Effect of progesterone receptor on cervical patency in canine pyometra

Introduction

Vermeirsch et al. (2000) [11] stated that because of progesterone (P4) action, cytosol and nuclear PR were reduced and uterine cytosol PR concentration was lower in cystic endometrial hyperplasia (CEH-P) bitches than in the normal ones during dioestrus which indicated a strong P4 action and an intense PR inactivation in the uterus. Vermeirsch et al. (1999) [11] stated that PR expression was badly associated with the serum P4 level, but the potency and total scores were significantly and positively correlated with the testosterone and oestradiol-17ᵦ level. Kovacs et al. (2004) [5] concluded that oestradiol-17ᵦ induced upregulation of estrogen receptor (ER) and PR and P4 down-regulated the ER. Ververidis et al. (2000) [11] reported that high serum P4 levels helped in over-activation of uterine PR, which led to higher than usual suppression of ER and PR. In CEH-P cases, high serum oestradiol-17ᵦ levels didn't perform during dioestrus, also when P4 was withdrawn it activated ERs during early anoestrus. Also, they stated that in CEH-P cases, which occurred during early anoestrus, uterine cytosol ER concentration was lower than the normal. On the contrary, Prapaiwan et al. (2017) [9] reported that in cyclic dogs, cervical dilation was influenced by these hormone receptors expression. Also, they stated that dilation of the cervix in both pyometric and dioestrous bitches was possibly regulated by different mechanisms. They concluded that the variation of gene localization and expression of the OTR in association with ER and PR in reproductive tissues of pyometric bitches, showed different OTR localisation and expression of the ER and PRs might be involved in pathogenesis of pyometra. Also they experimented that in affected bitches, ERα localisation has been reduced in the uterus and also integrated with suppression of E2 and/or prolonged P4 levels for triggering uterine infection. Identically, an increased PR localization and decreased ERα expression were noticed in both segments of the cervical tissue of pyometra cases. Darko et al. (2018) [2] stated that uterine endometrial cells with CEH expressed a moderate reaction on PR and a strong reaction on ER. But on comparison between the sex steroid receptors expression of uteri from pyometra cases and normal dioestrus bitches, ER was expressed lesser in all the uterine layers of pyometra affected bitches. But PR was expressed higher in almost all uterine layers of pyometra cases. Based on the above findings, it was assumed that changes in ERα and progesterone receptor expression in uterus of bitch had a vital function in pyometra pathogenesis.

Materials and Methods

Female dogs of various breeds showing clinical signs of pyometra were selected for this study. Six healthy female dogs in the dioestrous stage were included as the control group (Group III, n = 6). Pyometra was confirmed using trans-abdominal ultrasonography. Dogs diagnosed with pyometra were classified into open cervix (Group I, n = 6) and closed cervix (Group II, n = 6) based on clinical signs. All these bitches were subjected to OHE under standard anaesthetic protocols. After OHE, part of the cervix was collected and stored in the RNA later for gene expression studies. The samples were kept in ice box (4 °C) and transported to the laboratory and stored at-80 °C in deep freezer. After thawing, 30 mg of the collected tissue was removed from RNA later (Origin) and sectioned into small pieces using scissors and immediately processed for RNA isolation. The total RNA isolation was carried out using RNeasy fibrous tissue mini kit (Qiagen) as per manufacturer's instructions. Quality and integrity of isolated total RNA was checked by agarose gel (one percent W/V) electrophoresis. The concentration and quality of RNA was checked by NanoDrop (Thermo Scientific, NanoDrop TM 2000 Spectrophotometer, USA) method. Complementary DNA (cDNA) was synthesised from isolated total RNA using RevertAid first strand cDNA synthesis kit (Thermo Scientific, Cat# K1621). Exon spanning primers were designed using online primer design software NCBI. GenBank Accession number were NM_001003074.1, and AF021873.2for amplification of PR and β-actin (Table 1). The specificity of the primers was checked using BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers were custom-synthesized commercially (Sigma Aldrich Chemicals Pvt. Ltd., Bangalore) and obtained in lyophilized form. . PCR was carried out to amplify 258 bp and 141 bp fragments of PR and β-actin genes from canine cervix tissue. The annealing temperatures were optimised by using different temperatures ranging from 60-68 °C. Quantitative real time PCR was used to quantify mRNA in both relative and absolute terms. In the present study, this technique was used to find relative expression of genes PR in canine cervical tissue using β-actin as a reference gene. The relative quantification of gene expression was carried out using Illumina Eco ® qRT-PCR system using SYBR green chemistry.

Quantitative Real Time PCR

The details of reaction mix and reaction protocol are given in table 2 and 3. The reagents required for real time PCR were thawed and kept ready. Illumina Eco ® qRT-PCR system was set ready with pre-programmed plate layout and thermal cycling conditions before the onset of the experiment. The exact calculations for finding relative quantification by Livak [7]

Statistical analysis

Independent t-test was used for the statistical analysis of relative expression of ER-α, genes in cervix of pyometric and normal dioestrous bitches in three groups under study.

The data were analysed with one way ANOVA using SPSS software version 24.0 (IBM Corp. 2016) [6] .

Results and Discussion

The qRT-PCR amplification plot and derivative melt curve of PR and β-actin are shown in Plate 1, 2 and 3, 4 respectively. Melt curve analysis showed a single peak for each gene confirming absence of any non-specific product or primer dimer during qRT-PCR.

Relative Quantification of PR gene expression between dioestrous and pyometric bitches

The expression of target gene (PR) and reference gene (βactin) was compared to calculate ΔCT (Table 4, 5 &6) and the ΔCT of the control and experimental groups were compared to calculate ΔΔCT. A simple formula, 2 -∆∆Ct is used to calculate the relative fold gene expression of the samples In the present study, expression of PR was comparatively higher in open (14 folds) cervix pyometra than control (1 fold) group and this difference was found to be statistically significant (p value < 0.05). The expression of PR was comparatively higher in closed cervix pyometra (14 folds) than control group (1-fold) and this difference was found to be statistically significant (p value < 0.05). The expression of PR was similar in closed (1.01 folds) and open (1-fold) cervix pyometra and found to be statistically nonsignificant (p value > 0.05). The sequence obtained was blast using BLASTn suite available online and found 100% sequence similarity with PR gene Canis lupus familiaris gene. Thus, giving confirmative that the amplicon amplified for real time PCR study was specific to PR gene.

Progesterone receptor (PR) expression in uterine cervix

In the present study, expression of PR in closed and open cervix pyometra cases was more or less similar and nonsignificance was observed at five percent level. Whereas, expression of PR in closed cervix pyometra was 14 folds higher than the dioestrous group and this difference was found to be statistically significant (p value <0.05). Also, expression of PR in open cervix pyometra was 14 folds higher than dioestrous dogs and this variation was found to be statistically significant at five percent level. Hence, from the above observations it was inferred that PR expression was upregulated in the cervix of both open and closed cervix pyometra. The higher P4 levels (27.36 ng/mL) in normal dioestrous dogs, recorded in the study would have downregulated the PR expression in the uterine cervix which finds agreement with the findings of Amso et al. (1994) [1] who reported that PR were down-regulated by circulating serum P4. Also, Vermeirsch et al. (2000) [11] stated that the PR expression was inversely related to the serum P4 level. Higher expression of PRs (14 folds) observed in pyometric dogs could be attributed to the relatively lower P4 levels (17.19 and 22.38 ng/mL) in open and closed pyometric dogs respectively, this would have a reduced effect on the downregulation the PR expression in the uterine cervix as has been observed by Vermeirsch et al. (2000) [11] who stated the inhibitory effect of circulating P4 on its receptors. The PR expression between open and closed pyometric dogs was similar when compared against dioestrous bitches, which signifies the effect of PR in the pathogenesis of CEHpyometra. Corroborative to the findings of increased expression of PR in pyometric dogs, Darko et al. (2018) [2] also reported a strong reaction to PR in the uterus of the pyometric bitches. However, the PR expression did not vary between open and closed indicating the non-existence of its influence on the patency of cervix. On agreement to the present results, Tamada et al. (2012) [10] stated that no association was found between PR expression and cervical patency of pyometra bitches. Prapaiwan et al. (2017) [9] stated that the mechanism of cervical dilation in pyometric bitches might not be involved with the expression of PR.Similar study in pyometric uterus by Bosschere et al. (2003) [3] inferred that expression of PR in pyometricbitches uteri were more than that of dioestrous bitches. Also, Darko et al. (2018) [2] reported that the uterus with pyometra cases showed a moderate to strong reaction on PR, and a weak reaction on ER.

Kunkitti et al. (2011) [6] reported that PR concentration in the uterine cervix showed non-significance in difference between closed and open pyometric dogs regardless of oestrous stage. They also gave a different opinion regarding the expression of PR in pyometric bitches and stated that PR concentration in pyometric bitch's cervix were lower than dioestrous dogs which was contrary to our study. However, they also opined that PR expression didn't affect the cervical patency in pyometra affected bitches neither in anoestrus nor dioestrus. On contrary to the present findings, Volpato et al. (2012) [14] opined that concentrations of PR were comparatively higher in closed pyometric dogs when compared to open ones, however the difference in the expression of PR between closed and open pyometric dogs was without significance.

Conclusion

In this study it is concluded that there is no association between PRs and cervical patency in pyometric bitches. Even though the expression of PR was high in the open pyometric bitches, statistically no significance could be noticed. Hence the cervical patency is regulated by some other factors in canine pyometra.

References

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