Age-Dependent Alterations in Semen Parameters and Human Sperm MicroRNA Profile.

The trend to delay parenthood is increasing, impacting fertility and reproductive outcomes. Advanced paternal age (APA), defined as men's age above 40 years at conception, has been linked with testicular impairment, abnormal semen parameters, and poor reproductive and birth outcomes. Recently, the significance of sperm microRNA for fertilization and embryonic development has emerged. This work aimed to investigate the effects of men's age on semen parameters and sperm microRNA profiles. The ejaculates of 333 Portuguese men were collected between 2018 and 2022, analyzed according to WHO guidelines, and a density gradient sperm selection was performed. For microRNA expression analysis, 16 normozoospermic human sperm samples were selected and divided into four age groups: ≤30, 31-35, 36-40, and >40 years. microRNA target genes were retrieved from the miRDB and TargetScan databases and Gene Ontology analysis was performed using the DAVID tool. No significant correlation was found between male age and conventional semen parameters, except for volume. Fifteen differentially expressed microRNAs (DEMs) between groups were identified. Enrichment analysis suggested the involvement of DEMs in the sperm of men with advanced age in critical biological processes like embryonic development, morphogenesis, and male gonad development. Targets of DEMs were involved in signaling pathways previously associated with the ageing process, including cellular senescence, autophagy, insulin, and mTOR pathways. These results suggest that although conventional semen parameters were not affected by men's age, alterations in microRNA regulation may occur and be responsible for poor fertility and reproductive outcomes associated with APA.

Smoking Induces a Decline in Semen Quality and the Activation of Stress Response Pathways in Sperm.

Male infertility is a prevalent concern affecting couples worldwide. While genetic factors, hormonal imbalances, and reproductive system defects play significant roles, emerging evidence suggests that lifestyle choices also profoundly impact male fertility. This study aimed to explore the effects of several lifestyle factors, including tobacco and alcohol consumption, physical activity, and dietary habits, on semen quality parameters and molecular biomarkers. Thirty healthy male volunteers were recruited in the Urology service at Hospital Infante D. Pedro, Aveiro, Portugal. Participants completed lifestyle questionnaires and provided semen samples, which were analyzed according to the World Health Organization criteria by experienced technicians. We also analyzed the expression levels of antioxidant enzymes and heat-shock response-related proteins to explore the activation of signaling pathways involved in stress response within sperm cells. Our results revealed that tobacco consumption reduced semen volume and total sperm count. Although the changes in the percentage of total motility and normal morphology in the smokers' group did not reach statistical significance, a slight decrease was observed. Moreover, we identified for the first time a significant association between tobacco consumption and increased levels of heat shock protein 27 (HSP27) and phosphorylated HSP27 (p-HSP27) in sperm cells, indicating the potential detrimental effects of tobacco on the reproductive system. This study highlights that lifestyle factors reduce semen quality, possibly by inducing stress in sperm, raising awareness about the effects of these risk factors among populations at risk of male infertility.

Organoids of the male reproductive system: Challenges, opportunities, and their potential use in fertility research.

Organoids are units of function of a given organ able to reproduce, in culture, a biological structure similar in architecture and function to its counterpart in vivo. Today, it is possible to develop an organoid from a fragment of tissue, a stem cell located in an adult organ, an embryonic stem cell, or an induced pluripotent stem cell. In the past decade, many organoids have been developed which mimic stomach, pancreas, liver and brain tissues, optic cups, among many others. Additionally, different male reproductive system organs have already been developed as organoids, including the prostate and testis. These 3D cultures may be of great importance for urological cancer research and have the potential to be used in fertility research for the study of spermatozoa production and maturation, germ cells-somatic cells interactions, and mechanisms of disease. They also provide an accurate preclinical pipeline for drug testing and discovery, as well as for the study of drug resistance. In this work, we revise the current knowledge on organoid technology and its use in healthcare and research, describe the male reproductive system organoids and other biomaterials already developed, and discuss their current application. Finally, we highlight the research gaps, challenges, and opportunities in the field and propose strategies to improve the use of organoids for the study of male infertility situations. This article is categorized under: Reproductive System Diseases > Stem Cells and Development Reproductive System Diseases > Biomedical Engineering.

PP1, PP2A and PP2B Interplay in the Regulation of Sperm Motility: Lessons from Protein Phosphatase Inhibitors.

Male fertility relies on the ability of spermatozoa to fertilize the egg in the female reproductive tract (FRT). Spermatozoa acquire activated motility during epididymal maturation; however, to be capable of fertilization, they must achieve hyperactivated motility in the FRT. Extensive research found that three protein phosphatases (PPs) are crucial to sperm motility regulation, the sperm-specific protein phosphatase type 1 (PP1) isoform gamma 2 (PP1γ2), protein phosphatase type 2A (PP2A) and protein phosphatase type 2B (PP2B). Studies have reported that PP activity decreases during epididymal maturation, whereas protein kinase activity increases, which appears to be a requirement for motility acquisition. An interplay between these PPs has been extensively investigated; however, many specific interactions and some inconsistencies remain to be elucidated. The study of PPs significantly advanced following the identification of naturally occurring toxins, including calyculin A, okadaic acid, cyclosporin, endothall and deltamethrin, which are powerful and specific PP inhibitors. This review aims to overview the protein phosphorylation-dependent biochemical pathways underlying sperm motility acquisition and hyperactivation, followed by a discussion of the PP inhibitors that allowed advances in the current knowledge of these pathways. Since male infertility cases still attain alarming numbers, additional research on the topic is required, particularly using other PP inhibitors.

Effects of Age and Lifelong Moderate-Intensity Exercise Training on Rats' Testicular Function.

Aging is associated with testicular morphological and functional alterations, but the underlying molecular mechanisms and the impact of physical exercise are poorly understood. In this study, we examined the effects of age and lifelong moderate-intensity exercise on rat testis. Mature adults (35 weeks) and middle-aged (61 weeks) Wistar Unilever male rats were maintained as sedentary or subjected to a lifelong moderate-intensity treadmill training protocol. Testis weight and histology, mitochondrial biogenesis and function, and proteins involved in protein synthesis and stress response were evaluated. Our results illustrate an age-induced testicular atrophy that was associated with alterations in stress response, and mitochondrial biogenesis and function. Aging was associated with increased testicular levels of heat shock protein beta-1 (HSP27) and antioxidant enzymes. Aging was also associated with decreased mRNA abundance of the nuclear respiratory factor 1 (Nrf1), a key transcription factor for mitochondrial biogenesis, which was accompanied by decreased protein levels of the oxidative phosphorylation system (OXPHOS) complexes subunits in the testes of older animals. On the other hand, exercise did not protect against age-induced testicular atrophy and led to deleterious effects on sperm morphology. Exercise led to an even more pronounced decrease in the Nrf1 mRNA levels in testes of both age groups and was associated with decreased mRNA abundance of other mitochondrial biogenesis markers and decreased protein levels of OXPHOS complexes subunits. Lifelong moderate-intensity exercise training was also associated with an increase in testicular oxidative stress markers and possibly with reduced translation. Together, our results indicate that exercise did not protect against age-induced testicular atrophy and was not associated with beneficial changes in mitochondria and stress response, further activating mechanisms of protein synthesis inhibition.

G-Protein Coupled Receptors in Human Sperm: An In Silico Approach to Identify Potential Modulatory Targets.

G protein-coupled receptors (GPCRs) are involved in several physiological processes, and they represent the largest family of drug targets to date. However, the presence and function of these receptors are poorly described in human spermatozoa. Here, we aimed to identify and characterize the GPCRs present in human spermatozoa and perform an in silico analysis to understand their potential role in sperm functions. The human sperm proteome, including proteomic studies in which the criteria used for protein identification was set as <5% FDR and a minimum of 2 peptides match per protein, was crossed with the list of GPCRs retrieved from GLASS and GPCRdb databases. A total of 71 GPCRs were identified in human spermatozoa, of which 7 had selective expression in male tissues (epididymis, seminal vesicles, and testis), and 9 were associated with male infertility defects in mice. Additionally, ADRA2A, AGTR1, AGTR2, FZD3, and GLP1R were already associated with sperm-specific functions such as sperm capacitation, acrosome reaction, and motility, representing potential targets to modulate and improve sperm function. Finally, the protein-protein interaction network for the human sperm GPCRs revealed that 24 GPCRs interact with 49 proteins involved in crucial processes for sperm formation, maturation, and fertilization. This approach allowed the identification of 8 relevant GPCRs (ADGRE5, ADGRL2, GLP1R, AGTR2, CELSR2, FZD3, CELSR3, and GABBR1) present in human spermatozoa that can be the subject of further investigation to be used even as potential modulatory targets to treat male infertility or to develop new non-hormonal male contraceptives.

All you need to know about sperm RNAs.

BACKGROUND: Spermatogenesis generates a small and highly specialised type of cell that is apparently incapable of transcription and translation. For many years, this dogma was supported by the assumption that (i) the compact sperm nucleus, resulting from the substitution of histones by protamine during spermatogenesis, renders the genome inaccessible to the transcriptional machinery; and (ii) the loss of most organelles, including endoplasmic reticulum and ribosomes, limits or prevents translational activity. Despite these observations, several types of coding and non-coding RNAs have been identified in human sperm. Their functional roles, particularly during fertilisation and embryonic development, are only now becoming apparent. OBJECTIVE AND RATIONALE: This review aimed to summarise current knowledge of the origin, types and functional roles of sperm RNAs, and to evaluate the clinical benefits of employing these transcripts as biomarkers of male fertility and reproductive outcomes. The possible contribution of sperm RNAs to intergenerational or transgenerational phenotypic inheritance is also addressed. SEARCH METHODS: A comprehensive literature search on PubMed was conducted using the search terms 'sperm' AND 'RNA'. Searches focussed upon articles written in English and published prior to August 2020. OUTCOMES: The development of more sensitive and accurate RNA technologies, including RNA sequencing, has enabled the identification and characterisation of numerous transcripts in human sperm. Though a majority of these RNAs likely arise during spermatogenesis, other data support an epididymal origin of RNA transmitted to maturing sperm by extracellular vesicles. A minority may also be synthesised by de novo transcription in mature sperm, since a small portion of the sperm genome remains packed by histones. This complex RNA population has important roles in paternal chromatin packaging, sperm maturation and capacitation, fertilisation, early embryogenesis and developmental maintenance. In recent years, additional lines of evidence from animal models support a role for sperm RNAs in intergenerational or transgenerational inheritance, modulating both the genotype and phenotype of progeny. Importantly, several reports indicate that the sperm RNA content of fertile and infertile men differs considerably and is strongly modulated by the environment, lifestyle and pathological states. WIDER IMPLICATIONS: Transcriptional profiling has considerable potential for the discovery of fertility biomarkers. Understanding the role of sperm transcripts and comparing the sperm RNA fingerprint of fertile and infertile men could help to elucidate the regulatory pathways contributing to male factor infertility. Such data might also provide a molecular explanation for several causes of idiopathic male fertility. Ultimately, transcriptional profiling may be employed to optimise ART procedures and overcome some of the underlying causes of male infertility, ensuring the birth of healthy children.

Bovine semen sexing: Sperm membrane proteomics as candidates for immunological selection of X- and Y-chromosome-bearing sperm.

The use of sexed semen in dairy and beef farms ensures the production of animals of the desired sex, resulting in a reduction of costs and an improvement of environmental sustainability. Several methods have been developed over the years, but most of them were abandoned due to their limited efficacy. Currently, the only commercially available method for the separation of X- and Y-chromosome-bearing sperm is fluorescence-activated cell sorting. However, this technique is expensive and has limited usefulness for the industry, considering that it cannot produce doses of sexed semen with the desired number of sperm for artificial insemination. Immunological methods have emerged as an attractive alternative to flow cytometry and proteomic knowledge of X- and Y-sperm could be useful to the development of a new method. In this review, we identify the main applications of sexed semen, describe the existing methods and highlight future research opportunities in the field. We consider that immunological methods, based on sperm cell's surface proteins differentially expressed between X- and Y-sperm, could be an interesting and promising approach to semen sexing.

Fighting Bisphenol A-Induced Male Infertility: The Power of Antioxidants.

Bisphenol A (BPA), a well-known endocrine disruptor present in epoxy resins and polycarbonate plastics, negatively disturbs the male reproductive system affecting male fertility. In vivo studies showed that BPA exposure has deleterious effects on spermatogenesis by disturbing the hypothalamic-pituitary-gonadal axis and inducing oxidative stress in testis. This compound seems to disrupt hormone signalling even at low concentrations, modifying the levels of inhibin B, oestradiol, and testosterone. The adverse effects on seminal parameters are mainly supported by studies based on urinary BPA concentration, showing a negative association between BPA levels and sperm concentration, motility, and sperm DNA damage. Recent studies explored potential approaches to treat or prevent BPA-induced testicular toxicity and male infertility. Since the effect of BPA on testicular cells and spermatozoa is associated with an increased production of reactive oxygen species, most of the pharmacological approaches are based on the use of natural or synthetic antioxidants. In this review, we briefly describe the effects of BPA on male reproductive health and discuss the use of antioxidants to prevent or revert the BPA-induced toxicity and infertility in men.

Disruption of protein phosphatase 1 complexes with the use of bioportides as a novel approach to target sperm motility.

OBJECTIVE: To design protein phosphatase 1 (PP1)-disrupting peptides covalently coupled to inert cell-penetrating peptides (CPPs) as sychnologically organized bioportide constructs as a strategy to modulate sperm motility. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S)/ANIMAL(S): Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Effect of the bioportides on the activity and interactions of PP1γ2-a PP1 isoform expressed exclusively in testicular germ cells and sperm-and on sperm vitality and motility. RESULT(S): PP1-disrupting peptides were designed based on the sequences from: 1) a sperm-specific PP1 interactor (A kinase anchor protein 4); and 2) a PP1 inhibitor (protein phosphatase inhibitor 2). Those sequences were covalently coupled to inert CPPs as bioportide constructs, which were successfully delivered to the flagellum of sperm cells to induce a marked impact on PP1γ2 activity and sperm motility. Molecular modeling studies further facilitated the identification of an optimized PP1-binding sequence and enabled the development of a modified stop-sperm bioportide with reduced size and increased potency of action. In addition, a bioportide mimetic of the unique 22-amino acid C-terminus of PP1γ2 accumulated within spermatozoa to significantly reduce sperm motility and further define the PP1γ2-specific interactome. CONCLUSION(S): These investigations demonstrate the utility of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon spermatozoa motility, a key prognostic indicator of male fertility.

First Insights on the Presence of the Unfolded Protein Response in Human Spermatozoa.

The unfolded protein response (UPR) is involved in protein quality control and is activated in response to several stressors. Although in testis the UPR mechanisms are well described, their presence in spermatozoa is contentious. We aimed to investigate the presence of UPR-related proteins in human sperm and the impact of oxidative stress induction in UPR activation. To identify UPR-related proteins in human sperm, a bioinformatic approach was adopted. To explore the activation of UPR, sperm were exposed to hydrogen peroxide (H2O2) and motility, vitality, and the levels of UPR-related proteins were assessed. We identified 97 UPR-related proteins in human sperm and showed, for the first time, the presence of HSF1, GADD34, and phosphorylated eIF2α. Additionally, the exposure of human sperm to H2O2 resulted in a significant decrease in sperm viability and motility and an increase in the levels of HSF1, HSP90, HSP60, HSP27, and eIF2α; all proteins involved in sensing and response to unfolded proteins. This study gave us a first insight into the presence of UPR mechanisms in the male gamete. However, the belief that sperm are devoid of transcription and translation highlight the need to clarify if these pathways are activated in sperm in the same way as in somatic cells.

Testicular Aging: An Overview of Ultrastructural, Cellular, and Molecular Alterations.

The trend in parenthood at an older age is increasing for both men and women in developed countries, raising concerns about the reproductive ability, and the consequences for the offspring's health. While reproductive activity in women stops with menopause, a complete cessation of the reproductive potential does not occur in men. Although several studies have been published on the effects of aging on semen parameters and spermatozoa DNA integrity, literature on impact of aging on the testis, particularly cellular, and molecular alterations, has been, so far, limited and controversial. This work discusses the current knowledge on testicular aging in humans and other mammals, covering topics from tissue ultrastructure, to cellular and molecular alterations. Aging affects male reproductive function at multiple levels, from sperm production and quality, to the morphology and histology of the male reproductive system. The morphological and functional changes that occur in the testes result in variations in the levels of many hormones, changes in molecules involved in mitochondrial function, receptors, and signaling proteins. Despite knowing that these age-related alterations occur, their real impact on male fertility and reproductive health are still far from being fully understood, highlighting that research in the field is crucial.

The Generality of Interview-Informed Functional Analyses: Systematic Replications in School and Home.

Behavioral interventions preceded by a functional analysis have been proven efficacious in treating severe problem behavior associated with autism. There is, however, a lack of research showing socially validated outcomes when assessment and treatment procedures are conducted by ecologically relevant individuals in typical settings. In this study, interview-informed functional analyses and skill-based treatments (Hanley et al. in J Appl Behav Anal 47:16-36, 2014) were applied by a teacher and home-based provider in the classroom and home of two children with autism. The function-based treatments resulted in socially validated reductions in severe problem behavior (self-injury, aggression, property destruction). Furthermore, skills lacking in baseline-functional communication, denial and delay tolerance, and compliance with adult instructions-occurred with regularity following intervention. The generality and costs of the process are discussed.

Improving the Conversational Skills of a College Student with Peer-Mediated Behavioral Skills Training.

We used a multiple baseline design across behaviors to evaluate peer-mediated behavioral skills training to improve a complex repertoire of conversational skills of an undergraduate student diagnosed with a learning disability NOS. Following treatment, we observed a decrease in interrupting and content specificity and an increase in questioning. Treatment effects maintained with naïve peers during unstructured conversations and outcomes compared favorably with normative data on the conversational skills of three undergraduates without learning disabilities.