Cervical Ripening With PGE2 for Transcervical Embryo Transfer in Sheep: Studies of EP3 Receptor mRNA

Veterinary science, Endocrinology

Audicana, Lina

University:University of Glasgow

关键词: Veterinary science, Endocrinology;   

Others  :  http://theses.gla.ac.uk/75415/1/13815598.pdf

来源: University of Glasgow

【 摘 要 】

Artificial insemination (AI) and embryo transfer (ET) in the ewe are severely restricted by the rigid, impassable cervix that resists catheterization. This thesis deals with the ripening effect of intravaginal prostaglandin E2 (PGE2) in the luteal phase ovine cervix. The potential of this drug to induce cervical ripening has been extensively studied in periparturient sheep and other mammalian species to facilitate labour, but not as a mean of softening the cervix during the cycle. Physiological cervical ripening is characterised by macroscopical, ultrasonographical, microscopical, mechanical and biochemical changes. Studies during pregnancy and at term suggest that PGE2 mimics these changes, but there are no studies during the cycle. The sensitivity of the cervix to PGE2 during the cycle may be lower than at term, but there have been no attempts to characterise the PGE2 receptors (EP1- EP4) in the cervix. The ultrasonographic appearance of the cervix has been described in other species, but not in sheep. In chapter V, ultrasound is evaluated in the monitoring of cervical changes during the cycle, the ovine cervix was imaged at oestrus and during the early luteal phase. This was achieved employing the transrectal technique and two probes of 5 MHz and 7.5 MHz (Toshiba) and a linear scanner (Capasee, Toshiba). In chapter III, several sheep in the luteal phase and early pregnancy (n=15), were treated with different regimes of PGE2 alone or together with oestradiol, to induce cervical ripening. Catheterization was incomplete, but deeper scores (4 cm) than in control sheep (0.1-3 cm) were achieved. Following PGE2 treatment on day 6 of the cycle, microscopical changes similar to an inflammatory response and therefore similar to those during labour were observed. There was activation of polymorphonuclear leukocytes (PML), eosinophils, mast cells and platelets. Vasodilatation and extravasation of red blood cells were also prominent. The findings in chapter III, suggested that there was a response to PGE2 during the luteal phase and that receptors for PGE2 may be present in the cervix. The observation of PML degranulation is in line with current theories of physiological cervical ripening. These cells, when activated, release collagenolytic enzymes, like elastase, MMP-8 and MMP-9 capable of softening the cervix by degrading the extracellular matrix. It was hypothesised here that the EP3R, one of the four PGE2 receptor subtypes, may be involved in the activation of PMLs. At the beginning of these studies, the only reagents available for the characterisation of the EP3R in the ovine cervix were a few cDNA sequences in other mammalian species. The first objective therefore was to isolate and sequence cDNA encoding for the ovine EP3R, using RT-PCR techniques and bovine primers. The successful isolation and sequencing of a cDNA from kidney, using RT-PCR, is described in the first part of chapter IV. The sequence of the fragment isolated was highly homologous to the bovine cDNA sequence, indicating very strongly that it corresponded to the ovine homologue. In the second part of chapter IV, mRNA expression studies are described. The same primers as the cDNA isolation were used to study mRNA expression, DNAse I incubations of the RNA were necessary because the fragment amplified was found to be intronless. Using this method, the presence of mRNA was demonstrated in kidney, liver, uterus, adrenal gland and skin. This distribution pattern is similar to other species and further confirms the identity of the cDNA fragment as the ovine EP3 receptor. Expression of this message was also found for the first time in the oestrous and luteal cervix, by RT-PCR and on the peri parturient cervix by Southern analysis. These preliminary data suggest that the EP3R may have a function in the ovine cervix. It is reasoned, based on these findings and other publications, that EP3R may act as a proinflammatory receptor, activating the cellular response observed in chapter III.