DNA degradation in mural granulosa cells of non- and slightly atretic follicles of fresh and cold-stored domestic cat ovaries

Apoptosis of granulosa cells is associated with follicular atresia and may occur before atresia becomes morphologically evident. Detection of DNA fragmentation by in situ end-labeling (ISEL) with terminal transferase allows the histological assessment of apoptotic cells on conventional histological sections. Degradation of DNA also may occur after prolonged cold storage of ovaries caused by the release of lysosomal enzymes. The objectives of this study were to assess follicle atresia and the impact of cold storage for 8, 12, 24, and 48 hr after ovarian excision by assessing DNA degradation in mural granulosa cells of cat ovaries. Follicles were distinguished by morphological criteria as nonatretic (NA), slightly atretic (SA), or atretic, and the mean number (±SEM) of granulosa cells labeled by ISEL was determined. About 50% of follicles showed some sign of atresia independent from the stage of the reproductive cycle of the ovarian donor. Number of ISEL-stained granulosa cells for NA and SA, freshly collected follicles was 7.5 ± 0.6 and 9.3 ± 0.8 cells/field, respectively, compared to 16.2 ± 0.8 cells/field in the wall of atretic follicles (P < 0.001). Fresh NA follicles from luteal phase ovaries had more (P < 0.05) labeled granulosa cells (9.2 ± 0.7 cells/field) than measured in follicles of cats in a follicular phase (5.7 ± 0.7). During cold storage, DNA degradation began within 12 hr (NA, 12.2 ± 0.7 cells/field; SA, 13.3 ± 0.5), both values being different (P < 0.05) from fresh controls. By 24 hr, DNA degradation was at the level of a positive control subjected to DNAse treatment. In summary, results reveal that granulosa cell DNA degeneration precedes the loss of developmental capacity of cat oocytes during atresia and postexcision storage. Finding irreversible changes in granulosa cell DNA after storage of cat ovaries for >12 hr may be important for developing oocyte rescue protocols for rare felids in cases where prolonged storage and transport may be required. Mol. Reprod. Dev. 48:350–355, 1997. © 1997 Wiley-Liss, Inc.     

Bovine oocyte cytoplasm supports nuclear remodeling but not reprogramming of murine fibroblast cells

Nuclear transfer (NT) is used to elucidate fundamental biological issues such as reversibility of cell differentiation and interactions between the cytoplasm and nucleus. To obtain an insight into interactions between the somatic cell nucleus and oocyte cytoplasm, nuclear remodeling and gene expression were compared in bovine oocytes that had received nuclei from bovine and mouse fibroblast cells. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from mouse fibroblasts did not develop beyond the 8-cell stage. Similar nuclear remodeling procedures were observed in oocytes reconstructed with mouse and bovine fibroblast cells. Foreign centrosomes during NT were introduced into embryos reconstructed with both fibroblast cell types. A number of housekeeping mouse genes (hsp70, bax, and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells.     

PDGFRb+ mesenchymal cells,but not NG2+ mural cells,contribute to cardiac fat

1. Download : Download high-res image (119KB)
2. Download : Download full-size image

     

Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.     

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.     

Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicle.

Mural and cumulus granulosa cells synthesize hyaluronic acid (HA) and expand in vitro in response to follicle-stimulating hormone and a soluble factor(s) produced by fully grown oocytes. In the present study we examined HA synthesis and extracellular matrix organization by the two cell populations in vivo during the preovulatory period. After injection of human chorionic gonadotropin into pregnant mares' serum gonadotropin-primed animals, a progressive increase in HA synthesis was observed by the cumulus cell-oocyte complex (COC), and by the mural granulosa cells adjacent to the antrum (antral granulosa cells). The outermost layers of mural granulosa cells (peripheral granulosa cells) did not synthesize HA. Net HA synthesis was approximately 4 pg/cell for COCs isolated after full expansion induced either in vivo or in vitro, whereas the total HA content and cell number in the ovulated COC (approximately 11 ng HA and approximately 3000 cells per COC) were about threefold higher than for COCs expanded in vitro (approximately 4 ng HA and approximately 1000 cells per COC). The increased cell content of ovulated COCs appears to be primarily the result of inclusion of proximal mural granulosa cells which synthesize HA in response to the oocyte factor(s) and become incorporated in the expanded COC extracellular matrix mass. Media conditioned by oocytes enclosed in the cumulus cell mass (intact COCs) contained only 10-20% of the HA-stimulatory activity of media conditioned by an equal number of isolated oocytes when tested on mural granulosa cell cultures. Further, HA-stimulatory activity of media conditioned by isolated oocytes was dramatically reduced (approximately 70%) by preincubation for 5 hr with cumulus cells compared to preincubation in the absence of cells. The results suggest that differences in HA synthesis between subregions of membrana granulosa depend on a diffusion gradient of the oocyte factor(s).     

Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells

This report describes the detection and partial characterization of preovulatory human cumulus oophorus and mural granulosa cell-associated activity capable of initiating the human sperm acrosome reaction (AR) in vitro. Fragments of preovulatory human cumulus (cells plus extracellular matrix) were washed 3 times, incubated for 24 hr and the spent media and washes assayed for their ability to initiate the human sperm acrosome reaction (AR) in vitro. AR activity was present in the first two washes but not the third wash; however, AR activity was recovered in the spent medium after 3 X-washed fragments were incubated for 24 hr under conditions which maintained the viability of the cumulus cells. The spent media of preovulatory human mural granulosa cells contained AR-initiating activity after 1-3, 3-6, and 6-9 days of culture. The properties of the AR activity present in spent media of human cumulus fragments included resistance to loss of activity during treatment with pronase; resistance to loss of activity during treatment with chondroitinase ABC or bacterial hyaluronidase; heat stability after overnight incubation; lack of extraction by chloroform-methanol; an apparent molecular weight (MW) of 50,000, as determined by Sephadex G-75 column chromatography; conversion to a lower apparent MW activity by incubation with pronase. These properties are also characteristic of a fraction derived by Sephadex G-75 chromatography of preovulatory human follicular fluid which also has been shown to stimulate the human sperm acrosome reaction in vitro. The AR activity from spent media of human mural granulosa cells is also found in a 50,000 MW Sephadex G-75 fraction. We propose that the sources of the 50,000 MW human follicular fluid AR activity are the cumulus oophorus and the mural granulosa cells.     

Pepstatin A-sensitive aspartic proteases in lysosome are involved in degradation of the invariant chain and antigen-processing in antigen presenting cells of mice infected with Leishmania major

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2(d)) and in susceptible BALB/c mice (H-2(d)), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.     

Cytokine production by skin-derived mast cells: endogenous proteases are responsible for degradation of cytokines

The current study characterizes the cytokine protein (ELISA) and mRNA (gene array and RT-PCR) profiles of skin-derived mast cells cultured under serum-free conditions when activated by cross-linking of Fc epsilonRI. Prior to mast cell activation, mRNA only for TNF-alpha was detected, while after activation mRNA for IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF substantially increased, and for IL-4 it minimally increased. However, at the protein level certain recombinant cytokines, as measured by ELISAs, were degraded by proteases released by these skin-derived mast cells. IL-6 and IL-13 were most susceptible, followed by IL-5 and TNF-alpha; GM-CSF was completely resistant. These observations also held for the endogenous cytokines produced by activated mast cells. By using protease inhibitors, chymase and cathepsin G, not tryptase, were identified in the mast cell releasates as the likely culprits that digest these cytokines. Their cytokine-degrading capabilities were confirmed with purified chymase and cathepsin G. Soy bean trypsin inhibitor, when added to mast cell releasates, prevented the degradation of exogenously added cytokines and, when added to mast cells prior to their activation, prevented degradation of susceptible endogenous cytokines without affecting either degranulation or GM-CSF production. Consequently, substantial levels of IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF were detected 24-48 h after mast cells had been activated, while none were detected 15 min after activation, by which time preformed granule mediators had been released. IL-4 was not detected at any time point. Thus, unless cytokines are protected from degradation by endogenous proteases, cytokine production by human mast cells with chymase and cathepsin G cells may be grossly underestimated.     

Reptile embryos are not capable of behavioral thermoregulation in the egg

Reptile embryos have recently been observed moving within the egg in response to temperature, raising the exciting possibility that embryos might behaviorally thermoregulate analogous to adults. However, the conjecture that reptile embryos have ample opportunity and capacity to adaptively control their body temperature warrants further discussion. Using turtles as a model, we discuss the spatiotemporal constraints to movement in reptile embryos. We demonstrate that, as embryos grow, the internal egg space rapidly diminishes such that the temporal window for appreciable displacement is confined to stages that feature incomplete neuromuscular differentiation. During this time, muscles are insufficiently developed to actively and consistently control movement. These constraints are well illustrated by the Chinese softshelled turtle (Pelodiscus sinensis), the first reptile reported to behaviorally thermoregulate. Furthermore, sporadic embryo activity peaks after the temperature‐sensitive period in species with temperature‐dependent sex determination, thus nullifying the opportunity for embryos to exhibit control over this important phenotype. These embryonic constraints add to previously‐identified environmental constraints on behavioral thermoregulation by reptile embryos. We discuss alternative hypotheses to explain previously reported patterns of behavioral thermoregulation. Based on a holistic consideration of embryonic limitations, we conclude that reptile embryos are generally unable to adaptively behaviorally thermoregulate within the egg.     

IGF-I, IGFBP-2 and -3 but not GH concentrations are different in normal and poor growing piglets.

In this study we investigated the somatotropic axis in piglets with evident growth delay. Female Suffolk crossbred piglets (30 days old; N = 12) were divided into normal weight (10 +/- 0.9 kg) and poor growing subjects (7 +/- 0.5 kg) and bled for growth hormone (GH), Insulin-like growth factor-I (IGF-I), Insulin-like growth factor binding protein 2 and 3 (IGFBP-2 and -3) determination. Basal and induced-GH levels were not different in the groups. Plasma IGF-I concentrations were significantly different (p < 0.001): 101.8 +/- 9.8 ng x mL(-1) (normal weight group) and 39.5 +/- 4.0 ng x mL(-1) (poor growing group). IGFBP-2 and -3 concentrations were significantly (p < 0.001) lower in poor growing than in normal piglets. Piglet weight was positively correlated (r = 0.98, p < 0.001) with IGF-I and IGFBP-2 or -3 concentrations. Our data indicate that growth rate was not correlated to basal or secretagogue-induced GH secretion.     

Comparison of protein synthesis patterns in mouse cumulus cells and mural granulosa cells: effects of follicle-stimulating hormone and insulin on granulosa cell differentiation in vitro.

Successful development of mammalian oocytes requires correct interactions between developing oocytes and associated granulosa cells. Development of oocyte-granulosa cell complexes from preantral follicles in vitro does not produce oocytes competent to develop to blastocysts at the same frequency as for oocytes that develop in vivo. Addition of either FSH or insulin to cultures of oocyte-granulosa cell complexes does not improve the frequency of blastocyst development, and the combination of both insulin and FSH is deleterious. Here, high-resolution 2-dimensional PAGE (2D-PAGE) and computerized gel image analysis were used to compare patterns of protein synthesis in cumulus cells and mural granulosa cells of small antral follicles, and then to assess effects of FSH and insulin on the differentiation of oocyte-associated granulosa cells (OAGCs) in vitro. Culture of OAGCs without FSH or insulin resulted in failure to synthesize many proteins at rates characteristic of cumulus cells. Either hormone used alone caused many cumulus cell proteins that were decreased in control cultures to be synthesized at nearly normal cumulus cell rates, and also caused the synthesis of other proteins to be increased or decreased. The two hormones added together produced the greatest change in protein synthetic pattern, including overexpression or underexpression of many proteins not affected by either hormone alone. Addition of these hormones to culture media thus appeared insufficient to elicit a normal cumulus cell phenotype in OAGCs and could lead to complex changes in protein synthesis that may be deleterious to oocyte development. The high-resolution 2D-PAGE approach described here should be a valuable tool in studies on oocyte and granulosa cell development in vitro, since phenotype can be evaluated globally through the display of over 1000 newly synthesized proteins rather than relying upon the expression of just a few genes.     

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.     

Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor

Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro-dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35–40-day-old C57BL6/CBA F₁ hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/μl). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/μl for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing. © 1995 wiley-Liss, Inc.     

Free IGF-I, IGFBP-1, and the binary complex of IGFBP-1 and IGF-I are increased during human pregnancy

BACKGROUND/AIMS: To investigate changes in free insulin-like growth factor I (IGF-I) and IGF-binding protein 1 (IGFBP-1) complexed IGF-I during human pregnancy. METHODS: Overnight fasting serum was obtained in a longitudinal design from 11 women with non-complicated pregnancy at gestation weeks 6-10, 16-20, 24-28 and 35-38 and, for comparison, 5 weeks post-partum. All samples were analyzed for total and free IGF-I and IGF-II, IGFBP-3 and IGFBP-3 proteolysis, total and non-phosphorylated (np-) IGFBP-1, and IGFBP-1 complexed IGF-I. RESULTS: Total IGF-I was increased in late pregnancy (week 35-38) (p < 0.001), whereas free IGF-I was significantly increased by 77% already at week 6-10 (p = 0.004) and by 140% (p = 0.002) at week 34-38, when compared to post-partum levels. At weeks 16-20 and 24-28, levels of free IGF-I were not significantly different from post-partum levels. Significant IGFBP-3 proteolysis was detectable from week 6-10 and throughout pregnancy (p < 0.05). Total and np-IGFBP-1 were significantly increased from 16-20 weeks of pregnancy (both p < 0.05) and IGFBP-1 complexed IGF-I was increased 2-fold from week 16-20 and throughout pregnancy (p < 0.05). However, the saturation of IGFBP-1 remained constant at 27-29% during the study. CONCLUSION: We found evidence of increased free IGF-I and increased IGF-I in binary complexes during pregnancy, possibly caused by IGFBP-3 proteolysis and decreased ternary complex formation.     

Naive human CD4+ T cells are a major source of lymphotoxin alpha

It is generally accepted that immunologically naive T cells display a very restricted cytokine production profile consisting mainly of IL-2, which is used as an autocrine growth factor. Here we report that activated naive CD4+ T cells, of neonatal or adult origin, express very high levels of soluble lymphotoxin (LT) alpha (LTalpha3), as determined by ELISA, RNase protection assay, and intracytoplasmic staining. Besides LTalpha3 and IL-2, these cells also produce high levels of TNF-alpha together with significant amounts of IFN-gamma and IL-13. Naive cells also express LTbeta mRNA and the membrane form of LTalpha (LTalphabeta). On average, naive CD4+ T cells secrete four times more LTalpha3 than Th1-like cells, twice more than naive CD8+ T cells, and ten times more than B cells. Thus, naive T cells express a large spectrum of cytokines, mainly of the Th1 type, and the very high levels of LTalpha3/TNF-alpha that they release may play an hitherto unsuspected role in the early stage of T cell-dependent immune responses.     

Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope

Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat- shock proteins. Nuclear uptake studies were performed by microinjecting 125I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were approximately 24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120A in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with less than 10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm.