MicroRNA as an Important Mediator in the Regulation of Male Gallus gallus domesticus Reproduction: Current State of the Problem

The XIV Workshop on the Development and Function of Reproductive Organs was held in Rome, from 14-17 September, 2008, at the Congress Centre of the University of Tor Vergata of Villa Mondragone (Rome, Italy). The Workshop was conceived to be a survey of the events from the formation of the gamete precursors, the primordial germ cells, to the development of the preimplantation embryo through female and male gametogenesis in the mouse experimental model. It was divided into six topics including classic and highly-debated current subjects in the field of the biology of reproduction: mammalian primordial germ cells, the formation of the gonads, stem cells and germ cells, gametogenesis from birth to adult (male), gametogenesis from birth to adult (female), and finally fertilization and preimplantation embryo.

The germ cell lineage is a fundamental component of the metazoan life cycle, ensuring the perpetuation and substantialdiversification of genetic information across generations. Recent advances in the understanding of mousegerm cell development have culminated in the ability to reconstitute gametogenesis in vitro, thereby enabling the biochemical and molecular analyses of germ cell specification and subsequent development in mice. Similar advances in reconstituting human germ cells in vitro would provide critical insight into the etiology of various reproductive conditions and disorders, including infertility. This review presents the mechanisms leading to germ cell development in mammals, particularly in mice and non-human primates, as well as the applicability of these animal models to human germ cell development.The induction methods performed to recapitulate germ cell development in vitroare alsodiscussed in this review, specifically focusing on in vitro gametogenesis from pluripotent stem cells. This review compiles the key methods and findings of various references relevant to the above-mentioned topic. Murine models have provided essential mechanistic insight into the processof germ cell lineage development. However, there are several structural differences between mice and humans during early embryogenesis that hinder the extrapolation of findings made in murine models to what may occur in humans. Recent studies using human or non-human primate embryos and human-induced pluripotent stem cell (hiPSC)-derived germ cells shed light on key cellular and genetic mechanisms governing germ cell development in humans. Utilizing the knowledge obtained from studying germ cell development in different animal models, induction methods established by various laboratories now permit partial reconstitution of human gametogenesis in vitro. In vitro gametogenesis will constitute an emergent new field in human reproductive medicine in the near future, although legal and ethical considerations must be taken into account.

The thymus, a primary lymphoid organ, provides a complex environment essential for the generation of the T-cell repertoire. Thymic alterations occur during life either in the context of thymic involution upon aging or the pathophysiological context of Myasthenia Gravis (MG). These changes involve complicated regulatory networks, in which microRNAs (miRNAs) are key players. Here, we analyzed the role of miRNAs in thymocyte maturation and differentiation sustained by thymic epithelial cells. We compared data from the literature regarding the role of mouse thymic miRNAs and original data obtained from a human thymic miRnome study. We identified a set of highly expressed miRNAs defined as ThymiRs and investigated miRNA expression in infants as compared to adults to determine those associated with human thymic involution. Thymic changes are also frequently observed in MG, an autoimmune disease which results in the production of anti-acetylcholine receptor (AChR) antibodies that lead to muscle weaknesses. Alterations such as thymoma in late-onset MG patients and hyperplasia with ectopic germinal centers (GCs) in early-onset (EOMG) patients are found. Thymic miRNA expression has been studied in AChR-MG patients both in thymoma-associated MG (TAMG) and EOMG, and their function through their mRNA targets investigated. Most of the dysregulated thymic miRNAs in EOMG are associated with GC development, such as miR-7, miR-24, miR-139, miR-143, miR-145, miR-146, miR-150, miR-452, miR-548 or thymic inflammation, such as miR-125b, miR-146, or miR-29. Understanding these pathways may provide therapeutic targets or biomarkers of disease manifestations.

Introduction: Repeated heat shock (HS) stresses reduce the reproduction rate of Drosophila flies. Heat shock proteins (HSPs) protect cells against irreversible damages inducing heatinduced.Oxidative stress declines protective function of HSPs. Diploschistes ocellatus lichen aqueous extract possesses a strong antioxidant potential in vitro. Antioxidants can preserve HSPs function. Therefore, the present study for the first time investigated the cytoprotective effects of D. ocellatus aqueous extract against HS-mediated deleterious effects on reproductive function in Drosophila melanogaster. Methods: Three different types of culture media including control, 30% lichen extract, and 60%lichen extract were prepared. Adult D. melanogaster flies were placed on Delcour medium and allowed to lay eggs for 2 hours. Then the eggs were equally distributed between the culture media. After flies completed their life cycle, the adult enclosed flies were exposed to HS. To assess reproductive function, the newly emerged adult flies were transferred to the freshly prepared regular culture medium every three days for 3 times and finally adult offspring born to these flies were enumerated.Results: HS negatively affected the reproduction rate in flies in control group. Quantification of adult enclosed flies born to the D. ocellatus extract treated flies showed that lichen extract could negate the deleterious effects of HS on reproduction function of D. melanogaster in a dose-dependent manner.Conclusion: Diploschistes ocellatus aqueous extract attenuated the harmful effects of HS stress on reproductive function of D. melanogaster. The secondary metabolites present in D. ocellatus can be considered as a bona fide candidate in novel drug development to target reproductive diseases in which oxidative stress is involved. Moreover, it can be concluded that D. melanogaster is an ideal model organism to induce cellular stress in vitro and study therapeutic potential of lichen extracts.

T-2 toxin, an inevitable environmental pollutant, is the most toxic type A trichothecene mycotoxin. Reproductive disruption is a key adverse effect of T-2 toxin. Herein, this paper reviews the reproductive toxicity of T-2 toxin and its mechanisms in male and female members of different species. The reproductive toxicity of T-2 toxin is evidenced by decreased fertility, disrupted structures and functions of reproductive organs, and loss of gametogenesis in males and females. T-2 toxin disrupts the reproductive endocrine axis and inhibits reproductive hormone synthesis. Furthermore, exposure to T-2 toxin during pregnancy results in embryotoxicity and the abnormal development of offspring. We also summarize the research progress in counteracting the reproductive toxicity of T-2 toxin. This review provides information toward a comprehensive understanding of the reproductive toxicity mechanisms of T-2 toxin.

Limitation of food availability (starvation) is known to influence the reproductive ability of animals. Autophagy is a lysosomal driven degradation process that protects the cell under metabolic stress conditions, such as during nutrient shortage. Whether, and how starvation-induced autophagy impacts on the maturation and function of reproductive organs in animals are still open questions. In this study, we have investigated the effects of starvation on histological and cellular changes that may be associated with autophagy in the ovary of the giant freshwater prawn, Macrobachium rosenbergii. To this end, the female prawns were daily fed (controls) or unfed (starvation condition) for up to 12 days, and the ovary tissue was analyzed at different time-points. Starvation triggered ovarian maturation, and concomitantly increased the expression of autophagy markers in vitellogenic oocytes. The immunoreactivities for autophagy markers, including Beclin1, LC3-II, and Lamp1, were enhanced in the late oocytes within the mature ovaries, especially at the vitellogenic stages. These markers co-localized with vitellin in the yolk granules within the oocytes, suggesting that autophagy induced by starvation could drive vitellin utilization, thus promoting ovarian maturation.

Rhodtestolin is a cardio-inhibitor that was first discovered in testes extracts of the blood-feeding insect, Rhodnius prolixus. Its role in reproduction remains unconfirmed, but if delivered to the female during spermatophore formation, it may serve to calm the female and/or relax the vaginal muscles to facilitate delivery and storage of the spermatophore. We describe here the anatomy of reproductive organs in R. prolixus and show that rhodtestolin is present in a low-molecular weight fraction of testes extracts separated by gel filtration, as well as in spermatophores delivered to the female during spermatophore formation. We also report that a rhodtestolin-like factor is present in the testes of R. brethesi, Triatoma dimidiata, T. klugi and Nesotriatoma bruneri, other Reduviidae, which are vectors of Chagas disease. Male secretions in insects are known to modify female behavior after copulation, and the presence of rhodtestolin in several genera of Reduviidae suggests that it plays an important role in reproductive success. Determining this role could lead to developing additional population control strategies for these bugs.

Glyphosate (GLP) is an active agent of GLP-based herbicides(GBHs),i.e., broad-spectrum and postemergent weedkillers, commercializedby Monsanto as, e.g., Roundup and RangerPro formulants. The GBH cropspraying, dedicated to genetically engineered GLP-resistant crops,has revolutionized modern agriculture by increasing the productionyield. However, abusively administered GBHs’ ingredients, e.g.,GLP, polyoxyethyleneamine, and heavy metals, have polluted environmentaland industrial areas far beyond farmlands, causing global contaminationand life-threatening risk, which has led to the recent local bansof GBH use. Moreover, preclinical and clinical reports have demonstratedharmful impacts of GLP and other GBH ingredients on the gut microbiome,gastrointestinal tract, liver, kidney, and endocrine, as well as reproductive,and cardiopulmonary systems, whereas carcinogenicity of these herbicidesremains controversial. Occupational exposure to GBH dysregulates thehypothalamic–pituitary–adrenal axis, responsible forsteroidogenesis and endocrinal secretion, thus affecting hormonalhomeostasis, functions of reproductive organs, and fertility. On theother hand, acute intoxication with GBH, characterized by dehydration,oliguria, paralytic ileus, as well as hypovolemic and cardiogenicshock, pulmonary edema, hyperkalemia, and metabolic acidosis, mayoccur fatally. As no antidote has been developed for GBH poisoningso far, the detoxification is mainly symptomatic and supportive andrequires intensive care based on gastric lavage, extracorporeal bloodfiltering, and intravenous lipid emulsion infusion. The current reviewcomprehensively discusses the molecular and physiological basics ofthe GLP- and/or GBH-induced diseases of the endocrine and reproductivesystems, and cardiopulmonary-, nephro-, and hepatotoxicities, presentedin recent preclinical studies and case reports on the accidental orintentional ingestions with the most popular GBHs. Finally, they brieflydescribe modern and future healthcare methods and tools for GLP detection,determination, and detoxification. Future electronically powered,decision-making, and user-friendly devices targeting major GLP/GBH’smodes of actions, i.e., dysbiosis and the inhibition of AChE, shallenable self-handled or point-of-care professional-assisted evaluationof the harm followed with rapid capturing GBH xenobiotics in the bodyand precise determining the GBH pathology-associated biomarkers levels.

The effect of heavy metals (HMs) has been extensively studied. They cause diverse clinical manifestation through various mechanisms. Male fertility is among the most disturbing effect of HMs affecting family life in human and reproduction in animals. Notably among these effects is interference with the reproductive hormones, morphology and function of reproductive organs, sexual behaviors, and the spermiogram. Quercetin is a dietary flavanoid from edible plants and, has proven pharmacological properties in the treatment and management of many disease conditions. Quercetin ameliorates the adverse effects of HMs on male reproductive hormones by increasing the activity of 3β-hydroxysteroid dehydrogenase (3β- HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in the synthesis of testosterone. Quercetin chelates HMs, scavenge free radicals, and other cytotoxicant capable of disrupting the morphology and function of the male reproductive system. Apart from it neuroprotective activity on the pituitary gland and increased steroidogenesis, quercetin mitigate neurotransmitter that aid in copulation and improve histopathological changes in the brain due to HMs toxicity to improve sexual behavior. Quercetin was also found to be effective in increasing sperm count, daily sperm production, mortility, viability, and also decreased in the percentage of abnormal sperm morphology due to HMs toxicity. In conclusion quercetin was found to be effective in mitigating HMs toxicity that affects male fertility, and so, it is recommended to be incorporated into the treatment and management of HMs toxicity. Individuals who are at risk of HMs toxicity should take dietary plants that contain quercetin to minimize the effects of these metals.

This review presents analysis of the literature and our own research with respect to the role of miRNAs in the regulation of activity (expression) of genes controlling cellular homeostasis in human cells when exposed to ionizing radiation. Human cells, on one hand, can have increased resistance to radiation, which hinders the effectiveness of tumor treatment in radiotherapy. On the other hand, increased sensitivity to radiation may be accompanied by the development of several pathologies, including tumorigenesis. This paper examines the role of specific miRNAs in the formation of radioresistance and radiosensitivity of human cells and their impact on the respective target genes. Separate sections are devoted to the role of different miRNAs in radiation therapy of tumors of different localization, as well as their role in the bystander effect. A special section highlights features of gene activity and its regulators, miRNAs, in radiosensitive cells in patients with Down syndrome. The final section provides information about new approaches to change miRNA expression and, accordingly, their target genes by the action of plant and synthetic drugs (crown compounds) which reduce damaging effects of mutagens. It is assumed that antimutagens affecting the expression levels of miRNAs and structural genes may be used to correct the increase and decrease in cellular radioresponse, reducing the risk of development of pathological processes, including tumorigenesis.

Germline development from human pluripotent stem cells toward disease modeling of infertility

Background and AimsmicroRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. miRNAs have been implicated in regulating gene expression in embryonic developmental processes, including proliferation and differentiation. The liver is a multifunctional organ, which undergoes rapid changes during the developmental period and relies on tightly-regulated gene expression. Little is known regarding the complex expression patterns of both mRNAs and miRNAs during the early stages of human liver development, and the role of miRNAs in the regulation of this process has not been studied. The aim of this work was to study the impact of miRNAs on gene expression during early human liver development.MethodsGlobal gene and miRNA expression were profiled in adult and in 9–12w human embryonic livers, using high-density microarrays and quantitative RT-PCR.ResultsEmbryonic liver samples exhibited a gene expression profile that differentiated upon progression in the developmental process, and revealed multiple regulated genes. miRNA expression profiling revealed four major expression patterns that correlated with the known function of regulated miRNAs. Comparison of the expression of the most regulated miRNAs to that of their putative targets using a novel algorithm revealed a significant anti-correlation for several miRNAs, and identified the most active miRNAs in embryonic and in adult liver. Furthermore, our algorithm facilitated the identification of TGFβ-R1 as a novel target gene of let-7.ConclusionsOur results uncover multiple regulated miRNAs and genes throughout human liver development, and our algorithm assists in identification of novel miRNA targets with potential roles in liver development.

Recently, the research of animal microRNAs (miRNAs) has attracted wide attention for its regulatory effect in the development process and the response to abiotic stresses. Rainbow trout is a commercially and cold water fish species, and usually encounters heat stress, which affects its growth and leads to a huge economic loss. But there were few investigations about the roles of miRNAs in heat stress in rainbow trout. In this study, miRNAs of rainbow trout which were involved in heat stress were identified by high-throughput sequencing of six small RNA libraries from head kidney tissues under control (18°C) and heat-treated (24°C) conditions. A total of 392 conserved miRNAs and 989 novel miRNAs were identified, of which 78 miRNAs were expressed in different response to heat stress. Ten of these miRNAs were further validated by quantitative real-time PCR. In addition to, including 393 negative correlation miRNA-target gene pairs, several important regulatory pathways were involved in heat stress of the potential target genes, including protein processing in endoplasmic reticulum, NOD-like receptor signaling pathway, and phagosome. Our data significantly advance understanding of heat stress regulatory mechanism of miRNA in the head kidney of rainbow trout, which provide a useful resource for the cultivation of rainbow trout.

The long-term risks of pesticides to wildlife in the EU currently are assessed by comparing the lowest no-observed-effect concentration (NOEC) determined from the suite of endpoints measured in existing avian and mammalian laboratory reproduction tests with estimated exposure concentrations by calculating Toxicity to Exposure Ratios (TERs). Regulatory authorities experience difficulties when assessing long-term risks because of the lack of accepted methods to improve the ecological realism of exposure and toxicity estimates and understand risks at a population level. This paper describes an approach for interpreting existing avian and mammalian toxicity test data that divides breeding cycles into several discrete phases and identifies specific test endpoints as indicators of direct pesticide effects possible at each phase. Based on the distribution of breeding initiation dates for a species of concern and the dates of pesticide applications, this approach compares the phase-specific toxicity endpoint with the expected pesticide exposure levels during each of the breeding phases. The fate of each breeding attempt is determined through a series of decision points. The cumulative reproductive response of individuals in a breeding population based on this decision framework provides a means of examining the estimated risks over the course of the breeding season and deriving an overall metric of the impact of the pesticide on reproduction. Research needed to further improve the approach is discussed.

Urgency of the research. A municipality is an autonomous and independent from other state administration institution. The process of development that is being implemented by a district is a set of qualitative and quantitative transformations aimed at increasing the satisfaction level of the collective and individual needs of its inhabitants. Target setting. The evaluation of the action of local government should be directed at the utilization of endogenous potential. The results of the analysis should allow identifying the peripheral areas or key actions. Actual scientific researches and issues analysis. Scientific work of M. Bilski, M. Holstein-Beck, K. Cameron, R. Griffin, A. Hamroll, M. Kachniarz, P. Dziekanskigo, B. FIlipiak, S. Owsiak, - indicate the effectiveness of the action of local self-government and the measures to evaluate it. Uninvestigated parts of general matters defining. The process of social and economic development carried out by the municipality is a set of transformations aimed at meeting the needs of the local community. One of the fundamental factors of the development process is the economic base of individual units. The research objective. The aim of the study is to analyze the spatial diversification of economic efficiency of municipalities in the Świętokrzyskie voivodship in 2010 and 2015. The analysis is both static and dynamic. The source of statistical information was GUS Local Data Bank. The statement of basic materials. The synthetic measure indicates the poor financial performance of the surveyed units. Both in 2010 and 2015 the best unit turned out to be Sitkówka-Noiny 0,495 - 0.472. At the end of the ranking were respectively Dwikozy 0.239 - 2010; Bliżyn 0.257 - 2015. Conclusions. The indicated method provides information about the category of the object under consideration. The value of the measure depends on the economic character of the region as well as financial autonomy, level of own income, local taxes or infrastructure. Data received depend on the number and type of accepted variables to be tested.