Golgi associated RAB2 interactor protein family contributes to murine male fertility to various extents by assuring correct morphogenesis of sperm heads.

Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2-/- and Garin4-/- males are normal, Garin5a-/- and Garin5b-/- males are subfertile, and Garin3-/- males are infertile. Further analysis revealed that Garin3-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility.

Intraperitoneal administration of mouse kisspeptin-10 to mice during estrus stage induces pseudopregnancy.

A simple method for producing pseudopregnant mice supports pup production. In this study, pregnant ICR were obtained mice without mating with vasectomized mice via administration of mouse Kisspeptin-10 (mKp-10) and transferring blastocysts to the uterus. Blastocyst transfer after mKp-10 administration to mice with gapping and reddish pink vagina resulted in 65.2% (15/23) pregnancies, and 39.1% (34/87) of the transferred blastocysts showed full-term growth. Vaginal smears were observed for accurate estrus cycle determination, and subsequent administration of mKp10 to mice during the estrus stage and blastocyst transfer resulted in 95.2% (20/21) pregnancies and 50.7% (104/205) birth rates. Regarding 2-cell transfer after administration of mKp-10, 100% (8/8) of the mice became pregnant, and 45.0% (36/80) of the embryos were born. Administration of mKp-10 to mice during the estrus stage is a convenient way to generate pseudopregnant mice.

SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility in mice.

Calcineurin is a calcium-dependent phosphatase that plays roles in a variety of biological processes including immune responses. In spermatozoa, there is a testis-enriched calcineurin composed of PPP3CC and PPP3R2 (sperm calcineurin) that is essential for sperm motility and male fertility. Because sperm calcineurin has been proposed as a target for reversible male contraceptives, identifying proteins that interact with sperm calcineurin widens the choice for developing specific inhibitors. Here, by screening the calcineurin-interacting PxIxIT consensus motif in silico and analyzing the function of candidate proteins through the generation of gene-modified mice, we discovered that SPATA33 interacts with sperm calcineurin via a PQIIIT sequence. Spata33 knockout mice exhibit reduced sperm motility because of an inflexible midpiece, leading to impaired male fertility, which phenocopies Ppp3cc and Ppp3r2 knockout mice. Further analysis reveals that sperm calcineurin disappears from the mitochondria in the Spata33 knockout testis. In addition, immunoprecipitation analysis indicates that sperm calcineurin interacts with not only SPATA33 but also the mitochondrial protein VDAC2. These results indicate that SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility.

Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging.

In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the 40 chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Micronucleus formation during early cleavage division is a potential hallmark of preimplantation embryonic loss in cattle.

In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.

Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice.

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes.

The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off-target candidates (exact match 13 nucleotides from 3' target and NGG) found by Bowtie software was 9.2 ± 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes (including two genes previously tested) and obtained mutant mice at a 52.9 ± 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off-target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost-effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.

Gene-deficient mouse model established by CRISPR/Cas9 system reveals 15 reproductive organ-enriched genes dispensable for male fertility.

Since the advent of gene-targeting technology in embryonic stem cells, mice have become a primary model organism for investigating human gene function due to the striking genomic similarities between the two species. With the introduction of the CRISPR/Cas9 system for genome editing in mice, the pace of loss-of-function analysis has accelerated significantly. This has led to the identification of numerous genes that play crucial roles in male reproductive processes, including meiosis, chromatin condensation, flagellum formation in the testis, sperm maturation in the epididymis, and fertilization in the oviduct. Despite the advancements, the functions of many genes, particularly those enriched in male reproductive tissues, remain largely unknown. In our study, we focused on 15 genes and generated 13 gene-deficient mice [4933411K16Rik, Adam triple (Adam20, Adam25, and Adam39), BC048671, Cfap68, Gm4846, Gm4984, Gm13570, Nt5c1b, Ppp1r42, Saxo4, Sh3d21, Spz1, and Tektl1] to elucidate their roles in male fertility. Surprisingly, all 13 gene-deficient mice exhibited normal fertility in natural breeding experiments, indicating that these genes are not essential for male fertility. These findings have important implications as they may help prevent other research laboratories from duplicating efforts to generate knockout mice for genes that do not demonstrate an apparent phenotype related to male fertility. By shedding light on the dispensability of these genes, our study contributes to a more efficient allocation of research resources in the exploration of male reproductive biology.

Individual disruption of 12 testis-enriched genes via the CRISPR/Cas9 system does not affect the fertility of male mice.

More than 1200 genes have been shown in the database to be expressed predominantly in the mouse testes. Advances in genome editing technologies such as the CRISPR/Cas9 system have made it possible to create genetically engineered mice more rapidly and efficiently than with conventional methods, which can be utilized to screen genes essential for male fertility by knocking out testis-enriched genes. Finding such genes related to male fertility would not only help us understand the etiology of human infertility but also lead to the development of male contraceptives. In this study, we generated knockout mice for 12 genes (Acrv1, Adgrf3, Atp8b5, Cfap90, Cfap276, Fbxw5, Gm17266, Lrrd1, Mroh7, Nemp1, Spata45, and Trim36) that are expressed predominantly in the testis and examined the appearance and histological morphology of testes, sperm motility, and male fertility. Mating tests revealed that none of these genes is essential for male fertility at least individually. Notably, knockout mice for Gm17266 showed smaller testis size than the wild-type but did not exhibit reduced male fertility. Since 12 genes were not individually essential for male fertilization, it is unlikely that these genes could be the cause of infertility or contraceptive targets. It is better to focus on other essential genes because complementary genes to these 12 genes may exist.

Search for morphological indicators that predict implantation by principal component analysis using images of blastocyst.

Background: Although the current evaluation of human blastocysts is based on the Gardner criteria, there may be other notable parameters. The purpose of our study was to clarify whether the morphology of blastocysts has notable indicators other than the Gardner criteria. Methods: To find such indicators, we compared blastocysts that showed elevated human chorionic gonadotropin (hCG) levels after transplantation (hCG-positive group; n = 129) and those that did not (hCG-negative group; n = 105) using principal component analysis of pixel brightness of the images. Results: The comparison revealed that the hCG-positive group had grainy morphology and the hCG-negative group had non-grainy morphology. Classification of the blastocysts by this indicator did not make a difference in Gardner score. Interestingly, all embryos with ≥20% fragmentation were non-grainy. The visual classification based on this analysis was significantly more accurate than the prediction of implantation using the Gardner score ≥3BB. As graininess can be used in combination with the Gardner score, this indicator will enhance current reproductive technologies.

An explainable deep learning-based algorithm with an attention mechanism for predicting the live birth potential of mouse embryos.

In assisted reproductive technology (ART), embryos produced by in vitro fertilization (IVF) are graded according to their live birth potential, and high-grade embryos are preferentially transplanted. However, rates of live birth following clinical ART remain low worldwide. Grading is based on the embryo shape at a limited number of stages and does not consider the shape of embryos and intracellular structures, e.g., nuclei, at various stages important for normal embryogenesis. Here, we developed a Normalized Multi-View Attention Network (NVAN) that directly predicts live birth potential from the nuclear structure in live-cell fluorescence images of mouse embryos from zygote to across a wide range of stages. The input is morphological features of cell nuclei, which were extracted as multivariate time-series data by using the segmentation algorithm for mouse embryos. The classification accuracy of our method (83.87%) greatly exceeded that of existing machine-learning methods and that of visual inspection by embryo culture specialists. Our method also has a new attention mechanism that allows us to determine which values of multivariate time-series data, used to describe nuclear morphology, were the basis for the prediction. By visualizing the features that contributed most to the prediction of live birth potential, we found that the size and shape of the nucleus at the morula stage and at the time of cell division were important for live birth prediction. We anticipate that our method will help ART and developmental engineering as a new basic technology for IVF embryo selection.

Asynchronous division at 4-8-cell stage of preimplantation embryos affects live birth through ICM/TE differentiation.

To improve the performance of assisted reproductive technology, it is necessary to find an indicator that can identify and select embryos that will be born or be aborted. We searched for indicators of embryo selection by comparing born/abort mouse embryos. We found that asynchronous embryos during the 4-8-cell stage were predisposed to be aborted. In asynchronous mouse embryos, the nuclear translocation of YAP1 in some blastomeres and compaction were delayed, and the number of ICMs was reduced. Hence, it is possible that asynchronous embryos have abnormal differentiation. When the synchrony of human embryos was observed, it was confirmed that embryos that did not reach clinical pregnancy had asynchrony as in mice. This could make synchrony a universal indicator common to all animal species.

Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage.

In the pre-implantation embryo, aneuploidy resulting from chromosome segregation error is considered responsible for pregnancy loss. However, only a few studies have examined the relationship between chromosome segregation errors during early cleavage and development. Here, we evaluated this relationship by live-cell imaging using the histone H2B-mCherry probe and subsequent single blastocyst transfer using mouse embryos obtained by in vitro fertilization. We showed that some embryos exhibiting early chromosomal segregation error and formation of micronuclei retained their developmental potential; however, the error affected the blastocyst/arrest ratio. Further, single-cell sequencing after live-cell imaging revealed that all embryos exhibiting micronuclei formation during 1st mitosis showed aneuploidy at the 2-cell stage. These results suggest that early chromosome segregation error causing micronuclei formation affects ploidy and development to blastocyst but does not necessarily cause developmental failure after the blastocyst stage. Our result suggests the importance of the selection of embryos that have reached blastocysts.

3D convolutional neural networks-based segmentation to acquire quantitative criteria of the nucleus during mouse embryogenesis.

During embryogenesis, cells repeatedly divide and dynamically change their positions in three-dimensional (3D) space. A robust and accurate algorithm to acquire the 3D positions of the cells would help to reveal the mechanisms of embryogenesis. To acquire quantitative criteria of embryogenesis from time-series 3D microscopic images, image processing algorithms such as segmentation have been applied. Because the cells in embryos are considerably crowded, an algorithm to segment individual cells in detail and accurately is needed. To quantify the nuclear region of every cell from a time-series 3D fluorescence microscopic image of living cells, we developed QCANet, a convolutional neural network-based segmentation algorithm for 3D fluorescence bioimages. We demonstrated that QCANet outperformed 3D Mask R-CNN, which is currently considered as the best algorithm of instance segmentation. We showed that QCANet can be applied not only to developing mouse embryos but also to developing embryos of two other model species. Using QCANet, we were able to extract several quantitative criteria of embryogenesis from 11 early mouse embryos. We showed that the extracted criteria could be used to evaluate the differences between individual embryos. This study contributes to the development of fundamental approaches for assessing embryogenesis on the basis of extracted quantitative criteria.

Nuclear formation induced by DNA-conjugated beads in living fertilised mouse egg.

Reformation of a functional nucleus at the end of mitosis is crucial for normal cellular activity. Reconstitution approaches using artificial beads in frog egg extracts have clarified the molecules required for nuclear formation in vitro. However, the spatiotemporal regulation of these components, which is required for the formation of a functional nucleus in living embryos, remains unknown. Here we demonstrate that exogenous DNA introduced in the form of DNA-conjugated beads induces the assembly of an artificial nucleus in living mouse cleavage-stage embryos. Live-cell imaging and immunofluorescence studies revealed that core histones and regulator of chromosome condensation 1 (RCC1) assembled on the DNA, suggesting that nucleosomes were formed. Electron microscopy showed that double-membrane structures, partly extended from annulate lamellae, formed around the beads. Nuclear pore complex-like structures indistinguishable from those of native nuclei were also formed, suggesting that this membranous structure resembled the normal nuclear envelope (NE). However, the reconstituted NE had no nuclear import activity, probably because of the absence of Ras-related nuclear protein (Ran). Thus, DNA is necessary for NE reassembly in mouse embryos but is insufficient to form a functional nucleus. This approach provides a new tool to examine factors of interest and their spatiotemporal regulation in nuclear formation.

Mouse spermatozoa with higher fertilization rates have thinner nuclei.

BACKGROUND: Although spermatozoa with normal morphology are assumed to have uniform fertilization ability, recent data show that even normal spermatozoa have considerable variation in their head shape which is associated with differences in fertilization ability. Appropriate quantitative indicators for good sperm morphology, however, remain unidentified. METHODS: Therefore, in an effort to identify such an indicator, we compared the nuclear contour of normal mouse spermatozoa by quantitative multivariate analysis using elliptic Fourier descriptors combined with principal component analysis. The spermatozoa were obtained from different strains and collection sites which have been shown to be associated with different fertilization abilities. RESULTS: We found that the head was 5.7% thinner in spermatozoa from the B6D2F1 (BDF1) strain, known to have a higher fertilization rate, than in those from the C57BL/6N (B6N) strain, which has a lower fertilization rate. Moreover, zona-penetrated spermatozoa in the perivitelline space consistently had 5.4% thinner heads than those isolated from the epididymis before ejaculation. The aspect ratio, which represents the sperm head thinness, uniquely distinguished these sperm populations, confirming its validity as a morphological indicator. DISCUSSION: Because aspect ratio has also been shown to characterize human spermatozoa, this unique morphometric indicator might be applicable to compare normal spermatozoa among multiple patients, which will greatly facilitate and enhance current reproductive technologies.

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive.

Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis.

Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO.

In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.