Sergey Medvedev

In the Absence of the Mouse DNA/RNA-Binding Protein MSY2, Messenger RNA Instability Leads to Spermatogenic Arrest

Abstract MSY2 is a member of the Y-box family of proteins solely expressed in male and female germ cells. In the male, MSY2 serves as a coactivator of transcription by binding to a consensus promoter element present in many germ cell-specific genes. In the nucleus, MSY2 marks specific mRNAs for cytoplasmic storage, stabilization, and suppression of translation. The inactivation of MSY2 by gene targeting leads to spermatogenic arrest and infertility. In testes of mice lacking MSY2, incomplete nuclear condensation is prominent in later-stage spermatids at the time of massive spermatid loss. Because MSY2 interacts with DNA and mRNAs, there are several distinct sites of action, which could be disrupted in mice that lack MSY2, resulting in the arrest of spermatogenesis. To define the molecular cause(s) of the spermatogenic arrest in mice lacking MSY2, transcriptional and posttranscriptional processes were assayed. Transcription, mRNA processing, and mRNA intracellular transport appear normal in the absence of MSY2. However, a redistribution of mRNAs from ribonucleoprotein particles to polysomes and marked decreases were detected for many meiotic and postmeiotic germ cell mRNAs, including the mRNAs encoding the transition proteins and protamines. This suggests that increased mRNA instability is a likely cause of the male infertility in Msy2-null mice.

Absence of MSY2 in Mouse Oocytes Perturbs Oocyte Growth and Maturation, RNA Stability, and the Transcriptome

Messenger RNA is remarkably stable during oocyte growth, thus enabling mRNAs to accumulate during the growth phase and thereby provide mRNAs that support early embryonic development. MSY2, a germ cell-specific RNA-binding protein, is implicated in regulating mRNA stability. MSY2 is essential for development because female Msy2−/− mice are infertile. We describe here the characterization of Msy2−/− oocytes. Mutant oocytes grow more slowly during the first wave of folliculogenesis, and maturation to and arrest at metaphase II is severely compromised because of aberrant spindle formation and chromosome congression. Consistent with MSY2 conferring mRNA stability is that the amount of poly(A)-containing RNA is reduced by ∼25% in mutant oocytes. Stability of an exogenous mRNA injected into mutant oocytes is lower than when compared to their wild-type counterparts, and moreover, expression of wild-type MSY2 in mutant oocytes increases mRNA stability, whereas injection of a mutant form of MSY2 not capable of binding RNA does not. Transcription quiescence that normally occurs during the course of oocyte growth is not observed in mutant oocytes, and the transcriptome of mutant oocytes is markedly perturbed. These results, and those of previous studies, strongly implicate a central role of MSY2 in regulating mRNA stability. Msy2−/− oocytes exhibit numerous phenotypes including defects in oocyte growth and maturation, RNA stability, and gene expression.

CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers maternal mRNA degradation during mouse oocyte maturation.

Degradation of maternal mRNA is thought to be essential to undergo the maternal-to-embryonic transition. Messenger RNA is extremely stable during oocyte growth in mouse and MSY2, an abundant germ cell-specific RNA-binding protein, likely serves as a mediator of global mRNA stability. Oocyte maturation, however, triggers an abrupt transition in which most mRNAs are significantly degraded. We report that CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers this transition. Injecting Cdc2a mRNA, which activates CDC2A, overcomes milrinone-mediated inhibition of oocyte maturation, induces MSY2 phosphorylation and the maturation-associated degradation of mRNAs. Inhibiting CDC2A following its activation with roscovitine inhibits MSY2 phosphorylation and prevents mRNA degradation. Expressing non-phosphorylatable dominant-negative forms of MSY2 inhibits the maturation-associated decrease in mRNAs, whereas expressing constitutively active forms induces mRNA degradation in the absence of maturation and phosphorylation of endogenous MSY2. A positive-feedback loop of CDK1-mediated phosphorylation of MSY2 that leads to degradation of Msy2 mRNA that in turn leads to a decrease in MSY2 protein may ensure that the transition is irreversible.

Deletion of the DNA/RNA-binding protein MSY2 leads to post-meiotic arrest

Y-box proteins are a well-characterized family of nucleic acid binding proteins that are expressed from bacteria to human. This review will focus on MSY2, a member of the Y-box gene family that is exclusively expressed in male and female germ cells. MSY2 is the mouse ortholog of FRGY2, the Xenopus germ cell-specific protein and the human germ cell protein, Contrin. MSY2 functions as a co-activator of transcription in male germ cells and plays an important role in the translational repression and storage of both paternal and maternal mRNAs in spermatocytes, spermatids and oocytes. Following gene targeting, matings of heterozygotes produce a normal Mendelian ratio with equal numbers of phenotypically normal males and females. However, males and females lacking Msy2 are infertile. In Msy2-null males, spermatogenesis is disrupted in post-meiotic germ cells with many misshapen and multinucleated spermatids. No spermatozoa are found in the epididymis. The germ cell specificity and the critical functions played by this multifunctional DNA- and RNA-binding protein during spermatogenesis make Contrin, the human ortholog of MSY2, an attractive and novel target for male contraception.

MIWI-independent small RNAs (MSY-RNAs) bind to the RNA-binding protein, MSY2, in male germ cells

The germ cell-specific DNA/RNA-binding protein MSY2 binds small RNAs (MSY-RNAs) that are ≈25–31 nt in length, often initiate with a 5′ adenine, and are expressed in both germ cells and somatic cells. MSY-RNA levels do not decrease in Miwi or Msy2 null mice. Most MSY-RNAs map within annotated genes, but some are PIWI-interacting RNA (piRNA)-like and map to piRNA clusters. MSY-RNAs are in both nuclei and cytoplasm. In nuclei, MSY-RNAs are enriched in chromatin, and in the cytoplasm they are detected in both ribonucleoproteins and polysomes.

High-throughput sexing of mouse blastocysts by real-time PCR using dissociation curves.

Sex ratio is defined as the proportion ofmales to females in a population. Subdivisions of the sex ratio include the primary (ratio at fertilization) and the secondary (ratio at birth). The expected secondary sex ratio is 0.5, but biological, environmental, or occupational variables can shift the secondary sex ratio from this expectation. Economic and medical benefits also exist for altering the secondary sex ratio; for example, breeding cattle for milk production and developing a method to suppress or eliminate pest populations, like the malaria mosquito, could benefit from manipulating the secondary sex ratio. Mice are an ideal model to understand the biological mechanism of sex-ratio alterations because there are numerous wild-type and genetically altered strains. The secondary sex ratio of a mouse litter can be determined by visual inspection at birth or at weaning, but more sophisticated techniques are required to track the primary sex ratio as it evolves into the secondary sex ratio. Morphological distinction between males and females, for example, is possible after gonadal differentiation at embryonic day 12.5, while female embryos can be identified throughout embryogenesis via selective mating between a male with anXchromosomecontaininga fluorescent transgeneanda wild-type female (Kobayashi et al., 2006). Sexing mice using either a DNAor RNA-based PCR method is a less technically demanding protocol that is readily available to most laboratories. The stability during embryogenesis of DNA compared to RNA, which undergoes dynamic changes during the maternal-to-zygotic genome transition and the development of sex-specific gene expression inpreimplantationembryos (blastocysts), is one advantage of DNA-based sexing. Multiplex and simplex PCRmethods are two common approaches used to reveal an individual’s genotypic sex. Multiplex PCR simultaneously amplifies a Y chromosome gene (e.g., sex-determining region on the Y, Sry) in combination with an endogenous control gene (e.g., interleukin 2, Il2) that functions as an internal control of PCR amplification to confirm that the inability to amplify the Y chromosome gene is a true negative for that gene.SimplexPCR,which canbeeasier to optimize and validate than multiplex PCR, uses a single primer pair to amplify two highly homologous genes that have an intron of different lengths (McFarlane et al., 2013). We used simplex quantitative real-time PCR (qPCR) with a primer pair that amplifies a portion of the X-chromosome gene lysine-specific demethylase 5, Kdm5c (synonyms: Jarid1c, Smcx) and the corresponding Y-chromosome gene Kdm5d (synonyms: Jarid1d, Smcy) (Clapcote and Roder, 2005). The sizes of the fragments from the X (331 bp) and the Y (302 bp) chromosomes are distinct because the intron lengths are different on theXandY chromosome, resulting in distinguishable melting curves. We prepared template from single blastocysts (n1⁄4 11) for sexing using conventional multiplex PCR and simplex qPCR followed by melting-curve analysis (Supplemental Materials and Methods). Two PCR bands (Sry and Il2) are observed from male blastocysts (Fig. 1A) by agarose gel electrophoresis with two peaks on the melting-curve analysis (Kdm5c/Kdm5d), whereas a single band/peak is observed from female blastocysts (Fig. 1B) by both agarosegel (Il2 only) and melting-curve analysis (Kdm5c). The combined data from the 11 blastocysts represent six males and five females. Our melting-curve analysis protocol thus provides a high-throughput sexing method that eliminates the time-consuming steps of running, staining, and interpreting agarose gels of PCRproducts.Weenvision that this method will contribute to high-throughput screening for factors that contribute to alterations in the primary and secondary sex ratio.

Specificity of calcium/calmodulin-dependent protein kinases in mouse egg activation

CaMKIIγ, the predominant CaMKII isoform in mouse eggs, controls egg activation by regulating cell cycle resumption. In this study we further characterize the involvement and specificity of CaMKIIγ in mouse egg activation. Using exogenous expression of different cRNAs in Camk2g−/− eggs, we show that the other multifunctional CaM kinases, CaMKI, and CaMKIV, are not capable of substituting CaMKIIγ to initiate cell cycle resumption in response to a rise in intracellular Ca2+. Exogenous expression of Camk2g or Camk2d results in activation of nearly 80% of Camk2g−/− MII eggs after stimulation with SrCl2, which does not differ from the incidence of activation of wild-type eggs expressing exogenous Egfp. In contrast, none of the Camk2g−/− MII eggs expressing Camk1 or Camk4 activate in response to SrCl2 treatment. Expression of a constitutively active form of Camk4 (ca-Camk4), but not Camk1, triggers egg activation. EMI2, an APC/C repressor, is a key component in regulating egg activation downstream of CaMKII in both Xenopus laevis and mouse. We show that exogenous expression of either Camk2g, Camk2d, or ca-Camk4, but not Camk1, Camk4, or a catalytically inactive mutant form of CaMKIIγ (kinase-dead) in Camk2g−/− mouse eggs leads to almost complete degradation (~90%) of exogenously expressed EMI2 followed by cell cycle resumption. Thus, degradation of EMI2 following its phosphorylation specifically by CaMKII is mechanistically linked to and promotes cell cycle resumption in MII eggs.