bims-gamemb 2022-03-13 papers

Hum Reprod. 2022 Mar 08. pii: deac042. [Epub ahead of print]

Human sperm cells can form paracetamol metabolite AM404 that directly interferes with sperm calcium signalling and function through a CatSper-dependent mechanism.

A Rehfeld, H Frederiksen, R H Rasmussen, A David, J Chaker, B S Nielsen, J E Nielsen, A Juul, N E Skakkebæk, D M Kristensen.

STUDY QUESTION: Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function?
SUMMARY ANSWER: While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action.
WHAT IS KNOWN ALREADY: Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy.
STUDY DESIGN, SIZE, DURATION: This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments.
MAIN RESULTS AND THE ROLE OF CHANCE: Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells).
LIMITATIONS, REASONS FOR CAUTION: The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404.
WIDER IMPLICATIONS OF THE FINDINGS: We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy.
STUDY FUNDING/COMPETING INTEREST(S): D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests.
TRIAL REGISTRATION NUMBER: N/A.

Keywords: AM404; APAP; CatSper; endocrine-disrupting chemicals; sperm motility; time-to-pregnancy

DOI: https://doi.org/10.1093/humrep/deac042

iScience. 2022 Mar 18. 25(3): 103904

Mining RNAseq data reveals dynamic metaboloepigenetic profiles in human, mouse and bovine pre-implantation embryos.

Marcella Pecora Milazzotto, Michael James Noonan, Marcia de Almeida Monteiro Melo Ferraz.

Metaboloepigenetic regulation has been reported in stem cells, germ cells, and tumor cells. Embryonic metaboloepigenetics, however, have just begun to be described. Here we analyzed RNAseq data to characterize the metaboloepigenetic profiles of human, mouse, and bovine pre-implantation embryos. In embryos, metaboloepigenetic reprogramming was species-specific, varied with the developmental stage and was disrupted with in vitro culture. Metabolic pathways and gene expressions were strongly correlated with early embryo DNA methylation and were changed with in vitro culture. Although the idea that the in vitro environment may influence development is not new, there has been little progress on improving pregnancy rates after decades using in vitro fertilization. Hence, the present data will contribute to understanding how the in vitro manipulation affects the metaboloepigenetic status of early embryos, which can be used to establish culture strategies aimed at improving the in vitro environment and, consequently, pregnancy rates and offspring health.

Keywords: Biological sciences; Endocrinology; Molecular biology; Omics; Transcriptomics

DOI: https://doi.org/10.1016/j.isci.2022.103904

Front Physiol. 2022 ;13 844148

Exogenous Linoleic Acid Intervention Alters Hepatic Glucose Metabolism in an Avian Embryo Model.

Xiufen Zhang, Qilin Wu, Wenxuan Zheng, Chuang Liu, Liang Huang, Xin Zuo, Wenquan Xiao, Xiaofeng Han, Hui Ye, Wence Wang, Yongwen Zhu, Lin Yang.

In the present study, developmental changes of gluconeogenesis and glycolysis in an avian model were measured, and then the intervention effects of in ovo feeding (IOF) linoleic acid (LA) on hepatic glucose metabolism were evaluated. In Experiment 1, thirty fertilized eggs were sampled on embryonic days (E) of 16, 19, 22, 25, 28, 31, and thirty newly-hatched ducklings at hatch (E34 and E35). In Experiment 2, a total of 120 fertilized eggs (60 eggs for each group) were injected into the yolk sac with PBS as the control group and LA as the IOF LA group on E25. Twelve eggs were selected for sample collection on E28 and E31. Serum contents of glucose, pyruvate, and lactate increased ( p < 0.05) linearly or quadratically from E16 to hatch, as well as hepatic glycogen and pyruvate contents. Hepatic mRNA expression related to energy homeostasis, gluconeogenesis, and glycolysis increased ( p < 0.05) in embryogenesis, and the plateau period was presented on E25-E31. IOF LA decreased ( p < 0.05) serum contents of glucose, triacylglycerol, cholesterol, and hepatic oleic acid, unsaturated fatty acids on E28, as well as the gene expression relative to gluconeogenesis. IOF LA increased ( p < 0.05) pyruvate content in serum and liver, and hepatic gene expression relative to glycolysis on E31. In summary, hepatic gluconeogenesis and glycolysis were enhanced to meet the increasing energy demands of embryonic development during E25 - hatch. Exogenous LA intervention on E25 could inhibit hepatic gluconeogenesis and enhance glycolysis during the later developmental period, disrupting glucose embryonic homeostasis and energy status.

Keywords: avian embryo model; energy status; glucose homeostasis; in ovo feeding; linoleic acid

DOI: https://doi.org/10.3389/fphys.2022.844148

World J Mens Health. 2022 Feb 21.

Low Sperm Motility Is Determined by Abnormal Protein Modification during Epididymal Maturation.

Yoo-Jin Park, Byeong-Mu Lee, Won-Ki Pang, Do-Yeal Ryu, Md Saidur Rahman, Myung-Geol Pang.

PURPOSE: During epididymal sperm maturation, spermatozoa acquire progressive motility through dynamic protein modifications. However, the relationship between sequential protein modifications during epididymal sperm maturation and sperm motility and fertility has not yet been investigated. This study investigated whether sequential changes in fertility-related protein expression including that of enolase 1 (ENO1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and UQCRC2), and voltage-dependent anion channel 2 (VDAC2) in spermatozoa during epididymal maturation are related to bovine sperm motility. Moreover, we found that mitochondrial metabolism is closely related to fertility-related proteins. Therefore, we investigated how the sequential modification of mitochondrial proteins during epididymal maturation regulates sperm motility.
MATERIALS AND METHODS: To determine the differential protein expression in caput and cauda epididymal spermatozoa from low and high motility bulls, western blot analysis was performed. Moreover, signaling pathways were identified to understand the mechanisms of regulation of sperm motility through the differential protein expression associated with fertility-related proteins.
RESULTS: We found that ENO1 was substantially higher in the caput spermatozoa from low motility bulls than the caput and cauda spermatozoa from high motility bulls. However, ENO1 expression in low motility bull spermatozoa was downregulated to a level comparable to that in the high motility bull spermatozoa during epididymal maturation. Moreover, there was a lack of modification of mitochondrial proteins, including glutathione peroxidase 4 and NADH:Ubiquinone Oxidoreductase Core Subunit S8, in low motility bull spermatozoa during epididymal maturation, whereas active changes were detected in high motility bull spermatozoa.
CONCLUSIONS: Irregular modifications of mitochondrial proteins during epididymal sperm maturation may increase excessive ROS production and premature activation of spermatozoa during epididymal maturation. Consequently, spermatozoa may lose their motility by the earlier consumption of their energy source and may be damaged by ROS during epididymal maturation, resulting in a decline in sperm motility and bull fertility.

Keywords: Epididymis; Fertility; Proteome; Sperm maturation; Sperm motility

DOI: https://doi.org/10.5534/wjmh.210180

Theriogenology. 2022 Mar 03. pii: S0093-691X(22)00074-7. [Epub ahead of print]184 51-60

Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity.

Saulo Menegatti Zoca, Emmalee J Northrop-Albrecht, Julie A Walker, Robert A Cushman, George A Perry.

Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. The objective of this study was to identify differences in proteins between epididymis and ejaculated samples that are associated with longevity. Ejaculated semen was collected from mature Angus bulls (n = 9); bulls were slaughtered and epididymal semen was collected. Epididymal and ejaculated semen were centrifuged to separate sperm and fluid. Fluids were removed and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was removed; both fluid and sperm samples were snap frozen in liquid nitrogen and stored at -80 °C. Protein analysis was performed by LCMS/MS. A different group of yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) semen were diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 °C. Sperm were evaluated for motility and viability every 24 h until motility was lower than 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 reached motility below 20%. A total of 458 unique proteins were identified; 178, 298, 311, and 344 proteins were identified in ejaculated fluid, ejaculated sperm, epididymal fluid and epididymal sperm, respectively. There were 8, 24, 10, and 18 significant KEGG pathways (FDR <0.05) for ejaculated fluid, epididymal fluid, ejaculated sperm, and epididymal sperm, respectively. The metabolic pathway was identified as the most important KEGG pathway; glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significant among proteins only present in epididymal samples within the metabolic pathway. Other proteins identified that may be related to epididymal sperm's increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties. In summary, energy metabolism in the epididymis appears to be more glycolytic compared to ejaculated and epididymis sperm have a larger number of antioxidants available which may be helping to maintain sperm in a quiescent state. Epididymal sperm remained viable (membrane integrity) longer than ejaculated sperm when cultured at the same pH.

Keywords: Ejaculation; Epididymis; Protein; Spermatozoa

DOI: https://doi.org/10.1016/j.theriogenology.2022.02.021

Int J Mol Sci. 2022 Feb 28. pii: 2706. [Epub ahead of print]23(5):

LC-MS Analysis Revealed the Significantly Different Metabolic Profiles in Spent Culture Media of Human Embryos with Distinct Morphology, Karyotype and Implantation Outcomes.

Chupalav Eldarov, Alina Gamisonia, Vitaliy Chagovets, Luiza Ibragimova, Svetlana Yarigina, Veronika Smolnikova, Elena Kalinina, Nataliya Makarova, Victor Zgoda, Gennady Sukhikh, Mikhail Bobrov.

In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development.

Keywords: LC-MS; aneuploidy; exometabolomics; human embryo; implantation; metabolomics

DOI: https://doi.org/10.3390/ijms23052706

BJOG. 2022 Mar 11.

Sperm mitochondrial DNA copy number in relation to semen quality: A cross-sectional study of 1164 potential sperm donors.

Bin Sun, Jian Hou, Yi-Xiang Ye, Heng-Gui Chen, Peng Duan, Ying-Jun Chen, Cheng-Liang Xiong, Yi-Xin Wang, An Pan.

OBJECTIVE: To investigate the association between mitochondrial DNA copy number (mtDNAcn) and semen quality.
DESIGN: A cross-sectional study.
SETTING: Hubei Province Human Sperm Bank of China (from April 2017 to July 2018).
POPULATION: A total of 1164 healthy male sperm donors with 5739 specimens.
MAIN OUTCOME MEASURES: Real-time quantitative polymerase chain reaction (RT-PCR) was used to measure sperm mtDNAcn. We also determined semen volume, concentration, and motility parameters (progressive motility, nonprogressive motility, and immotility).
METHODS: Mixed-effect models and general linear models were performed.
RESULTS: After adjusting for relevant confounding factors, mixed-effect models revealed diminished sperm motility (progressive and total), concentration, and total count across the quartiles of mtDNAcn (all p < 0.05). Compared to men in the lowest quartile, men in the highest quartile of mtDNAcn had lower progressive sperm motility, total motility, concentration, and total count of -8.9% (95% CI: -12.7%, -5.0%), -8.0% (95% CI: -11.6%, -4.4%), -42.8% (95% CI: -47.7%, -37.4%), and -44.3% (95% CI: -50.1%, -37.7%), respectively. These inverse dose-response relationships were further confirmed in the cubic spline models, where mtDNAcn was modeled as continuous variables.
CONCLUSIONS: We found that mtDNAcn was inversely associated with semen quality in a dose-dependent manner. Our results provide novel clues that sperm mtDNAcn may serve as a useful predictor of human semen characteristics.

Keywords: Mitochondrial DNA copy number; Semen quality; mitochondrial biomarkers

DOI: https://doi.org/10.1111/1471-0528.17139