup, superovulated estrus; Met, metaestrus; Metsup, superovulated metaestrus; Die, diestrus; Estrus phase, Est + Estsup; Metaestrus phase, Met + Metsup. ** Significant differences between natural and superovulated estrus (P < 0.05).

a trend towards greater nuclear ER in the oviductal epithelium at the estrus phase. However, few studies involving these receptors were reported in bovine. Thus, van de Leemput et al. (2001) have demonstrated their presence during estrus of heifers; and Ulbrich et al. (2003) reported positive correlations between ER␣ and E2 , with greater ER␣ during early luteal phase. In the present study, the presence of ER␣ was demonstrated during the entire estrous cycle (Table 2); furthermore, no correlation was found between their presence and E2 and P4 circulating concentrations. Therefore, the epithelial in the oviduct does not seem to be associated to the oscillating concentrations of these hormones during the estrous cycle. Epithelial ER␣ were found in all oviductal segments (Table 2), which is in agreement with previous reports in rats (Pelletier et al., 2000; Wang et al., 2000). Based on the negative correlations found between PR intensity and P4 circulating concentrations, as well as on the absence of correlation between PR and E2 scores at the epithelia of ampullary and ampullary/isthmic transition, it is plausible to assume that P4 might inhibit PR,


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Table 4 Correlations between hormonal levels and scores for progesterone receptors staining in the oviducts of heifers* Region

E2 a rb

P4 a Pb

r

P

Infundibulum Epithelium Connective tissue

0.208 0.206

0.379 0.384

−0.387 −0.582

0.083 0.006

Ampulla Epithelium Connective tissue

−0.046 0.077

0.848 0.747

−0.546 −0.456

0.010 0.038

Ampulla/isthmus Epithelium Connective tissue Muscular layer

0.185 0.103 0.011

0.461 0.685 0.965

−0.534 −0.560 −0.721

0.018 0.013 0.000

Isthmus Epithelium Connective tissue Muscular layer

0.093 0.341 0.083

0.705 0.153 0.736

−0.378 −0.619 −0.604

0.101 0.004 0.005

a b *

Hormonal concentrations: E2 , 17␤-estradiol plasma concentration; P4 , progesterone plasma concentration. r, correlation coefficient; P, significance level. Significant when P < 0.05.

which would appear to be stimulated by concentrations of E2 that were less than those found in the present experiment. These results seem to favor the negative effect of P4 upon the induction of its own receptors and not a positive effect of E2 upon PR, as described by Ulbrich et al. (2003). Possibly, E2 concentrations in lesser amounts than those reported for animals with normal estrous cycles are enough to stimulate PR. In the connective tissue, ER␣ staining was more intense at the ampulla and infundibulum (Table 2) and PR staining at the ampulla (Table 3). PR intensity was less, however, at the metaestrus phase of the estrous cycle (Table 3). These results seem to confirm earlier studies in rats, in which both receptors were found along the entire oviduct but with less intensity during dioestrus (Pelletier et al., 2000; Wang et al., 2000). Less PR intensity after exogenous P4 treatment was reported in rabbits (Hyde et al., 1989). Indeed, a negative correlation was found between PR and P4 circulating concentrations, but not between PR and E2 circulating concentrations. These findings would corroborate that P4 inhibits amounts of PR in the connective tissue and, as occurred in the epithelia, no stimulation was associated to E2 concentrations. According to Brenner and Slayden (1994), the role of these receptors in the connective tissue of primate oviduct is to regulate the activity of the oviductal ciliary cells because the latter do not have ER and PR. In the present study, however, no differential staining was found between ciliary cells and secretory cells, thus demonstrating that both receptors are present in the two cell types. Regarding the ER␣ staining intensity in the oviductal muscle layer, no significant variation was observed in the present study (Table 2). This finding is similar to that reported in women, but in the latter an increase of ER␣ associated with time of ovulation was observed (Amso et al., 1994). The increased PR concentration in the muscle layer during the estrus phase (Table 3) was also similar to that reported in women (Amso et al., 1994). Following that observed in the connective tissue, a negative correlation was found between P4 circulating concentrations and PR in the muscle layer.

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Nuclear PR within the oviductal epithelium, connective tissue and muscular layer is apparently regulated by P4 (Table 4), which is in accordance with the findings by Devoto and Pino (1991) in women. However, based on the amounts of ER␣ found in the present study, E2 did not seem to affect the amount ER␣ and PR. The most plausible hypothesis for explaining the regulatory role exerted by P4 , but not by E2 , upon the oviduct, is that E2 basal concentrations, less than those normally found along the estrous cycle, would be enough to stimulate amount of ER␣ and PR. This could explain the lack of correlation between E2 circulating concentrations and the scores of ER␣ and PR in the oviduct, because that basal concentrations would always be present. A few studies might corroborate this latter hypothesis. Thus, Theofan and Notides (1984) have found PR in the uterus of calves; Tasende et al. (1996) have reported that the return of ER and PR within the postpartum uterus of sheep occurs after the return of ovarian activity with follicular growth; Wu et al. (1996) have shown the stimulatory effect of E2 upon ER within the myometrium and endometrium after exogenous injection of E2 in ovariectomized sheep, in which the circulating concentrations of E2 were possibly below the minimum necessary to stimulate amount of these receptors. In humans, Amso et al. (1994) have stated that supraphysiological concentrations of E2 would not cause any deleterious effect upon embryo survival in the oviduct, as opposed to that occuring in the uterus. In bovine, van de Leemput et al. (2001) did not find any effect of superovulation upon the intensity of ER and PR within the oviduct. The present study seems to confirm these previous findings, because only in the connective tissue of the isthmus an increased intensity of nuclear PR was observed in heifers under superovulated estrus (Estsup group) as compared to those under natural estrus (Est group, Table 3). In conclusion, it appears from the present data that P4 exerts an inhibitory role upon its own receptors in bovine oviducts. Furthermore, superovulation did not appear to affect the general pattern of ER␣ and PR in those organs. Acknowledgements The present study was supported by FAPEMIG (CAG 492/2001); we are grateful to Dr. Armando Leal do Norte and to Prof. Décio de Souza Graça for their collaboration. References Amso, N.N., Crow, J., Shaw, R.W., 1994. Comparative immunohistochemical study of oestrogen and progesterone receptors in the Fallopian tube and uterus at different stages of the menstrual cycle and the menopause. Hum. Reprod. 9, 1027–1037. Brenner, R.M., Slayden, O.D., 1994. Cyclic changes in the primate oviduct and endometrium. In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 541–569. Cassali, G.D., Silva, P., Rema, A., Gartner, F., Gobbi, H., Tafuri, W.L., Schmitt, F.C., 2001. A new methodology for the improvement of diagnostic immunohistochemistry in canine veterinary pathology: automated system using human monoclonal and polyclonal antibodies. Arq. Bras. Med. Vet. Zootec. 53, 326–331. Coppens, M.T., Boever, J.G., Dhont, M.A., Serreyn, R.F., Vandekerckhoue, D.A., Roels, H.J., 1993. Topographical distribution of oestrogen and progesterone receptors in the human endometrium and Fallopian tube. Histochemistry 99, 127–131. Devoto, L., Pino, A.M., 1991. Estradiol and progesterone receptors in the human oviduct. Arch. Biol. Med. Exp. 24, 295–300. Hyde, B.A., Blaunstein, J.D., Black, D.L., 1989. Differential regulation of progestin receptor immunoreactivity in the rabbit oviduct. Endocrinology 125, 1479–1483.


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