Influence of oestrogen receptor-α gene expression on cervical patency in pyometric bitches

Introduction

Chastain and Ganjam (1986) [2] stated that endometrial gland proliferation was induced by the oestrogen (E2) and the rise in serum progesterone (P4) level down regulated the ER in normal dogs. Oestrogen supported the proliferation of endometrial glands and P4 which aided in secretory activity. Hence, concurrent prolonged action of both E2 and P4 resulted in the proliferative cystic changes which finally resulted in cystic endometrial hyperplasia (CEH). Hardy et al. (1994) [5] reported that expression of ER was high in CEH-P cases. Cock et al. (1997) [1] concluded that the abnormalities in the ER played a crucial role in progress of pyometra in dog and weren't only factor responsible for the disease. Kovacs et al. (2004) [8] reported that increased concentrations of E2 led to increased PR and ER expression, while a decrease in concentrations of these receptors was related to increased plasma concentrations of P4. Reduced ER expression may contribute to cervical closure, resulting in retention of uterine contents and increased systemic illness (De Bosschere et al., 2003) [4] . Understanding the role of ER expression in cervical patency may help to explain the pathophysiology of pyometra and aid in better diagnosis and therapeutic decision-making.

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 ⁰C) and transported to the laboratory and stored at-80 ⁰C 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 were checked by agarose gel (one percent W/V) electrophoresis. The concentration and quality of RNA were checked by NanoDrop (Thermo Scientific, NanoDrop TM 2000 Spectrophotometer, USA) method.

Complementary DNA was synthesised from isolated total RNA using RevertAid first strand cDNA synthesis kit (Thermo Scientific, Cat# K1621). For cDNA synthesis, 500 ng of RNA from each sample was reverse transcribed. Exon spanning primers were designed using online primer design software NCBI. GenBank Accession number were NM_001286958.1 and AF021873.2 for amplification of ERα and β-actin (Table 1). The specificity of the primers was checked using BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). PCR was carried out to amplify 345 bp, and 141 bp fragments of ERα, and β-actin genes from canine cervix tissue. The amplified products were checked by two percent agarose gel electrophoresis.

Relative quantification using real time (qRT-PCR)

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 ERα 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. The details of reaction mix and reaction protocol are given in table 2 and 3. The exact calculations for finding relative quantification by the Livak (2001) [10] method the given below:

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

RNA samples having optical density ratio (OD) 260/280 above 1.8 were used for this study. Average values of OD 260/280 and 260/230 ratio for the cervical tissue samples were 2.06 ± 0.09 and 2.05 ± 0.18 respectively. Good quality total RNA was isolated from eighteen cervical tissue samples. Clear bands for 28S and 18S were obtained by 0.8 percent agarose gel electrophoresis of RNA. Total RNA isolated was converted into cDNA using random hexamer primer method.

Real time quantitative PCR of ER-α in cervical tissue

The composition of PCR, temperature and number of cycles were optimized for the efficient amplification of ER-α and β-actin in Illumina PCR machine. The qRT-PCR amplification plot and derivative melt curve of β-actin and ER-α are shown in plate 1, 2, 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. The sequences obtained were subjected to NCBI BLASTn software alignment and was found to have 100% sequence similarity with ER-α gene of Canis lupus familiaris gene. Thus, giving confirmative that the amplicon amplified for real time PCR study was specific to ER-α gene.

Relative Quantification of ER-α gene expression between dioestrous and pyometricbitches

The expression of target gene (ER-α) 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, there was no statistically significant difference in the expression of ER-α in control (1-fold) and open (1.05-fold) cervix pyometra at five percent level. Expression of ER-α was similar in control (1 fold) and closed (1.04 fold) cervix pyometra and found to be statistically non-significant (p value > 0.05). Expression of ER-α was similar in open (1 fold) and closed (0.99 fold) cervix pyometra and found to be statistically non-significant (p value > 0.05).

Oestrogen receptor (ER-α) expression in uterine cervix

In the present study, ER-α in the uterine cervix of control, open and closed cervix pyometra had no significant difference in expression signifying the non-existence of its influence in controlling the cervical patency. Corroborative to the findings of current study, indicating similarity in expression of ER-α in the cervix of dioestrous, closed, open cervix pyometric dogs, Tamada et al. (2012) [13] reported that there was no correlation observed between ER-α expression in cervix and its patency in closed and open cervix pyometric dogs. Similar study reports by Kunkitti et al. (2011) [9] also reported that ER-α concentration in the uterine cervix showed non-significance between closed and open cervix pyometric dogs. They also reported that quantification and identification of ER-α in the cervix and uterus of open and closed cervix pyometra groups did not showed any difference. They concluded that ER-α expression didn't affect the cervical patency in pyometra affected bitches neither in anoestrous nor dioestrous. Volpato et al. (2012) [15] concluded that ER-α and β concentration was same in closed and open cervix pyometric dogs and ruled out involvement of these receptors in controlling cervical patency. Kovac et al. (2004) [8] reported that E2 induced up-regulation of ER, but in the present study, though the E2 concentration was more, the high circulating P4 levels in pyometric and dioestrous bitches, would have down-regulated the ER expression in the uterine cervix. Similar observations were made by Johnston et al. (1985) [7] who reported that P4 suppressed ER synthesis and activation. Similar observations on the downregulations of ER in the uterus of dogs were also reported by Darko et al. (2018) [3] who observed a weak reaction on ER in dogs with pyometra during the dioestrous phase, signifying the down-regulatory effect of P4 on ER. Also, Ververidis et al. (2004) [14] had mentioned that eventhough serum oestradiol-17ᵦ levels were high it couldn't activate uterine ER, unless circulating P4 level was withdrawn. (2017) [12] stated that the mechanism of cervix dilation in pyometra bitches might not be involved with the expression of ER. In the present study, ER-α in the uterine cervix of control, open and closed cervix pyometra had no significant difference in expression signifying the non-existence of its influence in controlling the cervical patency.

Acknowledgement

AcknowledgementThe author sincerely thanks Dr. C. Jayakumar, Dr. K.Raji, COVAS, Mannuthy for providing research facilities, and KVASU for the assistantship and fellowship support.

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