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16 pages, 8631 KiB

Open AccessArticle

Anticancer Effects of Mitoquinone via Cell Cycle Arrest and Apoptosis in Canine Mammary Gland Tumor Cells

by Ran Lee, Won-Young Lee and Hyun-Jung Park

Int. J. Mol. Sci. 2024, 25(9), 4923; https://doi.org/10.3390/ijms25094923 - 30 Apr 2024

Cited by 1 | Viewed by 1462

Abstract

Treating female canine mammary gland tumors is crucial owing to their propensity for rapid progression and metastasis, significantly impacting the overall health and well-being of dogs. Mitoquinone (MitoQ), an antioxidant, has shown promise in inhibiting the migration, invasion, and clonogenicity of human breast [...] Read more.

Treating female canine mammary gland tumors is crucial owing to their propensity for rapid progression and metastasis, significantly impacting the overall health and well-being of dogs. Mitoquinone (MitoQ), an antioxidant, has shown promise in inhibiting the migration, invasion, and clonogenicity of human breast cancer cells. Thus, we investigated MitoQ’s potential anticancer properties against canine mammary gland tumor cells, CMT-U27 and CF41.Mg. MitoQ markedly suppressed the proliferation and migration of both CMT-U27 and CF41.Mg cells and induced apoptotic cell death in a dose-dependent manner. Furthermore, treatment with MitoQ led to increased levels of pro-apoptotic proteins, including cleaved-caspase3, BAX, and phospho-p53. Cell cycle analysis revealed that MitoQ hindered cell progression in the G1 and S phases in CMT-U27 and CF41.Mg cells. These findings were supported using western blot analysis, demonstrating elevated levels of cleaved caspase-3, a hallmark of apoptosis, and decreased expression of cyclin-dependent kinase (CDK) 2 and cyclin D4, pivotal regulators of the cell cycle. In conclusion, MitoQ exhibits in vitro antitumor effects by inducing apoptosis and arresting the cell cycle in canine mammary gland tumors, suggesting its potential as a preventive or therapeutic agent against canine mammary cancer. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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18 pages, 5135 KiB

Open AccessArticle

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects

by Štefan Baňas, Eva Tvrdá, Filip Benko, Michal Ďuračka, Natália Čmiková, Norbert Lukáč and Miroslava Kačániová

Int. J. Mol. Sci. 2024, 25(7), 4129; https://doi.org/10.3390/ijms25074129 - 8 Apr 2024

Cited by 1 | Viewed by 1592

Abstract

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen–thawed bovine [...] Read more.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen–thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 μM KAE and compared to native ejaculates (negative control—Ctrl_N) as well as semen samples cryopreserved in the absence of KAE (positive control—Ctrl_C). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to Ctrl_C. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with Ctrl_C. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 μM KAE (p < 0.01). At the same time, supplementation with 25 μM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg²⁺-ATPase (p < 0.05) and Na⁺/K⁺-ATPase (p < 0.0001) in comparison to Ctrl_C. Western blot analysis revealed that supplementation with 25 μM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze–thaw process. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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14 pages, 4279 KiB

Open AccessArticle

YTHDF2 as a Mediator in BDNF-Induced Proliferation of Porcine Follicular Granulosa Cells

by Kening Liu, Xu Zhou, Chunjin Li, Caomeihui Shen, Guitian He, Tong Chen, Maosheng Cao, Xue Chen, Boqi Zhang and Lu Chen

Int. J. Mol. Sci. 2024, 25(4), 2343; https://doi.org/10.3390/ijms25042343 - 16 Feb 2024

Cited by 1 | Viewed by 1417

Abstract

In female mammals, the proliferation and apoptosis of granulosa cells (GCs) are critical in determining the fate of follicles and are influenced by various factors, including brain-derived neurotrophic factor (BDNF). Previous research has shown that BDNF primarily regulates GC proliferation through the PI3K/AKT, [...] Read more.

In female mammals, the proliferation and apoptosis of granulosa cells (GCs) are critical in determining the fate of follicles and are influenced by various factors, including brain-derived neurotrophic factor (BDNF). Previous research has shown that BDNF primarily regulates GC proliferation through the PI3K/AKT, NF-kB, and CREB tumour pathways; however, the role of other molecular mechanisms in mediating BDNF-induced GC proliferation remains unclear. In this study, we investigated the involvement of the m⁶A reader YTH domain-containing family member 2 (YTHDF2) in BDNF-stimulated GC proliferation and its underlying mechanism. GCs were cultured in DMEM medium supplemented with varying BDNF concentrations (0, 10, 30, 75, and 150 ng/mL) for 24 h. The viability, number, and cell cycle of GCs were assessed using the CCK-8 assay, cell counting, and flow cytometry, respectively. Further exploration into YTHDF2’s role in BDNF-stimulated GC proliferation was conducted using RT-qPCR, Western blotting, and sequencing. Our findings indicate that YTHDF2 mediates the effect of BDNF on GC proliferation. Additionally, this study suggests for the first time that BDNF promotes YTHDF2 expression by increasing the phosphorylation level of the ERK1/2 signalling pathway. This study offers a new perspective and foundation for further elucidating the mechanism by which BDNF regulates GC proliferation. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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11 pages, 1635 KiB

Open AccessArticle

Serum Lipocalin-2 Levels as a Biomarker in Pre- and Post-Pubertal Klinefelter Syndrome Patients: A Pilot Study

by Roberto Paparella, Giampiero Ferraguti, Marco Fiore, Michela Menghi, Ginevra Micangeli, Francesca Tarani, Aurora Ligotino, Marisa Patrizia Messina, Mauro Ceccanti, Antonio Minni, Christian Barbato, Marco Lucarelli, Luigi Tarani and Carla Petrella

Int. J. Mol. Sci. 2024, 25(4), 2214; https://doi.org/10.3390/ijms25042214 - 12 Feb 2024

Cited by 1 | Viewed by 1233

Abstract

Klinefelter syndrome (KS) is a male genetic disease caused by the presence of an extra X chromosome, causing endocrine disorders mainly responsible for a high rate of infertility and metabolic disorders in adulthood. Scientific research is interested in identifying new biomarkers that can [...] Read more.

Klinefelter syndrome (KS) is a male genetic disease caused by the presence of an extra X chromosome, causing endocrine disorders mainly responsible for a high rate of infertility and metabolic disorders in adulthood. Scientific research is interested in identifying new biomarkers that can be predictive or prognostic of alterations strictly connected to KS. Lipocalin-2 (LCN-2, also known as NGAL) is a small protein initially identified within neutrophils as a protein related to innate immunity. Serum LCN-2 estimation seems to be a useful tool in predicting the metabolic complications caused by several pathological conditions. However, little is known about its potential role in infertility conditions. The present pilot study aims to investigate the presence of LCN-2 in the serum of a group of pre-pubertal and post-pubertal children affected by KS, compared to healthy controls. We demonstrated for the first time the presence of elevated levels of LCN-2 in the serum of KS patients, compared to controls. This increase was accompanied, in pre-pubertal KS patients, by the loss of correlation with LH and HDL, which instead was present in the healthy individuals. Moreover, in all KS individuals, a positive correlation between LCN-2 and inhibin B serum concentration was found. Despite the limited size of the sample analyzed, our preliminary data encourage further studies to confirm the findings and to extend the study to KS adult patients, to verify the predictive/prognostic value of LCN-2 as new biomarker for metabolic diseases and infertility associated with the pathology. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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14 pages, 1000 KiB

Open AccessArticle

Quantitative Analysis of the Human Semen Phosphorometabolome by ³¹P-NMR

by Rebeca Serrano, David Martin-Hidalgo, Jon Bilbao, Ganeko Bernardo-Seisdedos, Oscar Millet, Luis J. Garcia-Marin and Maria Julia Bragado

Int. J. Mol. Sci. 2024, 25(3), 1682; https://doi.org/10.3390/ijms25031682 - 30 Jan 2024

Viewed by 1476

Abstract

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by ³¹P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples [...] Read more.

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by ³¹P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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15 pages, 1221 KiB

Open AccessArticle

Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia

by Makchit Galadima, Mariana Teles, Josep Pastor, Javier Hernández-Losa, Joan Enric Rodríguez-Gil and Maria Montserrat Rivera del Alamo

Int. J. Mol. Sci. 2024, 25(2), 1170; https://doi.org/10.3390/ijms25021170 - 18 Jan 2024

Cited by 1 | Viewed by 1405

Abstract

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas [...] Read more.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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21 pages, 5140 KiB

Open AccessArticle

Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells

by Qiwei Yang, Somayeh Vafaei, Ali Falahati, Azad Khosh, Maria Victoria Bariani, Mervat M. Omran, Tao Bai, Hiba Siblini, Mohamed Ali, Chuan He, Thomas G. Boyer and Ayman Al-Hendy

Int. J. Mol. Sci. 2024, 25(2), 905; https://doi.org/10.3390/ijms25020905 - 11 Jan 2024

Cited by 4 | Viewed by 2215

Abstract

Bromodomain-containing proteins (BRDs) are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD dysfunction has been linked to many diseases, including tumorigenesis. However, the role of BRDs in the pathogenesis of uterine fibroids (UFs) is entirely unknown. The present [...] Read more.

Bromodomain-containing proteins (BRDs) are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD dysfunction has been linked to many diseases, including tumorigenesis. However, the role of BRDs in the pathogenesis of uterine fibroids (UFs) is entirely unknown. The present study aimed to determine the expression pattern of BRD9 in UFs and matched myometrium and further assess the impact of a BRD9 inhibitor on UF phenotype and epigenetic/epitranscriptomic changes. Our studies demonstrated that the levels of BRD9 were significantly upregulated in UFs compared to matched myometrium, suggesting that the aberrant BRD expression may contribute to the pathogenesis of UFs. We then evaluated the potential roles of BRD9 using its specific inhibitor, I-BRD9. Targeted inhibition of BRD9 suppressed UF tumorigenesis with increased apoptosis and cell cycle arrest, decreased cell proliferation, and extracellular matrix deposition in UF cells. The latter is the key hallmark of UFs. Unbiased transcriptomic profiling coupled with downstream bioinformatics analysis further and extensively demonstrated that targeted inhibition of BRD9 impacted the cell cycle- and ECM-related biological pathways and reprogrammed the UF cell epigenome and epitranscriptome in UFs. Taken together, our studies support the critical role of BRD9 in UF cells and the strong interconnection between BRD9 and other pathways controlling the UF progression. Targeted inhibition of BRDs might provide a non-hormonal treatment option for this most common benign tumor in women of reproductive age. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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10 pages, 741 KiB

Open AccessCommunication

Immune Checkpoints in Recurrent Pregnancy Loss: New Insights into a Detrimental and Elusive Disorder

by Luca Marozio, Anna Maria Nuzzo, Eugenio Gullo, Laura Moretti, Emilie M. Canuto, Annalisa Tancredi, Margherita Goia, Stefano Cosma, Alberto Revelli, Alessandro Rolfo and Chiara Benedetto

Int. J. Mol. Sci. 2023, 24(17), 13071; https://doi.org/10.3390/ijms241713071 - 22 Aug 2023

Cited by 4 | Viewed by 1815

Abstract

Recurrent pregnancy loss (RPL) refers to two or more miscarriages before 20 weeks gestation. Its prevalence is 1–2%; its pathogenesis remains unexplained in more than 50% of cases, in which the cause is thought to be abnormal immune activity during placentation leading to [...] Read more.

Recurrent pregnancy loss (RPL) refers to two or more miscarriages before 20 weeks gestation. Its prevalence is 1–2%; its pathogenesis remains unexplained in more than 50% of cases, in which the cause is thought to be abnormal immune activity during placentation leading to a lack of pregnancy-induced immune tolerance. It is unknown whether immune activity is deranged in the endometrium of women with RPL. We studied the gene expression and the quantitative tissue protein levels of three immune checkpoints (CD276, which enhances cytotoxic T-cell activity, cytotoxic T-lymphocyte-associated antigen-4 [CTL-4], which reduces Th1 cytokine production, and lymphocyte activation gene-3 [LAG-3], which shows suppressive activity on Tregs and CD4+ T-cells) in endometrial samples from 27 women with unexplained RPL and in 29 women with dysfunctional uterine bleeding and previous uneventful pregnancies as controls. RNA isolation, real-time PCR, protein isolation, and ELISA were performed. CD276 gene expression and protein tissue levels were significantly lower in the endometrium of the RPL group than in the controls, whereas both CTL-4 and LAG-3 were significantly higher. This difference suggests defective endometrial immune regulation and overactivation of immune response in women with a history of RPL, at least in relation to controls with dysfunctional uterine bleeding and previous normal reproductive history. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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Review

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34 pages, 1370 KiB

Open AccessReview

Follicular Fluid and Blood Monitorization of Infertility Biomarkers in Women with Endometriosis

by Ana Teresa Brinca, Ana Maria Peiró, Pilar Matallín Evangelio, Irene Eleno, Antonio Helio Oliani, Vladimiro Silva, Luís F. Vicente, Ana Cristina Ramalhinho and Eugenia Gallardo

Int. J. Mol. Sci. 2024, 25(13), 7177; https://doi.org/10.3390/ijms25137177 - 29 Jun 2024

Cited by 2 | Viewed by 1702

Abstract

Infertility is recognized globally as a social disease and a growing medical condition, posing a significant challenge to modern reproductive health. Endometriosis, the third-most frequent gynecologic disorder, is one of the most common and intricate conditions that can lead to female infertility. Despite [...] Read more.

Infertility is recognized globally as a social disease and a growing medical condition, posing a significant challenge to modern reproductive health. Endometriosis, the third-most frequent gynecologic disorder, is one of the most common and intricate conditions that can lead to female infertility. Despite extensive research, the etiology, malignant transformation, and biological therapy of endometriosis remain unknown. Blood and follicular fluid are two matrices that have been carefully studied and can provide insights into women’s health. These matrices are clinically significant because they contain metabolites closely associated with women’s illness stage and reproductive outcomes. Nowadays, the application of metabolomic analysis in biological matrices may be able to predict the outcome of assisted reproductive technologies with greater precision. From a molecular viewpoint on reproductive health, we evaluate and compare the utilization of human follicular fluid and blood as matrices in analysis for diagnostic and assisted reproductive technology (ART) predictors of success for endometriosis patients. In the follicular fluid (FF), plasma, and serum of endometriosis-affected women, researchers identified dysregulations of oxidative stress, upregulation of several immune factors, and aberrations in energy metabolic pathways. The altered signatures negatively correlate with the overall oocyte and embryo quality and fertilization rate. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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29 pages, 1205 KiB

Open AccessReview

Phospholipase C Zeta in Human Spermatozoa: A Systematic Review on Current Development and Clinical Application

by Alessandra Parrella, Llanos Medrano, Jon Aizpurua and María José Gómez-Torres

Int. J. Mol. Sci. 2024, 25(2), 1344; https://doi.org/10.3390/ijms25021344 - 22 Jan 2024

Cited by 1 | Viewed by 1858

Abstract

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca²⁺) oscillations, a process known as oocyte activation. The sperm-specific factor responsible [...] Read more.

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca²⁺) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca²⁺ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes. In these patients, the use of assisted oocyte activation (AOA) technique can help to yield successful ICSI results and shorten the time of pregnancy. However, the production of a stable PLCζ recombinant protein represents a new powerful therapeutic approach to treating individuals with this condition. We aim to conduct a systematic review focusing on the expression, level, and localization of PLCζ, discussing the novel genetic mutation associated with its impairment. In addition, we highlight the benefits of AOA, looking at new and less invasive methods to diagnose and treat cases with PLCζ dysfunction. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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14 pages, 718 KiB

Open AccessReview

Human Papillomavirus and Male Infertility: What Do We Know?

by Arianna Sucato, Michela Buttà, Liana Bosco, Leonardo Di Gregorio, Antonio Perino and Giuseppina Capra

Int. J. Mol. Sci. 2023, 24(24), 17562; https://doi.org/10.3390/ijms242417562 - 16 Dec 2023

Cited by 6 | Viewed by 3362

Abstract

In recent years, increasing attention has been paid to understanding the causes of infertility, which is being recognized as a growing health problem affecting large numbers of couples worldwide. Male infertility is a contributing factor in approximately 30–40% of cases, and one of [...] Read more.

In recent years, increasing attention has been paid to understanding the causes of infertility, which is being recognized as a growing health problem affecting large numbers of couples worldwide. Male infertility is a contributing factor in approximately 30–40% of cases, and one of its etiological causes is sexually transmitted infections (STIs). Among sexually transmitted pathogens, human papillomavirus (HPV) can contribute in various ways to the failure of spontaneous and assisted reproduction, acting in the different phases of conception, especially in the early ones. In particular, HPV infection can affect sperm DNA integrity, sperm motility, count, viability, and morphology and can induce the production of anti-sperm antibodies (ASAs). In this narrative review, we aimed to provide an overview of existing research on the potential adverse effects of HPV infection on male reproductive health. Furthermore, we analyzed how limiting the spread of the infection, particularly with gender-neutral vaccination, could be a possible therapeutic tool to counteract male and female fertility problems. Full article

(This article belongs to the Special Issue A Molecular Perspective on Reproductive Health)

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