Immunolocalization of Progesterone Receptors in the Canine Oviduct around Ovulation

Authors

  • MZ Tahir,

    1. INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France
    2. ENVA, UMR1198, Maisons-Alfort, France
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  • K Reynaud,

    1. INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France
    2. ENVA, UMR1198, Maisons-Alfort, France
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  • G Mawa,

    1. INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France
    2. ENVA, UMR1198, Maisons-Alfort, France
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  • S Thoumire,

    1. INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France
    2. ENVA, UMR1198, Maisons-Alfort, France
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  • S Chastant-Maillard,

    1. INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France
    2. ENVA, UMR1198, Maisons-Alfort, France
    Current affiliation:
    1. INP-ENVT, Reproduction, Toulouse Cedex, France
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  • M Saint-Dizier

    Corresponding author
    1. ENVA, UMR1198, Maisons-Alfort, France
    2. AgroParisTech, UFR Génétique Elevage Reproduction, Paris Cedex 05, France
    • INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France
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Author's address (for correspondence): M Saint-Dizier, AgroParisTech, UFR Génétique Elevage Reproduction, 16 rue Claude Bernard, 75231 Paris Cedex 05, France. E-mail: stdizier@agroparistech.fr

Contents

In the bitch, oocyte maturation, sperm storage, fertilization and early embryo development take place within the oviducts under high and increasing circulating progesterone concentrations. To investigate the potential effects of progesterone on the canine oviduct, nuclear progesterone receptors (PR) were localized. Oviducts were collected by ovariectomy from adult Beagle bitches during anestrus, after the onset of proestrus but prior to the Luteinizing Hormone (LH) peak (Pre-LH), after the LH peak but before ovulation (Pre-ov) and on Days 1, 4 and 7 after ovulation (n = 3 bitches per stage). The cellular distribution of PR was studied by immunohistochemistry (IHC) in the ampulla, isthmus and tubal part of the utero-tubal junction (UTJ). Plasma progesterone and 17β-oestradiol were assayed on the day of surgery. PR were specifically expressed in the nuclei of epithelial, stromal and muscular cells in the ampulla, isthmus and UTJ. The IHC scores did not vary from one oviductal region to another. However, the epithelium displayed higher scores than the stroma at anestrus, Pre-ov, Days 4 and 7, and also higher scores than muscle at Days 4 and 7 (p < 0.05). Immunohistochemistry scores in the stroma and muscle decreased at Days 4 and 7 compared with previous stages (p < 0.05). Furthermore, muscular IHC scores were positively correlated with circulating 17β-oestradiol concentrations and negatively correlated with circulating progesterone concentrations (p < 0.05). In conclusion, PR were identified in the canine oviduct, with differences in expression between tissues and times around ovulation, suggesting that progesterone may regulate tubal functions and reproductive events in this species.

Introduction

The mammalian oviduct is the site of crucial events for reproduction, such as gamete transport, sperm capacitation and storage, fertilization and early embryo development (Croxatto 2002; Hunter 2005, 2012). The canine oviduct slightly differs from that of other mammals, in that it also provides a suitable environment for meiosis resumption of oocytes that occurs between 2 and 4 days after ovulation (Tsutsui 1989; Reynaud et al. 2005). Furthermore, the lifespan of gametes and embryos within the oviducts is particularly long in the bitch compared with other mammals: mature oocytes may remain fertilizable for 8 days (vs 8–18 h in most mammals; Betteridge 1995; Tsutsui et al. 2009), spermatozoa remain mobile up to 11 days (vs <3 days in most mammals; England et al. 2006; Hunter 2012), and embryos do not leave the oviduct earlier than the morula or blastocyst stage, 8–10 days after ovulation (vs 2 and 4 days in the sow and cow, respectively; Tsutsui 1989; Betteridge 1995; Reynaud et al. 2005). Anatomically, the proximal and distal parts of the oviduct, referred to as ampulla and isthmus respectively, may be involved in the maturation and fertilization of oocytes, whereas the UTJ seems to play important roles in sperm storage and capacitation around ovulation (Tsutsui 1989; England et al. 2006).

The tubal events described earlier take place in the presence of high and increasing circulating concentrations of progesterone (P4), because of pre-ovulatory luteinization of ovarian follicles (Concannon et al. 1989). In other mammals, P4 was shown to be an important regulator of the morphology and functions of the oviduct (Hunter 2005, 2012). In the bitch, the regulation of tubal functions by steroid hormones is still poorly understood. Important changes across the oestrous cycle were reported in the morphology and cellular activities of the canine oviduct epithelium (Verhage et al. 1973; Steinhauer et al. 2004; Urhausen et al. 2011). Some of those changes are correlated with circulating P4 and 17β-oestradiol (E2) concentrations (Verhage et al. 1973; Steinhauer et al. 2004; Urhausen et al. 2011). Specific P4 binding sites were evidenced in oviductal cytosols from Beagle bitches (Lessey and Gorell 1980; Lessey et al. 1981), and nuclear receptors for P4 (PR) were immunolocalized in the ampulla and fimbriae of canine oviducts at various stages of the oestrous cycle (Vermeirsch et al. 2002). However, to our knowledge, the presence of PR in the isthmus and UTJ has not been examined. Furthermore, no comprehensive study in the literature reports on PR distribution in the canine oviduct around the time of ovulation, oocyte meiosis resumption and fertilization. Thus, this study was designed to investigate the localization and distribution of PR in the whole canine oviduct at precise times relative to ovulation.

Materials and Methods

Animals

Eighteen Beagle bitches from our experimental kennel were included in this study. The protocol was approved by the ethical committee of the Alfort National Veterinary School. The monitoring of ovarian cycles was carried out using vaginal smears, plasma P4 assays (Elecsys® enhanced chemiluminescence kit; Roche Diagnostics, Meylan, France; intra- and inter-assay coefficients of variation < 2%) and transabdominal ovarian ultrasonography (MyLab, Biosound Easote, Saint-Germain-en-Laye, France), as previously described (Reynaud et al. 2005). On the day of surgery, plasma P4 and E2 were assayed (Elecsys® kit; Roche Diagnostics; intra- and inter-assay of variation were 1.2% and 6.5%, respectively). Ovariectomies were performed at various stages of the oestrous cycle (n = 3 bitches per stage): anestrus, after the onset of proestrus but prior to the LH peak (Pre-LH), after the LH peak and before ovulation (Pre-ov), 1 day (Day 1), four (Day 4) and seven (Day 7) days after ovulation. Following ovariectomy, one oviduct was trimmed free of surrounding tissues and divided into three regions (i.e. ampulla, isthmus and UTJ) after separation of the infundibulum. For immunohistochemical examination, the ampulla section consisted of 0.5 cm of the most cranial part of the oviduct, the isthmus section consisted of 0.5 cm in the mid to distal part of the oviduct, and the UTJ section consisted of 0.5 cm of the most distal part of the oviduct (i.e. the tubal part of the UTJ). These tissue sections were embedded in cryoembedding medium (OCT Embedding Matrix, CellPath, Newtown, UK), frozen in liquid nitrogen and stored at −20°C until use.

Western blot

For Western blot analysis, a canine ovary collected at the Pre-LH stage was homogenized in a lysis buffer for protein extraction. Proteins (50 μg) were heated at 95°C for 5 min, migrated on a 10% SDS-PAGE gel then transferred onto a nitrocellulose membrane (Trans-Blot®; Bio-Rad, Marnes La Coquette, France). Membranes were incubated with a monoclonal mouse anti-human PR antibody (MA1-410; Affinity BioReagents, Golden, CO, USA) diluted at 2 μg/ml at 37°C for 1 h. Membranes were then washed, incubated with an anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, Suffolk, UK), washed again then incubated with enhanced chemiluminescence reagent detection solution (Super Signal® West Pico, Thermo Scietific Pierce Protein Biology, Rockford, IL, USA) for film exposition.

Immunohistochemistry

For immunohistochemistry, frozen oviducts were serially sectioned (7 μm) using a cryostat (CM3050 S®; Leica Biosystems, Nussloch, Germany), and sections were fixed for 10 min in acetone at 4°C. Non-specific protein binding was inhibited by incubation with porcine serum (10% in PBS) for 30 min. Sections were incubated with the anti-PR antibody described earlier and diluted at 1 μg/ml, then washed and incubated with an anti-mouse biotinylated secondary antibody (LSAB® kit, Dako, Trappes, France). Negative controls were obtained by replacing the primary antibody by mouse IgG1 (Sigma, Saint-Quentin Fallavier, France) at the same dilution. Sections were then treated with 3% H2O2 and incubated with a streptavidin–horseradish peroxidase complex (LSAB kit). The signal was detected using a solution of diaminobenzidine (DAB). Finally, sections were counterstained with alcian blue and mounted for examination under light microscopy. The IHC staining was evaluated semi-quantitatively by the proportion of stained cells in the epithelium, stroma and muscular layer in each oviductal region, as previously described in the bovine oviduct (Valle et al. 2007). Only nuclear staining of the cells was considered. IHC score was graded on a scale of 0 (<10% stained cells), 1 (between 10% and 40%), 2 (between 40% and 80%) and 3 (>80%). The scores were determined independently by two observers, and the average of their scores was used for evaluation.

Statistical analysis

Results are presented as mean ± SD of three bitches per group. Comparisons among estrous stages were performed by using the nonparametric Kruskal–Wallis test, followed, when significant, by the Bonferroni test. The Friedman test was used for comparisons between regions of the oviduct and between tissue layers. The relationship between variables was identified by Spearman correlation coefficients. Statistical values of p < 0.05 were considered significant.

Results

Plasma P4 concentrations were low during anestrus (0.05 ± 0.02 ng/ml) and at Pre-LH (0.34 ± 0.2 ng/ml), then increased from Pre-ov (3.8 ± 0.4 ng/ml) to Day 4 (31.4 ± 13.4 ng/ml) and Day 7 (43.0 ± 8.3 ng/ml). Plasma E2 concentrations were high at Pre-LH (45.9 ± 6.9 pg/ml) and Pre-ov (53.5 ± 20.9 pg/ml), then decreased from Day 1 (15.9 ± 10.0 pg/ml) to Day 7 post-ovulation (5.4 ± 5.9 pg/ml). A significant negative correlation was found between P4 and E2 circulating concentrations (r = −0.732, p < 0.01).

Western blot analysis confirmed that both PR-A and PR-B isoforms were specifically detected in canine ovarian tissue as proteins at the expected weights of 95 and 120 kDa, respectively (Fig. 1).

Figure 1.

Western blot analysis of nuclear progesterone receptors in a canine ovary at the Pre-LH stage. Molecular weight markers are indicated on the left

Immunohistochemistry staining revealed the expression of PR in the nuclei of epithelial, stromal and muscular cells in the three regions of the canine oviduct, that is, the ampulla (Fig. 2, left column), isthmus (Fig. 2, middle column) and tubal part of the UTJ (Fig. 2, right column). No staining of the cytoplasm was observed in any cell group at any stage. Immunostaining was absent in all negative controls (Fig. 2–s–u).

Figure 2.

Immunohistochemical localization of nuclear progesterone receptors in the ampulla (left column), isthmus (middle column) and utero-tubal junction (right column). Various stages were analysed: anestrus (a–c), Pre-LH (d–f), Pre-ov (g–i), Day 1 (j–l), Day 4 (m–o) and Day 7 (p–r) after ovulation. Negative control (s–u). E, epithelium; S, stroma; M, smooth muscle. Scale bars = 75 μm

There were no differences in IHC scores between oviductal regions (Table 1). However, the scores did vary depending of the tissue layer and cycle stage (p < 0.01). The oviductal epithelium displayed a higher PR expression than the stroma at anestrus, Pre-ov, Days 4 and 7 (p < 0.05). PR were also expressed higher in the epithelium than in the oviduct muscle at Days 4 and 7 (p < 0.05). Furthermore, significant changes in PR expression were recorded between stages in the stromal and muscular layers (p < 0.01). Globally, PR expression in the stroma was significantly higher at Pre-LH, Pre-ov and Day 1 compared with anestrus, and decreased significantly at Days 4 and 7 compared with Pre-LH and Pre-ov (p < 0.05). In the muscular layer, PR expression was significantly higher at Pre-LH, Pre-ov and Day 1 compared with Days 4 and 7 (p < 0.05). Some of these differences remained significant in the stroma and muscle of the isolated isthmus and UTJ (Table 1). No significant correlations between circulating steroid concentrations and IHC scores were found either in the epithelium or in the stroma. However, the muscular scores were positively correlated with plasma E2 concentrations in the three regions of the oviduct (ampulla: r = 0.660, p < 0.01; isthmus: r = 0.763, p < 0.001; UTJ: r = 0.788, p < 0.001) and negatively correlated with plasma P4 concentrations in the isthmus and UTJ (r = −0.537 and −0.556, respectively, p < 0.05).

Table 1. Immunohistochemical scores for nuclear progesterone receptor expression in the canine oviduct during anestrus and at various stages around ovulation (means ± SD) In each tissue layer, values within a column with different superscripts are significantly different (p < 0.05)
Tissue layerStagesRegion
AmpullaIsthmusUtero-tubal junction
EpitheliumAnestrus2.7 ± 0.62.2 ± 1.02.2 ± 0.6
Pre-LH3.0 ± 0.03.0 ± 0.03.0 ± 0.0
Pre-ov3.0 ± 0.03.0 ± 0.02.3 ± 0.6
Day 12.7 ± 0.62.7 ± 0.62.2 ± 0.8
Day 42.8 ± 0.32.8 ± 0.32.8 ± 0.3
Day 72.8 ± 0.33.0 ± 0.03.0 ± 0.0
StromaAnestrus0.2 ± 0.30.3 ± 0.30.0 ± 0.0a
Pre-LH2.2 ± 0.82.5 ± 0.52.8 ± 0.3b
Pre-ov1.7 ± 0.31.5 ± 1.01.7 ± 1.2ab
Day 11.2 ± 0.81.2 ± 0.31.3 ± 0.8ab
Day 40.3 ± 0.30.2 ± 0.30.2 ± 0.3ab
Day 70.3 ± 0.30.7 ± 0.30.0 ± 0.0a
MuscleAnestrus0.2 ± 0.31.3 ± 0.8ab1.2 ± 1.0ab
Pre-LH2.0 ± 0.52.0 ± 0.9ab2.8 ± 0.3a
Pre-ov2.0 ± 0.02.5 ± 0.5a2.5 ± 0.5ab
Day 10.8 ± 0.61.7 ± 1.0ab1.8 ± 0.8ab
Day 40.0 ± 0.00.0 ± 0.0b0.0 ± 0.0b
Day 70.0 ± 0.00.3 ± 0.6ab0.0 ± 0.0b

Discussion

In the bitch, important reproductive events take place in the oviduct under high and increasing circulating P4 concentrations. In this work, the distribution of PR over the whole canine oviduct was examined for the first time at precise times around ovulation. PR were specifically expressed in the nuclei of several tissue layers in the ampulla, isthmus and tubal part of the UTJ. Changes in PR expression in specific tissue layers related to periovulatory stages were evidenced.

The cellular localization of PR observed in this study was in keeping with earlier results obtained by immunohistochemistry in the ampulla of bitches (Vermeirsch et al. 2002). In our findings, the expression of PR varied around ovulation in the stromal and muscular layers of the oviduct but remained constantly high in the epithelium. In contrast, Vermeirsch et al. (2002) observed variations in PR immunostaining scores in all tissue layers of the oviduct, including the epithelium. These conflicting findings may reflect differences in score calculation methods (scores combining staining intensity and proportion of stained cells in the study of Vermeirsch et al.) and to the time period considered after ovulation, that was much longer in the latter study (from proestrus to late metestrus). We observed a significant decrease in PR expression from Pre-LH to Day 7 post-ovulation in the stroma and muscle of the canine oviduct. Furthermore, expression changes in the muscular layer were correlated with the changes in plasma P4 and E2 concentrations. Vermeirsch et al. (2002) also reported a decrease in PR immunostaining in the canine ampulla from proestrus to late metestrus, which was negatively correlated with circulating E2 concentrations. These findings are in agreement with the current knowledge on regulation of PR expression: E2 induces the synthesis of PR, whereas P4 down-regulates its own receptor (Graham and Clarke 1997). Our results are also in keeping with radio-receptor studies conducted on canine oviduct cytosols, which reported a decrease in P4 binding sites when immature bitches were treated with both E2 and P4 (Lessey et al. 1981). Moreover, the present findings are consistent with previous data from our laboratory on PR gene expression in whole-oviduct sections from Beagle bitches at the same stages around ovulation (Tahir et al. 2011). Indeed, a significant decrease in PR mRNA levels was observed from Pre-LH to Day 7 in the three oviductal regions.

P4 regulates quantitatively and qualitatively the tubal fluid as well as the contractions of the muscular layer in the mammalian oviduct (Hunter 2005, 2012). The expression of PR in all tissue layers of the canine oviduct suggests that P4 may be an important regulator of tubal functions in this species too. Furthermore, the expression of PR in the muscle and stroma decreased significantly at Day 4 post-ovulation, that is, at the time of completion of oocyte meiosis and fertilization (Tsutsui 1989; Reynaud et al. 2005). As PR is differentially expressed in the smooth muscle of the bitch oviduct, it is likely that P4 modulates the response of muscular cells to myotrophic factors in a way that eventually modulates gamete and embryo transport after ovulation, as shown in other mammals (Croxatto 2002). It is noteworthy that the PR immunostaining in the canine oviduct epithelium displayed higher score values than in both the stroma and muscle, and did not decline over the entire time period examined. P4-associated variations in cell morphology and apoptotic activity were previously reported in the epithelium of the canine oviduct (Verhage et al. 1973; Steinhauer et al. 2004; Urhausen et al. 2011). Furthermore, sperm binding to and release from the sperm reservoir could be modulated by P4 around ovulation (England et al. 2006), probably through action on the oviductal epithelium, as in other domestic mammals (Hunter 2005, 2012).

In conclusion, the tissue- and stage-dependent expression of PR in the canine oviduct suggests that P4 regulates tubal functions in this species. The mechanisms by which P4 could indirectly modulate gamete storage and maturation around ovulation remain to be explored.

Acknowledgements

We are grateful to Marc Chodkiewicz for the critical review of this manuscript and to Bénédicte Grimard for her help in statistical analysis. Authors thank Ingrid Gruyer, Lucy Foucher and Cathy Claramonte for taking care of the animals.

Conflicts of interest

None of the authors have any conflicts of interest to declare.

Author contributions

MZT conducted experiments, analysed data and drafted the paper, KR designed and conducted experiments and revised the manuscript, GM and ST conducted experiments and analysed data, SCM designed experiments and revised the manuscript, MSD designed and condusted experiments, analysed data and revised the manuscript.

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