Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.

The present study was designed to determine if the dose of purified ovulation-inducing factor (OIF) from llama seminal plasma required to provoke an ovulatory response is physiologically relevant in terms of the proportion present in a normal ejaculate and to test the hypothesis that corpus luteum (CL) form and function are affected by OIF in a dose-dependent manner. Female llamas were assigned randomly to five groups (n = 10 per group) and given a single i.m. dose of 500, 250, 125, or 60 μg of purified OIF (representative of the amount present in 1/25th to 1/200th of a normal ejaculate) or 1 ml of PBS (control). Ovulation and CL development were monitored by transrectal ultrasonography. Blood samples were taken to measure plasma progesterone concentrations and to determine changes in plasma concentrations of luteinizing hormone (LH). The high dose of OIF (500 μg) was associated with the highest incidence of ovulation (P < 0.05), the greatest maximum CL diameter (P < 0.05), and the largest day-to-day profiles of CL diameter (P < 0.05) and plasma progesterone concentrations (P < 0.01). A rise in plasma LH concentration was apparent in all llamas that ovulated and was most rapid and highest in the high-dose group (P < 0.01). The low dose of OIF (60 μg) was minimally effective for induction of ovulation and the least luteotrophic, as evidenced by the smallest maximum CL diameter and the smallest day-to-day profiles for CL diameter and plasma concentrations of progesterone and LH. Responses were intermediate for the middle-dose groups (125 and 250 μg). We conclude that OIF from llama seminal plasma has a dose-dependent effect on ovulation rate and CL form and function in llamas and that the biological effect of OIF is evident at physiologically relevant doses (i.e., as little as 1/100th of that present in an ejaculate).

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Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus (ARC) play a key role in gonadotropin-releasing hormone (GnRH) pulse generation and gonadal steroid feedback, with kisspeptin driving GnRH release and neurokinin B and dynorphin acting as pulse start and stop signals, respectively. A separate cell group, expressing RFamide-related peptide-3 (RFRP-3) has been shown to be a primary inhibitor of GnRH release. Very little is known regarding these cell groups in the bovine. In this study, we examined the relative immunoreactivity of kisspeptin, dynorphin, and RFRP-3 and their possible connectivity to GnRH neurons in the hypothalami of periestrus and diestrus bovine. While GnRH and RFRP-3 immunoreactivity were unchanged, kisspeptin and dynorphin immunoreactivity levels varied in relation to plasma progesterone concentrations and estrous status. Animals with higher plasma progesterone concentrations in diestrus had lower kisspeptin and increased dynorphin immunoreactivity in the ARC. The percentage of GnRH cells with kisspeptin or RFRP-3 fibers in close apposition did not differ between estrous stages. However, the proportions of GnRH cells with kisspeptin or RFRP-3 contacts (∼49.8% and ∼31.3%, respectively) suggest direct communication between kisspeptin and RFRP-3 cells to GnRH cells in the bovine. The data produced in this work support roles for kisspeptin and dynorphin, within the KNDy neural network, in controlling GnRH release over the ovarian cycle and conveying progesterone-negative feedback onto GnRH neurons in the bovine.