A Study On T-type Ca²⁺ Channel And ENaC Of Mouse Spermatogenic Cells

Fertilization involves accurately choreographed cellular interactions required for sperm and egg to fuse and generate a new individual. Gametogenesis occurs in the gonads where sperm and egg are produced and where they partially mature. Mammalian sperm undergo further maturation within the female reproductive tract, in a process called capacitation, to become responsive to the physiological inducer of the AR, the zona pellucida. A great deal of researches indicated that ion channels, including T-type Ca²⁺ channels and epithelial sodium channels(ENaC), are important for male mammalian's spermatogenesis and fertilization. Therefore, we recorded and identified T-type Ca²⁺ channel and ENaC currents in mouse spermatogenic cells using whole cell patch clamp technique .And also we investigated the effects of trace element and estrogen on them. The Ca²⁺ current recorded from mouse spermatogenic cells showed characteristics as follow: the threshold for Ca²⁺ current activation was at about -60 mV, the peak amplitude occurred at -20 mV and the reversal potential was about +40 mV. Half activation value and steepness parameter were (-49.05±0.63) mV and (5.75±055) mV, respectively. Half inactivation value and steepness parameter averaged (-61.74±0.36) mV, (3.89±0.31) mV, respectively. The time constants of both the peak current and inactivation were reduced in voltage-dependent, the time constant of deactivation was >1 ms. The inhibitory action of Ni was more obvious than Cd²⁺, the similar permeability of Ba²⁺ and Ca²⁺. All the characteristics are according with the properties of T-type Ca²⁺ channels.We recorded the epithelial sodium channels current (I_ENaC) on spermatogenic cells with step and ramp stimulate, and further studyed the permeability with different external solution. We recorded the standard I_ENaC in Na⁺ external solution; but we did not record I_ENaC in K⁺ and ACh⁺ external solutions. Ion selectivity sequence of ENaC was Na⁺>>K⁺=ACh⁺. The ENaC current was inhibited by Amiloride. We found there were two types of Amiloride sensitive ENaC. At the end of Ami-sensitive I_ENaC, there was an inward current; we considered it as T-type Ca²⁺ current. The IC₅₀ of Amiloride to Amiloride-sensitive ENaC was 1μmol·L^-1; The inhibitory rate of 10μmol·L^-1 Amiloride to Amiloride- insensitive ENaC was about 30%. So we conclude there are two different Amiloride sensitive ENaCs to carry out different sperm function.Many studies indicated that a lot of trace elements kept tight correlation with mammalian's reproductive function, sex hormone and pathological changes of reproductive system. And it is now popular regarded that the elements of Zn²⁺, Pb²⁺ and Cu²⁺ were three more crucial effects, but their mechanisms are far from elucidated. The effects of Zn²⁺, Pb²⁺ to I_CaT with whole cell patch-clamp were investigated by predecessor. But the effect of Cu²⁺ is still ambiguous, so we observed Cu²⁺ to I_CaT and I_ENaC with whole cell patch-clamp.We found that Cu reversibly inhibited the amplitudes of I_CaT in a dose-dependent, non voltage-depedent manner. Cu²⁺(0.3, 1, 3, 10, 30)μmol·L^-1 inhibibited I_CaT significantly with IC₅₀ value of 12.06μmol·L^-1. Maintaining holding potential in -90 mV and stimulating potential in -20 mV, the inhibitory rates of Cu²⁺ to I_CaT were (9.32±0.74)%, (18.40±1.03)%, (33.60±1.73)%, (49.59±4.13)%, (59.96±3.60)%, respectively. After administering 10μmol·L^-1 Cu²⁺, we observed these phenomenons, including the activation and inactivation curves of T-type Ca²⁺ channels shifted to hyperpolarization slightly; the time of peak current, time constant of inactivation and deactivation reduced, and the time constant of reactivation enlarged.We observed the effects of Cu²⁺ to two different Amiloride sensitive ENaC channels. Cu²⁺ had no effect to Ami-insensitive ENaC current, but inhibited Ami-sensitive ENaC current with a dose-dependent and non voltage-dependent manner. The inhibition ratios of Cu²⁺ (0.1, 1, 10)μmol·L^-1 to Ami-sensitive ENaC current were (8.13±2.55)%, (22.25±2.28)%, (41.11±2.58)%, respectively.17β-estrodiol (E2) plays an important role in female reproduction function, as a fundamental estrogen. It also has a regulation in male reproduction function. Saberwal et al found that E2 can influence vitality and acrosome reaction (AR) of rat sperm through changing [Ca²⁺]_i; Estrogen antagonist could change the liveness of the relative mobile protein of rat sperm. The mechanism of the accumulation of [Ca²⁺]_i of E2 is still unclear. E2 whether affect the hyperpolarity in the process of capacitation through ENaC or not, there has been no conclusion up to now. We examined the effect of E2 to T-type Ca channels and ENaC of mouse spermatogenic cells with whole cell patch-clamp to clarity its mechanisms in mammal sperm function.The results showed E2 inhibited I_CaT significantly with the K₅₀ value of 8.89μmol·L^-1 in a dose and voltage-dependent manner. The inhibitory rates of E2 (1,10, 100)μmol·L^-1 to I_CaT were (13.48±1.86)%, (28.98±2.70) % and (52.93±3.42)%, respectively. After administering 100μmol·L^-1 E2, the current density of T-type Ca²⁺ channels decreased significantly, shifted to hyperpolarity; voltage of peak current, activation curve and inactivation curve shited to left; time to current peak, time constant of inactivation and deactivation decreased; but time constant of reactivation increased.Unlike Cu²⁺, E2 has no effect on both Ami-insensitive ENaC and Ami-sensitive ENaC currents. The inward current inhibited by E2 at the end of Ami-sensitive ENaC current was confirmed as T-type Ca²⁺ current.