The conserved molting/circadian rhythm regulator NHR-23/NR1F1 serves as an essential co-regulator ofC. elegansspermatogenesis

Abstract

In sexually reproducing metazoans, spermatogenesis is the process by which uncommitted germ cells give rise to haploid sperm. Work in model systems has revealed mechanisms controlling commitment to the sperm fate, but how this fate is subsequently executed remains less clear. While studying the well-established role of the conserved nuclear hormone receptor transcription factor, NHR-23/NR1F1, in regulating C. elegans molting, we discovered NHR-23/NR1F1 is also constitutively expressed in developing 1° spermatocytes and is a critical regulator of spermatogenesis. In this novel role, NHR-23/NR1F1 functions downstream of the canonical sex determination pathway. Degron-mediated depletion of NHR-23/NR1F1 within hermaphrodite or male germlines causes sterility due to an absence of functional sperm as depleted animals produce arrested primary spermatocytes rather than haploid sperm. These spermatocytes arrest in prometaphase I and fail to either progress to anaphase or attempt spermatid-residual body partitioning. They make sperm-specific membranous organelles (MOs) but fail to assemble their major sperm protein into fibrous bodies. NHR-23/NR1F1 appears to function independently of the known SPE-44 gene regulatory network, revealing the existence of an NHR-23/NR1F1-mediated module that regulates the spermatogenesis program.

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans

“…Additionally, our sun-1p::TIR1::F2A::BFP::AID*::NLS transgene produced equivalent mNG::AID*::PAR-3 depletion and resultant synchronous cell division as compared to a sun-1p::TIR1::mRuby2 transgene ( Figure 2, C and D ). Similarly, using a mex-5p::TIR1::F2A::BFP::AID*::NLS transgene we observed complete sterility following NHR-23 or SPE-44 depletion, identical to that observed using pie-1p or sun-1p::TIR1::mRuby2 transgenes ( Kasimatis et al 2018 ; Ragle et al 2020 ). However, there may be cases where depletion rates affect phenotypic preference, so this point should be taken into consideration when choosing which TIR1 transgene to use.…”

“…AID*-tagged BFP is degraded in the presence of auxin, confirming TIR1 activity via degradation of an internal control ( Figures 2 and 3 , and Supplementary Figure S2 ). Recent work from our lab shows that our new TIR1 strains are effective for experiments where red FP imaging is desired ( Ragle et al 2020 ).…”

“…This system has allowed for rapid depletion of proteins in tissues that are refractory to RNA interference approaches, such as the germline ( Pelisch et al 2017 ; Shen et al 2018 ; Zhang et al 2018 b), vulval precursor cells ( Matus et al 2014 ), and neurons ( Liu et al 2017 ; Patel and Hobert 2017 ; Serrano-Saiz et al 2018 ). The system is also powerful for studying rapid developmental events such as molting ( Zhang et al 2015 ; Joseph et al 2020 ), organogenesis ( Martinez et al 2020 ), developmental timing ( Azzi et al 2020 ), meiosis ( Zhang et al 2015 ), and spermatogenesis ( Ragle et al 2020 ). Improvements to the auxin ligand have enhanced protein degradation in the embryo ( Negishi et al 2019 ) and removed the need for ethanol solubilization, allowing the auxin derivative to be dissolved in any aqueous buffer ( Martinez et al 2020 ).…”

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

“…These results suggest a sexual dimorphic difference in auxin sensitivity in C. elegans , where male worms may absorb and process auxin differently than hermaphrodite worms. Although, recent study found that depletion of a different AID*-tagged protein (NHR-23) in the germline was equivalent between males and hermaphrodites on 4-mM auxin plates ( Ragle et al 2020 ). Thus, future studies focused on auxin processing in both C. elegans sexes may reveal the mechanisms behind this intriguing sexual dimorphism that may vary depending on the tissue assayed and AID*-tagged protein.…”

Circadian Clock-Controlled Checkpoints in the Pathogenesis of Complex Disease

“…The specific knockdown of Rora in Sertoli cells at puberty leads to declined sperm count in rats ( Mandal et al, 2018 ). In C. elegans , NHR-23/NR1F1 (RORA) depletion causes infertility due to the arrest of primary spermatocytes rather than haploid sperm ( Ragle et al, 2020 ).…”