Elevated Temperatures Cause Transposon-Associated DNA Damage in C. elegans Spermatocytes

Kurhanewicz, Nicole; Dinwiddie, Devin; Bush, Zachary D.; Libuda, Diana E.

doi:10.1016/j.cub.2020.09.050

Cited by 43 publications

(48 citation statements)

References 52 publications

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“…Our conditional immobilization tool works to efficiently immobilize both males and hermaphrodites for live imaging worms for sex-specific comparison experiments. For the C. elegans germline, an increasing number of studies are indicating hermaphrodites and males have multiple sexually dimorphic features that lead to differential regulation of genome integrity germline (JARAMILLO-LAMBERT et al 2007;CAHOON AND LIBUDA 2019;KURHANEWICZ et al 2020;LI et al 2020). To our knowledge, the live images of the SC in males may represent the first time the SC has been observed within living C. elegans spermatocytes.…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Slides were washed three times in PBS+0.1% Tween20 (PBST) for 5 minutes each and incubated in block (0.7% bovine serum albumin in PBST) for 1 hour. Primary antibodies (rabbit anti-RAD-51, 1:1500 (KURHANEWICZ et al 2020;TORAASON et al 2021)) were added and incubated overnight in a humid chamber with a parafilm cover. Slides were then washed three times in PBST for 10 minutes each and incubated with secondary antibodies (goat anti-rabbit AlexaFluor488, ThermoFisher, cat.…”

Section: Dlw118mentioning

confidence: 99%

“…Consequently, many methods that immobilize the hermaphrodites fail to immobilize males. C. elegans is an excellent model system for sexual dimorphism studies with many sex-specific differences having been identified related to germline processes, nervous system development, and animal aging (JARAMILLO-LAMBERT et al 2007;BARR et al 2018;HOTZI et al 2018;CAHOON AND LIBUDA 2019;KURHANEWICZ et al 2020;LI et al 2020).…”

Section: Introductionmentioning

confidence: 99%

“…2018; H otzi et al . 2018; C ahoon and L ibuda 2019; K urhanewicz et al . 2020; L i et al .…”

Section: Introductionmentioning

confidence: 99%

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Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion

Cahoon

Libuda

2021

Preprint

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The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin inducible degron (AID) system to immobilize both hermaphrodites and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Dlw118mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…2018; H otzi et al . 2018; C ahoon and L ibuda 2019; K urhanewicz et al . 2020; L i et al .…”

Section: Introductionmentioning

confidence: 99%

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Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion

Cahoon

Libuda

2021

Preprint

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The protein tyrosine phosphatase PPH‐7 is required for fertility and embryonic development in C. elegans at elevated temperatures

Franziscus,

Ritz,

Kappel

et al. 2024

FEBS Open Bio

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Post‐translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post‐translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH‐7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH‐7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel–Lindau (VHL) E3 ligase, suggesting a potential role for PPH‐7 in the regulation of VHL.

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NBMA Promotes Spermatogenesis by Mediating Oct4 Pathway

et al. 2022

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Non‐obstructive azoospermia is one of the most common causes of male infertility, but there is still no specific treatment drug. Given that the Oct4 (Octamer‐binding transcription factor 4) has an important regulatory effect on spermatogenesis, activating it can effectively promote spermatogenesis, so it is of great value to develop Oct4‐targeted drug design and elucidating its mechanism of action. Here, we screened out the Oct4‐targeted drug molecule NBMA ( N ‐benzyl‐4‐methoxy‐2‐(1‐(4‐(trifluoromethyl)phenyl)vinyl)aniline) by computer‐assisted technology, and found that it has a significant promoting effect on spermatogenesis in the established mouse azoospermia model. Subsequently, through transcriptome sequencing and enrichment analysis, real‐time fluorescent quantitative PCR (qPCR) and western blot experiments revealed that NBMA promotes the differentiation of spermatogonial stem cells by activating the Oct4 pathway, thereby promoting spermatogenesis. This study proves that NBMA is a molecule with great potential to be developed as a therapeutic drug for azoospermia. It also shows that computer‐assisted, chemical and biological multidisciplinary methods play a very important role in innovative drug discovery.

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Elevated Temperatures Cause Transposon-Associated DNA Damage in C. elegans Spermatocytes

Cited by 43 publications

References 52 publications

Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion

Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion

The protein tyrosine phosphatase PPH‐7 is required for fertility and embryonic development in C. elegans at elevated temperatures

NBMA Promotes Spermatogenesis by Mediating Oct4 Pathway

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