The antibiotic streptomycin assessed in a battery of in vitro tests for reproductive toxicology.

Streptomycin is one of the most widely used antibiotics and is frequently added to cell culture media to prevent bacterial growth. We tested streptomycin in a battery of in vitro assays for assessment of reproductive toxicity. The follicle bio-assay (FBA) is a multiparametric long-term follicle culture system mimicking ovarian function; in vitro fertilisation (IVF) of exposed oocytes enables gamete quality determination through fecundability; the mouse embryo assay (MEA) analyses pre-implantation embryo development whereas the embryonic stem cell test (EST) studies post-implantation embryotoxicity. The FBA revealed a concentration-dependent decrease in oocyte nuclear maturation during continuous exposure from 50 microg/ml streptomycin onwards, characterised by a significantly reduced polar body-rate (40% vs. 92% in the control group). Oocytes that remained arrested in metaphase I (germinal vesicle breakdown) had aberrant spindle formation. IVF of long-term exposed oocytes in the FBA to 50 microg/ml streptomycin resulted in a significantly lower fertilisation rate of 23% vs. 74% in the control group and were unable to develop to the blastocyst stage. The MEA revealed no effect at pre-implantation embryo development and quality. Furthermore, no embryo-toxic effects of streptomycin were observed in the EST. In conclusion, oocytes are vulnerable to streptomycin treatment. Long-term exposure might cause fertility problems in the female and caution should be taken using streptomycin in cell culture media for assisted reproductive technology (ART).

L-selectin and chemotaxis throughout bone marrow granulocyte maturation in the bovine.

Polymorphonuclear neutrophilic leukocytes (PMNL) play a pivotal role during inflammation. Bone marrow (BM) reserves are depleted as cells are released into circulation for recruitment to infection sites. Expression of L-selectin on the cell membrane allows neutrophils to roll along the activated endothelium. Whereas mechanisms leading to recruitment to infection sites are well established, expression of BM adhesion molecules in cows is limited. In this study, we assessed L-selectin expression and chemotactic response to zymosan-activated serum (ZAS) in bovine BM cells and in circulating neutrophils. Isolated blood PMNL and BM cells were used from 9 dairy cows, for quantifying L-selectin expression using flow cytometry, and from 12 dairy cows for chemotaxis studies. All granulocytic maturation stages expressed L-selectin. The percentage of cells fluorescing increased significantly in BM band and mature granulocytes and reached maximal expression on circulating neutrophils. Bone marrow band and segmented cells showed the highest L-selectin density. Chemotaxis through micropore filters in response to zymosan-activated fetal bovine serum was first observed in the myelocytic and metamyelocytic stages, and it increased with maturation and release into the blood stream. From these results, we conclude that L-selectin expression varies among stages of granulocytic maturation within the BM and differs from circulating PMNL. Further, BM cells are capable of migration starting at the metamyelocytic stage, and compared with BM cells, circulating neutrophils are more chemotactively active.

Differential effects of steroids and retinoids on bovine myelopoiesis in vitro.

Pregnancy and parturition are associated with physiological changes caused by steroid hormones. Alterations in number, maturity, and function of polymorphonuclear leukocytes observed in dairy cows at parturition suggest a common causative relationship with steroid hormones. This study was designed to investigate the effects of progesterone, 17-beta-estradiol, and hydrocortisone on the proliferation of bovine progenitor cells. An in vitro culturing system was used, and colonies were scored after 7 d of incubation. At low concentrations, 17-beta-estradiol inhibited proliferation of granulocyte progenitor cells. Hydrocortisone reduced growth of granulocyte and monocyte colonies, whereas myelopoiesis was not altered by progesterone. Furthermore, we studied the effect of retinoids on colony formation of bovine bone marrow cells. All-trans- and 9-cis-retinoic acid stimulated growth of granulocyte colonies and inhibited proliferation of the monocyte lineage. The addition of the 13-cis-isomer also increased numbers of granulocyte colony-forming units. This study indicates that steroid hormones may be responsible for alterations in the bovine hematopoietic profiles observed in circulation during the postpartum period. White blood cells, especially polymorphonuclear leukocytes, which are derived from bone marrow, are an important first line defense against mastitis. Therefore, these effects of steroids might contribute to the increased susceptibility of dairy cows to Escherichia coli mastitis. We furthermore hypothesize that an important role might be attributed to retinoic acid in its regulation of bovine myelopoiesis. Modulation of myelopoiesis in favor of the granulocyte lineage during the acute-phase reaction may be an adaptive mechanism designed to increase the capacity of first-line defense to intramammary infections.

All-trans retinoic acid is increased in the acute phase-related hyporetinemia during Escherichia coli mastitis.

Blood vitamin A profiles, including concentrations of retinol and its active metabolite retinoic acid, were assessed during the peripartum period and during experimentally induced Escherichia coli mastitis in heifers. Serum retinol decreased in all animals in the immediate postpartum period and normalized within 1 wk after parturition. No significant changes were detectable in the concentrations of retinoic acid isomers during puerperium. Following intramammary E. coli infusion, all cows showed moderate symptoms of systemic disease besides the local signs of inflammation. The presence of a systemic acute-phase reaction was documented by fever, increase in serum amyloid A, and decrease in serum albumin. Retinol concentration in serum also decreased spectacularly during coliform mastitis, and the decline was clearly related to the timing of the acute-phase response. Moreover, a significant increase of all-trans retinoic acid, mirrored by a lowering of 13-cis retinoic acid, was detected during the same time period. The 9-cis isomer of retinoic acid was present in all samples, but it remained below the quantification limit. Results confirmed the decrease in serum retinol during the peripartum period of dairy cows. Furthermore, the study established that profound changes in vitamin A metabolism occur during the acute-phase reaction of coliform mastitis in heifers. The bovine infection model reproduced the acute phase-related hyporetinemia, as previously observed in humans and rats. In addition, all-trans retinoic acid was found to be the most abundant circulating acid isomer during mastitis, providing an indication for a possible key role of all-trans retinoic acid in the modulation of the immune response.

Functional maturation during bovine granulopoiesis.

Granulocytic precursor cells undergo morphologic changes in the nucleus and the cytoplasm during the process of granulopoiesis, which takes place in the bone marrow. These changes are associated with the development of stage-specific proteins necessary for the highly specialized roles of polymorphonuclear leukocytes in phagocytosis, bacterial killing, and in mediating the inflammatory process. The objective of the current study was to sequence the various events that occur upon functional development of granulocytic bone marrow cells in the bovine species. Cells were obtained from the bone marrow of clinically healthy cows and separated into different stages of maturation using density gradient centrifugation. Three cellular fractions were obtained that were enriched for either early immature, late immature or mature granulocytic cells. Functions and receptor expressions assessed in the three maturation stages were:Fc-IgG2 receptor and CD11b expression, phagocytosis of Escherichia coli, respiratory burst activity, and cellular myeloperoxidase activity. Immature cells expressed already Fc-IgG2 receptor and CD11b on their cytoplasma membrane. Phagocytic ability was acquired in the myelocytic stage, but only the more mature forms were readily capable of phagocytosis. Promyelocytes, myelocytes and metamyelocytes showed no respiratory burst activity. Only band and segmented cells produced reactive oxygen species. Myeloperoxidase was present at all stages of maturity. Thus, each of the maturation stages was characterized by a selective expression of one or more functions and receptors. Therefore, sequential biochemical maturation is postulated during bovine granulopoiesis.

Separation of bovine bone marrow into maturation-related myeloid cell fractions.

A prerequisite for studies on bovine myeloid cells in relation to maturity is a reliable separation method, in order to obtain enriched and partially purified cell fractions of different maturation stages. Since current techniques for bovine bone marrow cell isolation fall short of this requirement, a technique for fractionating bovine bone marrow using a three-layer discontinuous Percoll gradient was developed. Three maturation-dependent myeloid cell fractions were obtained at specific densities, as maturation of cells is accompanied with a progressive density increase. Early immature myeloid cells, i.e. myeloblasts and promyelocytes, were found at a density of 1.060g/ml. Late immature myeloid cells, i.e. myelocytes and metamyelocytes, were retrieved at 1.080g/ml. Bands and segmented cells, representing the mature fraction, accumulated in the high-density pellet (>1.080g/ml). Myeloid cell populations were identified in each fraction by flow cytometry based on their forward and side scatter pattern. Confirmation was provided by light microscopy of flow cytometrically sorted myeloid populations, using morphological characteristics. The developed method provides a unique tool for studying maturation-dependent functions in bovine bone marrow.

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.