CBs of <i>Bmal1</i> KO round spermatids display morphological alterations.

<p>(A) Morphological parameters were measured in WT and <i>Bmal1</i> KO round spermatids, such as total area, number of inner vacuoles, vacuolar total area, length, width and number of outer vesicles. Data were compared using test t Student and the asterisks (*) indicate statistical differences between WT and <i>Bmal1</i> KO CB. Statistical significance was considered with a p≤0.05. (B) Morphological parameters were measured in <i>Bmal1</i> KO round spermatids possessing 2 CB spots, such as total area, number of inner vacuoles, vacuolar total area, length, width and number of outer vesicles, and distance between the two CB spots. Data were compared using test t Student and the asterisks (*) indicate statistically significant differences between the large main CB (1^st) and the ‘satellite’ CB (2^nd). Statistical significance was considered with a p≤0.05.</p

Circadian Proteins CLOCK and BMAL1 in the Chromatoid Body, a RNA Processing Granule of Male Germ Cells

<div><p>Spermatogenesis is a complex differentiation process that involves genetic and epigenetic regulation, sophisticated hormonal control, and extensive structural changes in male germ cells. RNA nuclear and cytoplasmic bodies appear to be critical for the progress of spermatogenesis. The chromatoid body (CB) is a cytoplasmic organelle playing an important role in RNA post-transcriptional and translation regulation during the late steps of germ cell differentiation. The CB is also important for fertility determination since mutations of genes encoding its components cause infertility by spermatogenesis arrest. Targeted ablation of the <em>Bmal1</em> and <em>Clock</em> genes, which encode central regulators of the circadian clock also result in fertility defects caused by problems other than spermatogenesis alterations. We show that the circadian proteins CLOCK and BMAL1 are localized in the CB in a stage-specific manner of germ cells. Both BMAL1 and CLOCK proteins physically interact with the ATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog), a hallmark component of the CB. BMAL1 is differentially expressed during the spermatogenic cycle of seminiferous tubules, and <em>Bmal1</em> and <em>Clock</em> deficient mice display significant CB morphological alterations due to BMAL1 ablation or low expression. These findings suggest that both BMAL1 and CLOCK contribute to CB assembly and physiology, raising questions on the role of the circadian clock in reproduction and on the molecular function that CLOCK and BMAL1 could potentially have in the CB assembly and physiology.</p> </div

CLOCK and BMAL1 co-localize with CB proteins.

<p>(A) Squash preparations of stages IV-VI WT, <i>Bmal1</i> KO and <i>Clock</i> KO seminiferous tubules were stained with anti-CLOCK antibody (Green) and anti-MVH antibody (Red). The co-localization between CLOCK and MVH was observed in the WT and <i>Bmal1</i> KO round spermatids. (B) Squash preparations of stages IV-VI WT, <i>Bmal1</i> KO and <i>Clock</i> KO seminiferous tubules were stained with anti-BMAL1 antibody (Green) and anti-EIF4e antibody (Red). The co-localization between BMAL1 and EIF4e was observed only in the WT round spermatids. DNA was counter-stained by DRAQ5 (Blue). Scale bar: 10 µm.</p

Alteration in the number of CB spots in the round spermatids from <i>Bmal1</i> KO and <i>Clock</i> KO mice.

<p>(A) Electron microscopy was carried out with WT and <i>Bmal1</i> KO seminiferous tubules that had been fixed in glutaraldehyde. Ultrastructural analyses show a CB with its regular sponge-like structure, and regions that have different electron-density levels, in WT spermatids (1). Conversely, CBs from <i>Bmal1</i> KO spermatids display altered shape (2), and fragmentation of their structure (3 and 4). In most cases, a large CB was found adjacent to a small ‘satellite’ fragment. (B) The number of WT, <i>Bmal1</i> KO and <i>Clock</i> KO round spermatids possessing 1 or 2 or more CBs was determined. Most of the spermatids have one single CB spot. There was no statistical difference among the number of WT, <i>Bmal1</i> KO and <i>Clock</i> KO round spermatids possessing 1 single CB spots or between the number of <i>Bmal1</i> KO and <i>Clock</i> KO round spermatids possessing 2 or more CB spots. Scale bars: 1 and 2∶1 µm; 3 and 4∶0.5 µm.</p

BMAL1 and CLOCK may play an important role during the spermatogenesis process.

<p>(A) BMAL1 is differentially expressed during the development of seminiferous tubules. Total proteins from stage-specific seminiferous tubules segments, which were identified by transilluminating dissection under transilumination microscope, were extracted from WT and <i>Bmal1</i> KO mice. Extracts were immunoblotted with anti-BMAL1, anti-CLOCK, anti-MVH, anti-MIWI, anti-DICER and anti-DCP1a antibodies. A decrease in BMAL1 levels was detected in stages VII-IX. The amount of CB proteins of <i>Bmal1</i> KO specific-stages segments did not follow the same expression pattern as compared to WT mice. Tubulin served as a loading control. (B) Immunopurified (IP) Flag-MVH by M2 Agarose from HEK 293 cells was probed by western blot with the indicated antibodies. Total lysates after co-immunoprecipitation were used as input. (C) In vivo immunoprecipitation (IP) was performed from cytoplasmic and nuclear fractions from seminiferous tubules of <i>Bmal1</i> WT and <i>Bmal1</i> KO mice with anti-CLOCK antibody. Samples were immunoblotted with the indicated antibodies. Cytoplasmic and nuclear fractions after co-immunoprecipitation were used as input.</p

Alteration in the number of CB spots in <i>Bmal1</i> KO and <i>Clock</i> KO round spermatids.

<p>(a) and (d): Wild type (WT) round spermatids. (b) and (e): <i>Bmal1</i> knockout (KO) round spermatids. (c) and (f): <i>Clock</i> knockout (KO) round spermatids. Squash preparations of stages IV–VI seminiferous tubules were stained with anti-MVH antibody (a–c) (Green) and with anti-MIWI antibody (d–f) (Green). Two or three CB spots were observed in the cytoplasm of <i>Bmal1</i> KO and <i>Clock</i> KO spermatids but not in the cytoplasm of WT spermatids. DNA was counter-stained by DRAQ5 (Blue). Scale bar: 10 µm.</p

Relationship between the nucleolar cycle and chromatoid body formation in the spermatogenesis of Phrynops geoffroanus (Reptilia Testudines)

The nucleolus is a distinct nuclear territory involved in the compartmentalization of nuclear functions. There is some evidence of a relationship between nuclear fragmentation during spermatogenesis and chromatoid body (CB) formation. The CB is a typical cytoplasmic organelle of haploid germ cells, and is involved in RNA and protein accumulation for later germ-cell differentiation. The goal of this study was to qualitatively and quantitatively describe the nucleolar cycle during the spermatogenesis of Phrynops geoffroanus (Reptilia Testudines), and compare this nucleolar fragmentation with CB formation in this species through the use of cytochemical and ultrastructural analysis. Qualitative analysis showed a fragmentation of the nuclear material after pachytene of the first meiotic division in the primary spermatocytes. Quantitative analysis of the nucleolar cycle revealed a significant difference in the number of nucleoli and in the size of the nucleolus between spermatogonia and early spermatids. Using ultrastructural analysis, we recorded the beginning of the CB formation process in the cytoplasm of primary spermatocytes at the same time as when nuclear fragmentation occurs. In the cytoplasm of primary spermatocytes, the CB was observed in association with mitochondrial aggregates and the Golgi complex. In the cytoplasm of early spermatids, the CB was observed in association with lipid droplets. In conclusion, our data show that the nucleolus plays a role in the CB formation process. During spermatogenesis of P. geoffroanus, the CB is involved in some important biological processes, including acrosome formation and mitochondrial migration to the spermatozoon tail and middle piece region.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq