No abstract

No abstract

BackgroundIn the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer.Methods and FindingsWe show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo.ConclusionsThese findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

The aim of this study was to investigate the direct effect of leptin on GH gene expression and secretion and the role of nitric oxide as a possible mediator in pig anterior pituitary cells. Pituitary cells from adult sows were treated for 4 or 24 h with rhleptin (from 0.1 nM to 1 microM) alone or in association with GHRH (10 nM) or hexarelin (10 nM). At the end of incubation, medium was collected for GH and nitric oxide determination by ELISA and Griess test, respectively. Total RNA was collected from cells, and GH gene expression was measured by RT-PCR. Leptin significantly (P < 0.001) stimulated GH secretion in both incubation periods. The maximum response was induced by 10 nM leptin; furthermore, a significant interaction (P < 0.002) between leptin and GHRH (P < 0.03) and between leptin and hexarelin was observed when the molecules were used in association. GH gene expression was significantly increased (at least P < 0.05) by hexarelin, GHRH, and leptin (1000 and 100 nM) after 24 h of treatment. Leptin (10 nM and 1 microM) significantly (P < 0.05) increased nitric oxide production, whereas S-nitroso-N-acetyl-penicillamine (from 0.01-1000 nM) significantly (P < 0.05) stimulated GH secretion. These data demonstrate that leptin directly influences GH regulation at the pituitary level, and nitric oxide may be involved in this function.

Nitric oxide (NO) is an important intra- and intercellular messenger controlling many biological processes. It is synthesized by NO synthases, which have been found also in granulosa cells. The present study examined whether NO is present in bovine follicular fluid and is produced by granulosa cells in culture. Secondly, it aimed to determine if NO affects the main parameters of granulosa cell function. The NO donor S-nitroso-L-acetyl-penicillamine (10(-3), 10(-4), 10(-5) M) was used to evaluate whether NO might influence steroidogenesis, proliferation and apoptosis in bovine granulosa cells collected from follicles divided according to their size in small (<5 mm) and large (>8 mm). The data demonstrate the presence of NO in follicular fluid and its production by granulosa cells in culture: the most active cells in producing NO are those from the small follicles, as confirmed by the NO levels in follicular fluid. This study also shows that NO donor significantly (P<0.001) inhibits progesterone (P4) and oestradiol 17beta (E2) production by the granulosa cells from both kinds of follicle; moreover, the highest concentration of NO donor significantly (P<0.001) inhibits DNA fragmentation in all the cells whereas the lowest concentration stimulates (P<0.001) cellular apoptosis only in granulosa cells from large follicles. NO donor does not seem to modify cell proliferation. Taken together these data lead point to NO as a local modulator of granulosa cell function.