Pharmaceutical contraception is still nonexistent for half the world’s population – people with testes – and nearly half of all pregnancies are still unintended, even in the United States. These unintended pregnancy rates remain stubbornly high despite increasing access to existing contraception, which suggests that new contraceptives are needed to give every person the tools to make independent, voluntary decisions about their own fertility. Spermatozoa are the only human cells that are created in one body and fulfill their function in another, so with proper pharmaceutical properties, the same sperm-impairing drug could be delivered either in bodies that produce or receive sperm, thereby creating the world’s first non-hormonal, unisex contraceptive. To enable this goal, we need a better biological understanding of these unique and understudied cells, to identify which sperm physiological processes and sperm-specific proteins are the most targetable for pharmaceutical inhibition. The studies in this dissertation improve the foundational biological understanding of multiple processes necessary for a spermatozoon to fertilize an egg, from energy metabolism and motility to sperm-egg fusion, using diverse approaches such as in vitro cell physiology, molecular biology, electrophysiology, and structural biology. The first of these studies found that human sperm can maintain their ATP content in the presence of small-molecule uncouplers, even though uncouplers can depolarize the mitochondrial and plasma membranes and impair sperm motility; these results also suggest that the FDA-approved uncoupler niclosamide might be a useful ingredient in spermicide gels. Another study described here revealed that the human sperm-specific protein TMEM95 has a binding partner on the egg membrane and that this interaction is involved in sperm-egg fusion but not binding. The final project produced extremely high-resolution cryo-electron tomography structures of the human and mouse sperm flagellar axonemes, which revealed several species-specific and asymmetrically distributed protein structures connecting various microtubule pairs, with likely significance in the creation of flagellar motility. Together, these findings set the stage for future investigations which may identify new ways to target sperm-specific proteins for the production of novel non-hormonal unisex contraceptives.

Mitochondria play a pivotal role in the cellular physiology. Besides orchestrating cellular energy generation, they are also crucial to numerous other processes, including the maintenance of intracellular Ca2+ homeostasis and the production of reactive oxygen species 1–4. This thesis focuses on the two aforementioned functions and their regulation by endogenous and exogenous factors.

Mitochondria sustain calcium homeostasis by actively buffering cytosolic calcium. Calcium uptake into the mitochondrial matrix, in turn, regulates metabolism and energy production1. It is now well established that the primary mechanism of Ca2+ uptake in mitochondria is carried via the mitochondrial calcium uniporter (MCU) 5–8. Interestingly, to date, only a few endogenous regulators of the MCU channel have been identified 9–12. By using a targeted lipid mass spectrometry analysis and direct patch-clamp recordings from the inner mitochondrial membrane, we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is a novel endogenous regulator of the MCU complex.

The byproduct of mitochondrial calcium uptake and subsequently increased metabolism is the generation reactive oxygen species (ROS)13. While low levels of ROS are essential for various physiological processes, ROS overproduction can destroy mitochondria and lead to cell death14–17.

A significant portion of this thesis deals with the effects of increased formation of ROS, triggered by environmental toxins, on sperm fertility. Specifically, we tested four ubiquitous chemicals found in plastics: bisphenol A (BPA), di-2-ethylhexyl phthalate (DEHP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) on sperm fertilizing ability. Of all the tested toxins, DEHP demonstrated the most damaging impact on sperm fertility by increasing ROS production, altering the sperm maturation process, and impairing the acrosome reaction. We have concluded that even acute exposure to phthalates, such as DEHP, presents a significant risk to male fertility.