Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Mouse oocytes regulate metabolic cooperativity between granulosa cells and oocytes: amino acid transport

A search for genes expressed more highly in mouse cumulus cells than mural granulosa cells by subtraction hybridization yielded Slc38a3. SLC38A3 is a sodium-coupled neutral amino acid transporter having substrate preference for l-glutamate, l-histidine, and l-alanine. Detectable levels of Slc38a3 mRNA were found by in situ hybridization in granulosa cells of large preantral follicles, but levels were higher in all granulosa cells of small antral follicles; expression became limited to cumulus cells of large antral follicles. Expression of Slc38a3 mRNA in granulosa cells was promoted by fully grown oocytes from antral follicles but not by growing oocytes from preantral follicles. Fully grown oocytes were dependent on cumulus cells for uptake of l-alanine and l-histidine but not l-leucine. Fully grown but not growing oocytes secreted one or more paracrine factors that promoted cumulus cell uptake of all three amino acids but of l-alanine and l-histidine to a much greater extent than l-leucine. Uptake of l-leucine appeared dependent primarily on contact-mediated signals from fully grown oocytes. Fully grown oocytes also promoted elevated levels of Slc38a3 mRNA and l-alanine transport by preantral granulosa cells, but growing oocytes did not. Therefore, fully grown oocytes secrete one or more paracrine factors that promote cumulus cell uptake of amino acids that oocytes themselves transport poorly. These amino acids are likely transferred to oocytes via gap junctions. Thus, oocytes use paracrine signals to promote their own development via metabolic cooperativity with cumulus cells. The ability of oocytes to mediate this cooperativity is developmentally regulated and acquired only in later stages of oocyte development.     

Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study

Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.     

Biochemical studies of mammalian oogenesis: Metabolic cooperativity between granulosa cells and growing mouse oocytes

Freeze fracture and lanthanum tracer experiments have shown that gap junctions exist throughout folliculogenesis between granulosa cells and growing mouse oocytes (Anderson and Albertini, J. Cell Biol.71, 680–686, 1976). The following lines of experimentation in the present study suggest that metabolic cooperativity exists between granulosa cells and their enclosed oocytes, i.e., gap junctions are functional, and that in most cases examined, greater than 85% of the metabolites present in follicle-enclosed oocytes were originally taken up by the granulosa cells and transferred to the oocyte via gap junctions: (1) When incubated with various radiolabeled compounds, follicle-enclosed oocytes contained more intracellular radioactivity than did oocytes with no attached granulosa cells (denuded oocytes); (2) for two radiolabeled ribonucleosides examined, the distribution of phosphorylated metabolites in follicle-enclosed oocytes resembled that of granulosa cells and differed significantly from that in denuded oocytes; (3) pulse-chase experiments with radiolabeled ribonucleosides revealed that during the chase period more radioactivity became associated with the follicle-enclosed oocyte; (4) treatments known to disrupt gap junctions in other cell types were effective in reversibly uncoupling metabolic cooperativity between granulosa cells and oocytes; and (5) a series of control experiments using (a) medium conditioned by granulosa cells and (b) cocultures of denuded oocytes and granulosa cells in which physical contact between the two cell types was not permitted demonstrated that contact between follicle cells and oocytes was necessary for observing metabolic cooperativity. Metabolic cooperativity was also found between follicle cells and oocytes in the two culture systems which support growth of mouse oocytes in vitro. The fact that oocytes do not grow well, if at all, in the absence of follicle cells and the large contribution of nutrients apparently furnished to the oocyte by the granulosa cells is consistent with the concept that gap junction mediated metabolic cooperativity between follicle cells and their enclosed oocytes is vital for mammalian oocyte growth.     

Studies on metabolism of lung fluke genus Paragonimus. VII. Action of bithionol on glycolytic and oxidative metabolism of adult worms

The present study was carried out in order to obtain information on the mechanism of action of bithionol on Paragonimus westermani (Kerbert 1878). Bithionol stimulated lactic acid production of intact adult worms above the level of the control worms, while it inhibited oxygen consumption of intact adult worms in vitro. Bithionol treatment of adult worms in vivo decreased glycolytic and oxidative metabolism of homogenates of uterine eggs and adult worms.Bithionol inhibited lactic acid formation except when fructose 1, 6-diphosphate (FDP) was used as a substrate in the homogenates of adult worms, and it also inhibited oxygen consumption of homogenates of eggs and adult worms. Bithionol inhibited reduction of methylene blue when succinate was used as a substrate. Bithionol inhibited oxidation of reduced cytochrome c in the 1000g supernatant of homogenates of adult worms. Bithionol inhibited activity of the succinate oxidation in homogenates of adult worms.     

Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: optimization using experimental design

As a basis for the development of predictive mathematical models in systems biology and a quantitative understanding of cellular metabolism, reliable experimental data sets of intracellular metabolites are indispensable. A prerequisite for the acquisition of such data is the identification of a suitable sample preparation method. In this work, the extraction procedure for the simultaneous measurement of a wide range of intracellular metabolites from adherent mammalian cells in culture was optimized. A screening of several commonly used extraction protocols with Madin-Darby canine kidney (MDCK) cells found the methanol/chloroform (MeOH/CHCl(3)) and MeOH/Boil methods to be promising candidates for further analysis by anion-exchange chromatography. Both methods were optimized based on experimental design techniques with four response variables: Nucleotide Content, Energy Charge, Fructose 1,6-Bisphosphate content (F16bP), and Absorption at 280 nm. After data evaluation and with the help of desirability functions, an overall optimum for the extraction conditions was found. Using optimal settings, the extraction performances for MDCK and Vero cell cultivations of both methods were compared. Both methods extracted nearly the same absolute amounts of intracellular metabolites, suggesting that these methods are equal. However, recoveries for nucleotide diphosphates were significantly above 100% for both methods. This most likely was due to remaining nucleotide kinase activity during extraction. After combining individual steps of both methods, recoveries close to 100% for all metabolites could be reached. Absolute values of intracellular metabolites extracted with this modified method are comparable to the results of the two previously optimized methods, indicating a good extraction procedure according to the chosen response variables.     

Influence of lysophosphatidic acid on estradiol production and follicle stimulating hormone action in bovine granulosa cells

The objective of the study was to examine the effect of lysophosphatidic acid (LPA) on 17β-estradiol (E₂) synthesis and follicle stimulating hormone (FSH) action in bovine granulosa cells. We found that granulosa cells in the bovine antral follicle, in addition to the uterus and the CL, are also the site of LPA synthesis and the target for LPA action in the bovine reproductive tract. Our findings suggest that LPA stimulates E₂ synthesis, probably via increased expression of FSHR and 17β-HSD genes.     

Expression of the intercellular adhesion molecule-1 on high endothelial venules and on non-lymphoid antigen handling cells: interdigitating cells,antigen transporting cells and follicular dendritic cells

Intercellular adhesion molecule-1 (ICAM-1)¹ has been implicated in the development of germinal center reactions in vitro, and the present study was undertaken to determine the distribution of ICAM-1 in active germinal centers in vivo and in murine secondary lymphoid tissues in general. Anti-ICAM-1-specific monoclonal antibodies were used in conjunction with immunohistochemistry at both the light and ultrastructural levels of resolution. Examination of cryostat sections of lymph nodes, spleens, and Peyer's patches revealed that anti-ICAM-1 distinctly labeled cells in the light zones of germinal centers, a few cells in the T cell zones (e.g. paracortex of lymph nodes), cells in the sinus floor of the subcapsular sinuses of lymph nodes, and high endothelial venules (HEV). Ultrastructural studies revealed that the cells labeling with anti-ICAM-1 in germinal centers were follicular dendritic cells (FDC) which appeared to have more ICAM-1 than any other cell type. The surfaces of well-developed, intricate, convoluted FDC processes were intensely labeled even under conditions where B cells appeared negative. Interdigitating cells (IDC) were also labeled as were certain endothelial cells in the HEV. The cells in the subcapsular sinus floor labeling with anti-ICAM-1 were the antigen transporting cells (ATC) that carry antigen-antibody complexes into lymph node follicles. We suspect ATC are FDC precursors which mature into FDC in the follicles. Interestingly, FDC, IDC, and ATC are 3 important accessory cells known to handle antigens in specific compartments of lymphoid tissues. The marked localization of this adhesion molecule on these critical antigen handling cells supports the concept that ICAM-1 is important in providing the intercellular adhesion necessary for optimal initiation of immune responses in vivo.Abbreviations ICAM-1 Intercellular adhesion molecule-1 - LFA-1 leukocyte functional antigen-1 - IDC interdigitating cells - ATC antigen transporting cells - FDC follicular dendritic cells - HEV high endothelial venules - DC dendritic cells - PBS phosphate-buffered saline - PLP periodate-lysine-4% paraformaldehyde - GPLP periodate-lysine-0.1% glutaraldehyde-2% paraformaldehyde - EM electron microscopy - HRP horseradish peroxidase - DAB diaminobenzidine tetrahydrochloride - HSA human serum albumin     

Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [¹⁴C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [³H]thymidine and biosynthesis of prostaglandin E₂(PGE₂). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE₂ production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE₂ in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE₂-dependent proliferation.     

Leishmania mexicana: Energy metabolism of amastigotes and promastigotes

The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO₂ by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO₂ and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.     

Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation

In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells.     

Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization

It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

The effect of glyceraldehyde on red cells: Haemoglobin status,oxidative metabolism and glycolysis

Glyceraldehyde induces changes in the flux of glucose oxidised through the hexose monophosphate pathway, the concentrations of intermediates in the Embden-Meyerhoff pathway, the oxidative status of haemoglobin and levels of reduced and oxidised pyridine nucleotides and glutathione in red cells. Glyceraldehyde autoxidises in the cellular incubations, consuming oxygen and producing glyoxalase I- and II-reactive materials. Major fates of glyceraldehyde in red cells appear to be: (i) adduct formation with reduced glutathione and cellular protein; (ii) autoxidation and reaction with oxyhaemoglobin and pyridine nucleotides, and (iii) phosphorylation of d-glyceraldehyde and entry into the glycolytic pathway as glyceraldehyde 3-phosphate. The production of glycerol from glyceraldehyde by red cell l-hexonate dehydrogenase appears not to be a major reaction of glyceraldehyde in red cells. These results indicate that high concentrations of glyceraldehyde (1–50 mM) may induce oxidative stress in red cells by virtue of the spontaneous autoxidation of glyceraldehyde, forming hydrogen peroxide and α-ketoaldehydes (glyoxalase substrates). The implications of glyceraldehyde-induced oxidative stress for the in vitro anti-sickling effect of dl-glyceraldehyde and for the polyol pathway metabolism of glyceraldehyde are discussed.     

Modulatory effect of linoleic and oleic acid on cell proliferation and lipid metabolism gene expressions in primary bovine satellite cells

This study was performed to elucidate the effects of linoleic acid (LA), oleic acid (OA) and their combination (LA?+?OA) on cell proliferation, apoptosis, necrosis, and the lipid metabolism related gene expression in bovine satellite cells (BSCs), isolated from bovine muscles. Cell viability was significantly increased with the OA and LA treatment. Furthermore, LA?+?OA enhanced cell proliferation in a dose-dependent manner (10 to 100?µM), whereas it lowered at 250?µM. In addition, a cell-cycle analysis showed that 100?µM of LA and OA markedly decreased the G0/G1 phase proportion (62.58% and 61.33%, respectively), compared to controls (68.02%), whereas the S-phase cells’ proportion was increased. The ratio of G2/M phase cells was not significantly different among the groups. Moreover, analyses with AO/EtBr staining showed that no apoptosis occurred. Necrosis were determined by flow cytometry using Annexin V-FITC/PI staining which revealed no early apoptosis in the cells pretreated with LA or OA, but occurred in the LA?+?OA group. We also analyzed the mRNA expression of lipid metabolizing genes such as peroxisome proliferator receptor alfa (PPARα), peroxisome proliferator receptor gamma (PPARγ), acyl-CoA oxidase (ACOX), lipoprotein lipase (LPL), carnitine palmitoyl transferase (CPT-1), and fatty-acid binding protein4 (FABP4), which were upregulated in LA or OA treated cells compared to the control group. In essence, LA and OA alone promote the cell proliferation without any apoptosis and necrosis, which might upregulate the lipid metabolism related gene expressions, and increase fatty-acid oxidation in the BSCs’ lipid metabolism.     

An ultrastructural study of relationships between the ovarian haemal system,follicle cells,and primary oocytes in the sea star,Asterias rubens

Summary The genital haemal sinus, present throughout the gonad wall of sea stars, is supposed to be the site of ultimate accumulation of nutrients for the germinal epithelium. Early vitellogenic pear-shaped oocytes are attached to this sinus by stalk-like processes. The ultrastructure of this association and of the oocyte-follicle cell complex is described with emphasis on mechanisms involved in oocyte nutrition.The genital haemal sinus, and sometimes portions of the surrounding genital coelomic sinus, contain a fine granular ground substance and amoeboid cells. Material similar to the haemal ground substance also fills vacuities in the inner basal laminae of the haemal sinus and intervenes between this layer and adjacent germinal and follicle cells in the ovarian lumen.Vitellogenesis is first detectable as numerous vacuoles accumulate within the oocyte-stalk near the haemal sinus; they contain flocculent material and often fuse with adjacent lysosome-like vacuoles. As vitellogenesis proceeds, oocytes develop complex and tenuous connections with the haemal sinus. These consist of a network of pseudopodia that interdigitate with thin sheet-like extensions of follicle cells. These cells are attached to the oolemma by microfilamentous processes and contain regularly arranged concentrations of glycogen granules and well developed rough endoplasmic reticulum.It is concluded, (1) that follicle cells provide each oocyte with a compartmentalized microenvironment within the ovarian lumen, (2) that such compartments are intimately associated with the nutrient laden haemal sinus, and (3) that nutritive and vitellogenic substances, derived extragonadally and stored temporarily in the ovarian wall, can pass through the oocyte-stalk.     

Chronic exposure to a 2 mT static magnetic field affects the morphology,the metabolism and the function of in vitro cultured swine granulosa cells

In recent years, the exposure of organisms to static magnetic fields (SMFs) is continuously increasing. Thus, we investigated the effect of chronic exposure to a 2 mT SMF on in vitro cultured swine granulosa cells (GCs). In particular, the culture expansion (cell viability and doubling time), the cell phenotype (cell morphology and orientation, actin and α-tubulin cytoskeleton), the cell metabolism (intracellular Ca²⁺ concentration [Ca²⁺]_i and mitochondrial activity) and the cell function (endocrine activity) were assessed. It has been found that the exposure to the field did not affect the cell viability, but the doubling time was significantly reduced (p < 0.05) in exposed samples after 72 h of culture. At the same time, the cell length and thickness significantly changed (p < 0.05), while the cell orientation was unaffected. Evident modifications were induced on actin and α-tubulin cytoskeleton after 3 days of exposure and, simultaneously, a change in [Ca²⁺]_i and mitochondrial activity started to become evident. Finally, the SMF exposure of GCs longer than 72 h determined a significant alteration of progesterone and estrogen production (p < 0.05). In conclusion, our results demonstrate that the chronic exposure of swine GCs to a 2 mT SMF exerts a negative effect on cell proliferation, morphology, biochemistry and endocrine function in an in vitro model.