Differential expression of prostaglandin F receptor in goat endometrium during estrous cycle and early pregnancy

Aim: Present study was undertaken to investigate profiling of prostaglandin F receptor (PGFR) mRNA expression during estrous cycle and early pregnancy. Materials and Methods: Female genitalia of goats (n=3) were collected from a local abattoir. Based on color, vasculature, size, and consistency of corpus luteum (CL), the uteri were classified into three stages of the estrous cycle: stage I: Days 3–5; stage II: Days 6–15, stage III: Days 16–21 and early pregnancy (<30 d foetus). Total RNA was isolated from endometrial scrapings of respective uteri and reverse transcribed followed by DNase treatment. cDNA was used as template to assess gene expression. Expression profiling was performed for PGFR gene using Real Time quantitative PCR (RT-qPCR). Results: At the time of luteolysis the expresion of PGFR was found to be high. However, during pregnancy the expression of PGFR was high as compared to stages of estrous cycle. Conclusion: Higher expression of PGFR at stage III showed luteolytic signal during estrous cycle. However, peaked expression during pregnancy revealed that the conceptus could be responsible for upregulated expression of PGFR in the endometrium during the implantation period.


Introduction
luteolysis [8] and establish/mainatanance of early pregnancy [9].Prostaglandins (PGs) act as a most important Expression of PGFR during estrous/mesuration regulator of female reproductive functions and cycle have been reported in human, horse, and cow associated pathologies [1].Among different classes of endometrium [6,10,11].However, during early pregn-PGs, PGF2α is one of the important prostanoids, ancy its expression was studied only in rat and pig produced by bovine and human endometrium [2,3].It endometrium [9,12].Although, goat is considered as regulates ovarian cycle through initiating the one of important livestock species with high economic regression (luteolysis) of CL and functions as major value, the information on expression profiles of PGFR luteolytic agent [4,5].Luteolysis has defined as has not been reported yet.Considering the importance functional and/or structural regression of CL regressed of PGFR in regulation of estrous cycle and embryo to permit a new ovarian cycle [4,5].Conversely, the survivability, the present study is proposed to function and integrity of the CL are required to investigate its expression profile in goat endometrium establish and maintain pregnancy.In domestic species, during estrous cycle and early pregnancy.luteolysis must be initiated to permit a new ovarian cycle to begin when conception does not occur.The

Materials and Methods
major route in PGF2α formation is via reduction of Tissue collection: Female goat genitalia were PGH2 by 9, 11-endoperoxidase reductase activity.
collected from a local abattoir and transported After biosynthesis, PGF2α is rapidly transported out of immediately to the laboratory on ice.Uteri specimens the cell, once released it acts in an autocrine or were washed with sterile phosphate buffer saline.paracrine manner.Prostaglandins mediate their effects Based on color, vasculature, size and consistency of CL through membrane-bound receptors in the uterus [6].
The age of foetus was determined (<30 d) on the basis Therefore, PGFR is found to be associated for both, of cranial length and weight.Uteri were opened longitudinally followed by scrapping of endometrial tissues from the uterus using RNase free glass slides.While in the gravid uteri, embryo/foetus along with the (n=3) foetal membranes was carefully removed and the ANOVA.All numerical data were expressed as a mean intercaruncular endometrium tissues were collected.
± SEM of 3 biological replicates from each reproduc-The specimens showing any abnormality or tive stages.inflammatory conditions were discarded.Tissue Results samples were collected from middle and cranial part of Expression profile of PGFS and PGFR: Relative quantithe ipsilateral and contralateral uterine horns.Tissues fication of PGFR transcripts was determined by Real were kept in RNA later (Ambion, USA) at -80°C until Time PCR.We observed significantly lower mRNA used.
expression of PGFR at stage I and II, in comparison to RNA isolation and cDNA synthesis: Total RNA was stage III (P<0.01).However, its expression was isolated using TRI reagent (Sigma, USA) following the approximately fifteen fold higher (P<0.01) in manufacturer's instructions.The RNA samples were pregnancy than stage III of estrous cycle.Similarly in treated with DNase using DNA-free DNase kit horse, the expression was found to be increased (Ambion, USA).The concentration and purity of RNA towards luteal phase, but its expression was constantly was determined spectrophotometrically at OD and 260 low during early pregnancy [6].In contrast, the PGFR OD and the integrity was examined by electro-280 was expressed at low levels and there were no changes phoresis.Total RNA was reverse transcribed using in its level during the estrous cycle in bovine Revert Aid cDNA synthesis kit (Fermentas, USA) endometrium [17].following manufacturer's instructions.Briefly, the cDNA was synthesized from 1 µg total RNA using random hexamer primers in a final volume of 20 µl.The resultant first strand cDNA was stored at -80°C until further use.

Real time quantitative PCR (RT-qPCR):
The mRNA expressions of the target and reference gene was quantified using RT-qPCR.The glyceraldehyde 3phosphate dehydro-genase (GAPDH) was used as an endogenous reference gene.The gene specific primers were designed using Primer3Plus online software tool (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/)(Table -1).The RT-qPCR was carried out using iQ5 real-time PCR system (Bio-Rad, USA).Luteolysis is mediated by the release of luteolytic 95°C for 30 sec, 60°C for 20 sec, 72°C for 20 sec, pulses of PGF2α by the uterus at the end of diestrus, followed by a dissociation stage of 95°C for 1 min, which must be suppressed by the conceptus to permit 60°C for 20 sec and 95°C for 30 sec.In order to rule out maternal recognition of pregnancy.Presence of PGFR the possible DNA contamination for each RNA sample in the caprine endometrium is in agreement with a no-RT control reaction was set up in which reverse detection of this receptor in uterus of ovine [18], equine transcriptase enzyme was omitted during cDNA [6], bovine [19], murine [12], and porcine [9].We synthesis.To determine the specificity of the PCR observed a high expression of PGFR mRNA in goat at reaction, a dissociation curve was generated after the stage III as compared to stage I and II during estrous completion of amplification.
cycle, which corresponds to elevated level of PGFS Statistical analysis: Levels of expression of caprine reported in several species [9,12].However, its -∆∆CT expression was much higher during pregnancy than PGFR mRNA were analyzed using 2 method stages of estrus cycle.(Livak and Schmittgen, 2001) and expressed as fold High expression of PGFR in the goat endometrium change relative stage III.Subsequently, differences corresponds to luteolytic signal of PGF2α as reported between groups were statistically analyzed by one way Expression of PGFR mRNA during the estrous cycle and pregnancy of goat.Uncommon superscripts represent a statistical difference (P < 0.05) between stages of estrous cycle and gestation.early pregnancy amongst cyclic stages was in

Table - 1
. Primers used in real time PCR Harvey N., Parent J., Chapdelaine P., Arosh J. A.CL the expression of PGFS and its receptor PGFR turnand Fortier, M. A. (2003) An Aldose Reductase with 20αout to be more at luteolytic stage/late diestrous (stage Hydroxysteroid dehydrogenase Activity is Most Likely the enzyme responsible for the production of Prostaglandin F2α III).The upregulated transcripts of PGFR may indicate in the Bovine Endometrium, J. Biol.Chem., 278,13-28.a coordination of events which result in biosynthesis 3. Chapdelaine, P., Kang, J., Boucher-Kovalik, S., Caron, N., and action of PGF2α in the endometrium leading to Tremblay, J. P. and Fortier, M. A. (2006) Decidualization luteolysis in goats as reported earlier in equine also [6].and maintenance of a functional prostaglandin system in human endometrial cell lines following transformation with Higher expression of PGFR in goat endometrium at SV40 large T antigen.Mol.H. Reprod., 12: 309-319.