Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging

Abstract

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

Per capita sperm metabolism is density dependent

“…FAD, NAD(P)H, ATP production] and studies (e.g. Massino et al, 2021 ) could not be included. We used oxygen consumption rate as our measure of metabolism because it is the most widely used metabolic parameter for measuring metabolism and allowed us to include the greatest number of species and studies, increasing the reliability and generality of our findings.…”

Autofluorescent Biomolecules in Diptera: From Structure to Metabolism and Behavior

“…These data support the idea that sperm undergo metabolic alteration(s) after transfer to the female seminal receptacle, a specialized organ where sperm can be stored and released for fertilization [ 106 ], and open valuable paths to investigate sperm functionality. The possibility to assay sperm metabolism in a label-free manner, in vivo and directly in its physiological environment, is particularly relevant for comparative studies on sperm metabolism across insect species, as shown in Hemiptera [ 107 , 108 ], but also to set-up protocols to apply to the seminal fluid of higher animals and humans. In these regards, FLIM was recently used to assess the rate of oxidative phosphorylation in Drosophila sperm [ 109 ].…”

Changes in body size and fertility due to artificial and natural feeding of laboratory common bed bugs (Heteroptera: Cimicidae)

“…In terms of basic research, the common bed bug ( Cimex lectularius ) is studied for genetic diversification between 2 naturally occurring host lineages, i.e., human-associated (HL) and bat-associated (BL) lineage (Balvín et al 2012, Booth et al 2015) and reproductive compatibility of the lineages addressing possible incipient sympatric speciation (Wawrocka et al 2015, Křemenová et al 2021). Further interests encompass rather general questions like traumatic insemination (Stutt and Siva-Jothy 2001, Reinhardt et al 2003), sperm metabolism (Massino et al 2022), reproductive immunity and sexually transmitted infections (Bellinvia et al 2020, Bellinvia et al 2020a, Thongprem et al 2020), the relationship with a primary symbiont, the Wolbachia bacteria (Hosokawa et al 2010, Balvín et al 2018), heteroplasmy (Robison et al 2015) or insecticide resistance-associated mutations (Dang et al 2015). Finally, the olfactory communication is studied in the context of (i) sexual (Siljander et al 2007), (ii) aggregation behavior (Weeks et al 2011, Olson et al 2017), and (iii) trap development (Gries et al 2015).…”