Production of insulin-like growth factor-I by granulosa cells but not thecal cells is hormonally responsive in cattle

To determine whether the hormonal regulation of IGF-I production differs between granulosa and thecal cells in cattle, granulosa and thecal cells from bovine follicles were collected, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 24 h in serum-free medium with various hormones. In Exp. 1, granulosa cells were treated with 0 or 100 ng/mL of insulin and(or) 50 ng/mL of follicle-stimulating hormone (FSH), insulin plus 10 ng/mL of epidermal growth factor, or insulin plus 10 ng/mL of basic fibroblast growth factor. In Exp. 2, thecal cells were treated as described in Exp. 1 except that 100 ng/mL of luteinizing hormone (LH) was used instead of 50 ng/mL of FSH. In Exp. 3, granulosa and thecal cells were treated with 0 or 30 ng/mL of cortisol with or without 100 ng/mL of insulin, 300 pg/mL of glucagon, or glucagon plus insulin. In Exp. 4, granulosa and thecal cells were treated with 0 or 300 ng/mL of estradiol with or without 100 ng/mL of insulin and(or) 100 ng/mL of LH. At the end of treatment, medium was collected, concentrated with Centricon-3 concentrators, and assayed for IGF-I by radioimmunoassay. Cell numbers were determined by Coulter counting at the end of culture. Thecal cells produced low amounts of IGFI (0.48 +/- 0.04, 0.63 +/- 0.03, and 0.82 +/- 0.03 ng per 100,000 cells per 24 h in Exp. 2, 3, and 4, respectively), and this production was not influenced (P > 0.05) by the various treatments. In contrast, IGF-I production by granulosa cells (2.0 to 6.2 ng per 100,000 cells per 24 h) was influenced by treatment in Exp. 1, 3, and 4 and was greater than IGF-I production by thecal cells (Exp. 2, 3, and 4). Alone, insulin, FSH, LH, and cortisol (but not estradiol) each decreased (P < 0.05) granulosa-cell IGF-I production by 20 to 57%; combined treatments of insulin plus FSH or insulin plus cortisol decreased IGF-I production to levels seen with insulin alone. Glucagon had no effect (P > 0.10) on IGF-I production in the absence or presence of insulin. In the presence of insulin, epidermal growth factor, basic fibroblast growth factor, and estradiol decreased (P < 0.05) IGF-I production below that observed for insulin alone. These results indicate that, during follicular development in cattle, changes in intrafollicular levels of IGF-I may be due to hormonally-induced changes in granulosa-cell, but not thecal-cell, IGF-I production.     

Caffeine in fertilization medium is not essential for bovine IVF by fully capacitated spermatozoa

The purpose of this study was to examine: 1) whether caffeine in the fertilization medium under mineral oil is essential for bovine in vitro fertilization by fully capacitated spermatozoa, 2) the minimum concentration of caffeine that shows an adverse effect on the motility of preincubated spermatozoa. Cumulus-oocyte complexes with heterogeneous-appearing ooplasm were matured in in vitro culture for 24 h and used for insemination. The fertilization rates of the preincubated spermatozoa introduced into the fertilization medium containing 0 mM or 5 mM caffeine were examined. The fertilization rate of the spermatozoa introduced into the medium without caffeine (final concentration of caffeine at fertilization was 0.27-0.35 mM) was significantly higher than that in the medium with 5 mM caffeine (82.4% vs 55.2%, P<0.05). When the final concentration of caffeine at fertilization was reduced ten-fold (0.02-0.03 mM), the fertilization rate was not significantly improved (86.0%). The motility of the preincubated spermatozoa introduced into the fertilization medium containing 0-5 mM caffeine was examined. The sperm motility in the fertilization medium without caffeine was significantly higher than that in the fertilization medium with more than 2 mM caffeine. These results indicate that caffeine in the fertilization medium is not essential for bovine in vitro fertilization by fully capacitated spermatozoa, and that more than 2 mM caffeine has an adverse effect on preincubated (capacitated) sperm motility.     

Results of endocrinologic studies in culture media after coculture of bovine oocytes with granulosa cells

The steroid hormones progesterone (P.), and testosterone (T.) were radio-immunologically determined in 108 medium samples, following co-culturing of bovine oocytes with granulosa cells. P. and T. values recorded from a control group were lower with significance than those recorded from co-culturing groups, that is 72 +/- 21 ng/ml and 264 +/- 84 pg/ml as compared to 208 +/- 138 ng/ml and 2,168 +/- 1,595 pg/ml in the oocyte plus fresh granulosa cell co-culturing group as well as 364 +/- 215 ng/ml and 825 +/- 233 pg/ml in the oocyte plus pre-incubated granulosa cell co-culturing group. These rises were accompanied by decline in maturation rate, increase in oocyte degeneration, and rises in the rates of fertilisation and segmentation.     

Kisspeptin在牛卵巢颗粒细胞的表达及功能研究

为明确kisspeptin在牛卵巢颗粒细胞的表达和功能,本试验首先利用免疫荧光技术研究了kisspeptin在牛卵巢颗粒细胞的表达,随后利用MTT和ELISA技术研究了不同浓度的kisspeptin-10(Kp10)对牛卵巢颗粒细胞增殖和孕酮分泌的影响。结果表明,kisspeptin在牛卵巢颗粒细胞有表达,10、100、1 000nmol/L的Kp10处理24h,均可以极显著提高牛卵巢颗粒细胞活力(P0.01);100nmol/L的Kp10处理24、72h,可极显著提高牛卵巢颗粒细胞的活力(P0.01),但处理48h未能显著提高细胞活力(P0.05)。100nmol/L的Kp10处理24h,可显著提高牛卵巢颗粒细胞孕酮的分泌水平(P0.05),而10、1 000nmol/L的Kp10对孕酮分泌无显著影响(P0.05)。研究结果提示kisspeptin可促进牛卵巢颗粒细胞增殖和孕酮分泌。     

Gonadotropin-releasing hormone inhibition of LH stimulated progesterone secretion by porcine granulosa cells in vitro

GnRH has several direct actions on rat granulosa cells. Specific receptors for GnRH have been demonstrated on rat and human ovaries. Whether the porcine ovary has specific receptors for GnRH is still debated and the physiological actions of GnRH on porcine granulosa cells have not yet been clarified. Consequently, we have examined the actions of a GnRH agonist (GnRHa) on basal and LH stimulated progesterone secretion by porcine granulosa cells. GnRHa inhibited both basal and LH stimulated progesterone secretion by granulosa cells from medium (3-5 mm) and large (6-10 mm) antral follicles during 3 day incubations. LH stimulated progesterone secretion was more sensitive to inhibition than basal progesterone secretion. Studies on the time course for GnRHa inhibition of progesterone secretion indicated that the decrease in progesterone secretion occurred 48 to 72 hr after first exposure to GnRHa. Earlier inhibition occurred in only a fraction of the experiments. GnRHa did not have to be present during the time when inhibition occurred. Incubations of 2 days with GnRHa were just as effective as 3 day incubations at inhibiting progesterone secretion on day 3. Furthermore, a 30 min exposure to GnRHa on day 1 was just as inhibitory as a full 2 day incubation with GnRHa in inhibiting LH stimulated progesterone secretion on day 3. Incubation of the cells for 3 days prior to exposure of the cells to GnRHa did not alter the time course for GnRHa action. GnRHa did not alter the DNA content of the cultures in up to 6 day incubations or the number of viable cells attached to the wells in up to 3 day incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the mammalian target of rapamycin pathway and the effect of rapamycin on target expression and cellular proliferation in osteosarcoma cells from dogs

OBJECTIVE: To investigate activation of the mammalian target of rapamycin (mTOR) pathway and the antitumor effect of rapamycin in canine osteosarcoma cells. SAMPLE POPULATION: 3 established primary canine osteosarcoma cell lines generated from naturally developing tumors. PROCEDURES: Expression of total and phosphorylated mTOR and p70S6 kinase was assessed by use of western blot analysis in canine osteosarcoma cells with and without the addition of rapamycin. A clonogenic assay was performed to determine the surviving fraction of osteosarcoma cells at various concentrations of rapamycin. RESULTS: Total and phosphorylated mTOR and p70S6 kinase expression was evident in all 3 cell lines evaluated, which was indicative of activation of this pathway. Treatment with rapamycin resulted in a time-dependent decrease in phosphorylated mTOR expression and a lack of detectable phosphorylated p70S6 kinase. No detectable change in expression of total mTOR and total p70S6 kinase was identified after rapamycin treatment. The clonogenic assay revealed a significant dose-dependent decrease in the surviving fraction for all 3 cell lines when treated with rapamycin. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicated that mTOR and its downstream product are present and active in canine osteosarcoma cells. The pathway can be inhibited by rapamycin, and treatment of cells with rapamycin decreased the surviving tumor cell fraction. These data support the molecular basis for further investigation into the use of mTOR inhibitors as an antineoplastic approach for dogs with osteosarcoma.     

Timing of preovulatory LH surge and ovulation in superovulated sheep are affected by follicular status at start of the FSH treatment.

The aims of this study were to evaluate the chronology of periovulatory events (oestrus behaviour, LH surge and ovulation) in 16 superovulated Manchega sheep and to determine whether follicular status at start of the FSH supply might affect their occurrence. Mean timing for onset of oestrus behaviour was detected at 28.1 +/- 0.7 h after sponge withdrawal; the preovulatory LH surge and ovulation started at 37.2 +/- 0.7 h and 65.4 +/- 0.7 h after progestagen withdrawal, respectively. The intervals between oestrus, LH surge and ovulation were affected by a high individual variability, which might be the cause for reported decreased efficiency in embryo production. Current results also addressed the role of follicular status at start of the superovulatory treatment on the preovulatory LH surge and the ovulation. The interval LH surge-ovulation was increased in ewes with a growing dominant follicle at starting the FSH treatment (32.3 +/- 0.9 vs 28.6 +/- 0.5 h, p < 0.05). The developmental stage of the largest follicle at starting the superovulatory treatment also affected occurrence of LH surge and ovulation; follicles in growing phase advanced the occurrence of the LH surge and ovulation when compared to decreasing follicles (33.0 +/- 1.0 vs 43.5 +/- 1.1 h, p < 0.05, for LH peak and 60.7 +/- 1.1 vs 72.8 +/- 1.2 h, p < 0.05, for ovulation). Thus, only ewes with growing follicles ovulated prior to 55 h after sponge withdrawal; conversely, no sheep with decreasing follicles ovulated earlier than 67 h, when an 85.7% of the ewes bearing growing follicles has ovulated at 63 h.     

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200~ implanted heifers

Background： Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments： non-implanted （n = 5）, Revalor 200 for 28 d （n = 5）, or Revalor 200 for 84 d （n = 6）. Small follicle （1 to 5 mm） granulosa cells were isolated from each pair and incubated with phosphate buffered saline （n = 16） or 100 ng/mL follicle stimulating hormone （n = 16） for 24 h. Results： Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar （P = 0.22） to non-implanted heifers, while medium estradiol concentrations were increased （P 〈 0.10） at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein （P = 0.05） mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 （P 〈 0.10） or 84 d （P 〈 0.05） compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase （P= 0.57） and aromatase （P = 0.23） were demonstrated in implanted or non-implanted heifers. Conclusions： These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an alter     

Insights into the mechanism by which kisspeptin stimulates a preovulatory LH surge and ovulation in seasonally acyclic ewes: Potential role of estradiol

We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48 h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43 h. Blood samples were taken every 10 min for 15 h, starting 5 h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22 h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (± SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 ± 0.09 vs 2.83 ± 0.49 ng/mL (P = 0.004) for LH, 0.43 ± 0.05 vs 0.55 ± 0.03 ng/mL (P = 0.015) for FSH, and 9.34 ± 1.01 vs 11.51 ± 0.92 ng/mL (P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E₂) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E₂ concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24 h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28 h (beginning 5 h before the start of infusion), then every 2 h for the following 22 h. Kisspeptin infused for 24 h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12 h = 50%; 6 h = 12.5%). The plasma concentration of E₂ was greater in ewes with an LH surge compared to those without LH surges; mean (± SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 ± 0.16 vs 1.27 ± 0.13 pg/mL (P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E₂-positive feedback on gonadotropin secretion in acyclic ewes.     

Equine herpesvirus type 1 tegument protein VP22 is not essential for pathogenicity in a hamster model,but is required for efficient viral growth in cultured cells

VP22 is a major tegument protein of Equine herpesvirus type 1 (EHV-1) that is a conserved protein among alphaherpesviruses. However, the roles of VP22 differ among each virus, and the roles of EHV-1 VP22 are still unclear. Here, we constructed an EHV-1 VP22 deletion mutant and a revertant virus to clarify the role of VP22. We found that EHV-1 VP22 was required for efficient viral growth in cultured cells, but not for virulence in a hamster model.     

Hyaluronidase is not essential for the lethality of Erysipelothrix rhusiopathiae infection in mice

To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.     

Selenium stimulates estradiol production in bovine granulosa cells: possible involvement of nitric oxide

Reduction in fertility is well known to be possibly related to selenium deficiencies, even if target organ for selenium action is, at present, unclear. The present study was aimed to examine whether selenium directly influences granulosa cells. Bovine granulosa cells from different size follicles were used to investigate the effect of selenium (5 ng/ml), with or without bovine follicle-stimulating hormone (bFSH) (100 ng/ml), on proliferation and steroidogenesis. In addition, we sought to determine if selenium modulates the production of nitric oxide, which is known to play an important role in ovarian activity. Our data demonstrate that selenium significantly (P < 0.001) stimulates the proliferation of the cells from small follicles; moreover, it further potentiates the stimulatory effect of the gonadotropin in the same cells. Furthermore, selenium significantly (P < 0.01) augments E2 output by cells from both kinds of follicles. bFSH increases E2 production (P < 0.01) by cells from large follicles, whereas it exerts a stimulatory (P < 0.01) effect only in the presence of selenium in the cells from the small ones. The production of nitric oxide is significantly increased (P < 0.001) by bFSH, but only in cells from small follicles. Selenium inhibits (P < 0.001) nitric oxide production in cells from both kinds of follicles and significantly decreases (P < 0.001) bFSH-induced nitric oxide production in cells from the small ones. We conclude that selenium acts on granulosa cells by modulating their proliferation and E2 synthesis; moreover, its effect could be mediated, at least in part, through an inhibition of nitric oxide.     

Further phenotypic characterization of target cells for bovine leukemia virus experimental infection in sheep

To determine the phenotype of target cells for bovine leukemia virus (BLV) infection in sheep, we analyzed blood lymphocytes from BLV-infected clinically healthy and leukemic sheep by use of monoclonal antibodies. In clinically healthy and leukemic sheep that were BLV-infected, the blood concentration of T lymphocytes was within normal values, but the number of B lymphocytes was increased in several cases. In addition, the number of blood lymphocytes expressing the BLV antigen correlated well with that of B lymphocytes. Double immunofluorescence staining demonstrated that lymphocytes expressing BLV antigens bore B-cell but not T-cell surface markers. Moreover, neoplastic cells in the lymph nodes of leukemic sheep were stained immunohistochemically with an anti-B monoclonal antibody but not with any of anti-T monoclonal antibody tested, indicating that tumor cells are of B-lymphocyte origin. Collectively, these results show that BLV antigen-positive cells obtained from BLV-infected sheep that have no clinical signs and BLV-induced lymphosarcoma cells belong to the B-lymphocyte lineage.     

奶牛乳腺上皮细胞中Bta-miR-877靶基因的初步验证

利用高通量测序的方法筛选出在高脂奶牛和低脂奶牛中差异表达的miR-877和其预测的靶基因。通过生物信息学方法预测出edn1、ptgis可能是miR-877的靶基因,之后通过荧光定量检测miR-877以及其靶基因的表达量。结果显示,转染miR-877mimics组miR-877的相对表达量显著高于转染miR-877inhibitor组;靶基因的相对表达量,转染miR-877mimics组的edn1、ptgis基因表达水平均显著低于转染miR-877inhibitor组(P<0.01)。经GraphPad Prism6分析显示,edn1、ptgis基因的相对表达量与miR-877的表达量呈负相关,初步验证edn、ptgis是miR-877的靶基因。本试验为深入了解miR-877在乳脂质代谢调节中的作用提供依据,从而为生产实践提供一定的理论基础。     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Granzyme B-mRNA expression by equine lymphokine activated killer (LAK) cells is associated with the induction of apoptosis in target cells

Lymphokine-activated killer (LAK) cells are a subset of cytotoxic cells capable of lysing freshly isolated tumor cells. While LAK activity is typically measured using the (51)Cr-release assay, here we used a non-radioactive flow cytometric method to demonstrate equine LAK activity. Equine peripheral blood mononuclear cells (PBMC) were stimulated in vitro with recombinant human interleukin 2 (hIL-2) to generate LAK cells. An equine tumor cell line, EqT8888, labeled with carboxyfluorescein succinimidyl ester (CFSE) was used as target cells. Following incubation of the targets with different concentrations of LAK cells, Annexin V was added to identify the early apoptotic cells. With increasing effector to target cell ratios, EqT8888 apoptosis was increased. We also measured interferon-gamma, granzyme B and perforin mRNA expression in the LAK cell cultures as possible surrogate markers for cytotoxic cell activity and found granzyme B mRNA expression correlated best with LAK activity. Also, we found that the reduced LAK activity of young horses was associated with decreased granzyme B mRNA expression. Our results indicate that fluorescence-based detection of LAK cell activity provides a suitable non-radioactive alternative to (51)Cr-release assays and mRNA expression of granzyme B can be used as surrogate marker for these cytotoxic cells.