Effects of Sperm Conjugation and Dissociation on Sperm Viability In Vitro

Sperm conjugation is an unusual variation in sperm behavior where two or more spermatozoa physically unite for motility or transport through the female reproductive tract. Conjugation has frequently been interpreted as sperm cooperation, including reproductive altruism, with some sperm advancing their siblings toward the site of fertilization while ostensibly forfeiting their own ability to fertilize through damage incurred during conjugate break-up. Conversely, conjugation has been proposed to protect sensitive regions of spermatozoa from spermicidal conditions within the female reproductive tract. We investigated the possibility of dissociation-induced sperm mortality and tested for a protective function of conjugation using the paired sperm of the diving beetle, Graphoderus liberus. Sperm conjugates were mechanically dissociated and exposed to potentially damaging tissue extracts of the female reproductive tract and somatic tissue. We found no significant difference in viability between paired sperm and dissociated, single sperm. The results further indicate that the reproductive tract of female G. liberus might not be spermicidal and conjugation is not protective of sperm viability when damaging conditions do exist. Our results support the interpretation that, at least in some taxa, sperm conjugation is neither protective nor damaging to sperm viability

Identification of ejaculated proteins in the house mouse (Mus domesticus) via isotopic labeling

<p>Abstract</p> <p>Background</p> <p>Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (<it>Mus domesticus</it>), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with ¹⁵N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins.</p> <p>Results</p> <p>We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract.</p> <p>Conclusion</p> <p>Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract.</p

The evaluation of skin sensitization potential of the UVCB substance diisopentyl phthalate by in silico and in vitro methods.

Diisopentyl phthalate (DiPeP) is primarily used as a plasticizer or additive within the production of polyvinyl chloride (PVC), and has many additional industrial applications. Its metabolites were recently found in urinary samples of pregnant women; thus, this substance is of concern as relates to human exposure. Depending upon the nature of the alcohol used in its synthesis, DiPeP may exist either as a mixture consisting of several branched positional isomers, or as a single defined structure. This article investigates the skin sensitization potential and immunomodulatory effects of DiPeP CAS No. 84777-06-0, which is currently marketed and classified as a UVCB substance, by in silico and in vitro methods. Our findings showed an immunomodulatory effect for DiPeP in LPS-induced THP-1 activation assay (increased CD54 expression). In silico predictions using QSAR TOOLBOX 4.5, ToxTree, and VEGA did not identify DiPeP, in the form of a discrete compound, as a skin sensitizer. The keratinocyte activation (Key Event 2 (KE2) of the adverse outcome pathway (AOP) for skin sensitization) was evaluated by two different test methods (HaCaT assay and RHE assay), and results were discordant. While the HaCaT assay showed that DiPeP can activate keratinocytes (increased levels of IL-6, IL-8, IL-1α, and ILA gene expression), in the RHE assay, DiPeP slightly increased IL-6 release. Although inconclusive for KE2, the role of DiPeP in KE3 (dendritic cell activation) was demonstrated by the increased levels of CD54 and IL-8 and TNF-α in THP-1 cells (THP-1 activation assay). Altogether, findings were inconclusive regarding the skin sensitization potential of the UVCB DiPeP-disagreeing with the results of DiPeP in the form of discrete compound (skin sensitizer by the LLNA assay). Additional studies are needed to elucidate the differences between DiPeP isomer forms, and to better understand the applicability domains of non-animal methods in identifying skin sensitization hazards of UVCB substances