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CDKN1A Deficiency in Zebrafish Promote Follicle Maturation and Degeneration Mediated by CRISPR/Cas9 Targeted Disruption

ABSTRACT: CDKN1A plays multiple roles in distinctive biological processes, such as cellular proliferation, apoptosis, DNA repairing, and etc. In mouse, Cdkn1a deficiency led to cell arrest at G1 phase, and also reported that Cdkna1-deficient mice were susceptible to tumorigenesis. However, the functions of Cdkn1a in zebrafish were still elusive due to few studies. Our previous study indicated that Cdkn1a might be closely associated with folliculogenesis, and to further investigate its specific role during folliculogenesis, Cdkn1a was targeted disrupted by CRISPR/Cas9. Interestingly, Cdkn1a-deficient zebrafish were embryonic lethality, resulted in approximate 2% homozygous mutants in adult stage. Furthermore, homozygous mutants have accelerated follicle maturation that faster than WT, and were fertile at young age (2 month). However, the matured or large follicles in mutant soon become dysfunctional due to abnormal oocyte that led to infertile at 3 months. Gradually, the abnormal follicles were undertaken follicle degeneration, resulted in PG follicle enrichment in mutant ovary at 6 months. Similar follicle degeneration phenomenon was also observed in heterozygous mutant ovary at 8 months. The degenerating follicles in mutant were convinced by in situ apoptosis detection kit. To further explore molecular mechanism, RNA-seq and qPCR validation were adopted to identify underlying molecular pathway that involved in Cdkn1a deficient apoptosis. This study firstly reported the functions of Cdkn1a in zebrafish, particular in its critical role in maintenance of normal follicle development, and the special phenotypes in folliculogenesis perfectly interpretation of its dual roles on both proliferation and apoptosis at two different phases, which replenished its novel role in folliculogenesis that expanded our insights.

ORGANISM(S): Danio rerio

PROVIDER: GSE239622 | GEO | 2024/10/27

REPOSITORIES: GEO

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Project description:Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are three phthalates commonly found in consumer products, including the plastic coating of pharmaceuticals and personal care products. Folliculogenesis, a tightly regulated process occurring in the ovary, is the maturation of an immature primordial follicle to a mature antral follicle. Follicles house the oocyte and antral follicles specifically play a crucial role in ovarian steroidogenesis and ovulation. DBP, BBP, and DEHP have been associated with inhibited antral follicle growth in vitro, decreased ovulation rates in vitro, and decreased antral follicle counts in women. However, little is known about the effects of a three-phthalate mixture on antral follicles in vivo. The objective of this study was to evaluate the effects of a human relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. CD-1 female mice (60 days old) were pipet fed tocopherol stripped corn oil (vehicle control) only or a phthalate mixture (52% DBP, 36% DEHP, and 12% BBP dissolved in vehicle) which modeled human follicular fluid concentrations. The mice were treated with 32µg/kg/day (PHT Mix 32; cumulative estimate in general population) and 500µg/kg/day (PHT Mix 500; cumulative estimate in occupationally exposed individuals) for 10 consecutive days. Proteome profiling of antral follicles (>250µm) was performed via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were identified in the three groups, of which 194 were differentially abundant between the vehicle and PHT Mix 32 group, and 136 between the vehicle and PHT Mix 500 group. Gene ontology analysis revealed that the two treatments of the phthalate mixture upregulate and downregulate distinctive processes, supporting non-monotonic effects of phthalates on the antral follicle proteome. Taken together, these results reveal that a human relevant mixture of DBP, BBP, and DEHP alters the antral follicle proteome and merits further evaluation to elucidate the molecular mechanisms by which phthalates cause negative reproductive outcomes.

Project description:In mammals, ovarian folliculogenesis leading to the ovulation of completely mature oocytes is a long and complex process that is regulated at different levels. The mechanisms that underlie the selection of one or several dominant follicles as well as the regulation of the number of ovulating follicles are largely unknown. In this project, we proposed to study the genetic determinism that underlies the difference of ovulation rate between species (cattle and pigs), by studying two processes that exist in ovary: follicular development and follicular atresia. Towards this purpose, we made a comparative transcriptomics study on granulosa cells. Pig and cattle comparison was achieved by a transcriptome analysis with a 9K nylon pig microarray (GPL3729) on granulosa cells from either small healthy antral follicles (SHF), small atretic follicles (SAF) or large healthy antral follicles (LHF). The images were quantified using AGscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Transcriptomic analysis on pig and cattle evidenced 997 differentially expressed genes (FDR1%) between the three follicle classes and/or the two species. This research project which implicated three laboratories from INRA: "Laboratoire de Genetique Cellulaire" (UMR444-LGC) , "Station d'Amelioration Genetique des Animaux" (UR 631-SAGA) and "Physiologie des Comportements et de la reproduction" (UMR 85-PRC) benefited from both European funding through SABRE project and French ANR funding through GenOvul project. Keywords: transcriptome analysis, pig, cattle, ovary, folliculogenesis, gene expression, cDNA microarray The data were obtained from 34 RNA samples: 10 small healthy follicles samples (6 for sows and 4 for cows), 13 small atretic follicles samples (7 for sows and 6 for cows) and 11 samples for large healthy follicles (6 for sows and 5 for cows). They were hybridized on a 9K pig nylon microarray (GPL3729).

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CDKN1A Deficiency in Zebrafish Promote Follicle Maturation and Degeneration Mediated by CRISPR/Cas9 Targeted Disruption

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