Cumulus Matrix Research Articles

Cumulus matrix selection leads to isolation of spermatozoa with better motility, morphology, and lower DNA fragmentation.

The objective of this study was to compare the motility, morphology, and levels of DNA fragmentation of spermatozoa subjected to conventional swim-up or cumulus matrix (CM) sperm selection. Semen samples were collected from 60 normozoospermic men at a private hospital between December 2021 and March 2022. After liquefaction, semen samples were separated into two portions - one part was subjected to conventional swim-up preparation and the remaining spermatozoa were subjected to CM selection. The CM was obtained by mechanical isolation from healthy donor oocytes. Semen analysis and evaluation of sperm were performed according to WHO 6th Edition Laboratory Manual and Kruger's strict criteria, respectively. Sperm DNA fragmentation (SDF, %) of the two preparations was evaluated using Halosperm G2 detection kit (Halotech, Madrid, Spain). Wilcoxon rank-sum test was used to compare the characteristics of spermatozoa obtained by the two preparations. Spermatozoa selected by CM showed significantly better rapidly progressive motility (43.5% vs. 30.6%, respectively, p<0.001), higher percentage of morphologically normal forms (14.0% vs. 9.0%, respectively, p<0.05), and lower levels of SDF (26.0% vs. 45.0%, p<0.05) in comparison to those prepared by conventional swim-up. Moreover, the incidence of multiple sperm defects was considerably lower in the samples that underwent CM selection compared to those that did not (30.0% vs. 49.0%, respectively, p<0.05).The selection by CM significantly increases sperm motility and reduces morphologically abnormal spermatozoa and DNA fragmentation rates in comparison to the conventional swim-up preparation. The application of this selection technique may increase chances of successful IVF outcomes.

Activin A promotes hyaluronan production and upregulates versican expression in human granulosa cells†.

Hyaluronan is a structural component of the expanded cumulus matrix, and hyaluronan synthase 2 is the major enzyme for the synthesis of hyaluronan in humans. Versican cross-links the hyaluronan-rich matrix to cumulus cells and is critical for successful ovulation. Activin A is a critical intrafollicular regulator of ovarian function. Although activin A has been shown to promote cumulus matrix expansion in mice, the functional role of activin A in the regulation of cumulus expansion in the human ovary remains to be elucidated. Using primary and immortalized human granulosa-lutein cells as study models, we provide the first data showing that activin A increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 in these cells. Additionally, activin A also promoted the expression of the hyaluronan-binding protein versican. Moreover, using inhibitor- and small interfering RNA-mediated inhibition approaches, we found that these stimulatory effects of activin A are most likely mediated through the type I receptor activin receptor-like kinase (ALK4)-mediated Sma- and Mad-related protein (SMAD2)/SMAD3-SMAD4 signaling pathway. Notably, the chromatin immunoprecipitation analyses demonstrated that SMAD4 could bind to human hyaluronan synthase 2 and VERSICAN promoters. The results obtained from this in vitro study suggest that locally produced activin A plays a functional role in the regulation of hyaluronan production and stabilization in human granulosa-lutein cells.

Functional Characterization of Fusion Proteins of the Tumor Necrosis Factor Alpha‐Induced Protein 6 (TNFAIP6) Domains

TNFAIP6 is an extracellular protein that is essential for cumulus cell extracellular matrix formation and female fertility in mice. A mammalian expression system was used to generate recombinant proteins of the full length TNFAIP6 and its individual domains, Link and CUB. These proteins contained a short amino terminal sequence essential for catalytic function and a carboxy‐terminal Fc region of mouse IgG2a for easy detection, and were termed TNFAIP6Fc, LinkFc and CUBFc, respectively. Hyaluronan‐binding of the fusion proteins was measured with an enzyme‐linked immunosorbent assay. The catalytic activity of the proteins was assessed by their ability to remove heavy chain 1 (HC1) from inter‐alpha‐trypsin inhibitor (IαI) using western‐blot. The ability of the fusion proteins to restore cumulus cell extracellular matrix formation was tested in a cell culture assay of Tnfaip6−/− cumulus cell‐oocyte complexes (COCs). Both TNFAIP6Fc and LinkFc were able to bind hyaluronan, with LinkFc showing much stronger binding at equimolar concentrations. CUBFc showed no significant binding to hyaluronan even at high concentrations. TNFAIP6Fc catalysed the removal of HC1 from IαI in a concentration dependent manner, indicating that the Fc tag did not interfere with catalytic function. LinkFc, CUBFc, and their combination were unable to remove the HC1 from IαI at high concentrations even though they contained the essential short amino‐terminal region. TNFAIP6Fc successfully restored cumulus cell extracellular matrix formation in Tnfaip6−/− COCs, while LinkFc, CUBFc, and their combination were unable to do so. LinkFc, CUBFc, and their combination did not disrupt normal cumulus cell extracellular matrix formation of Tnfaip6+/− COCs. Our results suggest that the catalytic function of TNFAIP6 requires both of its domains in addition to the previously identified serine residue in the amino‐terminal region, because neither domain alone is able to remove HC1 from IαI or support cumulus cell extracellular matrix formation in Tnfaip6−/− COCs. Furthermore, the absence of catalytic activity of both domains when combined together could imply a pivotal conformational interaction normally achieved by their covalent attachment. The finding that the LinkFc has stronger hyaluronan binding than TNFAIP6Fc could support the presence of such a conformational interaction that weakens the hyaluronan‐binding of the full‐length protein compared to the Link domain alone.Support or Funding InformationSupported by internal funding of Touro University Nevada

Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability

Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.

Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors - A practical approach.

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling.

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

Sperm Tail would Cause of Fertilization Failure due to the Genetic Problem

they suggested that human CatSper mutations with infertility phenotype are quite rare because the patient must have homozygous mutations (the identical mutation in both alleles) for CatSper-less phenotype. Therefore, they only have seen two patients like this. The patient from PNAS paper had homozygous microdeletion in chromosome 15 which also affected CatSper2 gene, two of his brothers had the same deletions and all three brothers were infertile. They only examined sperm cells from one of the brothers. The men with affected CatSper, just like CatSper knockout mice and they theoretically should have normal sperm count and normal motility. However, sperm hyperactivation will be affected. Their patients had severe oligo-therato-asthenozoospermia, but probably due to the removal of other genes, adjacent to CatSper 2 locus. They never ever seen fertile men with CatSper locuses affected on both alleles. If only one is affected, the other one will rescue the phenotype. Heterozygous mice are perfectly normal, hence they assumed that humans should have similar trend. Additionally, three brothers indeed have very similar genetics with identical microdeletion [2]. Turning to the role of progesterone in sperm, progesterone is definitely required for fertilization process. In general, progesterone, which is present throughout the female genital tract with peaks of levels in the cumulus matrix surrounding the oocyte, stimulates several sperm functions, including hyperactivation and acrosome reaction. However, from recent findings, there are the similar trends at progesterone concentration in insemination media of human IVF used was detected in both patients with or without fertilization failure, while the patient exhibiting fertilization failure shows the induction of hyperactivation. Therefore, this demonstrated that fertilization failure is not due to hyperactivation and progesterone concentration producing by cumulus cells in human [3]. After I told about that, they said that it is very interesting. This means that other factors are involved, such as an ability to undergo acrosome reaction and so on. In addition, I did not have a case when sperm did not respond to progesterone and did not hyperactivate, I am not sure that the sperm still can fertilize an egg. In conclusion, while lack of CatSper gene in patient could cause male infertility, semen quality of the patient might normal. Furthermore, progesterone can boost sperm motility at fertilization, however there was no clinical evidence that the lack of progesterone cause fertilization failure in human IVF.

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.

Molecular analysis of the cumulus matrix: insights from mice with O-glycan-deficient oocytes.

During follicle development, oocytes secrete factors that influence the development of granulosa and cumulus cells (CCs). In response to oocyte and somatic cell signals, CCs produce extracellular matrix (ECM) molecules resulting in cumulus expansion, which is essential for ovulation, fertilisation, and is predictive of oocyte quality. The cumulus ECM is largely made up of hyaluronan (HA), TNF-stimulated gene-6 (TSG-6, also known as TNFAIP6), pentraxin-3 (PTX3), and the heavy chains (HCs) of serum-derived inter-α-inhibitor proteins. In contrast to other in vivo models where modified expansion impairs fertility, the cumulus mass of C1galt1 Mutants, which have oocyte-specific deletion of core 1-derived O-glycans, is modified without impairing fertility. In this report, we used C1galt1 Mutant (C1galt1 FF:ZP3Cre) and Control (C1galt1 FF) mice to investigate how cumulus expansion is affected by oocyte-specific deletion of core 1-derived O-glycans without adversely affecting oocyte quality. Mutant cumulus–oocyte complexes (COCs) are smaller than Controls, with fewer CCs. Interestingly, the CCs in Mutant mice are functionally normal as each cell produced normal levels of the ECM molecules HA, TSG-6, and PTX3. However, HC levels were elevated in Mutant COCs. These data reveal that oocyte glycoproteins carrying core 1-derived O-glycans have a regulatory role in COC development. In addition, our study of Controls indicates that a functional COC can form provided all essential components are present above a minimum threshold level, and thus some variation in ECM composition does not adversely affect oocyte development, ovulation or fertilisation. These data have important implications for IVF and the use of cumulus expansion as a criterion for oocyte assessment.

The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition.

One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI), recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2), as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF) success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

Characterization of pig sperm hyaluronidase and improvement of the digestibility of cumulus cell mass by recombinant pSPAM1 hyaluronidase in an in vitro fertilization assay

Although sperm hyaluronidase is thought to play an important role in mammalian fertilization, the molecular function underlying these steps remains largely unknown. In mouse models, sperm-specific SPAM1 and HYAL5 hyaluronidase are believed to function in both sperm penetration of the cumulus matrix and sperm-ZP binding. However, gene-targeting studies for SPAM1 or HYAL5 show that hyaluronidases are not essential for fertilization, despite the fact that exogenous hyaluronidase can disrupt the cumulus matrix. Therefore, to evaluate whether sperm hyaluronidase is essential for mammalian fertilization, it is necessary to generate HYAL5/SPAM1 double-knockout mice. However, generating double-knockout mice is very difficult because these two genes exist on the same chromosome. Recently, investigators have begun to employ the pig model system to study human disease due to its similarities to human anatomy and physiology. In this study, we confirmed that pig SPAM1 exists as a single copy gene on chromosome 18 and is specifically expressed in the testis. In addition, we expressed recombinant pig SPAM1 in human embryonic kidney 293 cells and showed that these enzymes possess hyaluronidase activity. We also demonstrated that a polyclonal antibody against pig sperm hyaluronidase inhibits sperm–egg interactions in an in vitro fertilization (IVF) assay. Our results suggest that pig SPAM1 may play a critical role in pig fertilization and that recombinant SPAM1 can disperse the oocyte–cumulus complex in an IVF assay.

Evidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase A activation during human sperm capacitation.

Sperm capacitation involves an increase in intracellular Ca(2+) concentration as well as in protein kinase A (PKA)-dependent protein tyrosine (Tyr) phosphorylation. Interestingly, in humans, a decrease in extracellular Ca(2+) concentration ([Ca(2+)]e) during capacitation induces an increase in Tyr phosphorylation indicating the complexity of Ca(2+) signaling during this process. In view of this, in the present study we further investigated the Ca(2+)-mediated signaling pathways implicated in Tyr phosphorylation during human sperm capacitation. Results revealed that sperm incubation in a medium without added Ca(2+) (⊖ Ca(2+)) increased Tyr phosphorylation but did not modify PKA-mediated phosphorylation. Moreover, inhibition of either PKA or Src family kinase signaling cascades in ⊖ Ca(2+) down-regulated both PKA substrate and Tyr phosphorylations, indicating that the [Ca(2+)]e effects on Tyr phosphorylation depend on PKA targets. Inhibition of calmodulin or Ser/Thr protein phosphatase 2B also increased Tyr phosphorylation without affecting PKA-mediated phosphorylation, supporting the potential role of these Ca(2+) downstream effectors in the increase in Tyr phosphorylation observed in ⊖ Ca(2+). Experiments aimed to identify the kinase responsible for these observations revealed the presence of proline-rich tyrosine kinase 2 (PYK2), a focal adhesion kinase (FAK) family member, in human sperm, and the use of PF431396, an FAK inhibitor, supported the involvement of PYK2 in Tyr phosphorylation downstream of PKA activation. Results also showed that PYK2 was activated in ⊖ Ca(2+) as well as during capacitation and that PF431396 affected capacitated sperm motility, acrosome reaction and ability to penetrate both mouse cumulus matrix and zona-free hamster eggs. Together, our observations support PYK2 as an intermediary component of Ca(2+) signaling between PKA-mediated and Tyr phosphorylations that is required for achieving functional human sperm capacitation.

Comparison of outcomes from conventional intracytoplasmic sperm injection and selected hyaluronan-bound sperm injection

Mature sperm that bind hyaluronan, a major constituent of the cumulus matrix surrounding the oocyte, have been reported to have lower DNA fragmentation and potentially lower aneuploidy. The present study evaluated the use of selected hyaluronan-bound (HB) sperm in IVF-ICSI cycles.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of Heat shock protein 90

What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Versatile Action of Picomolar Gradients of Progesterone on Different Sperm Subpopulations

High step concentrations of progesterone may stimulate various sperm physiological processes, such as priming and the acrosome reaction. However, approaching the egg, spermatozoa face increasing concentrations of the hormone, as it is secreted by the cumulus cells and then passively diffuses along the cumulus matrix and beyond. In this context, several questions arise: are spermatozoa sensitive to the steroid gradients as they undergo priming and the acrosome reaction? If so, what are the functional gradual concentrations of progesterone? Do spermatozoa in different physiological states respond differentially to steroid gradients? To answer these questions, spermatozoa were confronted with progesterone gradients generated by different hormone concentrations (1 pM to 100 µM). Brief exposure to a 10 pM progesterone gradient stimulated priming for the acrosome reaction in one sperm subpopulation, and simultaneously induced the acrosome reaction in a different sperm subpopulation. This effect was not observed in non-capacitated cells or when progesterone was homogeneously distributed. The results suggest a versatile role of the gradual distribution of very low doses of progesterone, which selectively stimulate the priming and the acrosome reaction in different sperm subpopulations.

Involvement of the Serine Protease Inhibitor, SERPINE2, and the Urokinase Plasminogen Activator in Cumulus Expansion and Oocyte Maturation

The serpin peptidase inhibitor, clade E, member 2 (SERPINE2) inhibits urokinase-type plasminogen activator (PLAU) and tissue-type plasminogen activator. Higher SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes. The objective of this study was to evaluate whether high SERPINE2 levels in cumulus cells are associated with oocyte immaturity. Using the mouse cumulus–oocyte complex as an experimental model, the effects of elimination and overexpression of SERPINE2 in cumulus cells on cumulus expansion and oocyte maturation were assayed by in vitro maturation. Serpine2 and PLAU transcripts were the most highly expressed serpins and plasminogen activators, respectively. Their expression was coordinately regulated in cumulus cells during gonadotropin-induced oocyte maturation. Silencing of Serpine2 expression using small interfering RNAs or blockage of SERPINE2 protein using a specific antibody had no effect on oocyte maturation. However, overexpression of Serpine2 or exogenous supplementation with high levels of SERPINE2 impaired cumulus expansion and oocyte maturation, probably by decreasing hyaluronan synthase 2 (Has2) and versican (Vcan) mRNA expression. Amiloride, a specific PLAU inhibitor, also suppressed these processes. PLAU supplementation of the oocyte in vitro maturation medium caused earlier and more extensive expansion of cumulus cells and oocyte maturation that may be mediated by increased Has2 mRNA expression. However, these effects were neutralized by coincubation with SERPINE2 or amiloride and PLAU. In conclusion, SERPINE2 and PLAU are involved in cumulus expansion and oocyte maturation. High SERPINE2 levels impair these processes, probably by decreasing cumulus matrix gene expression as well as reducing cumulus hyaluronan contents and inhibiting PLAU activity. These findings may explain why cumulus cells surrounding immature human oocytes express high SERPINE2 levels.

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm*

[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium

To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC). Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling. A reproductive biology laboratory. Not applicable. Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib. Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA. In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 μM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 μM). Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/LH-supplemented medium.

Investigation of the mechanisms by which the molecular chaperone HSPA2 regulates the expression of sperm surface receptors involved in human sperm-oocyte recognition

A unique characteristic of mammalian spermatozoa is that, upon ejaculation, they are unable to recognize and bind to an ovulated oocyte. These functional attributes are only realized following the cells' ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as 'capacitation'. We have previously shown that this functional transformation is, in part, engineered by the modification of the sperm surface architecture leading to the assembly and/or presentation of multimeric sperm-oocyte receptor complexes. In this study, we have extended our findings through the characterization of one such complex containing arylsulfatase A (ARSA), sperm adhesion molecule 1 (SPAM1) and the molecular chaperone, heat shock 70kDa protein 2 (HSPA2). Through the application of flow cytometry we revealed that this complex undergoes a capacitation-associated translocation to facilitate the repositioning of ARSA to the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida (ZP) interactions. Conversely, SPAM1 appears to reorient away from the sperm surface, possibly reflecting its primary role in cumulus matrix dispersal preceding sperm-ZP recognition. The dramatic relocation of the complex was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol or broad spectrum protein kinase A (PKA) and tyrosine kinase inhibitors suggesting that it may be driven by alterations in membrane fluidity characteristics and concurrently by the activation of a capacitation-associated signal transduction pathway. Collectively these data afford novel insights into the sub-cellular localization and potential functions of multimeric protein complexes in human spermatozoa.

Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro

In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 μM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm- or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1-TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.