Molecular Human Reproduction, Vol.22, No.5 pp. 350–363, 2016 Advanced Access publication on February 7, 2016 doi:10.1093/molehr/gaw011

ORIGINAL RESEARCH

Statins inhibit blastocyst formation by preventing geranylgeranylation Vernadeth B. Alarcon* and Yusuke Marikawa Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, USA *Correspondence address. Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii, 651 Ilalo Street, BSB 163, Honolulu, HI 96813, USA. Tel: +1-808-692-1417; E-mail: [email protected]

Submitted on July 4, 2015; resubmitted on January 24, 2016; accepted on January 29, 2016

study hypothesis: 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. study finding: HMG-CoA reductase activity is required for trophectoderm specification, namely blastocyst cavity formation and Yesassociated protein (YAP) nuclear localization, through the production of isoprenoid geranylgeranyl pyrophosphate (GGPP) and the action of geranylgeranyl transferase. what is known already: 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. study design, samples/materials, methods: 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. main results and the role of chance: 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. limitations, reasons for caution: 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. wider implications of the findings: 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.

& The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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large scale data: Not applicable. study funding and competing interests: 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. Key words: CDX2 / HIPPO pathway / mevalonate pathway / trophectoderm / Yes-associated protein / preimplantation / pluripotent / 3-hydroxy-3-methylglutaryl-coenzyme A reductase / RHO / fertility

Introduction In preimplantation development, the conceptus begins as a ball of totipotent cells or morula that segregate into the first two cell lineages, the extra-embryonic and pluripotent tissues known as trophectoderm (TE) and inner cell mass (ICM), respectively. The morula then forms a structure called the blastocyst, with TE on the surface surrounding a fluidfilled cavity and the ICM. ICM produces the fetal body, whereas TE plays a number of roles that are critical to producing pregnancy, namely expansion of the cavity to increase blastocyst surface area, hatching, and attachment to the uterine endometrium for implantation (Marikawa and Alarcon, 2012). Initial signs of TE specification manifest around the 16-cell stage involving cellular and molecular changes. Cells differ from one another in terms of position and polarity, i.e. outside cells possess an apical-basal polarity whereas inside cells are non-polar. Cell polarity regulators, such as PARD6B (partitioning defective 6B) of the PAR-aPKC (atypical protein kinase C) system and RHO-ROCK (Rho-associated kinase) signaling, are required for TE specification by modulating the HIPPO signaling pathway (Alarcon, 2010; Hirate et al., 2013; Kono et al., 2014; Cao et al., 2015). HIPPO pathway regulates cell– cell interactions in diverse developmental contexts and tissue homeostasis (Halder and Johnson, 2011). In the mouse conceptus, the actions of HIPPO pathway components, including large tumor suppressor 1/2 (LATS1/2) kinases, angiomotin (AMOT), and neurofibromatosis 2 (NF2), control the localization of Yes-associated protein (YAP) in a cell position-specific manner (Nishioka et al., 2009; Cockburn et al., 2013; Hirate et al., 2013; Leung and Zernicka-Goetz, 2013). YAP localizes to the nucleus in outside cells, where it acts as a co-activator of transcriptional enhancer activator (TEA)-domaincontaining transcription factor TEAD4. The nuclear action of YAPTEAD4 complex leads to transcriptional activation of TE-specific Cdx2 gene, whereas ICM-specific Pou5f1 gene is down-regulated in outside cells. Knockdown of PARD6B or pharmacological inactivation of ROCK or RHO diminishes nuclear localization of YAP, resulting in defective TE formation (Alarcon, 2010; Hirate et al., 2013; Kono et al., 2014; Cao et al., 2015). The emergent mechanisms of cell lineage specification are complex, and other regulators are to be discovered to gain further understanding of the mechanisms of blastocyst formation (Frum and Ralston, 2015). Hypercholesterolemia, a condition of high levels of cholesterol in the blood, is a risk factor for cardiovascular disease (Yeganeh et al., 2014). It is often treated with the class of drugs called statins that inhibit the enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. HMG-CoA reductase catalyzes conversion of HMG-CoA to mevalonic acid, which is a substrate for the synthesis of cholesterol. Cholesterol modulates membrane fluidity as a component of cell

membranes, and serves as precursor for steroid hormones. The conversion of HMG-CoA to mevalonic acid is the rate-limiting step of cholesterol synthesis, and inhibition of HMG-CoA reductase with statins effectively lowers cholesterol level. Mevalonic acid is also crucial for the synthesis of isoprenoids. Both cholesterol and isoprenoids have important functions in development by serving as lipid attachment for posttranslational modification of signaling proteins (Eisa-Beygi et al., 2014). Surani et al. (1983) have previously shown that treatment of early cleaving mouse embryos with mevastatin prevents blastocyst formation. However, how HMG-CoA reductase is involved in blastocyst formation is unknown. More recently, the mevalonate pathway has been shown to regulate YAP activity to mediate self-renewal and survival in human cell lines and eye growth in fruitfly (Sorrentino et al., 2014). These studies raise the possibility that in preimplantation development, statins may act through HIPPO pathway to interfere with TE lineage specification and blastocyst formation. Investigation on how statins act as a developmental toxicant bears relevance to fertility in women. The United States Food and Drug Administration (FDA) places statins under Pregnancy Risk Category X, i.e. contraindicated for use in pregnancy, and it is advisable for statin treatment to be suspended in pregnant patients. However, a recent epidemiological study showed that statin use in the first trimester is not associated with increased risk of major congenital malformations in the offspring (Bateman et al., 2015), raising the question of whether statins can be safely used before or after the first trimester. In relation to this issue, no studies to date have examined the effects of statin use on the ability of women to successfully conceive. Due to the rising prevalence of hypercholesterolemia in younger populations and the trend to delay in conceiving until later years, women of reproductive age will increasingly be taking statins (Heffner, 2004; Arnett et al., 2005). Moreover, about half of pregnancies are unplanned (Finer and Zolna, 2014). Thus, there is increasing likelihood of exposure to statins which may have detrimental effects on the early conceptus. Knowledge of the effects of statins on preimplantation development would be beneficial for women who are trying to conceive. Here, we further clarified the effects of statins by examining the relationship between mevalonate pathway and blastocyst development. Our results showed that the HMG-CoA reductase activity was required for TE specification, namely YAP nuclear localization and blastocyst cavity formation, through the production of isoprenoid geranylgeranyl pyrophosphate and the action of geranylgeranyl transferase. The wider implication of our study is that statins have potential to prevent the conceptus from reaching the blastocyst stage and to promote early conceptus demise. In light of the changing demographics of statin users, further investigations on the effects of various types of statins on the fertility of women are necessary.

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Materials and Methods Animals and embryo collection The protocol for animal use was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Hawaii at Manoa. F1 (C57BL/6 × DBA/2) mice were used (Charles River Laboratories, Frederick, MD, USA). Female mice were injected with pregnant mare serum gonadotrophin and human chorionic gonadotrophin (hCG) (EMD Millipore, Temecula, CA, USA) at 48 h apart, and mated with males. At approximately 42 h post-hCG injection, oviducts were removed from females and 2-cell stage embryos were collected using standard protocol (Behringer et al., 2014). For each experiment, embryos were collected from three or four females and were grouped together as a single batch. Embryos were transferred into 20 ml drops of KSOM-AA culture medium (MR-121-D, EMD Millipore) covered with mineral oil and incubated in a CO2 incubator (378C, 5% CO2 in humidified air) for approximately 27 h when it reached the 8-cell stage.

Pharmacological treatment of embryos in hanging drop culture All reagents used in the study were purchased from Sigma-Aldrich (St. Louis, MO, USA; lovastatin [PHR1285], cerivastatin [SML0005], pravastatin [P3510], mevalonic acid [M4667], squalene [S3626], cholesterol [C8667], geranylgeranyl pyrophosphate [G6025], farnesyl pyrophosphate [F6892], GGTI-298 [G5169]), or from Santa Cruz Biotechnology (Dallas, TX, USA; atorvastatin [sc-337542], YM-53601 [sc-205543]), or from Cytoskeleton (Denver, CO, USA; RHO inhibitor I [CT04]). Stock solutions were prepared by dissolving in dimethyl sulfoxide for all reagents, except for mevalonic acid, geranylgeranyl pyrophosphate, farnesyl pyrophosphate (these were provided by the vendor in a liquid format), cholesterol (dissolved in ethanol), and RHO inhibitor I (dissolved in water). Pharmacological inhibitors used in the present study and their IC50 values are listed in Table I. All treatments were performed in hanging drop culture, starting from 8-cell stage. Most pharmacological agents used in the present study are cellpermeable and relatively hydrophobic, so that the conventional embryo culture format with covered mineral oil may absorb these agents, which

potentially lowers drug concentrations in the culture medium. In contrast, hanging drop culture is free of mineral oil, so that concentrations of agents are more likely to be maintained. Specific drug dilutions were prepared in KSOM-AA, and 20 ml drops were placed on the inner surface of the lid of a 6 cm Petri dish. The lid was then flipped over and placed on the bottom portion of the Petri dish that was filled with 6 ml of phosphate-buffered saline. The prepared dishes were equilibrated in a CO2 incubator prior to the transfer of embryos at the 8-cell stage. After embryo transfer, the dish was incubated in a CO2 incubator for 48 h or up to E4.5. Embryos were photographed using AxioCam MRm digital camera attached to Axiovert 200 inverted microscope with Hoffman modulation contrast optics and controlled by AxioVision software (Carl Zeiss, Thornwood, NY, USA). AxioVision image files were converted to JPG format, which were then opened in ImageJ program (http://rsb.info.nih.gov/ij) to analyze the cavity size of embryos. The periphery of embryo and the lining of cavity were manually traced using the Polygon selections tool to measure the areas occupied by the embryo and the cavity, respectively, and the cavity size was calculated as a percentage of the latter to the former for each embryo. The numbers of total embryos examined for experimental treatments are indicated in the legends of corresponding figures.

Immunofluorescent staining Procedure was conducted as described previously (Laeno et al., 2013). Embryos were incubated in primary antibody at 48C and in secondary antibody at 258C. Primary antibodies were mouse anti-CDX2 (1:800) (#CDX2-88, BioGenex, Fremont, CA), goat anti-POU5F1 (1:100) (#N-19, Santa Cruz Biotechnology), and mouse anti-YAP1 (1:300) (#2F12, Novus Biologicals, Littleton, CO). Secondary antibodies were rabbit anti-mouse Alexa 488, rabbit anti-goat Alexa 546, and goat anti-mouse Alexa 488 which were all used at 1:1000 (Life Technologies, Grand Island, NY). Embryos were mounted in ProLong Gold medium containing 4′ ,6-diamidino-2-phenyl-indole (DAPI) to stain nuclei (Life Technologies).

Confocal microscopy All embryos from the same experiment were imaged in a single session with FV1000 confocal laser scanning microscope (Olympus, Center Valley, PA,

Table I Pharmacological inhibitors used in the present study. Drug name

Molecular target

IC50 (test system)

References

............................................................................................................................................................................................. Lovastatin

HMGCR

90 nM (rat liver microsome) 300 nM (human liver microsome) 2–4 nM (cultured hepatocytes)

Dansette et al. (2000)

Atorvastatin

HMGCR

6 nM (rat liver microsome) 40 nM (human liver microsome) 1.16 nM (cultured hepatocytes)

Dansette et al. (2000)

Cerivastatin

HMGCR

2 nM (rat liver microsome) 6 nM (human liver microsome)

Dansette et al. (2000)

Pravastatin

HMGCR

100 nM (rat liver microsome) 200 nM (human liver microsome) 4 nM (cultured hepatocytes)

Dansette et al. (2000)

GGTI-298

GGTase

4 mM (human lung adenocarcinoma growth) 15 mM (Rap1A geranylgeranylation) .20 M (human colon cancer growth)

Miquel et al. (1997) Kusama et al. (2003)

YM-53601

SQS

79 nM (human HepG2 cells) 90 nM (rat liver microsome)

Ugawa et al. (2000)

RHO inhibitor I

RHO

0.5– 2.0 mg/ml (RHO activity assay)

Vendor’s brochure (Cytoskeleton Inc.)

Sirtori (2014)

Sirtori (2014)

Sirtori (2014)

IC50: Concentration needed to decrease rate by 50%; HMGCR: HMG-CoA reductase; GGTase: geranylgeranyl transferase; SQS: squalene synthase.

Blastocyst formation requires geranylgeranylation

USA). The microscope was under the control of Fluoview software (Olympus), which used identical configurations for all embryos in a single session of imaging, as described previously (Kono et al., 2014). Serial optical sections of entire embryos were obtained at 2 mm intervals under a 40× oil objective lens. Total cell number per embryo was determined by examining every optical section and counting DAPI-positive nuclei. Total number of POU5F1positive, CDX2-positive, and YAP-positive nuclei per embryo was determined by examining every optical section. Mitotic nuclei were identified as intense DAPI staining with the shape of condensed chromosomes.

Quantitative reverse transcription polymerase chain reaction (qRT–PCR) Total RNA was extracted from each sample of 15– 25 E3.5 embryos with TRI reagent (Sigma-Aldrich) and used for cDNA synthesis, as previously described (Alarcon, 2010). Quantitative PCR was performed, using iCycler Thermal Cycler with MyiQ Single Color Real-Time PCR Detection System with iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). The following primers were used: Cdx2, F-GAC TTC CTG TCC CTT CCC TCG TCT, R-CCT CCC GAC TTC CCT TCA CCA TAC; Gata3, F-CAT GCT CTG TGA ATC AGT CCC TGT, R-AAC CCT CCA GAG TAC ATC CAC CTT; Pou5f1, F-AGG CAG GAG CAC GAG TGG AAA GCA, R-GGA GGG CTT CGG GCA CTT CAG AAA; Nanog, F-ATA ACT TCG GGG AGG ACT TTC TGC, R-CCC TGA CTT TAA GCC CAG ATG TTG; Sox2, F-CCA TGC AGG TTG ATA TCG TTG GTA, R-GCC AGC CTG ATT CCA ATA AGA GAG; and Actb (encoding a house-keeping b-actin), F-GAG AGG GAA ATC GTG CGT GAC ATC, R-CAG CTC AGT AAC AGT CCG CCT AGA. Each set of experiment consisted of three culture conditions: control, treatment with lovastatin (1 mM), and treatment with lovastatin (1 mM) and GGPP (10 mM), and three sets of experiments were conducted using different batches of embryos as biological replicates. The expression levels of Cdx2, Gata3, Pou5f1, Nanog, and Sox2 were normalized with Actb in each experiment.

Statistics All experiments were conducted at least twice using different batches of pooled embryos as biological replicates, and compiled data were presented. When three or more experimental groups were compared, the one-way analysis of variance (ANOVA) was used to determine whether there were any significant differences among their means, which was then followed by post-hoc two-sample t-test to compare between two specific groups.

Results Inhibition of HMG-CoA reductase impairs blastocyst formation Various types of statins have been approved by the FDA as cholesterollowering medications. Mevastatin is the founding member of the statin class of drugs, and it has been shown to impair mouse blastocyst formation (Surani et al., 1983). However, developmental effects of other statins are unknown. Here, we examined four statins, namely lovastatin, atorvastatin, cerivastatin, and pravastatin, for their impact on mouse preimplantation development (see Supplementary Table for known serum concentrations for each statin in human). Embryos were cultured in the presence of each statin at various concentrations (i.e. 0.2, 1, and 5 mM for lovastatin, atorvastatin, and cerivastatin, and 1, 5, and 20 mM for pravastatin) from the 8-cell stage (E2.5), and their gross morphology was examined up to the late blastocyst stage (E4.5). After one day of treatment (E3.5), many embryos in the control (i.e. no statin) group had already formed blastocyst cavities (Fig. 1A and B). In contrast,

353 many of the statin-treated embryos failed to form a cavity by E3.5, although the potency to diminish cavity formation differed depending on the type of statin. Significant reduction (P , 0.01; 2-sample t-test) in the mean cavity size was observed for lovastatin, atorvastatin, and cerivastatin at the lowest concentration tested (0.2 mM), whereas a much higher concentration (5 mM) was required for pravastatin to reduce the cavity size significantly. Statin-treated embryos that had failed to form a cavity at E3.5 appeared as intact morula with no sign of fragmentation, cell detachment, or cell lysis at a gross morphological level. By E4.5, all control embryos had formed a blastocyst cavity. Many of the lovastatin-treated embryos at 0.2 mM formed a cavity by E4.5 in a manner comparable to the control, and their mean cavity size was not significantly different from control (Fig. 1B), suggesting that cavitation was delayed rather than permanently impaired. On the other hand, cerivastatin at 0.2 mM was more potent so that essentially no cavity formed by E4.5. At a higher concentration (1 mM), lovastatin and atorvastatin also reduced the mean cavity size significantly compared with the control by about 65 and 45%, respectively (Fig. 1B). At this concentration, lovastatin appeared more potent than atorvastatin, although the difference was not statistically significant. The mean cavity size was also significantly lower for embryos that were treated with pravastatin at 20 mM (Fig. 1B). Most of the statin-treated embryos that failed to form a cavity by E4.5 appeared disintegrated, based on their gross morphology showing indistinct embryo surface and granular appearance (Fig. 1A). Statins are inhibitors of HMG-CoA reductase that catalyzes conversion of HMG-CoA into mevalonic acid (MVA). To confirm that the adverse impact of statins on blastocyst formation was solely due to reduction in MVA production, statin-treated embryos were supplemented with exogenous MVA. Embryos at 8-cell stage were cultured with lovastatin (1 mM) or cerivastatin (1 mM) in the presence of MVA (100 mM), and their gross morphology was observed at E3.5 and E4.5. While lovastatin alone reduced the mean cavity size to zero at both time points, supplementation of MVA fully restored cavitation in a manner similar to control (Fig. 1C). The rescued embryos adopted the morphology of expanded blastocysts, which were indistinguishable from control (Fig. 1A). The same kind of rescue was also observed for cerivastatin (Fig. 1C). To further assess the normalcy of the MVA-rescued blastocysts, we examined expression patterns of POU5F1 and CDX2, transcription factors that are specific to ICM and TE lineages, respectively, by immunostaining. At E4.5, control blastocysts displayed POU5F1 localized to ICM nuclei, whereas CDX2 localized to TE nuclei. Blastocysts derived from lovastatin-plus-MVA-treated embryos also exhibited similar localization patterns of POU5F1 and CDX2 (Fig. 1D). The average numbers of POU5F1-positive nuclei and CDX2-positive nuclei were 17.5 + 5.2 (standard deviation) and 71.0 + 9.4, respectively, for control (n ¼ 6), whereas 17.3 + 3.4 and 77.5 + 6.4, respectively, for lovastatin plus MVA (Fig. 1E). With the inclusion of mitotic nuclei, which had no POU5F1 or CDX2 localization, the total numbers of nuclei were 91.8 + 13.9 for control and 97.8 + 6.4 for lovastatin plus MVA. Thus, the adverse developmental impact of statins was essentially circumvented by MVA supplementation at the morphological and molecular level.

Production of geranylgeranyl pyrophosphate is essential for blastocyst formation MVA, the end product of HMG-CoA reductase reaction, serves as the precursor of various bioactive lipid compounds, one of which is

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Figure 1 Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, impair blastocyst formation. (A) Photos of embryos at E3.5 and E4.5 that had been cultured from E2.5 (8-cell stage) with or without lovastatin (1 mM) and mevalonic acid (MVA; 100 mM). Scale bar ¼ 50 mm. (B) Inhibition of cavity formation by various statins. Bars represent the mean + standard deviation of cavity size as a percent of embryo area. Control (n ¼ 15), lovastatin (n ¼ 9 for each concentration), atorvastatin (n ¼ 9 for each concentration), cerivastatin (n ¼ 10 for each concentration), and pravastatin (n ¼ 10 for each concentration). (C) MVA (100 mM) rescues the adverse impact of lovastatin (Lova; 1 mM) and cerivastatin (Ceriva; 1 mM). Bars represent the mean + standard deviation of cavity size as a percent of embryo area. Left graph: control (n ¼ 20), lovastatin (n ¼ 20), and lovastatin + MVA (n ¼ 25). Right graph: control (n ¼ 7), cerivastatin (n ¼ 7), and cerivastatin + MVA (n ¼ 10). (D) Projected confocal images of embryos at E4.5 that were stained with 4′ ,6-diamidino-2-phenyl-indole (DAPI; total nuclei), anti-POU5F1, and anti-CDX2. Scale bar ¼ 50 mm. (E) Average numbers of mitotic (gray), POU5F1-positive (red), and CDX2-positive (green) nuclei. Control (n ¼ 6) and lovastatin + MVA (n ¼ 6). (B, C) Asterisks indicate statistically significant reduction in the mean cavity size in comparison with the control of the same stage (P , 0.01; 2-sample t-test).

cholesterol. Cholesterol results from cyclization of squalene, which is synthesized from two molecules of farnesyl pyrophosphate (FPP) by the action of squalene synthase (SQS) (Fig. 2A). To test whether reduced production of squalene or cholesterol was responsible for the adverse developmental effect of statins, we treated embryos with lovastatin (1 mM) together with squalene (100 mM) or cholesterol (100 mM). However, no sign of rescue in cavitation was observed, and squalene- or

cholesterol-supplemented embryos appeared morphologically indistinguishable from embryos treated with lovastatin alone (Fig. 2B and C). Another major compound derived from MVA is geranylgeranyl pyrophosphate (GGPP), which results from conjugation of FPP with isopentenyl pyrophosphate (IPP) (Fig. 2A). GGPP is covalently attached to various proteins, the process known as prenylation, to modulate their activity. Thus, we tested whether reduction in GGPP, or its immediate

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Figure 2 Geranylgeranyl pyrophosphate (GGPP) production is essential for blastocyst formation. (A) Schematic diagram of mevalonate pathway, focusing on the components and pharmacological inhibitors that are investigated in the present study. HMGCR: 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase; SQS: squalene synthase; GGTase: geranylgeranyl transferase; MVA: mevalonic acid; IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl pyrophosphate; GPP: geranyl pyrophosphate; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate. (B) Photos of embryos at E3.5 and E4.5 that had been cultured from E2.5 with or without lovastatin (Lova; 1 mM), squalene (100 mM), and cholesterol (100 mM). Scale bar ¼ 50 mm. (C) Neither squalene (Squ; 100 mM) nor cholesterol (Cho; 100 mM) can rescue the cavitation defects caused by lovastatin (1 mM). Control (n ¼ 8), lovastatin (n ¼ 8), lovastatin + squalene (n ¼ 15), and lovastatin + cholesterol (n ¼ 15). (D) Photos of embryos at E3.5 and E4.5 that had been cultured from E2.5 with or without lovastatin (1 mM), FPP (10 mM), and GGPP (10 mM). Scale bar ¼ 50 mm. (E) GGPP (10 mM) and FPP (10 mM) rescue the adverse impact of lovastatin (1 mM). Bars represent the mean + standard deviation of cavity size. Control (n ¼ 16), lovastatin (n ¼ 16), lovastatin + FPP (n ¼ 26), and lovastatin + GGPP (n ¼ 20). (F) GGPP (10 mM) and FPP (10 mM) rescue the adverse impact of cerivastatin (1 mM). Bars represent the mean + standard deviation of cavity size as a percent of embryo area. Control (n ¼ 9), cerivastatin (n ¼ 9), cerivastatin + FPP (n ¼ 21), and cerivastatin + GGPP (n ¼ 16). (G) Projected confocal images of embryos at E4.5 that were stained with 4′ ,6-diamidino-2-phenyl-indole (DAPI; total nuclei), anti-POU5F1, and anti-CDX2. Scale bar ¼ 50 mm. (H) Average numbers of mitotic (gray), POU5F1-positive (red), and CDX2-positive (green) nuclei. Control (n ¼ 6) and lovastatin + GGPP (n ¼ 6). (C, E, F) Asterisks indicate statistically significant reduction in the mean cavity size in comparison with the control of the same stage (P , 0.01; 2-sample t-test).

356 precursor FPP, is the cause of developmental defect in statin-treated embryos. When embryos were cultured with lovastatin (1 mM) together with FPP (10 mM) or GGPP (10 mM), a significant recovery in cavity formation was observed at E3.5 (Fig. 2D). Their mean cavity sizes were similar to control (Fig. 2E). Cavitation at E3.5 was also rescued in embryos treated with cerivastatin (1 mM) by supplementation of FPP (10 mM) or GGPP (10 mM) (Fig. 2F). At E4.5, GGPP-supplemented embryos became expanded blastocysts, which were morphologically indistinguishable from control (Fig. 2D). The mean cavity sizes were also similar between control and lovastatin plus GGPP or cerivastatin plus GGPP treatments (Fig. 2E and F). Furthermore, expression patterns of the lineage markers POU5F1 and CDX2 in lovastatin-plusGGPP-treated embryos were similar to control (Fig. 2G). The average numbers of POU5F1-positive, CDX2-positive and total nuclei were 17.5 + 5.2, 71.0 + 9.4 and 91.8 + 13.9, respectively, for control (n ¼ 6), whereas 12.7 + 6.0, 70.2 + 8.0 and 87.5 + 11.1, respectively, for lovastatin plus GGPP (n ¼ 6) (Fig. 2H). Thus, essentially, blastocyst formation was fully restored in statin-treated embryos by supplementation of GGPP. In contrast, rescue by FPP supplementation was not as robust as GGPP. The mean cavity sizes in lovastatin-plus-FPP- or cerivastatin-plus-FPP-treated embryos were significantly lower (P , 0.01; 2-sample t-test) than control at E4.5 (Fig. 2E and F). These results

Alarcon and Marikawa

suggest that the adverse developmental impact of statins is largely due to insufficient production of GGPP.

Inhibition of geranylgeranyl transferase or RHO impairs blastocyst formation For protein prenylation, geranylgeranyl moiety of GGPP is transferred to substrates by geranylgeranyl transferase (GGTase). To test whether the activity of GGTase is required for blastocyst formation, 8-cell stage embryos were cultured in the presence of a GGTase inhibitor, GGTI-298 (10 mM). At E3.5, none of the GGTI-298-treated embryos formed a cavity, but there was no sign of fragmentation or cell lysis at a gross morphological level (Fig. 3A). By E4.5, all of the treated embryos appeared dead. Thus, GGTI-298-treated embryos appeared morphologically similar to statin-treated embryos (Fig. 1A). However, supplementation of GGPP (10 mM), which fully rescued statin-treated embryos, was unable to restore cavity formation in GGTI-298-treated embryos (Fig. 3A and B), in line with the notion that GGPP requires the activity of GGTase for prenylation. One of the well-known targets of geranylgeranylation is RHO (Hori et al., 1991; Adamson et al., 1992). Treatment of 8-cell stage embryos with RHO inhibitor I (1 mg/ml) prevented blastocyst cavity formation

Figure 3 Inhibition of geranylgeranyl transferase (GGTase) or RHO impairs blastocyst formation. (A) Photos of embryos at E3.5 and E4.5 that had been cultured from E2.5 with or without GGTI-298 (10 mM) and geranylgeranyl pyrophosphate (GGPP; 10 mM). Scale bar ¼ 50 mm. (B) Cavity sizes of embryos treated with GGTI-298 and GGPP. Bars represent the mean + standard deviation of cavity size. Control (n ¼ 14), GGTI-298 (n ¼ 14), and GGTI-298 + GGPP (n ¼ 14). (C) Photos of embryos at E3.5 and E4.5 that had been cultured from E2.5 with or without RHO inhibitor I (1 mg/ml) and GGPP (10 mM). Scale bar ¼ 50 mm. (D) Cavity sizes of embryos treated with RHO inhibitor I and GGPP. Bars represent the mean + standard deviation of cavity size. Control (n ¼ 13), RHO inhibitor I (n ¼ 13), and RHO inhibitor I + GGPP (n ¼ 13). (B, D) Asterisks indicate statistically significant reduction in the mean cavity size in comparison with the control of the same stage (P , 0.01; 2-sample t-test).

Blastocyst formation requires geranylgeranylation

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Figure 4 Yes-associated protein (YAP) nuclear localization is dependent on geranylgeranyl pyrophosphate (GGPP) and geranylgeranyl transferase (GGTase). (A) Projected confocal images of embryos that were stained with DAPI (total nuclei) and anti-YAP. Embryos had been treated with lovastatin (Lova; 1 mM), mevalonic acid (MVA; 100 mM), and/or GGPP (10 mM) for 20 h. Scale bar ¼ 50 mm. (B) MVA (100 mM) and GGPP (10 mM) rescue YAP nuclear localization in lovastatin-treated embryos (1 mM). Average numbers of mitotic (gray), YAP-negative (white), and YAP-positive (green) nuclei. Control (n ¼ 12), lovastatin (n ¼ 12), lovastatin + MVA (n ¼ 11), and lovastatin + GGPP (n ¼ 12). (C) GGPP (10 mM) rescues YAP nuclear localization in cerivastatin-treated embryos (1 mM). Average numbers of mitotic (gray), YAP-negative (white), and YAP-positive (green) nuclei. Control (n ¼ 12), cerivastatin (n ¼ 10), and cerivastatin + GGPP (n ¼ 10). (D) Quantitative RT – PCR analysis shows that lovastatin treatment (1 mM) reduces the expression levels of Cdx2 and Gata3, which is circumvented by supplementation with GGPP (10 mM). Expression levels of pluripotency regulators Pou5f1, Nanog and Sox2 are mostly unaffected. The vertical axis represents gene expression levels relative to untreated control embryos set as 100. Bars indicate average values of three sets of experiments, and circles represent values for individual experiments. Asterisks indicate statistically significant reduction in the average expression level in comparison with the control (P , 0.05; 2-sample t-test). (E) Projected confocal images of embryos that were stained with 4′ ,6-diamidino-2-phenyl-indole (DAPI; total nuclei) and anti-YAP. Embryos had been treated with GGTI-298 (10 mM) for 20 h. Scale bar ¼ 50 mm. (F) Average numbers of mitotic (gray), YAP-negative (white), and YAP-positive (green) nuclei. Control (n ¼ 10) and GGTI-298 (n ¼ 10). (B, C, F) Green asterisks indicate statistically significant reduction in YAP-positive nuclear number in comparison with the control (P , 0.01; 2-sample t-test).

(Fig. 3C), confirming the previous study (Kono et al., 2014). The cavitation defect by RHO inhibition was not reversed by supplementation of GGPP (10 mM) (Fig. 3C and D). The result is consistent with the notion that activation of RHO is required for blastocyst formation downstream of GGPP but not upstream of the mevalonate pathway.

Nuclear localization of YAP is dependent on GGPP and GGTase Formation of the blastocyst cavity depends on the action of TE (Marikawa and Alarcon, 2012). One of the key molecular events that specify the TE

lineage is nuclear localization of YAP, a downstream effector of HIPPO signaling (Nishioka et al., 2009). Thus, we examined how YAP localization is affected by manipulations of the mevalonate pathway. Embryos at 8-cell stage (E2.5) were cultured in the presence of lovastatin (1 mM) with or without MVA (100 mM) or GGPP (10 mM), and YAP localization was examined 20 h later by immunostaining. In control embryos, YAP-positive nuclei were observed in cells near the embryo surface (Fig. 4A). In contrast, YAP nuclear localization was largely absent in lovastatin-treated embryos, indicating that activation of YAP is dependent on the HMG-CoA reductase activity. However, supplementation of MVA or GGPP restored nuclear localization of YAP to

358 the level that was indistinguishable from control (Fig. 4A and B). The average numbers of YAP-positive nuclei were 14.6 + 2.6 for control (n ¼ 12), 1.9 + 2.0 for lovastatin (n ¼ 12), 15.9 + 3.2 for lovastatin plus MVA (n ¼ 11), and 16.1 + 3.1 for lovastatin plus GGPP (n ¼ 12). Importantly, the total number of nuclei, which includes YAP-positive, YAP-negative and mitotic nuclei, was similar among the four groups, indicating that lovastatin, MVA, or GGPP did not impact cell viability or proliferation. Similarly, treatment with cerivastatin (1 mM) significantly reduced the number of YAP-positive nuclei, which was restored by supplementation of GGPP (10 mM) (Fig. 4C). The average numbers of YAP-positive nuclei were 15.5 + 2.9 for control (n ¼ 12), 4.3 + 2.7 for cerivastatin (n ¼ 10), and 17.0 + 1.6 for cerivastatin plus GGPP (n ¼ 10). To verify the impact of HMG-CoA reductase inhibition on YAP nuclear localization, we examined the expression levels of Cdx2 and Gata3, because transcriptional up-regulation of these TE-specific genes is mediated by the nuclear activity of the YAP/TEAD4 complex (Nishioka et al., 2009; Ralston et al., 2010; Rayon et al., 2014). Quantitative RT –PCR analysis showed that the levels of Cdx2 and Gata3 were consistently reduced by lovastatin treatment but not when GGPP was supplemented (Fig. 4D). Thus, transcriptional activation of the TE-specific genes was dependent on the production of GGPP through the mevalonate pathway. In contrast, no significant alteration was observed in the expression levels of pluripotency regulators, Pou5f1, Nanog, and Sox2, by lovastatin treatment (Fig. 4D). These results further support the notion that the activity of HMG-CoA reductase is primarily essential for TE lineage formation. Because formation of the blastocyst cavity was also dependent on the activity of GGTase, we examined the impact of GGTI-298 (10 mM) treatment on YAP localization. The number of YAP-positive nuclei was significantly reduced by GGTI-298, as the average numbers were 17.7 + 2.6 for control (n ¼ 10), and 3.2 + 3.4 for GGTI-298 (n ¼ 10) (Fig. 4E and F). The total number of nuclei was similar between control and GGTI-298-treated embryos, indicating no significant impairment in cell viability or proliferation by inhibition of GGTase. These results suggest that statins impact HIPPO signaling by diminishing YAP nuclear localization, which is possibly due to attenuation of geranylgeranylation-dependent activation of RHO.

YM-53601, an inhibitor of squalene synthase, impairs blastocyst formation The adverse developmental impact of statins was fully reversed by supplementation of GGPP (Figs 2 and 4), which raises the possibility that synthesis of other MVA derivatives, namely squalene and cholesterol, is dispensable for blastocyst formation. To test this, we treated 8-cell stage embryos with various concentrations of YM-53601, an inhibitor of squalene synthase (Fig. 2A), and morphology was examined at E3.5 and E4.5. Significant reduction (P , 0.01; 2-sample t-test) in the mean cavity size was observed with 1 mM and higher but not with lower concentrations (Fig. 5A and B). Most embryos that were treated with YM-53601 at 1 mM did not have a cavity at E4.5, but in terms of gross morphology they were not totally disintegrated, unlike statin-treated or GGTI-298treated embryos (Figs 1A and 3A). However, at higher concentration (2 mM), YM-53601-treated embryos appeared disintegrated by E4.5. To assess whether the adverse impact of YM-53601 treatment (1 mM) is due to inhibition of squalene or cholesterol synthesis, the

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treated embryos were supplemented with squalene (100 mM) or cholesterol (100 mM). However, there was no significant rescue in cavitation neither with squalene nor cholesterol supplementation (Fig. 5C and D). Thus, blastocyst impairment by YM-53601 may not be due to lack of squalene or cholesterol synthesis.

Discussion In the present study, we examined the impact of statins, inhibitors of HMG-CoA reductase that are commonly prescribed as cholesterollowering medications, on mouse blastocyst formation. All four FDAapproved statins tested, namely lovastatin, atorvastatin, cerivastatin, and pravastatin, interfered with blastocyst cavity formation, consistent with the previous observation that mevastatin impairs blastocyst development (Surani et al., 1983). The key features of normal blastocyst formation, including cavity formation, expression of TE-specific transcription factors, and nuclear accumulation of YAP, were restored in statin-treated embryos by supplementation of MVA or GGPP. An inhibitor of GGTase also interfered with blastocyst formation and YAP nuclear accumulation. These results suggest that the adverse impact of HMG-CoA reductase inhibitors is largely due to reduction in GGPP production and geranylgeranylation of target molecules, possibly RHO family proteins. Based on these findings along with the previous studies (Alarcon, 2010; Hirate et al., 2013, 2015; Leung and Zernicka-Goetz, 2013; Kono et al., 2014; Cao et al., 2015), we speculate a model of how the mevalonate pathway regulates the formation of TE, as depicted in Fig. 6. Essentially, the action of HMG-CoA reductase is to generate sufficient amount of GGPP to covalently modify (i.e. prenylate) and activate RHO proteins, which in turn enable proper apical-basal polarization in outer cells to suppress HIPPO signaling, leading to YAP-dependent expression of TE-specific transcription factor genes, Cdx2 and Gata3. Several key steps in this model need to be verified or further investigated in future studies, e.g. whether RHO proteins are the sole geranylgeranylation targets essential for TE formation and how activation of RHO proteins is involved in apical-basal polarization in preimplantation development. Most statins, including the four that were evaluated in the present study, are currently classified by the FDA as Pregnancy Risk Category X drugs, i.e. they are contraindicated for use during pregnancy due to their potential developmental toxicity. Nonetheless, whether the use of statins during pregnancy increases the frequency of birth defects or embryo loss in human is unclear and controversial (Godfrey et al., 2012; Bateman et al., 2015). Even with animal studies, the in vivo effects of statins on embryo development are still elusive. For example, treatment of female rats with atorvastatin has shown no adverse effects on reproductive outcome (Dostal et al., 1996), whereas treatment with simvastatin has caused significant alterations in endocrine signals critical for normal reproduction (Guldvang et al., 2015). Such apparent discordance may be due to the different types of statins that were tested, as each statin has unique pharmacokinetic properties that influence its absorption, distribution, metabolism, and excretion. Although concentrations of statins that are found in human blood plasma are generally low (Supplementary Table), the data regarding actual concentrations of statins in the reproductive tract, where embryos develop, are currently scarce. The evaluation of various statins in different animal models and detailed assessment of key reproductive parameters, such as ovulation, fertilization, and pre- and post-implantation development,

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359

Figure 5 YM-53601, an inhibitor of squalene synthase, impairs blastocyst formation. (A) Photos of embryos at E3.5 and E4.5 that had been cultured from E2.5 with various concentrations of YM-53601. Scale bar ¼ 50 mm. (B) Cavitation of embryos treated with YM-53601. Bars represent the mean + standard deviation of cavity size. Control (n ¼ 11), 0.25 mM (n ¼ 16), 0.5 mM (n ¼ 17), 1 mM (n ¼ 14), and 2 mM (n ¼ 16). (C) Photos of embryos treated with YM-53601 (1 mM), squalene (100 mM), and/or cholesterol (100 mM). Scale bar ¼ 50 mm. (D) Neither squalene (Squ; 100 mM) nor cholesterol (Cho; 100 mM) can rescue the cavitation defects caused by YM-53601 (1 mM). Bars represent the mean + standard deviation of cavity size. Control (n ¼ 18), YM-53601 (n ¼ 18), YM-53601 + squalene (n ¼ 21), and YM-53601 + cholesterol (n ¼ 21). (B, D) Asterisks indicate statistically significant reduction in the mean cavity size in comparison with the control of the same stage (P , 0.01; 2-sample t-test).

are crucial to obtain robust information on their in vivo reproductive impact, in conjunction with further human epidemiological studies. Moreover, in vitro studies, such as those presented in this report, are also informative to predict potential developmental toxicity of drugs, because they enable determination of specific concentrations that impair embryo development, which can be compared with drug concentrations that may be found in the human reproductive tract. Each of the four statins tested displayed different potency in inhibiting blastocyst cavity formation, among which pravastatin was the least potent and cerivastatin was the most potent (Fig. 1B). Structurally, pravastatin differs from other statins because of the hydroxyl group attached to its decalin ring, which makes pravastatin hydrophilic and less permeable to the plasma membrane (Quion and Jones, 1994). Thus, to inhibit HMG-CoA reductase, pravastatin needs to enter the cell through specific transporters (Yamazaki et al., 1993). Such transporters for pravastatin are enriched in hepatocytes, accounting for its selective inhibition of cholesterol synthesis in the liver (van Vliet et al., 1995). Pravastatin is generally considered non-embryotoxic in humans and animals due to its impermeability through the placental barrier (Edison and Muenke, 2004; Riebeling et al., 2012). However, the present study showed that pravastatin was the least potent inhibitor of blastocyst formation even though it was directly exposed to embryos, suggesting that preimplantation embryos lack membrane transporters to uptake pravastatin. On the other hand, cerivastatin was the most potent in inhibiting

blastocyst formation, which is in line with other studies demonstrating the highest potency of cerivastatin to inhibit HMG-CoA reductase in liver microsome fractions (Bischoff et al., 1997) and cholesterol synthesis in arterial myocytes (Corsini et al., 1996). High potency of cerivastatin may have contributed to the serious side effect of myotoxicity along with fatality in a number of cases, which resulted in its withdrawal from the market (Tobert, 2003). The present study was conducted with mouse embryos, and further investigations are pivotal to examine whether human preimplantation embryos are also susceptible to statins in the same manner. Generally, preimplantation development of mouse resembles that of human at the gross morphological level in terms of cleavage, compaction, and TE epithelialization (Cockburn and Rossant, 2010; Wong et al., 2010). Orthologs of OCT4 and CDX2 are also expressed in a lineage-specific manner in the human late blastocyst (Cauffman et al., 2006; Roode et al., 2012; Niakkan and Eggan, 2013), and RNAs encoding various components of the mevalonate and HIPPO signaling pathways are present in human preimplantation embryos (Yan et al., 2013). If these molecules interact with each other in a conserved manner, statins may also interfere with human blastocyst formation, which would lead to embryo loss before implantation. Hmgcr gene, which encodes HMG-CoA reductase, has been knocked out, and homozygous knockout (Hmgcr 2/2) embryos die around the time of implantation (Ohashi et al., 2003). It is unclear whether the phenotype of Hmgcr 2/2 embryos is related to that of statin-treated

360

Figure 6 Model for regulation of trophectoderm (TE) lineage specification by mevalonate pathway. 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) converts HMG-CoA into mevalonic acid (MVA), which is transformed into farnesyl pyrophosphate (FPP) and then into geranylgeranyl pyrophosphate (GGPP). Geranylgeranyl transferase (GGTase) attaches the geranylgeranyl (GG) moiety of GGPP onto RHO. Geranylgeranylated RHO becomes active by binding to guanosine triphosphate (GTP), which is essential for apicalbasal cell polarization. Proper apical-basal cell polarity is required to suppress HIPPO signaling, which leads to nuclear localization of YAP. YAP forms a complex with TEAD4, and acts as a transcriptional activator, which directly or indirectly up-regulates transcription of trophectodermspecific genes, Cdx2 and Gata3. See the text for further discussion on this model.

embryos that is described in the present study. In the knockout study, there was no sign of implantation by Hmgcr 2/2 embryos, suggesting that they failed to form functional blastocysts. However, Hmgcr 2/2 embryos at E3.5 were recovered from intercrosses of heterozygous mice, and they were referred to as ‘blastocysts’. Thus, Hmgcr 2/2 embryos appear to form a blastocyst cavity, suggesting that they develop further than statin-treated embryos, although morphological descriptions or photographs of Hmgcr 2/2 blastocysts were not included in the study. There are several possibilities that account for the potential differences between the phenotypes of Hmgcr 2/2 and statin-treated embryos. First, statins, as pharmacological agents, may have off-targets other than HMG-CoA reductase to compromise blastocyst formation. However, the adverse impact of statins was solely due to diminished production of mevalonic acid, because supplementation of mevalonic acid alone was able to restore blastocyst formation. Therefore, the target of statins to interfere with blastocyst formation is specific to HMG-CoA reductase. Interestingly, the presence of another isoform of HMG-CoA reductase that is not encoded by the Hmgcr gene is speculated (Breitling and Krisans, 2002). If statins are to inhibit such HMG-CoA reductase isoform, it may account for the severer impact of statins than the Hmgcr knockout. Secondly, a significant amount of HMG-CoA reductase may be present in the oocytes derived from Hmgcr +/2 females as maternal supplies that persist after fertilization to support blastocyst formation in Hmgcr 2/2 embryos. Statins are likely to inhibit both maternal and zygotic HMG-CoA reductase, which may cause severer impact on blastocyst formation. Thirdly, the fluid in the fallopian tube or uterus may contain a certain amount of mevalonic acid or its derivatives, which may support blastocyst formation in Hmgcr 2/2 embryos. Indeed, when pregnant Hmgcr +/2 mice were administered

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with mevalonic acid subcutaneously, development of Hmgcr 2/2 embryos was significantly improved, indicating that availability of mevalonic acid in the external environment of embryos greatly influences developmental potential. It is of particular interest to examine how well Hmgcr 2/2 zygotes can develop in vitro where no mevalonic acid or its derivatives are present in the culture medium. We showed in the present study that nuclear accumulation of YAP, a prerequisite for TE lineage formation (Nishioka et al., 2009), was dependent on the production of GGPP through the mevalonate pathway and on the activity of GGTase. Because active RHO-ROCK signaling is essential for nuclear accumulation of YAP in mouse preimplantation embryos (Kono et al., 2014), RHO proteins, which are well-known substrates for geranylgeranylation (Zhang and Casey, 1996; Allal et al., 2000), are likely to be the key targets of GGTase to enable TE formation. This notion is consistent with the recent study that GGPP-dependent activation of YAP is mediated by RHO proteins in human cell lines (Sorrentino et al., 2014). Nonetheless, RHO proteins are not the only substrates for geranylgeranylation, and other small GTPases (e.g. RAP and RAL) as well as non-GTPases (e.g. 2′ ,3′ -cyclic nucleotide 3′ -phosphodiesterase) are also geranylgeranylated (De Angelis and Braun, 1994; Reid et al., 2004; Wright and Philips, 2006; Maurer-Stroh et al., 2007). Therefore, it is possible that targets of GGTase other than RHO may also be involved in YAP regulation and TE formation in mouse embryos. Furthermore, it has been shown that Pggt1b gene, which encodes GGTase, is dispensable for activation of RHO in macrophages (Khan et al., 2011, 2013), raising the possibility that the role of geranylgeranylation in RHO activation is dependent on cell types or biological contexts. Further investigations on the relationship between the mevalonate pathway and RHO activation are warranted in mouse preimplantation development. We showed in the present study that supplementation of GGPP was sufficient to fully reverse the effect of statins, and that blastocyst formation was impaired by GGTase inhibitor GGTI-298. These results suggest that reduction in geranylgeranylation was responsible for the adverse impact of statins in preimplantation development. Interestingly, however, supplementation of FPP was also able to reverse the effect of statins partially (Fig. 2). The nature of this ‘partial rescue’ by FPP is unclear. It is possible that a small amount of exogenous FPP was converted into GGPP in the embryo, which in turn partially restored cavity formation. Synthesis of GGPP is catalyzed by geranylgeranyl diphosphate synthase from FPP and isopentenyl pyrophosphate (IPP) (Fig. 2A; Kainou et al., 1999). Thus, for this reaction to happen in statin-treated embryos, a certain level of HMG-CoA reductase activity should be present to produce a small amount of IPP. Because statins inhibited blastocyst cavitation in a dose-dependent manner (Fig. 1B), pharmacological inhibition of HMG-CoA reductase was probably not 100% at the concentration tested (e.g. 1 mM lovastatin). Also, although the present study underscores the importance of geranylgeranylation in blastocyst formation, roles of several well-known targets of farnesylation have been studied in mouse preimplantation development, including RAC1, CDC42, and PRICKLE2 (Natale and Watson, 2002; Cui et al., 2007; Tao et al., 2012). It is of particular interest to examine whether these targets were impacted by statin treatment and whether supplementation of FPP boosted their activities, which in turn contributed to the partial rescue of statin-treated embryos. Inhibition of HMG-CoA reductase lowers production of GGPP as well as squalene and cholesterol (Fig. 2A; Park et al., 2014). The adverse

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Blastocyst formation requires geranylgeranylation

impact of statins on blastocyst formation was bypassed by supplementation of GGPP, suggesting that the squalene synthesis pathway is dispensable in preimplantation development. Nonetheless, YM-53601, an inhibitor of squalene synthase (SQS), interfered with blastocyst formation at the dose of 1 mM. IC50 of YM-53601 for SQS inhibition is estimated to be 45 –170 nM in hepatic microsome preparations (Ugawa et al., 2000), but how effectively SQS is inhibited in the mouse embryo by YM-53601 is currently unknown. It is possible that YM-53601 at 1 mM can affect other yet unknown targets to impair blastocyst formation. This notion is in line with our observation that supplementation of squalene or cholesterol did not reverse the effect of YM-53601, indicating that the lack of these lipids was not the cause of blastocyst impairment. Consistently, homozygous knockout of Fdft1 gene, which encodes SQS, exhibits embryonic lethality only after implantation, although maternal supply of SQS in the oocyte may be sufficient to support preimplantation development in the knockout embryos (Tozawa et al., 1999). It is also possible that SQS has another function besides the conversion of FPP into squalene, which may be essential for blastocyst formation. Indeed, the lethality of homozygous Fdft1-knockout embryos cannot be rescued by dietary supplementation of squalene or cholesterol. Alternatively, the deleterious effects of SQS knockout or its pharmacological inhibition may be due to build-up of the immediate precursor of squalene, i.e. FPP, which is then converted to potentially toxic farnesol-derived dicarboxylic acids (Gonzalez-Pacanowska et al., 1988; Bostedor et al., 1997). Regardless, further investigations are crucial to determine the role of SQS in preimplantation development, because use of SQS inhibitors has been explored to treat hypercholesterolemia as well as other diseases (Seiki and Frishman, 2009; Shang et al., 2014; Saito et al., 2015), while their reproductive and developmental toxicity is largely unknown.

Supplementary data Supplementary data are available at http://molehr.oxfordjournals.org/.

Acknowledgements We are grateful to the Animal and Veterinary Service staff for taking care of our mice.

Authors’ roles V.B.A. and Y.M. designed the research study, performed the research, analyzed the data, and wrote the paper.

Funding This work was supported by the George F. Straub Trust of the Hawaii Community Foundation [13ADVC-60315 to V.B.A.]; and the National Institutes of Health [P20GM103457 to V.B.A.].

Conflict of interest None declared.

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