Proper oocyte maturation is crucial for subsequent embryo development; however, oocyte mitochondrial and lipid-droplet behaviour are still poorly understood. Although excessive lipid accumulation during in vitro production (IVP) of bovine embryos has been linked with impaired cryotolerance, lipid oxidation is essential for adequate energy supply. Fetal bovine serum (FBS) and bovine serum albumin (BSA) are supplements used during IVP, containing high and low lipid content, respectively. This study aimed to understand how these supplements influence oocyte mitochondrial and lipid behaviour during in vitro maturation (IVM) in comparison to in vivo maturation, as well as their influence on development rates and embryo lipid accumulation during IVP. We demonstrate that only in vivo-matured oocytes maintained correlation between lipid content and active mitochondria. IVM media containing FBS increased total lipid content 18-fold and resulted in higher lipid accumulation in oocytes when compared with media with BSA. IVM using a lower FBS concentration combined with BSA resulted in satisfactory maturation and embryo development and also reduced lipid accumulation in blastocysts. In conclusion, IVM causes changes in mitochondrial and lipid dynamics, which may have negative effects on oocyte development rates and embryo lipid accumulation. Moreover, decreasing FBS concentrations during IVM may reduce embryo lipid accumulation without affecting production rates.

In cardiac research, single-cell experimental models have been extensively used to study the molecular mechanisms of intracellular Ca 2+ homeostasis. The results of these studies are usually extrapolated to the tissue level assuming that the phenomena studied at the cellular level are either similar in the intact organ, or only slightly modified by variables that exist at the whole-heart level. The validity of these assumptions has rarely been confirmed experimentally. Common obstacles associated with the study of intracellular Ca 2+ signals in beating hearts include motion artifacts and spatio-temporal limitations of the recording system. In this work, action potentials and intracellular Ca 2+ signals were measured in beating hearts from young rats, with spatio-temporal resolutions similar to cellular studies using a novel pulsed local-field fluorescence technique. This method was based on maximizing emitted fluorescence to increase the signal-to-noise ratio (S/N). The fluorescence emission of the indicator molecules was synchronized with brief (<1 ns), high-power (400 W) laser pulses, and the common mode noise of the fluorescence signal was differentially cancelled. To follow rapidly evolving signals, a highly sensitive and fast detection system was used (10 kHz). The spatial resolution was improved using a small (50-200 m diameter) multimode fiberoptic.Mechanical artifacts were effectively reduced by inserting the fiberoptic into a "floating" glass micropipette sealed to the heart wall with negative pressure. Our results demonstrate that local-field fluorescence microscopy offers an outstanding experimental approach for studying physiological signals at the whole-organ level with the high spatio-temporal resolution common to normal cellular approaches.

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X 1 -P2X 7 ) and seven heteromeric receptors (P2X 1/2 , P2X 1/4 , P2X 1/5 , P2X 2/3 , P2X 2/6 , P2X 4/6 , P2X 4/7 ) have been described. ATP treatment of Leydig cells leads to an increase in [Ca 2+ ] i and testosterone secretion, supporting the hypothesis that Ca 2+ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X 2 . In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X 2 , P2X 4 , P2X 6 , and P2X 7 subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2±5.9%) and 3 μM ivermectin a decrease (64.2±4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X 4 subunits. P2X 7 receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X 2/4/6 , is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X 2 subunit.

Leydig cells are responsible for the synthesis and secretion of testosterone, processes controlled by luteinizing hormone (LH). Binding of LH to a G protein-coupled receptor in the plasma membrane results in an increase in cAMP and in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here we show, using immunofluorescence, that Leydig cells express ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Measurements of intracellular calcium changes using the fluorescent calcium-sensitive dye fluo-3 and confocal microscopy show that both types of receptors are involved in a calcium-induced calcium release (CICR) mechanism, which amplifies the initial Ca(2+) influx through plasma membrane T-type calcium channels (Ca(V)3). The RyRs and IP(3)Rs are functional, as judged from both their activation by caffeine and IP(3) and block by ryanodine and 2-aminoethoxydiphenyl borate (2-APB), respectively. RyRs are the principal players involved in the release of Ca(2+) from the endoplasmic reticulum, as evidenced by the fact that global Ca(2+) changes evoked by LH are readily blocked by 100 muM ryanodine but not by 2-APB or xestospongin C. Finally, steroid production by Leydig cells is inhibited by ryanodine but not by 2-APB. These results not only broaden our understanding of the role played by calcium in Leydig cells but also show, for the first time, that RyRs have an important role in determining testosterone secretion by the testis.

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