title: Effects of the histone deacetylase inhibitor 'Scriptaid' on the developmental competence of mouse embryos generated through round spermatid injection.
abstract: Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model?
  
  
  
  Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos.
  
  
  
  The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species.
  
  
  
  Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times.
  
  
  
  Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment.
  
  
  
  The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn.
  
  
  
  N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required.
  
  
  
  Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI.
  
  
  
  The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare.
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China.|Center of Reproductive Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, 2699 West Gaoke, 200040 Shanghai, China.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Kong
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju road, 200011 Shanghai, China.
authors.forename: Mingru
authors.initials: M
authors.lastname: Yin
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China.
authors.forename: Dongbao
authors.initials: D
authors.lastname: Chen
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China.
authors.forename: Shangang
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China.
authors.forename: Fengying
authors.initials: F
authors.lastname: Xing
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China.
authors.forename: Manxi
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China.
authors.forename: Zhenfu
authors.initials: Z
authors.lastname: Fang
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju road, 200011 Shanghai, China chenxuejin_6@163.com lyuqifeng@126.com.
authors.forename: Qifeng
authors.initials: Q
authors.lastname: Lyu
authors.affiliation: Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing, 200025 Shanghai, China chenxuejin_6@163.com lyuqifeng@126.com.
authors.forename: Xuejin
authors.initials: X
authors.lastname: Chen
mesh_terms: D000818:Animals; D004624:Embryo Transfer; D047108:Embryonic Development / Q000187:drug effects*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006898:Hydroxylamines / Q000494:pharmacology*; D000090062:Kruppel-Like Factor 4; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009865:Oocytes / Q000187:drug effects; D011804:Quinolines / Q000494:pharmacology*; D020554:Sperm Injections, Intracytoplasmic; D013087:Spermatids / Q000187:drug effects*
chemical_list: D056572:Histone Deacetylase Inhibitors; D006898:Hydroxylamines; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D011804:Quinolines; C410733:scriptaid
keywords: DNA methylation; Scriptaid; development-related genes; imprinted genes; round spermatid injection (ROSI)