Reassessment of the proteomic composition and function of extracellular vesicles in the seminal plasma

Seminal plasma contains a high concentration of extracellular vesicles (EVs). The heterogeneity of small EVs or the presence of non-vesicular extracellular matter (NV) pose major obstacles in understanding the composition and function of seminal EVs. In this study, we employed high-resolution density gradient fractionation to accurately characterize the composition and function of seminal EVs and NV. We found that the seminal EVs could be divided into three different subtypes, namely high-density EV (EV-H), medium-density EV (EV-M), and low-density EV (EV-L) after purification using iodixanol,while NV was successfully isolated. EVs and NV display different features in size, shape and expression of some classic exosome markers. Both EV-H and NV could markedly promote sperm motility and capacitation compared with EV-M and EV-L, whereas only the NV fraction induced sperm acrosome reaction. Proteomic analysis results showed that EV-H, EV-M, EV-L, and NV had different protein components and were involved in different physiological functions. Further study showed that EV-M might reduce the production of sperm intrinsic reactive oxygen species (ROS) through Glutathione S-transferase Mu 2 (GSTM2).This study provides novel insights into important aspects of seminal EVs constituents and sounder footing to explore their functional properties in male fertility.

Discrete dynamic model of the mammalian sperm acrosome reaction: the influence of acrosomal pH and biochemical heterogeneity

The acrosome reaction (AR) is an exocytotic process essential for mammalian fertilization. It involves diverse biochemical and physiological changes that culminate in the release of the acrosomal content to the extracellular medium as well as a reorganization of the plasma membrane (PM) that allows sperm to interact and fuse with the egg. In spite of many efforts, there are still important pending questions regarding the molecular mechanism regulating the AR. Particularly, the contribution of acrosomal alkalinization to AR triggering in physiological conditions is not well understood. Also, the dependence of the proportion of sperm capable of undergoing AR on the biochemical heterogeneity within a sperm population has not been studied. Here we present a discrete mathematical model for the human sperm AR, based on the biophysical and biochemical interactions among some of the main components of this complex exocytotic process. We show that this model can qualitatively reproduce diverse experimental results, and that it can be used to analyze how acrosomal pH (pHa) and cell heterogeneity regulate AR. Our results confirm that pHa increase can on its own trigger AR in a subpopulation of sperm, and furthermore, it indicates that this is a necessary step to trigger acrosomal exocytosis through progesterone, a known physiological inducer of AR. Most importantly, we show that the proportion of sperm undergoing AR is directly related to the detailed structure of the population biochemical heterogeneity.

Expression and Possible Role of Nicotinic Acetylcholine Receptor ε Subunit (AChRe) in Mouse Sperm

The nicotinic acetylcholine receptor (nAChR) is one of the receptors of acetylcholine (ACh), and nicotine (NIC) acts as an agonist of this receptor. Among the nAChR subunits, we found that the ε subunit (AChRe) had approximately 10 to 1000 times higher level of mRNA expression in mouse testes than the other subunits. In this study, we aimed to elucidate the expression and localization of AChRe in the testes and spermatozoa of mice and clarify the effect of AChRe on sperm function. Immunocytochemistry showed that AChRe was expressed in the murine testes and spermatozoa. We found that AChRe was localized only in elongated spermatids from step 12 onwards in the testes. In spermatozoa, AChRe was localized in the head, especially in the anterior region of the acrosome, but only approximately 50% of spermatozoa showed this immunoreactivity. Additionally, we analyzed the effects of ACh and NIC on sperm acrosome reaction (AR) and found that both ACh and NIC suppressed the AR rate, which was restored by an AChRe-specific antagonist. These results suggest that AChRe may be a regulator of mammalian sperm AR.