bims-cebooc 2024-09-22 papers

bims-cebooc

Biomed News

on Cell biology of oocytes

Issue of 2024–09–22
fiveteen papers selected by
Gabriele Zaffagnini, Centre for Genomic Regulation

CtIP regulates G2/M transition and bipolar spindle assembly during mouse oocyte meiosis.
VPS34 Governs Oocyte Developmental Competence by Regulating Mito/Autophagy: A Novel Insight into the Significance of RAB7 Activity and Its Subcellular Location.
Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.
The structural role of Skp1 in the synaptonemal complex is conserved in nematodes.
OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.
Artificial kinetochore beads establish a biorientation-like state in the spindle.
Nutritional vitamin B12 regulates RAS/MAPK-mediated cell fate decisions through one-carbon metabolism.
Dynamics and necessity of SIRT1 for maternal-zygotic transition.
Oocytes maintain low ROS levels to support the dormancy of primordial follicles.
MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos.
Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse.
Temporal BMP4 effects on mouse embryonic and extraembryonic development.
Epigenetic inheritance and gene expression regulation in early Drosophila embryos.
Optimization of lipofection protocols for CRISPR/Cas9 delivery in porcine zona pellucida intact oocytes: A study of coincubation duration and reagent efficacy.
Non-cell-autonomous regulation of germline proteostasis by insulin/IGF-1 signaling-induced dietary peptide uptake via PEPT-1.

J Genet Genomics. 2024 Sep 12. pii: S1673-8527(24)00242-X. [Epub ahead of print]

CtIP regulates G2/M transition and bipolar spindle assembly during mouse oocyte meiosis.

Wei Yue, Hong-Yong Zhang, Heide Schatten, Tie-Gang Meng, Qing-Yuan Sun.

  CtBP-interacting protein (CtIP) is known for its multifaceted roles in DNA repair and genomic stability, directing the homologous recombination-mediated DNA double-stranded break (DSBs) repair pathway via DNA end resection, an essential error-free repair process vital for genome stability. Mammalian oocytes are highly prone to DNA damage accumulation due to prolonged G2/prophase arrest. Here, we explore the functions of CtIP in meiotic cell cycle regulation via a mouse oocyte model. Depletion of CtIP by siRNA injection results in delayed germinal vesicle breakdown and failed polar body extrusion. Mechanistically, CtIP deficiency increases DNA damage and decreases the expression and nuclear entry of CCNB1, resulting in marked impairment of meiotic resumption, which can be rescued by exogenous CCNB1 overexpression. Furthermore, depletion of CtIP disrupts MTOCs coalescence at spindle poles as indicated by failed accumulation of γ-tubulin, p-Aurora kinase A, Kif2A, and TPX2, leading to abnormal spindle assembly and prometaphase arrest. These results provide valuable insights into the important roles of CtIP in the G2/M checkpoint and spindle assembly in mouse oocyte meiotic cell cycle regulation.

Keywords:  CtIP; G2/M transition; Meiosis; Oocyte; Spindle assembly

DOI:  https://doi.org/10.1016/j.jgg.2024.09.005
Adv Sci (Weinh). 2024 Sep 17. e2308823

VPS34 Governs Oocyte Developmental Competence by Regulating Mito/Autophagy: A Novel Insight into the Significance of RAB7 Activity and Its Subcellular Location.

Wenwen Liu, Kehan Wang, Yuting Lin, Lu Wang, Xin Jin, Yuexin Qiu, Wenya Sun, Ling Zhang, Yan Sun, Xiaowei Dou, Shiming Luo, Youqiang Su, Qingyuan Sun, Wenpei Xiang, Feiyang Diao, Jing Li.

  Asynchronous nuclear and cytoplasmic maturation in human oocytes is believed to cause morphological anomalies after controlled ovarian hyperstimulation. Vacuolar protein sorting 34 (VPS34) is renowned for its pivotal role in regulating autophagy and endocytic trafficking. To investigate its impact on oocyte development, oocyte-specific knockout mice (ZcKO) are generated, and these mice are completely found infertile, with embryonic development halted at 2- to 4-cell stage. This infertility is related with a disruption on autophagic/mitophagic flux in ZcKO oocytes, leading to subsequent failure of zygotic genome activation (ZGA) in derived 2-cell embryos. The findings further elucidated the regulation of VPS34 on the activity and subcellular translocation of RAS-related GTP-binding protein 7 (RAB7), which is critical not only for the maturation of late endosomes and lysosomes, but also for initiating mitophagy via retrograde trafficking. VPS34 binds directly with RAB7 and facilitates its activity conversion through TBC1 domain family member 5 (TBC1D5). Consistent with the cytoplasmic vacuolation observed in ZcKO oocytes, defects in multiple vesicle trafficking systems are also identified in vacuolated human oocytes. Furthermore, activating VPS34 with corynoxin B (CB) treatment improved oocyte quality in aged mice. Hence, VPS34 activation may represent a novel approach to enhance oocyte quality in human artificial reproduction.

Keywords:  VPS34; autophagy; mitophagy; oocyte; retromer

DOI:  https://doi.org/10.1002/advs.202308823
Proc Natl Acad Sci U S A. 2024 Sep 24. 121(39): e2407083121

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.

Stella E Cho, Wei Li, Andrew M Beard, Jonathan A Jackson, Risa Kiernan, Kazunori Hoshino, Adam C Martin, Jianjun Sun.

  Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

Keywords:  Rho1; actomyosin contraction; follicle rupture; mechanical force; ovulation

DOI:  https://doi.org/10.1073/pnas.2407083121
Genetics. 2024 Sep 18. pii: iyae153. [Epub ahead of print]

The structural role of Skp1 in the synaptonemal complex is conserved in nematodes.

Lisa E Kursel, Kaan Goktepe, Ofer Rog.

  The synaptonemal complex (SC) is a meiotic interface that assembles between parental chromosomes and is essential for gamete formation. While the dimensions and ultrastructure of the SC are conserved across eukaryotes, its protein components are highly divergent. Recently, an unexpected component of the SC has been described in the nematode C. elegans: the Skp1-related proteins SKR-1/2, which are components of the Skp1, Cullin, F-box (SCF) ubiquitin ligase. Here, we find that the role of SKR-1 in the SC is conserved in P. pacificus. The P. pacificus Skp1 ortholog, Ppa-SKR-1, colocalizes with other SC proteins throughout meiotic prophase, where it occupies the middle of the SC. Like in C. elegans, the dimerization interface of Ppa-SKR-1 is required for its SC function. A dimerization mutant, ppa-skr-1F105E, fails to assemble SC and produces almost no progeny. Interestingly, the evolutionary trajectory of SKR-1 contrasts with other SC proteins. Unlike most SC proteins, SKR-1 is highly conserved in nematodes. Our results suggest that the structural role of SKR-1 in the SC has been conserved since the common ancestor of C. elegans and P. pacificus, and that rapidly evolving SC proteins have maintained the ability to interact with SKR-1 for at least 100 million years.

Keywords:   P. pacificus; SCF; Skp1; evolution; meiosis; nematodes; synaptonemal complex

DOI:  https://doi.org/10.1093/genetics/iyae153
Elife. 2024 Sep 18. pii: RP94631. [Epub ahead of print]13

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.

Leif Benner, Savannah Muron, Jillian G Gomez, Brian Oliver.

  Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO's role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

Keywords:  D. melanogaster; chromosomes; developmental biology; embryonic development; gene expression; gene regulation; germ cells; transcription factor

DOI:  https://doi.org/10.7554/eLife.94631
Science. 2024 Sep 20. 385(6715): 1366-1375

Artificial kinetochore beads establish a biorientation-like state in the spindle.

Kohei Asai, Yuanzhuo Zhou, Osamu Takenouchi, Tomoya S Kitajima.

Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.

DOI: https://doi.org/10.1126/science.adn5428
Nat Commun. 2024 Sep 18. 15(1): 8178

Nutritional vitamin B12 regulates RAS/MAPK-mediated cell fate decisions through one-carbon metabolism.

Ana Cristina Laranjeira, Simon Berger, Tea Kohlbrenner, Nadja R Greter, Alex Hajnal.

Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin B12 enhances via the choline/phosphatidylcholine metabolism vulval induction by down-regulating fat biosynthesis genes and increasing H3K4 tri-methylation, which results in increased expression of RAS/MAPK target genes. Furthermore, the nucleoside metabolism and H3K4 tri-methylation positively regulate germ cell apoptosis and oocyte production. Using mammalian cells carrying different activated KRAS and BRAF alleles, we show that the effects of methionine on RAS/MAPK-regulated phenotype are conserved in mammals. Our findings suggest that the vitamin B12-dependent one-carbon metabolism is a limiting factor for diverse RAS/MAPK-induced cellular responses.

DOI: https://doi.org/10.1038/s41467-024-52556-3
Sci Rep. 2024 09 16. 14(1): 21598

Dynamics and necessity of SIRT1 for maternal-zygotic transition.

Jan Nevoral, David Drutovic, Michaela Vaskovicova, Michal Benc, Frantisek Liska, Iveta Valentova, Sara Stachovicova, Jan Kubovciak, Jirina Havrankova, Miki Shavit, Ladan Monsef, Maria Iniesta-Cuerda, Tereza Zalmanova, Petr Hosek, Frantisek Strejcek, Milena Kralickova, Jaroslav Petr.

  Dynamic changes in maternal‒zygotic transition (MZT) require complex regulation of zygote formation, maternal transcript decay, embryonic genome activation (EGA), and cell cycle progression. Although these changes are well described, some key regulatory factors are still elusive. Sirtuin-1 (SIRT1), an NAD+-dependent histone deacetylase, is a versatile driver of MZT via its epigenetic and nonepigenetic substrates. This study focused on the dynamics of SIRT1 in early embryos and its contribution to MZT. A conditional SIRT1-deficient knockout mouse model was used, accompanied by porcine and human embryos. Embryos across mammalian species showed the prominent localization of SIRT1 in the nucleus throughout early embryonic development. Accordingly, SIRT1 interacts with histone H4 on lysine K16 (H4K16) in both mouse and human blastocysts. While maternal SIRT1 is dispensable for MZT, at least one allele of embryonic Sirt1 is required for early embryonic development around the time of EGA. This role of SIRT1 is surprisingly mediated via a transcription-independent mode of action.

Keywords:  Embryo; Embryonic genome activation; Epigenetics; Histone deacetylase; Oocyte, zygote

DOI:  https://doi.org/10.1038/s41598-024-72595-6
Aging Cell. 2024 Sep 19. e14338

Oocytes maintain low ROS levels to support the dormancy of primordial follicles.

Shaogang Qin, Xinyue Chi, Zijian Zhu, Chuanhe Chen, Tuo Zhang, Meina He, Meng Gao, Ting Zhao, Jingwen Zhang, Lifan Zhang, Wenying Zheng, Ziqi Chen, Wenji Wang, Bo Zhou, Guoliang Xia, Chao Wang.

  Primordial follicles (PFs) function as the long-term reserve for female reproduction, remaining dormant in the ovaries and becoming progressively depleted with age. Oxidative stress plays an important role in promoting female reproductive senescence during aging, but the underlying mechanisms remain unclear. Here, we find that low levels of reactive oxygen species (ROS) are essential for sustaining PF dormancy. Compared to growing follicles, oocytes within PFs were shown to be more susceptible to ROS, which accumulates and damages PFs to promote reproductive senescence. Mechanistically, oocytes within PFs were shown to express high levels of the intracellular antioxidant enzyme superoxide dismutase 1 (SOD1), counteracting ROS accumulation. Decreased SOD1 expression, as a result of aging or through the experimental deletion of the Sod1 gene in oocytes, resulted in increased oxidative stress and triggered ferroptosis within PFs. In conclusion, this study identified antioxidant defense mechanisms protecting PFs in mouse ovaries and characterized cell death mechanisms of oxidative stress-induced PF death.

Keywords:  ROS; SOD1; ferroptosis; primary ovarian insufficiency; primordial follicle

DOI:  https://doi.org/10.1111/acel.14338
Theriogenology. 2024 Sep 10. pii: S0093-691X(24)00375-3. [Epub ahead of print]230 81-90

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos.

Xiao-Han Li, Song-Hee Lee, Qin-Yue Lu, Cheng-Lin Zhan, Gyu-Hyun Lee, Ji-Dam Kim, Jae-Min Sim, Hyeon-Ji Song, Xiang-Shun Cui.

  Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

Keywords:  Embryo development; Histone methylation; MAT2A; Porcine; SAM; ZGA

DOI:  https://doi.org/10.1016/j.theriogenology.2024.09.006
Geroscience. 2024 Sep 16.

Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse.

Farners Amargant, Carol Magalhaes, Michele T Pritchard, Francesca E Duncan.

  The female reproductive system is one of the first to age in humans, resulting in infertility and endocrine disruptions. The aging ovary assumes a fibro-inflammatory milieu which negatively impacts gamete quantity and quality as well as ovulation. Here, we tested whether the systemic delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs attenuates ovarian aging in mice. We first evaluated the ability of these drugs to decrease the expression of fibro-inflammatory genes in primary ovarian stromal cells treated with a pro-fibrotic or a pro-inflammatory stimulus. Whereas Etanercept did not block Tnf expression in ovarian stromal cells, Pirfenidone significantly reduced Col1a1 expression. We then tested Pirfenidone in vivo where the drug was delivered systemically via mini-osmotic pumps for 6 weeks. Pirfenidone mitigated the age-dependent increase in ovarian fibrosis without impacting overall health parameters. Ovarian function was improved in Pirfenidone-treated mice as evidenced by increased follicle and corpora lutea number, AMH levels, and improved estrous cyclicity. Transcriptomic analysis revealed that Pirfenidone treatment resulted in an upregulation of reproductive function-related genes at 8.5 months and a downregulation of inflammatory genes at 12 months of age. These findings demonstrate that reducing the fibroinflammatory ovarian microenvironment improves ovarian function, thereby supporting modulating the ovarian environment as a therapeutic avenue to extend reproductive longevity.

Keywords:  Fibrosis; Healthspan; Inflammation; Ovarian function; Reproductive aging

DOI:  https://doi.org/10.1007/s11357-024-01322-w
Nature. 2024 Sep 18.

Temporal BMP4 effects on mouse embryonic and extraembryonic development.

Ron Hadas, Hernan Rubinstein, Markus Mittnenzweig, Yoav Mayshar, Raz Ben-Yair, Saifeng Cheng, Alejandro Aguilera-Castrejon, Netta Reines, Ayelet-Hashahar Orenbuch, Aviezer Lifshitz, Dong-Yuan Chen, Michael B Elowitz, Magdalena Zernicka-Goetz, Jacob H Hanna, Amos Tanay, Yonatan Stelzer.

The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro.

DOI: https://doi.org/10.1038/s41586-024-07937-5
EMBO Rep. 2024 Sep 16.

Epigenetic inheritance and gene expression regulation in early Drosophila embryos.

Filippo Ciabrelli, Nazerke Atinbayeva, Attilio Pane, Nicola Iovino.

  Precise spatiotemporal regulation of gene expression is of paramount importance for eukaryotic development. The maternal-to-zygotic transition (MZT) during early embryogenesis in Drosophila involves the gradual replacement of maternally contributed mRNAs and proteins by zygotic gene products. The zygotic genome is transcriptionally activated during the first 3 hours of development, in a process known as "zygotic genome activation" (ZGA), by the orchestrated activities of a few pioneer factors. Their decisive role during ZGA has been characterized in detail, whereas the contribution of chromatin factors to this process has been historically overlooked. In this review, we aim to summarize the current knowledge of how chromatin regulation impacts the first stages of Drosophila embryonic development. In particular, we will address the following questions: how chromatin factors affect ZGA and transcriptional silencing, and how genome architecture promotes the integration of these processes early during development. Remarkably, certain chromatin marks can be intergenerationally inherited, and their presence in the early embryo becomes critical for the regulation of gene expression at later stages. Finally, we speculate on the possible roles of these chromatin marks as carriers of epialleles during transgenerational epigenetic inheritance (TEI).

Keywords:  Chromatin Establishment After Fertilization; Epigenetic Inheritance; H3K27me3; H3K9me3

DOI:  https://doi.org/10.1038/s44319-024-00245-z
Theriogenology. 2024 Sep 14. pii: S0093-691X(24)00383-2. [Epub ahead of print]230 121-129

Optimization of lipofection protocols for CRISPR/Cas9 delivery in porcine zona pellucida intact oocytes: A study of coincubation duration and reagent efficacy.

Celia Piñeiro-Silva, Joaquín Gadea.

  A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes. We compared these reagents with the electroporation method and a control group using two sgRNAs targeting the CAPN3 and CD163 genes. The detrimental effects on cleavage rates were observed in both lipofection treatments compared to the control and electroporated groups. However, blastocyst rates were higher in the Lipofectamine 3000 group than in the electroporated group for both genes. Mutation parameters varied by target gene, with Lipofectamine 3000 achieving higher mutation rates for CD163, while all groups were similar for the CAPN3 gene. Overall efficiency was similar for both lipofectamines, confirming their feasibility for use. In addition, we evaluated the effect of coincubation time (4, 8, and 24 h) on IVF outcomes, embryo development, and mutation parameters. Results indicated that an 8-h coincubation period optimized fertilization and mutation efficiency without significant toxic effects. This study demonstrates that lipofection with either Lipofectamine 3000 or CRISPRMAX during IVF is an effective method for generating genetically modified porcine embryos without the need for specialized equipment or trained personnel, with efficiencies similar to or greater than electroporation. This study also highlights the importance of optimizing reagent selection and coincubation times. There is no difference between Lipofectamine 3000 and CRISPRMAXTM in terms of embryo development and mutation efficiency, and under our experimental conditions, the optimal coincubation time with lipofectamine is 8 h.

Keywords:  Electroporation; Gene editing; Human disease model; Lipofection; Swine; Viral disease resistance

DOI:  https://doi.org/10.1016/j.theriogenology.2024.09.014
EMBO J. 2024 Sep 16.

Non-cell-autonomous regulation of germline proteostasis by insulin/IGF-1 signaling-induced dietary peptide uptake via PEPT-1.

Tahir Muhammad, Stacey L Edwards, Allison C Morphis, Mary V Johnson, Vitor De Oliveira, Tomasz Chamera, Siyan Liu, Ngoc Gia Tuong Nguyen, Jian Li.

  Gametogenesis involves active protein synthesis and is proposed to rely on proteostasis. Our previous work in C. elegans indicates that germline development requires coordinated activities of insulin/IGF-1 signaling (IIS) and HSF-1, the central regulator of the heat shock response. However, the downstream mechanisms were not identified. Here, we show that depletion of HSF-1 from germ cells impairs chaperone gene expression, causing protein degradation and aggregation and, consequently, reduced fecundity and gamete quality. Conversely, reduced IIS confers germ cell resilience to HSF-1 depletion-induced protein folding defects and various proteotoxic stresses. Surprisingly, this effect was not mediated by an enhanced stress response, which underlies longevity in low IIS conditions, but by reduced ribosome biogenesis and translation rate. We found that IIS activates the expression of intestinal peptide transporter PEPT-1 by alleviating its repression by FOXO/DAF-16, allowing dietary proteins to be efficiently incorporated into an amino acid pool that fuels germline protein synthesis. Our data suggest this non-cell-autonomous pathway is critical for proteostasis regulation during gametogenesis.

Keywords:  Germline Proteostasis; Heat Shock Factor 1; Insulin and IGF-1 Signaling; Peptide Uptake; Protein Synthesis

DOI:  https://doi.org/10.1038/s44318-024-00234-x