Altered actin cytoskeleton in ageing eggs of starfish affects fertilization process
Graphical abstract
Introduction
Optimal fertilization requires that oocytes should be fertilized at a specific meiotic stage, characteristic of the given species. For example, mammalian oocytes retain full competence for development only for several hours after ovulation during which their meiotic cycle is arrested at metaphase II [1]. If not fertilized within a certain stage or time of the maturation process, oocytes are destined to undergo time-dependent deterioration of their gamete quality [2,3]. The declining competence of the oocytes comes with alterations of the subcellular structures and improper interaction with the sperm at fertilization, which often leads to an aberrant embryonic development. At birth, a female body already possesses more than one million progenitor egg cells. Throughout life, the number of follicles decreases progressively, and only a few hundred will be released as mature eggs, whilst the remaining cells will eventually degrade and die [4]. This gradual decrease in gamete quantity is accompanied by a decline in quality, which is associated with a progressive decrease in female fertility, higher rate of fetal pathologies, and increased risk to develop diseases in adulthood [[5], [6], [7]]. However, the physiological process of ‘oocyte ageing’ has remained largely unknown, and it is of fundamental importance to understand the cause of the deleterious cellular and molecular changes associated with this phenomenon.
In this regard, starfish provide an invaluable experimental model system in which to analyze the molecular events taking place in the ageing oocyte. It has long been known that the optimal time frame for monospermic fertilization in starfish eggs is between the germinal vesicle breakdown (GVBD) and the extrusion of the first polar body [[8], [9], [10], [11], [12], [13]]. For the sake of discussion, the oocytes in this interval will be referred to as “mature eggs” in this study. Fertilization outside this time frame often displays polyspermy that inevitably leads to abnormal embryonic development. Indeed, when inseminated, the “immature” oocytes arrested at the GV-stage (prophase of the first meiotic division) are penetrated by multiple spermatozoa. During meiotic maturation, however, the cortex and cytoplasm of the oocytes undergo significant biochemical and morphological changes, enabling the eggs to show a normal Ca2+ response and monospermic incorporation. It has also been demonstrated that maturing oocytes of starfish become more sensitive to the Ca2+-mobilizing second messenger 1,4,5-inositol trisphosphate (InsP3) presumably owing to the extensive restructuring of the endoplasmic reticulum, the major intracellular Ca2+ store, which is concomitant with the significant reorganization of the actin cytoskeleton [[14], [15], [16]] in the cytoplasm and cortex of the maturing oocyte [[17], [18], [19]]. In line with the cortical restructuring, cortical granules are positioned just below the plasma membrane, thereby facilitating exocytosis of their contents into the perivitelline space at fertilization. The latter event leads to the separation of the vitelline coat and formation of the fertilization envelope (FE), which has been considered as the “mechanical block to polyspermy” [[20], [21], [22]]. However, it has been pointed out that starfish eggs treated with 1-MA for a prolonged period of time (i.e., overripe/ageing gametes) incorporate more than one spermatozoon despite the full elevation of the FE [11,23,24].
In the eggs of all animal species, interaction with the fertilizing spermatozoon gives rise to increases of intracellular Ca2+, which play a variety of roles toward successful egg activation [25,26]. Although the precise mechanism by which Ca2+ is increased has not been completely clarified, it is well known that the Ca2+ increase follows a precise spatio-temporal pattern characteristic of the species [[27], [28], [29]], which is necessary for the subsequent embryonic development [[30], [31], [32]]. In this regard, several studies have suggested that the reduced success rate of embryonic development after the fertilization of ageing oocytes could arise from abnormal calcium homeostasis [33] such as variations in the modality and frequency of the Ca2+ release at fertilization [34,35].
In echinoderm eggs, the cortical actin cytoskeleton plays a fundamental role in the modulation of the intracellular Ca2+ release, as judged by the experimental data obtained with various agents perturbing the structural organization of the actin filaments, i.e. actin drugs, the actin-severing/depolymerizing protein cofilin, anti-depactin antibody, heparin, and the sequester of PIP2 [[36], [37], [38], [39], [40]]. Accelerated actin polymerization at the time of Ca2+ increase after ionomycin treatment may also buffer the increase of Ca2+ [41]. The alteration of the actin cytoskeleton in the egg cortex with these agents commonly increased the rate of polyspermy and deregulated embryonic development. In particular, the latter consequence of ionomycin casts a warning sign to the use of this Ca2+ ionophore in the practice of artificial egg activation after intracellular sperm injection technique (ICSI) as a part of the protocol in advanced assisted reproductive technology (ART), which is now widely used to boost the successful implantation rate [[42], [43], [44], [45]]. During this procedure, mature oocytes remain in the oviduct or incubation media for several hours before fertilization or activation, and the oocyte ‘ageing’ in this context of procedural handling could be one of the main causes of fertilization failure, increased embryo mortality and birth defects in most commonly used ART [46]. It has also been shown that the cleavage failure and the formation of multiple asters in equine zygotes following ICSI can be associated with alterations of F-actin in the ageing oocytes [47].
In this study, we simulated the ageing process simply by leaving the oocytes in seawater for 24 or 48 hours before the addition of 1-MA, or by inducing overmaturation of freshly collected oocytes (1-MA, 6 h). After that, we investigated the effects of the ‘ageing’ on the cellular and molecular events of fertilization. To this end, we examined whether and how the changes of the actin cytoskeletal organization in the ageing eggs are linked to the rate of polyspermy and failed development, as well as to the alteration of the Ca2+ signaling patterns. We found that the ageing eggs at fertilization manifest an altered pattern of the Ca2+ release and deregulated actin dynamics, as well as an increased rate of polyspermy. These results underscore the importance of a spatio-temporal differentiation of the actin cytoskeleton structure of the egg necessary to ensure successful monospermic fertilization.
Section snippets
Preparation of oocytes and reagents
Starfish (Astropecten aranciacus) were collected in the Gulf of Gaeta and maintained in circulating cold seawater (16 °C). Female gonads were dissected from the central dorsal area near the arms and transferred to filter-sterilized seawater. Free individual oocytes were obtained by passing through gauze and rinsing in filtered seawater. After 30 min, only the fully grown immature oocytes containing the large nucleus (germinal vesicle, GV) were selected for the experiments. During the ‘ageing’
Incorporation of a single spermatozoon occurs only when the eggs are fertilized at the specific maturation stage
To verify the correlation between polyspermy and the oocyte maturation stage, we examined the number of spermatozoa that were incorporated into the oocytes and eggs upon insemination. As expected, GV-stage oocytes, which do not elevate the FE, were invariably polyspermic at fertilization, and many spermatozoa (7.98 ± 1.88, n = 80) penetrated the oocyte surface and entered the cytoplasm (Fig. 1 A). In sharp contrast, the eggs matured for 70 min with 1-MA incorporated only a single spermatozoon
Discussion
In this study, we examined the functional consequences of the structural changes of starfish eggs caused by overmaturation and by a condition simulating ageing. As aforementioned, the morphology of the oocyte drastically changes during the course of the meiotic progression, as evidenced by altered ultrastructure of the cortex and the actin cytoskeleton. According to the prevailing view, immature oocytes arrested at GV-stage are prone to polyspermy since they are unable to lift out the FE (Fig. 1
Declarations of interest
None.
Funding
This work was supported by the Stazione Zoologica Anton Dohrn Napoli (SZN), Italy. N. Limatola and F. Vasilev have been financially supported by a SZN postdoctoral fellowship (Assemble Plus project) and a SZN postdoctoral fellowship, respectively.
Acknowledgements
The authors are grateful to D. Caramiello for the careful maintenance of A. aranciacus and to G. Gragnaniello, F. Iamunno R. Graziano and G. Lanzotti for their technical assistance at the Morpho-Functional Analysis and Bioimaging Unit of the Stazione Zoologica Anton Dohrn in Napoli, Italy.
References (81)
- et al.
Fertilization and early cleavage in vitro of ageing bovine oocytes after maturation in culture
Theriogenology
(1992) - et al.
Female reproductive ageing: current knowledge and future trends
Trends Endocrinol. Metabol.
(2007) - et al.
Fetal origins of adult disease
Curr. Probl. Pediatr. Adolesc. Health Care
(2011) - et al.
Fertilization in echinoderms
Biochem. Biophys. Res. Commun.
(2012) - et al.
Calcium and actin in the saga of awakening oocytes
Biochem. Biophys. Res. Commun.
(2015) - et al.
Development of calcium release mechanisms during starfish oocyte maturation
Dev. Biol.
(1990) - et al.
Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization
Dev. Biol.
(1994) - et al.
The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton
J. Biol. Chem.
(2003) - et al.
Cortical granule translocation during maturation of starfish oocytes requires cytoskeletal rearrangement triggered by InsP3-mediated Ca2+ release
Exp. Cell Res.
(1999) - et al.
Morphological changes during maturation of starfish oocytes: surface ultrastructure and cortical actin
Dev. Biol.
(1983)
Maturation and fertilization of echinoderm eggs: role of actin cytoskeleton dynamics
Biochem. Biophys. Res. Commun.
Comparative biology of calcium signaling during fertilization and egg activation in animals
Dev. Biol.
Egg activation: upstream of the fertilization calcium signal
Biol. Cell
Calcium and fertilization: the beginning of life
Trends Biochem. Sci.
Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term
Dev. Biol.
Modulation of calcium signaling by the actin-binding protein cofilin
Biochem. Biophys. Res. Commun.
Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes
Dev. Biol.
Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules
Biochem. Biophys. Res. Commun.
The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle
Reprod. Biomed. Online
On behalf of the Oocyte Activation Study Group, Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study
Reprod. Biomed. Online
Assisted yes, but where do we draw the line?
Reprod. Biomed. Online
Assembly-disassembly of actin bundles in starfish oocytes: an analysis of actin-associated proteins in the isolated cortex
Dev. Biol.
Novel Ca2+ increases in the maturing oocytes of starfish during the germinal vesicle breakdown
Cell Calcium
Maturation and fertilization in starfish oocytes
Int. Rev. Cytol.
Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish
Dev. Biol.
Mechanical properties of the endoplasm in starfish oocytes
Exp. Cell Res.
Cytoplasmic cycle in meiotic division of starfish oocytes
Dev. Biol.
Germinal vesicle contents are required for the cytoplasmic cycle during meiotic division of starfish oocytes
Dev. Biol.
Fast polyspermy block and activation potential. Correlated changes during oocyte maturation of a starfish
Dev. Biol.
Hormone-induced loss of surface membrane during maturation of starfish oocytes: differential effects on potassium and calcium channels
Dev. Biol.
Antibody against the actin-binding protein depactin attenuates Ca2+ signaling in starfish eggs
Biochem. Biophys. Res. Commun.
Mechanisms underlying oocyte activation and postovulatory ageing
Reproduction
Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility
Hum. Reprod. Update
A model conforming the decline in follicle numbers to the age of menopause in women
Humanit. Rep.
The fetal and infant origins of disease
Eur. J. Clin. Investig.
Recherches sur la fecondation et le commencement de I'henogenie chez divers animaux
Geneve Soc Phys Mem
Etudes experimentales sur la maturation cytologigue et sur la parthenogenese artificielle ches les echinoderms
Arch Zool Exp 3 ser
The Biology of the Cell Surface
Differences in starfish oocyte susceptibility to polyspermy during the course of maturation
Biol. Bull.
Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte
Development
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Current affiliation: Centre de Recherche du Centre Hospitalier de l’Université de Montréal, (CRCHUM) Montréal, Canada.