Distribution of ryanodine receptors in rat ventricular myocytes.

Ryanodine receptors (RyRs) are the major ion channels in the sarcoplasmic reticulum responsible for Ca2+ release in muscle cells. Localization of RyRs is therefore critical to our understanding of Ca2+ cycling and Ca2+-dependent processes within ventricular cells. Recently, RyRs were reportedly found in non-classical locations in the middle of the sarcomere, between perinuclear mitochondria and in the inner mitochondrial membrane of cardiac mitochondria. However, for multiple reasons these reports could not be considered conclusive. Therefore, we modified immunogold labeling to visualize the distribution of RyRs in ventricular myocytes. Using antibodies to the voltage-dependent anion channel (i.e. VDAC) or cytochrome c along with our labeling method, we showed that these mitochondrial proteins were appropriately localized to the mitochondrial outer and inner membrane respectively. Immunogold labeling of ultrathin sections of intact and permeabilized ventricular myocytes with antibodies to three types of RyRs confirmed the existence of RyRs between the Z-lines and around the perinuclear mitochondria. However, we did not find any evidence to support localization of RyRs to the mitochondrial inner membrane.

Identification of the lipophilic factor produced by macrophages that stimulates steroidogenesis.

Macrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility. The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC columns. A single peak was observed when MDF, obtained from the final HPLC column, was analyzed by gas chromatography. The MS fragmentation pattern of purified material from both peritoneal and testicular macrophages was identical to that of a reference preparation of 25-hydroxycholesterol. Also, the nuclear magnetic resonance spectrum of MDF was similar to that of authentic 25-hydroxycholesterol. When 25-hydroxycholesterol was subjected to the identical purification scheme as MDF, it was found to elute at the same times as MDF on all three columns and elicited activity in the Leydig cell bioassay as expected. Control medium purified identically did not contain 25-hydroxycholesterol or have biological activity. Ether extracts of testis contained 25-hydroxycholesterol, indicating that this compound is present under physiological conditions. Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h. Taken together, these data indicate that the lipophilic factor produced by macrophages that stimulates steroidogenesis is 25-hydroxycholesterol.

Effects of clodronate-containing liposomes on testicular macrophages and Leydig cells in vitro.

We undertook the present studies to determine if clodronate-containing liposomes have direct effects on Leydig cells. Macrophages and Leydig cells were isolated and maintained separately in culture. Following treatment with clodronate-containing liposomes, macrophages were killed in a dose-response fashion over a range of 5-200 microliters liposomes. By comparison, a 500 microliters dose was required to kill Leydig cells, but this was not dependent upon clodronate since liposomes containing buffer elicited an identical response. The concentration of testosterone in medium from Leydig cells treated with clodronate-containing liposomes was significantly reduced compared with untreated cells. However, we subsequently found that liposomes can adsorb testosterone. Therefore, testosterone production was determined at various times following removal of liposomes from Leydig cells, thereby circumventing this complication. It was found that testosterone production was not altered by liposomes under these conditions. Finally, free clodronate had no effect on testosterone production, even at doses representing the amount present within the 500 microliters dose of liposomes. In summary, clodronate-containing liposomes killed testicular macrophages at a far smaller dose than required to kill Leydig cells. Most importantly, neither liposomes no free clodronate had a direct effect on testosterone production. Thus, clodronate-containing liposomes represent a valuable tool to study Leydig cell-macrophage interactions.

Regulation of Leydig cells through a steroidogenic acute regulatory protein-independent pathway by a lipophilic factor from macrophages.

The purpose of this investigation was to study the mechanism of action of a macrophage-derived factor that stimulates steroid production by Leydig cells. This factor increased testosterone production within 30 min, and reached a half-maximal response by 6-8 h. At a maximal dose, it stimulated testosterone production 20-fold at 24 h. Its efficacy was consistently higher than that achieved with a maximal dose of human chorionic gonadotropin (hCG). However, Leydig cells treated with a maximal dose of both the macrophage-derived factor and hCG secreted the same amount of testosterone as when given a maximal dose of only the macrophage-derived factor. The macrophage-derived factor did not require new protein synthesis to stimulate testosterone production, nor did it alter the amount of steroidogenic acute regulatory protein (StAR). While the macrophage-derived factor required an active cholesterol side-chain cleavage complex system, it did not alter the capacity of this enzyme complex. Finally, the macrophage-derived factor was unable to stimulate the production of progesterone by isolated mitochondria. In summary, the macrophage-derived factor is a highly active, acute regulator of steroidogenesis that acts through a high capacity StAR-independent pathway.

Pregnenolone synthesis in immature rat Sertoli cells.

It has been reported that testicular Sertoli cells can be induced to synthesize the steroidogenic acute regulatory (StAR) protein. StAR mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. Since Sertoli cells are thought to be unable to utilize cholesterol for the synthesis of steroids the role of StAR in these cells was questioned. In the present studies we have corroborated the induction of StAR protein in immature cultured Sertoli cells in response to either trophic hormone or cAMP analog stimulation. Further, we have shown that long term stimulation of Sertoli cells with cAMP analog results in the induction of P450scc enzyme and increased pregnenolone production. In this manner, the Sertoli cell may resemble its ovarian homolog, the granulosa cell, more closely than previously thought with regards to its steroidogenic capacity. Thus, StAR may play the same role in Sertoli cells as it does in other steroidogenic tissues.

FSH does not directly influence testicular macrophages.

We have previously demonstrated that conditioned medium from testicular macrophages stimulates testosterone production by Leydig cells. It was also reported that conditioned medium from macrophages treated with follicle-stimulating hormone (FSH) had an even greater amount of Leydig cell-stimulating activity than medium from untreated macrophages, indicating that this factor is under the regulation of FSH. However, most other laboratories have been unable to reproduce this effect of FSH. We have recently purified and partially characterized the stimulatory factor from macrophage-conditioned medium that stimulates Leydig cells. The purpose of the present investigation was to reinvestigate the effect of FSH by determining whether it regulates the production of this purified factor and by determining whether macrophages have mRNA for the FSH receptor. Testicular macrophages were isolated from adult rats and incubated 24 hours with human recombinant FSH (20 units/ml), ovine FSH (200 ng/ml), fetal bovine serum (2%), or dibutyryl cyclic adenosine monophosphate (1 mM). The macrophage-derived factor (MDF) was then purified from conditioned medium of the various treatment groups and added to Leydig cells. The concentration of testosterone in the Leydig cell medium was then measured after 16 hours. It was found that serum significantly stimulated production of the MDF. However, FSH had no effect on production of the MDF in the presence or absence of serum. Dibutyryl cyclic adenosine monophosphate exerted a slight inhibitory effect on production of the macrophage-derived factor. Most importantly, testicular macrophages did not express detectable levels of FSH receptor mRNA, either in vivo or in vitro, when evaluated using either in situ hybridization or northern analysis, under identical conditions that clearly demonstrated FSH receptor mRNA in Sertoli cells. We conclude that testicular macrophages are not a direct target for FSH.

Probing the outer mitochondrial membrane in cardiac mitochondria with nanoparticles.

The outer mitochondrial membrane (OMM) is the last barrier between the mitochondrion and the cytoplasm. Breaches of OMM integrity result in the release of cytochrome c oxidase, triggering apoptosis. In this study, we used calibrated gold nanoparticles to probe the OMM in rat permeabilized ventricular cells and in isolated cardiac mitochondria under quasi-physiological ionic conditions and during permeability transition. Our experiments showed that under control conditions, the OMM is not permeable to 6-nm particles. However, 3-nm particles could enter the mitochondrial intermembrane space in mitochondria of permeabilized cells and isolated cardiac mitochondria. Known inhibitors of the voltage-dependent anion channel (VDAC), König polyanion, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited this entrance. Thus, 3-nm particles must have entered the mitochondrial intermembrane space through the VDAC. The permeation of the isolated cardiac mitochondria OMM for 3-nm particles was approximately 20 times that in permeabilized cells, suggesting low availability of VDAC pores within the cell. Experiments with expressed green fluorescent protein showed the existence of intracellular barriers restricting the VDAC pore availability in vivo. Thus, our data showed that 1), the physical diameter of VDAC pores in cardiac mitochondria is >or=3 nm but

Production of 25-hydroxycholesterol by testicular macrophages and its effects on Leydig cells.

Testicular macrophages secrete 25-hydroxycholesterol, which can be converted to testosterone by neighboring Leydig cells. The purposes of the present studies were to determine the mode of production of this oxysterol and its long-term effects on Leydig cells. Because oxysterols are produced both enzymatically and by auto-oxidation, we first determined if testicular macrophages possess cholesterol 25-hydroxylase mRNA and/or if macrophage-secreted products oxidize cholesterol extracellularly. Rat testicular macrophages had 25-hydroxylase mRNA and converted 14C-cholesterol to 14C-25-hydroxycholesterol; however, radiolabeled cholesterol was not converted to 25-hydroxycholesterol when incubated with medium previously exposed to testicular macrophages. Exposure of Leydig cells to 10 microg/ml of 25-hydroxycholesterol, a dose within the range known to result in high basal production of testosterone when tested from 1 to 6 h, completely abolished LH responsiveness after 2 days of treatment. Because 25-hydroxycholesterol is toxic to many cell types at 1-5 microg/ml, we also studied its influence on Leydig cells during 4 days in culture using a wide range of doses. Leydig cells were highly resistant to the cytotoxic effects of 25-hydroxycholesterol, with no cells dying at 10 microg/ml and only 50% of cells affected at 100 microg/ml after 2 days of treatment. Similar conditions resulted in 100% death of a control lymphocyte cell line. These results demonstrate that 1) testicular macrophages have mRNA for cholesterol 25-hydroxylase and can convert cholesterol into 25-hydroxycholesterol, 2) macrophage-conditioned medium is not capable of auto-oxidation of cholesterol, 3) Leydig cells are highly resistant to the cytotoxic influences of 25-hydroxycholesterol, and 4) long-term treatment with high doses of 25-hydroxycholesterol results in loss of LH responsiveness. These results support the concept that testicular macrophages enzymatically produce 25-hydroxycholesterol that not only is metabolized to testosterone by Leydig cells when present at putative physiological levels but also may exert inhibitory influences on Leydig cells when present for extended periods at very high concentrations that may occur under pathological conditions.

Specificity of a new lipid mediator produced by testicular and peritoneal macrophages on steroidogenesis.

Macrophage-derived factor (MDF) is a lipophilic factor produced by rat testicular and peritoneal macrophages that maximally stimulates testosterone production by rat Leydig cells through a steroidogenic acute regulatory protein independent mechanism. The purpose of the present study was to determine whether MDF is also produced by human macrophages, and/or if it acts on human steroidogenic cells. We also studied the tissue-specific functions of MDF by determining if it also acts on steroidogenic cells of the ovary and adrenal glands and, if so, does it require new protein synthesis. It was found that MDF was produced by human peritoneal macrophages, and was capable of stimulating human steroidogenic cells. In terms of tissue specificity, it was found that primary cultures of rat adrenocortical cells respond to MDF with increased secretion of aldosterone and corticosterone, as did rat granulosa cells by producing progesterone. MDF acted in the presence of cycloheximide, indicating that it does not require new protein synthesis. These results indicate that MDF may have significant therapeutic potential and provide a basis for future studies concerning its physiological role in humans. These results further suggest that MDF is not only involved in paracrine regulation of Leydig cells, but also has the potential for the local regulation of steroidogenesis in both granulosa and adrenal cortical cells.

Short-term leptin infusion does not affect circulating levels of LH, testosterone or cortisol in food-restricted pubertal male rhesus macaques.

OBJECTIVES: Although the adipocyte protein leptin has been implicated in the control of reproductive function in rodents, its role in primate reproductive physiology is poorly understood. Because primates in puberty show nighttime LH secretion and there is considerable evidence that the fertile state requires adequate nutrition, we reasoned that animals on the verge of reproductive competence would respond to leptin infusions by secreting LH. Food restriction reduces circulating leptin levels and slows or stops the GnRH pulse generator. Therefore, we examined the endocrine effects of leptin infusions in food-restricted male pubertal primates during the night when they normally secrete LH. In addition, we investigated the effect of leptin on in vitro testosterone production by Leydig cells. SUBJECTS: Four pubertal male rhesus macaques (Macaca mulatta), 4-5.5 kg in weight (2.5-4-year-old) were examined in this study. Leydig cells from adult male rats were to investigate in vitro effects of leptin. DESIGN: To document that animals had entered puberty, blood samples were collected from each of the four animals at 15-minute intervals for 15 h both during the day and at night. Since at this age animals secrete LH mainly at night, blood samples were collected at 15-minute intervals from each of the four animals on two separate occasions for 15 h between 1500 and 0600h. During the experiment, animals were feeding from 0800 to 0830h, cages were completely cleaned of food at 0900h and the afternoon meal was not given to individual animals on the day they were studied. One of the studies served as the control (food restricted group) and during the other, 2 mg (n = 4) or 0.3 mg (n = 3) of recombinant human leptin was administered intravenously during 2000-0100h (food restricted plus leptin group). Blood samples (1 ml) were collected through the indwelling catheter and immediately transferred from the plastic syringe into chilled glass tubes containing 10 microl 14% EDTA. The samples were centrifuged at 5-h intervals and the plasma withdrawn and stored frozen at - 20 degrees C in polypropylene vials until assays were performed. MEASUREMENTS: Bioactive LH was determined and testosterone, cortisol and leptin were measured by radioimmunoassay. RESULTS: During daytime experiments in these animals, LH pulses were sometimes observed late in the day and generally continued for 12-15 h. Food-restricted pubertal males showed delayed or absent LH pulses. Short-term leptin administration to food-restricted male rhesus macaques had no effect on LH, testosterone, or cortisol levels either during or after the infusion. Leptin also had no direct effect on basal or LH-stimulated testosterone production in Leydig cells. CONCLUSIONS: Our data support the notion that leptin is not the missing signal for the acute suppression of reproductive hormones secretion in food-restricted primates.