Influence of Extracellular Vesicles from the Follicular Fluid of Young Women and Women of Advanced Maternal Age with Different miRNA Profiles on Sperm Functional Properties.

We studied the effect of co-culturing of extracellular vesicles in the follicular fluid of young women and women of advanced maternal age on sperm motility. Vesicles were obtained by differential centrifugation. The sperm fraction was isolated from the seminal fluid of 18 patients (age 28-36 years). The spermatozoa were incubated with vesicles (1:2 ratio) for 60 or 120 min at 37°C in a CO2 incubator. A fraction of spermatozoa incubated without vesicles served as the control. After the incubation, the sperm samples were sedimented by centrifugation, fixed in 2.5% glutaraldehyde, and analyzed by transmission electron microscopy. RNA was isolated from the follicular fluid vesicles by column method followed by cDNA synthesis in a reaction mixture according to miScript II RT Kit protocol (Qiagen). After 60-min incubation with extracellular vesicles from the follicular fluid of women of advanced maternal age, the sperm motility and hyperactivation slightly changed in comparison with the group where incubation was performed with follicular fluid vesicles from young women and control group. Follicular fluid miRNA profiles in women of different ages varied, which suggests different functional compositions and effects of follicular fluid vesicles of different age groups on sperm characteristics. Transmission electron microscopy revealed differences in the interaction of follicular fluid vesicles from women of different age groups with spermatozoa. Further study of the effect of extracellular vesicles from the follicular fluid and analysis of their transcriptomic, proteomic, and metabolomic composition on sperm mobility and fertilizing ability will improve the effectiveness of assisted reproductive technology programs in patients with male infertility.

Influence of Extracellular Vesicles of the Follicular Fluid on Morphofunctional Characteristics of Human Sperm.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

Bilateral occlusion of carotid artery in awake hypertensive rats (SHR-SP) as a model of global cerebral ischemia.

Bilateral occlusion of carotid arteries in awake hypertensive rats (SHR-SP) was used as a model of global brain ischemia (duration of occlusion--until appearance of seizures). In normotensive rats (WKY), no seizures developed over 60 min. We revealed swelling of mitochondria in dendrites of hippocampal CA1 pyramidal cells, which was more pronounced in SHP-SP than in WKY rats. Blood pressure and heart rate in SHR-SP rats increased starting from the first minutes of occlusion, while in WKY rats these parameters remained unchanged. We proved that bilateral occlusion of the carotid arteries in awake SHR-SP rats can be used as an adequate model of global cerebral ischemia.

Biogenic amine content in the brains of rats with different levels of resistance to emotional stress.

The levels of biogenic amines and of a number of the products of their metabolism were studied in the hypothalamic nuclei in Wistar and August rats, which have different levels of resistance to emotional stress; levels were also studied in structures functionally and anatomically associated with the hypothalamic nuclei, i.e., the reticular formation of the midbrain, the amygdaloid body, the septum, the locus ceruleus, the dorsal cervical nucleus, and the ventral region of the tegmentum. The genotype was found to determine the level of metabolism of biogenic amines in structures of the central nervous system in conditions of emotional stress. In August rats, the activities of the dopaminergic and serotoninergic systems, which are stress-limiting, decreased to a greater extent during 24-hour immobilization stress. Adrenaline levels in structures of the central nervous system in August rats were higher during stress. Changes in the contents of biogenic amines in the paraventricular and ventromedial nuclei of the hypothalamus in Wistar and August rats could affect the preganglionic neurons of the autonomic nervous system.

[Blood catecholamine levels in rabbits with varying resistance of the cardiovascular system to emotional stress]. / Kontsentratsiia katekholaminov v krovi krolikov s raznoi ustoichivost'iu serdechno-sosudistoi sistemy k émotsional'nomu stressu.

The content of catecholamines and some products of their metabolism in the blood of rabbits with different resistance of the cardiovascular system to acute emotional stress was determined by high performance liquid chromatography with electrochemical detection. It was shown that in rabbits with mild arterial pressure (AP) changes in noradrenaline (NA) concentration increased by 145% by the end of the 3rd hour of the experiment. In rabbits with progressively reducing AP the blood NA concentration increased (by 272%) 10 minutes after the beginning of stimulation of the ventromedial nuclei of the hypothalamus and remained increased (to 500%) to the end of the experiment. The concentration of adrenaline and 3-4 dioxyphenylacetic acid was also increased in these animals.

[Changes in the catecholamine content of brain structures in rats subjected to immobilization stress]. / Izmeneniia soderzhaniia katekholaminov v strukturakh mozga krys, perenesshikh immobilizatsionnyi stress.

The changes in DOPA and catecholamine (adrenaline, noradrenaline, dopamine) levels were investigated in noradrenaline- and dopamine-synthesizing brain nuclei of Wistar rats after prolonged immobilization stress on catecholamine analyzer (BAS, USA) using HPLC technique. Distinct DOPA and catecholamine changes were observed in locus ceruleus + nucleus subceruleus (1. c + n. sc) and substantia nigra at any stage after immobilization (right after immobilization and 15 and 30 days later). The most prominent alterations in noradrenaline content were detected in 1. c + n. sc. 30 days after immobilization NA level in these nuclei was 1.5 times higher, as compared to the control one. It is suggested that the increasing noradrenaline level in 1. c + n. sc. plays a defensive role in survival of rats after immobilization stress.