title: Statins inhibit blastocyst formation by preventing geranylgeranylation.
abstract: Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway and prescription drugs that treat hypercholesterolemia, compromise preimplantation mouse development via modulation of HIPPO signaling.
  
  
  
  HMG-CoA reductase activity is required for trophectoderm specification, namely blastocyst cavity formation and Yes-associated protein (YAP) nuclear localization, through the production of isoprenoid geranylgeranyl pyrophosphate (GGPP) and the action of geranylgeranyl transferase.
  
  
  
  Previous studies have shown that treatment of mouse embryos with mevastatin prevents blastocyst formation, but how HMG-CoA reductase is involved in preimplantation development is unknown. HIPPO signaling regulates specification of the trophectoderm lineage of the mouse blastocyst by controlling the nuclear localization of YAP. In human cell lines, the mevalonate pathway regulates YAP to mediate self-renewal and survival through geranylgeranylation of RHO proteins. These studies suggest that in preimplantation development, statins may act through HIPPO pathway to interfere with trophectoderm specification and thereby inhibit blastocyst formation.
  
  
  
  Eight-cell stage (E2.5) mouse embryos were treated in hanging drop culture with chemical agents, namely statins (lovastatin, atorvastatin, cerivastatin and pravastatin), mevalonic acid (MVA), cholesterol, squalene, farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranylgeranyltransferase inhibitor GGTI-298, RHO inhibitor I, and squalene synthase inhibitor YM-53601, up to the late blastocyst stage (E4.5). Efficiency of blastocyst formation was assessed based on gross morphology and the measurement of the cavity size using an image analysis software. Effects on cell lineages and HIPPO signaling were analyzed using immunohistochemistry with confocal microscopy based on the expression patterns of the lineage-specific markers and the nuclear accumulation of YAP. Effects on cell lineages were also examined by quantitative RT-PCR based on the transcript levels of the lineage-specific marker genes. Data were analyzed using one-way ANOVA and two-sample t-test.
  
  
  
  All four statins examined inhibited blastocyst formation. The adverse impact of statins was rescued by supplementation of MVA (P < 0.01) or GGPP (P < 0.01) but not squalene nor cholesterol. Blastocyst formation was also prevented by GGTI-298 (P < 0.01). These results indicate that HMG-CoA reductase activity is required for blastocyst formation mainly through the production of GGPP but not cholesterol. Inhibition of RHO proteins, known targets of geranylgeranylation, impaired blastocyst formation, which was not reversed by GGPP supplementation. Nuclear localization of YAP was diminished by statin treatment but fully restored by supplementation of MVA (P < 0.01) or GGPP (P < 0.01). This suggests that HIPPO signaling is regulated by GGPP-dependent mechanisms, possibly geranylgeranylation of RHO, to enable trophectoderm formation. YM-53601 prevented blastocyst formation (P < 0.01), but its adverse impact was not rescued by supplementation of squalene or cholesterol, suggesting that squalene synthesis inhibition was not the cause of blastocyst defects.
  
  
  
  Analyses were conducted on embryos cultured ex vivo, but they enable the determination of specific concentrations that impair embryo development which can be compared with drug concentrations in the reproductive tract when testing in vivo impact of statins through animal experimentations. Also, analyses were conducted in only one species, the mouse. Epidemiological studies on the effects of various types of statins on the fertility of women are necessary.
  
  
  
  Our study reveals how the mevalonate pathway is required for blastocyst formation and intersects with HIPPO pathway to provide a mechanistic basis for the embryotoxic effect of statins. This bears relevance for women who are taking statins while trying to conceive, since statins have potential to prevent the conceptus from reaching the blastocyst stage and to cause early conceptus demise.
  
  
  
  Not applicable.
  
  
  
  This study was supported by grants from the George F. Straub Trust of the Hawaii Community Foundation (13ADVC-60315 to V.B.A.) and the National Institutes of Health, USA (P20GM103457 to V.B.A.). The authors have no conflict of interest to declare.
authors.affiliation: Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, USA vernadet@hawaii.edu.
authors.forename: Vernadeth B
authors.initials: VB
authors.lastname: Alarcon
authors.affiliation: Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, USA.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Marikawa
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001755:Blastocyst / Q000187:drug effects / Q000378:metabolism*; D018797:Cell Cycle Proteins; D005260:Female; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D008798:Mevalonic Acid / Q000494:pharmacology; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D017035:Pravastatin / Q000494:pharmacology; D054876:Prenylation / Q000187:drug effects*; D011812:Quinuclidines / Q000494:pharmacology; D012717:Sesquiterpenes / Q000494:pharmacology; D000091102:YAP-Signaling Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001549:Benzamides; D018797:Cell Cycle Proteins; C102521:GGTI 298; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D010750:Phosphoproteins; D011106:Polyisoprenyl Phosphates; D011812:Quinuclidines; D012717:Sesquiterpenes; D000091102:YAP-Signaling Proteins; C414830:YM 53601; C486223:Yap1 protein, mouse; C012258:mevastatin; C004808:farnesyl pyrophosphate; D008148:Lovastatin; D017035:Pravastatin; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid
keywords: 3-hydroxy-3-methylglutaryl-coenzyme A reductase; CDX2; HIPPO pathway; RHO; Yes-associated protein; fertility; mevalonate pathway; pluripotent; preimplantation; trophectoderm