Reconstruction of the birth of a male sex chromosome present in Atlantic herring

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an similar to 500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics

<div><p>Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.</p></div

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons

Lack of GBA2 causes globozoospermia and cytoskeletal defects in the testis.

<p><b>(A)</b> Immunofluorescent labeling of wild-type (+/+) and GBA2 knockout-sperm (-/-). Fluorescent peanut lectin (green), a mitotracker (red), and DAPI (blue) was used to label the acrosome, the mitochondria in the sperm flagellum, and the DNA, respectively. Scale bars are indicated. <b>(B)</b> Immunofluorescent labeling of the cytoskeleton in adult wild-type (+/+) and GBA2 knockout-testis (-/-). Microtubules were labeled using an anti-beta tubulin antibody (red), F-actin using Alexa Fluor 488 Phalloidin (green), and the DNA using DAPI (blue). Defects in the F-actin structure are highlighted with arrows. Scale bars are indicated. <b>(C)</b> Immunofluorescent labeling of germ and Sertoli cells isolated from adult wild-type (+/+) and GBA2 knockout-testis (-/-). Cells were labeled with an anti-beta tubulin III antibody (red) as a marker for Sertoli cells, Alexa Fluor 488 Phalloidin (green) to label F-actin, and DAPI to label the DNA (blue). Scale bars are indicated. <b>(D)</b> See (C) for P7 Sertoli cells.</p

Acrosome development.

<p>Immunofluorescent labeling of the acrosome using fluorescent peanut lectin (green) in P21, P23, and P34 testes. Microtubules were labeled with an anti-beta tubulin antibody (red), DAPI (blue) has been used to stain the DNA. Scale bars are indicated.</p

Lack of GBA2 results in accumulation of GlcCer.

<p><b>(A)</b> Quantitative analysis of neutral sphingolipids in adult testis. +/+: wild-type;-/-: GBA2 knockout. LCB: long-chain bases, Cer: ceramides, HexCer: hexosylceramides, Spm: sphingomyelins. <b>(B)</b> See (A) for P7 Sertoli cells. <b>(C)</b> See (A) for sperm. <b>(D)</b> Quantitative analysis of HexCer in adult testis. Lipids are classified according to their acyl chain-length. <b>(E)</b> See (D) for sperm. <b>(F)</b> See (D) for very long chain fatty acids. All data are presented as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p

Dermal fibroblasts from GBA2 knockout-mice also display cytoskeletal defects.

<p><b>(A)</b> GBA2 expression in dermal fibroblasts from adult mice. Total protein lysates were probed with a GBA2-specific antibody (2F8) on a Western blot. Heterologously expressed HA-tagged GBA2 was used as a positive control, calnexin (Clnx) as a loading control. <b>(B)</b> Accumulation of GlcCer in GBA2 knockout-fibroblasts. Thin layer chromatography (TLC) analyzing glycosphingolipids from wild-type (+/+) and GBA2 knockout-fibroblasts (-/-). Representative TLC analysis for neutral sphingolipids. GlcCer: glucosylceramide, LacCer: lactosylceramide, Spm: sphingomyelin. GlcCer levels were quantified by densitometry and are presented as mean ± S.D. The fold change in GlcCer levels in GBA2 knockout-fibroblasts was calculated. <b>(C)</b> Fluorescent labeling of the cytoskeleton in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were transfected with lifeact (green) to visualize F-actin and with EB3-cherry to visualize microtubules. Scale bars are indicated. <b>(D)</b> Fluorescent labeling of F-actin in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were seeded on CYTOO chips with micropatterns that are coated with fluorescently-labeled fibronectin (purple). F-actin was stained using Alexa Fluor Phalloidin 488 (green) and the DNA was stained with DAPI (blue). Scale bars are indicated. <b>(E)</b> Analysis of cytoskeletal structures. Cells were seeded on the crossbow shape. The number of cells containing filopodia or lamellipodia (left) and the average number of filopodia or lamellipodia per cell (right) were determined. <b>(F)</b> Gene expression-analysis. The mRNA expression level of <i>Cdc42</i>, <i>Rac1</i>, and <i>Rho</i> was analyzed by qRT-PCR. <b>(G)</b> Protein expression-analysis. Total protein lysates were probed with a GBA2- (2F8), a Cdc42-, and a Rac1-specific antibody on a Western blot. Calnexin (Clnx) was used as a loading control. <b>(H)</b> Quantification of protein expression based on (G). <b>(I)</b> Quantification of actin turnover in dermal fibroblasts. Expression levels of G- and F-actin in wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were determined using Western blot-analysis. Ratio of F-actin/G-actin for wild-type and GBA2 knockout-fibroblasts is expressed relative to the control. <b>(J)</b> See (I) for testis. <b>(K-M)</b> Analysis of microtubule dynamics in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). <b>(K)</b> Expression of EB3-cherry in dermal fibroblasts. Cells were transfected with EB3-cherry and microtubule dynamics were analyzed. Representative tracks of growing microtubule plus-ends are indicated with white lines. <b>(L)</b> Microtubule growth rate. Wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were transfected with EB3-cherry and the growth rate of growing plus-ends was analyzed. Per genotype, n = 3 animals with a minimum of 7 cells and 10 tracks per cell were analyzed. Data are presented as mean ± S.D. <b>(M)</b> see (L) for microtubule persistence. For all bar graphs, data are shown as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p

Cytoskeletal defects in GBA2 knockout-testis already occur during the first spermatogenic wave.

<p><b>(A)</b> Immunofluorescent labeling of the cytoskeleton in wild-type (+/+) and GBA2 knockout-testis (-/-) at P7. Microtubules have been labeled using an anti-beta tubulin III antibody (red), F-actin using Alexa Fluor Phalloidin 488 (green), and the DNA using DAPI (blue). Scale bars are indicated. <b>(B)</b> See (A) for P21. <b>(C)</b> See (A) for P23. <b>(D)</b> See (A) for P34. <b>(E)</b> Development of the manchette in spermatids. The manchette was stained with beta-tubulin (red), DNA was labeled with DAPI (blue). Different developmental stages are indicated. <b>(F)</b> Manchette length. The manchette length of spermatids from wild-type (+/+) and GBA2 knockout-mice (-/-) was determined using ImageJ. At least 30 cells have been analyzed per genotype. Data are shown as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p

The absence of GBA2 affects cell migration.

<p><b>(A-B)</b> Wound-healing assay to analyze cell migration of dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). <b>(A)</b> Representative images at different time points after initiating the assay. Scale bars are indicated. <b>(B)</b> Analysis of cell migration. The rate of cell migration has been analyzed. Average data points for wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) for different time points are shown; n numbers and p values using One-Way ANOVA are indicated.</p