Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene.

Liver directed gene transfer with adenoviral vectors is being considered for the treatment of several metabolic diseases, including familial hypercholesterolaemia (FH). Gene replacement therapy of human low density lipoprotein (LDL) receptor gene into the murine model of FH transiently corrected the dyslipidaemia; however, humoral and cellular immune responses to LDL receptor developed--possibly contributing to the associated hepatitis and extinguishing of transgene expression. We evaluated an alternative strategy of ectopic expression in the liver of the very low density lipoprotein (VLDL) receptor, which is homologous to the LDL receptor but has a different pattern of expression. Infusion of recombinant adenoviruses containing the VLDL receptor gene corrected the dsylipidaemia in the FH mouse and circumvented immune responses to the transgene leading to a more prolonged metabolic correction.

Impact of FTO genotypes on BMI and weight in polycystic ovary syndrome: a systematic review and meta-analysis.

AIMS/HYPOTHESIS: FTO gene single nucleotide polymorphisms (SNPs) have been shown to be associated with obesity-related traits and type 2 diabetes. Several small studies have suggested a greater than expected effect of the FTO rs9939609 SNP on weight in polycystic ovary syndrome (PCOS). We therefore aimed to examine the impact of FTO genotype on BMI and weight in PCOS. METHODS: A systematic search of medical databases (PubMed, EMBASE and Cochrane CENTRAL) was conducted up to the end of April 2011. Seven studies describing eight distinct PCOS cohorts were retrieved; seven were genotyped for SNP rs9939609 and one for SNP rs1421085. The per allele effect on BMI and body weight increase was calculated and subjected to meta-analysis. RESULTS: A total of 2,548 women with PCOS were included in the study; 762 were TT homozygotes, 1,253 had an AT/CT genotype, and 533 were AA/CC homozygotes. Each additional copy of the effect allele (A/C) increased the BMI by a mean of 0.19 z score units (95% CI 0.13, 0.24; p = 2.26 × 10(-11)) and body weight by a mean of 0.20 z score units (95% CI 0.14, 0.26; p = 1.02 × 10(-10)). This translated into an approximately 3.3 kg/m(2) increase in BMI and an approximately 9.6 kg gain in body weight between TT and AA/CC homozygotes. The association between FTO genotypes and BMI was stronger in the cohorts with PCOS than in the general female populations from large genome-wide association studies. Deviation from an additive genetic model was observed in heavier populations. CONCLUSIONS/INTERPRETATION: The effect of FTO SNPs on obesity-related traits in PCOS seems to be more than two times greater than the effect found in large population-based studies. This suggests an interaction between FTO and the metabolic context or polygenic background of PCOS.

Directed vascular expression of the thromboxane A2 receptor results in intrauterine growth retardation.

Thromboxane (Tx) A2 is a platelet agonist, smooth muscle cell constrictor, and mitogen. Urinary Tx metabolite (Tx-M) excretion is increased in syndromes of platelet activation and early in both normal pregnancies and in pregnancy-induced hypertension. A further increment occurs in patients presenting with severe preeclampsia, in whom Tx-M correlates with other indices of disease severity. TxA2 exerts its effects through a membrane receptor (TP), of which two isoforms (alpha and beta; refs. 5,6) have been cloned. Overexpression of TP in the vasculature under the control of the pre-proendothelin-1 promoter results in a murine model of intrauterine growth retardation (IUGR), which is rescued by timed suppression of Tx synthesis with indomethacin. IUGR is commonly associated with maternal diabetes or cigarette smoking, both conditions associated with increased TxA2 biosynthesis.

Connective tissue and related disorders and preterm birth: clues to genes contributing to prematurity.

To identify candidate genes contributing to preterm birth, we examined the existing literature on the association between known disorders of connective tissue synthesis and metabolism and related diseases and prematurity. Our hypothesis was that abnormal matrix metabolism contributes to prematurity by increasing risk of preterm premature rupture of membranes (PPROM) and cervical incompetence. Based on this review, we identified gene mutations inherited by the fetus that could predispose to preterm birth as a result of PPROM. The responsible genes include COL5A1, COL5A2, COL3A1, COL1A1, COL1A2, TNXB, PLOD1, ADAMTS2, CRTAP, LEPRE1 and ZMPSTE24. Marfan syndrome, caused by FBN1 mutations, and polymorphisms in the COL1A1 and TGFB1 genes have been associated with cervical incompetence. We speculate that an analysis of sequence variation at the loci noted above will reveal polymorphisms that may contribute to susceptibility to PPROM and cervical incompetence in the general population.

Uptake of lipoproteins by in situ perfused rat ovaries: identification of binding sites for high density lipoproteins.

We have examined the uptake and distribution of 125I-labeled human high density lipoprotein, apolipoprotein E-free (hHDL3), 125I-rat high density lipoprotein (HDL), and human HDL (hHDL) reconstituted with [3H]cholesteryl linoleate after their in situ vascular perfusion to ovaries of gonadotropin-primed immature rats on days 6-9 post human chorionic gonadotropin (hCG)-injection. Some rats were treated with 4-aminopyrazolopyrimidine to reduce plasma lipoproteins and ovarian cholesteryl ester stores. Perfused ovaries were analyzed biochemically and autoradiographically, and progestin content of the ovarian effluent was quantified. Infusion of ovine luteinizing hormone and hHDL increased ovarian progestin secretion severalfold, indicating that the perfused ovary was functional. After perfusion with HDL reconstituted with [3H]cholesteryl linoleate, radioactive progestin appeared in the effluent; thus, sterol carried by exogenous HDL was converted to steroid. At 37 degrees C, uptake of 125I-hHDL3 was greatest after 15 min of perfusion with label. This was decreased by 80% when the perfusion was carried out at 4 degrees C and by 70-95% when excess unlabeled hHDL, but not human low density lipoprotein (hLDL), was included in the perfusate with 125I-hHDL. Aminopyrazolopyrimidine treatment enhanced 125I-hHDL uptake twofold. After perfusion for 15 min with 125I-hHDL3, radioactivity in the ovary was high for 3-30 min of HDL-free wash, then declined 75% by 30-60 min. With light and electron microscope autoradiography, 125I-hHDL3 was localized to corpora lutea, both along luteal cell surfaces and over their cytoplasm. The plasma membrane grains appeared to be associated with segments that lacked bristle coats. Perfusion with 125I-rat HDL produced a similar pattern of labeling. In ovaries perfused with 125I-BSA, silver grains were concentrated over macrophage-like cells but were sparse over luteal cells. We conclude that the in situ perfused rat ovary takes up 125I-hHDL3 by a temperature-dependent, lipoprotein-specific process, and that this lipoprotein is accumulated by luteal cells.

Uptake of gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein by cultured rat granulosa cells: cellular mechanisms involved in lipoprotein metabolism and their importance to steroidogenesis.

We used electron microscopy, acid hydrolase cytochemistry, and biochemistry to analyze the uptake and metabolism of colloidal gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein (LDL) by cultured rat granulosa cells. The initial interaction of gold-LDL conjugates with granulosa cells occurred at binding sites diffusely distributed over the plasma membrane. After incubation with ligand in the cold, 99.9% of the conjugates were at the cell surface but less than 4% lay over coated pits. Uptake was specific since it was decreased 93-95% by excess unconjugated LDL and heparin, but only 34-38% by excess unconjugated human high density lipoprotein. LDL uptake was related to granulosa cell differentiation; well-luteinized cells bound 2-3 times as much gold-LDL as did poorly luteinized cells. Ligand internalization was initiated by warming and involved coated pits, coated vesicles, pale multivesicular bodies (MVBs), dense MVBs, and lysosomes. A key event in this process was the translocation of gold-LDL conjugates from the cell periphery to the Golgi zone. This step was carried out by the pale MVB, a prelysosomal compartment that behaves like an endosome. Granulosa cells exposed to LDL labeled with gold and [3H]cholesteryl linoleate converted [3H]sterol to [3H]progestin in a time-dependent manner. This conversion was paralleled by increased gold-labeling of lysosomes and blocked by chloroquine, an inhibitor of lysosomal activity. In brief, granulosa cells deliver LDL to lysosomes by a receptor-mediated mechanism for the hydrolysis of cholesteryl esters. The resulting cholesterol is, in turn, transferred to other cellular compartments, where conversion to steroid occurs. These events comprise the pathway used by steroid-secreting cells to obtain the LDL-cholesterol vital for steroidogenesis.

PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1.

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML-/- cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1-modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.

Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis.

Congenital lipoid adrenal hyperplasia is an autosomal recessive disorder that is characterized by impaired synthesis of all adrenal and gonadal steroid hormones. In three unrelated individuals with this disorder, steroidogenic acute regulatory protein, which enhances the mitochondrial conversion of cholesterol into pregnenolone, was mutated and nonfunctional, providing genetic evidence that this protein is indispensable normal adrenal and gonadal steroidogenesis.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Human chorionic gonadotropin and 8-bromo cyclic adenosine monophosphate promote an acute increase in cytochrome P450scc and adrenodoxin messenger RNAs in cultured human granulosa cells by a cycloheximide-insensitive mechanism.

Treatment of human granulosa cells with human chorionic gonadotropin (hCG) or an analogue of its second messenger, cyclic AMP (cAMP), promotes a rapid accumulation of the messenger RNAs (mRNAs) for cytochrome P450 side-chain cleavage (scc) and adrenodoxin. A twofold increase in the cellular content of these mRNAs was observed within 4 h of exposure to 8-bromo-cAMP, and was maintained for up to 48 h. Inhibition of protein synthesis by cycloheximide did not prevent the hCG- or 8-bromo-cAMP-stimulated accumulation of either cytochrome P450scc or adrenodoxin mRNAs. We conclude that human granulosa cells respond rapidly to hCG and cAMP analogues with a coordinate increase in levels of the mRNAs encoding two key proteins of the steroidogenic machinery, and that this stimulation does not require synthesis of a protein intermediate.

Mechanisms subserving the trophic actions of insulin on ovarian cells. In vitro studies using swine granulosa cells.

Direct actions of insulin on gonadal tissues have been difficult to demonstrate in vivo. We have developed an in vitro system in which swine ovarian cells remain highly responsive to trophic actions of insulin. Purified porcine insulin significantly augmented the biosynthesis and secretion of progesterone by cultured granulosa cells. These stimulatory actions of insulin were dose- and time-dependent and saturable. Under serum-restricted conditions, insulin also significantly amplified the capacity of estradiol and 8-bromo cyclic AMP to stimulate progesterone production. Inhibitors of protein and RNA synthesis (cycloheximide, actinomycin D, and alpha-amanatin) inhibited insulin action. The stimulation of progesterone production by insulin was attributable to increased biosynthesis of pregnenolone, rather than diminished catabolism of progesterone to its principal metabolite, 20alpha-hydroxypregn-4-en-3-one. Insulin also enhanced progesterone production in the presence of a soluble sterol substrate, 5-cholesten-3beta,25-diol, which readily gains access to the mitochondrial cholesterol side-chain cleavage system. Moreover, exposure of granulosa cells to insulin produced a three- to sevenfold increase in mitochondrial content of cytochrome P-450 measured by difference spectroscopy, with a corresponding increase in mitochondrial cholesterol side-chain cleavage activity. The capacity of insulin to facilitate progesterone biosynthesis by ovarian cells was mimicked by the insulinlike somatomedin, multiplication stimulating activity, but not by epidermal growth factor, fibroblast growth factor, or porcine relaxin. Insulin's augmentation of progesterone production reflected a selective action on progestin biosynthesis, since insulin significantly suppressed estrogen biosynthesis by granulosa cells.Thus, our investigations indicate that insulin acts on ovarian cells selectively to stimulate pregnenolone (but not estrogen) biosynthesis. The actions of insulin are exerted by processes that require protein and RNA synthesis, and by mechanisms that augment mitochondrial cytochrome P-450 content and facilitate the utilization of cholesterol in the side-chain cleavage reaction. The striking mimicry of insulin effect by multiplication stimulating activity suggests that insulin action may be mediated through somatomedin receptors. Moreover, in view of the high concentrations of somatomedin in ovarian follicles in vivo, our in vitro observations suggest that specific trophic actions of insulin or insulinlike growth factors are likely to significantly regulate the differentiated function of the Graafian follicle in vivo.

Spontaneous puberty in 46,XX subjects with congenital lipoid adrenal hyperplasia. Ovarian steroidogenesis is spared to some extent despite inactivating mutations in the steroidogenic acute regulatory protein (StAR) gene.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. We report here the clinical, endocrinological, and molecular analyses of two unrelated Japanese kindreds of 46,XX subjects affected with lipoid CAH who manifested spontaneous puberty. Phenotypic female infants with 46,XX karyotypes were diagnosed with lipoid CAH as newborns based on a clinical history of failure to thrive, hyperpigmentation, hyponatremia, hyperkalemia, and low basal values of serum cortisol and urinary 17-hydroxycorticosteroid and 17-ketosteroid. These patients responded to treatment with glucocorticoid and 9alpha-fludrocortisone. Spontaneous thelarche occurred in association with increased serum estradiol levels at the age of 10 and 11 yr, respectively. Pubic hair developed at the age of 12 yr 11 mo in one subject and menarche was at the age of 12 yr in both cases. Both subjects reported periodic menstrual bleeding and subsequently developed polycystic ovaries. To investigate the molecular basis of the steroidogenic lesion in these patients, the StAR gene was characterized by PCR and direct DNA sequence analyses. DNA sequence analysis revealed that one patient is homozygous for the Gln 258 Stop mutation in exon 7 and that the other patient is a compound heterozygote with the Gln 258 Stop mutation and a single A deletion at codon 238 in the other allele causing a frame-shift, which renders the StAR protein nonfunctional. These findings demonstrate that ovarian steroidogenesis can be spared to some extent through puberty when the StAR gene product is inactive. This is in marked contrast to the early onset of severe defects in testicular and adrenocortical steroidogenesis which are characteristics of this disease.

A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor.

Fetal membranes usually rupture during the process of labor. Premature fetal membrane rupture occurs not infrequently and is associated with significant fetal and maternal morbidity. The mechanisms of normal and pathologic fetal membrane rupture are not well understood. We have examined structural and biochemical changes in the rat amnion as labor approaches in order to characterize this process in normal pregnancy. Here we report that before the onset of active labor the amnion epithelial cells undergo apoptotic cell death which encompasses degradation of 28S ribosomal subunit RNA and associated P proteins and fragmentation of nuclear DNA. Concurrent with these cellular changes, the amnion type I collagen matrix is degraded with the accumulation of three-quarter length type I collagen fragments in extraembryonic fluid, characteristic of the cleavage of fibrillar collagen by interstitial collagenase. Western blot and immunohistochemical analyses confirmed that interstitial collagenase protein appears in association with the loss of amnion type I collagen. We conclude that amnion epithelial cells undergo a process of programmed cell death associated with orchestrated extracellular matrix degradation which begins before the onset of active labor. Thus, fetal membrane rupture is likely to be the result of biochemical changes as well as physical forces.

Fine mapping of genetic susceptibility to polycystic ovary syndrome on chromosome 19p13.2 and tests for regulatory activity.

CONTEXT: Little is known about genes that contribute to polycystic ovary syndrome (PCOS). We previously found linkage and association of PCOS with the dinucleotide marker D19S884 in two independent sets of families; allele 8 of D19S884 confers increased risk. OBJECTIVE/DESIGN: The objectives of the study were: 1) use the transmission/disequilibrium test (TDT) to assess linkage and association between PCOS and D19S884 (and nearby markers) in a third set of families; and 2) test D19S884 and surrounding DNA sequence for in vitro regulatory activity in lymphoblastoid cell lines (LCLs) and granulosa cells. SETTING/SUBJECTS: We studied 98 new families with a PCOS proband, father, mother, and other available offspring. We analyzed data from these families separately and in combination with data obtained previously. INTERVENTIONS: Interventions were venipuncture. MAIN OUTCOME MEASURES: Measures were transmission frequencies and in vitro functional studies. RESULTS: The first result we found was that in the 98 new families, the TDT was significant for allele 8 of D19S884 (P = 0.043). In the total collection of 465 families, the TDT evidence is very strong (nominal P < 7 x 10(-5)). Results for all other genetic markers near D19S884 were nonsignificant after correction for multiple testing. The second result was that an approximately 800-bp fragment containing various alleles of D19S884 showed modest but reproducible promoter activity in LCLs. However, no allelic differences were detected. No activity of this fragment was detected in granulosa cells. CONCLUSIONS: This is the second independent confirmation of linkage and association of D19S884 with PCOS. We found in addition that some sequence in the region of D19S884 confers in vitro promoter activity in LCLs.

Enhanced translation: a novel mechanism of mdm2 oncogene overexpression identified in human tumor cells.

The cellular mdm2 gene, which has potential transforming activity that can be activated by overexpression, is amplified in a significant percentage of human sarcomas and in other mammalian tumors. Proteins encoded by the mdm2 gene can bind to, and inhibit the function of, the protein product of the p53 tumor suppressor gene. As reported here, we have identified human choriocarcinoma cell lines that express high levels of mdm2 proteins as well as the p53 protein. Several lines of evidence demonstrate that the p53 in these tumor cells has a wild-type nucleotide sequence, although the protein exhibits an extended half-life. Further, the more than 100-fold overexpression of mdm2 proteins in these cells cannot be explained by gene amplification, elevated RNA expression, or altered protein stability; rather our data indicate that elevated mdm2 protein levels in these choriocarcinoma cell lines result from enhanced translation. This mechanism has not previously been implicated in the regulation of mdm2 gene expression, and it represents a novel means by which the potential transforming activity of the mdm2 oncogene could be activated.

Steroidogenic acute regulatory protein (StAR) and the intramitochondrial translocation of cholesterol.

The steroidogenic acute regulatory (StAR) protein regulates the rate limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. Insight into the structure and function of StAR was attained through molecular genetic studies of congenital lipoid adrenal hyperplasia, a rare disease caused by mutations in the StAR gene. Subsequent functional analysis defined two major domains within the StAR protein, the N-terminal mitochondrial targeting sequence and the C-terminus, which promotes the translocation of cholesterol between the two mitochondrial membranes. Two models of StAR's mechanism of action, (1) stimulation of cholesterol desorption from the outer mitochondrial membrane and (2) an intermembrane shuttle hypothesis, are discussed with respect to the known biochemical and biophysical events associated with the process of steroidogenesis and the structure of StAR. StAR gene expression is regulated primarily at the transcriptional level, and the roles of transcription factors that govern basal and cAMP-dependent StAR expression including SF-1, C/EBP beta, Sp1 and GATA-4 are reviewed.

Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries.

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.

Effect of incubation of guinea-pig taenia coli in potassium-free media on arachidonate release and lipid metabolism.

We studied the effects of immersion of guinea-pig taenia coli strips in potassium-free media on arachidonate stores and other lipid fractions. Control studies obtained with the strips in Krebs solution showed that greater than 97% of arachidonate was found esterified in phospholipid with the following distribution: phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol. 30 min incubation of the strips with [3H]arachidonate complexed to albumin resulted in incorporation of this isotope into phospholipid and neutral lipid fractions, phosphatidylcholine greater than neutral lipid greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. 30 min incubations with 32PO4(2-)-resulted in an isotope incorporation into phospholipids, phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. After 'loading' with [3H]arachidonate and 32P, placing the strips in potassium-free media caused the following: there was an increased release of [3H]arachidonate from the tissue into the bathing solution. [3H]Arachidonate and 32P radioactivity in phosphatidylinositol fell without a change in phosphatidylinositol content. [3H]Arachidonate and 32P radioactivity in other phospholipid fractions was unchanged. Arachidonate specific activity fell and arachidonate content increased in the phosphatidylserine plus phosphatidylinositol fraction. [3]Arachidonate in neutral lipid did not change significantly. We conclude that exposure of taenia coli to potassium-free media activates turnover of phosphatidylinositol, which results in release of arachidonate.

Lysosomal phospholipase A activities of rat ovarian tissue.

1.1. Lysosome-enriched fractions were prepared by differential centrifugation of homogenates of luteinized rats ovaries. Acid phospholipase A activities were characterized with [U-14C]diacyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-[9,10-3H]- or [1-14C]oleoyl-sn-glycero-3-phosphocholine as substrates. Acid phospholipase A1 activity had properties similar to other hydrolases of lysosomal origin; subcellular distribution, latency and acidic pH optimum. Acid phospholipase A2 activity with similar characteristics was also tentatively identified. We were unable to exclude the possibility that the combined action of phospholipase A1 and lysophospholipase contributed to the release of acyl moieties from the 2-position of the synthetic substrates. 2. Lysophospholipase activity was present in the lysosome-enriched fractions. This activity had an alkaline pH optimum. 3. Phospholipase A1 and A2 activities solubilized from lysosome fractions by freeze-thawing were inhibited by Ca2+ and slightly activated by EDTA. A Ca2+- stimulated phospholipase A2 activity, with an alkaline pH optimum, remained in the particulate residue of freeze-thawed lysosome preparations. This activity is believed to represent mitochondrial contamination. 4. Activities of acid phospholipase A, as well as other acid hydrolases, increased approx. 1.5-fold between 1 and 4 days following induction of luteinizatin, suggesting a hormonal influence on lysosomal enzyme activities.