The morphology of neoptile feathers: Ancestral state reconstruction and its phylogenetic implications

Avian neoptile feathers are defined as the first feather generation, which covers the chick after hatching, and usually described as simple structures consisting of numerous downy barbs which are radially symmetrically arranged and come together in a short calamus. In contrast, in some birds (e.g., Anas platyrhynchos, Dromaius novaehollandiae) the neoptile feathers have a prominent rhachis, and therefore display clear bilateral symmetry. Because the symmetrical variety found in neoptile feathers is poorly understood, their morphology was studied in a more comprehensive and phylogenetic approach. Neoptile body feathers from over 22 bird species were investigated using light microscopy, SEM, and MicroCT. Characters such as an anterior–posterior axis, a central rhachis, medullary cells, and structure of the calamus wall were defined and mapped onto recent phylogenetic hypotheses for extant birds. It can be shown that bilaterally symmetric neoptile feathers (with a solid calamus wall) were already present in the stem lineage of crown‐group birds (Neornithes). In contrast, simple radially symmetric neoptile feathers (with a fragile calamus wall) are an apomorphic character complex for the clade Neoaves. The simple morphology of this feather type may be the result of a reduced period of development during embryogenesis. To date, embryogenesis of neoptile feathers from only a few bird species was used as a model to reconstruct feather evolution. Because this study shows that the morphology of neoptile feathers is more diverse and even shows a clear phylogenetic signal, it is necessary to expand the spectrum of “model organisms” to species with bilaterally symmetric neoptile feathers and compare differences in the frequency of feather development from a phylogenetic point of view. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TTPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive 86Rb uptake and amiloride-sensitive 24Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na+/H+ and Na+/K+ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na+/H+ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na+/H+ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na+ pump, Na+/H+ exchange) without eliciting corresponding regulatory mechanisms (Na+ stat, pH stat).     

Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of cAMP levels, DNA synthesis and cell division

Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca²⁺-free sea water (a treatment that bypasses the cortical reaction and the Ca²⁺ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.     

Effects of a phorbol ester on mouse eggs: dissociation of sperm receptor activity from acrosome reaction-inducing activity of the mouse zona pellucida protein, ZP3

Biologically active phorbol esters or a diacylglycerol induce mouse eggs to modify the zona pellucida such that sperm receptor activity is retained but the ability of bound sperm to undergo a complete acrosome reaction is lost (Y. Endo, R.M. Schultz, and G.S. Kopf. 1987. Dev. Biol. 119, 199-209). We now show that purified ZP3 from 12-O-tetradecanoyl phorbol-13-acetate (TPA)-treated eggs possesses full sperm receptor activity but has lost its ability to induce a complete acrosome reaction. The modification of the acrosome reaction-inducing activity of ZP3 from these TPA-treated eggs differs from ZP3 isolated from two-cell embryos, which cannot initiate the acrosome reaction. These results demonstrate the dissociation of the two biological activities of ZP3 and may provide a system to assess the components of ZP3 involved in the acrosome reaction.     

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs.

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.     

Size transformations of intermediate and low density lipoproteins induced by unesterified fatty acids

Plasma from individual human subjects is known to contain multiple discrete subpopulations of low (LDL) and intermediate (IDL) density lipoproteins that differ in particle size and density. The metabolic origins of these subpopulations are unknown. Transformation of IDL and larger LDL to smaller, denser LDL particles had been postulated to occur as a result of the combined effects of triglyceride hydrolysis and lipid transfer. However, the presence of multiple small LDL subspecies has been described in patients lacking cholesteryl ester transfer protein. We have characterized an alternative pathway in which size decrements in IDL or LDL are produced in the presence of unesterified fatty acids and a source of apolipoprotein (apo) A-I. Incubation of IDL or LDL subfractions with palmitic acid and either high density lipoproteins (HDL), apoHDL, or purified apoA-I gives rise to apoA-I, apoB-containing complexes that can dissociate into two particles, an apoB-containing lipoprotein with particle diameter 10-30 A smaller than the starting material, and a still smaller species (apparent peak particle diameter 140-190 A) containing lipid and apoA-I but no apoB. The newly formed IDL or LDL are depleted in phospholipid and free cholesterol with no change in apoB-100 as assessed by SDS gel electrophoresis. We hypothesize that this reaction may contribute to the formation of discrete IDL and LDL subpopulations of varying size during the course of hydrolysis of triglyceride-rich lipoproteins in plasma.     

PKC对小鼠受精卵发育的调控作用

为研究 TPA及 PKC的反义寡核苷酸对 1 -细胞期鼠受精卵发育的影响 ,采用免疫细胞化学法标记 PKC(α及 β亚型 ) ,并用激光扫描共聚焦显微镜测定卵内 PKC荧光强度 ;同时利用显微注射法注射 PKC的反义寡核苷酸 ,观察其对受精卵分裂的影响 . 1 0 0 μg/ L TPA对 1 -细胞期受精卵的发育具有完全抑制作用 .TPA处理 1 2 h后 ,对照组受精卵停留在 1 -细胞期 ,而未经 TPA处理的1 -细胞期卵可以分裂到 2 -细胞期 .共焦激光显示实验组与对照组相比 ,PKC(α、β亚型 )荧光强度均有下降 (P<0 .0 1 ) .显微注射 PKC antisenseα及 antisenseβ的受精卵 ,分别只有 1 4 .2 %和 3.33%的卵可以发育到 2 -细胞期 .与对照组 (注射 M2培养液 )差异显著 (P<0 .0 1 ) .结果表明 ,(1 ) TPA长期处理 1 -细胞期受精卵 ,抑制 1 -细胞期卵分裂到 2 -细胞期 ;(2 ) PKC的反义寡核苷酸 (α及β亚型 )可以抑制小鼠 1 -细胞期卵的发育     

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive ⁸⁶Rb uptake and amiloride-sensitive ²⁴Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na⁺/H⁺ and Na⁺/K⁺ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na⁺/H⁺ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na⁺/H⁺ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na⁺ pump, Na⁺/H⁺ exchange) without eliciting corresponding regulatory mechanisms (Na⁺ stat, pH stat).     

小鼠受精卵早期发育过程中PKC对cdc2和cdc25C活性的影响

为研究小鼠受精卵细胞早期发育过程中PKC对cdc2和cdc2 5C活性的影响 ,采用免疫印迹和电泳迁移率差异分析的方法 ,观察PKC的激活剂TPA及其抑制剂星形孢子素对小鼠受精卵一细胞期cdc2和cdc2 5C活性的影响 .10nmol L的TPA作用 10min后 ,小鼠受精卵一细胞期卵裂率明显大于对照组 (P <0 0 5 ) ,而星形孢子素作用后卵裂率显著下降 (P <0 0 1) .TPA处理后 ,受精卵中呈去磷酸化状态的活性cdc2明显增加 ,没有活性呈磷酸化状态的cdc2 5C明显减少 ;而星形孢子素处理的受精卵中没有活性的cdc2明显增加 ,有活性的cdc2 5C明显减少 .结果表明 ,TPA短时间作用可以促进小鼠一细胞期受精卵分裂 ,星形孢子素抑制受精卵的分裂 ;TPA可以促进cdc2的去磷酸化以及cdc2 5C的磷酸化 ,从而促进G2 M转换 ,星形孢子素则抑制cdc2和cdc2 5C的活性 ,阻止受精卵由G2 期进入M期     

Tumor promoters induce changes in the chick embryo fibroblast cytoskeleton

We have examined the effect of the tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), on the actin-containing elements of the cytoskeleton of chick embryo fibroblasts (CEF). TPA at concentrations as low as 7.3 times 10-10M indices a reversible change in the cytoskeleton as visualized by indirect immunofluorescence using anti-actin antibodies. Cells incubated with TPA lose the ordered actin-containing structures found in normal cells and resemble Rous sarcoma virus-transformed cells in that the immunofluorescent actin pattern is diffuse. The TPA effects are both dose-and time-dependent. Analogs of TPA which are inactive as tumor promoters do not induce cytoskeletal changes at the concentrations tested, while a second tumor promoter, PDD, is also able to cause alterations in actin-containing structures. The action of TPA requires de novo synthesis of both RNA and protein. The direct cytoskeletal changes are neither plasmin-dependent nor subject to inhibition by incubating the cells with high levels of protease inhibitors during the exposure to TPA. However, plasminogen does increase the sensitivity of cells to TPA.     

ELECTRON MICROSCOPIC STUDIES OF INDUCED CARTILAGE DEVELOPMENT AND CALCIFICATION

Cultured, human, amniotic cells (FL strain) injected into the thigh muscles of cortisone-conditioned mice proliferated to form discrete colonies which, over a period of 5 days, became invested by numerous fibroblasts. Cartilage cells and matrix appeared within the fibroblastic zones during the succeeding 2–4 days. Cartilage matrix calcified within 12 days following FL-cell injection. Cartilage cells closely resembled fibroblasts from which they appeared to be derived, and were readily distinguished from FL cells by their prominent ergastoplasm and Golgi complexes. Cartilage matrix was composed of a distinctive feltwork of randomly arranged, collagen fibrils (~600 A axial period and ~250 A width) from which small electron-opaque, leaflike matrix particles extended. Matrix calcification occurred with the deposition of radially arranged needle-like structures resembling hydroxyapatite. Dense centers were often identified within these clusters. Examination of heavily calcified areas revealed confluent masses of apatite-like material. In general, the fine structure of induced cartilage formation and calcification resembled that of cartilage development and calcification as previously described in the normal epiphysis.     

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.     

Epithelial heterogeneity in the murine thymus: fucose-specific lectins bind medullary epithelial cells

By means of histochemical techniques, two lectins with nominal specificity for L-fucose, Tetragonolobus purpureas agglutinin (TPA), and Ulex europeus agglutinin (UEA) were found to specifically label the medullary area of murine thymuses. Although the binding of both lectins was restricted to the medullary area of the thymus, each staining pattern was unique. Cells binding UEA formed a reticular network throughout the medulla, whereas cells binding TPA occurred as single cells or small clumps of cells and resembled Hassall's corpuscles. The cells binding either lectin were identified as epithelial on the basis of ultrastructural features (tonofilaments, desmosomes, and keratohyalin bodies) and resembled Ia+ medullary epithelial cells described previously. An age-related decline in UEA binding was observed, whereas labeling with TPA remained unchanged. On the basis of the labeling patterns obtained with UEA and TPA and the reported specificities of these two lectins, it is suggested that the majority of the fucose detected is associated with type 1 carbohydrate chains.     

Effects of phorbol esters and a diacylglycerol on the mouse sperm acrosome reaction induced by the zona pellucida

Capacitated mouse sperm undergo the spontaneous acrosome reaction in suspension and the zona-induced acrosome reaction when bound to isolated, intact zonae pellucidae. The zona-induced acrosome reaction in the mouse resembles, in part, ligand-receptor-mediated exocytotic processes that occur in some somatic cells. Since such processes have been shown to be mediated in part by protein kinase C-catalyzed protein phosphorylation, the effects of phorbol esters, which are potent activators of this kinase, on both the spontaneous and the zona-induced acrosome reaction were examined. At concentrations up to 10 microM, 12-tetradecanoyl phorbol-13-acetate (TPA) had no effect on the time course of the spontaneous acrosome reaction as scored by the chlortetracycline (CTC) fluorescence assay. Capacitated, acrosome-intact sperm display Pattern B in the CTC assay with fluorescence on the anterior head; fully acrosome-reacted sperm display Pattern AR with no fluorescence on the head. The time course of the loss of Pattern B in the zona-induced acrosome reaction was markedly accelerated by 65 nM TPA as compared to controls, whereas the appearance of Pattern AR was retarded. The appearance of Pattern S, which is characterized by punctate fluorescence on the head and which marks an intermediate state between Pattern B and Pattern AR in the controls, was accelerated by 65 nM TPA to the same extent as the loss of Pattern B at early times post-binding to zonae. The disappearance of Pattern S at later times post-binding to zonae was retarded by 65 nM TPA to the same extent as the appearance of Pattern AR. The transitions between the fluorescence patterns, designated the B-to-S and the S-to-AR transitions, therefore define two stages of the zona-induced acrosome reaction, which are affected in opposite directions by TPA. The effects of 65 nM TPA are mimicked by 60 nM 4-beta-phorbol-12,13-didecanoate (4-beta-PDD) while the 4-alpha isomer is without effect. Such stereospecificity is similar to that reported for the activation of protein kinase C. The diacylglycerol, 1-oleyl-2-acetylglycerol, which is also known to activate protein kinase C, mimicked the effects of TPA and 4-beta-PDD on the time courses of the B-to-S and S-to-AR transitions. These results suggest that protein kinase C may play an intermediary role in the zona-induced mouse sperm acrosome reaction.     

Tumor promoters and diacylglycerol activate the Na+/H+ antiporter of sea urchin eggs

Various tumor promoters (TPA, lyngbyatoxin and aplysiatoxin) and diacylglycerol induced cytoplasmic alkalinization of sea urchin eggs independently of intracellular Ca2+ release. This response stimulated protein synthesis and was blocked by amiloride or a lack of extracellular Na+, procedures which inhibit the Na+/H+ antiporter. These results suggest that the antiporter which is responsible for cytoplasmic alkalinization in sea urchin eggs is activated directly or indirectly by protein kinase C in a Ca2+-independent manner.     

Regulation of c-Myc and Max in megakaryocytic and monocytic-macrophagic differentiation of K562 cells induced by protein kinase C modifiers: c-Myc is down-regulated but does not inhibit differentiation.

We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.     

Studies on the metabolism of apolipoprotein B in hypertriglyceridemic subjects using simultaneous administration of tritiated leucine and radioiodinated very low density lipoprotein

To study the metabolic pathways of apolipoprotein B (apoB), a series of studies were carried out in which both radioiodinated very low density lipoproteins (VLDL) and tritiated leucine were simultaneously injected into three hypertriglyceridemic subjects. The appearance and disappearance of tritium activity in VLDL apoB, intermediate density lipoprotein (IDL) apoB, and low density lipoprotein (LDL) apoB were followed as was the disappearance of iodine activity from VLDL and the appearance and disappearance of iodine activity in IDL apoB and LDL apoB. It was found that a delipidation chain could describe the kinetics of both endogenously and exogenously labeled VLDL. A slow component of VLDL was necessary to fit the VLDL 131I-labeled apoB data and was consistent with the observed VLDL [3H]apoB kinetics. In addition, the estimated rate of conversion of VLDL apoB to LDL exceeded that which appeared to pass through the measured IDL pools, suggesting that a fraction of the IDL was not directly observed. It was also found that a higher percentage of VLDL 131I-labeled apoB was converted to LDL apoB than was VLDL [3H]apoB. To evaluate possible causes of this apparent anomaly, simultaneous examination of all kinetic data was performed. This exercise resulted in the resolution of removal pathways from multiple compartments in the VLDL delipidation chain and the conversion of slowly metabolized VLDL to IDL and LDL. The wide spectrum of this loss pathway indicates that previous estimates of VLDL apoB production rate using the radioiodinated methodology probably represent lower bounds for the true physiologic variable. It is important to note that these direct losses were apparent only when the combination of endogenous and exogenous labeling was used.     

Effects of 1,2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.     

Control of bovine placental progestin synthesis: calcium dependent steroidogenesis is modulated at the site of the cholesterol side chain cleavage enzyme

We have previously reported that progesterone synthesis in the bovine placenta is regulated by Ca2+ dependent and cyclic nucleotide independent mechanism. In studies conducted to further define the role of Ca2+ in the synthesis of progestins in bovine placental tissue, it was found that both protein kinase C (PKC), as determined by phosphorylation, and cytochrome P-450 side chain cleavage, as determined by Western blot analysis, were detectable in the steroidogenetically active portion of the placentome. To determine the site of action of PKC, fetal cotyledon cells were incubated in media containing 25-hydroxycholesterol in the absence or or presence of 10 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It was found that TPA significantly (P less than 0.05) increased the conversion of the exogenous cholesterol analog to progesterone. To determine if the TPA could act synergistically with calcium activators, fetal cotyledon cells were incubated with either methyl isobutyl xanthine (MIX), an activator of intracellular calcium, or the calcium ionophore, A23187, which increases extracellular calcium influx, or both of these agents, in the presence or absence of TPA. It was found that TPA synergistically increased the conversion of sterol to progestins induced by submaximal concentrations of either MIX or A23187. In the presence of both compounds, TPA induced an even more dramatic increase in progestin synthesis. In experiments in which cyanoketone, an agent that inhibits the conversion of pregnenolone to progesterone, was added, TPA addition resulted in increased pregnenolone production, indicating that side chain cleavage of cholesterol is the site of action. The data, therefore, suggest that: (a) Ca2+ affects mechanisms regulating placental steroidogenesis; (2) one locus of Ca2+ is the cholesterol side chain cleavage reaction; and (3) PKC found in this tissue has a role in the Ca activated progestin production.