Natasa Sebkova

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer. DOI: http://dx.doi.org/10.7554/eLife.22187.001

Sperm-egg fusion: a molecular enigma of mammalian reproduction.

The mechanism of gamete fusion remains largely unknown on a molecular level despite its indisputable significance. Only a few of the molecules required for membrane interaction are known, among them IZUMO1, which is present on sperm, tetraspanin CD9, which is present on the egg, and the newly found oolema protein named Juno. A concept of a large multiprotein complex on both membranes forming fusion machinery has recently emerged. The Juno and IZUMO1, up to present, is the only known extracellular receptor pair in the process of fertilization, thus, facilitating the essential binding of gametes. However, neither IZUMO1 nor Juno appears to be the fusogenic protein. At the same time, the tetraspanin is expected to play a role in organizing the egg membrane order and to interact laterally with other factors. This review summarizes, to present, the known molecules involved in the process of sperm-egg fusion. The complexity and expected redundancy of the involved factors makes the process an intricate and still poorly understood mechanism, which is difficult to comprehend in its full distinction.

The slower the better: how sperm capacitation and acrosome reaction is modified in the presence of estrogens

In order for mammalian sperm to obtain a fertilizing ability, they must undergo a complex of molecular changes, called capacitation. During capacitation, steroidal compounds can exert a fast nongenomic response in sperm through their interaction with plasma membrane receptors, and activate crucial signaling pathways leading to time-dependent protein tyrosine phosphorylation (TyrP). Estrogen receptor beta was detected in epididymal mouse sperm; therefore, the effect of 17B-estradiol, estrone, estriol, and 17A-ethynylestradiol on mouse sperm capacitation in vitro was investigated. The effect was evaluated by positive TyrP in sperm heads and in the whole sperm lysates. Simultaneously, the state of the acrosome after the calcium ionophore-induced acrosome reaction was assessed. Generally, estrogens displayed a time and concentration-dependent stimulatory effect on sperm TyrP during capacitation. In contrast, the number of sperm that underwent the acrosome reaction was lower in the experimental groups. It has been demonstrated that both natural and synthetic estrogens can modify the physiological progress of mouse sperm capacitation. The potential risk in the procapacitation effect of estrogens can also be seen in the decreased ability of sperm to undergo the acrosome reaction. In conclusion, the capacitating ability of sperm can be significantly lowered by increasing the level of estrogens in the environment.

In vivo exposure to 17β-estradiol triggers premature sperm capacitation in cauda epididymis

Estrogens play a crucial role in spermatogenesis and estrogen receptor α knock-out male mice are infertile. It has been demonstrated that estrogens significantly increase the speed of capacitation in vitro; however this may lead to the reduction of reproductive potential due to the decreased ability of these sperm to undergo the acrosome reaction. To date the in vivo effect of estrogens on the ability of sperm to capacitate has not been investigated. Therefore, in this study, we exposed mice (n=24) to 17β-estradiol (E2) at the concentration of 20 ng/ml either during puberty from the fourth to seventh week of age (n=8), or continuously from birth for a period of 12 weeks (n=8) at which age the animals from both groups were killed. The capacitation status of epididymal and testicular sperm was analysed by tyrosine phosphorylation (TyrP) antibody (immunofluorescence and western blot) and chlortetracycline (CTC) assay. According to our results, in vivo exposure to increased E2 concentrations caused premature sperm capacitation in the epididymis. The effect of E2, however, seems reversible because after the termination of the exposure premature epididymal sperm capacitation is decreased in animals treated during puberty. Furthermore the changes in epididymal sperm capacitation status detected by TyrP and CTC positively correlate with plasma levels of E2 and the expression of the estrogen-dependent trefoil factor 1 (Tff1) gene in testicular tissue. Therefore, our data implicate that in vivo exposure to E2 under specific conditions leads to the premature capacitation of mouse sperm in epididymis with a potential negative impact on the sperm reproductive fitness in the female reproductive tract.

Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

The acrosome reaction (AR) is a process of membrane fusion and lytic enzyme release, which enables sperm to penetrate the egg surroundings. It is widely recognized that specific sperm proteins form an active network prior to fertilization, and their dynamic relocation is crucial for the sperm-egg fusion. The unique presence of the membrane cofactor protein CD46 in the sperm acrosomal membrane was shown, however, its behaviour and connection with other sperm proteins has not been explored further. Using super resolution microscopy, we demonstrated a dynamic CD46 reorganisation over the sperm head during the AR, and its interaction with transmembrane protein integrins, which was confirmed by proximity ligation assay. Furthermore, we propose their joint involvement in actin network rearrangement. Moreover, CD46 and β1 integrins with subunit α3, but not α6, are localized into the apical acrosome and are expected to be involved in signal transduction pathways directing the acrosome stability and essential protein network rearrangements prior to gamete fusion.

Characterization of tetraspanin protein CD81 in mouse spermatozoa and bovine gametes.

Sperm-egg interaction and fusion represent a key moment of fertilization. In mammals, it is not possible without the interaction of the tetraspanin superfamily proteins including CD81. A detailed immunohistochemical localization of CD81 was monitored in bovine oocytes during different maturation stages, as well as during early embryogenesis. In addition, characterization of CD81 was carried out in bovine and mouse sperm. In bovine eggs, CD81 was detected on the plasma membrane of the germinal vesicle, metaphase I and metaphase II oocytes. During fertilization, accumulation of CD81 molecules in the perivitelline space of fertilized oocytes, which appeared as vesicles associated with plasma membrane, was observed. In majority of bull-ejaculated sperm and caput, corpus and cauda epididymal sperm, as well as mouse cauda epididymal sperm, CD81 was found on the plasma membrane covering the apical acrosome. Although the process of capacitation did not influence the localization of CD81, it was lost from the surface of the acrosome-reacted spermatozoa in bull, in contrast to mouse sperm where there was a relocalization of the CD81 protein during acrosome reaction across the equatorial segment and later over the whole sperm head. The presented results highlight conservative unifying aspects of CD81 expression between cattle and mouse, together with mouse-specific traits in sperm CD81 behaviour, which emphasizes certain species-specific mechanisms of fertilization to be considered.

Kinetic analysis of decreased sperm fertilizing ability by fluorides and fluoroaluminates: a tool for analyzing the effect of environmental substances on biological events

Fluorides and fluoroaluminates decrease mouse sperm fertilizing potential by modifying the process of sperm preparation for fertilization, so-called capacitation, followed by acrosome reaction (AR). Capacitation was monitored by protein tyrosine phosphorylation (pTyr), and AR was induced consequently. The aim of this study was to apply kinetic analysis to the previously obtained dependences of pTyr and AR at capacitation times, and propose a mathematical theory for a mechanism when sperm maturation ability is amended by external stimuli. The experimental input data, previously obtained, are consistent with the proposed theory and the results of kinetic analysis show that sperm capacitation runs as two subsequent first-order steps. Firstly, an unstable intermediate is formed and then gradually decomposes. The time corresponding to the maximal production of the unstable intermediate is probably most suitable for sperm obtaining the ability to fertilize the egg. The presented calculations indicate that the application of kinetic analysis can serve as a tool to predict or confirm a course of biological events that are modified by external factors, and therefore the proposed theory shall be of interest to a broad scientific audience.