Document Type : Original Article
Authors
1 Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
2 Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran;Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3 Laboratory Animal Center, Shiraz University of Medical Sciences, Shiraz, Iran
4 4Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
5 5Department of Animal Sciences, School of Agriculture, Shiraz University, Shiraz, Iran
6 Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran;6Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
7 7Biotechnology Institute, College of Agriculture, Shiraz University, Shiraz, Iran
Abstract
Keywords
There is no follicular development during pregnancy in the rat compared to the changes during the estrous cycle (
Kisspeptins belong to a family of peptides which are encoded by the
Gonadotropin-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide was discovered in birds as an inhibitory factor for gonadotropin release (
The aim of the present study was to evaluate the relative expression of RFRP-3 and KiSS-1 mRNAs in the hypothalamus of pregnant rats on days 7, 14 and 21 after mating. To achieve this goal, we need to determine pregnancy earlier than day 7 after mating with high accuracy and without using hormonal estrous synchronization. Hitherto, different non-invasive methods such as vaginal plug observation (day 1 after mating) (
In a randomized controlled experimental study, 40 mature female Sprague-Dawley rats (body weight 150-250 g) were selected and housed in The Laboratory Animal Center of Shiraz University of Medical Sciences, Shiraz, Iran. The animals were housed in standard cages, six per cage, in a controlled temperature room (22℃), with a 12 hours light and 12 hours dark cycle. Standard laboratory chow and tap water were available ad libitum.
Phases of their estrous cycle were determined by microscope observation of their vaginal smears (
The rats were assigned into three groups. The first group was the rats with diestrous cell characteristics in day 4 and metestrous/diestrous cell characteristics in day 5. The second group was the rats with metestrous cell characteristics in day 4 and metestrous/diestrous cell characteristics in day 5. The other cell characteristics in days 4 and 5 were assigned to the third group.
Twenty two adult (3-4 months old) female Sprague-Dawley rats (
Four ovariectomized rats, selected randomly, were used as the control group. The rats were anesthetized by an intraperitoneal injection of ketamine (100 mg/kg, Woerden, Netherlands) and xylazine (7 mg/kg, Alfazyne, Woerden, Netherlands) and ovariectomized through ventral midline incision. Further procedures were carried out after a 2-week recovery period.
The pregnant and ovariectomized rats were decapitated and brains were removed immediately. Pregnancy of 18 rats was confirmed with certainty by observing their pregnant uterus. The diencephalon was dissected out by an anterior coronal section, anterior to the optic chiasm, and a posterior coronal cut at the posterior border of the mammillary bodies. To separate ARC from AVPV, a third coronal cut was made through the middle of the optic tract, just rostral to infundibulum (
Total RNA was extracted, using the RNX-Plus buffer (Cinnagen, Tehran, Iran). Briefly, the tissue (100 mg) was ground in liquid nitrogen, transferred to RNX-Plus buffer (1 mL) in an RNase-free microtube, mixed thoroughly, and kept at room temperature for 5 minutes. Chloroform (0.2 mL) was added to the slurry and mixed gently. The mixture was centrifuged at 12,000 ×g (4℃) for 20 minutes and the supernatant was transferred to another tube and precipitated with an equal volume of isopropanol for 15 minutes. The RNA pellet was washed with 75% ethanol and quickly dried and re-suspended in 50 µL RNase-free water. The purified total RNA was quantified by Nano-Drop ND 1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). The DNase treatment was carried out using the DNase kit (Fermentas, St. Leon-Roth, Germany) according to the manufacturer¡¯s instructions. The DNase-treated RNA (3 µg) was used for the first strand cDNA synthesis, using 100 pmol oligo-dT, 15 pmol dNTPs, 20 U RNase inhibitor, and 200 U M-Mulv reverse transcriptase (Fermentas, St. Leon-Roth, Germany) in a 20 µL final volume. Primers were designed using Allele ID 7 software (Premier Biosoft International, Palo Alto, USA) for the reference gene, KiSS-1 (NM_181692) and RFRP-3 (NM_023952). The rat
Sequences of real time polymerase chain reaction (PCR) primers for evaluation of the relative expression of RFRP-3 and KiSS1 genes in rat
Primer | Sequence | Amplicon length (bp) |
---|---|---|
TGCTGCTTCTCCTCTGTG | 116 | |
CCAGGCATTAACGAGTTCC | ||
CTCAGCAGCCAACCTTCC | 165 | |
AAACCAGCCAGTGTCTTG | ||
AAGAAGGTGGTGAAGCAGGCATC | 112 | |
CGAAGGTGGAAGAGTGGGAGTTG | ||
KiSS1; Kisspeptin, RFRP-3; RFamide-related peptide-3 and GAPDH; Glyceraldehyde-3-phosphate dehydrogenase.
In study 1, relationship between the cell characteristics of vaginal smear (proestrus, estrus, metestrus and diestrus) on days 4 and 5 after mating and positive and negative results of pregnancy were compared using the Chi-square test (SPSS for Windows, version 11.5, SPSS Inc, Chicago, Illinois). Percent of pregnant rats between three groups were analyzed using one-way analysis of variance (ANOVA) (SAS 9.1 SAS Institute Inc., Cary, NC). Tukey post hoc test was used for comparison of means within the groups. Values of p≤0.05 were considered significant.
In study 2, the data on relative expression of KiSS-1 and RFRP-3 genes were subjected to the test of normality and analyzed by one-way ANOVA, and mean separation was performed by Tukey’s test at p=0.01. Group means and their standard errors are reported in the text and figures (GraphPad Prism v 5.01, GraphPad software Inc., San Diego, CA, USA).
In study 1, there was a positive association between pregnancy of rats and vaginal smear cell characteristics of metestrus (the same proportion among leukocytes, cornified and nucleated epithelial cells) or diestrous (a predominance of leukocytes) stages observed in days 4 and 5 after mating (p=0.001). If diestrous cell characteristics were observed in vaginal smear on day 4 after mating, and metestrous or diestrous cell characteristics were detected on day 5 after mating, 78.4% of rats would be pregnant. The cell characteristics of vaginal smear on days 4 and 5 were compared with cell characteristics of vaginal smear in different stages of estrous cycle in rat is shown in fig 1. Moreover, if metestrous cell characteristics were observed on day 4 after mating and metestrous or diestrous cell characteristics were detected on day 5 after mating, accuracy of pregnancy detection dropped to 60% which was not significantly different with the previous case (diestrous on day 4 and metestrous/diestrous on day 5). Only 16.7% of pregnant rats showed other cases of cellular characteristics of estrous cycle on days 4 and 5 after mating with chances to be pregnant was less than the two previous cases (p<0.05).
Accuracy of rat pregnancy detection by vaginal smear evaluation on days 4 and 5 after mating. a, b; Bars labeled with different letters are significantly different from each other at p<0.05.
In study 2, the mean and standard error of relative expression of RFRP-3 mRNA in DMH did not change during pregnancy (p>0.01,
In study 2, the mean and standard error of relative expression of RFRP-3 mRNA in DMH did not change during pregnancy (p>0.01,
Effect of pregnancy on the relative expression of KiSS-1 gene (mean ± SE) in the hypothalamic arcuate nucleus (ARC) of rats (n=6 for each pregnancy day). a, b; Different letters indicate significant difference (p<0.01).
The relative expression of KiSS-1 mRNA in ARC was at its peak in the first week of pregnancy and decreased 4-fold in the third week of pregnancy in rat. In contrast to our results, Roa et al. (
It has been shown that the number of GnIH neurons have a positive correlation with plasma progesterone concentration (
Decrease of GnRH and LH secretion during rat pregnancy may be controlled by constant expression of RFRP-3 mRNA and reduced expression of KiSS-1 mRNA. On the other hand, methods of early detection of pregnancy and exact determination of pregnancy time in rats are widely applied on pregnant rats. Our presented method, in addition to increasing the probability of rat pregnancy after the first mating event, can detect pregnancy with an accuracy of more than 60% on day 5.