- Title
- Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis
- Creator
- Zhou, Wei; Anderson, Amanda L.; Turner, Adrian P.; De Iuliis, Geoffry N.; McCluskey, Adam; McLaughlin, Eileen A.; Nixon, Brett
- Relation
- Funding BodyNHMRC.Grant Number1103176 http://purl.org/au-research/grants/nhmrc/1103176
- Relation
- Molecular Human Reproduction Vol. 23, Issue 10, p. 657-673
- Publisher Link
- http://dx.doi.org/10.1093/molehr/gax044
- Publisher
- Oxford University Press
- Resource Type
- journal article
- Date
- 2017
- Description
- Does dynamin regulate human sperm acrosomal exocytosis? Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDKI-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDKI elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis.
- Subject
- acrosome reaction; dynamin; human spermatozoa; infertility; sperm maturation
- Identifier
- http://hdl.handle.net/1959.13/1396200
- Identifier
- uon:34014
- Identifier
- ISSN:1360-9947
- Rights
- This is a pre-copyedited, author-produced version of an article accepted for publication in the Molecular Human Reproduction following peer review. The version of the above record is available online at: http://dx.doi.org/10.1093/molehr/gax044.
- Language
- eng
- Full Text
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