Sarcoplasmic reticulum (SR) Ca release was studied at 13-16~ in cut fibers (sarcomere length, 3.4-3.9 I~m) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action-potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Ca] is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the ApH signal measured with phenol red by -13/2. The value of 13, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting voltage step; its mean value was 2,685 I~M in the action-potential experiments and 2,544 IxM in the voltage-clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of,'~5 %/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release was produced by the first action potential. During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of~3 %/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release was studied with small step depolarizations that open no more than one SR Ca channel in 7

Cut muscle fibers from Rana teraporaria (sarcomere length, 3.4-4.2 p.m) were mounted in a double and equilibrated with end-pool solutions that contained 20 mM EGTA and 1.76 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from changes in pH (Pape, P. C., D.-S.Jong, and W. I~ Chandler. 1995.Journal of GeneralPhysiology. 106:000-000). Although the amplitude and duration of the [Ca] transient, as well as its spatial spread from the release sites, are reduced by EGTA, SR Ca release elicited by either depolarizing voltage-clamp pulses or action potentials behaved in a manner consistent with Ca inactivation of Ca release. After a step depolarization to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca depletion, reached a peak value within 5-15 ms and then rapidly decreased to a quasi-steady level that was about half the peak value; the time constant of the last half of the decrease was usually 2--4 ms. Immediately after an action potential or a 10-15 ms prepulse to -20 mV, the peak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The rising phase of the rate of release was also reduced, suggesting that at least 0.9 of the ability of the SR to release Ca had been inactivated by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were not caused by changes in its voltage activation. These effects of a first stimulation on the rate of SR Ca release elicited by a second stimulation recovered during repolarization to -90 mV; the time constant of recovery was ~25 ms in the action-potential experiments and ~50 ms in the voltage-clamp experiments. Fura-2, which is able to bind Ca more rapidly than EGTA and hence reduce the amplitude of the [Ca] transient and its spatial spread from release sites by a greater amount, did not prevent Ca inactivation of Ca release, even at con-

Cut muscle fibers from Rana temporaria (sarcomere length, 3.3-3.5 microns; temperature, 13-16 degrees C) were mounted in a double Vaseline-gap chamber and equilibrated for at least an hour with an internal solution that contained 20 mM EGTA and phenol red and an external solution that contained predominantly TEA-gluconate; both solutions were nominally Ca-free. The increase in total myoplasmic concentration of Ca (delta[CaT]) produced by sarcoplasmic reticulum (SR) Ca release was estimated from the change in pH produced when the released Ca was complexed by EGTA (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:259-336). The resting value of SR Ca content, [CaSR]R (expressed as myoplasmic concentration), was taken to be equal to the value of delta[CaT] obtained during a step depolarization (usually to -50 to -40 mV) that was sufficiently long (200-750 ms) to release all of the readily releasable Ca from the SR. In ten fibers, the first depolarization gave [CaSR]R = 839-1,698 microM. Progressively smaller values were obtained with subsequent depolarizations until, after 30-40 depolarizations, the value of [CaSR]R had usually been reduced to < 10 microM. Measurements of intramembranous charge movement, Icm, showed that, as the value of [CaSR]R decreased, ON-OFF charge equality held and the amount of charge moved remained constant. ON Icm showed brief initial I beta components and prominent I gamma "humps", even after the value of [CaSR]R was < 10 microM. Although the amplitude of the hump component decreased during depletion, its duration increased in a manner that preserved the constancy of ON charge. In the depleted state, charge movement was steeply voltage dependent, with a mean value of 7.2 mV for the Boltzmann factor k. These and other results are not consistent with the idea that there is one type of charge, Q beta, and that I gamma is a movement of Q beta caused by SR Ca release, as proposed by Pizarro, Csernoch, Uribe, Rodriguez, and Rios (1991. Journal of General Physiology. 97:913-947). Rather, our results imply that Q beta and Q gamma represent either two distinct species of charge or two transitions with different properties of a single species of charge, and that SR Ca content or release or some related event alters the kinetics, but not the amount of Q gamma. Many of the properties of Q gamma, as well as the voltage dependence of the rate of SR Ca release for small depolarizations, are consistent with predictions from a simple model in which the voltage sensor for SR Ca release consists of four interacting charge movement particles.

Cut fibers (striation spacing, 3.6-4.2 wm) were mounted in a double Vaseline-gap chamber and studied at 14-15°C. One or both of the Ca indicators fura-2 and purpurate-3,3'diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (

Kisspeptin acts as an upstream regulator of the hypothalamus-pituitary-gonad axis, which is one of the main regulatory systems for mammalian reproduction. Kiss1 and its receptor Kiss1r (also known as G protein-coupled receptor 54 (Gpr54)) are expressed in various organs, but their functions are not well understood. The purpose of this study was to investigate the expression profiles and functions of kisspeptin and KISS1R in the reproductive tissues of imprinting control region mice. To identify the expression pattern and location of kisspeptin and KISS1R in gonads, testes and ovarian tissues were examined by immunohistochemical or immunofluorescent staining. Kisspeptin and KISS1R were expressed primarily in Leydig cells and seminiferous tubules respectively. KISS1R was specifically localized in the acrosomal region of spermatids and mature spermatozoa. Kisspeptin, but not KISS1R, was expressed in the cumulus-oocyte complex and oviductal epithelium of ovarian and oviductal tissues. The sperm intracellular calcium concentrations significantly increased in response to treatment with kisspeptin 10 in Fluo-4-loaded sperm. The IVF rates decreased after treatment of sperm with the kisspeptin antagonist peptide 234. These results suggest that kisspeptin and KISS1R might be involved in the fertilization process in the female reproductive tract. In summary, this study indicates that kisspeptin and KISS1R are expressed in female and male gametes, respectively, and in mouse reproductive tissues. These data strongly suggest that the kisspeptin system could regulate mammalian fertilization and reproduction.