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Chad S Driscoll Department of Animal Science, Developmental Epigenetics Laboratory, Reproductive and Developmental Sciences Program, Michigan State University, East Lansing, Michigan, USA

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Jaehwan Kim Department of Animal Science, Developmental Epigenetics Laboratory, Reproductive and Developmental Sciences Program, Michigan State University, East Lansing, Michigan, USA
Department of Animal Sciences, University of Missouri, Columbia, Missouri, USA

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Mohamed Ashry Department of Animal Science, Developmental Epigenetics Laboratory, Reproductive and Developmental Sciences Program, Michigan State University, East Lansing, Michigan, USA

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Jason G Knott Department of Animal Science, Developmental Epigenetics Laboratory, Reproductive and Developmental Sciences Program, Michigan State University, East Lansing, Michigan, USA

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Transcription factor AP2 gamma (TFAP2C) is a well-established regulator of the trophoblast lineage in mice and humans, but a handful of studies indicate that TFAP2C may play an important role in pluripotency. Here, we hypothesize and provide new evidence that TFAP2C functions as an activator of trophoblast and pluripotency genes during preimplantation embryo development.

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Chuan-Mei Qin The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Xiao-Wei Wei Shanghai Jiao Tong University School of Medicine Affiliated Sixth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Jia-Yi Wu The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Xue-Qing Liu The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Yi Lin Shanghai Jiao Tong University School of Medicine Affiliated Sixth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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In brief

The proliferation of the endometrium is regulated by histone methylation. This study shows that decreased NSD2 impairs proliferative-phase endometrial stromal cell proliferation in patients with recurrent implantation failure via epigenetic reprogramming of H3K36me2 methylation on the promoter region of MCM7.

Abstract

Recurrent implantation failure (RIF) is a formidable challenge in assisted reproductive technology because of its unclear molecular mechanism. Impaired human endometrial stromal cell (HESC) proliferation disrupts the rhythm of the menstrual cycle, resulting in devastating disorders between the embryo and the endometrium. The molecular function of histone methylation enzymes in modulating HESC proliferation remains largely uncharacterized. Herein, we found that the levels of histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2) and the dimethylation of lysine 36 on histone H3 are decreased significantly in the proliferative-phase endometrium of patients with RIF. Knockdown of NSD2 in an HESC cell line markedly impaired cell proliferation and globally reduced H3K36me2 binding to chromatin, leading to altered expression of many genes. Transcriptomic analyses revealed that cell cycle-related gene sets were downregulated in the endometrium of patients with RIF and in NSD2‑knockdown HESCs. Furthermore, RNA-sequencing and CUT&Tag sequencing analysis suggested that NSD2 knockdown reduced the binding of H3K36me2 to the promoter region of cell cycle marker gene MCM7 (encoding minichromosome maintenance complex component 7) and downregulated its expression. The interaction of H3K36me2 with the MCM7 promoter was verified using chromatin immunoprecipitation–quantitative real-time PCR. Our results demonstrated a unifying epigenome-scale mechanism by which decreased NSD2 impairs endometrial stromal cell proliferation in the proliferative-phase endometrium of patients with RIF.

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Vakil Ahmad Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA

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Sai Goutham Reddy Yeddula Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA

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Bhanu P Telugu Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA

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Thomas E Spencer Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
Department of Obstetrics, Gynecology, and Women’s Health, University of Missouri, Columbia, Missouri, USA

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Andrew M Kelleher Department of Obstetrics, Gynecology, and Women’s Health, University of Missouri, Columbia, Missouri, USA

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In brief

Polarity-reversed endometrial epithelial organoids exhibit histological and physiological characteristics resembling uterine epithelium in vivo, respond to hormones, and undergo secretory cell transformation. The ability to modify the polarity without impairing functionality, coupled with successful coculture with microbial and embryonic entities, paves the way for investigating complex interactions at the endometrial epithelial surface.

Abstract

The uterine epithelium comprises a single layer of hormone-responsive polarized epithelial cells that line the lumen and form tubular glands. Endometrial epithelial organoids (EEO) can be generated from uterine epithelia and recapitulate cell composition and hormone responses in vitro. As such, the development of EEO represents a significant advance in facilitating mechanistic studies in vitro. However, a major limitation of the use of EEO cultured in basement membrane extract and other hydrogels is the inner location of the apical membrane (apical-in EEO), thereby hindering direct access to the apical surface of the epithelium to study interactions with the embryo or infectious agents such as viruses and bacteria. To address this challenge, we developed a suspension culture method to reverse the polarity of EEO. The result is an apical-out organoid that preserves a distinct apical–basolateral orientation and remains responsive to ovarian steroid hormones. Apical-out EEO were positive for the gland marker, FOXA2, and exhibited appropriate hormonal regulation of steroid hormone receptor expression. Notably, progesterone treatment resulted in secretory transformation in apical-out EEO, including a decrease in microvilli and cilia, and an increase in secretory granules. Likewise, reflective of in vivo conditions, ENPP3, a P4-regulated gene, was localized apically in steroid hormone-treated organoids. Coculture experiments with apical out EEO demonstrate the model’s utility in studying uterine epithelium interactions with bacteria (E. coli) and blastocysts. The apical out EEO model lays the foundation for developing new in vitro functional assays, particularly regarding epithelial interactions with embryos during pregnancy or other luminal constituents in a pathological or diseased state.

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Mathieu Douhard Laboratoire de Biométrie & Biologie Evolutive, CNRS, UMR 5558, Université Lyon, Villeurbanne, France

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Victor Ronget Laboratoire de Biométrie & Biologie Evolutive, CNRS, UMR 5558, Université Lyon, Villeurbanne, France

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Frédéric Douhard GenPhySE, Université de Toulouse, INRAE, ENVT, Castanet-Tolosan, France

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In brief

In litter-bearing species, developing offspring can be exposed to different concentrations of androgens and oestrogens according to the sex of neighbouring fetuses. However, the relationships between litter sex composition and subsequent reproductive performance are discordant and complex.

Abstract

Laboratory studies with rodents indicate that in utero proximity of a female to male fetus can affect female’s subsequent reproduction due to elevated testosterone exposure during early development. It remains unknown whether these findings can be generalised to non-laboratory species because the need for caesarean section makes it difficult to determine the intrauterine position outside laboratory conditions. As an alternative, some studies have compared the reproductive performance of individuals born in male-biased litters to those born in female-biased litters. We identified 44 of those studies in 28 viviparous species for a total of 176 relationships between litter sex composition around the time of birth and subsequent reproductive performance (fertility, fecundity, age at first reproduction, interbirth intervals or post-natal survival of offspring). Those relationships are discordant and complex both within and across species. Some factors can mask an actual association between litter sex composition and reproductive performance. Conversely, a part of significant relationships between litter sex composition and reproductive performance likely arises via pathways other than androgen- and oestrogen-transfer between fetuses of different sexes.

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Bridget M Arman Therapeutics Discovery and Vascular Function in Pregnancy Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia
Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Australia

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Natalie K Binder Therapeutics Discovery and Vascular Function in Pregnancy Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia
Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Australia

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Natasha de Alwis Therapeutics Discovery and Vascular Function in Pregnancy Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia
Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Australia

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Sally Beard Therapeutics Discovery and Vascular Function in Pregnancy Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia

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Anjali Garg Therapeutics Discovery and Vascular Function in Pregnancy Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia

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Tu’uhevaha J Kaitu’u-Lino Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Australia
Translational Obstetrics Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia

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Natalie J Hannan Therapeutics Discovery and Vascular Function in Pregnancy Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia
Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Australia

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In brief

Preterm birth is the leading cause of perinatal morbidity and mortality, and new therapies that delay preterm birth and improve neonatal outcomes are urgently needed. This study investigates whether ticagrelor inhibits uterine contractility and inflammation in preclinical in vitro, ex vivo (human) and in vivo (mouse) studies, to explore the potential of repurposing ticagrelor for the prevention of preterm birth.

Abstract

Preterm birth remains a significant global health challenge, affecting approximately 10% of pregnancies and resulting in one million deaths globally every year. Tocolytic agents, used to manage preterm labour, have considerable limitations including lack of efficacy, and adverse side effects, emphasising the urgent need for innovative solutions. Here, we explore repurposing an antiplatelet cardioprotective drug, ticagrelor, as a potential treatment to prevent preterm birth. Ticagrelor has demonstrated pleiotropic actions beyond platelet inhibition, including relaxant effects on smooth muscle cells and anti-inflammatory effects in models of diabetes and sepsis. As preterm birth is underscored by inflammatory processes triggering uterine contractions, these actions position ticagrelor as an attractive candidate for prevention or delay of preterm birth. Utilising primary human myometrial tissue, human myometrial cells, and a mouse model of preterm birth, we investigated ticagrelor’s potential as a safe and effective therapy for preterm birth. We showed that ticagrelor did not reduce the frequency or strength of spontaneous muscle contractions of ex vivo myometrial tissue nor did it reduce in vitro inflammation-induced contractility in myometrial cells. Additionally, ticagrelor did not exhibit the anticipated anti-inflammatory effects in myometrial cell culture experiments. In our mouse model of preterm birth, ticagrelor neither improved the preterm birth rate or fetal survival outcomes. Gene expression of pro-inflammatory cytokines and contraction-associated proteins in postpartum mouse uteri were unaltered by ticagrelor. In conclusion, ticagrelor is not a strong candidate to continue investigations in clinical trial for the treatment of preterm labour and prevention of preterm birth.

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Luisa Gioia Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy

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Luca Palazzese Department of Veterinary Medicine, University of Teramo, Teramo, Italy

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Marta Czernik Department of Veterinary Medicine, University of Teramo, Teramo, Italy

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Domenico Iuso Department of Veterinary Medicine, University of Teramo, Teramo, Italy

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Helena Fulka Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic, Institute of Animal Science, Prague, Czech Republic

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Josef Fulka Jr Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic, Institute of Animal Science, Prague, Czech Republic

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Pasqualino Loi Department of Veterinary Medicine, University of Teramo, Teramo, Italy

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The fertilizing spermatozoa induce a Ca2+ oscillatory pattern, the universal hallmark of oocyte activation, in all sexually reproducing animals. Assisted reproductive technologies (ARTs) like intracytoplasmic sperm injection (ICSI) bypass the physiological pathway; however, while a normal Ca2+ release pattern occurs in some species, particularly humans, artificial activation is compulsory for ICSI-fertilized oocytes to develop in most farm animals. Unlike the normal oscillatory pattern, most artificial activation protocols induce a single Ca2+ spike, undermining proper ICSI-derived embryo development in these species. Curiously, diploid parthenogenetic embryos activated by the same treatments develop normally at high frequencies and implant upon transfer in the uterus. We hypothesized that, at least in ruminant embryos, the oscillatory calcium waves late in the first cell cycle target preferentially the paternal pronucleus and are fundamentally important for paternal nuclear remodeling. We believe that Ca2+ signaling is central to full totipotency deployment of the paternal genome. Research in this area could highlight the asymmetry between the parental genome reprogramming timing/mechanisms in early development and impact ARTs like ICSI and cloning.

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Meng Ma Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Shutian Jiang Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Wei Jin Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Wenzhi Li Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Chen Chen Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Kaibo Lin Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Xiaoyu Liao Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Danjun Li Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Yanping Kuang Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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Qifeng Lyu Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

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In brief

The impact of HVJ-E employed in mitochondrial replacement techniques (MRTs) on embryonic development remains uncertain. This study has exhibited the influence of HVJ-E utilized in MRTs on embryonic development and has devised a novel HVJ-E-induced fusion approach to curtail the amount of HVJ-E employed in MRTs.

Abstract

Mitochondrial replacement techniques (MRTs) provide a viable option for women carrying pathogenic mitochondrial DNA (mtDNA) variants to conceive disease-free offspring with a genetic connection. In comparison to electrofusion, HVJ-E-induced fusion has been identified as the most promising approach for clinical translation of MRTs due to its absence of electrical interference. However, despite confirmation of the absence of RNA activity in HVJ-E, a reduction in blastocyst quality has been observed in various MRTs studies utilizing the HVJ-E-induced fusion scheme. Recent investigations have revealed a dose-dependent elevation of reactive oxygen species (ROS) levels in various cancer cells incubated with HVJ-E. However, the impact of HVJ-E as a sole determinant on embryonic development in MRTs remains unverified. This investigation establishes that the augmented concentration of HVJ-E utilized in the conventional HVJ-E fusion protocol is an autonomous variable that influences embryonic development in MRTs. This effect may be attributed to amplified DNA damage resulting from heightened levels of ROS in reconstructed embryos. To mitigate the presence of HVJ-E in reconstructed zygotes while maintaining optimal fusion efficiency in MRTs, a novel HVJ-E-induced fusion approach was devised, namely, press-assisted fusion. This technique offers potential advantages in reducing detrimental factors that impede embryo development in MRTs.

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Xiaotong Wu Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Yan Shi Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Bingjie Hu Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Panpan Zhao Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Shuang Li Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Lieying Xiao Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Shaohua Wang Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Kun Zhang Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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In brief

Lineage specification plays a vital role in preimplantation development. TEAD4 is an essential transcription factor for trophectoderm lineage specification in mice but not in cattle.

Abstract

Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development.

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Tomomi Kawamura Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Yidan Dai Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Masanori Ono Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Takayuki Kikuchi Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Akina Yamanaka Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Keiko Ueno Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Junya Kojima Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Tomoko Fujiwara Department of Social Work and Life Design, Kyoto Notre Dame University, Kyoto, Japan

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Takiko Daikoku Division of Animal Disease Model, Research Center for Experimental Modeling of Human Disease, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan

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Yoshiko Maida Department of Nursing, College of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kanazawa, Japan

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Hitoshi Ando Department of Cellular and Molecular Function Analysis, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan

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Hiroshi Fujiwara Department of Obstetrics and Gynecology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan

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Naoaki Kuji Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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Hirotaka Nishi Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

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In brief

In this study, we examined the relationship between BMAL1 expression and the genes regulating steroid biosynthesis in human luteinized granulosa cells. BMAL1 function is crucial for steroid production and proper ovarian function, highlighting the importance of circadian clock regulation in female reproductive health.

Abstract

Human luteinized granulosa cells were collected to analyze circadian clock gene expression and its effect on the genes regulating steroid biosynthesis. We used siRNA to knock down the expression of BMAL1 in KGN cells. We measured the expression levels of genes regulating steroid biosynthesis and circadian clock RT-qPCR. We demonstrated that BMAL1 expression positively correlates with genes regulating steroid biosynthesis (CYP11A1, CYP19A1, STAR, and ESR2). The knockdown of BMAL1 in KGN cells revealed a significant decrease in steroid synthase expression. In contrast, when BMAL1 was overexpressed in KGN and HGL5 cells, we observed a significant increase in the expression of steroid synthases, such as CYP11A1 and CYP19A1. These results indicated that BMAL1 positively controls 17β-estradiol (E2) secretion in granulosa cells. We also demonstrated that dexamethasone synchronization in KGN cells enhanced the rhythmic alterations in circadian clock genes. Our study suggests that BMAL1 plays a critical role in steroid biosynthesis in human luteinized granulosa cells, thereby emphasizing the importance of BMAL1 in the regulation of reproductive physiology.

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Yoloxochitl Sánchez-Guevara Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México

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Enrique I Oliver Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México

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Takuya Nishigaki Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México

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In brief

Free Ca2+ concentrations within mitochondria ([Ca2+]mt) in mouse spermatozoa were reported in a micromolar range based on the result obtained with Calcium Green 5N. The results obtained with Rhod-2, Fluo-3, and Fluo-5N suggest that the [Ca2+]mt are comparable to the cytosolic levels at the resting condition and under the condition stimulated by ATP.

Abstract

Mitochondria are important organelles in eukaryotic cells and play an essential role in energy production and cell signaling. However, the importance of mammalian sperm mitochondria as an energy source remains to be elucidated because glycolysis is known to be dominant. In this context, one of the functions of mammalian sperm mitochondria is considered as a calcium ion (Ca2+) homeostasis. Previously, the Ca2+ level within the mitochondria of mouse sperm under resting conditions was reported to be high (in the micromolar range) using the fluorescent Ca2+ indicator Calcium Green-5N (CG-5N). To confirm this fact, we performed the semiquantitative determination of Ca2+ concentration with several Ca2+ indicators. Although we reproduced the previous report of CG-5N, other Ca2+ indicators do not support the result obtained with CG-5N. The results obtained with Rhod-2, Fluo-3, and Fluo-5N indicate that the free Ca2+ concentration in mitochondria is comparable to that of the cytosol at the resting condition and under the condition stimulated by ATP. Although we still do not understand why CG-5N exhibits a distinct result from other indicators, the regulation of Ca2+ concentration in murine sperm mitochondria is analogous to that observed in somatic cells. Namely, the Ca2+ concentrations within sperm mitochondria fluctuate in response to changes in cytosolic Ca2+ levels. Our results contribute to a revised understanding of the role of mitochondria in Ca2+ homeostasis in mammalian sperm.

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