117 Nerve growth factor in seminal plasma and its association with extracellular vesicles in South American camelids

Nerve growth factor (NGF) is a neurotrophin present in the seminal plasma of several mammalian species. In camelids, NGF of prostatic origin is deposited into the uterus at the time of mating and is the stimulus for inducing ovulation. It is hypothesised that seminal NGF reaches the brain via an endocrine route to trigger ovulation, but how and in what form NGF is transported from the endometrium to the central nervous system remains unknown. The objective of this study was to determine whether NGF is secreted into the seminal plasma in the form of extracellular vesicles. Semen was collected from llamas and alpacas (n = 5) by artificial vagina, and seminal plasma was separated from sperm by centrifugation. Semen was liquefied by treatment with collagenase at 37°C for 30 min. Extracellular vesicles were isolated from seminal plasma by sequential ultracentrifugation at 100 000 × g using a sucrose gradient. The pellet was then resuspended in PBS and herein will be referred as the EV preparation. The EV preparation was examined by transmission electron microscopy for extracellular vesicle visualisation, as well as by western blot (CD63 and NGF) and immunogold labelling (CD63). To discern whether NGF was free (soluble) or associated with EVs, immunoprecipitation followed by western blot against NGF on the EV preparation was used. Last, we used an in vitro PC12 cell bioassay to test the presence of NGF-like activity in the EV preparation vs. purified NGF (n = 3 wells/treatment). Ultracentrifugation in a sucrose gradient led to the identification of vesicle-like structures measuring 80 to 338 nm (169.9 ± 62.1 nm; mean ± s.d.) in the pellet. Western blot and immunogold labelling confirmed that the vesicles contained the protein CD63, a marker of Evs. In addition, western blot analysis identified NGF in EV preparations as well as in post-centrifugation seminal plasma (positive control). NGF was not immunoprecipitated from the EV preparation; instead, NGF was retained in the post-immunoprecipitation EV preparation, likely associated with EVs. As expected, incubation of PC12 cells with purified NGF induced a high rate of differentiation to a neuron-like phenotype. Incubation with 100 μg of the seminal EV preparation in the culture medium also triggered differentiation of the PC12 cells, but at a lower rate than free NGF (P < 0.05). Control wells remained undifferentiated (P < 0.05). Results document that NGF is secreted into the seminal plasma in a free form and in the form of extracellular vesicles, or becomes associated with extracellular vesicles during ejaculation into the seminal plasma of camelids. The abundance of seminal NGF in extracellular vesicles provides a rationale for the hypothesis that extracellular vesicles play an important role in the systemic absorption and transport of NGF to the central nervous system to elicit a preovulatory LH surge in the mated female camelid.

Research was supported by the Natural Sciences and Engineering Research Council of Canada.