O-294 Spindle transfer rescues impaired fertilization linked to Patl2-mediated dysregulation of maternal mRNA translation

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Abstract Study question Can single-cell, mass spectrometry-based proteomics reveal proteins underlying the reduced fertilizing capacity of Patl2-/- MII mouse oocytes? Is it possible to treat Patl2-related infertility? Summary answer Reduced protein synthesis from maternal mRNAs is detected in Patl2-/- MIIs, which can be rescued by spindle transfer (ST), but not assisted oocyte activation (AOA). What is known already PATL2 is an RNA-binding protein that represses maternal mRNA translation during oocyte maturation. PATL2 mutations in humans often cause GV arrest, although some patients produce MII oocytes with reduced fertilization and developmental potential. Consequently, oocyte donation is required. The Patl2-/- knockout mouse model offers a unique opportunity to study Patl2-related infertility and potential treatments. To gain a deeper understanding of the role of Palt2 at the MII stage, we evaluated various oocyte quality markers and conducted single-cell proteomic analysis. Proposed treatments to overcome low fertilization, including AOA, targeting calcium release, and ST, replacing suboptimal cytoplasm, were investigated. Study design, size, duration Palt2 -/- mice (C57BL/6NTac-Patl2tm1a) with a deletion of Patl2 exon 7 were used. Breeding of heterozygous mice from April 2021 to October 2023 produced 271 pups, of which 36 were homozygous Palt2-/- females. MII oocytes were collected from Patl2-/- and Patl2+/+ females for the evaluation of oocyte quality markers, and for the assessment of AOA and ST efficacy. Moreover, some MII oocytes were vitrified for subsequent single-cell proteomics. At least three replicates were conducted per experiment. Participants/materials, setting, methods Four- to 12-week-old mice underwent superovulation and oocyte collection to assess maturation rate. Spindle configurations, calcium releasing capacity after SrCl2 exposure, as well as activation (AR) and blastocyst rates (BR) after PIEZO-ICSI were evaluated in MII oocytes. AOA was performed by SrCl2 exposure, and ST involved transferring the Patl2-/- spindle to Patl2+/+ enucleated cytoplasm followed by SrCl2 exposure. Vitrified-warmed oocytes were used for single-cell proteomics using a timsTOF Ultra mass spectrometer operated in diaPASEF mode. Main results and the role of chance The MII rates were higher in Patl2+/+ (89.39%) than Patl2-/- mice (79.63%, p = 0.015). Nuclear maturation was not compromised, as normal spindle rates were similar in Patl2+/+ (86.00%) and Patl2-/- oocytes (78.57%, p = 0.414) The total calcium released during oocyte activation did not significantly differ between Patl2+/+ (AxF=4.59) and Patl2-/- oocytes (AxF=6.15, p = 0.145) . After PIEZO-ICSI with wild-type sperm, reduced activation (Patl2-/-=31.71%, Patl2+/+=76.74%, p < 0.0001) and blastocyst rates (Patl2-/-=7.69%, Patl2+/+=42.42%, p = 0.035) were observed in knockout oocytes. Single-cell proteomics identified 4939 proteins and confirmed absence of Patl2 in knockout oocytes, analyzing 25 Patl2+/+ and 27 Palt2-/- MIIs. After filtering, 3777 proteins were used for statistical analysis, revealing 1511 differentially expressed proteins (q-value < 0.05; 999 downregulated, 512 upregulated in Patl2-/- oocytes). Multiple proposed Patl2 interactors (Cpeb1, Eif4e1b) and maternal effect genes (MEGs) implicated in RNA regulation (Zar1), meiosis progression (Wee2), cell division (Cdk1), and microtubule organization (Tuba4a) were significantly reduced. Additionally, proteins involved in calcium dynamics were upregulated (Pde1b, Tmco1, Micu). Consistent with these observations, AOA did not lead to comparable activation (Patl2-/-=75.95%, Patl2+/+=95.97%, p < 0.0001) or blastocyst rates (Patl2-/-=45.00%, Patl2+/+=79.83%, p < 0.0001). However, ST treatment in Patl2-/- oocytes restored activation (100%) and blastocyst rates (75.00%) to levels observed in Patl2+/+ oocytes (AR = 96.15%, p = 1 and BR = 96.00%, p = 0.049). Limitations, reasons for caution The Patl2-/- mice exhibits a less severe phenotype compared to patients carrying PATL2 variants. Patl2-/- oocytes display high MII rate without significant spindle abnormalities, which contrasts with a previous published report. Additionally, the comparison between AOA and ST treatments was conducted using parthenogenetically activated oocytes, rather than biparental embryos. Wider implications of the findings ST could treat PATL2-related female-infertility in patients with MII oocytes, whereas AOA is unlikely effective, as calcium release in Patl2-/- oocytes is slightly increased. Single-cell proteomics reveals cytoplasmic deficiencies in Patl2-/- MII oocytes linked with abnormal regulation of MEGs, some already associated to fertilization failure and embryo arrest in humans. Trial registration number No

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