Uptake of exogenous bovine GH–pmKate2–N expression vector by rams spermatozoa

Abstract

Abstract Background Sperm-mediated gene transfer (SMGT) is a technique that utilizes the ability of the spermatozoa to take up exogenous DNA. Growth hormone is anabolic hormone that plays an important role in muscle-building process and milk production in all animals. High blood concentration of growth hormones (GH) was observed for animals that were genetically selected for high milk production or for low carcass fatness levels. The present study aimed to investigate and enhance the capacity of ovine spermatozoa to uptake exogenous growth hormone cDNA and its impact on sperm motility. The current study is an introduction for further future studies to produce transgenic Egyptian sheep characterized with high productive performance. Methods The growth hormone cDNA sequence was extracted from pituitary gland of Egyptian × Holstein (EH_GH) cattle and subcloned into the pmKate2–N vector to construct the EH_GH–pmKate2–N expression vector. The complete sequence of EH_GH mRNA was registered in GenBank (AC: KP221576). A total of three groups were assessed for the sperm uptake experiment, namely, negative control, positive control, and dimethyl sulfoxide (DMSO) groups; all treated groups were incubated with the EH_GH–pmKate2–N vector. The expression of EH_GH protein was detected in DH10B cells using a fluorescence microscope and the SDS polyacrylamide gel electrophoresis. Results The EH_GH–pmKate2–N vector was expressed in cultured Escherichia coli cells, and the molecular weight of EH_GH protein was 24,558 Da. The EH_GH–pmKate2–N vector was introduced efficiently into the heads of the spermatozoa in the DMSO and positive control groups. Incubation of the spermatozoa with the vector caused a significant reduction in progressive motility compared to the negative control. Conclusion The present results demonstrated the ability of ovine spermatozoa to take up the exogenous vector without notable deleterious effects on sperm motility. In subsequent studies, the successful introduction of the exogenous GH expression vector into the sperm head allows for the production of GH-transgenic sheep characterized by a high growth rate in order to reduce the meat shortage in Egypt.

Keywords

• Bovine growth hormone
• pmKate2–N expression vector
• Sperm uptake