Incompatibility in cell adhesion constitutes a barrier to interspecies chimerism.

Interspecies blastocyst complementation holds great potential to address the global shortage of transplantable organs by growing human organs in animals. However, a major challenge in this approach is the limited chimerism of human cells in evolutionarily distant animal hosts due to various xenogeneic barriers. Here, we reveal that human pluripotent stem cells (PSCs) struggle to adhere to animal PSCs. To overcome this barrier, we developed a synthetic biology strategy that leverages nanobody-antigen interactions to enhance interspecies cell adhesion. We engineered cells to express nanobodies and their corresponding antigens on their outer membranes, significantly improving adhesion between different species' PSCs during in vitro assays and increasing the chimerism of human PSCs in mouse embryos. Studying and manipulating interspecies pluripotent cell adhesion will provide valuable insights into cell interaction dynamics during chimera formation and early embryogenesis.

MYCBPAP is a central apparatus protein required for centrosome-nuclear envelope docking and sperm tail biogenesis in mice.

The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap-knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap-knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins, such as CFAP65 and CFAP70, which constitute the C2a projection, and centrosome-associated proteins, such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.

Multiple genes in the Pate5-13 genomic region contribute to ADAM3 processing†.

Sperm proteins undergo post-translational modifications during sperm transit through the epididymis to acquire fertilizing ability. We previously reported that the genomic region coding Pate family genes is key to the proteolytic processing of the sperm membrane protein ADAM3 and male fertility. This region contains nine Pate family genes (Pate5-13), and two protein-coding genes (Gm27235 and Gm5916), with a domain structure similar to Pate family genes. Therefore, in this study, we aimed to identify key factors by narrowing the genomic region. We generated three knockout (KO) mouse lines using CRISPR/Cas9: single KO mice of Pate10 expressed in the caput epididymis; deletion KO mice of six caput epididymis-enriched genes (Pate5-7, 13, Gm27235, and Gm5916) (Pate7-Gm5916 KO); and deletion KO mice of four genes expressed in the placenta and epididymis (Pate8, 9, 11, and 12) (Pate8-12 KO). We observed that the fertility of only Pate7-Gm5916 KO males was reduced, whereas the rest remained unaffected. Furthermore, when the caput epididymis-enriched genes, Pate8 and Pate10 remained in Pate7-Gm5916 KO mice were independently deleted, both KO males displayed more severe subfertility due to a decrease in mature ADAM3 and a defect in sperm migration to the oviduct. Thus, our data showed that multiple caput epididymis-enriched genes within the region coding Pate5-13 cooperatively function to ensure male fertility in mice.

Large-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal cross talk.

Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. One limitation of our first protocol for human blastoid generation was relatively low efficiency. We now report an optimized protocol for the efficient generation of large quantities of high-fidelity human blastoids from naive pluripotent stem cells. This enabled proteomics analysis that identified phosphosite-specific signatures potentially involved in the derivation and/or maintenance of the signaling states in human blastoids. Additionally, we uncovered endometrial stromal effects in promoting trophoblast cell survival, proliferation, and syncytialization during co-culture with blastoids and blastocysts. Side-by-side single-cell RNA sequencing revealed similarities and differences in transcriptome profiles between pre-implantation blastoids and blastocysts, as well as post-implantation cultures, and uncovered a population resembling early migratory trophoblasts during co-culture with endometrial stromal cells. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, and tractable model for human blastocysts.

Recent advances in stem cell-based blastocyst models.

Early embryo development is a highly dynamic process that plays a crucial role in determining the health and characteristics of an organism. For many years, embryonic and extraembryonic stem cell lines representing various developmental stages have served as valuable models for studying early embryogenesis. As our understanding of stem cell culture and embryo development has advanced, researchers have been able to create more sophisticated 3D structures mimicking early embryos, such as blastocyst-like structures (blastoids). These innovative models represent a significant leap forward in the field. In this mini-review, we will discuss the latest progress in stem cell-based embryo models, explore potential future directions, and examine how these models contribute to a deeper understanding of early mammalian development.

Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids.

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

A small secreted protein NICOL regulates lumicrine-mediated sperm maturation and male fertility.

The mammalian spermatozoa produced in the testis require functional maturation in the epididymis for their full competence. Epididymal sperm maturation is regulated by lumicrine signalling pathways in which testis-derived secreted signals relocate to the epididymis lumen and promote functional differentiation. However, the detailed mechanisms of lumicrine regulation are unclear. Herein, we demonstrate that a small secreted protein, NELL2-interacting cofactor for lumicrine signalling (NICOL), plays a crucial role in lumicrine signalling in mice. NICOL is expressed in male reproductive organs, including the testis, and forms a complex with the testis-secreted protein NELL2, which is transported transluminally from the testis to the epididymis. Males lacking Nicol are sterile due to impaired NELL2-mediated lumicrine signalling, leading to defective epididymal differentiation and deficient sperm maturation but can be restored by NICOL expression in testicular germ cells. Our results demonstrate how lumicrine signalling regulates epididymal function for successful sperm maturation and male fertility.

TSKS localizes to nuage in spermatids and regulates cytoplasmic elimination during spermiation.

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.

Evaluation of the efficacy of creatine chemical exchange saturation transfer imaging in assessing testicular maturity.

Purpose: Microscopic testicular sperm extraction is the most effective treatment for NOA, but the sperm retrieval rate is low and depends on testicular maturity. However, there are limited useful tests to assess testicular maturity. Chemical exchange saturation transfer (CEST) imaging is a new magnetic resonance imaging (MRI) technique that can image the distribution of trace substances in vivo. We focused on the potential role of creatine (Cr) in testes and hypothesized that Cr-CEST could indicate intratesticular spermatogenesis. Methods: We performed Cr-CEST by using 7T MRI on wild-type C57B6/J mice and several types of male infertility models such as Sertoli-cell only (SCO) (Kitw/Kitwv), maturation arrest (MA) (Zfp541 knockout mouse and Kctd19 knockout mouse), and teratozoospermia (Tbc1d21 knockout mouse). After performing Cr-CEST, histological analysis was performed. Results: The SCO and MA models showed decreased CEST signal intensity (p < 0.05), while no reduction was observed in the teratozoospermia model (p = 1.0). CEST signal intensity increased as the spermatogenesis stage progressed from the SCO model to the MA and teratozoospermia models. Furthermore, CEST signal intensity was reduced in 4-week-old wild-type mice with immature testes (p < 0.05). Conclusions: This study suggests that Cr-CEST evaluates intratesticular spermatogenesis noninvasively and provides a new therapeutic strategy for treating male infertility.

Testis-specific proteins, TSNAXIP1 and 1700010I14RIK, are important for sperm motility and male fertility in mice.

BACKGROUND: TSN (translin), also called testis brain RNA-binding protein, binds to TSNAX (translin-associated factor X) and is suggested to play diverse roles, such as RNA metabolism and DNA damage response. TSNAXIP1 (Translin-associated factor X-interacting protein 1) was identified as a TSNAX-interacting protein using a yeast two-hybrid system, but its function in vivo was unknown. OBJECTIVE: To reveal the function of TSNAXIP1 in vivo in mice. MATERIALS AND METHODS: We generated Tsnaxip1 knockout mice using the CRISPR/Cas9 system and analyzed their fertility and sperm motility. Further, we generated 1700010I14Rik knockout mice, because 1700010I14RIK is also predominantly expressed in testes and contains the same Pfam (protein families) domain as TSNAXIP1. RESULTS: Reduced male fertility and impaired sperm motility with asymmetric flagellar waveforms were observed in not only Tsnaxip1 but also 1700010I14Rik knockout mice. Unlike Tsn knockout mice, no abnormalities were found in testicular sections of either Tsnaxip1 or 1700010I14Rik knockout mice. Furthermore, TSNAXIP1 was detected in the sperm tail and fractionated with axonemal proteins. DISCUSSION AND CONCLUSION: Unlike the TSN-TSNAX complex, whose disruption causes abnormal vacuoles in mouse testes, TSNAXIP1 and 1700010I14RIK may play roles in regulating sperm flagellar beating patterns.

Proximity-dependent biotin labeling in testicular germ cells identified TESMIN-associated proteins.

Characterization of protein-protein interactions (PPI) is a key to understanding the functions of proteins of interest. Recently developed proximity-dependent biotin identification (BioID) has been actively investigated as an alternative PPI mapping method because of its usefulness in uncovering transient PPI. Here, as an example of proximity labeling proteomics application in the testis, we generated two transgenic mouse lines expressing two biotin ligases (BioID2 or TurboID) fused with TESMIN, which translocates from the cytosol to the nucleus during meiotic progression and is required for reproduction. The BioID2 transgene, albeit not the TurboID transgene, rescued fertility defects of the Tesmin KO male mice, indicating that the TESMIN-BioID2 fusion can physiologically replace TESMIN. Furthermore, biotinylated protein pull-down and affinity-purification followed by mass spectrometry using the TESMIN-BioID2 transgenic mice captured components of the MYBL1-MuvB complex that regulate cell-cycle gene expression. Thus, our study shows that proximity labeling proteomics can be applied in male germ cells, although the choice of biotin ligase needs to be carefully tested.

Trim41 is required to regulate chromosome axis protein dynamics and meiosis in male mice.

Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.

Sperm membrane proteins DCST1 and DCST2 are required for sperm-egg interaction in mice and fish.

The process of sperm-egg fusion is critical for successful fertilization, yet the underlying mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. melanogaster. Mouse Dcst1 and Dcst2 single knockout (KO) sperm are able to undergo the acrosome reaction and show normal relocalization of IZUMO1, an essential factor for sperm-egg fusion, to the equatorial segment. While both single KO sperm can bind to the oolemma, they show the fusion defect, resulting that Dcst1 KO males become almost sterile and Dcst2 KO males become sterile. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO sperm are motile and can approach the egg, but are defective in binding to the oolemma. Furthermore, we find that DCST1 and DCST2 interact with each other and are interdependent. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species, highlighting their crucial role as conserved factors in fertilization.

FAM71F1 binds to RAB2A and RAB2B and is essential for acrosome formation and male fertility in mice.

The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation.

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.

Precise CAG repeat contraction in a Huntington's Disease mouse model is enabled by gene editing with SpCas9-NG.

The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a research hotspot in gene therapy. However, the widely used Streptococcus pyogenes Cas9 (WT-SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting targetable disease mutations. To address this issue, we recently reported an engineered SpCas9 nuclease variant (SpCas9-NG) recognizing NGN PAMs. Here, as a feasibility study, we report SpCas9-NG-mediated repair of the abnormally expanded CAG repeat tract in Huntington's disease (HD). By targeting the boundary of CAG repeats with SpCas9-NG, we precisely contracted the repeat tracts in HD-mouse-derived embryonic stem (ES) cells. Further, we confirmed the recovery of phenotypic abnormalities in differentiated neurons and animals produced from repaired ES cells. Our study shows that SpCas9-NG can be a powerful tool for repairing abnormally expanded CAG repeats as well as other disease mutations that are difficult to access with WT-SpCas9.

KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice.

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.

ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility.

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

Cfap97d1 is important for flagellar axoneme maintenance and male mouse fertility.

The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An axoneme is comprised of two central microtubules surrounded by nine doublets, the nexin-dynein regulatory complex, radial spokes, and dynein arms. Failure to properly assemble components of the axoneme in a sperm flagellum, leads to fertility alterations. To understand this process in detail, we have defined the function of an uncharacterized gene, Cfap97 domain containing 1 (Cfap97d1). This gene is evolutionarily conserved in mammals and multiple other species, including Chlamydomonas. We have used two independently generated Cfap97d1 knockout mouse models to study the gene function in vivo. Cfap97d1 is exclusively expressed in testes starting from post-natal day 20 and continuing throughout adulthood. Deletion of the Cfap97d1 gene in both mouse models leads to sperm motility defects (asthenozoospermia) and male subfertility. In vitro fertilization (IVF) of cumulus-intact oocytes with Cfap97d1 deficient sperm yielded few embryos whereas IVF with zona pellucida-free oocytes resulted in embryo numbers comparable to that of the control. Knockout spermatozoa showed abnormal motility characterized by frequent stalling in the anti-hook position. Uniquely, Cfap97d1 loss caused a phenotype associated with axonemal doublet heterogeneity linked with frequent loss of the fourth doublet in the sperm stored in the epididymis. This study demonstrates that Cfap97d1 is required for sperm flagellum ultra-structure maintenance, thereby playing a critical role in sperm function and male fertility in mice.

PHF7 Modulates BRDT Stability and Histone-to-Protamine Exchange during Spermiogenesis.

Spermatogenesis is a complex process of sperm generation, including mitosis, meiosis, and spermiogenesis. During spermiogenesis, histones in post-meiotic spermatids are removed from chromatin and replaced by protamines. Although histone-to-protamine exchange is important for sperm nuclear condensation, the underlying regulatory mechanism is still poorly understood. Here, we identify PHD finger protein 7 (PHF7) as an E3 ubiquitin ligase for histone H3K14 in post-meiotic spermatids. Generation of Phf7-deficient mice and Phf7 C160A knockin mice with impaired E3 ubiquitin ligase activity reveals defects in histone-to-protamine exchange caused by dysregulation of histone removal factor Bromodomain, testis-specific (BRDT) in early condensing spermatids. Surprisingly, E3 ubiquitin ligase activity of PHF7 on histone ubiquitination leads to stabilization of BRDT by attenuating ubiquitination of BRDT. Collectively, our findings identify PHF7 as a critical factor for sperm chromatin condensation and contribute to mechanistic understanding of fundamental phenomenon of histone-to-protamine exchange and potential for drug development for the male reproduction system.