Reproductive biology
Abstract
Proper action of the female sex steroids, 17β-estradiol (E2) and progesterone (P4) on endometrium is essential for fertility. Beyond its role in regulating the cell cycle, cyclin A2 (CCNA2) also mediates E2 and P4 signaling in vitro, but a potential role in modulating steroid action for proper endometrial tissue development and function is unknown. To fill this gap in our knowledge, we examined human endometrial tissue from fertile and infertile women for CCNA2 expression and correlated this with pregnancy outcome. Functional assessment of CCNA2 was validated in vivo using a conditional Ccna2 uterine deficient mouse model while in vitro function was assessed using human cell culture models. We found that CCNA2 expression was significantly reduced in endometrial tissue, specifically the stromal cells, from women undergoing in vitro fertilization who failed to achieve pregnancy. Conditional deletion of Ccna2 from mouse uterine tissue resulted in an inability to achieve pregnancy which appears to be due to alterations in the process of decidualization, which was confirmed using in vitro models. From these studies, we conclude that CCNA2 expression during the proliferative/regenerative stage of the menstrual cycle acts as a safeguard allowing for proper steroid responsiveness, decidualization and pregnancy. When CCNA2 expression levels are insufficient there is impaired endometrial responsiveness, aberrant decidualization and loss of pregnancy.
Authors
Fatimah Aljubran, Katelyn Schumacher, Amanda Graham, Sumedha Gunewardena, Courtney Marsh, Michael Lydic, Kristin Holoch, Warren B. Nothnick
Abstract
Endometriosis is a debilitating, chronic inflammatory disease affecting ~10% of reproductive age women worldwide with no cure. While macrophages have been intrinsically linked to the pathophysiology of endometriosis, targeting them therapeutically has been extremely challenging due to their high heterogeneity and because these disease-associated macrophages (DAMs) can be either pathogenic or protective. Here, we reported identification of pathogenic macrophages characterized by TET3 overexpression in human endometriosis lesions. We showed that factors from the disease microenvironment upregulated TET3 expression transforming macrophages into pathogenic DAMs. TET3 overexpression stimulated pro-inflammatory cytokine production via a feedback mechanism involving inhibition of let-7 miRNA expression. Remarkably, these cells relied on TET3 overexpression for survival, hence vulnerable to TET3 knockdown. We demonstrated that Bobcat339, a synthetic cytosine derivative, triggered TET3 degradation both in human and mouse macrophages. This degradation was dependent on a VHL E3 ubiquitin ligase whose expression was also upregulated in TET3-overexpressing macrophages. Furthermore, depleting TET3-overexpressing macrophages either through myeloid-specific Tet3 ablation or using Bobcat339 strongly inhibited endometriosis progression in mice. Our results defined TET3-overexpressing macrophages as key pathogenic contributors to and attractive therapeutic targets for endometriosis. Our findings may also be applicable to other chronic inflammatory diseases where DAMs have important roles.
Authors
Haining Lv, Beibei Liu, Yangyang Dai, Feng Li, Stefania Bellone, Yuping Zhou, Ramanaiah Mamillapalli, Dejian Zhao, Muthukumaran Venkatachalapathy, Yali Hu, Gordon G. Carmichael, Da Li, Hugh S. Taylor, Yingqun Huang
Abstract
Reproduction is safeguarded by multiple, often cooperative regulatory networks. Kisspeptin signaling, via KISS1R, plays a fundamental role in reproductive control, primarily by regulation of hypothalamic GnRH neurons. We disclose herein a pathway for direct kisspeptin actions in astrocytes that contributes to central reproductive modulation. Protein-protein-interaction and ontology analyses of hypothalamic proteomic profiles after kisspeptin stimulation revealed that glial/astrocyte markers are regulated by kisspeptin in mice. This glial-kisspeptin pathway was validated by the demonstrated expression of Kiss1r in mouse astrocytes in vivo and astrocyte cultures from humans, rats and mice, where kisspeptin activated canonical intracellular signaling-pathways. Cellular co-expression of Kiss1r with the astrocyte markers, GFAP and S100-β, occurred in different brain regions, with higher percentage in Kiss1- and GnRH-enriched areas. Conditional ablation of Kiss1r in GFAP-positive cells, in the G-KiRKO mouse, altered gene expression of key factors in PGE2 synthesis in astrocytes, and perturbed astrocyte-GnRH neuronal appositions, as well as LH responses to kisspeptin and LH pulsatility, as surrogate marker of GnRH secretion. G-KiRKO mice also displayed changes in reproductive responses to metabolic stress induced by high-fat diet, affecting female pubertal onset, estrous cyclicity and LH-secretory profiles. Our data unveil a non-neuronal pathway for kisspeptin actions in astrocytes, which cooperates in fine-tuning the reproductive axis and its responses to metabolic stress.
Authors
Encarnacion Torres, Giuliana Pellegrino, Melissa Granados-Rodríguez, Antonio C. Fuentes-Fayos, Inmaculada Velasco, Adrian Coutteau-Robles, Amandine Legrand, Marya Shanabrough, Cecilia Perdices-Lopez, Silvia Leon, Shel H. Yeo, Stephen M. Manchishi, Maria J. Sánchez-Tapia, Victor M. Navarro, Rafael Pineda, Juan Roa, Frederick Naftolin, Jesús Argente, Raúl M. Luque, Julie A. Chowen, Tamas L. Horvath, Vicent Prevot, Ariane Sharif, William H. Colledge, Manuel Tena-Sempere, Antonio Romero-Ruiz
Abstract
Early gestational loss occurs in approximately 20% of all clinically recognized human pregnancies and is an important cause of morbidity. Either embryonic or maternal defects can cause loss, but a functioning and receptive uterine endometrium is crucial for embryo implantation. We report that the switch/sucrose nonfermentable (SWI/SNF) remodeling complex containing polybromo-1 (PBRM1) and Brahma-related gene 1 (BRG1) is essential for implantation of the embryonic blastocyst on the wall of the uterus in mice. Although preimplantation development is unaffected, conditional ablation of Pbrm1 in uterine stromal cells disrupts progesterone pathways and uterine receptivity. Heart and neural crest derivatives expressed 2 (Hand2) encodes a basic helix-loop-helix (bHLH) transcription factor required for embryo implantation. We identify an enhancer of the Hand2 gene in stromal cells that requires PBRM1 for epigenetic histone modifications/coactivator recruitment and looping with the promoter. In Pbrm1cKO mice, perturbation of chromatin assembly at the promoter and enhancer sites compromises Hand2 transcription, adversely affects fibroblast growth factor signaling pathways, prevents normal stromal-epithelial crosstalk, and disrupts embryo implantation. The mutant female mice are infertile and provide insight into potential causes of early pregnancy loss in humans.
Authors
Qiliang Xin, Iris Feng, Guoyun Yu, Jurrien Dean
Abstract
Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway — KYNU, HAAO, and NADSYN1 — have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain–specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/– mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1–/– mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.
Authors
Justin O. Szot, Hartmut Cuny, Ella M.M.A. Martin, Delicia Z. Sheng, Kavitha Iyer, Stephanie Portelli, Vivien Nguyen, Jessica M. Gereis, Dimuthu Alankarage, David Chitayat, Karen Chong, Ingrid M. Wentzensen, Catherine Vincent-Delormé, Alban Lermine, Emma Burkitt-Wright, Weizhen Ji, Lauren Jeffries, Lynn S. Pais, Tiong Y. Tan, James Pitt, Cheryl A. Wise, Helen Wright, Israel D. Andrews, Brianna Pruniski, Theresa A. Grebe, Nicole Corsten-Janssen, Katelijne Bouman, Cathryn Poulton, Supraja Prakash, Boris Keren, Natasha J. Brown, Matthew F. Hunter, Oliver Heath, Saquib A. Lakhani, John H. McDermott, David B. Ascher, Gavin Chapman, Kayleigh Bozon, Sally L. Dunwoodie
Abstract
Aneuploidy, a deviation from the normal chromosome copy number, is common in human embryos and is considered a primary cause of implantation failure and early pregnancy loss. Meiotic errors lead to uniformly abnormal karyotypes, while mitotic errors lead to chromosomal mosaicism: the presence of cells with at least two different karyotypes within an embryo. Knowledge about mosaicism in blastocysts mainly derives from bulk DNA sequencing of multicellular trophectoderm (TE) and/or inner cell mass (ICM) samples. However, this can only detect an average net gain or loss of DNA above a detection threshold of 20-30%. To accurately assess mosaicism, we separated the TE and ICM of 55 good quality surplus blastocysts and successfully applied single-cell whole genome sequencing (scKaryo-seq) on 1057 cells. Mosaicism involving numerical and structural chromosome abnormalities was detected in 82% of the embryos, where most abnormalities affected less than 20% of the cells. Structural abnormalities, potentially caused by replication stress and DNA damage, were observed in 69% of the embryos. In conclusion, our findings indicated that mosaicism is prevalent in good-quality blastocysts, while these blastocysts would likely be identified as normal with current bulk DNA sequencing techniques used for preimplantation genetic testing for aneuploidy (PGT-A).
Authors
Effrosyni A. Chavli, Sjoerd J. Klaasen, Diane Van Opstal, Joop S.E. Laven, Geert J.P.L. Kops, Esther B. Baart
Abstract
The infertility of many couples rests on an enigmatic dysfunction of the man’s sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.
Authors
Samuel Young, Christian Schiffer, Alice Wagner, Jannika Patz, Anton Potapenko, Leonie Herrmann, Verena Nordhoff, Tim Pock, Claudia Krallmann, Birgit Stallmeyer, Albrecht Röpke, Michelina Kierzek, Cristina Biagioni, Tao Wang, Lars Haalck, Dirk Deuster, Jan N. Hansen, Dagmar Wachten, Benjamin Risse, Hermann M. Behre, Stefan Schlatt, Sabine Kliesch, Frank Tüttelmann, Christoph Brenker, Timo Strünker
Abstract
In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F1 offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F1 offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation–ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F2 generation. Furthermore, although these F1 offspring appeared normal, F2 offspring produced by IVF using F1 sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.
Authors
Mito Kanatsu-Shinohara, Yusuke Shiromoto, Narumi Ogonuki, Kimiko Inoue, Satoko Hattori, Kento Miura, Naomi Watanabe, Ayumi Hasegawa, Keiji Mochida, Takuya Yamamoto, Tsuyoshi Miyakawa, Atsuo Ogura, Takashi Shinohara
Abstract
Three sisters, born from consanguineous parents, manifested a unique Mullerian anomaly characterized by uterine hypoplasia with thin estrogen-unresponsive endometrium, primary amenorrhea, but spontaneous tubal pregnancies. Through whole-exome sequencing followed by comprehensive genetic analysis, a missense variant was identified in the OSR1 gene. We therefore investigated OSR1/OSR1 expression in postpubertal human uteri, and the prenatal and postnatal expression pattern of Osr1/Osr1 in murine developing Mullerian ducts (MDs) and endometrium, respectively. We then investigated whether Osr1 deletion would affect MD development, using wild-type and genetically engineered mice. Human uterine OSR1/OSR1 expression was found primarily in the endometrium. Mouse Osr1 was expressed prenatally in MDs and Wolffian ducts (WDs), from rostral to caudal segments, in E13.5 embryos. MDs and WDs were absent on the left side and MDs were rostrally truncated on the right side of E13.5 Osr1-/- embryos. Postnatally, Osr1 was expressed in mouse uteri throughout lifespan, peaking at postnatal days 14 and 28. Osr1 protein was present primarily in uterine luminal and glandular epithelial cells and in the epithelial cells of mouse oviducts. Through this translational approach, we demonstrated that OSR1/Osr1 is important for MD development and endometrial receptivity and may be implicated in uterine factor infertility.
Authors
Adriana Lofrano-Porto, Sidney Alcântara Pereira, Andrew Dauber, Jordana C.B. Bloom, Audrey N. Fontes, Naomi Asimow, Olívia Laquis de Moraes, Petra Ariadne T. Araujo, Ana Paula Abreu, Michael H. Guo, Silviene F. De Oliveira, Han Liu, Charles Lee, Wendy Kuohung, Michella S. Coelho, Rona S. Carroll, Rulang Jiang, Ursula B. Kaiser
Abstract
Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. Here, we identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. Our study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.
Authors
Jingping Li, Huimei Zheng, Jiaru Hou, Jianhua Chen, Fengbin Zhang, Xiaohang Yang, Fan Jin, Yongmei Xi
Copyright © 2024 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)