bims-cebooc 2024-08-04 papers

bims-cebooc

Biomed News

on Cell biology of oocytes

Issue of 2024–08–04
seventeen papers selected by
Gabriele Zaffagnini, Centre for Genomic Regulation

Maternal age enhances purifying selection on pathogenic mutations in complex I genes of mammalian mtDNA.
An egg-sabotaging mechanism drives non-Mendelian transmission in mice.
Zygotic spindle orientation defines cleavage pattern and nuclear status of human embryos.
Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building.
Improved low-invasive mRNA electroporation method into immature mouse oocytes visualizes protein dynamics during development.
Declining translational capacity as a potential driver for oocyte meiotic instability.
KIT in oocytes: a key factor for oocyte survival and reproductive lifespan.
HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos.
Meiosis-specific decoupling of the pericentromere from the kinetochore.
Local nuclear to cytoplasmic ratio regulates chaperone-dependent H3 variant incorporation during zygotic genome activation.
MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclin B1 and Securin stabilization in meiotic oocyte.
The Molecular Mechanism of Aurora-B Regulating Kinetochore-Microtubule Attachment in Mitosis and Oocyte Meiosis.
Genetic circuitry controlling Drosophila female germline overgrowth.
Katanin, kinesin-13, and ataxin-2 inhibit premature interaction between maternal and paternal genomes in C. elegans zygotes.
In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans.
Conditional loss of Brca1 in oocytes causes reduced litter size, ovarian reserve depletion and impaired oocyte in vitro maturation with advanced reproductive age in mice.
X centromeric drive may explain the prevalence of polycystic ovary syndrome and other conditions: Genomic structure of the human X chromosome pericentromeric region is consistent with meiotic drive associated with PCOS and other conditions.

Nat Aging. 2024 Jul 29.

Maternal age enhances purifying selection on pathogenic mutations in complex I genes of mammalian mtDNA.

Yanfei Ru, Xiaoling Deng, Jiatong Chen, Leping Zhang, Zhe Xu, Qunyu Lv, Shiyun Long, Zijian Huang, Minghua Kong, Jing Guo, Min Jiang.

Mitochondrial diseases, caused mainly by pathogenic mitochondrial DNA (mtDNA) mutations, pose major challenges due to the lack of effective treatments. Investigating the patterns of maternal transmission of mitochondrial diseases could pave the way for preventive approaches. In this study, we used DddA-derived cytosine base editors (DdCBEs) to generate two mouse models, each haboring a single pathogenic mutation in complex I genes (ND1 and ND5), replicating those found in human patients. Our findings revealed that both mutations are under strong purifying selection during maternal transmission and occur predominantly during postnatal oocyte maturation, with increased protein synthesis playing a vital role. Interestingly, we discovered that maternal age intensifies the purifying selection, suggesting that older maternal age may offer a protective effect against the transmission of deleterious mtDNA mutations, contradicting the conventional notion that maternal age correlates with increased transmitted mtDNA mutations. As collecting comprehensive clinical data is needed to understand the relationship between maternal age and transmission patterns in humans, our findings may have profound implications for reproductive counseling of mitochondrial diseases, especially those involving complex I gene mutations.

DOI: https://doi.org/10.1038/s43587-024-00672-6
Curr Biol. 2024 Jul 24. pii: S0960-9822(24)00901-1. [Epub ahead of print]

An egg-sabotaging mechanism drives non-Mendelian transmission in mice.

Frances E Clark, Naomi L Greenberg, Duilio M Z A Silva, Emily Trimm, Morgan Skinner, R Zaak Walton, Leah F Rosin, Michael A Lampson, Takashi Akera.

  Selfish genetic elements drive in meiosis to distort their transmission ratio and increase their representation in gametes, violating Mendel's law of segregation. The two established paradigms for meiotic drive, gamete killing and biased segregation, are fundamentally different. In gamete killing, typically observed with male meiosis, selfish elements sabotage gametes that do not contain them. By contrast, killing is predetermined in female meiosis, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here, we show that a selfish element on mouse chromosome 2, Responder to drive 2 (R2d2), drives using a hybrid mechanism in female meiosis, incorporating elements of both killing and biased segregation. We propose that if R2d2 is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy, which is subsequently lethal in the embryo, ensuring that surviving progeny preferentially contain R2d2. In heterozygous females, R2d2 orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with R2d2 or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, R2d2 heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of R2d2. In contrast to typical gamete killing of sisters produced as daughter cells in a single meiosis, R2d2 prevents production of any viable gametes from meiotic divisions in which it should have been excluded from the egg.

Keywords:  aneuploidy; chromosome segregation; female meiotic drive; meiosis; mouse oocyte; selfish genetic element

DOI:  https://doi.org/10.1016/j.cub.2024.07.001
Nat Commun. 2024 Jul 29. 15(1): 6369

Zygotic spindle orientation defines cleavage pattern and nuclear status of human embryos.

Volodymyr Porokh, Drahomíra Kyjovská, Martina Martonová, Tereza Klenková, Pavel Otevřel, Soňa Kloudová, Zuzana Holubcová.

The first embryonic division represents a starting point for the development of a new individual. In many species, tight control over the first embryonic division ensures its accuracy. However, the first division in humans is often erroneous and can impair embryo development. To delineate the spatiotemporal organization of the first mitotic division typical for normal human embryo development, we systematically analyzed a unique timelapse dataset of 300 IVF embryos that developed into healthy newborns. The zygotic division pattern of these best-quality embryos was compared to their siblings that failed to implant or arrested during cleavage stage. We show that division at the right angle to the juxtaposed pronuclei is preferential and supports faithful zygotic division. Alternative configurations of the first mitosis are associated with reduced clustering of nucleoli and multinucleation at the 2-cell stage, which are more common in women of advanced age. Collectively, these data imply that orientation of the first division predisposes human embryos to genetic (in)stability and may contribute to aneuploidy and age-related infertility.

DOI: https://doi.org/10.1038/s41467-024-50732-z
iScience. 2024 Aug 16. 27(8): 110451

Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building.

Cecilia S Blengini, Michaela Vaskovicova, Jan Schier, David Drutovic, Karen Schindler.

  Meiotic spindles are critical to ensure chromosome segregation during gamete formation. Oocytes lack centrosomes and use alternative microtubule-nucleation mechanisms for spindle building. How these mechanisms are regulated is still unknown. Aurora kinase A (AURKA) is essential for mouse oocyte meiosis because in pro-metaphase I it triggers microtubule organizing-center fragmentation and its expression compensates for the loss of the two other Aurora kinases (AURKB/AURKC). Although knockout mouse models were useful for foundational studies, AURK spatial and temporal functions are not yet resolved. We provide high-resolution analyses of AURKA/AURKC requirements during meiotic spindle-building and identify the subcellular populations that carry out these functions: 1) AURKA is required in early spindle assembly and later for spindle stability, whereas 2) AURKC is required in late pro-metaphase, and 3) Targeted AURKA constructs expressed in triple AURK knockout oocytes reveal that spindle pole-localized AURKA is the most important population controlling spindle building and stability mechanisms.

Keywords:  cell biology

DOI:  https://doi.org/10.1016/j.isci.2024.110451
Biol Reprod. 2024 Jul 29. pii: ioae116. [Epub ahead of print]

Improved low-invasive mRNA electroporation method into immature mouse oocytes visualizes protein dynamics during development.

Yuhkoh Satouh, Emiko Suzuki, Keisuke Sasaki, Ken Sato.

  One of the major causes of oocyte quality deterioration, chromosome segregation abnormalities manifest mainly during meiosis I, which occurs before and during ovulation. However, currently, there is a technical limitation in the introduction of mRNA into premature oocytes without impairing embryonic developmental ability. In this study, we established a low-invasive electroporation (EP) method to introduce mRNA into pre-ovulatory, germinal vesicle (GV) mouse oocytes in an easier manner than the traditional microinjection method. The EP method with an optimized impedance value resulted in the efficient introduction of mRNAs encoding enhanced green fluorescent protein (EGFP) into the GV oocytes surrounded by cumulus cells at a survival rate of 95.0%. Furthermore, the introduction of histone H2B-EGFP mRNA into the GV oocytes labeled most of the oocytes without affecting the blastocyst development rate, indicating the feasibility of the visualization of oocyte chromosomal dynamics that enable us to assay chromosomal integrity in oocyte maturation and cell count in embryonic development. The establishment of this EP method offers extensive assays to select pre-implantation embryos and enables the surveying of essential factors for mammalian oocyte quality determination.

Keywords:  Live-cell imaging; in vitro maturation; isolated chromosome; premature oocyte

DOI:  https://doi.org/10.1093/biolre/ioae116
Reproduction. 2024 Jul 01. pii: REP-24-0198. [Epub ahead of print]

Declining translational capacity as a potential driver for oocyte meiotic instability.

Katie Jordanna Danielson, Kayla Lee Judson, Ethan Joseph Greenblatt.

Errors during female meiosis lead to embryonic aneuploidy and miscarriage and occur with increasing frequency during aging. The underlying molecular changes that drive female meiotic instability remain a subject of debate. Developing oocytes undergo a tremendous increase in cytoplasmic volume during follicle development and rely on long-lived mRNAs and ribosomes accumulated during this growth phase for subsequent meiotic maturation. In this perspective article, we discuss how the unique reliance on stores of long-lived mRNAs and ribosomes may represent an Achilles' heel for oocyte function, and that alterations that reduce the translational capacity of oocytes could be a factor significantly contributing to meiotic instability. Understanding these mechanisms could lead to new diagnostic and therapeutic strategies to improve fertility outcomes.

DOI: https://doi.org/10.1530/REP-24-0198
EBioMedicine. 2024 Jul 26. pii: S2352-3964(24)00299-8. [Epub ahead of print]106 105263

KIT in oocytes: a key factor for oocyte survival and reproductive lifespan.

Yi Luan, Wonmi So, Rosemary Dong, Amirhossein Abazarikia, So-Youn Kim.

   BACKGROUND: The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage.
METHODS: We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes.
FINDINGS: Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells.
INTERPRETATION: These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan.
FUNDING: This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).

Keywords:  Folliculogenesis; Infertility; KIT; Oocyte; Primary ovarian insufficiency; Survival

DOI:  https://doi.org/10.1016/j.ebiom.2024.105263
bioRxiv. 2024 Jul 19. pii: 2023.09.04.556218. [Epub ahead of print]

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos.

Estefania Sanchez-Vasquez, Marianne E Bronner, Magdalena Zernicka-Goetz.

  Human fertility is suboptimal, partly due to error-prone divisions in early cleavage-stages that result in aneuploidy. Most human pre-implantation are mosaics of euploid and aneuploid cells, however, mosaic embryos with a low proportion of aneuploid cells have a similar likelihood of developing to term as fully euploid embryos. How embryos manage aneuploidy during development is poorly understood. This knowledge is crucial for improving fertility treatments and reducing developmental defects. To explore these mechanisms, we established a new mouse model of chromosome mosaicism to study the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to generate aneuploidy in embryos. Here, we found that treatment with the more specific Mps1 inhibitor AZ3146 induced chromosome segregation defects in pre-implantation embryos, similar to reversine. However, AZ3146-treated embryos showed a higher developmental potential than reversine-treated embryos. Unlike reversine-treated embryos, AZ3146-treated embryos exhibited transient upregulation of Hypoxia Inducible-Factor-1A (HIF1A) and lacked p53 upregulation. Pre-implantation embryos develop in a hypoxic environment in vivo, and hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in the mosaic epiblast. Inhibiting HIF1A in mosaic embryos also decreased the proportion of aneuploid cells in mosaic embryos. Our work illuminates potential strategies to improve the developmental potential of mosaic embryos.

Keywords:  Aneuploidy; DNA repair; Embryogenesis; Hypoxia; Mosaic embryos

DOI:  https://doi.org/10.1101/2023.09.04.556218
bioRxiv. 2024 Jul 22. pii: 2024.07.21.604490. [Epub ahead of print]

Meiosis-specific decoupling of the pericentromere from the kinetochore.

Bo Pan, Melania Bruno, Todd S Macfarlan, Takashi Akera.

The primary constriction site of the M-phase chromosome is an established marker for the kinetochore position, often used to determine the karyotype of each species. Underlying this observation is the concept that the kinetochore is spatially linked with the pericentromere where sister-chromatids are most tightly cohered. Here, we found an unconventional pericentromere specification with sister chromatids mainly cohered at a chromosome end, spatially separated from the kinetochore in Peromyscus mouse oocytes. This distal locus enriched cohesin protectors, such as the Chromosomal Passenger Complex (CPC) and PP2A, at a higher level compared to its centromere/kinetochore region, acting as the primary site for sister-chromatid cohesion. Chromosomes with the distal cohesion site exhibited enhanced cohesin protection at anaphase I compared to those without it, implying that these distal cohesion sites may have evolved to ensure sister-chromatid cohesion during meiosis. In contrast, mitotic cells enriched CPC only near the kinetochore and the distal locus was not cohered between sister chromatids, suggesting a meiosis-specific mechanism to protect cohesin at this distal locus. We found that this distal locus corresponds to an additional centromeric satellite block, located far apart from the centromeric satellite block that builds the kinetochore. Several Peromyscus species carry chromosomes with two such centromeric satellite blocks. Analyses on three Peromyscus species revealed that the internal satellite consistently assembles the kinetochore in both mitosis and meiosis, whereas the distal satellite selectively enriches cohesin protectors in meiosis to promote sister-chromatid cohesion at that site. Thus, our study demonstrates that pericentromere specification is remarkably flexible and can control chromosome segregation in a cell-type and context dependent manner.

DOI: https://doi.org/10.1101/2024.07.21.604490
bioRxiv. 2024 Jul 16. pii: 2024.07.15.603602. [Epub ahead of print]

Local nuclear to cytoplasmic ratio regulates chaperone-dependent H3 variant incorporation during zygotic genome activation.

Anusha D Bhatt, Madeleine G Brown, Aurora B Wackford, Yuki Shindo, Amanda A Amodeo.

  Early embryos often have relatively unstructured chromatin that lacks active and inactive domains typical of differentiated cells. In many species, these regulatory domains are established during zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 fast, reductive, syncytial nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. These divisions incorporate maternally-loaded, cytoplasmic pools of histones into chromatin. Previous work found that chromatin incorporation of replication-coupled histone H3 decreases while its variant H3.3 increases in the cell cycles leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription as well as heterochromatin, suggesting a link between H3.3 incorporation and ZGA. Here, we examine the factors that contribute to H3.3 incorporation at ZGA. We identify a more rapid decrease in the nuclear availability of H3 than H3.3 over the final pre-ZGA cycles. We also observe an N/C ratio-dependent increase in H3.3 incorporation in mutant embryos with non-uniform local N/C ratios. We find that chaperone binding, not gene expression, controls incorporation patterns using H3/H3.3 chimeric proteins at the endogenous H3.3A locus. We test the specificity of the H3.3 chaperone pathways for H3.3 incorporation using Hira (H3.3 chaperone) mutant embryos. Overall, we propose a model in which local N/C ratios and specific chaperone binding regulate differential incorporation of H3.3 during ZGA.

Keywords:  Zygotic genome activation; chromatin; heterochromatin; histones; nuclear to cytoplasmic ratio; transcription

DOI:  https://doi.org/10.1101/2024.07.15.603602
Histochem Cell Biol. 2024 Aug 02.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclin B1 and Securin stabilization in meiotic oocyte.

Li-Hua Fan, Shu-Tao Qi, Zhen-Bo Wang, Ying-Chun Ouyang, Wen-Long Lei, Yue Wang, Ang Li, Feng Wang, Jian Li, Li Li, Yuan-Yuan Li, Yi Hou, Heide Schatten, Wei-Hua Wang, Qing-Yuan Sun, Xiang-Hong Ou.

  Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosis I. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphase I (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclin B1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphase II (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

Keywords:  Cyclin B1; MEIKIN; PLK1; SAC; Securin

DOI:  https://doi.org/10.1007/s00418-024-02316-7
Cytogenet Genome Res. 2024 Jul 27.

The Molecular Mechanism of Aurora-B Regulating Kinetochore-Microtubule Attachment in Mitosis and Oocyte Meiosis.

Shanshan Chen, Qiqi Sun, Bo Yao, Yanping Ren.

BACKGROUND: Aurora kinase B (Aurora-B), a member of the chromosomal passenger complex (CPC), is involved in correcting kinetochore-microtubule (KT-MT) attachment errors and regulating sister chromatid condensation and cytoplasmic division during mitosis.
SUMMARY: However, few reviews have discussed its mechanism in oocyte meiosis and the differences between its role in mitosis and meiosis. Therefore, in this review, we summarize the localization, recruitment, activation, and functions of Aurora-B in mitosis and oocyte meiosis. The accurate regulation of Aurora-B is essential for ensuring accurate chromosomal segregation and correct KT-MT attachments. Aurora-B regulates the stability of KT-MT attachments by competing with cyclin-dependent kinase 1 (CDK1) to control the phosphorylation of the SILK and RVSF motifs on kinetochore scaffold 1 and by competing with protein phosphatase 1 (PP1) to influence the phosphorylation of NDC80 which is the substrate of Aurora-B. In addition, Aurora-B regulates the spindle assembly checkpoint (SAC) by promoting the recruitment and activation of mitotic arrest deficient 2 (MAD2).
KEY MESSAGES: This review provides a theoretical foundation for elucidating the mechanism of cell division and understanding oocyte chromosomal aneuploidy.

DOI: https://doi.org/10.1159/000540588
Dev Biol. 2024 Jul 25. pii: S0012-1606(24)00196-9. [Epub ahead of print]515 160-168

Genetic circuitry controlling Drosophila female germline overgrowth.

Qian Zhang, Le Li, Qi Zhang, Yang Zhang, Lizhong Yan, Yanfang Wang, Yuejia Wang, Shaowei Zhao.

  Germ cells mutant for bam or bgcn are locked in a germline stem cell (GSC)-like state, leading to tumor-like overgrowth in Drosophila ovaries. Our previous studies have demonstrated that germline overgrowth in bam mutants can be suppressed by defects in the miRNA pathway but enhanced by a null mutation in hippo. However, the genetic epistasis between the miRNA and Hippo pathways still remains unknown. Here, we determined that the miRNA pathway acts downstream of the Hippo pathway in regulating this process. Germ cells mutant for bam or bgcn and defective in both pathways divide very slowly, phenocopying those defective only in the miRNA pathway. In addition, we found that Yki, a key oncoprotein in the Hippo pathway, promotes the growth of both wild-type germ cells and bam mutant GSC-like cells. Like wild-type GSCs, bam mutant GSC-like cells predominantly stay in the G2 phase. Remarkably, many of those defective in the miRNA pathway are arrested before entering this phase. Furthermore, our studies identified bantam as a critical miRNA promoting germline overgrowth in bam or bgcn mutants. Taken together, these findings establish a genetic circuitry controlling Drosophila female germline overgrowth.

Keywords:  Drosophila; Hippo pathway; bam; bantam; bgcn; miRNA pathway

DOI:  https://doi.org/10.1016/j.ydbio.2024.07.016
Elife. 2024 Jul 30. pii: RP97812. [Epub ahead of print]13

Katanin, kinesin-13, and ataxin-2 inhibit premature interaction between maternal and paternal genomes in C. elegans zygotes.

Elizabeth A Beath, Cynthia Bailey, Meghana Mahantesh Magadam, Shuyan Qiu, Karen L McNally, Francis J McNally.

  Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.

Keywords:  C. elegans; cell biology; fertilization; meiosis; sperm

DOI:  https://doi.org/10.7554/eLife.97812
J Vis Exp. 2024 Jul 12.

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans.

Michaela Foley, Katie Toperzer, Sara Sornat, Jesse Morgan, Savannah Brennan, Eric Guisbert, Karen Kim Guisbert.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 °C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter "CaFE" for Calcium during Fertilization in C. elegans. The CaFE reporter was engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

DOI: https://doi.org/10.3791/67141
EBioMedicine. 2024 Jul 30. pii: S2352-3964(24)00298-6. [Epub ahead of print]106 105262

Conditional loss of Brca1 in oocytes causes reduced litter size, ovarian reserve depletion and impaired oocyte in vitro maturation with advanced reproductive age in mice.

Amy L Winship, Lauren R Alesi, Jessica M Stringer, Yujie Cao, Yasmin M Lewis, Lisa Tu, Elyse O K Swindells, Saranya Giridharan, Xuebi Cai, Meaghan J Griffiths, Nadeen Zerafa, Leslie Gilham, Martha Hickey, Karla J Hutt.

   BACKGROUND: An estimated 1 in 350 women carry germline BRCA1/2 mutations, which confer an increased risk of developing breast and ovarian cancer, and may also contribute to subfertility. All mature, sex steroid-producing ovarian follicles are drawn from the pool of non-renewable primordial follicles, termed the 'ovarian reserve'. The clinical implications of early ovarian reserve exhaustion extend beyond infertility, to include the long-term adverse health consequences of loss of endocrine function and premature menopause. We aimed to determine whether conditional loss of Brca1 in oocytes impacts ovarian follicle numbers, oocyte quality and fertility in mice with advancing maternal age. We also aimed to determine the utility of AMH as a marker of ovarian function, by assessing circulating AMH levels in mice and women with BRCA1/2 mutations, and correlating this with ovarian follicle counts.
METHODS: In this study, we addressed a longstanding question in the field regarding the functional consequences of BRCA1 inactivation in oocytes. To recapitulate loss of BRCA1 protein function in oocytes, we generated mice with conditional gene deletion of Brca1 in oocytes using Gdf9-Cre recombinase (WT: Brca1fl/flGdf9+/+; cKO: Brca1fl/flGdf9cre/+).
FINDINGS: While the length of the fertile lifespan was not altered between groups after a comprehensive breeding trial, conditional loss of Brca1 in oocytes led to reduced litter size in female mice. Brca1 cKO animals had a reduced ovarian reserve and oocyte maturation was impaired with advanced maternal age at postnatal day (PN)300, compared to WT animals. Serum anti-Müllerian hormone (AMH) concentrations (the gold-standard indirect marker of the ovarian reserve used in clinical practice) were not predictive of reduced primordial follicle number in Brca1 cKO mice versus WT. Furthermore, we found no correlation between follicle number or density and serum AMH concentrations in matched samples from a small cohort of premenopausal women with BRCA1/2 mutations.
INTERPRETATION: Together, our data demonstrate that BRCA1 is a key regulator of oocyte number and quality in females and suggest that caution should be used in relying on AMH as a reliable marker of the ovarian reserve in this context.
FUNDING: This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. This work was supported by funding from the Australian Research Council (ALW - DE21010037 and KJH - FT190100265), as well as the National Breast Cancer Foundation (IIRS-22-092) awarded to ALW and KJH. LRA, YML, LT, EOKS and MG were supported by Australian Government Research Training Program Scholarships. LRA, YML and LT were also supported by a Monash Graduate Excellence Scholarship. YC, SG and XC were supported by Monash Biomedicine Discovery Institute PhD Scholarships. LRA was also supported by a Monash University ECPF24-6809920940 Fellowship. JMS was supported by NHMRC funding (2011299). MH was supported by an NHMRC Investigator Grant (1193838).

Keywords:  AMH; Ageing; BRCA1; DNA repair; Oocyte; Primordial follicle

DOI:  https://doi.org/10.1016/j.ebiom.2024.105262
Bioessays. 2024 Jul 29. e2400056

X centromeric drive may explain the prevalence of polycystic ovary syndrome and other conditions: Genomic structure of the human X chromosome pericentromeric region is consistent with meiotic drive associated with PCOS and other conditions.

Tom Moore.

  X chromosome centromeric drive may explain the prevalence of polycystic ovary syndrome and contribute to oocyte aneuploidy, menopause, and other conditions. The mammalian X chromosome may be vulnerable to meiotic drive because of X inactivation in the female germline. The human X pericentromeric region contains genes potentially involved in meiotic mechanisms, including multiple SPIN1 and ZXDC paralogs. This is consistent with a multigenic drive system comprising differential modification of the active and inactive X chromosome centromeres in female primordial germ cells and preferential segregation of the previously inactivated X chromosome centromere to the polar body at meiosis I. The drive mechanism may explain differences in X chromosome regulation in the female germlines of the human and mouse and, based on the functions encoded by the genes in the region, the transmission of X pericentromeric genetic or epigenetic variants to progeny could contribute to preeclampsia, autism, and differences in sexual differentiation.

Keywords:  X chromosome; autism; centromere; meiotic drive; menopause; miscarriage; oocyte aneuploidy; polycystic ovary syndrome; preeclampsia; sexual antagonism; sexual differentiation

DOI:  https://doi.org/10.1002/bies.202400056