The genetic program of oocytes can be modifiedin vivoin the zebrafish ovary

Abstract

Oocytes, the irreplaceable gametes for generating a new organism, are matured in the ovary of living female animals. It is unknown whether any genetic manipulations can be applied to immature oocytes inside the living ovaries. As a proof-of-concept, we here demonstrate genetic amendments of zebrafish immature oocytes within the ovary. Oocyte microinjection in situ (OMIS) stimulates tissue repair responses, but some of microinjected immature oocytes are matured, ovulated and fertilizable. By OMIS-mediated Cas9 approach, ntla and gata5 loci of oocytes arrested at prophase I of meiosis are successfully edited before fertilization. Through OMIS, high efficiency of biallelic mutations in single or multiple loci using Cas9/gRNAs allows immediate manifestation of mutant phenotypes in F0 embryos and multiple transgenes can co-express the reporters in F0 embryos with patterns similar to germline transgenic embryos. Furthermore, maternal knockdown of dnmt1 by antisense morpholino via OMIS results in a dramatic decrease of global DNA methylation level at the dome stage and causes embryonic lethality prior to segmentation period. Therefore, OMIS opens a door to efficiently modify the genome and provides a possibility to repair genetically abnormal oocytes in situ.

Widespread Enhancer Dememorization and Promoter Priming during Parental-to-Zygotic Transition

“…An initial attempt to deplete these three proteins by injecting morpholinos against these transcripts at the 1-cell stage failed to decrease histone acetylation levels, again likely because of abundant maternal proteins (data not shown). Therefore, we microinjected triple morpholinos into oocytes via OMIS (Wu et al, 2018b). Immunofluorescence staining showed that the level of H3K27ac in 256-cell embryos derived from the triple knockdown (TKD) oocytes (hereafter referred to as HAT mTKD embryos) was significantly reduced ( Figure 6A).…”

“…Oocyte microinjection in situ (OMIS) Briefly, on the first day of OMIS (Wu et al, 2018b), adult females at 5-12 month old were anesthetized in 550 mg/ml tricaine (Sigma, Cat # A5040) in a Petri dish. Then the fish was placed on a damp sponge with specific buffer (5.4 mM KCl,136.8 mM NaCl, 4.2 mM NaHCO 3 , 0.44 mM KH 2 PO 4 , 0.25 mM Na 2 HPO 4 , and 0.5% (wt/vol) BSA).…”

Mini-III RNase-based dual-color system for in vivo mRNA tracking

“…Immunofluorescence was performed as previously described ( Wu et al, 2018 ) using the following antibodies: anti-dsRNA (Merck Millipore, MABE1134, 1:50), anti-mCherry (Easybio, BE2027, 1:200), anti-GFP (Abcam, ab13970, 1:500).…”

Zebrafish as an emerging model to study gonad development

“…While no signs of mitotic activity are typically recorded until 15 dpf [9] , early diplotene oocytes can be observed with a clear juvenile ovarian structure [7] , [10] , suggesting early vertebrate oogenesis. Selman et al described the oogenesis process in zebrafish as having five stages, defined according to their morphology and karyotype in the ovary [11] , [12] . Based on this, sex differentiation is marked by the appearance of perinuclear oocytes, which occurs as early as 17 dpf [13] .…”