Endogenous biosynthesis of docosahexaenoic acid(DHA)regulates fish oocyte maturation by promoting pregnenolone production

Omega-3 polyunsaturated fatty acids(n-3 PUFAs),particularly docosahexaenoic acid(22:6n-3,DHA),play crucial roles in the reproductive health of vertebrates,including humans.Nevertheless,the underlying mechanism related to this phenomenon remains largely unknown.In this study,we employed two zebrafish genetic models,i.e.,elovl2^(-/-)mutant as an endogenous DHAdeficient model and fat1(omega-3 desaturase encoding gene)transgenic zebrafish as an endogenous DHA-rich model,to investigate the effects of DHA on oocyte maturation and quality.Results show that the elovl2^(-/-)mutants had much lower fecundity and poorer oocyte quality than the wild-type controls,while the fat1 zebrafish had higher fecundity and better oocyte quality than wildtype controls.DHA deficiency in elovl2^(-/-)embryos led to defects in egg activation,poor microtubule stability,and reduced pregnenolone levels.Further study revealed that DHA promoted pregnenolone synthesis by enhancing transcription of cyp11a1,which encodes the cholesterol side-chain cleavage enzyme,thereby stabilizing microtubule assembly during oogenesis.In turn,the hypothalamic-pituitary-gonadal axis was enhanced by DHA.In conclusion,using two unique genetic models,our findings demonstrate that endogenously synthesized DHA promotes oocyte maturation and quality by promoting pregnenolone production via transcriptional regulation of cyp11a1.

A landscape of differentiated biological processes involved in the initiation of sex differentiation in zebrafish

Zebrafish(Danio rerio)has been used as a promising animal model to study gonadal development and gametogenesis.Although previous studies have identified critical molecules participating in zebrafish gonad differentiation,a landscape view of the biological processes involved in this process is still lacking.Here we isolated intact zebrafish differentiating gonads,at 25 days post-fertilization(dpf)and 30 dpf and conducted RNA-seq analyses on the juvenile gonads that tended to develop into ovaries or testes.Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the biological process level.During ovary differentiation,the biological processes related to metabolic activities in the production of energy and maternal substances,RNA degradation,and DNA repair were enriched.During testis differentiation,the biological processes related to cell proliferation,differentiation,and morphogenesis were enriched,with a total of 15 signaling pathways.Notably,we reveal that the immune-related processes are extensively involved in the regulation of testis development.Overall,this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells.Interestingly,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.

Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells

The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell(PGC) transplantation(PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic(MZ) mutants of tcf7l1a(gene essential for head development), pou5f3(gene essential for zygotic genome activation) and chd(gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.