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PFOA is mainly employed in products with water and oil repellent properties. Due to its persistence, bioaccumulation and critical effects on health, its use has been restricted in several countries. This research was intended to explore PFOA action on the main functions of swine ovarian granulosa cells, a valuable model for translational medicine. Moreover, since we previously demonstrated a disruptive effect on free radical generation we sought to explore PFOA effects on the main antioxidant enzymes. PFOA inhibited cell proliferation (p < 0.001), assessed by BrdU uptake. Steroidogenesis was disrupted: PFOA also stimulated 17β-estradiol production (p < 0.05), increased progesterone production (p < 0.05) at the lowest dose while it displayed an inhibitory effect at higher concentrations (p < 0.05). SOD (p < 0.001), catalase (p < 0.05) and peroxidase (p < 0.01) activities were stimulated. Therefore, our study supports a disruptive effect of PFOA in cultured swine granulosa cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Although Glyphosate (GLY) is a widely used pesticide, its effects on ovarian function and stem cell differentiation are still largely unknown. Therefore, as a contribution on this subject, the present work reports an investigation of the in vitro effects of GLY on swine granulosa cells and adipose stromal cells (ASCs). The effect of GLY at different doses (0.2, 4 and 16 μg/mL) was evaluated on granulosa cells growth (BrDU incorporation and ATP production), steroidogenesis (17-β estradiol and progesterone secretion) and redox status (superoxide and nitric oxide production and non-enzymatic scavenging activity). GLY has been shown to inhibit cell growth, 17-β estradiol and non-enzymatic scavenging activity and to increase progesterone and nitric oxide secretion (P < 0.05). In addition, GLY significantly decreased the viability of ASCs (P < 0.001), and inhibited their adipogenic differentiation. These data indicate that GLY alters the main features of granulosa cells and ASCS thus suggesting that GLY could affect both reproductive function and adipose tissues homeostasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Orexins are neuropeptides with pleiotropic functions, involved in the coordination of multiple versatile physiological processes, in particular related to food intake and several aspects of the reproductive process. Their actions are carried out through the bond with the related Orexin 1 (OXR1) and Orexin 2 (OXR2) G-protein-coupled receptors. Studies on the expression of the orexinergic system in the female genital organs are scarce and limited to preovulatory gametogenic follicles and corpora lutea isolated from the rest of the ovary. As the description of only these structures is insufficient to provide a complete picture of the organ, the present study is aimed to give a panoramic view of all the ovarian structures and cells expressing Orexin A (OXA) and its receptors in their original localization. Double labeling immunofluorescent methods, applied on frozen sections of the whole organ in both follicular and luteal phase, were used to highlight the particular distribution and colocalization of the proteins. For a better recognition of cellular morphology and a better distinction between gametogenic (healthy) and atretic follicles, also a single labeling immunolocalization of OXA on formalin fixed paraffin embedded tissues and a TUNEL staining were performed. The results indicate that OXA and its two receptors subtypes are expressed in all the different structures composing the swine ovary, albeit in different ways, in both phases of the ovarian cycle. In general, OXA and OXR2 appear diffusely distributed within "health", proliferating and steroid producing cells, while has granular appearance, being presumably associated to cytoplasmic vesicles, in degenerating cells, independently if apoptotic or not. The immunoreactivity for OXR1, instead, is often associated with the nuclear envelope but it is also detectable, to a lesser extent, diffusely distributed in the cytoplasm of growing or steroid producing cells. When cells undertake the path leading to degeneration, also OXR1 immunoreactivity assumes a granular appearance in the cytoplasm and is colocalized with OXA and OXR2. Different roles for the two receptors in the same cell and a different regulation of their expression remain to be investigated. Their comprehension could help studies of follicle development in pig, as part of in vitro oocyte maturation and fertilization programs in livestock., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Melatonin exerts well-known reproductive effects, mainly acting on hypothalamic gonadotrophin-releasing hormone release. More recent data suggest that melatonin acts directly at the ovarian level, even if, at present, these aspects have been only partly investigated. Swine follicular fluid contains melatonin and its concentration is significantly reduced during follicular growth. Therefore, the present study was undertaken to examine the effects of melatonin, used at physiological concentrations, on cultured swine granulosa cells collected from small (<3mm) and large (>5mm) follicles on the main parameters of granulosa cell function such as proliferation and steroidogenesis, namely oestradiol 17β and progesterone (P4) production. Moreover, the effects of melatonin on superoxide anion and nitric oxide (NO) generation by swine granulosa cells were also investigated. Finally, since angiogenesis is crucial for follicle growth, the effects of melatonin on new vessel growth were studied. Collected data indicate that melatonin interferes with cultured granulosa cell proliferation and steroidogenesis, specifically in terms of P4 production and NO output. In addition, the events of physiological follicular angiogenesis were stimulated by melatonin as evidenced by angiogenesis bioassay. Therefore, we suggest that physiological melatonin concentrations could potentially be involved in local modulation of swine ovarian follicle function.

Simple Summary: Melatonin is a hormone known for regulating the sleep–wake cycle and as an indicator of the photoperiod in seasonally reproducing mammals. Its direct role in ovarian function has been described in the several studies reviewed in the present paper. Melatonin is a hormone mainly produced by the pineal gland in the absence of light stimuli. The light, in fact, hits the retina, which sends a signal to the suprachiasmatic nucleus, which inhibits the synthesis of the hormone by the epiphysis. Mostly by interacting with MT1/MT2 membrane receptors, melatonin performs various physiological actions, among which are its regulation of the sleep–wake cycle and its control of the immune system. One of its best known functions is its non-enzymatic antioxidant action, which is independent from binding with receptors and occurs by electron donation. The hormone is also an indicator of the photoperiod in seasonally reproducing mammals, which are divided into long-day and short-day breeders according to the time of year in which they are sexually active and fertile. It is known that melatonin acts at the hypothalamic–pituitary–gonadal axis level in many species. In particular, it inhibits the hypothalamic release of GnRH, with a consequent alteration of FSH and LH levels. The present paper mainly aims to review the ovarian effect of melatonin. [ABSTRACT FROM AUTHOR]

Simple Summary: Triclosan is widely used in cosmetics and hygiene products, including anti-bacterial hand sanitizers and disinfectants whose utilization has been greatly increased during the COVID-19 pandemic. Since critical health effects have been demonstrated for this substance we tested its potential harmful action on ovarian cells collected from pigs, a valuable animal model. We demonstrated that Triclosan impairs cell function, thus suggesting an impairment of reproductive function. Triclosan is a chlorinated biphenolic with a broad spectrum of antiseptic activities used in cosmetics and hygiene products. Continuous exposure can lead to absorption and bioaccumulation of this substance with harmful health effects. In fact, previous studies have shown that Triclosan acts as an endocrine-disrupting chemical on reproductive organs, with consequent negative effects on reproductive physiology. Therefore, to assess potential adverse impacts on fertility, we tested Triclosan on swine granulosa cells, a model of endocrine reproductive cells. We examined its effects on the main features of granulosa cell functions such as cell growth (BrdU incorporation and ATP production) and steroidogenesis (17-β estradiol and progesterone secretion). Moreover, since oxidant–antioxidant balance plays a pivotal role in follicular function, redox status markers (superoxide, hydrogen peroxide and nitric oxide production, enzymatic and non-enzymatic scavenging activity) were studied. Our results show that Triclosan significantly inhibits cell growth (p < 0.001), steroidogenesis (p < 0.001), superoxide and nitric oxide production (p < 0.001), while it increases (p < 0.05) enzymatic defense systems. Collectively, these data suggest a disruption of the main granulosa cell functions, i.e., proliferation and hormone production, as well as an imbalance in redox status. On these bases, we can speculate that Triclosan would impair granulosa cell functions, thus exerting negative effects on reproductive function. Further studies are needed to explore lower Triclosan concentrations and to unravel its mechanisms of action at gene level. [ABSTRACT FROM AUTHOR]

Nanoplastics (NPs) affect fertility. We evaluated the effects of NPs treatment on luteal and endothelial cells. We examined crucial markers of growth and redox status. NPs treatment did not induce changes in ATP levels in luteal cells, while it increased (p< 0.05) their proliferation. In endothelial cells, no change in proliferation was detected, while an increase (p<0.05) in ATP levels was observed. The increase of reactive oxygen species, superoxide anion (p<0.05) and nitric oxide (p<0.001) was detected in both cell types, which also showed changes in superoxide dismutase enzyme activity as well as an increase of non-enzymatic antioxidant power (p<0.05). A decrease (p<0.05) in progesterone production as well as an increase of vascular endothelial growth factor A levels were detected (p<0.05). In addition, a dose-dependent accumulation of NPs in endothelial cells was shown, that likely occurred through adhesion and internalization. Results underline potential risk of NPs for corpus luteum functionality. • Corpus luteum was studied as a model for reproductive effects of nanoplastics (NPs). • NPs dose-dependently accumulate in corpus luteum endothelial cells. • NPs affected growth and redox status of both cell types. • NPs inhibited progesterone and stimulated VEGF productions. • Corpus luteum function can be impaired by NPs. [ABSTRACT FROM AUTHOR]

Perfluorooctanoic acid (PFOA) is employed in the production and processing of several plastic materials, mainly during the production of waterproof fabrics or nonstick cookware. PFOA is identified as a substance of very high concern, as it is classified as a persistent, bioaccumulative, and toxic (PBT) substance because of its persistence in the environment and its potential accumulation in organisms. Thus, safe levels of exposure cannot be established, and PFOA emissions should be minimized. PFOA has recently been linked to several health concerns in humans. In particular, a disruptive effect on redox status homeostasis has been documented, with a potential impairment of normal reproductive function that requires adequate oxidative balance. Therefore, the aim of the present study was to evaluate the effects of PFOA (2, 20, and 200 ng/mL) on ovarian granulosa cells, a model of reproductive cells. The obtained results reveal that PFOA stimulated cell viability (p < 0.05). Regarding the effects on free radical production, O2−, NO, and H2O2 were significantly inhibited (p < 0.05), while the nonenzymatic antioxidant power was not significantly modified. Collectively, the present results deserve attention since free radical molecules play a crucial role in ovarian follicle development leading to a successful ovulation. [ABSTRACT FROM AUTHOR]

Atrazine is one of the most widely employed herbicides. Due to its environmental persistence, it can be detected in ground and water thus becoming the subject of a serious concern because of its potential endocrine disrupting activity. In particular, several in vitro and in vivo studies point out adverse effects on reproduction. However, these data were mainly collected in the male, while studies on females are lacking. Present work was therefore set up on swine ovarian granulosa cells to investigate the effect of atrazine on steroidogenesis and proliferation. Moreover, since vessel growth is fundamental for reproductive function, we evaluated the herbicide's effect on two of the main angiogenesis signaling molecules, VEGF and NO. Our data show that atrazine markedly interferes with steroidogenesis while it does not modify cell proliferation; in addition, the herbicide has also been found to affect the production of the examined angiogenesis molecules. Collectively, these results indicate for the first time a potential negative effect of atrazine on ovarian functions in the swine species. [Copyright &y& Elsevier]

Abstract: We previously demonstrated the presence of 2-methoxyestradiol (2-ME) in swine follicular fluid. Present study was aimed first of all to investigate if swine granulosa cell produce 2-ME; in addition, we tried to assess a potential effect of hypoxia in modulating 2-ME output. Finally, we explored the effect of 2-ME in an angiogenesis bioassay set up in our lab. Our data show that cultured granulosa cells are able to produce 2-ME; interestingly, the secretion of the hormone appeared to be stimulated by hypoxia. Angiogenesis bioassay points out that 2-ME displays an inhibitory effect on neovascularisation. Therefore our data suggest that 2-ME could be a local effector in determining the fine tuning responsible for follicle angiogenesis. These data deserve special attention since the ovary is a valuable experimental model in angiogenesis research. [Copyright &y& Elsevier]

Abstract: Stanniocalcin 1 (STC1) is a glycoprotein hormone expressed in different mammalian tissues. In previous studies, we showed STC1 expression in swine ovarian follicles and we demonstrated that STC1 may be a physiological regulator of follicular function. Since reactive oxygen species (ROS) are important signal transducers in the ovary, the present study was undertaken to investigate STC1 action on ROS generation and on the activity of the major enzymatic and non-enzymatic scavengers in swine granulosa cells. O2− generation, catalase activity and FRAP levels were increased by STC1, whereas H2O2 generation and peroxidase activity were decreased by STC1. Taken together, our data show that STC1 modulates redox status in swine granulosa cells. [Copyright &y& Elsevier]

Abstract: Sanguinarine (SA) has been used in toothpastes and oral rinse products and has been recently applied to livestock nutrition. This study was undertaken to examine the reproductive-related consequences of this feeding supplementation, evaluating SA effects (10, 100 and 300nM) on swine granulosa cell steroidogenesis, oxidative enzyme activities (peroxidase, catalase and superoxide dismutase), and proliferation. Since angiogenesis is fundamental for follicle development, we also tested the impact of SA exposure on vascular endothelial growth factor (VEGF) production. SA had no effect on proliferation and did not alter progesterone production, although it significantly reduced estradiol synthesis at the two highest concentrations tested (100 and 300nM). SA addition to granulosa cell culture inhibited VEGF production and increased peroxidase and catalase activities at all tested concentrations while superoxide dismutase activity was increased only at 300nM. Our data suggest that SA can negatively affect some key biochemical parameters of ovarian granulosa cells. [Copyright &y& Elsevier]

Follicular growth is characterized by an augmented vascularization, possibly driven by a fall in the oxygen supply. The present study was undertaken to investigate the effects of hypoxia on swine granulosa cells. At first, we quantified oxygen partial pressure (pO2) in follicular fluid from different size follicles; the granulosa cells collected from large follicles (>5 mm) were subjected for 18 h to normoxia (19% O2), partial (5% O2) or total hypoxia (1% O2). The effects of these conditions were tested on the main parameters of granulosa cell function, steroidogenesis and cell proliferation, and on vascular endothelial growth factor (VEGF), nitric oxide (NO) and superoxide anion (O2-) production. Oxygen tension in follicular fluid was negatively related to follicular size, pointing out a gradual reduction during follicular growth. Severe hypoxic conditions determined a reduction of both 17β estradiol and progesterone production, while partial hypoxia did not seem to affect them. Hypoxia increased VEGF as well as O2- production in swine granulosa cells without impairing cell growth; in addition, it decreased NO output.We may conclude that physiological hypoxia could play a pivotal role in the follicular angiogenic process stimulating VEGF synthesis by granulosa cells. ROS are possibly involved in hypoxic signalling. [Copyright &y& Elsevier]

During follicle growth swine granulosa cells are physiologically exposed to a progressive oxygen shortage. It has already been shown that hypoxia stimulates angiogenesis through an increase of VEGF production, however, despite considerable progress in the understanding of the final events induced by cellular hypoxia, the signal transduction pathway remains elusive. Recent evidence suggest a role for Reactive Oxygen Species (ROS) as hypoxia signal transducer. Granulosa cells were isolated from pig follicles (> 5 mm) and cultured for 18 h in normoxic (19% O_2), hypoxic (5% O_2) or anoxic (1% O_2) conditions. Following the incubation ROS (O_2^- and _2O_2) production and the activity of scavenging enzymes (SOD, catalase and peroxidase) were determined. It was apparent from our data that ROS generation was reduced by hypoxia. On the contrary, SOD and peroxidase, but not catalase, increased their activity. Further studies are needed to verify whether ROS are involved in signalling hypoxia. [ABSTRACT FROM AUTHOR]