Fertilization Competence Research Articles

Sneaker Male Squid Produce Long-lived Spermatozoa by Modulating Their Energy Metabolism

Spermatozoa released by males should remain viable until fertilization. Hence, sperm longevity is governed by intrinsic and environmental factors in accordance with the male mating strategy. However, whether intraspecific variation of insemination modes can impact sperm longevity remains to be elucidated. In the squid Heterololigo bleekeri, male dimorphism (consort and sneaker) is linked to two discontinuous insemination modes that differ in place and time. Notably, only sneaker male spermatozoa inseminated long before egg spawning can be stored in the seminal receptacle. We found that sneaker spermatozoa exhibited greater persistence in fertilization competence and flagellar motility than consort ones because of a larger amount of flagellar glycogen. Sneaker spermatozoa also showed higher capacities in glucose uptake and lactate efflux. Lactic acidosis was considered to stabilize CO2-triggered self-clustering of sneaker spermatozoa, thus establishing hypoxia-induced metabolic changes and sperm survival. These results, together with comparative omics analyses, suggest that postcopulatory reproductive contexts define sperm longevity by modulating the inherent energy levels and metabolic pathways.

Ovarian ageing: the role of mitochondria in oocytes and follicles.

There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in the deterioration of oocyte quality in terms of competence and of the risk of transmitting mitochondrial abnormalities to the offspring. In contrast, some mtDNA haplogroups are protective against the decline of ovarian reserve. Quantitatively, mitochondrial biogenesis is crucial during oogenesis for constituting a mitochondrial pool sufficiently large to allow normal early embryonic development and to avoid the untimely activation of mitochondrial biogenesis. Ovarian ageing also seriously affects the dynamic nature of mitochondrial biogenesis in the surrounding granulosa cells that may provide interesting alternative biomarkers of oocyte quality. A fuller understanding of the involvement of mitochondria in cases of infertility linked to ovarian ageing would contribute to a better management of the disorder in the future.

Effect of atrazine on sperm mitochondrial function, acrosome reaction and fertilization competence: The bovine model

Effect of atrazine on sperm mitochondrial function, acrosome reaction and fertilization competence: The bovine model

Gamete activation: basic knowledge and clinical applications.

BackgroundThe first clues to the process of gamete activation date back to nearly 60 years ago. The mutual activation of gametes is a crucial event during fertilization. In the testis and ovaries, spermatozoa and oocytes are in a state of meiotic and metabolic quiescence and require reciprocal signals in order to undergo functional changes that lead to competence for fertilization. First, the oocyte activates sperm by triggering motility, chemoattraction, binding and the acrosome reaction, culminating with the fusion of the two plasma membranes. At the end of this cascade of events, collectively known as sperm capacitation, sperm-induced oocyte activation occurs, generating electrical, morphological and metabolic modifications in the oocyte.Objective and rationaleThe aim of this review is to provide the current state of knowledge regarding the entire process of gamete activation in selected specific animal models that have contributed to our understanding of fertilization in mammals, including humans. Here we describe in detail the reciprocal induction of the two activation processes, the molecules involved and the mechanisms of cell interaction and signal transduction that ultimately result in successful embryo development and creation of a new individual.Search methodsWe carried out a literature survey with no restrictions on publication date (from the early 1950s to March 2016) using PubMed/Medline, Google Scholar and Web of Knowledge by utilizing common keywords applied in the field of fertilization and embryo development. We also screened the complete list of references published in the most recent research articles and relevant reviews published in English (both animal and human studies) on the topics investigated.OutcomesLiterature on the principal animal models demonstrates that gamete activation is a pre-requisite for successful fertilization, and is a process common to all species studied to date. We provide a detailed description of the dramatic changes in gamete morphology and behavior, the regulatory molecules triggering gamete activation and the intracellular ions and second messengers involved in active metabolic pathways in different species. Recent scientific advances suggest that artificial gamete activation may represent a novel technique to improve human IVF outcomes, but this approach requires caution.Wider implicationsAlthough controversial, manipulation of gamete activation represents a promising tool for ameliorating the fertilization rate in assisted reproductive technologies. A better knowledge of mechanisms that transform the quiescent oocyte into a pluripotent cell may also provide new insights for the clinical use of stem cells.

Adenosine Modulates the Oocyte Developmental Competence by Exposing Stages and Synthetic Blocking during In Vitro Maturation.

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence..

Roles of epidermal growth factor (EGF)-like factor in the ovulation process.

Luteinizing hormone (LH) surge stimulates preovulatory follicles to induce the ovulation process, including oocyte maturation, cumulus expansion, and granulosa cell luteinization. The matured oocytes surrounded by an expanded cumulus cell layer are released from follicles to the oviduct. However, LH receptors are dominantly expressed in granulosa cells, but less in cumulus cells and are not expressed in oocytes, indicating that the secondary factors expressed and secreted from LH-stimulated granulosa cells are required for the induction of the ovulation process. Prostaglandin and progesterone are well-known factors that are produced in granulosa cells and then stimulate in both granulosa and cumulus cells. The mutant mice of prostaglandin synthase (Ptgs2KO mice) or progesterone receptor (PRKO mice) revealed that the functions were essential to accomplish the ovulation process, but not to induce the ovulation process. To identify the factors initiating the transfer of the stimuli of LH surge from granulosa cells to cumulus cells, M. Conti's lab and our group performed microarray analysis of granulosa cells and identified the epidermal growth factor (EGF)-like factor, amphiregulin (AREG), epiregulin (EREG), and β-cellulin (BTC) that act on EGF receptor (EGFR) and then induce the ERK1/2 and Ca2+-PLC pathways in cumulus cells. When each of the pathways was down-regulated using a pharmacological approach or gene targeting study, the induction of cumulus expansion and oocyte maturation were dramatically suppressed, indicating that both pathways are inducers of the ovulation process. However, an in vitro culture study also revealed that the EGFR-induced unphysiological activation of PKC in cumulus cells accelerated oocyte maturation with low cytostatic activity. Thus, the matured oocytes are not arrested at the metaphase II (MII) stage and then spontaneously form pronuclei. The expression of another type of EGF-like factor, neuregulin 1 (NRG1), that does not act on EGFR, but selectively binds to ErbB3 is observed in granulosa cells after the LH surge. NRG1 supports EGFR-induced ERK1/2 phosphorylation, but reduces PKC activity to physiological level in the cumulus cells, which delays the timing of meiotic maturation of oocytes to adjust the timing of ovulation. Thus, both types of EGF-like factor are rapidly induced by LH surge and then stimulate cumulus cells to control ERK1/2 and PKC pathways, which results in the release of matured oocytes with a fertilization competence.

Dicalcin, a zona pellucida protein that regulates fertilization competence of the egg coat in Xenopus laevis.

Fertilization is a highly coordinated process whereby sperm interact with the egg-coating envelope (called the zona pellucida, ZP) in a taxon-restricted manner, Fertilization triggers the resumption of the cell cycle of the egg, ultimately leading to generation of a new organism that contains hereditary information of the parents. The complete sperm-ZP interaction comprises sperm recognition of the ZP, the acrosome reaction, penetration of the ZP, and fusion with the egg. Recent evidence suggests that these processes involve oligosaccharides associated with a ZP constituent (termed ZP protein), the polypeptide backbone of a ZP protein, and/or the proper three-dimensional filamentous structure of the ZP. However, a detailed description of the molecular mechanisms involved in sperm-ZP interaction remains elusive. Recently, I found that dicalcin, a novel ZP protein-associated protein, suppresses fertilization through its association with gp41, the frog counterpart of the mammalian ZPC protein. This review focuses on molecular aspects of sperm-ZP interaction and describes the fertilization-suppressive function of dicalcin and associated molecular mechanisms. The amount of dicalcin in the ZP significantly correlates with alteration of the lectin-staining pattern within the ZP and the orientation pattern of ZP filaments, which may assist in elucidating the complex molecular mechanisms that underlie sperm-ZP interaction.

Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3.

Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised six and nine amino acid residues within dicalcin and twenty-three within gp41. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

Mammalian sperm interactions with the female reproductive tract.

The mammalian female reproductive tract interacts with sperm in various ways in order to facilitate sperm migration to the egg while impeding migrations of pathogens into the tract, to keep sperm alive during the time between mating and ovulation, and to select the fittest sperm for fertilization. The two main types of interactions are physical and molecular. Physical interactions include the swimming responses of sperm to the microarchitecture of walls, to fluid flows, and to fluid viscoelasticity. When sperm encounter walls, they have a strong tendency to remain swimming along them. Sperm will also orient their swimming into gentle fluid flows. The female tract seems to use these tendencies of sperm to guide them to the site of fertilization. When sperm hyperactivate, they are better able to penetrate highly viscoelastic media, such as the cumulus matrix surrounding eggs. Molecular interactions include communications of sperm surface molecules with receptors on the epithelial lining of the tract. There is evidence that specific sperm surface molecules are required to enable sperm to pass through the uterotubal junction into the oviduct. When sperm reach the oviduct, most bind to the oviductal epithelium. This interaction holds sperm in a storage reservoir until ovulation and serves to maintain the fertilization competence of stored sperm. When sperm are released from the reservoir, they detach from and re-attach to the epithelium repeatedly while ascending to the site of fertilization. We are only beginning to understand the communications that may pass between sperm and epithelium during these interactions.

To ICSI or Not to ICSI.

Intracytoplasmic sperm injection (ICSI) is the most effective assisted reproductive procedure enabling fertilization in severe forms of male factor indications and male gamete dysfunction. Reliability of ICSI has allowed the expansion of its application to other forms of infertility rendering it the most popular assisted reproduction technology (ART) insemination method worldwide. The concern related to the invasiveness of ICSI together with the arbitrary selection of the inseminating spermatozoon has induced the execution of studies to compare the performance of ICSI in non-male factor infertility with standard in vitro insemination approach. Not surprisingly, the outcome has evidenced that ICSI does not yield higher pregnancy rates than in vitro fertilization but functions invariably as a normalizer of fertilization mollifying the absent or low fertilization. The follow-up studies on ICSI children have evidenced that the procedure is safe and the slightly higher incidences of neonatal malformations or de novo gonosomal abnormalities are related to the genetics of the infertile couples. Furthermore, ICSI is accepted for some specific indications such as low number and poor morphology oocytes, thicker zona, excess polyspermia, PGD/PGS/PGT (preimplantation genetic diagnosis/preimplantation genetic screening/preimplantation genetic testing), discordant HCV/HIV (hepatitis C virus/human immunodeficiency virus) couples, in vitro maturation (IVM), and oocyte cryopreservation. Only the advent of new biomarkers in combination with routine semen analysis capable of identifying the fertilization competence of the spermatozoon can guide the reproductive physician toward the proper insemination method.

SLO3 auxiliary subunit LRRC52 controls gating of sperm KSPER currents and is critical for normal fertility

Following entry into the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that results in competence to fertilize ova. Associated with capacitation is an increase in membrane conductance to both Ca(2+) and K(+), leading to an elevation in cytosolic Ca(2+) critical for activation of hyperactivated swimming motility. In mice, the Ca(2+) conductance (alkalization-activated Ca(2+)-permeable sperm channel, CATSPER) arises from an ensemble of CATSPER subunits, whereas the K(+) conductance (sperm pH-regulated K(+) current, KSPER) arises from a pore-forming ion channel subunit encoded by the slo3 gene (SLO3) subunit. In the mouse, both CATSPER and KSPER are activated by cytosolic alkalization and a concerted activation of CATSPER and KSPER is likely a common facet of capacitation-associated increases in Ca(2+) and K(+) conductance among various mammalian species. The properties of heterologously expressed mouse SLO3 channels differ from native mouse KSPER current. Recently, a potential KSPER auxiliary subunit, leucine-rich-repeat-containing protein 52 (LRRC52), was identified in mouse sperm and shown to shift gating of SLO3 to be more equivalent to native KSPER. Here, we show that genetic KO of LRRC52 results in mice with severely impaired fertility. Activation of KSPER current in sperm lacking LRRC52 requires more positive voltages and higher pH than for WT KSPER. These results establish a critical role of LRRC52 in KSPER channels and demonstrate that loss of a non-pore-forming auxiliary subunit results in severe fertility impairment. Furthermore, through analysis of several genotypes that influence KSPER current properties we show that in vitro fertilization competence correlates with the net KSPER conductance available for activation under physiological conditions.

Diagnosis and prognosis of male infertility in mammal: the focusing of tyrosine phosphorylation and phosphotyrosine proteins.

Male infertility refers to the inability of a man to achieve a pregnancy in a fertile female. In more than one-third of cases, infertility arises due to the male factor. Therefore, developing strategies for the diagnosis and prognosis of male infertility is critical. Simultaneously, a satisfactory model for the cellular mechanisms that regulate normal sperm function must be established. In this regard, tyrosine phosphorylation is one of the most common mechanisms through which several signal transduction pathways are adjusted in spermatozoa. It regulates the various aspects of sperm function, for example, motility, hyperactivation, capacitation, the acrosome reaction, fertilization, and beyond. Several recent large-scale studies have identified the proteins that are phosphorylated in spermatozoa to acquire fertilization competence. However, most of these studies are basal and have not presented an overall mechanism through which tyrosine phosphorylation regulates male infertility. In this review, we focus of this mechanism, discussing most of the tyrosine-phosphorylated proteins in spermatozoa that have been identified to date. We categorized tyrosine-phosphorylated proteins in spermatozoa that regulate male infertility using MedScan Reader (v5.0) and Pathway Studio (v9.0).

A bioassy to measure fertilization competence of human spermatozoa

OBJECTIVE: To test a biomarker-based assay to diagnose sperm function which specifically predicts the ability to capacitate and fertilize. DESIGN: We assessed ganglioside GM1 localization in sperm as a biomarker to quantify the sub-population that could respond to capacitating stimuli and become fertilization competent. Specific patterns of sperm GM1 localization were quantified in basal and capacitating conditions on semen samples of consenting men with the aim of predicting fertilizing ability. MATERIALS AND METHODS: Semen parameters were evaluated according to WHO 2010 criteria. Samples were scored via fluorescence microscopy for GM1 localization patterns reflecting capacitation status in at least 200 sperm incubated under both standard and capacitating media. Based on preliminary data and normal timing of human sperm capacitation within 4hrs, we made observations at 1, 2, and 3hrs. Men were categorized as having normal or abnormal capacitation based on pattern frequencies compared to our reference ranges, and then clinical outcomes followed to assess predictive ability. RESULTS: In all tested men (n 1⁄4 63), average semen parameters were 58.1 20 x10/ml, motility of 47.8 8%, and normal morphology of 2.7 1%. We identified 31 men with scores matching the normal reference group, with baseline GM1 patterns of 17%-22%-28% in standard and 26%31%-38% in capacitating media, respectively. We identified 32 men with below reference values of 15%-20%-24% in standard and 20%-25%-29% in capacitating media. Semen parameters were comparable between the two groups. The population with normal range GM1 patterns had an IUI pregnancy rate of 45.2% (14/31) of which 8 (25.8%) generated at least one fetal heartbeat. Three additional couples in this group became pregnant on their own. For men with below-reference GM1 patterns, the IUI clinical pregnancy rate was only 6.3% (2/32; P1⁄4 0.03). In this cohort, 13 underwent ICSI and 6 became pregnant (46.2%). CONCLUSION: The GM1 assay reflected sperm fertilizing ability and could identify men prone to IUI failure irrespective of semen parameters. Assay results may guide selection of optimal ART treatment. Supported by: BioAccelerate NYC Prize through the Partnership Fund of New York City (A.J. Travis, Cornell). Dr. Travis taught methods to the Palermo lab, who independently recruited patients, performed assays and analyzed results.

Activation of Sperm EGFR by Light Irradiation is Mediated by Reactive Oxygen Species

To acquire fertilization competence, spermatozoa must undergo several biochemical and motility changes in the female reproductive tract, collectively called capacitation. Actin polymerization and the development of hyperactivated motility (HAM) are part of the capacitation process. In a recent study, we showed that irradiation of human sperm with visible light stimulates HAM through a mechanism involving reactive-oxygen-species (ROS), Ca(2+) influx, protein kinases A (PKA), and sarcoma protein kinase (Src). Here, we showed that this effect of light on HAM is mediated by ROS-dependent activation of the epidermal growth factor receptor (EGFR). Interestingly, ROS-mediated HAM even when the EGFR was activated by EGF, the physiological ligand of EGFR. Light irradiation stimulated ROS-dependent actin polymerization, and this effect was abrogated by PBP10, a peptide which activates the actin-severing protein, gelsolin, and causes actin-depolymerization in human sperm. Light-stimulated tyrosine phosphorylation of Src-dependent gelsolin, resulting in enhanced HAM. Thus, light irradiation stimulates HAM through a mechanism involving Src-mediated actin polymerization. Light-stimulated HAM and in vitro-fertilization (IVF) rate in mouse sperm, and these effects were mediated by ROS and EGFR. In conclusion, we show here that irradiation of sperm with visible light, enhances their fertilization capacity via a mechanism requiring ROS, EGFR and HAM.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Effect of rat medicated serum containing you gui wan on mouse oocyte in vitro maturation and subsequent fertilization competence.

You Gui Wan (YGW) is a classic herbal formula in traditional Chinese medicine (TCM) used for the clinical treatment of infertility. This study was to explore whether YGW has an impact on mouse oocyte maturation in vitro and subsequent fertilization competence. Rat medicated serum containing YGW was prepared by orally administrating YGW. Mouse immature oocytes were cultured with YGW medicated serum and compared to those cultured with or without normal rat serum or follicle-stimulating hormone (FSH). YGW medicated serum significantly increased the percentages of matured oocytes when compared to the groups with or without normal rat serum (P < 0.01). Furthermore, YGW medicated serum increased the rate of in vitro fertilization (IVF) when compared to the groups treated with FSH and with or without normal rat serum (P < 0.001). YGW medicated serum also had significant effects on the mRNA expressions of PKA, CREB, MAPK, PKC, PKG, and MPF and the concentrations of cAMP, cGMP, and NO in matured oocytes. These results indicate that YGW can promote mouse oocyte maturation and IVF in vitro. Signaling pathways, such as the cAMP/PKA/MAPK, the PKC-MAPK, and the NO-cGMP-PKG pathway, which are similar to those induced by FSH, may be responsible for this action.

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3(-)-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca(2+) and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.

Antecedents of mammalian fertility: Lessons from the heat-stressed cow regarding the importance of oocyte competence for fertilization and embryonic development

An embryo arises when the pronucleus of the newly-fertilized oocyte fuses with the pronucleus of the spermatozoa. The fate of the newlyformed embryo is an uncertain one, and many fail to survive to term. Whether an embryo can develop successfully depends in part on the environment of the maternal reproductive tract in which it resides and in part on its inheritance of genetic and non-genetic factors from the oocyte and sperm from which it was derived. The process of formation of male and female gametes is prolonged. In cattle, the species focused on in this paper, spermatogenesis takes about 61 days. Oocyte growth is also a slow process. Once an ovarian follicle emerges from the pool of resting follicles and initiates growth, it takes approximately 100 days to reach the point where ovulation can occur and the oocyte contained within the follicle is released (Figure 1). Antecedents of mammalian fertility: Lessons from the heat-stressed cow regarding the importance of oocyte competence for fertilization and embryonic development

Sperm attachment and penetration competence in the human oocyte: a possible aetiology of fertilization failure involving the organization of oolemmal lipid raft microdomains influenced by the ΔΨm of subplasmalemmal mitochondria

The roles of oolemmal lipid raft microdomains enriched in the ganglioside GM1 and the tetraspanin protein CD9 were investigated as causative agents in fertilization failure in human IVF where spermatozoa progress to the oolemma but fail to attach or, if attached, to penetrate. The findings show that specific configurations of GM1 lipid raft microdomains are consistent with attachment and penetration, while microdomains composed of CD9 lipid rafts, a protein known to be critical for penetration, do not appear to have a central role in the initial stages of attachment. The relative magnitude of the potential difference across the inner membrane (ΔΨm) in mitochondria localized to a stable subplasmalemmal domain appears to influence the organization of GM1 but not CD9 lipid raft microdomains in the corresponding oolemma. The findings present a novel view of how fertilization competence may be established in the human oocyte and a means by which certain fertilization failures that occur after conventional clinical IVF can be identified and explained in the unfortunate instance of fertilization arrest at the oolemma.This study investigated the role of the human oocyte plasma membrane (oolemma) in a type of fertilization failure that occurs after conventional IVF where a sperm reaches the oolemma but fails to attach or, if attached, penetrate the cytoplasm. We examined whether differences in the organization of certain molecular microdomains in the oolemma that likely interact with sperm correlated with fertilizability after IVF. The results show that certain microdomain configurations are consistent with fertilization, others permissive for attachment but not penetration, and still others inconsistent with attachment. When carefully examined microscopically, a sperm head was present on the oolemma in ∼35% of normal-appearing but unfertilized oocytes. This suggests a type of fertilization defect that may be both common and, possibly, a cause of ‘unexplained’ infertility in women. While the analytical methods used are presently not clinically applicable to characterize the molecular architecture of the oolemma prior to insemination, use in instances of fertilization failure after IVF may be beneficial with respect to the identification of a cause.VIDEO LINK: http://sms.cam.ac.uk/media/1400893