N OCIETY' - the Society for Reproductive Biology

Plateletaactivating factor receptor in human and mouse spermatozoa and mouse preimplantation\.(~--. v~~J'"~e~~k- + h..............~~ j·v..... j..Ji.. t+T. Stojanov, C. Wu & C. 0 'NeillHuman Reproduction Unit, RNSH; Department ofPhysiology, University ofSydneyIntroductionPlatelet activating factor (PAF, 1-0-alkyl-2-acetyl-snglyceryl-3-phosphocholine)is a potent etherphospholipid. It improves motility and inducesacrosome reaction in spermatozoa and acts as anautocrine growth/survival factor for the preimplantationembryo. The concentration of PAF in spermatozoa andembryos is inversely related t6 their quality. Amechanism of action is yet to be defined. In this study,we examined the expression of a G-protein linkedreceptor to PAF (PAF-R).We have previously shown (1) that PAF-R mRNAtranscripts were detected in freshly fertilized mousezygotes. By the late I-cell stage and 2-cell stageexpression was less consistent but was observedconsistently again from the 4-cell stage up until theblastocyst stage. IVF and culture delayed the expressionof PAF-R mRNA until the 8-cell compacted stage.This study investigated: (i) the presence of PAF-RmRNA and protein in the human and mousespermatozoa; (ii) the presence of PAF-R protein inmouse preimplantation embryos and its pattern ofexpression following in vitro fertilisation and culture(IVF).Materials and methodsGamete collection and cultureSpermatozoa were obtained from three fertile donors(human) or were aspirated from cauda epididymis' offertile males (mouse). Motile spermatozoa were thenseparated by swim up into modified-RTF.Mouse embryos were collected fresh from reproductivetract (fresh) at various stages during preimplantationdevelopment. IVF embryos were cultured in modified­RTF. Embryos were immunostained 17, 27, 42, 66 and90 hours post hCG.RNA extraction and RT-PCRRNA was extracted from spermatozoa with TRIzolReagent (Gibco). RT-PCR was performed on spermsamples under standard Perkin-Elmer conditions usingspecific primers for PAF-R.ImmunofluorescenceSpermatozoa or embryos at various stages ofdevelopmental were fixed in 70% ethanol or 2%paraformaldehyde (respectively), permeabilised in 1%Tween 20 (embryos) and non specific binding blockedwith 30% sheep heat inactivated serum. Immunotaggingwas performed by incubation of gametes withmonoclonal antibody to PAF-R (human) (Alexis Corp.)followed by incubation in secondary fluorescent FITCanti-mouseIgG (Santa Cruz Biotechnology).Spermatozoa and embryos were viewed with anepifluorescent microscope (Nikon).ResultsPAF-R mRNA and protein were present in all samplesof human and mouse spermatozoa tested.Immunostained human spermatozoa showed specificstaining which was most prevalent at the midpiece and98distal head (Fig. 1). PAF-R location in mousespermatozoa was most prominent at the midpiece(Fig.!).Immunostaining of embryos collected fresh from thereproductive tract was significantly greater than isotypecontrols and the intensity was not different from the 1­cell up to blastocyst stage. During development thepattern of PAF-R localization changed from diffusecytoplasmic staining in zygotes to increasinglymembrane localized pat.tern of expression from the 2­cell stage onwards. There was no effect of IVF on thelevel and pattern ofprotein expression.Figure 1: PAF-R protein in human and mouse spennatozoa.i) human spennatozoa, ii) mouse spennatozoa, iii) mousespennatozoa incubated in non-immune IgGFigure 2: PAF-R protein in mouse preimplantation embryos.i) i-cell zygote, ii) 2-cell embryo, iii) 2-cell embryo incubatedin non-immune IgG iv) 4-cell embryo v) 8-cell embryo vi)blastocyst.ConclusionsThe results of this study showed that mRNA and proteinfor the G-protein linked PAF-R is present in human andmouse spermatozoa and within the mousepreimplantation embryo. Unlike the expression ofmRNA in embryos, the onset of PAF-R proteinexpression was not delayed following IVF and culture.(1) Stojanov T. & O'Neill C. (1999). Bioi Reprod 60(3):674-82Figure 1325~300.s 275o6 250u as 225u :u 200~ 175~ 150.5 125100o2 4Time (min)medium containing albumin (-, n=50) or no albumin(., n=50).8.- ~~ --b ~~~i- ~~f ", ~ ...;....JL-.\.d- ...Embryo-derived PAF induces Ca 2 + transients in the mouse preimplantation embryoR. Bathgate, M. Emerson, A. Travis and C. O'NeillRuman Reproduction, Department ofPhysiology, University of Sydney, Royal North Shore Hospital,St Leonards, NSW, 2065Introduction' .Platelet-activating factor (PAF) is an autocrine factor involved in regulation of embryo development. Its mecha~lsm ofaction remains to be defined. Transient increases in intracellular calcium are universal cues for cell-cycle progressIOn andregulation of differentiation and development. Exogenous PAF was S?own to induce a ~ansient rel.ease of i~trac~llularcalcium. In this study we investigate whether endogenous embryo-denved PAF can also Induce calCIUm tranSIents In the2-cell embryo.Meilio~ .Female mice were superovulated by Lp. injection of 10 IU eCG followed 48h l~ter by 10 ill hCG and mated overmght.Embryos were flushed from the oviducts of mice at the 2-cell stage of development 42h after eCG. Embryos wereincubated in Fura-2-AM (2uM) for 30min and then mounted on a coverslip coated with Cell-Tak and sealed onto aperspex perfusion chamber. Embryos were monitored with Ion Vision software package and changes in Fura-2 ratio(calculated using excitation wavelengths of 340 and 380 nm) recorded.Results and DiscussionRelease ofPAF from embryos requires albumin (1) therefore calcium measurements were made in media in the pres.en~eand absence of BSA. In the presence of albumin embryos showed characteristic calcium transients but did not do so In Itsabsence (Figure 1). It was determined that this transient was due to embryo-derived PAF by showing that addition ofrecombinant PAF:acetylhydrolase (it degrades PAF to an inactive metabolite) prevented the transient. These embryosstill responded to exogenous PAF with a calcium transient illustrating the specificity of the response. ~ PAF-r~cept~rantagonist (WEB 2086)' also blocked calcium transients. Depletion of Caj stores by -treatment WIth thapslgar~Ineliminated the observed calcium transient, suggesting that it is reliant on Caj stores (Figure 2). Embryos superfused WIthmedium containing 3ug/ml BSA responded more quickly and with a greater amplitude than those exposed to 3mg/mlBSA, suggesting that BSA competes with the PAF receptor for PAF.Figure 1.The change in [Ca 2 +]j in response to perfusion Figure 2.The [Ca 2 +]j response of 2-cell embryos to PAFFigure 2550~ 500.s450§U 400asU 350:s :03001 250:E 200••••••SHS&BB••eeBB B ••1 5 0 ~--r--.----r-..-----'2 4 6 8 10Tim e (m in)after depletion of internal stores by thapsigargin.Thapsigargin treated (-, n=63), control Ce, n=49).Embryos exhibit a transient release of internal calcium stores in response to embryo derived PAF during the 2-cell stage.It is dependent on the presence of BSA. Previous studies have routinely excluded albumin and have therefore not beencapable of detecting the actions of embryo-derived hydrophobic compounds.Reference(1) Ammit, AJ. and O'Neill, C. (1997) Studies of the nature of the binding by albumin of platelet-activating factorreleased from cells. Journal ofBiological Chemistry. 272: 18772-78.99