Ignacio López-González

In addition to its central role in cellular metabolism, adenosine 5′-triphosphate (ATP) is an important extracellular signalling molecule involved in various physiological processes. In reproduction, extracellular ATP participates in both autocrine and paracrine paths regulating gametogenesis, gamete maturation and fertilisation. This review focusses on how extracellular ATP modulates sperm physiology with emphasis on the mammalian acrosome reaction. The presence of extracellular ATP in the reproductive tract is primarily determined by the ion channels and transporters that influence its movement within the cells comprising the tract. The main targets of extracellular ATP in spermatozoa are its own transporters, particularly species-specific sperm purinergic receptors. We also discuss notable phenotypes from knock-out mouse models and human Mendelian inheritance related to ATP release mechanisms, along with immunological, proteomic, and functional observations regarding sperm purinergic receptors and their involvement in sperm signalling.

During spermatogenesis, fatty acids play an important role both as structural components and messengers that trigger male germ cell line differentiation. The spontaneous oxidation of fatty acids causes a decrease in mammalian fertility. Here, we examine the effects of nonenzymatically oxidized arachidonic acid (AA_ox) on mouse spermatogenic T‐type Ca²⁺ currents (I_CaT) due to their physiological relevance during spermatogenesis. AA_ox is 25‐fold more potent than AA at inhibiting I_CaT and it left shifts the I‐V curve peak and both activation and steady‐state inactivation curves. In addition, I_CaT deactivation kinetics and their recovery from inactivation are slower in the presence of AA_ox. Therefore, the fraction of inactivated Ca²⁺ channels is increased. AA_ox‐induced I_CaT inhibition could contribute to male infertility affecting Ca²⁺ regulation in spermatogenic cells.

There is growing evidence indicating that the pore structure of voltage-gated ion channels (VGICs) influences gating besides their conductance. Regarding low voltage-activated (LVA) Ca ²⁺ channels, it has been demonstrated that substitutions of the pore aspartate (D) by a glutamate (D-to-E substitution) in domains III and IV alter channel gating properties such as a positive shift in the channel activation voltage dependence. In the present report, we evaluated the effects of E-to-D substitution in domains I and II on the Ca _V 3.1 channel gating properties. Our results indicate that substitutions in these two domains differentially modify the gating properties of Ca _V 3.1 channels. The channel with a single mutation in domain I (DEDD) presented slower activation and faster inactivation kinetics and a slower recovery from inactivation, as compared with the WT channel. In contrast, the single mutant in domain II (EDDD) presented a small but significant negative shift of activation voltage dependence with faster activation and slower deactivation kinetics. Finally, the double mutant channel (DDDD) presented intermediate properties with respect to the two single mutants but with fastest deactivation kinetics. Overall, our results indicate that single amino acid modification of the selectivity filter of LVA Ca ²⁺ channels in distinct domains differentially influence their gating properties, suggesting a pore pseudo-symmetry. Statement of significance: Previous reports of low voltage-activated (LVA) Ca ²⁺ channels have demonstrated that pore aspartate (D) in domains III and IV equally modulates the channel gating properties, supporting a hypothesis of pore symmetry in LVA Ca ²⁺ channels. In the present report, we evaluated the effects of glutamate (E)-to-D pore substitution in domains I and II on the Ca _V 3.1 channel gating properties. Our results indicate that substitutions in these two domains differentially modify the gating properties of Ca _V 3.1 channels, therefore suggesting a pore pseudo-symmetry in them. Interestingly, our pore mutations affect inactivation of Ca _V 3.1 Ca ²⁺ channels indicating a selectivity filter contribution to this process similar to the recent proposed paradigm for high voltage-activated Ca ²⁺ channels.

During spermatogenesis, phospholipids and fatty acids (FAs) play an important role both as structural components of spermatogenic cell plasma membranes and as molecular messengers that trigger the differentiation of the male germ cell line. However, spontaneous oxidation of plasma membrane phospholipids and FAs causes a decrease in mammalian fertility. In the present report, we examine the effects of non-enzymatically oxidized arachidonic acid (AA _ox ) on mouse spermatogenic T-type Ca ²⁺ currents (I _CaT ) due to their physiological relevance during spermatogenesis. AA _ox effects on the biophysical parameters of I _CaT were significantly different from those previously reported for AA. AA _ox left shifted the I-V curve peak and both activation and steady-state inactivation curves. I _CaT deactivation kinetics were slower in presence of AA _ox and the time for its recovery from inactivation increased significantly. Therefore, the fraction of inactivated Ca ²⁺ channels of spermatogenic cells is increased at voltages where they are usually active. The inhibition of I _CaT by AA _ox could contribute to the infertility phenotype and to the observed apoptotic state of spermatogenic cells induced by oxidized FAs.

Zn²⁺ is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis, and prostate contain high Zn²⁺ concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express Ca_V3.2‐encoded T‐type Ca²⁺ currents (I_CaT) which are positively or negatively modulated by Zn²⁺ in other tissues. To explore whether I_CaT could be regulated by Zn²⁺ and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell I_CaT in the absence or presence of different Zn²⁺ concentrations. Zn²⁺ decreased I_CaT in a concentration‐dependent manner (IC₅₀ = 2 μM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, I_CaT did not show a tonic inhibition by Zn²⁺. Further analysis showed that Zn²⁺ did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady‐state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong I_CaT reduction. Using non‐stationary fluctuation analysis, we found that Zn²⁺ mainly reduced the number of available Ca²⁺ channels without changing the single channel current amplitude. I_CaT modulation by Zn²⁺ could be relevant for spontaneous Ca²⁺ oscillations during spermatogenesis and in pathophysiological conditions such as diabetes. J. Cell. Physiol. 231: 659–667, 2016.