Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

Compensatory endocytosis occurs after cortical granule exocytosis in mouse eggs.

Compensatory endocytosis (CE) is one of the primary mechanisms through which cells maintain their surface area after exocytosis. Considering that in eggs massive exocytosis of cortical granules (CG) takes place after fertilization, the aim of this study was to evaluate the occurrence of CE following cortical exocytosis in mouse eggs. For this purpose, we developed a pulse-chase assay to detect CG membrane internalization. Results showed internalized labeling in SrCl2 -activated and fertilized eggs when chasing at 37°C, but not at a nonpermissive temperature (4°C). The use of kinase and calcineurin inhibitors led us to conclude that this internal labeling corresponded to CE. Further experiments showed that CE in mouse eggs is dependent on actin dynamics and dynamin activity, and could be associated with a transient exposure of phosphatidylserine. Finally, CE was impaired in A23187 ionophore-activated eggs, highlighting once again the mechanistic differences between the activation methods. Altogether, these results demonstrate for the first time that egg activation triggers CE in mouse eggs after exocytosis of CG, probably as a plasma membrane homeostasis mechanism.

Mechanisms involved in the contraceptive effects of ulipristal acetate.

The use of emergency contraception (EC) methods is increasing worldwide as it constitutes an effective way to prevent unplanned pregnancy after unprotected sexual intercourse. During the last decade, ulipristal acetate (UPA), a selective progesterone receptor modulator, has emerged as the most effective EC pill, and it is now recommended as first-line hormonal treatment for EC in several countries. Its principal mechanism of action involves inhibition or delay of follicular rupture, but only when administered during the follicular phase before the luteinizing hormone (LH) peak. However, considering the high efficacy of UPA, it is possible that it also exerts contraceptive effects besides ovulation. In the present review, we summarize and discuss the existing evidence obtained on the effect of UPA on sperm function and post-ovulatory events as potential additional mechanisms to prevent pregnancy. The bulk of evidence collected so far indicates that UPA would not affect gamete function; however, it could impair embryo-uterine interaction. Thus, besides the described effects on ovarian function, UPA contraceptive effectiveness might also be attributed to post-ovulatory effects, depending on the moment of the female cycle in which the drug is administered.

A single post-ovulatory dose of ulipristal acetate impairs post-fertilization events in mice.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator used for emergency contraception that has proven to be highly effective in preventing pregnancy when taken up to 120 h after unprotected sexual intercourse. Even though it may act mainly by delaying or inhibiting ovulation, additional effects of UPA on post-fertilization events cannot be excluded. Therefore, the aim of this study was to determine whether a single post-ovulatory dose of UPA could prevent pregnancy using the mouse as a pre-clinical model. Mated females received a single dose of UPA (40 mg/kg) on Day E1.5 or E2.5 (E0.5: copulatory plug detection) and post-fertilization events were evaluated. Our studies revealed that UPA administration produced a significant decrease in the number of conceptuses compared to control. Moreover, UPA-treated females exhibited a lower number of early implantation sites on Day E5.5, despite normal in vivo embryo development and transport to the uterus at E3.5. Administration of UPA produced histological and functional alterations in the uterine horns, i.e., a dyssynchronous growth between endometrial glands and stroma, with non-physiological combination of both fractions compared to controls, and a completely impaired ability to respond to an artificial decidualization stimulus. Altogether, our results show that the administration of a single post-ovulatory dose of UPA impairs mouse pregnancy probably due to an effect on embryo-uterine interaction, supporting additional effects of the drug on post-fertilization events. Although these studies cannot be performed with human samples, our results with the mouse model provide new insights into the mechanism of action of UPA as an emergency contraception method.

In vitro and in vivo effects of ulipristal acetate on fertilization and early embryo development in mice.

STUDY QUESTION: Does ulipristal acetate (UPA), a selective progesterone receptor modulator used for emergency contraception (EC), interfere with fertilization or early embryo development in vitro and in vivo? SUMMARY ANSWER: At doses similar to those used for EC, UPA does not affect mouse gamete transport, fertilization or embryo development. WHAT IS KNOWN ALREADY: UPA acts as an emergency contraceptive mainly by inhibiting or delaying ovulation. However, there is little information regarding its effects on post-ovulatory events preceding implantation. STUDY DESIGN, SIZE, DURATION: This was an in vitro and in vivo experimental study involving the use of mouse gametes and embryos from at least three animals in each set of experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: For in vitro fertilization experiments, mouse epididymal spermatozoa capacitated in the presence of different concentrations of UPA (0-1000 ng/ml) were used to inseminate cumulus-intact or cumulus-free eggs in the presence or absence of UPA during gamete co-incubation, and the percentage of fertilized eggs was determined. For in vivo fertilization experiments, superovulated females caged with proven fertile males were injected with UPA (40 mg/kg) or vehicle just before or just after mating and the percentage of fertilized eggs recovered from the ampulla was determined. To investigate the effect of UPA on embryo development, zygotes were recovered from mated females, cultured in the presence of UPA (1000 ng/ml) for 4 days and the progression of embryo development was monitored daily. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro studies revealed that the presence of UPA during capacitation and/or gamete co-incubation does not affect fertilization. Whereas the in vivo administration of UPA at the same time as hCG injection produced a decrease in the number of eggs ovulated compared with controls (vehicle injected animals, P < 0.05), no effects on fertilization were observed when UPA was administered shortly before or after mating. No differences were observed in either the percentage of cleaved embryos or the cleavage speed when UPA was present during in vitro embryo culture. LIMITATIONS, REASONS FOR CAUTION: Considering the ethical and technical limitations inherent to the use of human gametes for fertilization studies, the mouse model was used as an approach for exploring the potential effects of UPA on in vivo sperm transport and fertilization. Nevertheless, the extrapolation of these results to humans requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: This study presents new evidence on the lack of effect of UPA on gamete interaction and embryo development, providing new insights into the mechanism of action of UPA as an emergency contraceptive method with potential clinical implications. These new findings could contribute to increase the acceptability and proper use of UPA as an emergency contraceptive method. STUDY FUNDING/COMPETING INTERESTS: This study was partially supported by a National Agency of Scientific and Technological Promotion (ANPCyT), Argentina grants PICT 2011-061 to D.J.C. and PICT 2011-2023 to P.S.C. None of the authors has any competing interests to declare.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization.

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after acrosomal exocytosis and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Although midpiece contraction may occur in a subset of cells that undergo acrosomal exocytosis, live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility. Significant statement: In this work, we demonstrate that the helical structure of polymerized actin in the flagellum undergoes a rearrangement at the time of sperm-egg fusion. This process is driven by intracellular calcium and promotes a decrease in the sperm midpiece diameter as well as the arrest in motility, which is observed after the fusion process is initiated.

Study of the effect of ulipristal acetate on human sperm ability to interact with tubal tissue and cumulus-oocyte-complexes.

OBJECTIVE: Ulispristal acetate (UPA) is a selective progesterone receptor modulator widely used for emergency contraception (EC). The described main mechanism of action is by inhibiting or delaying ovulation; however, the postovulatory effects of the drug are still on debate. Therefore, the aim of this study was to determine whether UPA could interfere with human sperm fertilizing ability. STUDY DESIGN: Human motile spermatozoa were incubated under capacitating conditions with or without UPA, and then used to inseminate human tubal explants, mouse cumulus-oocyte complexes and zona-free hamster eggs. The ability of UPA to interact with human sperm progesterone (P)-binding sites was investigated by incubating the cells with fluorescent-labeled P and analyzing them by fluorescence microscopy. RESULTS: UPA did not affect the ability of human sperm to bind to human tubal tissue explants surface or to penetrate the mouse cumulus mass and the zona-free hamster eggs. In addition, concentrations of UPA much higher than those present in the plasma of EC pill users were required to bind to human sperm P-binding sites. CONCLUSIONS: Our study supports a lack of an agonist or antagonist action of UPA on different functional parameters associated with the fertilizing ability of human sperm. IMPLICATIONS: This study provides new functional evidence supporting that the contraceptive action of UPA is not related to effects on human sperm cells, contributing to a better understanding of the mechanism of action of UPA as EC.

From the epididymis to the egg: participation of CRISP proteins in mammalian fertilization.

Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-RIch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.