A novel hypothesis for histone-to-protamine transition in Bos taurus spermatozoa

DNA compaction with protamines in sperm is essential for successful fertilization. However, a portion of sperm chromatin remains less tightly packed with histones, which genomic location and function remain unclear. We extracted and sequenced histone-associated DNA from sperm of nine ejaculates from three bulls. We found that the fraction of retained histones varied between samples, but the variance was similar between samples from the same and different individuals. The most conserved regions showed similar abundance across all samples, whereas in other regions, their presence correlated with the size of histone fraction. This may refer to gradual histone-protamine transition, where easily accessible genomic regions, followed by the less accessible regions are first substituted by protamines. Our results confirm those from previous studies that histones remain in repetitive genome elements, such as centromeres, and added new findings of histones in rRNA and SRP RNA gene clusters and indicated histone enrichment in some spermatogenesis-associated genes, but not in genes of early embryonic development. Our functional analysis revealed significant overrepresentation of cGMP-dependent protein kinase G (cGMP-PKG) pathway genes among histone-enriched genes. This pathway is known for its importance in pre-fertilization sperm events. In summary, a novel hypothesis for gradual histone-toprotamine transition in sperm maturation was proposed. We believe that histones may contribute structural information into early embryo by epigenetically modifying centromeric chromatin and other types of repetitive DNA. We also suggest that sperm histones are retained in genes needed for sperm development, maturation and fertilization, as these genes are transcriptionally active shortly prior to histone-to-protamine transition.Peer reviewe

Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility

Ablation of RIC8A Function in Mouse Neurons Leads to a Severe Neuromuscular Phenotype and Postnatal Death

<div><p>Resistance to inhibitors of cholinesterase 8 (RIC8) is a guanine nucleotide exchange factor required for the intracellular regulation of G protein signalling. RIC8 activates different Gα subunits via non-canonical pathway, thereby amplifying and prolonging the G protein mediated signal. In order to circumvent the embryonic lethality associated with the absence of RIC8A and to study its role in the nervous system, we constructed <i>Ric8a</i> conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (<i>SynCre</i>) into the floxed <i>Ric8a</i> (<i>Ric8a</i>^<i>F/F</i>) background ablated RIC8A function in most differentiated neuron populations. Mutant <i>SynCre</i>^<i>+/-</i><i>Ric8</i>^{<i>lacZ/F</i>} mice were born at expected Mendelian ratio, but they died in early postnatal age (P4-P6). The mutants exhibited major developmental defects, like growth retardation and muscular weakness, impaired coordination and balance, muscular spasms and abnormal heart beat. Histological analysis revealed that the deficiency of RIC8A in neurons caused skeletal muscle atrophy and heart muscle hypoplasia, in addition, the sinoatrial node was misplaced and its size reduced. However, we did not observe gross morphological changes in brains of <i>SynCre</i>^<i>+/-</i><i>Ric8a</i>^{<i>lacZ/F</i>} mutants. Our results demonstrate that in mice the activity of RIC8A in neurons is essential for survival and its deficiency causes a severe neuromuscular phenotype.</p> </div

GEF RIC8 co-localization with LGN during mouse oocyte maturation.

<p>(<b>A-F</b>) Mouse oocytes at meiosis I and (<b>G-I</b>) at metaphase block of meiosis II were double-labeled with RIC8 antibody (green) and LGN (red). DNA was stained with DAPI (blue). Localization of meiotic spindle is denoted with white arrows and yellow to orange colour in this area indicates the overlapping regions of RIC8 and LGN. Dotted white line indicates the borders of oocyte. Abbreviations: Ana/Tel I, anaphase/telophase of meiosis I; Met I or Met II, metaphase of meiosis I or meiosis II respectively; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.</p

Localization pattern of RIC8, Gα_i1/2, LGN and NuMA at early cleavage stage of mouse embryo.

<p>(<b>A-C</b>) One-cell (1-cell) embryo at metaphase (Met) of first mitosis. (<b>D-L</b>) Two-cell (2-cell) mouse embryos. Embryos were double-labeled with RIC8 antibody (green) and Gα_i1/2, LGN or NuMA antibodies respectively (red). DNA was stained with DAPI (blue). (<b>d-i</b>) Higher magnification of overlapping regions (yellow to orange) of RIC8 and Gα_i1/2 or LGN in cortex area of blastomere (indicated with white arrowhead). Abbreviations: ms, mitotic spindle; pb, polar body. Scale bar: (<b>A-L</b>) 20 μm, (<b>d-i</b>) 10 μm.</p

Neurospecific deletion of <i>Ric8a</i> in mice.

<p>(<b>A</b>) Schematic representation of neuron-specific deletion of the <i>Ric8a</i> gene. First four exons of floxed <i>Ric8a</i> are removed by Cre-recombinase which expression is under the control of <i>Synapsin I</i> promoter (SynCre). Numbered boxes represent <i>Ric8a</i> exons and arrows <i>loxP</i> sites. (<b>B</b>) PCR-based genotyping of mice using DNA from tail samples to detect <i>SynCre</i> transgene, floxed <i>Ric8a</i> allele and <i>LacZ</i> allele respectively. <i>Ric8a</i>^<i>CKO</i> genotype is emphasized with dotted line. (<b>C</b>) Representative PCR showing the deletion of floxed <i>Ric8a</i> in <i>Ric8a</i>^<i>CKO</i> mouse nervous system and no deletion in non-neural organs. (<b>D</b> and <b>E</b>) Comparison of Cre-recombinase (in <i>SynCre</i>^<i>+/-</i><i>R26R</i>) and <i>Ric8a</i> (in <i>Ric8a</i>^{<i>lacZ/+</i>}) expression in E12.5 embryos by X-gal staining. (<b>F</b>) Down regulation of <i>Ric8a</i> mRNA expression in <i>Ric8a</i>^<i>CKO</i> mice relative to littermate control in hippocampus (HIP), spinal cord (SC), cardiac muscle (CM) and liver (LIV). (<b>G</b>) Deficiency of RIC8A protein in <i>Ric8a</i>^<i>CKO</i> (CKO) mice compared to littermate control (LM) in hippocampus (HIP), spinal cord (SC), spinal ganglia (SG) and cardiac muscle (CM). GAPDH was used as a reference. Abbreviations: del, PCR fragment from the deleted allele Drg, dorsal root ganglia; F, floxed allele; Hb, hindbrain; Mb, midbrain; Nt, neural tube; SC, spinal cord; SG, spinal ganglia; Syt, sympathetic trunk; Vno, vomeronasal organ; wt, PCR fragment from the wild-type allele; V, trigeminal ganglion; X, vagus ganglion; ** <i>P</i> < 0,01. Error bars represent mean ± SEM scores. Scale bars: (D and E) 1 mm.</p

RIC8 in folliculogenesis and in the reproductive tract of adult mouse.

<p>RIC8 was visualized with RIC8 antibody (red), and cell nuclei were visualized with DAPI (blue). (<b>A-F</b>) Transversal cryosections of ovary with oocytes in different follicular stages starting from primordial follicle to Graafian follicle and (<b>G-I</b>) different regions of oviduct are shown. (<b>H</b>) Higher magnification of the region of ampulla and (<b>i</b>, indicated by white box) isthmus. Abbreviations: A, antrum; Amp, ampulla region of oviduct; Cb, basal layer of cilia; Cc, ciliated cell; Ci, cilia; Co, cumulus oophorus; Cr, corona radiata; Cx, cell cortex, Ec, epithelial cells; Fc, follicular cell; Gc, granulosa cells; Gf, Graafian follicle; GV, germinal vesicle; Ist, isthmus region of oviduct; Lp, lamina propria; Lu, lumen; Pc, peg cell; Pf, primary follicle; Pmf, primordial follicle; Po, primary oocyte; Sf, secondary follicle. Scale bars: 50 μm.</p

Distribution of RIC8, Gα_i1/2, LGN and NuMA in the fertilized oocyte at pronuclear stages.

<p>(<b>A-I</b>) Mouse fertilized oocyte at pronuclear stages were double-labeled with RIC8 antibody (green) and NuMA, LGN or Gα_i1/2 antibodies respectively (red). DNA was stained with DAPI (blue). (<b>a-i</b>) Higher magnification of female or male (indicated by sex symbols) pronucleus. Yellow arrowheads point to small RIC8 foci localized in the nucleoplasm. White arrowheads point to the meiotic spindle. Dotted white line indicates the borders of oocyte. Abbreviations: ms, meiotic spindle; pb, polar body; PN, pronuclear stage. Scale bar: 10 μm.</p

RIC8 protein expression pattern compared to NuMa or Gα_i1/2 proteins during mouse oocyte maturation.

<p>(<b>A-F</b>) Mouse oocytes at meiosis I were double-labeled with RIC8 antibody (green) and NuMA or Gα_i1/2 antibodies (red). DNA was stained with DAPI (blue). The overlapping regions of RIC8 and NuMA or Gα_i1/2 (yellow to orange) at meiotic spindle indicated with white arrows. White arrowheads indicated cell cortex regions, where RIC8 and Gα_i1/2 co-localize. Abbreviations: Ana/Tel I; anaphase/telophase of meiosis I; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.</p