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miR-23b-3p regulates human endometrial epithelial cell adhesion implying a role in implantation

Siena Barton, Wei Zhou, Leilani L Santos, Ellen Menkhorst, Guannan Yang, Wan Tinn Teh, Catarina Ang, Tarana Lucky, and Evdokia Dimitriadis

In brief

miR-23b-3p expression is increased in fertile endometrium during receptivity. This study investigates the function of miR-23b-3p on endometrial adhesion and its downstream targets.

Abstract

The human endometrium undergoes dramatic remodeling throughout the menstrual cycle that is essential for successful blastocyst attachment and implantation in the mid-secretory (receptive) phase. microRNA (miR) plays a role in the preparation of endometrial receptivity. miR-23b-3p expression is increased in fertile endometrium during receptivity. Here, we aimed to investigate miR-23b-3p function during receptivity. qPCR and in situ hybridization were used to investigate the expression and localization of miR-23b-3p in human endometrium, respectively. Ishikawa cells (endometrial epithelial cell line) and endometrial organoid-derived epithelial cells were transfected with miR-23b-3p mimic, and trophoblast progenitor spheroid (blastocyst surrogate) adhesion assay was used to determine effects on blastocyst adhesion to endometrial cells. We demonstrated that miR-23b-3p was significantly upregulated in the fertile endometrium of the receptive phase compared to the non-receptive, proliferative phase. No difference was identified for the expression of miR-23b-3p between fertile and infertile mid-secretory phase endometrium. miR-23b-3p localized to the epithelium and stroma in the mid-secretory phase but was undetectable in the proliferative phase of fertile endometrium. Functionally, miR-23-3p overexpression in Ishikawa cells and fertile endometrial organoid-derived epithelial cells significantly improved their adhesive capacity to trophoblast progenitor spheroids. miR-23b-3p overexpression in infertile endometrial organoid-derived epithelial cells did not improve adhesion. Among 10 miR-predicted gene targets examined, miR-23b-3p overexpression in Ishikawa cells significantly reduced the expression of MET, secreted frizzled-related protein 4 (SFRP4) and acyl-CoA dehydrogenase short/branched chain (ACADSB) compared to control. The reduction of SFRP4 after miR23b-3p overexpression was confirmed by immunoblotting in fertile organoid-derived epithelial cells. SFRP4 expression in fertile endometrium exhibited an inverse expression pattern compared to miR-23b-3p and was higher in the proliferative phase compared to the mid-secretory phase. Overall, miR-23b-3p is likely a critical regulator of endometrial epithelial adhesion and receptivity.

DOI:
https://doi.org/10.1530/REP-22-0338
Volume 165: Issue 4,
Pages:
407–416 (10 total)
Restricted access

OONO–/MMP2/MMP9 pathway-mediated apoptosis of porcine granulosa cells is associated with DNA damage

Kun Lei, Quanwei Wei, Ying Cheng, Zhe Wang, Haoze Wu, Fang Zhao, Wei Ding, and Fangxiong Shi

In brief

The apoptosis of granulosa cells (GCs) is the main reason for porcine follicular atresia. This study provides a novel mechanism for peroxynitrite anion-mediated GC apoptosis and follicular atresia in porcine ovary.

Abstract

Granulosa cells play a crucial role in the development of follicles, and their cell apoptosis in the porcine ovary is a major contributor to follicular atresia. Here, we provide a new mechanism for follicular atresia by describing a crucial mechanism by which peroxynitrite anion (OONO–) may cause GC death. We discovered that nitric oxide, oxidative stress level, and OONO– were positively correlated with porcine follicular atresia, which was accompanied by high expression of matrix metalloproteinase 2 (MMP2) and MMP9. We created a model of OONO–-induced apoptosis in GCs and discovered that OONO– could boost the expression of MMP2 and MMP9 and increase the expression of pro-apoptotic proteins and DNA damage. Furthermore, by inhibiting the activities of MMP2 and MMP9, we found that SB-3CT (a specific inhibitor for MMP2 and MMP9) alleviated the decrease in cell survival rates and DNA damage caused by OONO–, which may have been impacted by reducing the cleavage of PARP1 by MMP2 and MMP9. Therefore, our findings imply that OONO– can cause DNA damage to GCs, participating in mediating the expression of pro-apoptotic proteins and inhibiting DNA repair by preventing the activity of PARP1 through MMP2 and MMP9. These results help explain how OONO–/MMP2/MMP9 affects porcine follicular atresia and GC apoptosis.

DOI:
https://doi.org/10.1530/REP-22-0295
Volume 165: Issue 4,
Pages:
431–443 (13 total)
Restricted access

PCSK9 involves in the high-fat diet-induced abnormal testicular function of male mice

Zhi-hui Cui, Yong-dan Ma, Yi-cheng Wang, Huan Liu, Jia-wei Song, Li-xue Zhang, Wen-jing Guo, Xue-qin Zhang, Sha-sha Tu, Dong-zhi Yuan, Jin-hu Zhang, Li Nie, and Li-min Yue

In brief

Impaired spermatogenesis resulting from disturbed cholesterol metabolism due to intake of high-fat diet (HFD) has been widely recognized, however, the role of preprotein invertase subtilin 9 (PCSK9), which is a negative regulator of cholesterol metabolism, has never been reported. This study aims to reveal the role of PCSK9 on spermatogenesis induced by HFD in mice.

Abstract

Long-term consumption of a high-fat diet (HFD) is an important factor that leads to impaired spermatogenesis exhibiting poor sperm quantity and quality. However, the mechanism of this is yet to be elucidated. Disrupted cholesterol homeostasis is one of many crucial pathological factors which could contribute to impaired spermatogenesis. As a negative regulator of cholesterol metabolism, preprotein invertase subtilin 9 (PCSK9) mediates low density lipoprotein receptor (LDLR) degradation to the lysosome, thereby reducing the expression of LDLR on the cell membrane and increasing serum low-density lipoprotein cholesterol level, resulting in lipid metabolism disorders. Here, we aim to study whether PCSK9 is a pathological factor for impaired spermatogenesis induced by HFD and the underlying mechanism. To meet the purpose of our study, we utilized wild-type C57BL/6 male mice and PCSK9 knockout mice with same background as experimental subjects and alirocumab, a PCSK9 inhibitor, was used for treatment. Results indicated that HFD induced higher PCSK9 expression in serum, liver, and testes, and serum PCSK9 is negatively correlated with spermatogenesis, while both PCSK9 inhibitor treatment and PCSK9 knockout methodologies ameliorated impaired lipid metabolism and spermatogenesis in mice fed a HFD. This could be due to the overexpression of PCSK9 induced by HFD leading to dyslipidemia, resulting in testicular lipotoxicity, thus activating the Bcl-2–Bax–Caspase3 apoptosis signaling pathway in testes, particularly in Leydig cells. Our study demonstrates that PCSK9 is an important pathological factor in the dysfunction of spermatogenesis in mice induced by HFD. This finding could provide innovative ideas for the diagnosis and treatment of male infertility.

DOI:
https://doi.org/10.1530/REP-22-0216
Volume 165: Issue 4,
Pages:
457–474 (18 total)
Restricted access

Sperm utilize tumor necrosis factor alpha to shut down a 2-methoxyestradiol nongenomic pathway that accelerates oviductal egg transport in the rat

María L Oróstica, Patricia Reuquen, Emanuel Guajardo-Correa, Alexis Parada-Bustamante, Hugo Cardenas, and Pedro A Orihuela

In brief

Mating shuts down the 2-methoxyestradiol (2ME) nongenomic pathway that accelerates oviductal egg transport in the rat. This study shows that sperm cells, but not vaginocervical stimulation, utilize TNF-α to shut down this 2ME nongenomic pathway.

Abstract

The transport of oocytes or embryos throughout the oviduct to the implantation site in the uterus is defined as egg transport. In the rat, 2-methoxyestradiol (2ME) accelerates egg transport through the oviduct via a nongenomic pathway. Mating is known to shut down this 2ME pathway and then trigger an estradiol genomic pathway that accelerates egg transport. Here, we tested whether intrauterine insemination (IUI) or vaginocervical stimulation (VCS) shuts down the 2ME nongenomic pathway that accelerates egg transport, and if these mating components require tumor necrosis factor alpha (TNF-α). Levels of TNF-α and the mRNA for TNF-α receptors were measured in the oviduct of IUI or VCS rats. The tissue distribution of TNF-α receptor proteins and the concentration of the mRNA for catechol-O-methyl transferase (Comt) and 2ME were also analyzed in the oviduct. Finally, we assessed whether 2ME accelerates egg transport in IUI or VCS rats previously treated with the TNF-α antagonist W9P9QY. Results show that IUI, but not VCS, increased TNF-α and their receptors in the oviduct. IUI and VCS did not change the tissue distribution of TNF-α receptors; however, both decreased the oviductal concentration of Comt and 2ME. IUI and VCS each blocked the 2ME-induced egg transport acceleration; however, only the IUI was antagonized by the TNF-α antagonist. We concluded that IUI and VCS inhibit the 2ME nongenomic pathway that accelerates egg transport; however, the vias of action are distinct, with a TNF-α increase on spermatozoa presence being required for the shutdown of the 2ME pathway.

DOI:
https://doi.org/10.1530/REP-22-0289
Volume 165: Issue 4,
Pages:
383–393 (11 total)
Free access

Synthetic embryos: a new venue in ethical research

Adrian Villalba, Jon Rueda, and Íñigo de Miguel Beriain

In brief

Two independent groups have reported the development of ‘artificial embryos’. Those are in vitro models made of mouse embryonic stem cells, without the need for egg or sperm, and grown ex utero without requiring implantation. This system might open new venues in bioethical research if human cells show the ability to replicate this system.

Abstract

The recent publications reported in 2022 reveal the possibility of obtaining mouse embryos without the need for egg or sperm. These ‘artificial embryos’ can recapitulate some stages of development ex utero – from neurulation to organogenesis – without implantation. Synthetic mouse embryos might serve as a valuable model to gain further insights into early developmental stages. Indeed, it is expected for these models to be replicated by employing human cells. This promising research raises ethical issues and expands the horizon of ethics in regard to the development of the human embryo. From this point of view, we state some of the new open venues for bioethical research.

DOI:
https://doi.org/10.1530/REP-22-0416
Volume 165: Issue 4,
Pages:
V1–V3
Restricted access

Endometrial stromal cells from women with repeated implantation failure display impaired invasion towards trophoblastic spheroids

Qi Qiu, Yijing Li, Sze Wan Fong, Kai Chuen Lee, Andy Chun Hang Chen, Hanzhang Ruan, Kai-Fai Lee, Hang Wun Raymond Li, Ernest Hung Yu Ng, William Shu Biu Yeung, and Yin lau Lee

In brief

Implantation failure can occur even after the transfer of good-quality embryos. This study showed that the migration of human endometrial stromal cells towards embryonic trophoblasts is higher in women with live births in the first in vitro fertilization cycle than those with repeated implantation failure, suggesting that the chemotactic response of stroma cells is associated with successful pregnancy.

Abstract

The success rate of in vitro fertilization (IVF) remains limited in some women despite transfers of good-quality embryos in repeated attempts. There is no reliable tool for assessing endometrial receptivity. This study aimed to assess the interaction between decidualized human primary endometrial stromal cells (1°-EnSC) and human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) and to compare the invasion ability of decidualized 1°-EnSC towards BAP-EB between women attaining live birth in the first IVF cycle and those with repeated implantation failure (RIF). The invasion of the decidualized human endometrial cell line (T-HESC) and 1°-EnSC towards BAP-EB was studied. Real-time quantitative PCR and immunocytochemistry were employed to determine the expression of decidualization markers at mRNA and protein levels, respectively. Trophoblast-like BAP-EB-96h, instead of early trophectoderm (TE)-like BAP-EB-48h, facilitated the invasion ability of decidualized T-HESC and decidualized 1°-EnSC. Human chorionic gonadotropin at supra-physiological levels promoted the invasiveness of decidualized 1°-EnSC. The extent of BAP-EB-96h-induced invasion was significantly stronger in decidualized 1°-EnSC from women who had a live birth in the first IVF cycle when compared to those with RIF. While no difference was found in the expression of decidualization markers, PRL and IGFBP1 among two groups of women, significantly lower HLA-B was detected in the non-decidualized and decidualized 1°-EnSC from women with RIF. Collectively, the findings suggested that the invasion of decidualized 1°-EnSC towards trophoblast-like BAP-EB-96h was higher in women who had a live birth in the first IVF cycle than those with RIF.

DOI:
https://doi.org/10.1530/REP-22-0282
Volume 165: Issue 3,
Pages:
335–346 (12 total)
Open access

Functional role of GATA3 and CDX2 in lineage specification during bovine early embryonic development

Yan Shi, Bingjie Hu, Zizengchen Wang, Xiaotong Wu, Lei Luo, Shuang Li, Shaohua Wang, Kun Zhang, and Huanan Wang

In brief

The lineage specification during early embryonic development in cattle remains largely elusive. The present study determines the effects of trophectoderm-associated factors GATA3 and CDX2 on lineage specification during bovine early embryonic development.

Abstract

Current understandings of the initiation of the trophectoderm (TE) program during mammalian embryonic development lack evidence of how TE-associated factors such as GATA3 and CDX2 participate in bovine lineage specification. In this study, we describe the effects of TE-associated factors on the expression of lineage specification marker genes such as SOX2, OCT4, NANOG, GATA6, and SOX17, by using cytosine base editor system. We successfully knockout GATA3 or CDX2 in bovine embryos with a robust efficiency. However, GATA3 or CDX2 deletion does not affect the developmental potential of embryos to reach the blastocyst stage. Interestingly, GATA3 deletion downregulates the NANOG expression in bovine blastocysts. Further analysis of the mosaic embryos shows that GATA3 is required for NANOG in the TE of bovine blastocysts. Single blastocyst RNA-seq analysis reveals that GATA3 deletion disrupts the transcriptome in bovine blastocysts. Altogether, we propose that GATA3 plays an important role in maintaining TE lineage program in bovine embryos and the functional role of GATA3 is species-specific.

DOI:
https://doi.org/10.1530/REP-22-0269
Volume 165: Issue 3,
Pages:
325–333 (9 total)
Restricted access

Interferon-λ contributes to endometrial receptivity

Kezhen Yao, Yu Sun, Xiao-Qun Ye, and Ying Wu

Immunotherapy have been commonly used to prevent recurrent pregnancy loss in women with inadequate uterus receptivity or immunological imbalance. Many immune regulators are now identified as having crucial roles at the embryo–maternal interface. However, the clinical efficacy of immunity-related markers during peri-implantation period remains to be explored in depth. Here, we demonstrated that endometrial expression of Interferon-λ (IFN-λ), regarded as newer class of interferons, is aberrantly lower in women suffered from recurrent implantation failure than that in fertile control. We further uncovered genetic and biochemical evidence that IFN-λ is induced directly by estrogen in the endometrial cells, and IFN-λ pathway may play multiple roles involving in inflammatory response, uterine receptivity, cell migration and blastocyst adhesion. Furthermore, we indicated IFN-λ lessens the sensitivity of endometrium to FASL-mediated apoptosis. In addition to uncovering this IFN-λ as a novel nonredundant regulator that participates in the fetal-maternal immune interaction, our study helps to elucidate the underlying causes of spontaneous pregnancy loss in women.

DOI:
https://doi.org/10.1530/REP-22-0463
Accepted Manuscripts
Restricted access

Involvement of gap junctions in the increased contraction of bovine oviducts at pre-ovulation observed in vitro

Yuki Yamamoto, Maho Kurokawa, Taiji Ogawa, Sayaka Kubota, and Koji Kimura

In brief

Spontaneous contraction of oviductal smooth muscle is essential for gamete transport to the fertilization site in mammals. This study sheds light on the mechanism of elevated contraction amplitude in the bovine oviductal isthmus just before ovulation.

Abstract

Rhythmic contraction of the oviducts is essential for transporting gametes and embryos at peri-ovulation; however, its regulatory mechanism during the estrous cycle is unclear. Meanwhile, it is reported that ion currents regulate muscle contraction. Our study aimed to clarify the involvement of ion channels and gap junctions in regulating oviductal motility during the estrous cycle in cattle. The isthmic sections of bovine oviducts collected just after ovulation (0–4 days after ovulation), at the mid-late luteal stage (10–17 days), and at the follicular stage (1–3 days before ovulation) were used in the experiments. The frequency and amplitude of contraction of the oviductal strips in the longitudinal direction were examined using the Magnus system. The frequency was not different among the estrous stages. Conversely, the amplitude was significantly higher at the follicular stage. The blockers of voltage-dependent calcium channels, both IP3 receptor and ryanodine receptors, chloride channel, and gap junction reduced the amplitude. Additionally, mRNA and protein expression of GJA1, a component of the gap junction, in the smooth muscle tissues of the oviductal isthmus were significantly higher in the follicular stage. In addition, estradiol-17β (E2; 1.0 ng/mL) significantly increased GJA1 mRNA expression in cultured smooth muscle tissues after 24 h and GJA1 protein expression in cultured smooth muscle cells after 48 h. These results suggest that local levels of E2 in the oviductal isthmus ipsilateral to an ovary with a dominant follicle support the increased contraction amplitude of bovine ipsilateral oviducts by elevating the gap junction expression.

DOI:
https://doi.org/10.1530/REP-22-0174
Volume 165: Issue 3,
Pages:
301–312 (12 total)
Restricted access

Large animal models of developmental programming: Sustenance, stress and sex matter

Carolyn J Hammer, Joel S Caton, Carl Dahlen, Alison K Ward, Pawel P Borowicz, and Larry Reynolds

Developmental programming is the concept that certain health outcomes throughout life can be linked to early fetal or postnatal development. Progress in understanding concepts and mechanisms surrounding developmental programming is heavily leveraged by the use of large animal models. Numerous large animal models have been developed that apply a host of different maternal stressors, and more recently, paternal stressors. Maternal nutrition is the most researched maternal stressor applied during gestation and includes both global nutrient supply as well as models that target specific macro or micro nutrients. The focus of this review is to provide an overview of the many large animal models of developmental programming and to discuss the importance of sex effects (including paternal contributions) in study design and data interpretation.

DOI:
https://doi.org/10.1530/REP-22-0453
Accepted Manuscripts