Two Environmental Endocrine Disruptors On T-type Mouse Spermatogenic Cells Role Of Ca²⁺ Channel

Fusion of sperm and ovum is called fertilization, which is one of the basic characteristics of sexual reproduction. Sperm produced in the gonads develop into part of the mature . After it pasts the Vaginal, the decapacitation factor , Wrapped in sperm, is cleared while the proteins and membrane potential of the acrosome membrane changes, promoting sperm capacitation and fertilization. When the sperm contact with the egg envelope or zona pellucida, it specifically binding to the Glycoprotein, initiating acrosome reaction. The plasmalemma surrounding the acrosome disappeared while the outer acrosomal membrane become to invaginate or vesiculation and contents overflow which conduce to fertilization. A great deal of researches indicated that ion channels, including T-type Ca²⁺channels, is important for male mammalian's spermatogenesis and fertilization. Therefore, we recorded and identified T-type Ca²⁺ channel in mouse spermatogenic cells using whole cell patch clamp technique .And also we investigated the effects of Mancozeb and Bisphenol A 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(2+) was more obvious than Cd(2+), the similar permeability of Ba(2+) and Ca²⁺. All the characteristics are according with the properties of T-type Ca²⁺ channels.Environmental endocrine disruptors (EEDs) are a class of compounds that can simulate the physiological and biochemical effects of nature hormones, interference with the body's metabolic process of natural hormones, causing reversible or irreversible damage of the reproductive system,. Bisphenol A (BPA) and mancozeb are two kind of EEDs that catches the most attention in recent years. These EEDs can simulate the effect of 17β-estradiol, also they alter the endocrine function of the body and produce toxic effects to the offspring.It's an important reason of the rising incidence of some disorders such as Reproductive disorders, birth defects or development abnormalities.Bisphenol A (BPA) has an estrogen-like activity, which chemical structure is similar to estrogen,many studies found that BPA has an significantly damage effect on male reproductive systerm In recent years. The team of Vom Saal has shown that BPA has the ability to pass the blood-testis barrier and interfere with the growth and activity of sperm directly. BPA can change intracellular Ca2 + concentration of sperm. But the mechanism has not yet been clarified that the BPA increase of [Ca2 +]i on sperm through the T-type calcium channel. We used whole cell patch clamp technique to investigate the effort of BPA on T-type Ca2 + channels of mouse spermatogenic cells and speculated the mechanism of BPA on mammalian reproductive function.The results showed BPA inhibited ICaT significantly with the K50 value of 8.89μmol·L^-1 in a dose and voltage-dependent manner. The inhibitory rates of BPA (1,10,20 and 40)μmol·L^-1 to ICaT were (11.07±1.70)%,(37.10±2.43)%,(56.00±2.83)%,(69.92±3.98)%, respectively. After administering 20μmol·L^-1 BPA, 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.Many studies indicated that a lot of trace elements kept tight correlation with mammalian's reproductive function, sexual functionand sex hormone secretion. Mancozeb, as a important raw materials of Mixed pesticide is a kind of broad-spectrum protective fungicides containing zinc ion. It is widely considered that mancozeb can inhibit sperm motility of mice, cause male reproductive system damage at present, but mechanism is not clear. so we observed the effort of Mancozeb to ICaT and IENaC with whole cell patch-clamp.We found that Mancozeb reversibly inhibited the amplitudes of ICaT in a dose-dependent, non voltage-depedent manner. Mancozeb (10, 20, 40, 80 and 120)μmol·L^-1 inhibibited ICaT significantly with the K50 value of 36.50μmol·L^-1 and the inhibition rates of Mancozeb were(9.93±0.90)%,(20.93±2.50)%,(25.92±2.80)%, ( 42.64±3.10 ) % and ( 56.80±3.20 ) %, respectively. After administering 120μmol·L^-1 Mancozeb, 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.