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The CASR antagonist NPS2143 induces loss of acrosomal integrity and proteolysis of SPACA1 in boar spermatozoa

Beatriz Macías-García and Lauro González-Fernández

In brief

The mechanism by which p32 protein increases during capacitation in boar spermatozoa is unknown. This manuscript shows a new mechanism of induction of p32 in boar spermatozoa: the proteolysis of the phosphorylated and glycosylated form of SPACA1.

Abstract

Protein tyrosine phosphorylation (PY) induction is associated with sperm capacitation. We previously showed that calcium-sensing receptor (CASR) inhibition by NPS2143 induces the 32 kDa tyrosine-phosphorylated protein (p32) in boar spermatozoa. We showed that NPS2143 induced an increase in p32 and loss of acrosomal integrity in live and dead spermatozoa in capacitating conditions (Tyrode's complete medium); the p32 rise occurred in dead spermatozoa, as shown by flow cytometry sorting. EGTA addition blunted the increase in p32, the loss of acrosomal integrity, and the increase in dead spermatozoa induced by NPS2143, indicating that the effects of NPS2143 are calcium-dependent. Mass spectrometry was used to identify which tyrosine-phosphorylated proteins were induced by NPS2143, but only serine/threonine-phosphorylated proteins were found; among these, SPACA1 was identified with different molecular weights (18, 32, and 35–45 kDa). We confirmed tyrosine phosphorylation of SPACA1 at 32 and 35–45 kDa by immunoprecipitation and co-localization of PY and SPACA1 in the presence of NPS2143 by immunofluorescence. The molecular weight of SPACA1 (35–45 kDa) decreased after treatment with peptide-N-glycosidase F, indicating that this protein is N-glycosylated. The soybean trypsin inhibitor (STI), a serine protease inhibitor, suppressed the appearance of p32 and SPACA1 (30 and 32 kDa) induced by NPS2143. Also, 8-Br-cAMP and A23187 treatments induced an increase in p32 and SPACA1 (30–32 kDa) and a parallel induction of the acrosome reaction. These findings suggest that CASR inhibition induces loss of acrosomal integrity and proteolysis of the glycosylated and phosphorylated SPACA1 (35–45 kDa) resulting in a SPACA1 rise at 32 kDa (p32).

DOI:
https://doi.org/10.1530/REP-22-0299
Volume 165: Issue 5,
Pages:
475–489 (15 total)
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Conserved H3K27me3-associated chromatin remodelling allows STRA8 but not MEIOSIN expression in mammalian germ cells

Teruhito Ishihara, Jane C Fenelon, Oliver W Griffith, Kei-ichiro Ishiguro, and Marilyn B Renfree

In brief

Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other.

Abstract

In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

DOI:
https://doi.org/10.1530/REP-22-0286
Volume 165: Issue 5,
Pages:
507–520 (14 total)
Restricted access

Pendimethalin exposure disrupts mitochondrial function and impairs processes related to implantation

Miji Kim, Junho Park, Garam An, Whasun Lim, and Gwonhwa Song

In brief

Pendimethalin as a dinitroaniline herbicide is used to eliminate weeds during the cultivation of various crops such as grains, fruits, and vegetables. This study reveals that pendimethalin exposure at various concentrations led to disruption in Ca2+ homeostasis and mitochondrial membrane potential, as well as dysregulation of the mitogen-activated protein kinase signaling pathway and implantation-related genes in porcine trophectoderm and uterine luminal epithelial cells.

Abstract

The use of herbicides is a major control method in agriculture. Pendimethalin (PDM) has been increasingly used as a herbicide for approximately 30 years. PDM has been reported to cause various reproductive problems, but its toxicity mechanism in the pre-implantation stage has not been investigated in detail. Herein, we studied the effects of PDM on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells and identified a PDM-mediated anti-proliferative effect in both cell types. PDM exposure generated intracellular reactive oxygen species, induced excessive Ca2+ influx into mitochondria, and activated mitogen-activated protein kinase signaling pathway. Ca2+ burden resulted in the dysfunction of mitochondria and eventual disruption of Ca2+ homeostasis. Further, PDM-exposed pTr and pLE cells showed cell cycle arrest and programmed cell death. In addition, a decrease in migration ability and dysregulated expression of genes related to the functioning of pTr and pLE cells was evaluated. This study provides insight into time-dependent transitions within the cell environment after PDM exposure and elucidates a detailed mechanism of induced adverse effects. These results imply that PDM exposure can potentially cause toxic effects on the implantation-related process in pigs. Moreover, to the best of our knowledge, this is the first study to describe the mechanism by which PDM induces these effects, enhancing our understanding of the toxicity of this herbicide.

DOI:
https://doi.org/10.1530/REP-22-0397
Volume 165: Issue 5,
Pages:
491–505 (15 total)
Restricted access

bta-miR-183 targets EZRIN to regulate microvilli formation and improve early development of bovine embryos

Yanfang Wu, Zhenzi Zuo, Zheng Wang, Hanghang Liu, Qi Zhou, Subi Ren, Xinrui Lan, Yong Zhang, and Yongsheng Wang

In brief

Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer embryo. This paper reveals a need for us to further explore the roles of the paternal regulatory factors on embryonic development in early embryos.

Abstract

Mature sperm contain both coding and non-coding RNAs, which can be delivered into an oocyte with the sperm at fertilization. Accumulating evidences show that these sperm-borne RNAs play crucial roles in epigenetic reprogramming, cytoskeleton remodeling, embryonic development, and offspring phenotype. Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer (SCNT) embryo. bta-miR-183 was found to be highly expressed in bovine sperm and can be delivered into oocytes during fertilization in our previous study, and in this study, EZR was confirmed as a target gene of bta-miR-183 in early embryos by bioinformatics, luciferase, and gain-of-function and loss-of-function experiments. Scanning electron microscopy showed that the density of microvilli on the surface of SCNT embryos was significantly higher than that onin vitro fertilized embryos and was significantly reduced by injection of bta-miR-183 mimic. EZR-siRNA injected into SCNT embryos had a similar effect. This indicated that the lack of bta-miR-183 might lead to abnormal changes in microvilli by downregulating ezrin protein. In addition, gain-of-function studies showed that bta-miR-183 significantly improved developmental competence of SCNT embryo in terms of cleavage (76.63% vs 64.32%, P < 0.05), blastocyst formation (43.75% vs 28.26%, P < 0.05), apoptotic index (5.21% vs 12.64%, P < 0.05), and the trophoblast ratio (32.65% vs 25.58%, P < 0.05) in day 7 blastocysts. Thus, the present study indicated that bta-miR-183 might play crucial roles in the formation of microvilli and embryo development by regulating expression of EZR mRNA.

DOI:
https://doi.org/10.1530/REP-22-0361
Volume 165: Issue 4,
Pages:
363–371 (9 total)
Free access

Cross-species comparison of mouse and human preimplantation development with an emphasis on lineage specification

Savana Biondic, Jesica Canizo, Katherine Vandal, Cheng Zhao, and Sophie Petropoulos

In brief

Human embryogenesis still remains largely unexplored. This review helps identify some of our current gaps in knowledge pertaining to preimplantation development, which may have implications for understanding fundamental aspects of human development, assisted reproductive technologies, and stem cell biology.

Abstract

Preimplantation development is arguably one of the most critical stages of embryogenesis. Beginning with the formation of the totipotent zygote post-fertilization, a series of cell divisions, and a complex coordination of physical cues, molecular signals and changes in gene expression lead to the formation of the blastocyst, a structure capable of implanting into the uterine wall. The blastocyst is composed of more specified cellular lineages, which will give rise to every tissue of the developing organism as well as the extra-embryonic lineages which support fetal growth. While the mouse has been used as a model to understand the events of preimplantation development for decades, in recent years, an expanding body of work has been conducted using the human embryo. These studies have identified some crucial species differences, particularly in the transcriptional and spatio-temporal expression of lineage markers and responses to cell signaling perturbations. This review compares recent findings on preimplantation development in mouse and human, with a focus on the specification of the first cellular lineages. Highlighting differences and noting mechanisms that require further examination in the human embryo is of critical importance for both the accurate translation of results from the mouse model and our overall understanding of mammalian development. We further highlight the latest advancement in reproductive research, the development of the 3D stem cell-based models known as ‘blastoids’. The knowledge discussed in this review has major clinical implications for assisted reproductive technologies such as in vitro fertilization and for applications in stem cell biology.

DOI:
https://doi.org/10.1530/REP-22-0144
Volume 165: Issue 4,
Pages:
R103–R116
Restricted access

Downregulated ETV4 inhibits the proliferation, migration, and invasion of trophoblast cells in preeclampsia

Qiuling Jie, Lijun Chen, Jiangying Liang, Xiaohui Yang, Fei Sun, and Yanlin Ma

In brief

Preeclampsia is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes, but the mechanisms underlying the development of preeclampsia are not fully understood. This study shows that ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9 and is involved in the pathogenesis of preeclampsia.

Abstract

Preeclampsia (PE) is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes. However, the mechanisms underlying the development of PE are not fully understood. ETS Variant Transcription Factor 4 (ETV4) plays an important role in cell proliferation, migration, and invasion. In this study, we aimed to explore the potential function of ETV4 in placental trophoblast cells. We analyzed the expression and location of ETV4 in PE and uncomplicated placental tissues using RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence staining. The results showed that both the mRNA and protein levels of ETV4 were markedly decreased in PE placental tissues compared with placental tissues from women with uncomplicated pregnancies (P < 0.05). Then, the effects of ETV4 on HTR-8/SVneo and Bewo cell proliferation, migration, and invasion were evaluated by MTT, 5-ethynyl-2-deoxyuridine (EdU), wound healing, and Transwell assays, respectively. The results showed that ETV4 knockdown inhibited both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). Conversely, overexpression of ETV4 promoted both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). We then measured the expression of MMP-2 and MMP-9 in HTR8/SVneo cells. We found that ETV4 knockdown decreased the mRNA and protein expression of MMP-2 and MMP-9, while ETV4 overexpression increased MMP-2 and MMP-9 mRNA and protein expression (P < 0.05). In conclusion, ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9. Our findings provide novel insight into the mechanisms underlying the occurrence of PE.

DOI:
https://doi.org/10.1530/REP-22-0184
Volume 165: Issue 4,
Pages:
373–381 (9 total)
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Identification of circular RNAs as regulators and noninvasive biomarkers for placenta accreta spectrum

Junfang Shi, Mengtian Yang, Xin Cao, Qitao Huang, Fang He, You Peng, Jinru Cui, Wenqian Chen, Yiming Xu, Wenyan Geng, Laixin Xia, Dunjin Chen, and Shan Xiao

In brief

Placenta accreta spectrum (PAS) has an urgent need for reliable prenatal biomarkers. This study profiled the circular RNAs (circRNAs) in PAS placenta and maternal blood and identified two circRNAs can regulate trophoblast cells invasion and serve as noninvasive prenatal biomarkers for PAS prediction.

Abstract

PAS is one of the most alarming obstetric diseases with high mortality rates. The regulating mechanism underlying PAS remains to be investigated, and reliable blood biomarkers for PAS have not emerged. Circular RNAs (circRNAs) have become important regulators and biomarkers for disparate human diseases. However, the circRNA profiles of PAS were not reported, and the regulatory role and predictive value of circRNAs in PAS were unknown. Here, we comprehensively profiled the circRNAs in the placenta of PAS by transcriptome sequencing and analysis and uncovered 217 abnormally expressed circRNAs. Through competing endogenous RNA network analysis, we found that the target genes of upregulated circRNAs in PAS were enriched in placenta development-related pathways and further uncovered two circRNAs, circPHACTR4 and circZMYM4, that could regulate trophoblast cells invasion and migration in vitro. Finally, we verified that circPHACTR4 and circZMYM4 were also upregulated in the maternal peripheral blood of PAS women before delivery using transcriptome sequencing and RT-qPCR and evaluated their predictive value by ROC curves. We found that circPHACTR4 and circZMYM4 could serve as effective predicting biomarkers for PAS (area under the curve (AUC): 0.86 and 0.85) and propose an improved model for PAS prenatal prediction by combining the conventional ultrasound diagnosis with the new circRNA predictive factors (AUC: 0.91, specificity: 0.89, sensitivity: 0.82).Altogether, this work provides new resources for deciphering the biological roles of circRNAs in PAS, identified two circRNAs that could regulate trophoblast cells invasion during placentation, and revealed two noninvasive diagnostic markers for PAS.

DOI:
https://doi.org/10.1530/REP-22-0339
Volume 165: Issue 4,
Pages:
417–430 (14 total)
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Is seminal nerve growth factor-induced luteinizing hormone release in camelids mediated at the hypothalamus?

Rodrigo A Carrasco, Sergio Pezo, Eric M Zwiefelhofer, Emily E Lanigan, Jaswant Singh, Marco A Berland, Cesar Ulloa-Leal, Marcelo H Ratto, and Gregg P Adams

In brief

Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons.

Abstract

Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

DOI:
https://doi.org/10.1530/REP-22-0331
Volume 165: Issue 4,
Pages:
395–405 (11 total)
Restricted access

Low-moderate dietary phytoestrogens transiently disrupt spermatogenesis and the seminal plasma proteome in the ram

Kelsey R Pool, Tayler C Kent, Luoyang Ding, Callum Connolly, Kevin J Foster, Gereltsetseg Enkhbat, Megan H Ryan, and Dominique Blache

In brief

Dietary phytoestrogens disrupt a specific stage of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa. This paper demonstrates for the first time that ram reproduction is compromised by oestrogenic pasture, whilst also providing a longitudinal model for the impact of phytoestrogens on male fertility.

Abstract

Compounds with oestrogen-like actions are now common in both the Western diet. The long-term impacts and underlying mechanisms by which oestrogenic compounds alter male reproduction, however, are unclear. To investigate this, we used a longitudinal sheep model examining the impact of oestrogenic pasture consumption on semen quality and production, testicular size, sexual behaviour and the seminal plasma proteome of Merino rams (n = 20), over a full spermatogenic cycle and in the subsequent breeding season. Throughout the study period, sexual behaviour, sperm production and motility were similar between the exposed and non-exposed rams (P > 0.05). However, between 5 and 8 weeks of exposure to dietary phytoestrogens, rams produced a higher percentage of spermatozoa with a specific malformation of the sperm midpiece and reduced DNA integrity, compared to non-exposed rams (P < 0.001). Investigation into the seminal plasma proteome revealed 93 differentially expressed proteins between phytoestrogen-exposed and control rams (P < 0.05). Exposure to phytoestrogens increased the expression of proteins involved in cellular structure development, actin cytoskeleton reorganisation, regulation of cell function and decreased expression in those related to catabolic processes. The greatest fold changes were in proteins involved in the assembly of the sperm flagella, removal of cytoplasm, spermatid development and maintenance of DNA integrity. After returning to non-oestrogenic pasture, no differences in any measure were observed between treatment groups during the subsequent breeding season. We conclude that dietary phytoestrogens can transiently disrupt specific stages of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa.

DOI:
https://doi.org/10.1530/REP-22-0432
Volume 165: Issue 4,
Pages:
445–456 (12 total)
Restricted access

Maternal high-fat diet changes DNA methylation in the early embryo by disrupting the TCA cycle intermediary alpha ketoglutarate

Alexander Penn, Nicole McPherson, Tod Fullston, Bridget Arman, and Deirdre Zander-Fox

In brief

Maternal obesity can impair metabolism in the embryo and the resulting offspring. This study shows that metabolic disruptions through α-ketoglutarate may link altered metabolism with epigenetic changes in embryos.

Abstract

Maternal obesity can impair offspring metabolic health; however, the precise mechanism underpinning programming is unknown. Ten-Eleven translocase (TET) enzymes demethylate DNA using the TCA cycle intermediary α-ketoglutarate and may be involved in programming offspring health. Whether TETs are disrupted by maternal obesity is unknown. Five to six week-old C57Bl/6 female mice were fed a control diet (CD; 6% fat, n = 175) or a high-fat diet (HFD; 21% fat, n = 158) for 6 weeks. After superovulation, oocytes were collected for metabolic assessment, or females were mated and zygotes were cultured for embryo development, fetal growth, and assessment of global DNA methylation (5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC)) in the two-cell embryo. Zygotes collected from superovulated CBAF1 females were cultured in media containing α-ketoglutarate (0, 1.4, 3.5, or 14.0 mM) or with 2-hydroxyglutarate (2HG) (0 or 20 mM), a competitive inhibitor of α-ketoglutarate, with methylation and blastocyst differentiation assessed. After HFD, oocytes showed increased pyruvate oxidation and intracellular ROS, with no changes in Tet3 expression, while two-cell embryo global 5hmC DNA methylation was reduced and 5fC increased. Embryos cultured with 1.4 mM α-ketoglutarate had decreased two-cell 5mC, while 14.0 mM α-ketoglutarate increased the 5hmC:5mC ratio. In contrast, supplementation with 20 mM 2HG increased 5mC and decreased 5fC:5mC and 5caC:5mC ratios. α-ketoglutarate up to 3.5 mM did not alter embryo development, while culturing in 14.0 mM α-ketoglutarate blocked development at the two-cell. Culture with 2HG delayed embryo development past the four-cell and decreased blastocyst total cell number. In conclusion, disruptions in metabolic intermediates in the preimplantation embryo may provide a link between maternal obesity and programming offspring for ill health.

DOI:
https://doi.org/10.1530/REP-22-0302
Volume 165: Issue 4,
Pages:
347–362 (16 total)