Progesterone (PR), Oestrogen (ER-α and ER-β) and Oxytocin (OTR) Gene Expression in the Oviduct and Uterus of Pregnant and Non-pregnant Bitches
Author's address (for correspondence): AAP Derussi, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, São Paulo, Brazil. E-mail: email@example.com
The aim was to assess hormone receptor gene expression in the oviduct and uterus during canine pregnancy. Nineteen pregnant bitches divided into four groups were ovariohysterectomized (OVH) at either day 8, 12, 21 or 60 of pregnancy, and five non-pregnant females underwent OVH 12 days after the pre-ovulatory Luteinizant Hormone (LH) surge and served as controls. RT-qPCR for progesterone (PR), oestrogen (ER-α and ER-β) and oxytocin (OTR) receptors was performed on the oviduct and uterine tissue. The mRNA PR expression in the uterus during early stages of pregnancy and the luteal phase was higher than at other times. The mRNA ER-β expression in the oviduct during early pregnancy was less than in non-pregnant bitches. In the uterus, the mRNA ER-β expression was higher in the initial stages of pregnancy. The ER-α expression was higher in the oviduct and uterus in advanced stages of pregnancy. The mRNA OTR expression in the oviduct was lower than in the uterus in control group. The expression of this receptor in oviduct and the uterus was higher in the final stages of pregnancy, when compared with other phases. These data suggested that the serum progesterone concentrations probably exert a direct control on the PR and ER (α and β) expression and indirectly on OTR expression in the bitch oviduct and uterus.
The pre-implantation embryonic period corresponds to the migration of the embryo within the oviduct and uterus. During this phase, the interaction between the embryo and endometrium is influenced by progesterone and placental hormones that act in a paracrine manner on the endometrium, promoting the development of the female reproductive tract and causing changes in gene expression that participate in the development of the foetus and maintenance of pregnancy.
The mechanisms of action of these hormones involve interaction with specific receptor proteins present in cells that lead to activation and transcription of responsive genes to these steroids (Wang et al., 1999), allowing the synthesis of a wide variety of growth factors that participate in endometrial decidual cellular proliferation. Systematic study of gene expression of hormone receptors may clarify some mechanisms that control biological processes involved in early embryonic development. The identification and characterization of gene expression are important for the understanding of crucial events during the peri-implantation period (Cui et al., 2007). The aim of this study was to assess hormone receptor gene expression in the oviduct and uterus during canine pregnancy.
Materials and Methods
Animal and collection of tissue
Twenty-four healthy mature bitches were assigned to the experiment. Nineteen were inseminated and five served as control. Artificial insemination was performed based on monitoring vaginal cytology and progesterone concentration to determine the onset of the pre-ovulatory surge of LH (progesterone range, 1–1.9 ng/ml) and estimation of the time of ovulation time (progesterone 4 ng/ml). The first insemination was performed between days +1 and +2 after the pre-ovulatory LH surge, and the second insemination was performed 48-h later.
All females underwent ovariohysterectomy (OVH) on predetermined days: Group A (n = 5): 8 days after the LH surge, Group B (n = 5): 12 days after the LH surge, Group C (n = 5): 21 days after the LH surge, Group D (n = 4): 60 days after the LH surge. OVH was performed in the non-pregnant females (n = 5) 12 days after the LH surge (Group E).
Following OVH, the oviduct and uterus were isolated and flushed for the purpose of embryo recovery to determine the gestational age. Sections of the middle portion of uterine horn and oviduct were recovered and stored in 7.5-ml cryogenic tubes and frozen at −80°C.
RNA isolation and RT-qPCR
Total RNA was extracted using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's protocol. Total RNA amounts were determined using a NanoDrop® (Nanodrop Technologies, Wilmington, DE, USA). Total RNA (2 μg) was treated with Amplification Grade Deoxyribonuclease I (Life Technologies Corporation). The RNA structural integrity was assessed using a bioanalyzer (2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA, USA), calculating the RNA Integrity Number; for all samples, this was >7 (on a scale of 1–10).
The cDNA was synthesized using a High Capacity cDNA archive kit (Life Technologies), according to the manufacturer's protocols The target and reference gene expression levels were detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) using an ABI 7300 Real-Time PCR System (Life Technologies Corporation) and were amplified using Power SYBR Green PCR Master Mix 2x (Life Technologies Corporation). Primer pairs for all genes were designed using sequences published in GenBank using Primer Express 3.0 software (Life Technologies Corporation). The expression stability of reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA and Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was assessed through geNorm VBA applet for Microsoft Excel.
GrapPad3 (GrapPad Software Inc, San Diego, CA, USA) was used for statistical analysis. Two-tailed paired t-tests were used to examine the results between different tissue types and anova for analysing the results of the same tissue type from different groups. Data were expressed as mean ± SD. Differences were considered significant with a p-value of <0.05.
The values of mRNA expression of the hormone receptor (PR, ER-B, ER-L OTR) in the uterus and oviduct in the different groups are shown in Table 1. The PR expression in the oviduct was not different between any of the groups. However, the PR expression in the uterus during the early stages of pregnancy (Group A) and in non-pregnant bitches (Group E) was higher than in the other stages of pregnancy (Group B, C, D), indicating a change in the PR expression during pregnancy.
Table 1. Mean ± SD mRNA expression of the hormone receptors PR, ER-β, ER-α and OTR in the uterus and oviduct in non-pregnant bitches and bitches at different stages of pregnancy
|PR||1.82 ± 0.31A||1.1 ± 0.15B||1.05 ± 0.29C||0.07 ± 0.05AD||2.12 ± 0.17BCD|
|ER-β||3.24 ± 0.24||5.26 ± 0.31A||3.4 ± 0.89||0.01 ± 0.001AB||5.2 ± 1.2B|
|ER-α||0.33 ± 0.12A||0.33 ± 0.12B||0.55 ± 0.16C||0.94 ± 0.18ABCD||0.41 ± 0.13D|
|OTR||0.12 ± 0.07A||0.14 ± 0.06B||0.41 ± 0.29C||1.41 ± 0.22ABCD||0.17 ± 0.08D|
|PR||0.55 ± 0.29||1.14 ± 0.73||0.84 ± 0.26||0.59 ± 0.08||0.83 ± 0.32|
|ER-B||0.07 ± 0.028A||0.21 ± 0.096||0.13 ± 0.051||0.01 ± 0.002B||0.29 ± 0.051AB|
|ER-α||0.13 ± 0.10A||0.19 ± 0.03B||0.24 ± 0.05||0.59 ± 0.09ABC||0.13 ± 0.05C|
|OTR||0.16 ± 0.19||0.22 ± 0.09||0.38 ± 0.36||0.49 ± 0.18A||0.02 ± 0.01A|
The ER-β expression in the oviduct in the early pregnant females was less than in non-pregnant bitches. In the uterus, the ER-β expression was higher in the initial phases of pregnancy (group A and B) and in group E. The ER α expression was higher in the oviduct in advanced stages of pregnancy (Group D vs A). Similar results were observed in the uterus.
In the oviduct, the OTR expression did not change during pregnancy but was numerically higher at day 60; expression of OTR in late pregnancy was significantly higher than in the non-pregnant control bitches. In the uterus, the OTR expression was different between pregnant and non-pregnant bitches, and expression was highest in bitches at day 60 of pregnancy compared with any of the other groups.
In bitches, progesterone has an important role in the establishment and maintenance of pregnancy, because it prepares the uterine epithelium for receptivity and endometrial and stromal cell differentiation. These changes ensure that embryo development can be supported (Mulac-Jericevic and Conneely, 2004).
Progesterone has a self-regulatory action on the PRs in the endometrium and glandular epithelium. In pregnant and cyclic females, PR expression is generally thought to be negatively correlated to plasma progesterone concentrations. In our study, we observed similar results, although in oviduct there was no variation in PR expression associated with non-pregnancy or any stage of pregnancy. We believe that this occurred because there is different affinity between the tissues and the receptor; findings that have already been described (Jefferson et al., 2000).
In adult women, ERs are expressed in epithelial cell and uterine and vaginal stroma. Oestrogen is obligatory for normal epithelial morphogenesis, cyto-differentiation and secretory activity in these tissues (Cooke et al. 1998). In our study, changes in both ER-α and β within the oviduct and uterus were the opposite to those that have been previously described for ovarian tissue (Hatoya et al., 2003). We believe that this occurred because of the action of progesterone in these tissues. However, the ER-α results were similar to those obtained in women, in which ER-α was downregulated at the time of implantation when progesterone concentrations were greatest (Lessey et al., 2006).
The increase in OTR expression found in the uterus and a lesser degree the oviduct during late pregnancy may be due to the role of this hormone in parturition and lactation (Mitchell et al., 1998) and potentially because progesterone concentrations are basal such that there is no progesterone block (Vermeirsch et al. 2000).
We conclude that ER (α and β), PR and OTR gene expression differs throughout the various stages of pregnancy and is largely influences by concentrations of progesterone at the target tissues.
We thank FAPESP (Process 2009/50718-0) for financial support.
Conflicts of interest
None of the authors have any conflicts of interest to declare.