The subcellular distribution of beta-carotene in bovine corpus luteum

The distribution of beta-carotene was determined in various subcellular fractions of bovine corpus luteum. It was found in significant amounts in all subcellular fractions examined including nuclear, mitochondrial, microsomal, cytosolic, and floating lipid. Much of the beta-carotene found in the crude nuclear and mitochondrial fractions was loosely bound and could be removed with repeated washings. In contrast, the microsomal beta-carotene could only be removed by detergent extraction suggesting that it is an integral component of this membrane preparation. In the cytosol fraction beta-carotene was bound to high-molecular-weight protein(s), quite possibly a plasma-derived lipoprotein. The subcellular distribution of beta-carotene in corpus luteum is quite similar to the distribution of its metabolite, retinol, in liver. This finding coupled with other recently published data suggests that beta-carotene could play a distinct role in corpora lutea function.     

Enzymatic conversion of beta-carotene into beta-apo-carotenals and retinoids by human, monkey, ferret, and rat tissues

Whether the conversion of beta-carotene into retinoids involves an enzymatic excentric cleavage mechanism was examined in vitro with homogenates prepared from human, monkey, ferret, and rat tissue. Using high-performance liquid chromatography, significant amounts of beta-apo-12'-, -10'-, and -8'-carotenals, retinal, and retinoic acid were found after incubation of intestinal homogenates of the four different species with beta-carotene in the presence of NAD+ and dithiothreitol. No beta-apo-carotenals or retinoids were detected in control incubations done without tissue homogenates. The production of beta-apo-carotenals was linear for 30 min and up to tissue protein concentrations of 1.5 mg/ml. The rate of formation of beta-apo-carotenals from 2 microM beta-carotene was about 7- to 14-fold higher than the rate of retinoid formation in intestinal homogenates, and the rate of beta-apo-carotenal production was fivefold greater in primate intestine vs rat or ferret intestine (P less than 0.05). The amounts of beta-apo-carotenals and retinoids formed were markedly reduced when NAD+ was replaced by NADH, or when dithiothreitol and cofactors were deleted from the incubation mixture. Both beta-apo-carotenal and retinoid production from beta-carotene were inhibited completely by adding disulfiram, an inhibitor of sulfhydryl-containing enzymes. Incubation of beta-carotene with liver, kidney, lung, and fat homogenates from each species also resulted in the appearance of beta-apo-carotenals and retinoids. The identification of three unknown compounds which might be excentric cleavage products is ongoing. These data support the existence of an excentric cleavage mechanism for beta-carotene conversion.     

Optimized formation of detergent micelles of beta-carotene and retinal production using recombinant human beta,beta-carotene 15,15'-monooxygenase

The formation of beta-carotene detergent micelles and their conversion into retinal by recombinant human beta,beta-carotene 15,15'-monooxygenase was optimized under aqueous conditions. Toluene was the most hydrophobic among the organic solvents tested; thus, it was used to dissolve beta-carotene, which is a hydrophobic compound. Tween 80 was selected as the detergent because it supported the highest level of retinal production among all of the detergents tested. The maximum production of retinal was achieved in detergent micelles containing 200 mg/L of beta-carotene and 2.4% (w/v) Tween 80. Under these conditions, the recombinant enzyme produced 97 mg/L of retinal after 16 h with a conversion yield of 48.5% (w/w). The amount of retinal produced, which is the highest ever reported, is a result of the ability of our system to dissolve large amounts of beta-carotene.     

Effects of light and low oxygen tension on pigment biosynthesis in Halobacterium salinarum, revealed by a novel method to quantify both retinal and carotenoids

A novel method for analyzing halobacterial pigments was developed, in which retinal was liberated from halobacterial rhodopsins as retinal oxime by hydroxylamine, ethyl beta-apo-8'-carotenoate was introduced as an internal standard, and the pigments including bacterioruberin and beta-carotene were analyzed by HPLC at the same time. With this method, we revealed that light enhances the biosynthesis of bacterioruberin and the conversion of beta-carotene to retinal, but does not affect beta-carotene biosynthesis in Halobacterium salinarum strain Oyon Moussa-16. Low oxygen tension given in the light brought a slight increase in retinal accumulation, although its biosynthesis from beta-carotene is an oxygenation reaction. This paradox could be explained by the increase in beta-carotene biosynthesis.     

The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.

The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol.     

Cloning and expression of beta,beta-carotene 15,15'-dioxygenase

beta,beta-Carotene 15,15'-dioxygenase cleaves beta-carotene into two molecules of retinal and is therefore the key enzyme in beta-carotene metabolism to vitamin A. In the present study, it was possible to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent that partial amino acid sequence information could be obtained to design degenerate oligonucleotides. With RT-PCR a cDNA fragment could be obtained and used subsequently in a radioactive screening of a chicken duodenal expression library. We cloned the first eukaryotic beta,beta-carotene 15,15'-dioxygenase which symmetrically cleaves beta-carotene at the 15,15'-double bond.     

The metabolism of prostaglandins F2α and E2 by non-pregnant porcine endometrial and luteal tissue and early pregnant porcine endometrial tissue,luteal tissue and conceptuses in vitro

Slices of porcine endometrium and corpus luteum from both early pregnant and non-pregnant sows and conceptuses from early pregnant sows were incubated with radioactive PGF_2a and PGE₂ and the degree of metabolism of the prostaglandins measured. Prostaglandins and metabolites were separated by TLC, radioactive bands located with a Panax scanner and the radioactivity measured in a scintillation counter. Endometrial and luteal tissue from non-pregnant sows gave no significant metabolism at any stage of the oestrous cycle, and while similar tissue from pregnant sows metabolised both prostaglandins slightly, only the conceptuses gave any significant metabolism. The possible physiological significance of these results is discussed.     

The metabolism of prostaglandins F2α and E2 by non-pregnant porcine endometrial and luteal tissue and early pregnant porcine endometrial tissue, luteal tissue and conceptuses

Slices of porcine endometrium and corpus luteum from both early pregnant and non-pregnant sows and conceptuses from early pregnant sows were incubated with radioactive PGF_2a and PGE₂ and the degree of metabolism of the prostaglandins measured. Prostaglandins and metabolites were separated by TLC, radioactive bands located with a Panax scanner and the radioactivity measured in a scintillation counter. Endometrial and luteal tissue from non-pregnant sows gave no significant metabolism at any stage of the oestrous cycle, and while similar tissue from pregnant sows metabolised both prostaglandins slightly, only the conceptuses gave any significant metabolism. The possible physiological significance of these results is discussed.     

Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro

Slices of porcine endometrium and corpus luteum tissue obtained from mature sows throughout the luteal phase of the oestrous cycle were incubated in culture medium which was analysed at regular intervals over a period of 8 hours for prostaglandin F and progesterone. Prostaglandin F secretion was greatest by endometrium obtained during the mid III to late I luteal stage of the cycle and the increased levels secreted by this tissue were paralleled by high levels of secretion from corpus luteum tissue. The addition of indomethacin (10 μg/ml) to the culture medium completely abolished prostaglandin F secretion by both endometrium and luteal tissue indicating that the high levels of the prostaglandin were due to synthesis. Progesterone secretion by the corpus luteum was maximal from early luteal tissue and had declined to considerably lower levels by late stage tissue when prostaglandin secretion was greatest. The possible physiological significance of luteal prostaglandin F secretion is discussed.     

Coordinated distribution patterns of three enzyme activities involved in the absorption and metabolism of beta-carotene and vitamin A along the villus-crypt axis of chick duodenum.

The conversion of beta-carotene to retinal and the succeeding metabolic process of the retinal leading to production of retinol and retinyl esters are the prerequisite for the utilization of beta-carotene as a provitamin A. These processes are participated by beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzyme(s) in the small intestine. To examine whether these enzymes exhibit the coordinated distribution in the villus, we have used the cryostat sectioning technique to quantify the activities of beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzymes along the villus-crypt axis in 8-day-old chick duodenum. The beta-carotene cleavage enzyme activity was very low in the crypt and gradually increased, reaching a maximum in the mid-villus. The villus-crypt gradient of the beta-carotene cleavage enzyme activity corresponded with those of retinal reductase activity and lecithin: retinol acyltransferase (LRAT) activity, but distinct from that of acyl-CoA: retinol acyltransferase (ARAT) activity. Furthermore, the distribution of the content of retinyl esters was similar to that of LRAT activity. These results suggest that the beta-carotene cleavage enzyme is coordinately distributed along the villus-crypt axis with retinal reductase and LRAT, the two enzymes which require cellular retinol-binding protein, typeII (CRBPII) as the donor of the substrate.     

The effect of alpha-tocopherol on the oxidative cleavage of beta-carotene

Two cleavage pathways of beta-carotene have been proposed, one by central cleavage and the other by random (excentric) cleavage. The central cleavage pathway involves the metabolism of beta-carotene at the central double bond (15, 15') to produce retinal by beta-carotene 15, 15'-dioxygenase (E.C.888990988). The random cleavage of beta-carotene produces beta-apo-carotenoids, but the mechanism is not clear. To understand the various mechanisms of beta-carotene cleavage, beta-carotene was incubated with the intestinal postmitochondrial fractions of 10-week-old male rats for 1 h, and cleavage products of beta-carotene were analyzed using reverse-phase, high-performance liquid chromatography (HPLC). We also studied the effects of alpha-tocopherol and NAD(+)/NADH on beta-carotene cleavage. In addition to beta-carotene, we used retinal and beta-apo-14'-carotenoic acid as substrates in these incubations. Beta-apo-14'-carotenoic acid is the two-carbon longer homologue of retinoic acid. In the presence of alpha-tocopherol, beta-carotene was converted exclusively to retinal, whereas in the absence of alpha-tocopherol, both retinal and beta-apo-carotenoids were formed. Retinoic acid was produced from both retinal and beta-apo-14'-carotenoic acid incubations only in the presence of NAD(+). Our data suggest that in the presence of an antioxidant such as alpha-tocopherol, beta-carotene is converted exclusively to retinal by central cleavage. In the absence of an antioxidant, beta-carotene is cleaved randomly by enzyme-related radicals to produce beta-apo-carotenoids, and these beta-apo-carotenoids can be oxidized further to retinoic acid via retinal.     

Morphogenesis and histophysiology of the corpus luteum

A great amount of different problems on morphogenesis and histophysiology of the corpus luteum is presented, with an emphasis on light optic and ultrastructural data that characterize the developmental dynamics of the corpus luteum. The vascular reaction is described in details, beginning from the preovulatory period. The total high vascularization rate is demonstrated and certain information on ultrastructure of newly formed capillaries and macrophages is concerned with. For the first time the authors' data on intravascular macrophages are given. The role of macrophages in the function and structural dynamics of the corpus luteum is discussed. Owing to the results obtained histochemically, ultrastructurally and biochemically, the subject on dynamics of the corpus luteum hormonoproduction, on processes participating in the hormone secretion, as well as on the role of the interstitial tissue in the corpus luteum formation is considered. The data from the literature and those of the authors are presented concerning the means and ways of progesteron transport in the form of vesicles, granules, or by means of molecular diffusion. Participation of the corpus luteum macrophages (tissue and vascular ones) in processes of synthesis and transport of progesteron is analysed. The role of prostaglandins in the chain of regulation of development, function and involution of the corpus luteum is studied. The changes in balance of prostaglandins, when prostaglandin F2 is administered result in decreasing amount of progesterone in blood. In the experiment, synthesis of prostaglandins is blocked by indometacin administration and it causes certain disturbances in luteal transformation.     

GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells

Gonadotropin-releasing hormone (GnRH)-like proteins with anti-gonadotropic properties were recently discovered in the ovaries of several species, including humans. Since neither GnRH receptors nor GnRH are in bovine ovarian tissue, we examined, in the present studies, whether concentrations of GnRH-like proteins varied during development of the corpus luteum (CL) and whether GnRH-like proteins were selectively localized in ovarian cells of cows. For these studies, GnRH-like proteins were extracted from various ovarian and nonovarian tissues and fluids and fractionated for hydrophobic interaction chromatography. A highly specific and sensitive radioreceptor assay (RRA) was used to quantify concentrations of GnRH-like proteins. The major findings of these studies demonstrated that 1) the amount of GnRH-like proteins in the corpus luteum (CL) was proportional to the weight of the CL; 2) the concentration of GnRH-like proteins in luteal tissue decreased during development of the CL; 3) GnRH-like proteins were in ovarian and numerous nonovarian tissues, but were not in the heart, plasma, or follicular fluid; 4) the retention time for GnRH-like proteins following high-pressure liquid chromatography (HPLC) varied with the tissue source; and 5) compared with all other tissues, the greatest concentration of GnRH-like proteins was in granulosal cells. We concluded that the concentration of GnRH-like proteins in luteal cells decreased during development of the CL, and that a specific GnRH-like protein was selectively localized in bovine granulosal cells.     

Tumor necrosis factor production and accumulation of inflammatory cells in the corpus luteum of pseudopregnancy and pregnancy in rabbits

The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

Effect of cyclodextrin-encapsulated beta-carotene on progesterone production by bovine luteal cells

Experiments were conducted to examine the effect of cyclodextrin-encapsulated beta-carotene on basal or cholesterol (cyclodextrin-encapsulated), LH and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production by bovine corpus luteum cells isolated from mid-luteal heifer ovaries by collagenase digestion. Cells were cultured with serum-free DMEM/Ham's F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. Medium was replaced after 24h and thereafter every 48 h. Beta-carotene was added to cultures in a carrier molecule, dimethyl-beta-cyclodextrin, to facilitate dissolution. All treatments were started on day 3 of culture. Treatment of cells with 1 or 2 micromol/l beta-carotene resulted in sharp inhibition of progesterone production. On the contrary, treatment of cells with 0.1 micromol/l beta-carotene resulted in significant stimulation (P<0.05) of both basal and cholesterol-stimulated progesterone secretion. The effect of beta-carotene on LH or dbcAMP-stimulated progesterone production was also examined. Treatment of cells with LH or dbcAMP always resulted in stimulation of progesterone secretion (P<0.001). However, cells treated with LH plus beta-carotene or dbcAMP plus beta-carotene both produced significantly (P<0.01) less progesterone relative to those cells treated with LH or dbcAMP alone on days 7, 9 and 11 of culture. These results indicate that beta-carotene can enhance luteal steroidogenesis when present at low concentrations but is inhibitory at higher concentrations and that encapsulation of beta-carotene in cyclodextrin is an effective method of supplying it to cells in culture.     

Retinoic acid can be produced from excentric cleavage of beta-carotene in human intestinal mucosa.

The hypothesis that retinoic acid (RA) is produced from the excentric cleavage of beta-carotene was tested in human intestinal homogenates in vitro. Significant amounts of RA were identified by HPLC and derivatization after incubation of intestinal mucosal homogenates with retinal, beta-carotene, or beta-apocarotenals at 37 degrees C for 60 min. RA formation was inhibited, in a dose-dependent fashion, when retinal was incubated in the presence of 0.1-3.0 mM citral (3,7-dimethyl-2,6-octadienal) under identical experimental conditions. The formation of RA from both beta-carotene and beta-apocarotenals was dose and time dependent and RA was the major metabolite of both beta-apo-8'-carotenal and beta-apo-12'-carotenal after the incubation. However, citral (0.1 to 4 mM) did not inhibit the formation of beta-apocarotenals and RA from 2 microM beta-carotene (P greater than 0.05), which proves the existence of an excentric cleavage mechanism for beta-carotene conversion into retinoids. Furthermore, RA formation from both beta-apo-8'-carotenal and beta-apo-12'-carotenal in human intestinal homogenate occurred in the presence of citral, which demonstrates that RA can be produced from excentric cleavage of beta-carotene via a series of beta-apocarotenals as intermediates.     

Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

The relationship of oxygen free radicals to corpus luteum function in rabbits was explored during various stages of pseudopregnancy, including natural and induced luteal regression. Induced luteolysis was achieved during mid-pseudopregnancy by removal of an oestradiol capsule placed at the onset of pseudopregnancy, which suppressed ovarian oestradiol production. Activity of manganese superoxide dismutase (Mn SOD) was significantly and positively correlated with ovarian progesterone production (P < 0.01) throughout pseudopregnancy and during natural regression. Oestradiol deprivation for 12, 24 or 72 h resulted in declines in Mn SOD activity and progesterone secretion, although Mn SOD rose and corpus luteum steroidogenesis was restored to normal when the capsule was replaced for 48 h before assessment, having been removed for 24 h. Lipid peroxide and progesterone concentrations were not correlated, although a significant rise in lipid peroxides in the luteal tissue was detected after deprivation of oestradiol for 72 h. Changes in progesterone production and Mn SOD activity were not associated with alterations in concentration of prostaglandin F metabolite. These data suggest that Mn SOD may be involved in regulating function of the corpus luteum during pseudopregnancy in rabbits and that oxygen free radicals may play a role in regression of corpus luteum in this species.     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

Formation and fate of the corpus luteum in the Dusky leaf monkey (Presbytis obscura)

The formation and fate of the corpus luteum have been described for a previously un researched species of South-east Asian colobine, the Dusky leaf monkey, Presbytis obscura. Histological material from 44 wild female monkeys collected at various stages of the menstrual cycle, pregnancy and lactation over a 12-month period was available for study. The corpus luteum of the menstrual cycle was a cystic structure and consisted of a thin rim of luteal tissue surrounding a central cavity filled with a meshwork of fibrin. At the end of the luteal phase the corpus luteum either degenerated into a corpus albicans, or became transformed into a corpus aberrans. Corpora aberrantia have previously only been described in the ovaries of the rhesus monkey, where they may persist for many months. Ultimately the corpus aberrans may also degenerate into a corpus albicans. Small corpora lutea atretica were observed during early pregnancy but there was no evidence of corpora lutea accessoria. Anovulatory cycles were common amongst the females included in this study and may play a role in limiting the growth of troops in their natural environment. Comparisons have been drawn between the findings presented here and those published for other species of catarrhine primate.