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During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility., Competing Interests: Competing interest statement: T.O. and A.O. were co-authors on a paper published in February 2016 in Scientific Reports.

Mature male mice (aged 10-12 weeks or older) are conventionally used for in vitro fertilization (IVF) in order to achieve high fertilization rates (e.g., > 70%). Here, we sought to determine the earliest age at which male mice (C57BL/6J strain) can be used efficiently for producing offspring via IVF. Because we noted that the addition of reduced glutathione (GSH) to the IVF medium significantly increased the fertilizing ability of spermatozoa from prepubertal males, we used this IVF protocol for all experiments. Spermatozoa first reached the caudal region of the epididymides at day 35; however, they were unable to fertilize oocytes. Caudal epididymal spermatozoa first became competent for oocyte fertilization at day 37, albeit at a low rate (2.9%). A high fertilization rate (72.0%) was obtained at day 40, and 52.4% of the embryos thus obtained developed into offspring after embryo transfer. Moreover, we found that corpus epididymal spermatozoa in prepubertal mice could fertilize oocytes; however, the fertilization rates were always < 50%, regardless of the age of the males. Caput epididymal spermatozoa failed to fertilize oocytes irrespective of the age of the males. Therefore, we propose that caudal epididymal spermatozoa from male mice aged 40 days can be efficiently used for IVF, to obtain offspring in the shortest attainable time. This protocol will reduce the turnover time required for the generation of mice by ~1 month compared with that of the conventional IVF protocol.

Although it is known that the susceptibility of mouse spermatozoa to freezing-thawing varies greatly with genetic background, the underlying mechanisms remain to be elucidated. In this study, to map genetic regions responsible for the susceptibility of spermatozoa to freezing-thawing, we performed in vitro fertilization using spermatozoa from recombinant inbred mice derived from the C57BL/6J and DBA/2J strains, whose spermatozoa showed distinct fertilization abilities after freezing. Genome-wide interval mapping identified two suggestive quantitative trait loci (QTL) associated with fertilization on chromosomes 1 and 11. The strongest QTL on chromosome 11 included 70 genes at 59.237260-61.324742 Mb and another QTL on chromosome 1 included 43 genes at 153.969506-158.217850 Mb. These regions included at least 15 genes involved with testicular expression and possibly with capacitation or sperm motility. Specifically, the Abl2 gene on chromosome 1, which may affect subcellular actin distribution, had polymorphisms between C57BL/6J and DBA/2J that caused at least three amino acid substitutions. A correlation analysis using recombinant inbred strains revealed that the fertilization rate was strongly correlated with the capacitation rate of frozen-thawed spermatozoa after preincubation. This result is consistent with the fact that C57BL/6J frozen-thawed spermatozoa recover their fertilization capacity following treatment with methyl-β-cyclodextrin to enhance sperm capacitation. Thus, our data provide important clues to the molecular mechanisms underlying cryodamage to mouse spermatozoa.

In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

For normal fertilization in mammals, it is important that functionally mature sperm are motile and have a fully formed acrosome. The glycosyltransferase-like gene, human polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5), belongs to the polypeptide N-acetylgalactosamine-transferase (pp-GalNAc-T) gene family because of its conserved glycosyltransferase domains, but it uniquely truncates the C-terminal domain and is expressed exclusively in human testis. However, glycosyltransferase activity of the human GALNTL5 protein has not been identified by in vitro assay thus far. Using mouse Galntl5 ortholog, we have examined whether GALNTL5 is a functional molecule in spermatogenesis. It was observed that mouse GALNTL5 localizes in the cytoplasm of round spermatids in the region around the acrosome of elongating spermatids, and finally in the neck region of spermatozoa. We attempted to establish Galntl5-deficient mutant mice to investigate the role of Galntl5 in spermiogenesis and found that the heterozygous mutation affected male fertility due to immotile sperm, which is diagnosed as asthenozoospermia, an infertility syndrome in humans. Furthermore, the heterozygous mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes, and caused aberrant localization of the ubiquitin-proteasome system. By comparing the protein compositions of sperm from infertile males, we found a deletion mutation of the exon of human GALNTL5 gene in a patient with asthenozoospermia. This strongly suggests that the genetic mutation of human GALNTL5 results in male infertility with the reduction of sperm motility and that GALNTL5 is a functional molecule essential for mammalian sperm formation.

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80-500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen-thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13-15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96-1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.

Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation. The whole process, therefore, has never been reproduced in vitro in mammals, nor in any other species with a very few exceptions in some particular types of fish. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas-liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.

Microinsemination is the technique of delivering male germ cells directly into oocytes. The efficiency of fertilization after microinsemination and subsequent embryo development may vary with the animal species and male germ cells used. The present study was undertaken to observe the in vitro and in vivo developmental ability of rabbit embryos following microinsemination with male germ cells at different stages. First, we assessed their oocyte-activating capacity by injecting them into mouse and rabbit oocytes. The majority of mouse oocytes were activated irrespective of the type of rabbit male germ cell injected (61-77%), whereas rabbit oocytes were activated differently according to the type of male germ cells (89%, 75%, and 29% were activated by spermatozoa, elongated spermatids, and round spermatids, respectively; P < 0.05). After 120 hr in culture, 66%, 45%, and 13%, respectively, of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts (P < 0.05). Additional electric pulse stimulation of round spermatid-injected oocytes increased the blastocyst rate to 43%. After 24 hr in culture, some four to eight cell embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from spermatozoa and elongated spermatids, but not from round spermatids. Karyotypic analysis at the morula/blastocyst stage revealed that the majority of round spermatid-derived embryos had abnormal ploidy (8 out of 12 embryos). Our study indicates that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. As these differential developmental patterns are similar to those reported for humans in vitro and in vivo, rabbits may provide an alternative small animal model for studying the biological nature and molecular basis of human microinsemination techniques, especially those using immature male germ cells.

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to the next generation. These cells can be cultured for extended periods in the presence of serum and feeder cells. However, little is known about factors that regulate self-renewal division of spermatogonial stem cells. In this investigation we examined the possibility of establishing culture systems for spermatogonial stem cells that lack serum or a feeder cell layer. Spermatogonial stem cells could expand in serum-free conditions on mouse embryonic fibroblasts (MEFs), or were successfully cultivated without feeder cells on a laminin-coated plate. However, they could not expand when both serum and feeder cells were absent. Although the cells cultured on laminin differed phenotypically from those on feeder cells, they grew exponentially for at least 6 mo, and produced normal, fertile progeny following transplantation into infertile mouse testis. This culture system will provide a new opportunity for understanding the regulatory mechanism that governs spermatogonial stem cells.

Microinsemination has been widely used in basic reproductive research and in human-assisted reproductive technology for treating infertility. Historically, microinsemination in mammals started with research on the golden hamster; since then, it has provided invaluable information on the mechanisms of mammalian fertilization. Thanks to advances in animal genetic engineering and germ-cell technologies, microinsemination techniques are now used extensively to identify the biological significance of genes of interest or to confirm the genetic normality of gametes produced by experimental manipulations in vitro. Fortunately, in mice, high rates of embryo development to offspring can be obtained so long as postmeiotic spermatogenic cells are used as male gametes-that is, round spermatids, elongated spermatids, and spermatozoa. For some other mammalian species, using immature spermatogenic cells significantly decreases the efficiency of microinsemination. Physically unstable chromatin and low oocyte-activating capacity are the major causes of fertilization failure. The youngest male germ cells, including primordial germ cells and gonocytes, can be used in the construction of diploid embryos by nuclear-transfer cloning. The cloned embryos obtained in this way provide invaluable information on the erasure and reestablishment of genomic imprinting in germ cells.

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

Spermatogenesis is a highly specialized process of cellular differentiation to produce spermatozoa. This differentiation process accompanies morphological changes that are controlled by a number of genes expressed in a stage-specific manner during spermatogenesis. Here we show that in mice, the absence of a testis-specific, cytoplasmic polyadenylate [poly(A)] polymerase, TPAP, results in the arrest of spermiogenesis. TPAP-deficient mice display impaired expression of haploid-specific genes that are required for the morphogenesis of germ cells. The TPAP deficiency also causes incomplete elongation of poly(A) tails of particular transcription factor messenger RNAs. Although the overall cellular level of the transcription factor TAF10 is unaffected, TAF10 is insufficiently transported into the nucleus of germ cells. We propose that TPAP governs germ cell morphogenesis by modulating specific transcription factors at posttranscriptional and posttranslational levels.

Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.

Background: In laboratory mice and rats, congenic breeding is essential for analyzing the genes of interest on specific genetic backgrounds and for analyzing quantitative trait loci. However, in theory it takes about 3-4 years to achieve a strain carrying about 99% of the recipient genome at the tenth backcrossing (N10). Even with marker-assisted selection, the socalled 'speed congenic strategy', it takes more than a year at N4 or N5. Methodology/Principal Findings: Here we describe a new high-speed congenic system using round spermatids retrieved from immature males (22-25 days of age). We applied the technique to three genetically modified strains of mice: transgenic (TG), knockin (KI) and N-ethyl-N-nitrosourea (ENU)-induced mutants. The donor mice had mixed genetic backgrounds of C57BL/6 (B6):DBA/2 or B6:129 strains. At each generation, males used for backcrossing were selected based on polymorphic marker analysis and their round spermatids were injected into B6 strain oocytes. Backcrossing was repeated until N4 or N5. For the TG and ENU-mutant strains, the N5 generation was achieved on days 188 and 190 and the proportion of B6-homozygous loci was 100% (74 markers) and 97.7% (172/176 markers), respectively. For the KI strain, N4 was achieved on day 151, all the 86 markers being B6-homozygous as early as on day 106 at N3. The carrier males at the final generation were all fertile and propagated the modified genes. Thus, three congenic strains were established through rapid generation turnover between 41 and 44 days. Conclusions/Significance: This new high-speed breeding strategy enables us to produce congenic strains within about half a year. It should provide the fastest protocol for precise definition of the phenotypic effects of genes of interest on desired genetic backgrounds. [ABSTRACT FROM AUTHOR]

Primordial germ cells (PGCs) are derived from a population of pluripotent epiblast cells in mice. However, little is known about when and how PGCs acquire the capacity to differentiate into functional germ cells, while keeping the potential to derive pluripotent embryonic germ cells and teratocarcinomas. In this investigation, we show that epiblast cells and PGCs can establish colonies of spermatogenesis after transfer into postnatal seminiferous tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination using spermatozoa or spermatids derived from PGCs harvested from fetuses as early as 8.5 days post coitum. Thus, fetal male germ cell development is remarkably flexible, and the maturation process, from epiblast cells through PGCs to postnatal spermatogonia, can occur in the postnatal testicular environment. Primordial germ cell transplantation techniques will also provide a novel tool to assess the developmental potential of PGCs, such as those manipulated in vitro or recovered from embryos harboring lethal mutations. [ABSTRACT FROM AUTHOR]

Abstract: Faithful transcriptome regulation is important in development and also crucial for applications in reproductive and regenerative medicine. Intracytoplasmic sperm injection (ICSI), one of the human assisted reproductive technologies (ART), has long raised concerns about its influence on development. No clear consensus has been reached, however, in spite of many cohort studies carried out in the last two decades on the children conceived by ICSI and/or in vitro fertilization (IVF). In this study, the pre- and postnatal effects of ICSI were assessed using comprehensive transcriptome and phenotypic analyses in mice under strict conditions. Here we demonstrate that, in contrast to IVF, ICSI induces distinct long-lasting transcriptome change that remains at the neonatal stage. Importantly, no remarkable differences were observed in the ICSI adults in either the gene expression or phenotypic profiles, and there was no indication of transmission to the next generation via natural mating. Our results suggest there are no lifelong or transgenerational effects of ICSI, but the ICSI effects during neonatal period remain to be evaluated. [Copyright &y& Elsevier]

Investigates whether the species-specific exclusion system of sperm mitochondria present in egg cytoplasm could also exclude mitochondria from spermatids and from somatic cells of the same species. Estimation of amount of Mus musculus spretus mitochondrial DNA; Fate of spermatid or sperm mitochondrial DNA introduced into oocytes during embryogenesis.