Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Ovarian follicular atresia is mediated by heterophagy,autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

The roles of cellular reactive oxygen species,oxidative stress and antioxidants in pregnancy outcomes

Reactive oxygen species (ROS) are generated as by-products of aerobic respiration and metabolism. Mammalian cells have evolved a variety of enzymatic mechanisms to control ROS production, one of the central elements in signal transduction pathways involved in cell proliferation, differentiation and apoptosis. Antioxidants also ensure defenses against ROS-induced damage to lipids, proteins and DNA. ROS and antioxidants have been implicated in the regulation of reproductive processes in both animal and human, such as cyclic luteal and endometrial changes, follicular development, ovulation, fertilization, embryogenesis, embryonic implantation, and placental differentiation and growth. In contrast, imbalances between ROS production and antioxidant systems induce oxidative stress that negatively impacts reproductive processes. High levels of ROS during embryonic, fetal and placental development are a feature of pregnancy. Consequently, oxidative stress has emerged as a likely promoter of several pregnancy-related disorders, such as spontaneous abortions, embryopathies, preeclampsia, fetal growth restriction, preterm labor and low birth weight. Nutritional and environmental factors may contribute to such adverse pregnancy outcomes and increase the susceptibility of offspring to disease. This occurs, at least in part, via impairment of the antioxidant defense systems and enhancement of ROS generation which alters cellular signalling and/or damage cellular macromolecules. The links between oxidative stress, the female reproductive system and development of adverse pregnancy outcomes, constitute important issues in human and animal reproductive medicine. This review summarizes the role of ROS in female reproductive processes and the state of knowledge on the association between ROS, oxidative stress, antioxidants and pregnancy outcomes in different mammalian species.     

Short‐term heat stress impairs testicular functions in the American oyster,Crassostrea virginica: Molecular mechanisms and induction of oxidative stress and apoptosis in spermatogenic cells

Global temperature is increasing due to anthropogenic activities. Abnormal temperature has devastating effects on growth, reproduction, and development of aquatic organisms. In this study, we examined the effects of short‐term exposure to elevated temperatures (28 and 32°C for 1 week) on testicular functions, heat shock protein‐70 (HSP70), dinitrophenyl protein (DNP, a biomarker of reactive oxygen species [ROS]), and nitrotyrosine protein (NTP, an indicator of reactive nitrogen species [RNS]) expressions, protein carbonyl (PC, a measure of ROS) contents, nitrates/nitrites (NOx, a metabolite of nitric oxide) levels, extrapallial fluid (EPF) conditions, and cellular apoptosis in American oyster (Crassostrea virginica). Higher temperatures significantly decreased (~26%) sperm production in oysters compared with controls (24°C). HSP70, NTP, and DNP expressions were increased after heat exposure, consistent with increased EPF pH, and cellular apoptosis. The enhanced apoptosis in spermatogenic cells is associated with increased caspase 3/7 activity, PC contents, and NOx levels in testicular tissues. Together these results suggest that elevated temperature drastically increases oxidative stress and cellular apoptosis which in turn leads to decreased testicular functions in oysters. To the best of our knowledge, this study reports the first findings on the impacts of elevated temperatures on testicular functions in oysters.     

Roles of reactive oxygen species and antioxidants in ovarian toxicity

Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.     

Essential actions of melatonin in protecting the ovary from oxidative damage

Free radicals and other reactive species are involved in normal ovarian physiology. However, they are also highly reactive with complex cellular molecules (proteins, lipids, and DNA) and alter their functions leading to oxidative stress. Oxidative damage may play a prominent role in the development of disorders that considerably influence female fertility. Melatonin, because of its amphiphilic nature that allows for crossing morphophysiological barriers, is an effective antioxidant for protecting macromolecules against oxidative stress caused by reactive species. The balance between reactive oxygen species and antioxidants within the follicle seems to be critical to the function of the oocyte and granulosa cells and evidence has accumulated showing that melatonin is involved in the protection of these cells. Melatonin appears to have varied functions at different stages of follicle development, oocyte maturation, and luteal stage. Melatonin concentration in the growing follicle may be an important factor in avoiding atresia, because melatonin in the follicular fluid reduces apoptosis of critical cells. Melatonin also has protective actions during oocyte maturation reducing intrafollicular oxidative damage. An association between melatonin concentrations in follicular fluid and oocyte quality has been reported; this would allow a preovulatory follicle to fully develop and provide a competent oocyte for fertilization. The functional role of reactive species and the cytoprotective properties of melatonin on the ovary from oxidative damage are summarized in this brief review.     

Pigment-Epithelium Derived Factor (PEDF) and the human ovary: A role in the generation of ROS in granulosa cells

Aims

Pigment Epithelium Derived Factor (PEDF) is a multifunctional factor, which was found in mouse ovary and in human ovarian follicular fluid (FF). Its ovarian functions include anti-angiogenic actions. This study aimed to explore other PEDF-actions and the sites of PEDF expression in the human ovary.

Materials and methods

We used paraffin-embedded human ovarian sections for PEDF-immunohistochemistry and IVF-derived human granulosa cells (GCs) for RT-PCR, Western blotting and functional studies, including measurement of cell viability (ATP-assay), apoptosis (caspase-assay) and reactive oxygen species (ROS).

Key findings

Immunohistochemistry revealed PEDF in the cytoplasm of GCs of avascular follicles from the preantral to the antral stage and in FF. PEDF was also found in luteinized GCs of the highly vascularized corpus luteum, a result not in line with a sole anti-angiogenic action. Like GCs in vivo, cultured human luteinizing GCs express PEDF. They also responded to exogenous recombinant PEDF. In low concentrations PEDF did not affect cell viability but caused generation ROS. ROS-induction by PEDF was a concentration-dependent process and may be due to the activity of NADPH oxidase (NOX) type 4 and/or 5, which as we found are expressed by GCs. An antioxidant and apocynin, which inhibits NOX, blocked ROS generation. High levels of exogenous recombinant PEDF induced apoptosis of GCs, which was prevented by antioxidants, implying involvement of ROS.

Significance

PEDF is emerging as an ovarian factor, which has unexpected ROS-augmenting activities in the human ovary. It may be involved in ovarian ROS homeostasis and may contribute to oxidative stress.     

Chronic Exposure to Diquat Causes Reproductive Toxicity in Female Mice

Diquat is a bipyridyl herbicide that has been widely used as a model chemical for in vivo studies of oxidative stress due to its generation of superoxide anions, and cytotoxic effects. There is little information regarding the toxic effects of diquat on the female reproductive system, particularly ovarian function. Thus, we investigated the reproductive toxic effects of diquat on female mice. Chronic exposure to diquat reduced ovary weights, induced ovarian oxidative stress, resulted in granulosa cell apoptosis, and disrupted oocyte developmental competence, as shown by reactive oxygen species (ROS) accumulation, decreased polar body extrusion rates and increased apoptosis-related genes expression. Additionally, after diquat treatment, the numbers of fetal mice and litter sizes were significantly reduced compared to those of control mice. Thus, our results indicated that chronic exposure to diquat induced reproductive toxicity in female mice by promoting the ROS production of gruanousa cells and ooctyes, impairing follicle development, inducing apoptosis, and reducing oocyte quality. In conclusion, our findings indicate that diquat can be used as a potent and efficient chemical for in vivo studies of female reproductive toxicity induced by oxidative stress. Moreover, the findings from this study will further enlarge imitative research investigating the effect of ovarian damage induced by oxidative stress on reproductive performance and possible mechanisms of action in large domestic animals.     

Selection of antioxidants against ovarian oxidative stress in mouse model

Oxidative stress (OS) plays an important role in the process of ovarian granulosa cell apoptosis and follicular atresia. The aim of this study was to select antioxidant against OS in ovary tissue. Firstly, we chose the six antioxidants and analyzed the reactive oxygen species (ROS) level in the ovary tissue. The results showed that proanthocyanidins, gallic acid, curcumin, and carotene decrease the ROS level compared with control group. We further demonstrated that both proanthocyanidins and gallic acid increase the antioxidant enzymes activity. Moreover, change in the ROS level was not observed in proanthocyanidins and gallic acid group of brain, liver, spleen, and kidney tissues. Finally, we found that proanthocyanidins and gallic acid inhibit pro‐apoptotic genes expression in granulosa cells. Taken together, proanthocyanidins and gallic acid may be the most acceptable and optimal antioxidants specifically against ovarian OS and also may be involved in the inhibition of granulosa cells apoptosis in mouse ovary.     

Age-associated up-regulation of EGR1 promotes granulosa cell apoptosis during follicle atresia in mice through the NF-κB pathway

Follicular atresia is the main process responsible for the loss of follicles and oocytes from the ovary, and it is the root cause of ovarian aging. Apoptosis of granulosa cells (GCs) is the cellular mechanism responsible for follicular atresia in mammals. Recent advances have highlighted fundamental roles for EGR1 in age-related diseases via the induction of apoptosis. In the present study, we found that the expression of EGR1 was significantly increased in aged mouse ovaries compared with young ovaries. Immunohistochemical analysis revealed strongly positive EGR1 staining in atretic follicles, especially in apoptotic granulosa cells. We further showed that EGR1 up-regulation in mouse primary granulosa cells inhibited cell proliferation and promoted apoptosis. In addition, the promotion of apoptosis in GCs by EGR1 increases over time and with reactive oxygen species (ROS) stimulation. Our mechanistic study suggested that EGR1 regulates GC apoptosis in a mitochondria-dependent manner and that this mainly occurs through the NF-κB signaling pathway. In conclusion, our results suggested that age-related up-regulation of EGR1 promotes GC apoptosis in follicle atresia during ovarian aging.     

Effects of heat stress on ovarian development and the expression of HSP genes in mice

Heat stress reduces oocyte competence, thereby causing lower fertility in animals. Chronic and acute heat stresses cause extensive morphological damage in animals, but few reports have focused on the effects of chronic and acute heat stresses on ovarian function and heat shock protein (HSP) gene expression during ovarian injury. In this study, we subjected female mice to chronic and acute heat stresses; we then calculated the ovary index, examined ovary microstructure, and measured the expression of multiple HSP family genes. Chronic heat stress reduced whole-body and ovarian growth but had little effect on the ovarian index; acute heat stress did not alter whole-body or ovarian weight. Both chronic and acute heat stresses impaired ovary function by causing the dysfunction of granular cells. Small HSP genes increased rapidly after heat treatment, and members of the HSP40, HSP70, and HSP90 families were co-expressed to function in the regulation of the heat stress response. We suggest that the HSP chaperone machinery may regulate the response to heat stress in the mouse ovary.     

Heat shock prevents simulated ischemia-induced apoptosis in renal tubular cells via a PKC-dependent mechanism

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43 degrees C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl(2)) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl(2) expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.     

热胁迫对二化螟幼虫血淋巴细胞内活性氧、HSP90及细胞凋亡的影响

为探讨热激条件下二化螟Chilo suppressalis幼虫体内生理上的保护反应,本研究应用流式细胞术分析了热胁迫对二化螟幼虫血淋巴细胞内活性氧（ROS）、热休克蛋白90（HSP90）的产生和对细胞凋亡的影响。结果表明：暴露于33℃,36℃和39℃的二化螟5龄幼虫的ROS与对照（28℃）相比显著提高,分别增加了1.71,1.69和1.38倍;当温度达到33℃以后,ROS不再显著增加。实时定量PCR结果显示,二化螟HSP90基因在热胁迫诱导下表达。流式细胞术检测表明,HSP90的变化与在mRNA水平上的变化高度一致,热胁迫处理没有造成血淋巴细胞凋亡的显著变化。这些研究结果进一步证明热胁迫产生的ROS激活HSP90基因的表达,HSP90蛋白在保护机体免受ROS引起的伤害中起着重要作用,能够抑制血淋巴细胞凋亡的发生。     

Effects of heat stress on mammalian reproduction

Heat stress can have large effects on most aspects of reproductive function in mammals. These include disruptions in spermatogenesis and oocyte development, oocyte maturation, early embryonic development, foetal and placental growth and lactation. These deleterious effects of heat stress are the result of either the hyperthermia associated with heat stress or the physiological adjustments made by the heat-stressed animal to regulate body temperature. Many effects of elevated temperature on gametes and the early embryo involve increased production of reactive oxygen species. Genetic adaptation to heat stress is possible both with respect to regulation of body temperature and cellular resistance to elevated temperature.     

Role of oxidative stress in female reproduction

In a healthy body, ROS (reactive oxygen species) and antioxidants remain in balance. When the balance is disrupted towards an overabundance of ROS, oxidative stress (OS) occurs. OS influences the entire reproductive lifespan of a woman and even thereafter (i.e. menopause). OS results from an imbalance between prooxidants (free radical species) and the body's scavenging ability (antioxidants). ROS are a double-edged sword – they serve as key signal molecules in physiological processes but also have a role in pathological processes involving the female reproductive tract. ROS affect multiple physiological processes from oocyte maturation to fertilization, embryo development and pregnancy. It has been suggested that OS modulates the age-related decline in fertility. It plays a role during pregnancy and normal parturition and in initiation of preterm labor. Most ovarian cancers appear in the surface epithelium, and repetitive ovulation has been thought to be a causative factor. Ovulation-induced oxidative base damage and damage to DNA of the ovarian epithelium can be prevented by antioxidants. There is growing literature on the effects of OS in female reproduction with involvement in the pathophsiology of preeclampsia, hydatidiform mole, free radical-induced birth defects and other situations such as abortions. Numerous studies have shown that OS plays a role in the pathoysiology of infertility and assisted fertility. There is some evidence of its role in endometriosis, tubal and peritoneal factor infertility and unexplained infertility. This article reviews the role OS plays in normal cycling ovaries, follicular development and cyclical endometrial changes. It also discusses OS-related female infertility and how it influences the outcomes of assisted reproductive techniques. The review comprehensively explores the literature for evidence of the role of oxidative stress in conditions such as abortions, preeclampsia, hydatidiform mole, fetal embryopathies, preterm labour and preeclampsia and gestational diabetes. The review also addresses the growing literature on the role of nitric oxide species in female reproduction. The involvement of nitric oxide species in regulation of endometrial and ovarian function, etiopathogenesis of endometriosis, and maintenance of uterine quiescence, initiation of labour and ripening of cervix at parturition is discussed. Complex interplay between cytokines and oxidative stress in the etiology of female reproductive disorders is discussed. Oxidant status of the cell modulates angiogenesis, which is critical for follicular growth, corpus luteum formation endometrial differentiation and embryonic growth is also highlighted in the review. Strategies to overcome oxidative stress and enhance fertility, both natural and assisted are delineated. Early interventions being investigated for prevention of preeclampsia are enumerated. Trials investigating combination intervention strategy of vitamin E and vitamin C supplementation in preventing preeclampsia are highlighted. Antioxidants are powerful and there are few trials investigating antioxidant supplementation in female reproduction. However, before clinicians recommend antioxidants, randomized controlled trials with sufficient power are necessary to prove the efficacy of antioxidant supplementation in disorders of female reproduction. Serial measurement of oxidative stress biomarkers in longitudinal studies may help delineate the etiology of some of the diosorders in female reproduction such as preeclampsia.     

Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.     

Elevated seasonal temperature disrupts prooxidant-antioxidant homeostasis and promotes cellular apoptosis in the American oyster,Crassostrea virginica,in the Gulf of Mexico: a field study

One of the major impacts of climate change has been the marked rise in global temperature. Recently, we demonstrated that high temperatures (1-week exposure) disrupt prooxidant-antioxidant homeostasis and promote cellular apoptosis in the American oyster. In this study, we evaluated the effects of seasonal sea surface temperature (SST) on tissue morphology, extrapallial fluid (EPF) conditions, heat shock protein-70 (HSP70), dinitrophenyl protein (DNP, an indicator of reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, an indicator of RNS), catalase (CAT), superoxide dismutase (SOD) protein expressions, and cellular apoptosis in gills and digestive glands of oysters collected on the southern Texas coast during the winter (15 °C), spring (24 °C), summer (30 °C), and fall (27 °C). Histological observations of both tissues showed a notable increase in mucus production and an enlargement of the digestive gland lumen with seasonal temperature rise, whereas biochemical analyses exhibited a significant decrease in EPF pH and protein concentration. Immunohistochemical analyses showed higher expression of HSP70 along with the expression of DNP and NTP in oyster tissues during summer. Intriguingly, CAT and SOD protein expressions exhibited significant upregulation with rising seasonal temperatures (15 to 27 °C), which decreased significantly in summer (30 °C), leaving oysters vulnerable to oxidative and nitrative damage. qRT-PCR analysis revealed a significant increase in HSP70 mRNA levels in oyster tissues during the warmer seasons. In situ TUNNEL assay showed a significant increase in apoptotic cells in seasons with high temperature. These results suggest that elevated SST induces oxidative/nitrative stress through the overproduction of ROS/RNS and disrupts the antioxidant system which promotes cellular apoptosis in oysters.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01232-2.