Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model.

Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 - on E20 and P20, Hprt1, Actb, and Pgk1 - on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.

Age-Related COVID-19 Influence on Male Fertility.

The impact of coronavirus on the reproductive health of men attracts the special attention of many researchers. While studies suggest changes in sperm parameters and the possibility of testicular inflammation, further studies are needed to elucidate any potential age-related changes in these findings, which is the purpose of the present study. The semen quality parameters, cytokine concentration, and markers of the pro- and antioxidant system were assessed in 60 men five to seven months after the coronavirus infection and in 77 controls (without a history of coronavirus infection). Additionally, participants were divided into two age groups: less than 35 years and 35 years or older. Notably increased round cell count in ejaculate and reduced sperm hyaluronan binding ability were observed among post-infection patients younger than 35 years. In the same group, a decline in seminal plasma zinc levels and nitrotyrosine in the cell fraction was found. In men over 35 years of age, Coronavirus Disease 2019 (COVID-19) led to increased sperm DNA fragmentation, a decrease in the total antioxidant capacity, and an elevation in the levels of interleukin-1ß and interleukin-10. The concentration of interleukin-1ß decreased over time following recovery in all affected patients. The data obtained suggest the potential adverse impact of the coronavirus infection on male reproductive health; however, these effects appear to be age-dependent.

Imbalance of Angiogenic and Growth Factors in Placenta in Maternal Hyperhomocysteinemia.

Numerous studies have shown that various adverse factors of different nature and action mechanisms have similar negative influence on placental angiogenesis, resulting in insufficiency of placental blood supply. One of the risk factors for pregnancy complications with placental etiology is an increased level of homocysteine in the blood of pregnant women. However, the effect of hyperhomocysteinemia (HHcy) on the development of the placenta and, in particular, on the formation of its vascular network is at present poorly understood. The aim of this work was to study the effect of maternal HHcy on the expression of angiogenic and growth factors (VEGF-A, MMP-2, VEGF-B, BDNF, NGF), as well as their receptors (VEGFR-2, TrkB, p75NTR), in the rat placenta. The effects of HHcy were studied in the morphologically and functionally different maternal and fetal parts of the placenta on the 14th and 20th day of pregnancy. The maternal HHcy caused increase in the levels of oxidative stress and apoptosis markers accompanied by an imbalance of the studied angiogenic and growth factors in the maternal and/or fetal part of the placenta. The influence of maternal HHcy in most cases manifested in a decrease in the protein content (VEGF-A), enzymatic activity (MMP-2), gene expression (VEGFB, NGF, TRKB), and accumulation of precursor form (proBDNF) of the investigated factors. In some cases, the effects of HHcy differed depending on the placental part and stage of development. The influence of maternal HHcy on signaling pathways and processes controlled by the studied angiogenic and growth factors could lead to incomplete development of the placental vasculature and decrease in the placental transport, resulting in fetal growth restriction and impaired fetal brain development.

Maternal Hyperhomocysteinemia Disturbs the Mechanisms of Embryonic Brain Development and Its Maturation in Early Postnatal Ontogenesis.

Maternal hyperhomocysteinemia causes the disruption of placental blood flow and can lead to serious disturbances in the formation of the offspring's brain. In the present study, the effects of prenatal hyperhomocysteinemia (PHHC) on the neuronal migration, neural tissue maturation, and the expression of signaling molecules in the rat fetal brain were described. Maternal hyperhomocysteinemia was induced in female rats by per os administration of 0.15% aqueous methionine solution in the period of days 4-21 of pregnancy. Behavioral tests revealed a delay in PHHC male pups maturing. Ultrastructure of both cortical and hippocampus tissue demonstrated the features of the developmental delay. PHHC was shown to disturb both generation and radial migration of neuroblasts into the cortical plate. Elevated Bdnf expression, together with changes in proBDNF/mBDNF balance, might affect neuronal cell viability, positioning, and maturation in PHHC pups. Reduced Kdr gene expression and the content of SEMA3E might lead to impaired brain development. In the brain tissue of E20 PHHC fetuses, the content of the procaspase-8 was decreased, and the activity level of the caspase-3 was increased; this may indicate the development of apoptosis. PHHC disturbs the mechanisms of early brain development leading to a delay in brain tissue maturation and formation of the motor reaction of pups.

Oxidative Stress Markers and Sperm DNA Fragmentation in Men Recovered from COVID-19.

SARS-CoV-2 negatively affects semen characteristics, impairs various biochemical processes in seminal fluid and within spermatogenic cells ultimately leading to male fertility decline. However, the distinct mechanisms, in particular, the role of oxidative stress on the consequences of coronavirus infection, have not been well investigated, which is the purpose of the present study. The standard semen parameters, its pro- and antioxidant system state, as well as the level of sperm DNA fragmentation, were assessed in 17 semen samples of men five months after the coronavirus infection and in 22 age-matched control patients. We determined that the DNA fragmentation rate negatively correlated with the period after coronavirus recovery, as well as seminal fluid superoxide dismutase activity and uric acid level. It was demonstrated that COVID-19 is not always associated with increased DNA fragmentation, allowing them to be considered as two independent factors. Thus, the most significant changes were noted in the samples of men after COVID-19 and abnormal TUNEL results: increased round cell number, decreased seminal fluid's nitrotyrosine level, and total antioxidant capacity and Zn, as well as an increased 8-hydroxy-2'-deoxyguanosine level within spermatozoa. The data obtained indicate that increased DNA fragmentation and diminished semen quality in men can be the result of an imbalance in semen pro- and antioxidant components after COVID-19.

Maternal Hyperhomocysteinemia Produces Memory Deficits Associated with Impairment of Long-Term Synaptic Plasticity in Young Rats.

Maternal hyperhomocysteinemia (HCY) is a common pregnancy complication caused by high levels of the homocysteine in maternal and fetal blood, which leads to the alterations of the cognitive functions, including learning and memory. In the present study, we investigated the mechanisms of these alterations in a rat model of maternal HCY. The behavioral tests confirmed the memory impairments in young and adult rats following the prenatal HCY exposure. Field potential recordings in hippocampal slices demonstrated that the long-term potentiation (LTP) was significantly reduced in HCY rats. The whole-cell patch-clamp recordings in hippocampal slices demonstrated that the magnitude of NMDA receptor-mediated currents did not change while their desensitization decreased in HCY rats. No significant alterations of glutamate receptor subunit expression except GluN1 were detected in the hippocampus of HCY rats using the quantitative real-time PCR and Western blot methods. The immunofluorescence microscopy revealed that the number of synaptopodin-positive spines is reduced, while the analysis of the ultrastructure of hippocampus using the electron microscopy revealed the indications of delayed hippocampal maturation in young HCY rats. Thus, the obtained results suggest that maternal HCY disturbs the maturation of hippocampus during the first month of life, which disrupts LTP formation and causes memory impairments.

Prenatal Hyperhomocysteinemia Induces Glial Activation and Alters Neuroinflammatory Marker Expression in Infant Rat Hippocampus.

Maternal hyperhomocysteinemia is one of the common complications of pregnancy that causes offspring cognitive deficits during postnatal development. In this study, we investigated the effect of prenatal hyperhomocysteinemia (PHHC) on inflammatory, glial activation, and neuronal cell death markers in the hippocampus of infant rats. Female Wistar rats received L-methionine (0.6 g/kg b.w.) by oral administration during pregnancy. On postnatal days 5 and 20, the offspring's hippocampus was removed to perform histological and biochemical studies. After PHHC, the offspring exhibited increased brain interleukin-1ß and interleukin-6 levels and glial activation, as well as reduced anti-inflammatory interleukin-10 level in the hippocampus. Additionally, the activity of acetylcholinesterase was increased in the hippocampus of the pups. Exposure to PHHC also resulted in the reduced number of neurons and disrupted neuronal ultrastructure. At the same time, no changes in the content and activity of caspase-3 were found in the hippocampus of the pups. In conclusion, our findings support the hypothesis that neuroinflammation and glial activation could be involved in altering the hippocampus cellular composition following PHHC, and these alterations could be associated with cognitive disorders later in life.

Prenatal Stress in Maternal Hyperhomocysteinemia: Impairments in the Fetal Nervous System Development and Placental Function.

The article presents current views on maternal hyperhomocysteinemia (HHcy) as an important factor causing prenatal stress and impaired nervous system development in fetuses and newborns in early ontogenesis, as well as complications in adulthood. Experimental data demonstrate that prenatal HHcy (PHHcy) affects the morphological maturation of the brain and activity of its neurotransmitter systems. Cognitive deficit observed in the offspring subjected to PHHcy in experimental studies can presumably cause the predisposition to various neurodegenerative diseases, as the role of maternal HHcy in the pathogenesis such diseases has been proven in clinical studies. The review also discusses molecular mechanisms of the HHcy neurotoxic action on the nervous system development in the prenatal and early postnatal periods, which include oxidative stress, apoptosis activation, changes in the DNA methylation patterns and microRNA levels, altered expression and processing of neurotrophins, and neuroinflammation induced by an increased production of pro-inflammatory cytokines. Special attention is given to the maternal HHcy impact on the placenta function and its possible contribution to the brain function impairments in the offspring. Published data suggest that some effects of PHHcy on the developing fetal brain can be due to the disturbances in the transport functions of the placenta resulting in an insufficient supply of nutrients necessary for the proper formation and functioning of brain structures.