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16 pages, 2977 KiB

Open AccessArticle

Sperm Capacitation and Kinematics in Phodopus Hamsters

by Ana Sanchez-Rodriguez, Ingrid I. D. Idrovo, Juan Antonio Rielo and Eduardo R. S. Roldan

Int. J. Mol. Sci. 2023, 24(22), 16093; https://doi.org/10.3390/ijms242216093 - 8 Nov 2023

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Abstract

This study was designed to analyze changes in the spermatozoa of three species of Phodopus hamsters incubated under different conditions. Cauda epididymal sperm were incubated for 4 h in modified Tyrode’s medium containing albumin, lactate, pyruvate, and Hepes (mTALP-H), in the same medium [...] Read more.

This study was designed to analyze changes in the spermatozoa of three species of Phodopus hamsters incubated under different conditions. Cauda epididymal sperm were incubated for 4 h in modified Tyrode’s medium containing albumin, lactate, pyruvate, and Hepes (mTALP-H), in the same medium with the addition of bicarbonate (mTALP-BH), or with bicarbonate and 20 ng/mL of progesterone (mTALP-BH+P4). Media with bicarbonate are believed to promote capacitation in rodent species. Sperm motility, viability, capacitation patterns, and kinematics were assessed at different times. Capacitation in live cells was quantified after staining with Hoechst 33258 and chlortetracycline. Patterns believed to correspond to non-capacitated cells (F pattern), capacitated, acrosome-intact cells (B pattern), and acrosome-reacted cells (AR pattern) were recognized. Kinematics were examined via computer-assisted sperm analysis (CASA). The results showed a decrease in total motility in all three species in different media, with a sharp decrease in progressive motility in bicarbonate-containing media (without or with progesterone), suggesting hyperactivated motion. However, none of the other signs of hyperactivation described in rodents (i.e., decrease in STR or LIN, together with an increase in ALH) were observed. F pattern cells diminished with time in all media and were generally lower in P. roborovskii and higher in P. campbelli. B pattern cells increased in mTALP-BH media in all species. Progesterone did not enhance the percentage of B pattern cells. Finally, AR pattern cells increased in all species incubated in different media, showing the highest percentage in P. roborovskii and the lowest in P. campbelli. Comparisons between media revealed that there were higher percentages of F pattern cells and lower percentages of B pattern cells over time in medium without bicarbonate (mTALP-H) in comparison to media containing bicarbonate (mTALP-BH; mTALP-BH+P4). Overall, changes consistent with the acquisition of capacitation and development of hyperactivated motility were found; however, further studies are required to better characterize media necessary to support the pathways involved in these processes in Phodopus species. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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13 pages, 2777 KiB

Open AccessArticle

Involvement in Fertilization and Expression of Gamete Ubiquitin-Activating Enzymes UBA1 and UBA6 in the Ascidian Halocynthia roretzi

by Hitoshi Sawada, Shukumi Inoue, Takako Saito, Kei Otsuka and Maki Shirae-Kurabayashi

Int. J. Mol. Sci. 2023, 24(13), 10662; https://doi.org/10.3390/ijms241310662 - 26 Jun 2023

Viewed by 1436

Abstract

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is [...] Read more.

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the “sperm reaction”. However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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13 pages, 1179 KiB

Open AccessArticle

Spawning-Induced pH Increase Activates Sperm Attraction and Fertilization Abilities in Eggs of the Ascidian, Phallusia philippinensis and Ciona intestinalis

by Noburu Sensui, Yosinori Itoh, Nobuhiko Okura, Kogiku Shiba, Shoji A. Baba, Kazuo Inaba and Manabu Yoshida

Int. J. Mol. Sci. 2023, 24(3), 2666; https://doi.org/10.3390/ijms24032666 - 31 Jan 2023

Cited by 4 | Viewed by 2547

Abstract

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo [...] Read more.

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5–6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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19 pages, 3179 KiB

Open AccessArticle

Epicatechin Prevents Cryocapacitation of Bovine Spermatozoa through Antioxidant Activity and Stabilization of Transmembrane Ion Channels

by Štefan Baňas, Filip Benko, Michal Ďuračka, Norbert Lukáč and Eva Tvrdá

Int. J. Mol. Sci. 2023, 24(3), 2510; https://doi.org/10.3390/ijms24032510 - 28 Jan 2023

Cited by 7 | Viewed by 2366

Abstract

Epicatechin (EPC) is a flavonoid belonging to the family of catechins; it has been described as a powerful scavenger of a wide spectrum of reactive oxygen species (ROS) and a modulator of ex vivo sperm vitality. In this study, we assessed the potential [...] Read more.

Epicatechin (EPC) is a flavonoid belonging to the family of catechins; it has been described as a powerful scavenger of a wide spectrum of reactive oxygen species (ROS) and a modulator of ex vivo sperm vitality. In this study, we assessed the potential protective abilities of EPC on cryopreserved bovine spermatozoa. We focused on conventional quality parameters, as well as the oxidative profile of spermatozoa alongside capacitation patterns, and expression profiles of proteins involved in the process of capacitation. Semen samples were cryopreserved in the presence of 25, 50 or 100 μmol/L EPC and compared to native semen (negative control) as well as ejaculates frozen in the absence of EPC (positive control). A dose-dependent improvement of conventional sperm quality parameters was observed following EPC administration, particularly in case of the sperm motility, membrane, acrosome and DNA integrity in comparison to the positive control. Experimental groups exposed to all EPC doses presented with a significantly lower proportion of capacitated spermatozoa as opposed to the positive control. While no significant effects of EPC were observed in cases of superoxide production, a significant decrease in the levels of hydrogen peroxide and hydroxyl radical were recorded particularly in the experimental groups supplemented with 50 and 100 μmol/L EPC. Western blot analysis revealed that supplementation of particularly 100 μmol/L EPC to the semen extender prevented the loss of the cation channel of sperm (CatSper) isoforms 1 and 2, sodium bicarbonate cotransporter (NBC) and protein kinase A (PKA), which play important roles in the process of sperm capacitation. In summary, we may hypothesize that EPC is particularly effective in the stabilization of the sperm membrane during the freeze–thaw process through its ability to quench ROS involved in damage to the membrane lipids and to prevent the loss of membrane channels crucial to initiate the process of sperm capacitation. These attributes of EPC provide an additional layer of protection to spermatozoa exposed to low temperatures, which may be translated into a higher post-thaw structural integrity and functional activity of male gametes. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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15 pages, 3688 KiB

Open AccessArticle

Metabolic Signature of Energy Metabolism Alterations and Excess Nitric Oxide Production in Culture Media Correlate with Low Human Embryo Quality and Unsuccessful Pregnancy

by Romina Pallisco, Giacomo Lazzarino, Gabriele Bilotta, Francesca Marroni, Renata Mangione, Miriam Wissam Saab, Maria Violetta Brundo, Alessandra Pittalà, Giuseppe Caruso, Elena Capoccia, Giuseppe Lazzarino, Barbara Tavazzi, Pasquale Bilotta and Angela Maria Amorini

Int. J. Mol. Sci. 2023, 24(1), 890; https://doi.org/10.3390/ijms24010890 - 3 Jan 2023

Cited by 2 | Viewed by 2776

Abstract

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those [...] Read more.

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those in the highest class give rise to successful pregnancy. Previous studies highlighted the potentiality of the analysis of metabolites in human embryo culture media, useful for the selection of embryos for implantation. In the present study, we analyzed in blind 66 human embryo culture media at 5 days after in vitro fertilization with the aim of quantifying compounds released by cell metabolism that were not present as normal constituents of the human embryo growth media, including purines, pyrimidines, nitrite, and nitrate. Only some purines were detectable (hypoxanthine and uric acid) in the majority of samples, while nitrite and nitrate were always detectable. When matching biochemical results with morphological evaluation, it was found that low grade embryos (n = 12) had significantly higher levels of all the compounds of interest. Moreover, when matching biochemical results according to successful (n = 17) or unsuccessful (n = 25) pregnancy, it was found that human embryos from the latter group released higher concentrations of hypoxanthine, uric acid, nitrite, and nitrate in the culture media. Additionally, those embryos that developed into successful pregnancies were all associated with the birth of healthy newborns. These results, although carried out on a relatively low number of samples, indicate that the analysis of the aforementioned compounds in the culture media of human embryos is a potentially useful tool for the selection of embryos for implantation, possibly leading to an increase in the overall rate of ART. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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14 pages, 4069 KiB

Open AccessArticle

The Roles of Two CNG Channels in the Regulation of Ascidian Sperm Chemotaxis

by Kogiku Shiba and Kazuo Inaba

Int. J. Mol. Sci. 2022, 23(3), 1648; https://doi.org/10.3390/ijms23031648 - 31 Jan 2022

Cited by 5 | Viewed by 3090

Abstract

Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian Ciona intestinalis (type A; also called C. robusta), an egg-derived sulfated steroid [...] Read more.

Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian Ciona intestinalis (type A; also called C. robusta), an egg-derived sulfated steroid called SAAF (sperm activating and attracting factor), induces both sperm motility activation and chemotaxis. Two types of CNG (cyclic nucleotide-gated) channels, Ci-tetra KCNG (tetrameric, cyclic nucleotide-gated, K⁺-selective) and Ci-HCN (hyperpolarization-activated and cyclic nucleotide-gated), are highly expressed in Ciona testis from the comprehensive gene expression analysis. To elucidate the sperm signaling pathway to regulate flagellar motility, we focus on the role of CNG channels. In this study, the immunochemical analysis revealed that both CNG channels are expressed in Ciona sperm and localized to sperm flagella. Sperm motility analysis and Ca²⁺ imaging during chemotaxis showed that CNG channel inhibition affected the changes in flagellar waveforms and Ca²⁺ efflux needed for the chemotactic turn. These results suggest that CNG channels in Ciona sperm play a vital role in regulating sperm motility and intracellular Ca²⁺ regulation during chemotaxis. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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17 pages, 4714 KiB

Open AccessArticle

Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

by Kamila Dilimulati, Misaki Orita, Yoshiki Yonahara, Fabiana Lica Imai and Naoto Yonezawa

Int. J. Mol. Sci. 2022, 23(2), 762; https://doi.org/10.3390/ijms23020762 - 11 Jan 2022

Cited by 5 | Viewed by 1856

Abstract

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in [...] Read more.

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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25 pages, 5439 KiB

Open AccessArticle

Does the Rainbow Trout Ovarian Fluid Promote the Spermatozoon on Its Way to the Egg?

by Vitaliy Kholodnyy, Borys Dzyuba, Marek Rodina, Hermes Bloomfield-Gadêlha, Manabu Yoshida, Jacky Cosson and Sergii Boryshpolets

Int. J. Mol. Sci. 2021, 22(17), 9519; https://doi.org/10.3390/ijms22179519 - 1 Sep 2021

Cited by 5 | Viewed by 3382

Abstract

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we [...] Read more.

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the “turn-and-run” behavior involving asymmetric flagellar beating and Ca²⁺ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)–sperm interaction reflect the specific features of the spawning process in this species. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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10 pages, 1386 KiB

Open AccessArticle

In Silico Reconstruction of Sperm Chemotaxis

by Masahiro Naruse and Midori Matsumoto

Int. J. Mol. Sci. 2021, 22(17), 9104; https://doi.org/10.3390/ijms22179104 - 24 Aug 2021

Cited by 1 | Viewed by 2473

Abstract

In echinoderms, sperm swims in random circles and turns in response to a chemoattractant. The chemoattractant evokes transient Ca²⁺ influx in the sperm flagellum and induces turning behavior. Recently, the molecular mechanisms and biophysical properties of this sperm response have been clarified. [...] Read more.

In echinoderms, sperm swims in random circles and turns in response to a chemoattractant. The chemoattractant evokes transient Ca²⁺ influx in the sperm flagellum and induces turning behavior. Recently, the molecular mechanisms and biophysical properties of this sperm response have been clarified. Based on these experimental findings, in this study, we reconstructed a sperm model in silico to demonstrate an algorithm for sperm chemotaxis. We also focused on the importance of desensitizing the chemoattractant receptor in long-range chemotaxis because sperm approach distantly located eggs, and they must sense the chemoattractant concentration over a broad range. Using parameters of the sea urchin, simulations showed that a number of sperm could reach the egg from millimeter-order distances with desensitization, indicating that we could organize a functional sperm model, and that desensitization of the receptor is essential for sperm chemotaxis. Then, we compared the model with starfish sperm, which has a different desensitization scheme and analyzed the properties of the model against various disturbances. Our approach can be applied as a novel tool in chemotaxis research. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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15 pages, 2137 KiB

Open AccessArticle

Occurrence of Calcium Oscillations in Human Spermatozoa Is Based on Spatial Signaling Enzymes Distribution

by Julia Korobkin, Fedor A. Balabin, Sergey A. Yakovenko, Ekaterina Yu. Simonenko and Anastasia N. Sveshnikova

Int. J. Mol. Sci. 2021, 22(15), 8018; https://doi.org/10.3390/ijms22158018 - 27 Jul 2021

Cited by 4 | Viewed by 2692

Abstract

In human spermatozoa, calcium dynamics control most of fertilization events. Progesterone, present in the female reproductive system, can trigger several types of calcium responses, such as low-frequency oscillations. Here we aimed to identify the mechanisms of progesterone-induced calcium signaling in human spermatozoa. Progesterone-induced [...] Read more.

In human spermatozoa, calcium dynamics control most of fertilization events. Progesterone, present in the female reproductive system, can trigger several types of calcium responses, such as low-frequency oscillations. Here we aimed to identify the mechanisms of progesterone-induced calcium signaling in human spermatozoa. Progesterone-induced activation of fluorophore-loaded spermatozoa was studied by fluorescent microscopy. Two computational models were developed to describe the spermatozoa calcium responses: a homogeneous one based on a system of ordinary differential equations and a three-dimensional one with added space dimensions and diffusion for the cytosolic species. In response to progesterone, three types of calcium responses were observed in human spermatozoa: a single transient rise of calcium concentration in cytosol, a steady elevation, or low-frequency oscillations. The homogenous model provided qualitative description of the oscillatory and the single spike responses, while the three-dimensional model captured the calcium peak shape and the frequency of calcium oscillations. The model analysis demonstrated that an increase in the calcium diffusion coefficient resulted in the disappearance of the calcium oscillations. Additionally, in silico analysis suggested that the spatial distribution of calcium signaling enzymes governs the appearance of calcium oscillations in progesterone-activated human spermatozoa. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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8 pages, 1875 KiB

Open AccessCommunication

The Organization of the Golgi Structures during Drosophila Male Meiosis Requires the Citrate Lyase ATPCL

by Patrizia Morciano, Maria Laura Di Giorgio, Liliana Tullo and Giovanni Cenci

Int. J. Mol. Sci. 2021, 22(11), 5745; https://doi.org/10.3390/ijms22115745 - 27 May 2021

Cited by 1 | Viewed by 2152

Abstract

During spermatogenesis, the Golgi apparatus serves important roles including the formation of the acrosome, which is a sperm-specific organelle essential for fertilization. We have previously demonstrated that D. melanogaster ATP-dependent Citrate Lyase (ATPCL) is required for spindle organization, cytokinesis, and fusome assembly during [...] Read more.

During spermatogenesis, the Golgi apparatus serves important roles including the formation of the acrosome, which is a sperm-specific organelle essential for fertilization. We have previously demonstrated that D. melanogaster ATP-dependent Citrate Lyase (ATPCL) is required for spindle organization, cytokinesis, and fusome assembly during male meiosis, mainly due to is activity on fatty acid biosynthesis. Here, we show that depletion of DmATPCL also affects the organization of acrosome and suggest a role for this enzyme in the assembly of Golgi-derived structures during Drosophila spermatogenesis. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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Review

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17 pages, 1593 KiB

Open AccessReview

Role of Integrins in Sperm Activation and Fertilization

by Veronika Merc, Michaela Frolikova and Katerina Komrskova

Int. J. Mol. Sci. 2021, 22(21), 11809; https://doi.org/10.3390/ijms222111809 - 30 Oct 2021

Cited by 14 | Viewed by 4292

Abstract

In mammals, integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell–cell, cell–extracellular matrix, and cell–pathogen interactions. Integrin receptors are an important part of signalization pathways and have an ability to transmit signals into and out [...] Read more.

In mammals, integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell–cell, cell–extracellular matrix, and cell–pathogen interactions. Integrin receptors are an important part of signalization pathways and have an ability to transmit signals into and out of cells and participate in cell activation. In addition to somatic cells, integrins have also been detected on germ cells and are known to play a crucial role in complex gamete-specific physiological events, resulting in sperm-oocyte fusion. The main aim of this review is to summarize the current knowledge on integrins in reproduction and deliver novel perspectives and graphical interpretations presenting integrin subunits localization and their dynamic relocation during sperm maturation in comparison to the oocyte. A significant part of this review is devoted to discussing the existing view of the role of integrins during sperm migration through the female reproductive tract; oviductal reservoir formation; sperm maturation processes ensuing capacitation and the acrosome reaction, and their direct and indirect involvement in gamete membrane adhesion and fusion leading to fertilization. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

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