Prolactin, growth hormone and IGF-I in preimplantation mouse embryos

Preimplantation embryos develop normally in vitro, however, progression is slower than that observed in vivo, suggesting the absence of essential maternal factors such as growth factors. Growth hormone (GH) and its receptor (GHR) are expressed by the preimplantation embryo [Pantaleon, 1997 #548][Terada, 1996 #31], however the closely related prolactin (PRL) and its receptor (PRLR) had not been conclusively examined in the early embryo, despite the PRLR null mouse showing preimplantation deficiencies. It was therefore the aim of this thesis to examine the effects of GH in greater detail and to establish the expression pattern and physiological role of PRL in the preimplantation embryo. Human GH (hGH) not only binds and activates the murine GHR, but it also binds and activates the rodent PRLR. Thus to circumvent possible discrepancies present in earlier studies bovine GH (bGH) was used for its inability to bind the mouse PRLR. A 25% increase in blastocyst cell number was observed in embryos cultured from two-cells in the presence of 0.1 - 10 pg/ml of bGH. This increase in cell number was due to a selective increase in the TE cell population. When embryos were grown in groups of 5 embryos per 20 ml droplet, blastocysts had 15% fewer cells than those cultured as groups of 20 per 20 ml droplet. The blastocyst cell number of embryos cultured with anti-GH IgG was 8% smaller than that of embryos cultured in medium alone or in the presence of control IgG. Thus these results suggest that embryo-derived GH plays a role in promoting blastocyst cell number, at least in vitro. In many tissues the response to GH is mediated by IGF-I. As the preimplantation embryo also expresses the IGF-IR and IGF-I, the possibility of an IGF-I mediated response to GH was investigated. Confocal studies demonstrated the presence of the IGF-IR from the four-cell stage onwards. Culture of embryos from the twocell stage in the presence of IGF-IR neutralising antiserum resulted in blastocysts with a 12% smaller ICM. GH stimulated TE hyperplasia even in the presence of IGF-IR neutralisation, indicating that at least in vitro the effects of GH are mediated independently of IGF-1. RT-PCR studies demonstrated the presence of transcripts for the PRLR throughout preimplantation development, and identified the long isoform (PRLR-L) as the predominant receptor type present. The short isoforms of the PRLR were not evident at any stage. Confocal studies localised the PRLR from the oocyte stage onwards. Transcripts for PRL were only evident in the blastocyst, and confocal studies conducted on cultured blastocysts localised PRL in both the ICM and TE. The addition of ovine PRL (oPRL) to two-cell embryos in vitro resulted in a 10% increase in blastocyst cell number, with maximal response at 1-100 pg/ml of oPRL. Blastocysts treated with 0.1 - 1.0 ng/ml of oPRL demonstrated a 25% increase in the rate of glucose transport. The addition of either PRL or PRLR antiserum to embryos in vitro reduced blastocyst cell number confirming a functional role for embryo derived PRL and the PRLR. To examine a functional role for PRL in vivo, an examination of embryo development in PRL null mice was undertaken. The numbers of embryos present in vivo on Dl - D4 of pregnancy was not different between PRL null and wildtype females. However the proportion of embryos attaining the appropriate developmental stages was significantly reduced in the PRL null mothers from D3 onwards, even with progesterone (P4) treatment. Analysis of serum obtained from Dl to D4 of pregnancy showed an absence of early pregnancy factor (EPF) in the PRL and P4-treated PRL null females. EPF has been shown to be essential for embryo development in vivo [Athanasas-Platsis, 2000 #221; Athanasas-Platsis, 1991 #146]. When PRL null embryos were cultured from the two-cell stage blastocyst cell number was 20% smaller than that of wildtype embryos, and the rate of blastocyst formation after 50 hours of culture was almost half of that observed in the wildtype embryos. The development of prl -/- embryos in vitro was more advanced than that observed in vivo, suggesting that PRL is important in regulating the maternal environment. The results presented here illustrate the importance of GH and PRL in preimplantation development both in vivo and in vitro.n