Development of a non-invasive tool for mammalian sperm
Keywords: intragamete deliver
male infertility
mammalian sperm
exosomes

Background: Male-factor problems are responsible for 50% of all cases of infertility worldwide. With treatment and research skewed towards female infertility, many of the issues responsible for male infertility are not being addressed. The approach used for infertility treatment is crucial not only to the individuals suffering from this condition, but also for future generations. In vitro fertilization and intracytoplasmic sperm injection remain the gold standards for assisted reproduction; however, the manipulation of sperm, oocytes, and embryos, can induce structural and functional damage. Moreover, there is no optimal alternative for the introduction of therapeutic compounds into sperm. Therefore, there is an urgent need to develop an effective tool for the delivery of factors that can aid sperm function and help to combat infertility. Exosomes are a subtype of extracellular vesicle secreted by all cells, including commercial cell lines. The proven delivery potential and biocompatibility of exosomes have created a new opportunity for the non-invasive treatment of health conditions, such as male infertility. Hypothesis: Exosomes derived from Human Embryonic Kidney 293T (HEK293T) cells provide a safe, efficient, scalable, and versatile approach for the delivery of cargo into sperm, thus, making it possible to tackle sperm-borne deficiencies, which are among the most prevalent causes of male infertility, in a wide range of clinical scenarios. Aim: To develop a mammalian-based manufacturing system based on HEK293T cells to obtain highly enriched samples of exosomes for efficient and safe compound delivery into mammalian sperm under conditions that mimic the fertilization scenario in vitro. Results: The approach used to isolate exosomes from HEK293T cells was scalable and achieved high yields of exosomes with preserved exosome traits and cell adhesion functions. Five-hours in vitro exposure of boar sperm with HEK293T-derived exosomes under capacitating conditions did not exert a detrimental impact upon various aspects of sperm functionality, including sperm motility, sperm viability, membrane integrity, and mitochondrial membrane potential (P<0.05). Visualization of boar sperm samples treated with HEK293T-derived exosomes revealed a spontaneous interaction within only 10 minutes of in vitro co-incubation that was irrespective of the incubation conditions used (capacitating and non-capacitating). Moreover, the proportion of sperm featuring internalized exosomes significantly increased with the time of incubation (P>0.005). An enrichment in CONNEXIN- 43 (Cx-43) in the HEK293T-derived exosomes was confirmed, and hence, the further in-depth analysis may shed light on the specific role of Cx-43-constituted hemichannels in exosome-sperm communication. Conclusions: This thesis has proven that HEK293T-derived exosomes may represent a promising research tool for studying sperm biology and provide new options for the intra-gamete delivery of compounds to enhance sperm function, thus creating exciting options for the treatment of infertility. Further work is required before being able to translate this approach to the clinic. Such work may involve the enhancement of targeting efficiency via functionalization of the HEK293T-derived exosomes with cell-penetrating peptides, evaluating the delivery efficiency of the cargo of interest, and confirming the safety of this tool with human gametes.
Note to reader: The work presented in this thesis has been heavily impacted by the COVID- 19 outbreak and therefore this should be taken into consideration before evaluating the quality and quantity of the experiments described herein.