Presence Of Cell-conditioned Medium Research Articles

Uterosome-like vesicles prompt human sperm fertilizing capability.

Does the rapid transit through the uterine environment modulate the sperm physiological state? The uterosome-like vesicles (ULVs) secreted by endometrial epithelial cells (EECs) in vitro are able to fuse with human spermatozoa, prompting their fertilizing capacity. Early studies suggest that sperm capacitation begins in the uterus and ends in the oviduct, and that a synergistic effect of both female organs may accelerate this process. Although it has been reported that co-incubation of human spermatozoa with endometrial cell-conditioned medium (CM) stimulates sperm capacitation, the mechanism mediating this communication is unknown. Human ULVs secreted by EECs were characterized and their effect on human sperm physiology was analysed. Spermatozoa were incubated with EEC-derived CM or ULV, after which sperm capacitation was evaluated at different time points. In addition, the interaction of spermatozoa with ULV was analysed. ULVs were isolated by ultracentrifugation and identified using electron microscopy and Western blotting to assess the presence of specific protein markers. Following seminal plasma removal, human spermatozoa were incubated CM or ULV, after which sperm capacitation was evaluated as the ability of the sperm to undergo the induced acrosome reaction and the level of protein tyrosine phosphorylation (PY) determined by Western blot and immunocytochemistry. The interaction of spermatozoa with labelled ULV was analysed by fluorescence microscopy. In all cases, at least three biological replicates from different sperm donors were performed for each set of experiments. Significant differences between mean values were determined by one-way ANOVA followed by Tukey's post hoc test. Differences between treatments were considered statistically significant at P ≤ 0.05. The level of capacitated spermatozoa and those recruited by chemotaxis increased 3- to 4-fold when spermatozoa were incubated in the presence of CM for 4 h. Even a 15 min incubation of spermatozoa with CM was also enough to increase the level of capacitated cells 3- to 4-fold (P < 0.05). Furthermore, a short co-incubation of spermatozoa with ULV stimulates sperm capacitation, as determined by the increase in the level of induced acrosome reaction and the induction of PY. In addition, after the co-incubation of spermatozoa with fluorescent labelled ULV, the sperm cells acquired the fluorescent staining which indicates that ULV might be transferred to the sperm surface by a fusion mechanism. This is an in vitro study performed with human biological material, spermatozoa and endometrial derived cells; the latter being a cell line originally isolated from a uterine adenocarcinoma. The capability of spermatozoa to briefly interact with ULVs supports the hypothesis that any step of sperm transport may have physiological consequences, despite the interaction lasting for only a limited period of time. This way of communication of spermatozoa with cell products of uterine origin opens new frontiers of investigation (e.g. the signalling molecules involved), shedding light on the sperm processes that prepare the male gamete for fertilization, which might have implications for human infertility treatment. N/A. The project was financially supported by SECyT-UNC. The authors declareno conflict of interest.

Hypoxia Simultaneously Alters Satellite Cell‐Mediated Angiogenesis and Hepatocyte Growth Factor Expression

Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. Hepatocyte growth factor (HGF) plays a pivotal role in myogenesis by activating satellite cells (SC) in regenerating muscle and likely plays a role as a contributor to revascularization. Moreover, repair of a functional blood supply is critical to ameliorate tissue ischemia and restore skeletal muscle function, however effects of hypoxia on satellite cell-mediated angiogenesis remain unclear. The objective of this study was to examine the role of HGF and effect of hypoxia on the capacity of satellite cells to promote angiogenesis. To characterize the role of HGF, a microvascular fragment (MVF) culture model coupled with satellite cell conditioned media (CM) was employed. The activity of HGF was specifically blocked in SC CM reducing sprout length compared to control CM. In contrast, MVF sprout number did not differ between control or HGF-deficient SC CM media. Next, we cultured MVF in the presence of CM from satellite cells exposed to normoxic (20% O2 ) or hypoxic (1% O2 ) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF-1α protein abundance and VEGF mRNA abundance but decreased HGF mRNA abundance compared to normoxic satellite cells. Consistent with reduced HGF gene expression, HGF promoter activity decreased during hypoxia. Taken together, this data indicates that hypoxic modulation of satellite cell-mediated angiogenesis involves a reduction in satellite cell HGF expression.

Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes

Trout primary kidney monocyte-like cultures (T-PKM) were generated by incubating head kidney leukocytes in the presence of cell-conditioned medium (CCM). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the goldfish. Flow cytometric analysis of the initial T-PKM cultures, identified three cell sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. >8 days) in the presence of CCM. Functionally, reactive oxygen intermediate (ROI) production was detected following stimulation of T-PKM with PMA. However, these cells failed to produce reactive nitrogen intermediates (RNI) in response to immunological stimuli. In contrast, goldfish IVDKM were capable of producing both ROI and RNI. Using the dihydrorhodamine (DHR) assay and flow cytometry, we identified two ROI-producing sub-populations in goldfish IVDKM but only a single ROI-producing sub-population was present after extended cultivation of T-PKM. This T-PKM sub-population was subsequently sorted using the flow cytometer and shown to possess monocyte-like morphology by microscopic and cytometric analysis. Thus, acquisition of antimicrobial functions following cultivation of kidney leukocytes of rainbow trout and goldfish is markedly different, and may be due to the failure of trout monocyte-like cells to undergo a final differentiation step in vitro.

Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte–cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24 h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10 ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 μmol/l genistein (tyrosine kinase inhibitor), in the presence of 10 mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 μmol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10 ng/ml) failed to affect the progesterone synthesis during 72 h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.

Hormone-stimulated secretion of luteinization factor in porcine granulosa cells.

Luteinization stimulator (LS) is an intrafollicular compound which was shown to be released by granulosa cells under in vitro conditions with stimulatory effects on immature granulosa cell differentiation. This study was undertaken to determine the effects of various endocrine agents which are involved in the regulation of ovarian function on LS secretion by porcine granulosa cells isolated from 5-8-mm follicles (LGC). Cell conditioned media (CM) obtained after the 4-day culture of LGC were tested in the culture of immature (small) granulosa cells (SGC). The activity of LS released into the LGC conditioned medium was estimated by measuring progesterone (P4) produced by SGC in the presence of CM. Stimulation of P4 secretion was observed after addition of media from cultures treated by LHRH (10(-4) mol.l-1), epinephrine (10(-5) mol.l-1), LH (1 microgram.ml-1), dbcAMP (0.5 and 2.0 micrograms.ml-1) or insulin (1.0-5.0 micrograms.ml-1). Norepinephrine (10(-5) and 10(-7) mol.l-1), estradiol (0.1 and 1.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml-1) did not change steroidogenic activity of CM. Epinephrine and norepinephrine (10(-5) and 10(-7) mol.l-1), LH (1 microgram.ml-1), dbcAMP (2.0 microgram.ml-1) and estradiol (1 microgram.ml-1) alone enhanced P4 production by SGC, whereas LHRH (10(-3) and 10(-4) mol.l-1), insulin (1.0-5.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml.-1) did not have any effect. These observations suggest that the process of LS secretion in developing follicles is subject to a specific hormonal control.

Isolation of immortal cell lines from the first stage of murine leukemia virus-induced leukemia.

Friend murine leukemia virus (F-MuLV) is a replication-competent retrovirus that induces a rapidly fetal leukemia in susceptible mice (stage I disease). Leukemia cells obtained from these animals do not grow in cell culture using standard tissue culture conditions. However, in the presence of WEHI-3 cell-conditioned medium (CM), 100% of spleen or bone marrow explants from diseased mice yield immortal cell lines. These cell lines exhibit the same growth properties, produce the same viruses, and express the same oncogenes as the leukemia cells found in mice with stage I disease. No cell lines were obtained from leukemic mice in the absence of CM. No cell lines were obtained from uninfected adult, newborn, or phenylhydrazine-treated animals with or without CM. We conclude that some of the hematopoietic cells in F-MuLV-diseased mice will proliferate indefinitely in the presence of CM. The development of this abnormal response to CM is one of the early changes associated with F-MuLV-induced leukemia.