Functional changes in PNPLA6 (Patatin-like phospholipase domain-containing protein 6), caused by gene mutations or inhibition by organophosphates, affect the levels of various phospholipids. In humans, this leads to organophosphorus compound-induced delayed neurotoxicity syndrome (OPIDN) and a number of rare diseases. In this study, we analyze the role of the Swiss cheese gene (sws), an ortholog of PNPLA6, in spermatogenesis in Drosophila melanogaster. We report that the sws1 mutation affects membrane remodeling during spermatid individualization, as well as spermatid coiling during the late stages of spermatogenesis. In addition, the sws1 mutation leads to changes in the transcriptome in the testes of flies. We also demonstrate that sws is required for the proper functioning of important biological barriers in Drosophila.

The aim of this study was an attempt to prevent the decrease in sperm motility of the fruit fly Drosophila melanogaster that occurs in the early period of readaptation after space flight by adding phospholipids with polyunsaturated fatty acids in the tail groups (essential phospholipids) to the nutrient medium. The study was carried out as part of space flights on board the Russian segment of the ISS during the periods of September 15, 2023–September 27, 2023 (ISS-69) and March 23, 2024–April 6, 2024 (ISS-70). The content of cytoskeletal proteins in the testes was determined by Western blotting and sperm motility by video recording and subsequent analysis. The results of the study show that modification of the nutrient medium by adding essential phospholipids leads to a decrease in cholesterol content and an increase in actin content, which prevents a decrease in tubulin content and a decrease in the speed of movement of fruit fly sperm in the early period of readaptation (up to 16 hours) after space flight. The data we obtained, on the one hand, demonstrate a possible mechanism for triggering mechanotransduction, and on the other, allow us to propose essential phospholipids as one of the means of protection against negative changes associated with changes in the gravity field acting on the cell.

Exploring deep space and creation of bases on other bodies in the Solar System requires increased spaceflight duration and prolonged exposure to different gravity conditions. Most physiological changes under these conditions occur at the cellular level, but the long-term consequences of such effects remain unexplored. The structural and functional characteristics of cells are similar across animal species, but research into issues related to aging and lifespan, development and the reproductive system, the transgenerational inheritance of information about environmental factors is more conveniently conducted in animals with short developmental cycle. To date, the influence of spaceflight factors on these aspects is poorly understood, and the limited data available are often contradictory. Therefore, Drosophila melanogaster remains a promising object for future investigations in space biology.

Endometriosis is one of the leading pathologies of the reproductive system of women of fertile age, which shows changes in cell metabolism in the lesions. We conducted a study of the cellular respiration according to the polarography and the mRNA content of the main metabolic proteins using qRT-PCR of intraoperative endometrial biopsies from patients in the control group and with different localizations of endometriosis (adenomyosis, endometrioma, pelvic peritoneum). In biopsy samples of patients with endometriomas and pelvic peritoneum endometriotic lesions, the rate of oxygen absorption was significantly reduced, and, moreover, in the extragenital case, there was a shift to succinate utilization. The mRNA content of the cytochrome c, cytochrome c oxidase, and ATP synthase was also reduced, but hexokinase HK2 as well as pyruvate kinase were significantly higher than in the control. These oxidative phosphorylation and gene expression profiles suggest the Warburg effect and a shift in metabolism toward glycolysis. For adenomyosis, on the contrary, cellular respiration was significantly higher than in the control group due to the terminal region of the respiratory chain, ATP synthase, and its mRNA was increased as well. These data allow us to suggest that the therapeutic strategies of endometriosis based on modulation energy metabolism should take lesion localization into account.

The aim of this study was to prevent initial changes in Drosophila melanogaster oocytes under simulated weightlessness and hypergravity at the 2 g level. Phospholipids with polyunsaturated fatty acids in the tail groups (essential phospholipids) at a concentration of 500 mg/kg of nutrient medium were used as a protective agent. Cell stiffness was determined using atomic force microscopy, the change in the oocytes’ area was assessed as a mark of deformation, and the contents of cholesterol and neutral lipids were determined using fluorescence microscopy. The results indicate that the administration of essential phospholipids leads to a decrease in the cholesterol content in the oocytes’ membranes by 13% (p < 0.05). The stiffness of oocytes from flies that received essential phospholipids was 14% higher (p < 0.05) and did not change during 6 h of simulated weightlessness or hypergravity, and neither did the area, which indicates their resistance to deformation. Moreover, the exposure to simulated weightlessness and hypergravity of oocytes from flies that received a standard nutrient medium led to a more intense loss of cholesterol from cell membranes after 30 min by 13% and 18% (p < 0.05), respectively, compared to the control, but essential phospholipids prevented this effect.

Research into the mechanisms by which gravity influences spermatozoa has implications for maintaining the species in deep space exploration and may provide new approaches to reproductive technologies on Earth. Changes in the speed of mouse spermatozoa after 30 min exposure to simulated weightlessness (by 3D-clinostat) and 2 g hypergravity (by centrifugation) were studied using inhibitory analysis. Simulated microgravity after 30 min led to an increase in the speed of spermatozoa and against the background of an increase in the relative calcium content in the cytoplasm. This effect was prevented by the introduction of 6-(dimethylamino) purine, wortmannin, and calyculin A. Hypergravity led to a decrease in the speed of spermatozoa movement, which was prevented by sodium orthovanadate and calyculin A. At the same time, under microgravity conditions, there was a redistribution of proteins forming microfilament bundles between the membrane and cytoplasmic compartments and under hypergravity conditions—proteins forming networks. The obtained results indicate that even a short exposure of spermatozoa to altered gravity leads to the launch of mechanotransduction pathways in them and a change in motility.

With the increasing number of women-participants in piloted space programs, investigations of spaceflight effects on the female reproductive system are becoming vital. Purpose of the work is to determine thickness of the granulosa cells layer relative to the content of the luteinizing hormone (LH) receptor in antral follicles of the ovary, LH and a relative content of cytoskeletal proteins in the hypophysis of mice following suspension during the estrous cycle. SDS-PAGE and immunoblotting were used to define the relative proteins content. mRNAs were counted using RT-PCR. IHA was performed to measure thickness of the granulosa cells layer in ovarian sections. The results demonstrate an increase in the number of granulosa cells in the antral follicles of mice after 96-hr suspension, as well as in the relative content of the LH receptor and its mRNAs. However, the LH level in the hypophysis did not alter, even though we observed some changes in the content of cytoskeletal proteins, namely, an increase in the actin-binding proteins (alpha actinin 1 and 4) and a decrease in alpha tubulin and its acetylated form suggesting, probably, modification of the intracellular LH transport and release into blood.

Female germ cells provide the structural basis for the development of a new organism, while the main molecular mechanisms of the impact of weightlessness on the cell remain unknown. The aim of this work was to determine the relative content and distribution of the main proteins of microtubules and microfilaments, to assess the relative RNA content of genes in mouse oocytes after short-term exposure to simulated microgravity, and to determine the potential for embryo development up to the 3-cell stage. Before starting the study, BALB/c mice were divided into two groups. One group received water and standard food without any modifications. Before exposure to simulated microgravity, the oocytes of these animals were randomly divided into two groups – c and µg. The second group of animals additionally received essential phospholipids containing at least 80% phosphatidylcholines, per os for 6 weeks before the start of the experiment at a dosage of 350 mg/kg of the animal's body to modify the lipid composition of the oocyte membrane. The obtained oocytes of these animals were also randomly divided into two groups – ce and µge. To determine the protein distribution and its relative content, immunofluorescence analysis was performed, and the RNA content of genes was assessed using real-time PCR with reverse transcription. After cultivation under simulated microgravity, beta-actin and acetylated alpha-tubulin are redistributed from the cortical layer to the central part of the oocyte, and the relative content of acetylated alpha-tubulin and tubulin isoforms decreases. At the same time, the mRNA content of most genes encoding cytoskeletal proteins was significantly higher in comparison with the control level. The use of essential phospholipids led to a decrease in the content of cellular cholesterol in the oocyte and leveled changes in the content and redistribution of acetylated alpha-tubulin and beta-actin after cultivation under simulated microgravity. In addition, after in vitro fertilization and further cultivation under simulated weightlessness, we observed a decrease in the number of embryos that passed the stage of the 2-cell embryo, but while taking essential phospholipids, the number of embryos that reached the 3-cell stage did not differ from the control group. The results obtained show changes in the content and redistribution of cytoskeletal proteins in the oocyte, which may be involved in the process of pronucleus migration, the formation of the fission spindle and the contractile ring under simulated weightlessness, which may be important for normal fertilization and cleavage of the future embryo.

The participation of women in space programs of increasing flight duration requires research of their reproductive system from the perspective of subsequent childbearing and healthy aging. For the first time, we present hormonal and structural data on the dynamics of recovery after a 157-day space flight in a woman of reproductive age. There were no clinically significant changes in the reproductive system, but detailed analysis shows that weightlessness leads to an increase in the proportion of early antral follicles and granulosa cells in large antral follicles. Returning to Earth's gravity reduces the number and diameter of early antral follicles.

The review describes mechanisms of regulating the spermatozoa motor activity. The authors present the enzymes involved in phosphorylation of axonemal proteins, action of various inhibitors of the spermatozoa motility in animals, and discuss the results of investigations in simulated and space microgravity.

Endometriosis of the cervix is a rare form of genital endometriosis, which is characterized by the appearance of tissue on the vaginal part of the cervix, similar to the tissue of the mucous membrane of the uterine cavity. We describe a clinical case in which we compared the content of cytoskeletal proteins, H3 histone modifications and DNA methylation (total and 5-hydroxymethylcytosine content) in the eutopic endometrium and in tissue from endometriosis foci on the cervix. The patient had elevated levels of estradiol, interleukin-1β and interleukin-8. At the cellular level, the content of tubulin and the marker of stable microtubules were reduced in the ectopic endometrium (by 45% and 37%, p < 0.05, respectively), but the alpha-actinin-1 content was increased (by 75%, p < 0.05) with an increase in the expression of its gene. At the same time, the total level of DNA methylation in the endometriotic focus was reduced by more than 2 times with the accumulation of the intermediate product 5-hydroxymethylcytosine (the content increased by more than 3 times), probably due to an increase in the content of tet methylcytosine dioxygenase 1 (more than 4 times).

The purpose of this study was to assess oxidative phosphorylation (OXPHOS) in mouse ovaries, determine the relative content of proteins that form the respiratory chain complexes and the main structures of the cytoskeleton, and determine the mRNA of the corresponding genes after hindlimb suspension for 96 h. After hindlimb suspension, the maximum rate of oxygen uptake increased by 133% (p < 0.05) compared to the control due to the complex I of the respiratory chain. The content of mRNA of genes encoding the main components of the respiratory chain increased (cyt c by 78%, cox IV by 56%, ATPase by 69%, p < 0.05 compared with the control). The relative content of cytoskeletal proteins that can participate in the processes of transport and localization of mitochondria does not change, with the exception of an increase in the content of alpha-tubulin by 25% (p < 0.05) and its acetylated isoform (by 36%, p < 0.05); however, the mRNA content of these cytoskeletal genes did not differ from the control. The content of GDF9 mRNA does not change after hindlimb suspension. The data obtained show that short-term exposure to simulated weightlessness leads to intensification of metabolism in the ovaries.

Mechanism of the microgravity-induced changes in the cytoskeleton, reproductive system cells in particular, is still poorly known. We measured the relative content of cytoskeleton proteins and mRNAs in associated genes, levels of lysines 4 and 9 methylation, and lysines 9 and 27 acetylation in histone H3 in ovaries of 5-day Drosophila melanogaster after rotation in a random positioning machine with the aim to investigate the effects of microgravity on the course of hametogenesis. After the exposure in simulated microgravity, β-actin and α-tubulin in fly ovaries were found increased as compared to the control; content of associated mRNAs did not change. There was an increase in the relative content of histone H3 modifications, and also increases in both lysines 9 and 27 acetylation and lysines 4 and 9 methylation. These data shed light on the mechanisms of epigenetic regulation of gene expression and development of an adaptive pattern of the reproductive system cell structure in the condition of microgravity.

The hypothesis about the role of the cortical cytoskeleton as the primary mechanosensor was tested. Drosophila melanogaster oocytes were exposed to simulated microgravity (by 3D clinorotation in random directions with 4 rotations per minute-sµg group) and hypergravity at the 2 g level (by centrifugal force from one axis rotation-hg group) for 30, 90, and 210 min without and with cytochalasin B, colchicine, acrylamide, and calyculin A. Cell stiffness was measured by atomic force microscopy, protein content in the membrane and cytoplasmic fractions by Western blotting, and cellular respiration by polarography. The obtained results indicate that the stiffness of the cortical cytoskeleton of Drosophila melanogaster oocytes decreases in simulated micro- (after 90 min) and hypergravity (after 30 min), possibly due to intermediate filaments. The cell stiffness recovered after 210 min in the hg group, but intact microtubules were required for this. Already after 30 min of exposure to sµg, the cross-sectional area of oocytes decreased, which indicates deformation, and the singed protein, which organizes microfilaments into longitudinal bundles, diffused from the cortical cytoskeleton into the cytoplasm. Under hg, after 30 min, the cross-sectional area of the oocytes increased, and the proteins that organize filament networks, alpha-actinin and spectrin, diffused from the cortical cytoskeleton.

BackgroundAdenomyosis is characterized by overgrowth of endometrial glands and stroma in the myometrium and is associated with reduced apoptosis. One of the key participants in one of the pathways of apoptosis is cytochrome c, whose expression can be regulated by actin-binding proteins involved in the formation of structures that provide cell motility. The aim of the study was to determine the content of actin-binding proteins, cytochrome c, and terminal members of the mitochondrial respiratory chain in endometrial biopsies of patients with adenomyosis and the control group.Methods and resultsThe content of all studied proteins was determined by Western blotting, and the mRNA content of the corresponding genes was determined by quantitative RT-PCR. The relative content of alpha-actinin1 and mRNA of the gene encoding it in biopsy specimens from patients with adenomyosis was higher than in controls by 86 and 84% (p < 0.05), respectively. The relative content of alpha-actinin4 did not change, as did cytochrome c. The content of cytochrome-c-oxidase and ATPsynthase in the group with adenomyosis exceeded the control level by 270 and 121% (p < 0.05), respectively, but the relative content of mRNA of these genes did not change, which may indicate a change in regulation at the level of protein synthesis.ConclusionThe results may indicate a local increase in the synthesis of ATP in pathological endometrial cells, which indicates the possible effectiveness of local application of H+-ATP synthase inhibitors (for example, macrolide antibiotic) to reduce the severity of clinical symptoms of adenomyosis.

The paper presents evaluation of the effect of different doses of essential phospholipids on the cytoskeleton of mouse oocyte microtubes and microfilaments cultivated on a microgravity simulator. Total content of acetylated alpha-tubulin and beta-actin was determined by immunocytochemistry. After the cultivation in modeled microgravity, the relative content of acetylated alpha-tubulin was decreased, whereas the beta-actin level remained unchanged. Injection of essential phospholipids (350 mg/kg of body mass) caused recovery of this parameter in the conditions reproducing the effects of microgravity.

The impact of weightlessness on the female reproductive system remains poorly understood, although deep space exploration is impossible without the development of effective measures to protect women's health. The purpose of this work was to study the effect of a 5-day "dry" immersion on the state of the reproductive system of female subjects. On the fourth day of the menstrual cycle after immersion, we observed an increase in inhibin B of 35% (p < 0.05) and a decrease in luteinizing hormone of 12% (p < 0.05) and progesterone of 52% (p < 0.05) compared with the same day before immersion. The size of the uterus and the thickness of the endometrium did not change. On the ninth day of the menstrual cycle after immersion, the average diameters of the antral follicles and the dominant follicle were, respectively, 14% and 22% (p < 0.05) higher than before. The duration of the menstrual cycle did not change. The obtained results may indicate that the stay in the 5-day "dry" immersion, on the one hand, can stimulate the growth of the dominant follicle, but, on the other hand, can cause functional insufficiency of the corpus lutea.

The exploration of deep space or other bodies of the solar system, associated with a long stay in microgravity or altered gravity, requires the development of fundamentally new methods of protecting the human body. Most of the negative changes in micro- or hypergravity occur at the cellular level; however, the mechanism of reception of the altered gravity and transduction of this signal, leading to the formation of an adaptive pattern of the cell, is still poorly understood. At the same time, most of the negative changes that occur in early embryos when the force of gravity changes almost disappear by the time the new organism is born. This review is devoted to the responses of early embryos and stem cells, as well as terminally differentiated germ cells, to changes in gravity. An attempt was made to generalize the data presented in the literature and propose a possible unified mechanism for the reception by a single cell of an increase and decrease in gravity based on various deformations of the cortical cytoskeleton.

Maintenance of the human reproductive health is vital in context of preserving living species in remote space missions. Changes in the gene expression due to microgravity can lead to degradation of fertility; however, the mechanism that triggers these changes is not fully understood. In our investigation we determined mRNA levels in the prime cytoskeletal proteins and analyzed histone methylation and acetylation activities in Drosophila melanogaster ovaries during 79-hr microgravity modeled with the use of a random positioning machine (RPM), and per os introduction of essential phospholipids. The results showed that the fruit flies exposure to modeled microgravity over the entire oogenesis cycle increased levels of mRNA genes encoding the cytoskeletal proteins in oocytes and that this can be, probably, associated with increased acetylation of the histone H3 Lys9 and Lys27. Essential phospholipids seem to prevent growth and/or initiate mRNA reductions in the majority of cytoskeleton elements in the flies exposed to modeled microgravity; however, the tubulin and lamin B binding proteins increased in the respective controls. Essential phospholipids leveled histone 3 Lys9 acetylation in modeled microgravity but enhanced Lys27 acetylation in the control.