Expression of Chlamydomonas reinhardtii chloroplast diacylglycerol acyltransferase 3 is induced by light in concert with triacylglycerol accumulation.

Considerable progress has been made towards the understanding of triacylglycerol (TAG) accumulation in algae. One key aspect is finding conditions that trigger TAG production without reducing cell division. Previously, we identified a soluble diacylglycerol acyltransferase (DGAT), related to plant DGAT3, with heterologous DGAT activity. In this work, we demonstrate that Chlamydomonas reinhardtii DGAT3 localizes to the chloroplast and that its expression is induced by light, in correspondence with TAG accumulation. Dgat3 mRNAs and TAGs increase in both wild-type and starch-deficient cells grown with acetate upon transferring them from dark or low light to higher light levels, albeit affected by the particularities of each strain. The response of dgat3 mRNAs and TAGs to light depends on the pre-existing levels of TAGs, suggesting the existence of a negative regulatory loop in the synthesis pathway, although an effect of TAG turnover cannot be ruled out. Altogether, these results hint towards a possible role of DGAT3 in light-dependent TAG accumulation in C. reinhardtii.

Small RNAs and their protein partners in animal meiosis.

Meiosis is characterized by highly regulated transitions in gene expression that require diverse mechanisms of gene regulation. For example, in male mammals, transcription undergoes a global shut-down in early prophase I of meiosis, followed by increasing transcriptional activity into pachynema. Later, as spermiogenesis proceeds, the histones bound to DNA are replaced with transition proteins, which are themselves replaced with protamines, resulting in a highly condensed nucleus with repressed transcriptional activity. In addition, two specialized gene silencing events take place during prophase I: meiotic silencing of unsynapsed chromatin (MSUC), and the sex chromatin specific mechanism, meiotic sex chromosome inactivation (MSCI). Notably, conserved roles for the RNA binding protein (RBP) machinery that functions with small non-coding RNAs have been described as participating in these meiosis-specific mechanisms, suggesting that RNA-mediated gene regulation is critical for fertility in many species. Here, we review roles of small RNAs and their associated RBPs in meiosis-related processes such as centromere function, silencing of unpaired chromatin and meiotic recombination. We will discuss the emerging evidence of non-canonical functions of these components in meiosis.

Desmosterol Incorporation Into Ram Sperm Membrane Before Cryopreservation Improves in vitro and in vivo Fertility.

Pregnancy rates in ewes are markedly low after cervical insemination with frozen-thawed sperm. Sensitivity of ram sperm to freeze-thawing is related to the lipid composition of the membrane, particularly to its low sterol content. Recently, we proved that sterol content of ram sperm can be increased by treatment with methyl-ß-cyclodextrin-sterol complexes and we provided mechanistic based evidence on the differential behavior of cholesterol and desmosterol in the ram sperm membrane. In the present study, we evaluated the role of increasing cholesterol and desmosterol content of ram sperm before cryopreservation, on the extent and distribution of sterols, cryocapacitation status, acrosome integrity, DNA damage associated with apoptosis and fertility competence in vitro and in vivo of post-thawed sperm. After freeze-thawing, similar levels of sterol content were evidenced in control sperm cells and in those pre-incubated with either cholesterol or desmosterol. Still, moderately higher levels of sterols were registered in treated sperm compared to the control, indicating no physiological excess of sterols after thawing or sterol losses that exceed the control. Live cell imaging of fluorescent cholesterol evidenced the presence of sperm sub-populations differentially affected by freeze-thawing. Similar unimodal frequency profiles were observed between sterol-enriched groups, while the control exhibited a sub-population of sperm compatible with low sterol content. Tyrosine phosphorylation was significantly lower when ram sperm incorporated cholesterol compared to the control. No difference in this capacitation parameter was found between the latter and desmosterol-enriched sperm. The percentage of sperm with damaged acrosomes post-thawing, assessed by a fluorescent lectin, was reduced in sperm that incorporated sterols before freezing, irrespective of the sterol class. These results suggest that sterols exert a stabilizing effect on the acrosome. No differences were found in levels of apoptotic DNA fragmentation among experimental groups. As to fertility trials, desmosterol-enriched sperm gave rise to higher rates of in vitro activated oocytes by heterologous fertilization and to significantly lower pregnancy loss in vivo. Our research provides new insights on sterol incorporation into ram sperm prior to cryopreservation, in particular on the additional benefit of incorporating desmosterol as a strategy to improve fertility outcome.

Cholesterol and desmosterol incorporation into ram sperm membrane before cryopreservation: Effects on membrane biophysical properties and sperm quality.

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-ß-cyclodextrin (MßCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MßCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MßCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MßCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MßCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.