N OCIETY' - the Society for Reproductive Biology

humancowratsheepmouse102Ovarian steroid hormones and growth factors in the uterine control of early embryonicdevelopment in the tammar wallaby, Macropus eugenii.rat92%Cyrma M Hearn -and Marilyn B RenfreeDepartment of Zoology, University ofMelbourne, Parkville, Victoria 3052Embryonic diapause provides a powerful tool tounderstand the uterine control of early embryonicdevelopment. Whilst about 60 species of eutherian, and30 species of marsupial exhibit diapause, the control isbetter understood in the tammar wallaby, Macropuseugenii, than in most other species. Under the influenceof a sucking young in the pouch, development of thetammar embryo in utero ceases completely at the 100­cell. blastocyst stage. Diapause is maintained for at least11 months, during which time there is no cell division orblastocyst growth. Despite the long period of arrest thereis no loss of embryonic viability. Most attention to datehas focused on the reactivation of corpus luteum andembryonic growth after the long arrest, with little focuson the mechanisms that control the onset of diapause.The aim of this study is to investigate the interactionsbetween ovarian hormones, uterine secretions and earlyembryonic growth as the blastocyst approaches andenters diapause.It is clear from work on eutherian species that there aremany growth factors, including. leukaemia-inhibitingfactor (LIF), epidermal-growth factor (EOF) and insulinlikegrowth factors (IOF-l & IOF-2), produced in theuterine secretions which may potentially regulateembryonic development. LIF is essential for growth ofthe early mammalian embryo (1). The expression of LIFin mice is maternally controlled by oestrogen andcoincides with blastocyst formation (2). Similarly, inpigs (3) and cattle (4) oestradiol upregulates uterine LIFproduction. In rabbits by contrast, progesterone, notoestradiol, regulates LIF expression (5). Neither steroidinduces uterine LIF production in the skunk, but LIFmRNA is absent during diapause and high around thetime of implantation (6).Our hypothesis is that in the tammar the release ofoestradiol from the ovary, around the time of ovulation,induces the production of several growth factors,including LIF, which enhance cleavage and are essentialfor formation of the early blastocyst. Over the next fewdays, without continued oestradiol stimulation and with atransient rise in progesterone post partum, growth factorproduction is downregulated, forcing the blastocyst intodiapause. Thus there may be a direct interaction betweenovarian steroid' hormones and uterine" growth factorsecretion in initiation of diapause.human81%79%The endocrine changes and endometrial responses in theperiod between conception and the onset of diapause hasbeen poorly defined. Daily plasma samples weretherefore collected immediately after parturition until day10 after birth from females that retained their young(n=8), those who had their young removed at birth (n=8)and a group that carried a blastocyst in diapause (n=8).Plasma progesterone and oestradiol was measured byradioimmunoassay (RIA) as previously described (7),and a hormone profile for the three experimental groupsdefined. The genetic sequence of LIP in the tammar wasdetermined by nested polymerase chain reaction (PCR)using degenerate primers developed from conservedregions of known eutherian species, and probesdeveloped from mouse and human LIP plasmids. The 38amino acid sequence of the peR product shows highhomology with previously described species (Table 1).The tammar LIP (tLIP) gene has a higher homology tohuman LIP than other eutherian species. Using part ofthe tLIP sequence as a probe the expression of LIP inreproductive tissues and the early embryo will beaccurately determined by in situ hybridisation.Acknowledgments:We thank Prof. RR Behringer (Department of MolecularGenetics, MD Anderson Cancer Centre, Houston, Texas)for providing his expertise and laboratory facilities incloning tLIF. We are also grateful to Dr. D Hilton(Walter & Eliza Hall Institute, Victoria) for kindlyproviding the human and mouse LIF plasmids.References:1. Stewart CL (1994) Mol Reprod Dev 39:233-82. Bhatt H et al. (1991) Proc Natl Acad Sci 88:11408­123. Yelich IV et al. (1997) Biol Reprod 57:1256-654. Reinhart KC et al. (1998) Mol Hum Reprod 4:301-3085. Yang ZM et al. (1996) Mol Reprod Dev 43:470-66. Hirzel DJ et al. (1999) Biol Reprod 60:484-927. Fletcher TP & Renfree MB (1988) JRF 83:193-200Table 1. A cross species comparison of homology between the tLlF protein to known sequencessheep pig tammar87% 92% 88%84%74% 82% 790/076%71% 76% 73%TEMPORAL AND TISSUE SPECIFIC EXPRESSION OF MOUSE KALLIKREIN (mKlk) GENES ANDIDENTIFICATION OF A NOVEL mKlk mRNA TRANSCRIPT DURING EARLY DEVELOPMENT INTHE MOUSE.C.S. Chan, M.B. Harvey and J.A. Clements .Centre for Molecular Biotechnology, School ofLife Sciences, Queensland University ofTechnology, Bnsbane 400l.IntroductionThe tissue kallikreins are a highly conservedmultigene family of serine proteases that act on adiverse range of substrates including growth factors,extracellular matrix (ECM) glycoproteins andproteinases and are important regulators of ECMdegradation and cell proliferation (1). They havebeen implicated in the uterine function of otherspecies (2,3). The expression of five mKlk genesmKlk-1(tissue kallikrein), mKlk-3, mKlk-5, mKlk-9(epidermal growth factor binding protein) and mKlk­21- was detected in the non-pregnant mouse uterus(4). The aim of this study was to elucidate thetemporal and tissue specific expression pattern ofthese five mKlks during early development.Materials & MethodsFertilised eggs, two-cell embryos, blastocysts, day7.5, day 9.5 and day 11 embryos were collected fromsix-week old, superovulated, mated female BCBFlmice. Uterine tissue was collected at correspondingdevelopmental stages. Total RNA was extracted andexpression of each mKlk and ~-actin was detected byperforming reverse transcription (RT)-PCR, withgene specific primers, in the linear range to allowsemi-quantitative analysis. Southern hybridisationwith gene-specific probes and/or DNA sequencingconfirmed the specificity of the PCR.ResultsThe ubiquitous "house-keeping" gene, ~-actin, wasexpressed in all samples, confirming the integrityand quality of the cDNA (Figure la). Positivecontrols (salivary gland) yielded mKlk products ofthe predicted size. All mKlks displayed distinctexpression patterns. We observed the expression ofmKlk-1 (Figure Ib), mKlk-3, mKlk-9 and mKlk-21(Figure lc) in the fertilised egg to the two-cell stage.Only mKlk-21 continued to be expressed until day7.5 of pregnancy. mKlk-9 expression reappeared atday 7.5 and was consistently detected until day 11(data not shown). mKlk-1 was again expressed inthe embryo from day 9.5, with decreased levels byday 11 (Figure Ib). mKlk-5 was not detected in theembryo. In contrast, there was no consistentexpression in the uterus of mKlk-1 until day 7.5which then continued to day 11 (Figure Id). mKlk­21 expression in the uterus displayed a similarpattern but was detected at much lower levels(Figure Ie). Of interest, a novel mKlk-21-likemRNA·I3 bp larger was detected in the uterus, buta.310243234b.6 ......3 : -~~~-~ ~.-~3c. M - + f.e. 2-cell blast d 7.5 d 9.5 i..ll.31 • : ~ _ __~ .,~w ->20194d.603383310e. 31. .lG4~~tG\.;~not in the embryo (Figures lc,e). mKlk-3, mKlk-5and mKlk-9 were not consistently expressed in theuterus ~e! th~ tiT~: 2-cell blast d 7.5 d 9.5 ~2019- _st.iU-_ _llllliIU1l1l'l- _ i'$!