Reorganization of mouse sperm lipid rafts by capacitation

One of the hallmarks of mammalian sperm capacitation is the loss of cholesterol from the plasma membrane. Cholesterol has been associated with the formation of detergent insoluble membrane microdomains in many cell types, and sperm from several mammalian species have been shown to contain detergent‐resistant membranes (DRMs). The change in cholesterol composition of the sperm plasma membrane during capacitation raises the question of whether the contents of DRMs are altered during this process. In this study, we investigated changes in protein composition of DRMs isolated from uncapacitated or capacitated mouse sperm. TX‐100 insoluble membranes were fractionated by sucrose flotation gradient centrifugation and analyzed by Western and lectin blotting, and capacitation‐related differences in protein composition were identified. Following capacitation, the detergent insoluble fractions moved to lighter positions on the sucrose gradients, reflecting a global change in density or composition. We identified several individual proteins that either became enriched or depleted in DRM fractions following capacitation. These data suggest that the physiological changes in sperm motility, ability to penetrate the zona pellucida (ZP), ZP responsiveness, and other capacitation‐dependent changes, may be due in part to a functional reorganization of plasma membrane microdomains. Mol. Reprod. Dev. 73: 1541–1549, 2006. © 2006 Wiley‐Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/89579/1/20540_ftp.pd

Localizing Transcriptional Regulatory Elements at the Mouse Dlk1 Locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1) - Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression

Reorganization Of Mouse Sperm Lipid Rafts By Capacitation

One of the hallmarks of mammalian sperm capacitation is the loss of cholesterol from the plasma membrane. Cholesterol has been associated with the formation of detergent insoluble membrane microdomains in many cell types, and sperm from several mammalian species have been shown to contain detergent-resistant membranes (DRMs). The change in cholesterol composition of the sperm plasma membrane during capacitation raises the question of whether the contents of DRMs are altered during this process. In this study, we investigated changes in protein composition of DRMs isolated from uncapacitated or capacitated mouse sperm. TX-100 insoluble membranes were fractionated by sucrose flotation gradient centrifugation and analyzed by Western and lectin blotting, and capacitation-related differences in protein composition were identified. Following capacitation, the detergent insoluble fractions moved to lighter positions on the sucrose gradients, reflecting a global change in density or composition. We identified several individual proteins that either became enriched or depleted in DRM fractions following capacitation. These data suggest that the physiological changes in sperm motility, ability to penetrate the zona pellucida (ZP), ZP responsiveness, and other capacitation-dependent changes, may be due in part to a functional reorganization of plasma membrane microdomains. © 2006 Wiley-Liss, Inc

Localizing Transcriptional Regulatory Elements at the Mouse Dlk1 Locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1) - Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression

<i>CE4-lacZ</i> displays expression in a subset of <i>Dlk1</i>-expressing tissues.

<p>(A) Diagram of <i>lacZ</i> expression constructs used to produce <i>CE4-lacZ</i> and <i>CE8-lacZ</i> transgenic embryos. The arrow represents the direction of transcription, and CE stands for conserved element 4 or 8. (B–D) Ventral, dorsal and lateral views, respectively, of a representative whole mount transgenic embryo at e13.5. (E) Schematic representation of sagittal section planes used in these images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#pone.0036483-Kaufman1" target="_blank">[64]</a>. (F–M) Sagittal sections of embryos under low magnification (F, I, L) and high magnification (G, H, J, K, M). All sections are oriented with anterior on top and dorsal to the left. Expression was seen in (F–H) intercostal muscle, body wall muscle, and ribs; (I, J) dorsal root ganglia; (I, K) intrinsic tongue muscle; (L, M) thymus. The embryo shown in (F) displays <i>lacZ</i> expression in the pituitary gland and trigeminal ganglion, both sites of endogenous <i>Dlk1</i> expression, but this pattern was not seen in other embryos carrying <i>CE4-lacZ</i>. Scale bars represent 100 µm.</p

<i>Dlk1</i> is expressed but not imprinted from the 127H5 transgene.

<p>(A) Schematic of the <i>Dlk1</i>-<i>Meg3</i> BAC clones used to generate transgenic mice; the <i>28G5</i> transgene was described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#pone.0036483-Yevtodiyenko2" target="_blank">[46]</a>. The 127H5 BAC was linearized at a unique <i>Cla</i>I site. (B, C) Representative Northern blots for <i>Dlk1</i> mRNA in midgestation wild type (WT) and heterozygous transgenic (Tg) embryo (B) and placenta (C), after paternal (<i>127H5^Pat</i>) and maternal (<i>127H5^Mat</i>) inheritance. The mouse <i>β-actin</i> gene was used as a loading control. (D) Quantitative Northern data for blots shown in B & C; expression is normalized to <i>β-actin</i>. Gray bars represent wild type samples and black bars represent <i>127H5</i> transgenic samples upon paternal (<i>127H5^Pat</i>) or maternal (<i>127H5^Mat</i>) transmission in crosses to Cg12. (E) Direct sequencing assay for <i>Dlk1</i> imprinting in wild type (WT) and heterozygous transgenic (Tg) F₁ embryos after paternal (<i>127H5^Pat</i>) and maternal (<i>127H5^Mat</i>) inheritance. D indicates wild type animals carrying only the <i>M. domesticus</i> allele, while D×C or C×D indicates offspring of crosses to the Cg12 line carrying a <i>M. castaneus</i> allele, with the female genotype listed first. (F) Quantitative Northern blot analysis for <i>Dlk1</i> mRNA in 3–4 week old <i>127H5</i> tissues. Expression is normalized to <i>β-actin</i>, and each bar represents 8–10 animals. Gray bars represent wild type samples and the black bars represent <i>127H5</i> transgenic samples upon paternal (Pat) or maternal (Mat) transmission. In all figures asterisks indicate p≤0.05.</p

Embryonic expression of <i>CE4-lacZ</i>, <i>CE8-lacZ</i> and <i>Dlk1.</i>

<p>+ indicates expression; – indicates no expression.</p

Sequence conservation in the <i>Dlk1</i> upstream region.

<p>The sequence upstream of <i>Dlk1</i> was analyzed for regions of conservation across multiple species. The conserved elements chosen for further analysis are numbered CE1 to CE9. The region being displayed corresponds to the July 2007 mouse genome assembly; assembly dates for other species are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#s2" target="_blank">Methods</a> (adapted from the UCSC Genome Browser).</p

<i>CE8-lacZ</i> displays expression in a subset of <i>Dlk1</i>-expressing tissues

<p>. (A–C) Ventral, dorsal and lateral views, respectively, of a representative whole mount transgenic embryo at e13.5. (D) Schematic representation of sagittal section planes used in these images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#pone.0036483-Kaufman1" target="_blank">[64]</a>. (E–Q) Sagittal sections of embryos under low magnification (E, G, J, L, N, P) and high magnification (F, H, I, K, M, O, Q). All sections are oriented with anterior on top and dorsal to the left. Expression was seen in (E, F) skeletal muscle of the limb, (G, H) intervertebral cartilage; (G, I–K) ribs and costosternal junctions; (L, M) chromaffin cells of the adrenal gland; (N, O) dorsal root ganglia; (P, Q) spinal cord. Scale bars represent 100 µm.</p

Transcriptional activation by conserved elements is cell line-dependent.

<p>(A) Luciferase expression plasmids used to assay enhancer function in cell culture. “<i>Dlk1</i> Promoter” signifies the <i>Dlk1</i> basal promoter and CE signifies the individual conserved elements tested. Plasmid pGL3-B contains the promoterless luciferase gene, pGL3-<i>Dlk1</i>P contains the endogenous <i>Dlk1</i> promoter upstream of luciferase and pGL3-C contains the SV40 promoter/enhancer upstream of luciferase. (B) Enhancer activity observed in the NIH-3T3, C2C12, SVR and Y-1 cell lines. Expression is normalized to pGL3-<i>Dlk1</i>P for each cell type; the results are presented as mean ± SEM. P-values relative to pGL3-<i>Dlk1</i>P expression are indicated by *, p≤0.05; #, p≤0.01 (n = 3) (Student’s t-test).</p