
セルトリ細胞のmTOR経路の活性バランスが、精子のDNAメチル化の加齢変化を調節する。


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Over the past several decades, a trend toward delayed childbirth has led to increases in parental age at the time of conception. Sperm epigenome undergoes age-dependent changes increasing risks of adverse conditions in offspring conceived by fathers of advanced age. The mechanism(s) linking paternal age with epigenetic changes in sperm remain unknown. The sperm epigenome is shaped in a compartment protected by the blood-testes barrier (BTB) known to deteriorate with age. Permeability of the BTB is regulated by the balance of two mTOR complexes in Sertoli cells where mTOR complex 1 (mTORC1) promotes the opening of the BTB and mTOR complex 2 (mTORC2) promotes its integrity. We hypothesized that this balance is also responsible for age-dependent changes in the sperm epigenome. To test this hypothesis, we analyzed reproductive outcomes, including sperm DNA methylation in transgenic mice with Sertoli cell-specific suppression of mTORC1 (Raptor KO) or mTORC2 (Rictor KO). mTORC2 suppression accelerated aging of the sperm DNA methylome and resulted in a reproductive phenotype concordant with older age, including decreased testes weight and sperm counts, and increased percent of morphologically abnormal spermatozoa and mitochondrial DNA copy number. Suppression of mTORC1 resulted in the shift of DNA methylome in sperm opposite to the shift associated with physiological aging – sperm DNA methylome rejuvenation, and mild changes in sperm parameters. These results demonstrate for the first time that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. Thus, mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.

Ethics statement All procedures followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the approval for this study was received from the Institutional Animal Care and Use Committee at University of Massachusetts, Amherst.

### Competing Interest Statement

Alexander Suvorov is a CEO of ReGENE LLC. All other authors declare that they have no competing interests.

Mechanistic target of rapamycin (mTOR) pathway in Sertoli cells regulates age-dependent changes in sperm DNA methylation

父親の年齢が高くなるにつれ-精子のdnaメチル化が変化する仕組み


父親の年齢が高くなるにつれ、精子のDNAメチル化が変化する仕組み


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レテ・オバリーは卵巣の発達と機能に重要な役割を果たす分泌器官である。


The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Gray’s Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow towards the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.

### Competing Interest Statement

The authors have declared no competing interest.

Rediscovering the Rete Ovarii: a secreting auxiliary structure to the ovary

卵巣の補助構造であるレテ-オバリーの再発見


卵巣の補助構造であるレテ・オバリーの再発見



CRISP1 および CRISP3 は精子 DNA 完全性と初期胚発生に重要な役割を果たす新しい男性因子である。


Numerous reports show that the epididymis plays a key role in the acquisition of sperm fertilizing ability but less information exists on its contribution to embryo development. Evidence from our laboratory showed that mammalian CRISP (Cysteine-Rich Secretory Proteins), known to be expressed in the epididymis, to regulate calcium (Ca2+) channels and to participate in fertilization, may also be relevant for embryo development. More specifically, we found that males with simultaneous mutations in Crisp1 and Crisp3 genes exhibited normal in vivo fertilization but impaired embryo development. In the present work, aimed to investigate the mechanisms underlying this reproductive phenotype, we observed that embryo development failure was not due to delayed fertilization as no differences in sperm transport within the female tract nor in in vivo fertilization were found shortly after mating. The observation that impaired embryo development was also found in eggs fertilized by epididymal sperm either after uterine insemination or in vitro fertilization, revealed that the defects were already present at epididymal level. Of note, eggs fertilized in vitro by mutant sperm exhibited impaired meiotic resumption not due to defects in Ca2+oscillations during egg activation, prompting us to examine potential sperm DNA defects. Interestingly, higher levels of both DNA fragmentation and intracellular Ca2+ were observed for mutant than for control epididymal sperm, supporting sperm DNA damage, likely linked to a Ca2+ dysregulation, as the main responsible for the early development failure of mutant males. Together, our results support the contribution of the epididymis beyond fertilization, identifying CRISP1 and CRISP3 as novel male factors relevant for DNA integrity and early embryo development. Given the existence of human functional homologues of CRISP and the incidence of DNA fragmentation in infertile men, we believe these findings not only provide relevant information on the impact of epididymal factors on embryonic development but will also contribute to a better understanding, diagnosis and treatment of human infertility.

### Competing Interest Statement

The authors have declared no competing interest.

Contribution of the epididymis beyond fertilization: relevance of CRISP1 and CRISP3 for sperm DNA integrity and early embryo development

精子-dna-完全性と初期胚発生に関する-crisp1-および-crisp3-の重要性-受精以外での精巣上体の貢献


精子 DNA 完全性と初期胚発生に関する CRISP1 および CRISP3 の重要性 - 受精以外での精巣上体の貢献



IQCHはカルモジュリンと結合し、RNA結合タンパク質の発現を調節することで、精子形成に重要な役割を果たす。


IQ motif-containing proteins can be recognized by calmodulin (CaM) and are essential for many biological processes. However, the role of IQ motif-containing proteins in spermatogenesis is largely unknown. In this study, we identified a loss-of-function mutation in the novel gene IQ motif-containing H ( IQCH ) in a Chinese family with male infertility characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. To verify the function of IQCH, Iqch -knockout mice were generated via CRISPR-Cas9 technology, which reproduced human phenotypes. Mechanistically, IQCH can bind to CaM and subsequently regulate the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis. Overall, this study revealed the function of IQCH, expanded the role of IQ motif-containing proteins in reproductive processes, and provided important guidance for genetic counseling and genetic diagnosis of male infertility.

### Competing Interest Statement

The authors have declared no competing interest.

IQCH regulates spermatogenesis by interacting with CaM to promote the expression of RNA-binding proteins

男性不妊症の原因遺伝子iqch-の機能解明-カルモジュリンとの相互作用によるrna結合タンパク質の発現調節が精子形成に重要


男性不妊症の原因遺伝子IQCH の機能解明 - カルモジュリンとの相互作用によるRNA結合タンパク質の発現調節が精子形成に重要



サイクリン依存性キナーゼ8および19のダブルノックアウトにより、サイクリンCが枯渇し、精子形成と男性不妊が引き起こされる。


Paralogs CDK8 and CDK19 are regulatory kinases associated with the transcriptional Mediator complex. We have for the first time generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout. Cdk8/19 double knockout (DKO) males, but not single Cdk8 and Cdk19 KO, had an atrophic reproductive system and were infertile. The DKO males lacked postmeiotic spermatids and spermatocytes after meiosis I pachytene. Testosterone levels were decreased whereas the amounts of the luteinizing hormone were unchanged. Single cell RNA sequencing showed marked differences in the expression of steroidogenic genes (such as Cyp17a1, Star and Fads ) in Leydig cells concomitant with alterations in Sertoli cells and spermatocytes likely associated with impaired synthesis of steroids. Star and Fads were also downregulated in cultivated Leydig cells after DKO. The treatment of primary Leydig cells culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as DKO, and prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. DKO, in contrast to single knockouts or treatment with a CDK8/19 kinase inhibitor, led to depletion of cyclin C (CcnC), the binding partner of CDK8/19 that has been implicated in CDK8/19-independent functions. This suggests that the observed phenotype was likely mediated through kinase-independent activities of CDK8/19, such as CcnC stabilization.

### Competing Interest Statement

Igor B. Roninson is Founder and President and Mengqian Chen is consultant of Senex Biotechnology, Inc. Other authors declare no conflict of interest.

Knockout of cyclin dependent kinases 8 and 19 leads to depletion of cyclin C and suppresses spermatogenesis and male fertility in mice

マウスにおけるサイクリン依存性キナーゼ8および19のノックアウトによるサイクリンcの枯渇と精子形成および男性不妊の抑制


マウスにおけるサイクリン依存性キナーゼ8および19のノックアウトによるサイクリンCの枯渇と精子形成および男性不妊の抑制



BEND2の欠失は卵子の質の低下、卵胞形成の減少、生殖寿命の早期低下を引き起こす。BEND2は卵子形成と生殖老化の機構を理解する上で重要な遺伝子である。


Reproductive aging, characterized by a decline in female reproductive potential, is a significant biomedical challenge. A key factor in reproductive aging is the depletion of the ovarian reserve, the pool of primordial follicles in the ovary. Recent studies have implicated BEND2, a BEN domain-containing protein family member, in mammalian spermatogenesis. In the testis, Bend2 expresses two protein isoforms: full-length and truncated. Ablation of both proteins results in an arrested spermatogenesis. Because the Bend2 locus is on the X chromosome, and the Bend2-/y mutants are sterile, Bend2 ’s role in oogenesis remained elusive.

In this study, we employed a novel Bend2 mutation that completely blocks the expression of the full-length BEND2 protein but allows the expression of the truncated BEND2 isoform. However, this mutation does not confer male sterility, allowing us to investigate BEND2’s role in mice’s oocyte quality, follicular dynamics, and fertility. Our findings demonstrate that full-length BEND2 is dispensable for male fertility, and its ablation leads to impaired oocyte quality, reduced follicular formation, and an accelerated decline in fertility. These results reveal a critical role for BEND2 in oogenesis and provide insights into the mechanisms underlying reproductive aging. Furthermore, these findings hold relevance for the diagnostic landscape of human infertility.

### Competing Interest Statement

The authors have declared no competing interest.

BEND2 is a crucial player in oogenesis and reproductive aging

bend2は卵子形成と生殖老化において重要な役割を果たす


BEND2は卵子形成と生殖老化において重要な役割を果たす
