l-arginine alters the proteome of frozen-thawed bovine sperm during in vitro capacitation.

The aim of this work was to evaluate the proteomic changes that occurred in the frozen-thawed bovine spermatozoa after the addition of l-arginine (L-arg) during in vitro sperm capacitation. Aspects related to the sperm capacitation pattern like membrane integrity, mitochondrial activity, sperm motility and vigor, and the sperm proteome were determined. These were respectively assessed by chlortetracycline staining, H342/PI, JC-1, light microscopy, and the proteomic abundance by nUPLC-MS/MS analysis. Frozen-thawed sperm from three Nellore bulls were capacitated in vitro for 3 h in sp-TALP medium supplemented with 20 µg/mL heparin (Control) or with 20 µg/mL heparin plus 1 mM L-arg (L-arg group). Data were subjected to analysis of variance and means compared by SNK test at 5% probability. When compared to Control, the percentage of sperm motility was higher in the L-arg group (P < 0.05). For test data after 3 h of incubation, sperm capacitated with L-arg showed higher membrane integrity and mitochondrial potential when compared to Control (P < 0.05). Moreover, we observed an increase in the percentage of capacitated sperm pattern (P < 0.05). Protein abundance analysis identified 367 proteins. Forty proteins were differentially abundant between Control and L-arg group (P < 0.05), of which 11 were up-regulated, and 29 were down-regulated in L-arg group. In addition, we observed that one protein was uniquely abundant in the L-arg group. Our findings indicate that the addition of L-arg to the culture medium presented a differential protein abundance pattern and increased the bovine frozen-thawed sperm quality and the percentage of capacitated sperm. The proteomic changes observed may be linked to the molecular mechanisms involved in the action of L-arg on the in vitro sperm capacitation of cattle.

L-arginine affects the IVM of cattle cumulus-oocyte complexes.

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17ß-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control -ve). The addition of 5.0-mM L-arg increased (P < 0.05) the percentage of oocytes at the germinal vesicle breakdown stage after 7 hours of cultivation compared with control -ve. All concentrations of L-arg (2.5, 5.0, and 10.0 mM) increased the percentage of oocytes that reached the metaphase I (MI) at 15 hours (P < 0.05) but do not affect the progression from MI to metaphase II (P > 0.05) at 22 hours. All concentrations of L-arg tested increased (P < 0.05) the percentage of cumulus cells with plasma membrane integrity at 22 hours of cultivation. L-arginine did not change (P > 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control -ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control -ve and treatment with 5-mM L-arg progressively decreased (P < 0.05) after the first hour of cultivation; however, COCs treated with 5.0-mM L-arg had higher concentrations of cGMP at 1 hour of cultivation (P < 0.05). The cAMP concentration of COCs supplemented or not with 5.0-mM L-arg progressively increased until 3 hours of cultivation and at, 6 hours, decreased (P < 0.05). The results show, in using this system, that (1) the mechanisms that give the oocyte the ability to restart the meiosis until MI after adding 5.0-mM L-arg do not involve changes in the concentration of nitrate/nitrite, P4, and E2 in the culture medium and (2) L-arg acts on a pathway that involves changing the cGMP concentration but does not involve changing cAMP concentration. More studies are needed to assess whether the observed effects of L-arg during IVM using this system are via NO or not and what the role is in increasing the viability of cumulus cells in the resumption and progression of meiosis until MI.