Genetic loss of importin α4 causes abnormal sperm morphology and impacts on male fertility in mouse.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.

Reduction in BDNF from Inefficient Precursor Conversion Influences Nest Building and Promotes Depressive-Like Behavior in Mice.

We generated a knock-in mouse line in which the gene encoding brain-derived neurotrophic factor (Bdnf) was replaced with a sequence for proBDNF containing human single nucleotide polymorphisms encoding arginines proximal to the cleavage site (R125M and R127L). The ratio of the mature form of BDNF (mBDNF) to precursor BDNF (proBDNF) in hippocampal tissue lysates was decreased in a manner dependent on the number of copies of the mutant gene, indicating that the mutations inhibited proteolytic conversion of proBDNF into mBDNF. Although homozygous mice had a proBDNF/mBDNF ratio of ~9:1, they survived until adulthood. The levels of mBDNF were reduced by 57% in heterozygous mutant mice, which exhibited a depressive-like behavior in the tail suspension test and weight gain when housed in social isolation, showing that impaired proBDNF cleavage contributes to stress-induced depressive-like phenotypes. Furthermore, socially isolated heterozygous mice displayed a pronounced deficit in daily nest-building behaviors. These findings suggest that the decreased production of mBDNF by impaired proBDNF cleavage disturbs daily activities in mice.

NELL2-mediated lumicrine signaling through OVCH2 is required for male fertility.

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell-secreted epidermal growth factor-like protein, neural epidermal growth factor-like-like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which Nell2 had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.

Haprin-deficient spermatozoa are incapable of in vitro fertilization.

Haprin (TRIM36) is a ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin-deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility-related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin-deficient mice having poorer sperm morphology and motility than wild-type mice. Interestingly, haprin-deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.

CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.

Ventricular-subventricular zone fractones are speckled basement membranes that function as a neural stem cell niche.

Neural stem cells (NSCs) are retained in the adult ventricular-subventricular zone (V-SVZ), a specialized neurogenic niche with a unique cellular architecture. It currently remains unclear whether or how NSCs utilize basement membranes (BMs) in this niche. Here, we examine the molecular compositions and functions of BMs in the adult mouse V-SVZ. Whole-mount V-SVZ immunostaining revealed that fractones, which are fingerlike processes of extravascular BMs, are speckled BMs unconnected to the vasculature, and differ in their molecular composition from vascular BMs. Glial fibrillary acidic protein (GFAP)-positive astrocytes and NSCs produce and adhere to speckled BMs. Furthermore, Gfap-Cre-mediated Lamc1flox(E1605Q) knockin mice, in which integrin-binding activities of laminins are specifically nullified in GFAP-positive cells, exhibit a decreased number and size of speckled BMs and reduced in vitro neurosphere-forming activity. Our results reveal niche activities of fractones/speckled BMs for NSCs and provide molecular insights into how laminin-integrin interactions regulate NSCs in vivo.

Transgenic mice that accept Luciferase- or GFP-expressing syngeneic tumor cells at high efficiencies.

Jellyfish green fluorescent protein (GFP) and firefly luciferase can serve as versatile tracking markers for identification and quantification of transplanted cancer cells in vivo. However, immune reactions against these markers can hamper the formation of syngraft tumors and metastasis that follows. Here, we report two transgenic (Tg) mouse lines that express nonfunctional mutant marker proteins, namely modified firefly luciferase (Luc2) or enhanced GFP (EGFP). These mice, named as Tg-mLuc2 and Tg-mEGFP, turned out to be immunologically tolerant to the respective tracking markers and thus efficiently accepted syngeneic cancer cells expressing the active forms of the markers. We then injected intrarectally the F1 hybrid Tg mice (BALB/c × C57BL/6J) with Colon-26 (C26) colon cancer cells that originated from a BALB/c mouse. Even when C26 cells expressed active Luc2 or EGFP, they formed primary tumors in the Tg mice with only 104 cells per mouse compared with more than 106 cells required in the nontransgenic BALB/c hosts. Furthermore, we detected metastatic foci of C26 cells in the liver and lungs of the Tg mice by tracking the specific reporter activities. These results show the usefulness of the Tg mouse lines as recipients for transplantation experiments with the non-self tracking marker-expressing cells.

Beware of memes in the interpretation of your results - lessons from gene-disrupted mice in fertilization research.

For decades, researchers in the fertilization field reported various candidate factors involved in sperm-egg interaction through experiments using enzyme inhibitors and/or antibodies. However, almost all of these factors have been shown to be nonessential by gene disruption experiments. Recently, attention has focused on the low reproducibility of papers in many research fields. In this Review, I retrospectively revisit how fertilization factors were misinterpreted and led to wrong hypotheses in relation to the reportedly low reproducibility of scientific papers.

Sperm-egg interaction and fertilization: past, present, and future.

Fifty years have passed since the findings of capacitation and acrosome reaction. These discoveries and the extensive effort of researchers led to the success of in vitro fertilization, which has become a top choice for patients at infertility clinics today. The effort to understand the mechanism of fertilization is ongoing, but the small number of eggs and similarly small quantity of spermatozoa continue to hinder biochemical experiments. The emergence of transgenic animals and gene disruption techniques has had a significant effect on fertilization research. Factors considered important in the early years were shown not to be essential and were replaced by newly found proteins. However, there is much about sperm-egg interaction which remains to be learned before we can outline the mechanism of fertilization. In fact, our understanding of sperm-egg interaction is entering a new stage. Progress in transgenic spermatozoa helped us to observe the behavior of spermatozoa in vivo and/or at the moment of sperm-egg fusion. These advancements are discussed together with the paradigm-shifting research in related fields to help us picture the direction which fertilization research may take in the future.

A delayed sperm penetration of cumulus layers by disruption of acrosin gene in rats.

Acrosin, the trypsin-like serine protease in the sperm acrosome, was long viewed as a key enzyme required for zona pellucida penetration to fertilize eggs. However, gene disruption experiments in mice surprisingly showed that acrosin-disrupted males were fertile. Thus, the acrosin was considered to be not an essential enzyme for fertilization in mice. However, the involvement of acrosin in fertilization has been suggested in various species such as rat, bull, and pig. Moreover, it has been reported that serine protease (including acrosin) activity in mice is significantly weaker compared to other species, including rats. We analyzed the role of acrosin by disrupting the rat acrosin gene. It was found that, unlike in mice, acrosin was almost the sole source of serine protease in rat spermatozoa. Nevertheless, the acrosin-disrupted males were not infertile. However, the litter size from acrosin-disrupted males was decreased compared to heterozygous mutant rats. Further investigation using an in vitro fertilization system revealed that the acrosin-disrupted spermatozoa possessed an equal ability to penetrate the zona pellucida with wild-type spermatozoa, but the cumulus cell dispersal was slower compared to wild-type and heterozygous spermatozoa. This delay was presumed to be the cause of the small litter size of acrosin-disrupted male rats.

The mechanics clarifying counterclockwise rotation in most IVF eggs in mice.

In mammalian fertilization, a small spermatozoon interacts with an egg that is a few thousand times larger in volume. In spite of the big difference in size and mass, when spermatozoa are bound to eggs, they begin rotating the eggs in in vitro observation. This was dubbed the 'fertilization dance'. Interestingly, some papers reported that the rotation was counterclockwise, although the reason for this skewed rotation was not clarified. We focused on a chirality of helical beating of spermatozoa and found that eggs rotate counterclockwise in simulations under a certain geometrical condition where the eggs were situated. This theory of egg rotation was validated by demonstrating egg rotation in a clockwise direction by floating eggs to the upper surface of the IVF medium. The enigma of skewed rotation of IVF eggs was clarified.

STING in tumor and host cells cooperatively work for NK cell-mediated tumor growth retardation.

An interferon-inducing DNA sensor STING participates in tumor rejection in mouse models. Here we examined what mechanisms contribute to STING-dependent growth retardation of B16 melanoma sublines by NK cells in vivo. The studies were designed using WT and STING KO black mice, and B16D8 (an NK-sensitive melanoma line having STING) and STING KO B16D8 sublines established for this study. The results from tumor-implant studies suggested that STING in host immune cells and tumor cells induced distinct profiles of chemokines including CXCL10, CCL5 and IL-33, and both participated in NK cell infiltration and activation in B16D8 tumor. Spontaneous activation of STING occurs in host-immune and tumor cells of this NK-sensitive tumor, thereby B16D8 tumor growth being suppressed in this model. Our data show that STING induces tumor cytotoxicity by NK cells through tumor and host immune cell network to contribute to innate surveillance and suppression of tumors in vivo.

Live imaging of X chromosome reactivation dynamics in early mouse development can discriminate naïve from primed pluripotent stem cells.

Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naïve state, while one of the X chromosomes is inactivated in the primed state. Therefore, we aimed to distinguish the two states by monitoring X chromosome reactivation. Thus far, X chromosome reactivation has been analysed using fixed cells; here, we inserted different fluorescent reporter gene cassettes (mCherry and eGFP) into each X chromosome. Using these knock-in 'Momiji' mice, we detected X chromosome reactivation accurately in live embryos, and confirmed that the pluripotent states of embryos were stable ex vivo, as represented by embryonic and epiblast stem cells in terms of X chromosome reactivation. Thus, Momiji mice provide a simple and accurate method for identifying stem cell status based on X chromosome reactivation.

The Behavior and Acrosomal Status of Mouse Spermatozoa In Vitro, and Within the Oviduct During Fertilization after Natural Mating.

Although 90%-100% of mouse oocytes can be fertilized in vitro with capacitated spermatozoa within 1 h after insemination, oocytes within the oviduct are fertilized one by one over a period of several hours. In vitro experiments showed that both acrosome-intact and acrosome-reacted spermatozoa entered the cumulus oophorus, but that acrosome-reacted spermatozoa reached the surface of oocytes more readily than acrosome-intact spermatozoa. During the period of fertilization within the oviduct, acrosome-reacted spermatozoa were seen throughout the isthmus, but with higher incidence in the upper than in the mid- and lower segments of the isthmus. Very few spermatozoa were present in the ampulla, and almost all were acrosome reacted. Although the cumulus oophorus and zona pellucida are known to be able to induce or facilitate the acrosome reaction of spermatozoa, this picture makes it likely that almost all fertilizing mouse spermatozoa within the oviduct begin to react before ascending from the isthmus to the ampulla. We witnessed a reacted spermatozoon that stayed on the zona pellucida of a fertilized oocyte for a while; it then moved out of the cumulus before reaching the zona pellucida of the nearby unfertilized oocyte. We noted that only a few spermatozoa migrate from the isthmus to the ampulla during the progression of fertilization, and this must be one of the reasons why we do not see many spermatozoa swarming around a single oocyte during in vivo fertilization.

Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice.

Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

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.

The Acrosome Reaction: A Historical Perspective.

Acrosome reaction is often referred to as acrosomal exocytosis, but it differs significantly from normal exocytosis. While the vesicle membrane initially holding excreting molecules remains on the cell surface during exocytosis, the outer acrosomal membrane and plasma membrane are lost by forming vesicles during acrosome reaction. In this context, the latter process resembles a release of exosome. However, recent experimental data indicate that the most important roles of acrosome reaction lie not in the release of acrosomal contents (or "vesiculated" plasma and outer acrosomal membrane complexes) but rather in changes in sperm membrane. This review describes the mechanism of fertilization vis-a-vis sperm membrane change, with a brief historical overview of the half-century study of acrosome reaction.

Generation of Hprt-disrupted rat through mouse←rat ES chimeras.

We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production.

Behavior of Mouse Spermatozoa in the Female Reproductive Tract from Soon after Mating to the Beginning of Fertilization.

Using transgenic mice with spermatozoa expressing enhanced green fluorescent protein in their acrosome and red fluorescent protein in their midpiece mitochondria, we followed the behavior of spermatozoa within the female genital tract after natural mating. When examined 15 min after coitus, many spermatozoa were around the opening of the uterotubal junction. Spermatozoa that entered the uterotubal junction were seemingly not moving, yet they steadily migrated toward the isthmus at a speed only time-lapse video recording could demonstrate. Many spermatozoa reaching the lower isthmus were motile. The site where spermatozoa attached and detached from the isthmus epithelium shifted from the lower to the upper segment of the isthmus with time. Virtually all the live spermatozoa within the lower isthmus were acrosome intact, whereas many of the actively motile spermatozoa in the upper isthmus were acrosome reacted. As far as we could observe, all the spermatozoa we found within the lumen of the ampulla and the cumulus oophorus were acrosome reacted. Even though we saw only a very few spermatozoa within the ampulla during fertilization, all were associated with, or were already within, oocytes, indicating that mouse fertilization in vivo is extremely efficient.

Impaired female fertility in tubulointerstitial antigen-like 1-deficient mice.

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert's membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1(-/-) embryos were not lethal during development to term, homologous matings of Tinagl1(-/-) females and Tinagl1(-/-) males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1(-/-) and Tinagl1(flox/flox). In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1(-/-) oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.