Abstract
Current pest-management strategies rely on the use of 1080 poison, which only temporarily effects population sizes, and harms non-target indigenous species. Global appeal for fertility control, offering a more species-specific, permanent, and humane pest-management strategy, is growing. This research aimed to develop a sterility tool for managing mammalian pests in Aotearoa by evaluating the efficacy of a cell-targeting approach.
The proposed method involves delivery of two cytotoxins, saporin and D-(KLALAK)2, conjugated to targeting ligands (C6 and Kp*) with strong affinity for the kisspeptin receptor expressed on GnRH neurons, which regulate reproduction. This approach is hypothesized to induce apoptosis in GnRH neurons through internalization of the cytotoxin, impeding the animal’s downstream reproductive capacity.
Using key reproductive parameters, vaginal cytology and immunohistochemistry, estrous cycle patterns and GnRH cell survival were evaluated in female mice following central or peripheral injections of the conjugates. GnRH cell survival was analyzed in four target GnRH neuron-containing subregions of the preoptic area. Pilot studies employing a luteinizing hormone (LH) assay confirmed the abilities of the ligands and their conjugated forms to bind and activate the kisspeptin receptor, as evidenced by increased blood LH levels.
Initial assessments designed to demonstrate the process and reproductive consequences of GnRH cell death used a genetic mouse model which achieved GnRH cell ablation following administration of a diphtheria toxin. In treated mice, immunohistochemistry revealed a significant ~85% reduction in GnRH population size compared to untreated controls. However, estrous cycle patterns between control and treatment groups were not significantly different, implying that even a pronounced reduction in GnRH cells is insufficient to impede normal estrous cyclicity. Additionally, when the apoptosis marker⎯active caspase-3⎯was co-labelled with GnRH cells at 12h, 24h and 48h following diphtheria treatment in male mice, no apoptosis was detected within this timeline, suggesting later timepoints may be more appropriate.
Next, peripheral injections of C6-saporin and Kp*-saporin were tested for their effect on estrous cyclicity compared to controls. Kp*-saporin produced a stronger effect in disturbing normal frequency of estrous phases in the post-surgery period, suggesting superior targeting abilities of Kp* contrast to C6. However, neither of the conjugates abolished estrous cycles, so ix it was decided to deliver the conjugates directly to the region of the GnRH cell bodies in future experiments.
Finally, escalating doses (1μM, 10μM, 100μM) of Kp*-D-(KLALAK)2 were centrally administered, with no significant differences in estrous cycle patterns found. Furthermore, counting entire GnRH population collectively, no differences were observed in average GnRH cell numbers but interestingly, the GnRH population was significantly reduced specifically in the diagonal band nucleus at the 100μM dose. Additional analysis of reactive astrocyte numbers at 10μM and 100μM doses, to reveal signs of neuroinflammation, showed no significant differences compared to controls.
These findings show that despite a significant depletion of GnRH cells, female mice retained normal estrous cyclicity, implying retention of fertility. Additionally, they underscore the potential of high doses of targeted cytotoxin-conjugate Kp*-D-(KLALAK)2 to impede GnRH cell survival. This research provides valuable insights into the development of effective and humane pest-management for mammalian predators.