Transgenesis and Genome Editing of Mouse Spermatogonial Stem Cells by Lentivirus Pseudotyped with Sendai Virus F Protein

Spermatogonial stem cells (SSCs) serve as a resource for producing genetically modified animals. However, genetic manipulation of SSCs has met with limited success. Here, we show efficient gene transfer into SSCs via a lentivirus (FV-LV) using a fusion protein (F), a Sendai virus (SV) envelope protein involved in virion/cell membrane fusion. FV-LVs transduced cultured SSCs more efficiently than conventional LVs. Although SSCs infected with SV failed to produce offspring, those transduced with FV-LVs were fertile. In vivo microinjection showed that FV-LVs could penetrate not only the basement membrane of the seminiferous tubules but also the blood-testis barrier, which resulted in successful transduction of both spermatogenic cells and testicular somatic cells. Cultured SSCs transfected with FV-LVs that express drug-inducible CRISPR/Cas9 against Kit or Sycp3 showed impaired spermatogenesis upon transplantation and drug treatment in vivo. Thus, FV-LVs provide an efficient method for functional analysis of genes involved in SSCs and spermatogenesis

NMR Study of Quasicrystal Al_<70>Pd_<20>T_<M10> Alloy (T_M : Cr, Mn, Fe and Co)

^Al-, ^Mn- and ^Co-NMR measurements have been made on quasicrytalline phases of the Al_Pd_T_ alloy (T_M : Cr, Mn, Fe and Co) in the temperature range between 5K and room temperature. There is no marked temperature dependence on peak shift and line width of either ^Al- or ^Co- NMR spectra except ^Al- and ^Mn- spectra in the Al_Pd_Mn_. However, the peak shift of these spectra depends upon the constituent transition element. For the Al_Pd_Mn_, the negative ^Al- Knight shift and line broadening at low temperatures indicate that there exist two classes of Mn atoms, magnetic and nonmagnetic ones

An optimization method for designing high rate and high performance SCTCM systems with in-line interleavers

We present a method for designing high-rate, high-performance SCTCM systems with in-line interleavers. Using in-line EXIT charts and ML performance analysis, we develop criteria for choosing constituent codes and optimization methods for selecting the best ones. To illustrate our methods, we show that an optimized SCTCM system with an in-line interleaver for rate r = 5/6 and 64QAM has better performance than other turbo-like TCMs with the same parameters

Regulation of male germline transmission patterns by the Trp53-Cdkn1a pathway

A small number of offspring are born from the numerous sperm generated from spermatogonial stem cells (SSCs). However, little is known regarding the rules and molecular mechanisms that govern germline transmission patterns. Here we report that the Trp53 tumor suppressor gene limits germline genetic diversity via Cdkn1a. Trp53-deficient SSCs outcompeted wild-type (WT) SSCs and produced significantly more progeny after co-transplantation into infertile mice. Lentivirus-mediated transgenerational lineage analysis showed that offspring bearing the same virus integration were repeatedly born in a non-random pattern from WT SSCs. However, SSCs lacking Trp53 or Cdkn1a sired transgenic offspring in random patterns with increased genetic diversity. Apoptosis of KIT⁺ differentiating germ cells was reduced in Trp53- or Cdkn1a-deficient mice. Reduced CDKN1A expression in Trp53-deficient spermatogonia suggested that Cdkn1a limits genetic diversity by supporting apoptosis of syncytial spermatogonial clones. Therefore, the TRP53-CDKN1A pathway regulates tumorigenesis and the germline transmission pattern

On dissemination mechanism of corporate social responsibility (CSR): Analysis with agent simulation

Corporate Social Responsibility (CSR), such as pro-environmental behaviour and fair trade, is a kind of normative behaviour by private companies to provide a quasi-public good. We study dissemination mechanism of CSR with a multi-agent model in which corporation agents and consumer agents interact with each other. We show that the mechanism to disseminate CSR is a positive feedback between the corporations\u27 popularity seeking behaviour and the consumers\u27 social learning in which CSR-seeking preference is evaluated according to both the local average of the preferences of surrounding consumers and the global average of the investment in CSR by all corporations. We also discuss an institutional design to establish CSR from an objectionable social state

Cdc42 is required for male germline niche development in mice

精子形成促進分子GDNFの制御機構の解明 --男性不妊治療への応用に期待--. 京都大学プレスリリース. 2021-08-19.Spermatogonial stem cells (SSCs) are maintained in a special microenvironment called a niche. However, much is unknown about components that constitute the niche. Here, we report that Cdc42 is essential for germline niche development. Sertoli cell-specific Cdc42-deficient mice showed normal premeiotic spermatogenesis. However, germ cells gradually disappeared during haploid cell formation and few germ cells remained in the mature testes. Spermatogonial transplantation experiments revealed a significant loss of SSCs in Cdc42-deficient testes. Moreover, Cdc42 deficiency in Sertoli cells downregulated GDNF, a critical factor for SSC maintenance. Cdc42-deficient Sertoli cells also exhibited lower nuclear MAPK1/3 staining. Inhibition of MAP2K1 or depletion of Pea15a scaffold protein downregulated GDNF expression. A screen of transcription factors revealed that Cdc42-deficient Sertoli cells downregulate DMRT1 and SOX9, both of which are critical for Sertoli cell development. These results indicate that Cdc42 is essential for niche function via MAPK1/3-dependent GDNF secretion

Adenovirus-mediated gene delivery restores fertility in congenitally infertile female mice

Oogenesis depends on close interactions between oocytes and granulosa cells. Abnormal signaling between these cell types can result in infertility. However, attempts to manipulate oocyte-granulosa cell interactions have had limited success, likely due to the blood-follicle barrier (BFB), which prevents the penetration of exogenous materials into ovarian follicles. Here, we used adenoviruses (AVs) to manipulate the oocyte-granulosa cell interactions. AVs penetrated the BFB and transduced granulosa cells through ovarian microinjection. Although AVs caused transient inflammation, they did not impair fertility in wild-type mice. Introduction of Kitl-expressing AVs into congenitally infertile KitlSl-t/KitlSl-t mutant mouse ovaries, which contained only primordial follicles because of a lack of Kitl expression, restored fertility through natural mating. The offspring showed no evidence of AV integration and exhibited normal genomic imprinting patterns for imprinted genes. These results demonstrate the usefulness of AVs for manipulating oogenesis and suggest the possibility of gene therapies for human female infertility

Reconstitution of Mouse Spermatogonial Stem Cell Niches in Culture

SummarySpermatogonial stem cells (SSCs) reside in specific niches within seminiferous tubules. These niches are thought to secrete chemotactic factors for SSCs, because SSCs migrate to them upon transplantation. However, the identity of these chemotactic molecules remains unknown. Here, we established a testis feeder cell culture system and used it to identify SSC chemotactic factors. When seeded on testis cells from infertile mice, SSCs migrated beneath the Sertoli cells and formed colonies with a cobblestone appearance that were very similar to those produced by hematopoietic stem cells. Cultured cells maintained SSC activity and fertility for at least 5 months. Cobblestone colony formation depended on GDNF and CXCL12, and dominant-negative GDNF receptor transfection or CXCL12 receptor deficiency reduced SSC colonization. Moreover, GDNF upregulated CXCL12 receptor expression, and CXCL12 transfection in Sertoli cells increased homing efficiency. Overall, our findings identify GDNF and CXCL12 as SSC chemotactic factors in vitro and in vivo

Spermatogonial stem cell transplantation into nonablated mouse recipient testes

Spermatogonial transplantation has been used as a standard assay for spermatogonial stem cells (SSCs). After transplantation into the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) between Sertoli cells and settle in a niche. Unlike in the repair of other self-renewing systems, SSC transplantation is generally performed after complete destruction of endogenous spermatogenesis. Here, we examined the impacts of recipient conditioning on SSC homing. Germ cell ablation downregulated the expression of glial cell line-derived neurotrophic factor, which has been shown to attract SSCs to niches, implying that nonablated niches would attract SSCs more efficiently. As expected, SSCs colonized nonablated testes when transplanted into recipients with the same genetic background. Moreover, although spermatogenesis was arrested at the spermatocyte stage in Cldn11-deficient mice without a BTB, transplantation not only enhanced donor colonization but also restored normal spermatogenesis. The results show promise for the development of a new transplantation strategy to overcome male infertility