Embryonic ß-Catenin Is Required for Priming of the Uterus to Implantation.

Repeated implantation failure is a major cause of infertility among healthy women. Uterine ß-catenin (CTNNB1) plays a critical role in implantation. However, the role of embryonic CTNNB1 during implantation remains unclear. We addressed this topic by analyzing mice carrying Ctnnb1-deficient (Ctnnb1Δ/Δ) embryos. Ctnnb1Δ/Δ embryos were produced by intercrossing mice bearing Ctnnb1-deficient eggs and sperms. We found that Ctnnb1Δ/Δ embryos developed to the blastocyst stage; thereafter, they were resorbed, leaving empty decidual capsules. Moreover, leukemia inhibitory factor, a uterine factor essential for implantation, was undetectable in Ctnnb1Δ/Δ blastocysts. Furthermore, CDX2, a transcription factor that determines the fate of trophectoderm cells, was not observed in Ctnnb1Δ/Δ blastocysts. Intrauterine injection with uterine fluids (from control mice) and recombinant mouse leukemia inhibitory factor proteins rescued the uterine response to Ctnnb1Δ/Δ blastocysts. These results suggest that embryonic CTNNB1 is required for the secretion of blastocyst-derived factor(s) that open the implantation window, indicating that the uterine response to implantation can be induced using supplemental materials. Therefore, our results may contribute to the discovery of a similar mechanism in humans, leading to a better understanding of the pathogenesis of repeated implantation failure.

Regulatory role of CsuR (YiaU) in determination of cell surface properties of Escherichia coli K-12.

Genomic SELEX screening was performed to identify the binding sites of YiaU, an uncharacterized LysR family transcription factor, on the Escherichia coli K-12 genome. Five high-affinity binding targets of YiaU were identified, all of which were involved in the structures of the bacterial cell surface such as outer and inner membrane proteins, and lipopolysaccharides. Detailed in vitro and in vivo analyses suggest that YiaU activates these target genes. To gain insight into the effects of YiaU in vivo on physiological properties, we used phenotype microarrays, biofilm screening assays and the sensitivity against serum complement analysed using a yiaU deletion mutant or YiaU expression strain. Together, these results suggest that the YiaU regulon confers resistance to some antibiotics, and increases biofilm formation and complement sensitivity. We propose renaming YiaU as CsuR (regulator of cell surface).

Sperm acrosome status before and during fertilization in the Chinese hamster (Cricetulus griseus), and observation of oviductal vesicles and globules.

The present study was conducted to determine exact location where the acrosome reaction of fertilizing spermatozoa begins in the oviduct of the Chinese hamster. Unlike spermatozoa of other rodent species, Chinese hamster spermatozoa did not spontaneously undergo the acrosome reaction in fertilization-supporting media. In naturally mated females, spermatozoa in the uterus had intact acrosomes, whereas those in the lower oviductal isthmus had visibly thin acrosomal caps. The acrosomal cap was lost when spermatozoa passed through the cumulus oophorus. Thus, Chinese hamster spermatozoa begin the acrosome reaction in the lower isthmus and complete it in the cumulus oophorus. The mucosal epithelium of the oviductal isthmus released many "transparent" vesicles into the lumen, was very fragile and readily sloughed off by rough handling or rapid flushing with medium. Globular materials that oozed out of the dissected oviduct were most likely mucosa cells destroyed by rough handling. Although the oviducts of Chinese hamsters may be exceptionally delicate, this observation nevertheless warns us to cautiously handle the oviducts of any species when studying oviduct secretions that could be involved in inducing capacitation and the acrosome reaction of spermatozoa within the female genital tract.

Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

Men and women become infertile with age, but the mechanism of declining male fertility, more specifically, the decrease in in sperm quality, is not well known. Citrate synthase (CS) is a core enzyme of the mitochondrial tricarboxylic acid (TCA) cycle, which directly controls cellular function. Extra-mitochondrial CS (eCS) is produced and abundant in the sperm head; however, its role in male fertility is unknown. We investigated the role of eCS in male fertility by producing eCs-deficient (eCs-KO) mice. The initiation of the first spike of Ca2+ oscillation was substantially delayed in egg fused with eCs-KO sperm, despite normal expression of sperm factor phospholipase C zeta 1. The eCs-KO male mice were initially fertile, but the fertility dropped with age. Metabolomic analysis of aged sperm revealed that the loss of eCS enhances TCA cycle in the mitochondria with age, presumably leading to depletion of extra-mitochondrial citrate. The data suggest that eCS suppresses age-dependent male infertility, providing insights into the decline of male fertility with age.

Correction: Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human Semenogelin 1 Promotes Sperm Survival in the Mouse Female Reproductive Tract.

Semenogelin 1 (SEMG1), a main component of human seminal plasma, is a multi-functional protein involved in the regulation of sperm motility and fertility. SEMG1 is orthologous to mouse seminal vesicle secretion 2 (SVS2), required for sperm survival in the female reproductive tract after copulation; however, its in vivo function remains unclear. In this study, we addressed this issue by examining the effect of recombinant SEMG1 on intrauterine mouse sperm survival. SEMG1 caused a dose-dependent decrease in mouse sperm motility, similar to its effect on human sperm, but SVS2 had no effect on mouse sperm motility. Mouse epididymal sperm in the presence of 100 µM SEMG1, a concentration that does not affect mouse sperm motility, were injected into the mouse uterus (intrauterine insemination, IUI). IUI combined with SEMG1 significantly increased the survival rate of intrauterine mouse sperm. The effect of SEMG1 on intrauterine sperm survival was comparable with that of SVS2. For clinical applications, three potentially sperm-protecting polypeptides that are easy to handle were designed from SEMG1, but their individual use was unable to mimic the ability of SEMG1. Our results indicate that SEMG1 has potential clinical applications for effective IUI and thereby for safe, simple, and effective internal fertilization.

Suppression of Non-Random Fertilization by MHC Class I Antigens.

Hermaphroditic invertebrates and plants have a self-recognition system on the cell surface of sperm and eggs, which prevents their self-fusion and enhances non-self-fusion, thereby contributing to genetic variation. However, the system of sperm-egg recognition in mammals is under debate. To address this issue, we explored the role of major histocompatibility complex class I (MHC class I, also known as histocompatibility 2-Kb or H2-Kb and H2-Db in mice) antigens by analyzing H2-Kb-/-H2-Db-/-ß2-microglobulin (ß2M)-/- triple-knockout (T-KO) male mice with full fertility. T-KO sperm exhibited an increased sperm number in the perivitelline space of wild-type (WT) eggs in vitro. Moreover, T-KO sperm showed multiple fusion with zona pellucida (ZP)-free WT eggs, implying that the ability of polyspermy block for sperm from T-KO males was weakened in WT eggs. When T-KO male mice were intercrossed with WT female mice, the percentage of females in progeny increased. We speculate that WT eggs prefer fusion with T-KO sperm, more specifically X-chromosome-bearing sperm (X sperm), suggesting the presence of preferential (non-random) fertilization in mammals, including humans.

Membrane protein CD9 is repositioned and released to enhance uterine function.

Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.

Degradation of phosphate polymer polyP enhances lactic fermentation in mice.

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a "molecular fossil" due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

Deletion of a Seminal Gene Cluster Reinforces a Crucial Role of SVS2 in Male Fertility.

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2-deficient (Svs2-/-) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster (Svs2-6-/- mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2-/- male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.

Exosomes versus microexosomes: Shared components but distinct functions.

In multicellular organisms, cellular components are constantly translocated within cells and are also transported exclusively between limited cells, regardless of their physical distance. Exosomes function as one of the key mediators of intercellular transportation. External vesicles were identified 50 years ago in plants and now reconsidered to be exosome-like vesicles. Meanwhile, a well-known exosomal component, tetraspanin CD9, regulates sperm-egg fusion in mammals. A number of Arabidopsis tetraspanins are also expressed in reproductive tissues at fertilization, and are localized at the plasma membrane of protoplasts. Moreover, CD9-containing structures (or 'microexosomes') are released from mouse eggs during their maturation and promote the sperm-egg fusion. This phenomenon implies that two types of shared-component intercellular carriers might be released from multiple types of plant and animal cells, which widely regulate biological phenomena. We herein highlight their discrete structures, formation processes, and functions.

Seminal vesicle protein SVS2 is required for sperm survival in the uterus.

In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2(-/-) male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.

Birthweights and Down syndrome in neonates that were delivered after frozen-thawed embryo transfer: The 2007-2012 Japan Society of Obstetrics and Gynecology National Registry data in Japan.

Aim: To evaluate the use of frozen embryos on the outcome of assisted reproductive technology (ART), a retrospective study of the Japanese Assisted Reproductive Technology Registry data during the years 2007-2012 was conducted. Methods: A total of 124 946 singleton neonates who reached term gestation following ART from 2007-2012, with 80 660 achieved through frozen-thawed embryo transfer (ET) and 44 286 being achieved through fresh ET, were analyzed for their birthweights and chromosomal abnormalities. Results: The birthweight of the neonates from the frozen-thawed ETs was significantly higher than that of those from the fresh ETs throughout all the study years. The frequency of Down syndrome was 0.17% for the fresh ETs and 0.13% for the frozen-thawed ETs in the period 2007-2012. This study showed that frozen-thawed ETs result in a constant increase of the average birthweight between 37 and 41 weeks gestational age and lower frequencies of Down syndrome. Conclusion: Frozen-thawed ETs were comparable to the fresh ET method, with the exceptions of higher birthweights and a lower frequency of Down syndrome in the neonates that were born from frozen-thawed ET. The increase in birthweights was not proportional to the gestational ages. This cannot be explained with any well-known mechanism. The frequency of chromosomal abnormalities needs detailed data for analysis.

Seminal vesicle proteins SVS3 and SVS4 facilitate SVS2 effect on sperm capacitation.

Mammalian spermatozoa acquire their fertilizing ability in the female reproductive tract (sperm capacitation). On the other hand, seminal vesicle secretion, which is a major component of seminal plasma, inhibits the initiation of sperm capacitation (capacitation inhibition) and reduces the fertility of the capacitated spermatozoa (decapacitation). There are seven major proteins involved in murine seminal vesicle secretion (SVS1-7), and we have previously shown that SVS2 acts as both a capacitation inhibitor and a decapacitation factor, and is indispensable for in vivo fertilization. However, the effects of SVSs other than SVS2 on the sperm have not been elucidated. Since mouse Svs2-Svs6 genes evolved by gene duplication belong to the same gene family, it is possible that SVSs other than SVS2 also have some effects on sperm capacitation. In this study, we examined the effects of SVS3 and SVS4 on sperm capacitation. Our results showed that both SVS3 and SVS4 are able to bind to spermatozoa, but SVS3 alone showed no effects on sperm capacitation. On the other hand, SVS4 acted as a capacitation inhibitor, although it did not show decapacitation abilities. Interestingly, SVS3 showed an affinity for SVS2 and it facilitated the effects of SVS2. Interaction of SVS2 and spermatozoa is mediated by the ganglioside GM1 in the sperm membrane; however, both SVS3 and SVS4 had weaker affinities for GM1 than SVS2. Therefore, we suggest that separate processes may cause capacitation inhibition and decapacitation, and SVS3 and SVS4 act on sperm capacitation cooperatively with SVS2.

Seminal vesicle secretion 2 acts as a protectant of sperm sterols and prevents ectopic sperm capacitation in mice.

Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methyl-beta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo.

Critical role of exosomes in sperm-egg fusion and virus-induced cell-cell fusion.

In mammals, two integral membrane proteins, sperm IZUMO1 and egg CD9, regulate sperm-egg fusion, and their roles are critical, but yet unclear. Recent studies, however, indicate interesting connections between the sperm-egg fusion and virus-induced cell-cell fusion. First, CD9-containing exosome-like vesicles, which are released from wild-type eggs, can induce the fusion between sperm and CD9-deficient egg, even though CD9-deficient eggs are highly refractory to the fusion with sperm. This finding provides strong evidence for the involvement of CD9-containing, fusion-facilitating vesicles in the sperm-egg fusion. Secondly, there are similarities between the generation of retroviruses in the host cells and the formation of small cellular vesicles, termed exosomes, in mammalian cells. The exosomes are involved in intercellular communication through transfer of proteins and ribonucleic acids (RNAs) including mRNAs and microRNAs. These collective studies provide an insight into the molecular mechanism of membrane fusion events.

Proteasome-Associated Proteins, PA200 and ECPAS, Are Essential for Murine Spermatogenesis.

Proteasomes are highly sophisticated protease complexes that degrade non-lysosomal proteins, and their proper regulation ensures various biological functions such as spermatogenesis. The proteasome-associated proteins, PA200 and ECPAS, are predicted to function during spermatogenesis; however, male mice lacking each of these genes sustain fertility, raising the possibility that these proteins complement each other. To address this issue, we explored these possible roles during spermatogenesis by producing mice lacking these genes (double-knockout mice; dKO mice). Expression patterns and quantities were similar throughout spermatogenesis in the testes. In epididymal sperm, PA200 and ECPAS were expressed but were differentially localized to the midpiece and acrosome, respectively. Proteasome activity was considerably reduced in both the testes and epididymides of dKO male mice, resulting in infertility. Mass spectrometric analysis revealed LPIN1 as a target protein for PA200 and ECPAS, which was confirmed via immunoblotting and immunostaining. Furthermore, ultrastructural and microscopic analyses demonstrated that the dKO sperm displayed disorganization of the mitochondrial sheath. Our results indicate that PA200 and ECPAS work cooperatively during spermatogenesis and are essential for male fertility.

Long days restore regular estrous cyclicity in mice lacking circadian rhythms.

Many female mammals have recurring cycles of ovulation and sexual behaviors that are regulated by reproductive hormones and confer reproductive success. In addition to sexual behaviors, circadian behavioral rhythms of locomotor activity also fluctuate across the estrous cycle in rodents. Moreover, there is a bidirectional relationship between circadian rhythms and estrous cyclicity since mice with disrupted circadian rhythms also have compromised estrous cycles resulting in fewer pregnancies. In the present study, we assessed whether extending day length, which alters circadian rhythms, normalizes estrous cyclicity in mice. We found that Period (Per) 1/2/3 triple knockout (KO) mice, that have disabled canonical molecular circadian clocks, have markedly disrupted estrous cycles. Surprisingly, extending the day length by only 2 h per day restored regular 4- or 5-day estrous cycles to Per1/2/3 KO mice. Longer days also induced consistent 4-day, rather than 5-day, estrous cycles in wild-type C57BL/6J mice. These data demonstrate that extending daytime light exposure could be used for enhancing reproductive success.

Sodium Hexametaphosphate Serves as an Inducer of Calcium Signaling.

In bacteria, polymers of inorganic phosphates, particularly linear polyphosphate, are used as alternative phosphate donors for adenosine triphosphate production. A six-chain form of sodium metaphosphate, sodium hexametaphosphate (SHMP), is believed to have no physiological functions in mammalian cells. In this study, we explored the possible effects of SHMP on mammalian cells, using mouse oocytes, which are useful for observing various spatiotemporal intracellular changes. Fertilization-competent oocytes were isolated from the oviducts of superovulated mice and cultured in an SHMP-containing medium. In the absence of co-incubation with sperm, SHMP-treated oocytes frequently formed pronuclei and developed into two-cell embryos owing to the increase in calcium concentration in the cytoplasm. We discovered an intriguing role for SHMP as an initiator of calcium rise in mouse oocytes, presumably in a wide variety of mammalian cells.

CD9 protects the sperm from cytotoxic factors in the epididymis as extracellular components.

The mechanism by which seemingly normal sperm cause infertility is still under debate. Although CD9 is expressed in male reproductive tissues, its role in male fertility remains unclear. To address this, we investigated the role of CD9 in analyzing Cd9 -deficient ( Cd9 -KO) male mice. The litter size of Cd9 -KO males was comparable, regardless of mating experience. When Cd9 -KO males experienced their first mating chance, a considerable number of neonates died 48 hours after birth. Electron microscopy reveals the presence of CD9 in the epididymal space. Our results suggest that CD9 contributes to male fertility as an extracellular component.