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Biomedical subjects

D A Freeman

Publications and source records attributed to D A Freeman.

At least 73 records · Page 4Linked to original sources
PubMed1992

Accumulation and mobilization of triglycerides and cholesteryl esters in Leydig tumor cells.

Incubating MA-10 Leydig tumor cells with sodium oleate led to the accumulation of triglyceride within the cells. Triglycerides were deposited in a time- and dose-dependent fashion. Cellular triglyceride promoted storage of cholesteryl ester. As much cholesteryl ester was stored in oleate-treated cells as in cells treated with saturating concentrations of low density lipoprotein. Addition of both oleate and low density lipoprotein resulted in additive accumulation of cholesteryl esters. Cholesteryl esters in cells loaded with triglyceride by oleate treatment were mobilized in response to dibutyryl-cAMP to an extent similar to that in cells containing low triglyceride concentrations. Dibutyryl-cAMP stimulated cholesteryl ester mobilization under all conditions, and stimulated triglyceride mobilization when adequate fatty acid acceptors were available. The results indicate that while triglyceride accumulation in MA-10 cells promoted cholesteryl ester deposition, it did not impair cAMP-dependent cholesteryl ester hydrolysis or steroid hormone production.

Algestone↗
PubMed1991

Differential reproductive response to short photoperiod in deer mice: role of melatonin.

Inhibitory photoperiod differentially effects reproduction in deer mice (Peromyscus maniculatus nebrascensis). Pituitary-testicular function is arrested in about one-third of short-day exposed males (reproductively responsive mice), while an equal number remain fertile (reproductively nonresponsive mice). Both phenotypes are found in natural populations and their disparate reproductive responses have a genetic basis. To assess whether this difference is attributable to a prepineal/pineal or post-pineal mechanism, we compared spermatogenic responses of known and unknown phenotype to exogenous melatonin. Melatonin significantly reduced mean sperm number in long-day housed mice of unknown phenotype. But, individual responses ranged from azoospermia to normal spermatogenesis, and this range was not significantly different from that previously recorded for short-day exposed mice. Reproductively nonresponsive males were unaffected by melatonin administration when housed under long or short daylength. In contrast, melatonin significantly suppressed sperm production in reproductively responsive males housed under long photoperiod, but had no additional suppressive effect in short-day housed mice with regressed testes. These data demonstrate that melatonin is only effective in eliciting testicular regression in reproductively responsive males. Taken together, these results suggest that differential testicular response to photoperiod are caused by a post-pineal mechanism.

Animals↗
PubMed1991

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue.

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.

Journal Article↗
PubMed1991

Time of embryo transport through the mare oviduct.

The objectives of this study were 1) to determine the time of embryo transport through the mare oviduct, 2) to determine whether equine embryos increase in diameter prior to the time of oviductal transport, and 3) to assess the stage of equine embryonic development at the time of oviductal transport. The time of oviductal transport (interval from ovulation to uterine entry) was estimated by collecting embryos from the mare oviduct or uterus at 2-hour intervals from 120 to 168 h postovulation. The time of oviductal transport was 130 to 142 h, since 9 9 embryos were located in the oviduct from 120 to 128 h; 7 14 embryos were in the oviduct and 7 14 embryos were in the uterus from 130 to 142 h; and 13 14 embryos were in the uterus from 144 to 168 h postovulation. Embryos collected during the period of oviductal transport (130 h to 142 h) were not significantly larger (P>0.1) in diameter than embryos collected prior to the period of oviductal transport (162.5+/-3.7 vs 156.7+/-3.1 mum, respectively). During the period of oviductal transport, embryos collected from the uterus were not significantly larger (P>0.1) in diameter than embryos collected from the oviduct (160.7+/-3.2 vs 164.3+/-7.0 mum, respectively). During this same period 12 14 embryos were compact morulae, and 2 14 embryos were blastocysts.

Journal Article↗
PubMed1991

Prostaglandin E2 secretion by oviductal transport-stage equine embryos.

This study was conducted to identify embryonic products whose secretion was temporally associated with the oviductal transport period of the mare. Chemicals secreted by oviductal-transport-stage equine embryos were identified by incubating Day 6 or Day 7 early uterine embryos with 35S-methionine/cysteine, 3H-progesterone, or 3H-arachidonic acid for 24 h, and subsequently identifying radioactively labeled proteins (SDS-PAGE; n = 3 embryos), steroids (HPLC; n = 3 embryos), or prostaglandins (HPLC; n = 3 embryos) in the culture medium. Early uterine embryos secreted 116.1 +/- 45.5 pg of prostaglandin (PG) E2/embryo, 1.0 +/- 0.2 pg of 17 alpha-hydroxy progesterone/embryo, 4.8 +/- 0.6 pg of androstenedione/embryo, and 11.5 +/- 4.5 pg of PGF2 alpha/embryo. They did not secrete detectable quantities of protein, testosterone, or estradiol-17 beta. A second experiment was conducted to measure temporal changes in embryonic PGE2 secretion during the oviductal and early uterine period. Day 3, Day 4, Day 5, and Day 6 embryos (n = 8 embryos/day) were incubated with 3H-arachidonic acid for 24 h, and the concentration of 3H-PGE2 in the culture medium was subsequently measured by HPLC. Embryos did not secrete detectable amounts of PGE2 prior to the expected time of oviductal transport (Day 3 and Day 4). They secreted 5.7 +/- 1.0 pg of PGE2/embryo immediately before and during the expected time of oviductal transport (Day 5), and they secreted significantly of PGE2/embryo immediately before and during the expected time of oviductal transport (Day 5), and they secreted significantly (p less than 0.01) higher amounts (42.0 +/- 11.5 pg) of PGE2/embryo immediately after uterine entry (Day 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Prostaglandin E2 hastens oviductal transport of equine embryos.

The hypothesis that treatment of pregnant mares with prostaglandin E2 (PGE2) hastens the oviductal transport of equine embryos was tested by treating bred mares with PGE2 on Day 3 after ovulation and subsequently measuring the rate of hastened oviductal transport (estimated by the uterine embryo recovery rate on Day 4 after ovulation). In a preliminary, noncontrolled experiment, oviductal transport was apparently not hastened after intramuscular, intrauterine, or intraperitoneal PGE2 administration to bred mares (0/6, 0/3, and 0/3 mares, respectively). Oviductal transport appeared to be hastened in 1/13 mares after a single intraoviductal administration of PGE2, and in 2/2 mares after continuous intraoviductal administration of PGE2. In a subsequent, controlled experiment, treatment with a continuous intraoviductal infusion of PGE2 hastened oviductal transport in significantly more (p less than 0.01) mares versus a continuous intraoviductal infusion of vehicle or no treatment (6/11 vs. 0/11 or 0/11 mares, respectively). Unfertilized oocytes and oviductal masses were also recovered from mare uteri after continuous intraoviductal PGE2 administration, but were not recovered after vehicle administration or no treatment. These results support the hypothesis that PGE2 treatment hastens the oviductal transport of equine embryos, and suggest a role for embryonic PGE2 in the initiation of selective oviductal transport in the mare.

Animals↗
PubMed1991

Steroid hormone-producing tumors of the adrenal, ovary, and testes.

Steroid hormone-synthesizing tumors are rare and may be difficult to diagnose and treat. This article focuses on adrenal tumors as models for the even less common tumors of the ovary and testes, and emphasizes useful diagnostic procedures and pitfalls as well as treatment options. Ovarian and testicular steroidogenic tumors are categorized and discussed in terms of the generalizations that can be derived from adrenal neoplasia.

Adrenal Gland Neoplasms↗
PubMed1991

Cellular internalization, transport, and esterification of iodine-125-NP59 by MA-10 Leydig tumor cells.

The present studies were directed toward understanding the cellular processing of the cholesterol analogue, NP59. NP59 readily entered MA-10 Leydig tumor cells. The cholesterol analogue entered the cells by binding to the plasma membrane and becoming internalized along with plasma membrane cholesterol. Internalized NP59 was readily esterified to NP59 ester. Transport of NP59 within the cell was indistinguishable from transport of cholesterol. Cholesterol and NP59 transport were under the control of cAMP, however, only cholesterol entered the mitochondria and was converted into progesterone. Thus, internalized NP59 could not be removed from the cell by conversion into steroid hormones. Esterified NP59 was metabolically inert and could not be converted back to free NP59 and free fatty acid. Since NP59 was not a substrate for the cholesteryl ester hydrolase, it became trapped in the cell as NP59 ester.

Adosterol↗
PubMed1990

Cholesterol movement between the plasma membrane and the cholesteryl ester droplets of cultured Leydig tumour cells.

The present studies characterize the turnover of plasma membrane cholesterol in MA-10 Leydig tumour cells. Plasma membrane cholesterol of MA-10 cells was slowly internalized and converted into cholesteryl ester. Low-density lipoprotein (LDL) stimulated, in a dose- and time-dependent fashion, plasma membrane cholesterol conversion into intracellular esters. Stimulation of membrane internalization was not simply the consequence of accelerated uptake of membrane with LDL, since binding and internalization of epidermal growth factor and transferrin had no effect on turnover of plasma membrane cholesterol. The protein of LDL is unimportant as well, since delipidated LDL had no effect on membrane turnover. The action of LDL on cholesterol turnover was explained entirely by its contribution to cholesteryl ester stores. The degree of plasma membrane cholesterol internalization and esterification was directly proportional to the size of cellular ester stores.

Animals↗
PubMed1990

Effect of cholesterol transport inhibitors on steroidogenesis and plasma membrane cholesterol transport in cultured MA-10 Leydig tumor cells.

These studies were directed toward understanding the cellular actions of inhibitor drugs that affect steroidogenesis and cholesterol transport. We investigated the microfilament inhibitor cytochalasin-D, the microtubule inhibitor colchicine, the calmodulin antagonist trifluoperazine, and the inhibitor of acidic vesicle function nigericin. We found that all of these compounds caused dose-dependent inhibition of progesterone synthesis in the MA-10 cells. Each compound also inhibited (Bu)2cAMP-stimulated pregnenolone synthesis, indicating that each inhibited a fundamental process required for steroidogenesis. Each compound was next evaluated for inhibitory actions on cholesterol transport to and from the plasma membrane. On the basis of inhibitor sensitivity, two different categories of cholesterol transport were defined. Transport of newly synthesized or low density lipoprotein-derived cholesterol from the cell interior to the plasma membrane was inhibitor insensitive. Plasma membrane cholesterol internalization, however, was sensitive to all of the inhibitors and did not result because of any drug effect on the acyl-coenzyme-A-cholesterol acyl transferase. Cycling of cholesteryl ester-derived cholesterol through the plasma membrane appeared to occur before its use for steroidogenesis. Thus, inhibition of plasma membrane internalization would prevent utilization of both plasma membrane cholesterol and cholesteryl ester-derived cholesterol, the two major substrate sources for steroid hormone synthesis. Consistent with this interpretation was the finding that inhibition of plasma membrane cholesterol internalization by each inhibitor paralleled the inhibitor's effect on steroidogenesis.

Biological Transport↗
PubMed1989

Plasma membrane cholesterol: removal and insertion into the membrane and utilization as substrate for steroidogenesis.

The plasma membrane cholesterol content of MA-10 Leydig tumor cells is depleted by trophic hormone stimulation and repleted by incubating the cells with low density lipoprotein. The present studies used subcellular fractionation to investigate the membranes involved in steroid hormone synthesis. The results showed that the plasma membrane was the major source of cholesterol substrate and that the cholesterol content changed independently of any mass changes in membrane protein or phospholipid. Membrane phospholipid composition also did not change as membrane cholesterol content decreased or increased, a finding inconsistent with the proposal that phospholipid composition dictates the amount of cholesterol contained in a membrane. The mitochondria of the MA-10 cells were cholesterol rich, containing more cholesterol per unit protein or phospholipid than the plasma membrane. This cholesterol was presumably in the outer mitochondrial membrane, since virtually all of the cholesterol of intact mitochondria was accessible to cholesterol oxidase. Although there was a high concentration of mitochondrial cholesterol, this cholesterol was largely inert as a substrate for steroidogenesis, and plasma membrane cholesterol was incorporated into steroid hormones without ever equilibrating with the mitochondrial cholesterol pool.

Cell Membrane↗
PubMed1988

Studies on a possible molecular basis for the structure of mitochondrial cristae.

We have investigated a possible molecular basis for mitochondrial cristae formation. Proteoliposomes containing electron transport proteins, cytochrome oxidase, or complex III in their proper orientation bind to pig heart mitoplasts but not pig heart mitochondria. Using Leydig tumor cells, we have confirmed earlier reports that chloramphenicol causes a diminution in cristae content and a change in its characteristic lamellar form. We show that the proteoliposomes containing cytochrome oxidase or complex III in the proper orientation bind to mitoplasts from Leydig tumor cells but do not bind as well to mitoplasts from chloramphenicol-treated Leydig tumor cells. These experiments provide a possible mechanism to explain cristae formation.

Animals↗
PubMed1988

Paget's disease of bone.

Paget's disease is a relatively common bone disease. This review aims to present reasonable treatment recommendations with enough background to understand them. To accomplish this end, some aspects of basic bone cell biology, biochemistry, and pathology are presented, as are speculations about possible causes of this disease. Treatment of Paget's disease will be considered in three sections. The first two sections will review treatment with calcitonin and diphosphonates, respectively. These sections briefly will consider the mechanism of action of the drugs, review in detail clinical studies of drug effectiveness, and summarize the advantages and disadvantages of each drug. The third section details specific treatment recommendations for each of the six clinical settings in which treatment of Paget's disease is justified.

Calcitonin↗
PubMed1987

Cyclic AMP mediated modification of cholesterol traffic in Leydig tumor cells.

The level of nonesterified cholesterol within MA-10 Leydig Tumor cells is regulated acutely by trophic hormones (Freeman, D. A., and Ascoli, M. (1982) J. Biol. Chem. 257, 14231-14238). In the present studies, we localize the site of this steroidogenic cholesterol to the plasma membrane and characterize the means by which this membrane becomes cholesterol-depleted. It is possible to detect the translocation of both newly synthesized cholesterol and cholesterol derived from lipoproteins from the cell interior to the plasma membrane. Stimulated MA-10 cells that are actively producing steroid hormones divert cholesterol from the normal intracellular or plasma membrane acceptor sites into the steroid biosynthetic pathway. Another important effect of steroidogenic stimulation is to cause internalization of plasma membrane cholesterol. Changes in cholesterol traffic in stimulated cells can be blocked by preventing the utilization of cholesterol for steroidogenesis. This later finding indicates that the changes in cholesterol transport induced by trophic hormones are consequences rather than primary causes of steroidogenic stimulation.

Animals↗
PubMed1987

Regulation of the cholesterol ester cycle of cultured Leydig tumor cells.

The MA-10 Leydig tumor cells take up low-density lipoprotein (LDL) from the medium and store the LDL-derived cholesterol as cholesterol esters that can be subsequently mobilized and used for steroid hormone synthesis. The present studies investigate the mechanisms by which cAMP acutely regulates the cellular content of cholesterol esters. In the absence of cholesterol utilization for steroidogenesis, cAMP stimulates cholesterol ester hydrolysis and ester resynthesis proportionally. The augmentation of ester hydrolysis by cAMP is completely matched by increased activity of the acyl-coenzyme-A:cholesterol acyltransferase and thus does not regulate cellular cholesterol ester concentration per se. The more important action of cAMP is to interrupt the cycle of hydrolysis and ester resynthesis by decreasing cholesterol re-esterification. In cells actively synthesizing steroid hormones, cholesterol reesterification is decreased by 82%. The decrease in cholesterol re-esterification occurs because cAMP directs cholesterol normally destined for re-esterification into steroid synthesis; simply blocking the utilization of cholesterol for steroidogenesis completely prevents net cholesterol ester hydrolysis and increases the cellular rate of cholesterol esterification.

Animals↗
PubMed1987

Constitutive steroidogenesis in the R2C Leydig tumor cell line is maintained by the adenosine 3',5'-cyclic monophosphate-independent production of a cycloheximide-sensitive factor that enhances mitochondrial pregnenolone biosynthesis.

These studies were designed to characterize constitutive steroidogenesis in Leydig tumor cells. Constitutive steroidogenesis was investigated by comparing constitutively active R2C Leydig tumor cells to trophic hormone-responsive MA-10 Leydig tumor cells. Unlike the MA-10 cells, R2C cells appeared to synthesize steroid hormones independently of the cAMP-protein kinase pathway. Although the adenylate cyclase of R2C cells could be stimulated in the expected manner by cholera toxin, cAMP concentrations in these cells were low, and R2C cell steroidogenesis could be dissociated from other cAMP-dependent processes. Two cAMP-dependent processes in steroidogenic cells, protein kinase activation and lactate formation, showed low basal activities in R2C cells and could be stimulated by (Bu)2cAMP with a dose dependence similar to that detected in MA-10 cells. Steroid hormone biosynthesis parallelled these other cAMP-dependent processes in MA-10 cells, but not in R2C cells. Cycloheximide, however, caused similar dose-dependent inhibition of steroidogenesis in both the R2C and MA-10 cells. Using a cell component bioassay, it was shown that R2C cells constitutively synthesize an extramitochondrial cycloheximide-sensitive factor that is functionally identical to the factor produced in response to hCG in MA-10 cells. This factor enhanced mitochondrial pregnenolone biosynthesis. Thus, constitutive steroidogenesis in R2C cells could be explained by the cAMP-independent but cycloheximide-sensitive constitutive production of an extramitochondrial factor that activated mitochondrial pregnenolone biosynthesis.

Animals↗
PubMed1985

Estradiol acts as a competitive inhibitor of the 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase enzyme of cultured Leydig tumor cells.

To study the local regulatory mechanisms involving steroid hormones in steroidogenic cells, the effect of estradiol on steroidogenesis was investigated using MA-10 Leydig tumor cells. Estradiol inhibited progesterone biosynthesis in MA-10 cells in a dose-dependent manner. Inhibition of progesterone biosynthesis by estradiol was associated with a concomitant accumulation of pregnenolone in the incubation medium. Estradiol inhibited the activity of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase by a chemical mechanism which is not mediated through the cellular estrogen receptor. Thus, the estrogen receptor agonist diethylstilbestrol did not inhibit this enzyme activity, nor could this agent block the effect of estradiol on the enzyme. Furthermore, estradiol inhibited enzyme activity in isolated microsomes which do not contain estradiol receptor protein. Kinetic analysis of the inhibitory effect of estradiol on 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase revealed that this steroid hormone functions as a competitive inhibitor of the enzyme, with an average apparent Ki of 1.8 microM.

3-Hydroxysteroid Dehydrogenases↗