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Visualization of immunoreactive growth hormone in cultured peripheral bovine lymphocytes
1Department of Functional Genomics and Bioregulation, Institute of Farm Animal Genetics Mariensee, FLI, Höltystr. 10, 31535 Neustadt, Germany.
Insights
Growth hormone (GH) is synthesized by bovine lymphocytes, with its presence varying during pregnancy. Early pregnancy shows lower GH immunoreactivity, which is enhanced by PHA-M stimulation.
Area of Science:
- Veterinary Immunology
- Reproductive Immunology
- Endocrinology
Background:
- Growth hormone (GH) release from immune cells in vitro is documented.
- Intracellular localization of GH in bovine lymphocytes during pregnancy requires investigation.
Purpose of the Study:
- To investigate the intracellular confinement of GH immunoreactivity in cultured bovine lymphocytes.
- To analyze GH expression in lymphocytes from cows in early and mid-pregnancy.
- To assess the effect of phytohemagglutinin-M (PHA-M) on lymphocytic GH synthesis.
Main Methods:
- Culturing peripheral blood lymphocytes from pregnant cows.
- Immunocytochemistry using a homologous GH-antibody.
- Double staining with CD3-antibody and confocal laser scanning microscopy.
Main Results:
- GH immunoreactivity was detected in the cytoplasm of small lymphocytes, indicating synthesis.
- Lowest GH immunoreactivity was observed in early pregnancy.
- PHA-M stimulation increased GH-positive cells in early pregnancy but not in mid-pregnancy.
Conclusions:
- Lymphocytic GH synthesis is specifically regulated during bovine pregnancy.
- Cellular mechanisms for increased GH expression in mid-pregnancy require further study.
- Pregnancy stage significantly influences GH production by bovine lymphocytes.
Abstract:
Growth hormone (GH) has been shown to be released by immune cells in vitro. Thus, the intracellular confinement of GH immunoreactivity was investigated in cultured bovine lymphocytes using con-focal microscopy. Peripheral blood lymphocytes from cows in early pregnancy (10-20 days post insemination; pi) or during mid-pregnancy (day 110-140 pi) were harvested and cultured for 48 h in presence of phytohemagglutinin-M (PHA-M) or served as controls. Thereafter, immunocytochemistry was conducted using a homologous GH-antibody. Double staining (GH-antibody and directly DYE 549 labeled CD3-antibody) was performed to classify the cells. Con-focal laser scanning was applied verifying the immunofluorescence labeling. Interestingly, the presence of GH immunoreactivity in the cytoplasm, which indicates GH synthesis, was restricted to small cells. Whereas, few T-like cells revealed surface bound GH. Lowest immunoreactivity, concerning the number of the total labeled cells as well as the intensity of labeling was recorded in early pregnancy. Stimulation with PHA-M enhanced total labeled cells in early pregnancy. In contrast, PHA-M had no such effects in mid-pregnancy. The results confirm the specific regulation of synthesis of lymphocytic GH during pregnancy in the cow. The identification of cells producing GH and the elucidation of the mechanisms underlying the expression of GH in larger number of cells during mid-pregnancy than in the early pregnancy need further investigations.
