In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca 2؉ concentration ( (5), and nodulation (6) and are central to hormonal physiology (7-9). Changes in [Ca 2ϩ ] i influence ion channel gating (1, 9, 10), light-mediated gene expression (11), cell differentiation, elongation, and tip growth (3).In stomatal guard cells, one of the best-characterized plant cell models, increasing [Ca 2ϩ ] i is known to inactivate inwardrectifying K ϩ channels and to activate Cl Ϫ channels, biasing the plasma membrane for solute efflux, which drives stomatal closure (9). Changes in [Ca 2ϩ ] i have been associated with stimuli that lead to stomatal closure, notably abscisic acid (ABA) and CO 2 (9, 12). These changes in [Ca 2ϩ ] i depend on Ca 2ϩ release from intracellular stores (13-16) and on Ca 2ϩ entry across the plasma membrane (17, 18). Nonetheless, direct evidence for channels that could mediate Ca 2ϩ influx has been lacking. Indeed, little evidence has come forth for Ca 2ϩ channels at the plasma membrane of higher-plant cells until recently (19)(20)(21)(22)(23).One clue to a major pathway for Ca 2ϩ entry into guard cells has come from measurements of [Ca 2ϩ ] i and its elevation by ABA under voltage clamp (17). These studies indicated a voltage dependence to the [Ca 2ϩ ] i rise, suggesting that ABA stimulated a Ca 2ϩ channel, but that its activity also required membrane hyperpolarization. We have recorded single-channel currents from Vicia guard cell protoplasts under conditions that eliminate the background of current through K ϩ and Cl Ϫ channels. The results reported here demonstrate the presence of Ca 2ϩ channels at the plasma membrane that open on membrane hyperpolarization and are activated by ABA. Materials and MethodsPlant Material. Epidermal strips of Vicia faba L., cv. Bunyard Exhibition, were obtained and protoplasts were prepared as described (24,25). All operations were carried out on a Zeiss Axiovert microscope with 40ϫ LWD Nomarski DIC optics at 20-22°C. Solution was added (Ӎ20 chamber vol͞min) by gravity feed and removed by aspiration.Electrophysiology. Pipettes were pulled with a Narishige (Tokyo) PP-81 puller modified for three-stage pulls (input resistances, 30-50 M⍀) to reduce the number of channels under a patch. Pipettes were coated with Sigmacote (Sigma) to reduce capacitance. Connections to amplifier and bath were by a 0.1 M KCl͞Ag-AgCl liquid junctions, and junction potentials were taken into account (26). Single-channel currents were recorded with an Axopatch 200B patch amplifier (Axon Instruments, Foster City, CA) after filtering at 5 kHz and sampled at 44 kHz for analysis. Data were filtered at 1 kHz (Kemo, Beckenham, U.K.) offline and analyzed with N-PRO (Wye Science, Wye, Kent, U.K.), P/V CLAMP V. 6 (CED, Cambridge, U.K.) software. Channel amplitudes were calculated from point-amplitude histograms estimated from open events Ն5-ms duration (Fig. 1) beyond closed levels determined from periods of no channel activity (27). Channel numbers w...

Light and electron microscopic examination of tissues fixed in situ by perfusion of the gravid horn of the uteri of mares between 36 and 38 days of gestation revealed that the equine endometrial cups are composed of trophoblast cells which originate from the discrete annulate portion of the foetal membranes known as the chorionic girdle. This structure consists of closely opposed villous projections of elongated trophoblast cells and i t becomes firmly attached to the endometrium around the thirty-sixth day of pregnancy. The specialized girdle cells invade and phagocytose the endometrial epithelium and then migrate through the basal lamina into the endometrial stroma where they develop into endometrial cup cells.Measurement of pregnant mares serum gonadotrophin (PMSG) concentrations in foetal and maternal tissues of horses led Catchpole and Lyons ('34) to postulate that PMSG is secreted by the foetal chorion and stored in the endometrium. Subsequent experiments have conclusively dernonstrated, however, that PMSG is manufactured by discrete endometrial outgrowths present in the pregnant horn of the uterus of mares between the 38th and 150th days of gestation, the endometrial cups (Cole and Goss, '43; Clegg, Boda and Cole, '54).It has been widely considered in the past that the endometrial cups are entirely maternal in origin (Amoroso, '55; Gonzalez-Angullo and Hernandez-Jouregui, '71), although this hypothesis has been brought into question by the finding that foetal genotype profoundly influences PMSG levels in the maternal blood (Bielanski, Ewy and Pigoniowa, '55; Clegg, Cole, Howard and Pigon, '62; Allen, '69). Moreover, we have recently demonstrated that the only cells which possess the capacity to synthesize PMSG in vitro are those of the specialized area of the allantochorion known as the chorionic girdle (Allen and Moor, '72).The purpose of this paper is to describe the origin and histogenesis of endometrial cups at the fine-structure level. MATERIALS AND METHODSIn order to maintain the delicate anatomical relationship between foetal and maternal tissues, our experiments were carried out upon material fixed in situ by perfusion of the uterus.Laparotomies were performed upon three Welsh Pony mares at 36, 37 and 38 days of gestation respectively (day of ovulation = day 0). The anaesthetized animals were positioned on their backs and a large flap of the ventral wall of the abdomen was reflected to provide direct access to the uterus and ovaries. The uterine artery to the pregnant horn was cannulated with the minimum possible handling of the uterus and perfusion was commenced. The initial pre-wash fluid of approximately 5 ml of oxygenated bicarbonate-buffered Krebs-Ringer (PH 7.4) was followed immediately by 1000 ml of 5% s-collidine buffered glutaraldehyde (pH 7.2). At first the fixative was allowed to flow unimpeded but after about 200 rnl had been perfused, the flow rate was reduced so that the remainder took about fifteen minutes to pass through the organ.

Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3-. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3-, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.

The ultrastructure of the principal cells and intraepithelial leucocytes in the initial segment of the rat caput epididymidis was examined with the electron microscope. Specializations of the principal cells associated with absorption include numerous endocytic invaginations of the cell surface, numerous coated vesicles and multivesicular bodies in the apical cytoplasm. It was demonstrated that particulate tracers are taken into the cells and sequestered in secondary lysosomes and multivesicular bodies. Morphological features consistent with secretory activity are also found in the principal cells and include numerous cisternae of rough endoplasmic reticulum with a flocculent grey content and an extremely well-developed Golgi apparatus. The speculation that the principal cells are actively secretory despite the absence of secretory granules formed in the Golgi and of a visible mechanism for release of the product at the cell surface is discussed.The "halo cells" in the epididymal epithelium were also examined and it is shown that many of these cells are not typical migratory lymphocytes, Chief among the differences are their granule-containing multivesicular bodies and more abundant endoplasmic reticulum. Nonetheless, it is conceivable that the halo cells are lymphocytes and that the conditions they encounter as they leave the circulation and enter the epididymal epithelium may stimulate morphological changes. The possible immunological significance of these observations is discussed.There have been several descriptions of the ultrastructure of the epididymal epithelium in recent years (Nicander, '65; Friend and Farquhar, '67; Flickinger, '69; Hamilton, '72a,b). The improved preservation and higher resolution achieved in these studies have corrected a number of persistent misconceptions of early light microscopists who worked on the epididymis. Thus, irregular surface protrusions previously interpreted as a manifestation of apocrine secretion have been shown to be artifacts of fixation and certain granules formerly regarded as secretory products have been shown to be lysosomes. The structural differentiation of the cytoplasm has proven to be far more elaborate than previously supposed and it is quite evident that this eoithelium is by no means the metabolically inactive lining of an organ for sperm storage. The functional corre-ANAT. REC., 175: 169-202.lates of the cytological features are largely unknown, and, although considerable evidence is available on the morphology of the absorptive process in principal cells (Friend and Farquhar, '67; Nicander, '65; Burgos, '64), the morphology of secretion of glycerophosphorylcholine (Dawson, Mann and White, '57; Dawson and Rowlands, '59), carnitine (Marquis and Fritz, '65), sialic acid (Rajalakshmi and Prasad, '69) or steroids (Hamilton et al., '69; Hamilton, '72b) has not been elucidated.In the present paper we report the mor-