Inflammation, immune cells, and cellular senescence in the aging ovary.

Ovarian aging results in reduced fertility, disrupted endocrine signaling, and an increased burden of chronic diseases. The factors contributing to the natural decline of ovarian follicles throughout reproductive life are not fully understood. Nevertheless, local inflammation may play an important role in driving ovarian aging. Inflammation progressively rises in aged ovaries during the reproductive window, potentially affecting fertility. In addition to inflammatory markers, recent studies show an accumulation of specific immune cell populations in aging ovaries, particularly lymphocytes. Other hallmarks of the aging ovary include the formation and accumulation of multinucleated giant cells, increased collagen deposition, and increased markers of cellular senescence. Collectively, these changes significantly impact the quantity and quality of ovarian follicles and oocytes. This review explores recent literature on the alterations associated with inflammation, fibrosis, cell senescence, and the accumulation of immune cells in the aging ovary.

Participation of follicular superoxides, inflammatory modulators, and endocrine factors in zebrafish (Danio rerio) ovulation: Cross-talk between PKA and MAPK signaling in Pgr regulation of ovulatory markers.

The ovulatory response involves diverse molecular determinants, the interplay between which remains less investigated in fish. This study explores the temporal changes in the follicular microenvironment, regulatory factors, and underlying signaling events during ovulation in female zebrafish subjected to 14L:10D at 28 ± 1 °C in vivo vis-à-vis in hCG-stimulated full-grown (FG) follicles in vitro. Congruent with reduced GSH levels, SOD, and GPx activity, a graded increase in follicular free radicals, Nox4, and p38 MAPK phosphorylation in the morning hour groups (05:00 and 06:30) correlates positively with the ovulatory surge in inflammatory mediators (Tnf-α, Il-1ß, Il-6, Nos2, and Cox-2). Further, elevated Pgr expression and its nuclear translocation, congruent with follicular lhcgr, star, and hsd20b2 upregulation in vivo, corroborates well with the transcriptional activation of genes (pla2g4aa, ptgesl, ptger4b, mmp9, adamts9), triggering ovulation in this species. Mechanistically, an elevated ovulatory response in hCG-treated FG follicles in vitro involves the upregulation of inflammatory mediators, pgr and ovulation-associated genes in a manner sensitive to PKA- and MAPK3/1-mediated signaling.

Role of insulin-like growth factor 1 (IGF1) in the regulation of mitochondrial bioenergetics in zebrafish oocytes: lessons from in vivo and in vitro investigations.

Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished reproductive potential and impaired fertility. The role of the intra-ovarian IGF system in ovarian follicular dynamics has been implicated earlier. Although several studies have demonstrated the role of the IGF axis in facilitating mitochondrial function over a multitude of cell lines, its role in oocyte energy metabolism remains largely unexplored. Here using zebrafish, the relative importance of IGF1 in modulating oocyte mitochondrial bioenergetics has been investigated. A dramatic increase in ovarian lhcgr and igf1 expression accompanied heightened ATP levels and mitochondrial polarization in full-grown (FG) oocytes resuming meiotic maturation and ovulation in vivo. Concomitant with elevated igf1 expression and IGF1R phosphorylation, hCG (LH analog) stimulation of FG follicles in vitro prompted a sharp increase in NRF-1 and ATP levels, suggesting a positive influence of gonadotropin action on igf1 expression vis-à-vis oocyte bioenergetics. While recombinant IGF1 administration enhanced mitochondrial function, IGF1R immunodepletion or priming with PI3K inhibitor wortmannin could abrogate NRF-1 immunoreactivity, expression of respiratory chain subunits, ΔΨM, and ATP content. Mechanistically, activation of PI3K/Akt signaling in IGF1-treated follicles corroborated well with the rapid phosphorylation of GSK3ß at Ser9 (inactive) followed by PGC-1ß accumulation. While selective inhibition of GSK3ß promoted PGC-1ß, Akt inhibition could abrogate IGF1-induced p-GSK3ß (Ser9) and PGC-1ß immunoreactive protein indicating Akt-mediated GSK3ß inactivation and PGC-1ß stabilization. The IGF1-depleted follicles showed elevated superoxide anions, subdued steroidogenic potential, and attenuated G2-M1 transition. In summary, this study highlights the importance of IGF1 signaling in oocyte bioenergetics prior to resumption of meiosis.

Chronic exposure to nonylphenol induces oxidative stress and liver damage in male zebrafish (Danio rerio): Mechanistic insight into cellular energy sensors, lipid accumulation and immune modulation.

Nonylphenol (NP), an environmentally persistent and toxic endocrine-disrupting chemical with estrogenic properties, has severe implications on humans and wildlife. Accumulating evidence demonstrates the toxic response of NP on the developmental process, nervous system, and reproductive parameters. Although NP exposure has been implicated in chronic liver injury, the underlying events associated with hepatic pathophysiology remain less investigated. Using male zebrafish (Danio rerio) as the model, the present study investigates the impact of environmentally relevant concentrations of NP (50 and 100 µg/L, 21 days) on hepatic redox homeostasis vis-à-vis cellular energy sensors, inflammatory response, and cell death involving a mechanistic insight into estrogen receptor (ER) modulation. Our results demonstrate that congruent with significant alteration in transcript abundance of antioxidant enzymes (SOD1, SOD2, Catalase, GPx1a, GSTα1), chronic exposure to NP promotes ROS synthesis, more specifically superoxide anions and H2O2 levels, and lipid peroxidation potentially through elevated NOX4 expression. Importantly, NP perturbation of markers associated with fatty acid biosynthesis (srebf1/fasn) and cellular energy-sensing network (sirt1/ampkα/pgc1α) indicates dysregulated energy homeostasis, metabolic disruption, and macrovesicular steatosis, albeit with differential sensitivity at the dose level tested. Besides, elevated p38-MAPK phosphorylation (activation) together with loss of ER homeostasis at both mRNA (esr1, esr2a, esr2b) and protein (ERα, ERß) levels suggest that NP modulation of ER abundance may have a significant influence on hepatic events. Elevated expression of inflammatory markers (TLR4, p-NF-κB, TNF-α, IL-6, IL-1ß, and NOS2) and pro-apoptotic and necrotic regulators, e.g., Bax, caspase- 8, -9 and cleaved PARP1 (50 kDa), indicate chronic inflammation and hepatotoxicity in NP-exposed males. Collectively, elevated oxidative stress, metabolic dysregulation and immune modulation may lead to chronic liver injury in organisms exposed to metabolic disrupting chemicals.

Altered redox homeostasis in steroid-depleted follicles attenuates hCG regulation of follicular events: Cross-talk between endocrine and IGF axis in maturing oocytes.

Steroids and insulin-like growth factors (Igfs) are indispensable for folliculogenesis and reproductive fitness in the vertebrate ovary. The intrafollicular redox balance is also of immense importance for ovarian follicles wherein low levels of ROS are being utilized for cell signalling and regulation of gene expression; its excess may interfere with normal physiological processes leading to ovotoxicity. However, the functional relevance of ovarian steroidogenesis in maintaining the follicular microenvironment with coordinated redox homeostasis and intra-ovarian growth factors axis is relatively less understood. Using zebrafish full-grown (FG) ovarian follicles in vitro, our study shows that blocking steroid biosynthesis with anti-steroidal drugs, DL-aminoglutethimide (AG) or Trilostane (Trilo), prevents hCG (LH analogue)-induced StAR expression concomitant with a robust increase in intrafollicular ROS levels. Congruent with heightened intracellular levels of superoxide anions (O2•-) and hydrogen peroxide (H2O2), priming with AG or Trilo abrogates the transcript abundance of major antioxidant enzyme genes (SOD1, SOD2, and CAT) in hCG-stimulated follicles. Significantly, blocking steroidogenesis attenuates transcript abundance of HSP70 but elevates NOX4 expression potentially through ERα-mediated pathway. Importantly, disrupted redox balance in AG/Trilo pre-incubated FG follicles negatively impacts hCG-mediated activation of PKA/CREB signaling and transcriptional activation of igf ligands. Elevated ROS attenuation of antioxidant defense parameters and impaired endocrine and autocrine/paracrine homeostasis converge upon reduced p34cdc2 (Thr-161) phosphorylation, a reliable marker for MPF activation, and resumption of meiotic G2-M1 transition in hCG-treated follicles. Collectively, altered redox homeostasis in steroid-depleted follicles has a significant negative influence on GTH (LH) regulation of follicular events, specifically Igf synthesis, meiotic maturational competence and ovarian fitness.

Bisphenol A impairs reproductive fitness in zebrafish ovary: Potential involvement of oxidative/nitrosative stress, inflammatory and apoptotic mediators.

Bisphenol A (BPA) is a highly pervasive chemical in consumer products with its ascribed endocrine-disrupting properties. Several studies have shown the cytotoxic, genotoxic, and carcinogenic property of BPA over a multitude of tissues. Although BPA exposure has earlier been implicated in female infertility, the underlying molecular mechanisms explaining the toxicity of BPA in the ovary remains less understood. In the present study, a plausible correlation between redox balance or inflammatory signaling and reproductive fitness upon BPA exposure has been examined in zebrafish (Danio rerio) ovary. Congruent with significant alteration of major antioxidant enzymes (SOD1, SOD2, catalase, GPx1α, GSTα1) at the transcript level, 30 d BPA exposure at environmentally relevant concentrations (1, 10 and 100 µg L-1) promotes ovarian ROS/RNS synthesis, lipid peroxidation but attenuates catalase activity indicating elevated stress response. BPA promotes a sharp increase in ovarian p38 MAPK, NF-κB phosphorylation (activation), inducible nitric oxide synthase (Nos2a), and pro-inflammatory cytokines (TNF-α and IL-1ß) expression, the reliable markers for inflammatory response. Congruent to an increased number of atretic follicles, BPA-exposed zebrafish ovary reveals elevated Bax/Bcl2 ratio, activation of caspase-8, -3 and DNA breakdown suggesting heightened cell death. Importantly, significant alteration in nuclear estrogen receptor (ER) transcripts (esr1, esr2a, and esr2b) and proteins (ERα, ERß), gonadotropin receptors, and markers associated with steroidogenesis and growth factor gene expression in BPA-exposed ovary correlates well with impaired ovarian functions and maturational response. Collectively, elevated oxidative/nitrosative stress-mediated inflammatory response and altered ER expression can influence ovarian health and reproductive fitness in organisms exposed to BPA environment.

Bisphenol A-induced oxidative stress, hepatotoxicity and altered estrogen receptor expression in Labeo bata: impact on metabolic homeostasis and inflammatory response.

Bisphenol A (BPA), a weak estrogenic endocrine disruptor and a well-known plasticizer, has the potential to perturb diverse physiological functions; however, its impact on immune and metabolic function in aquatic vertebrates is relatively less understood. The present study aims to investigate the impact of BPA on hepatotoxicity, metabolic and immune parameters vis-à-vis estrogen receptor expression modulation in a freshwater teleost, Labeo bata (Cyprinidae, Cypriniformes). The 96-h median lethal concentration of BPA in L. bata has been determined as 4.79 mg/L. Our data demonstrate that congruent with induction of plasma vitellogenin (VTG), chronic exposure to sub-lethal BPA (2 and 4 µM/L) attenuates erythrocyte count, hemoglobin concentration, packed cell volume, mean corpuscular hemoglobin, but not leukocyte number. Further, a significant increase in MDA, concomitant with diminished catalase and heightened GST activity corroborates well with hepatic dystrophic changes, appearance of fatty liver (macrovesicular steatosis) and elevated serum lipids (triglyceride, cholesterol, LDL, VLDL) in BPA-treated groups. Interestingly, a differential regulation of estrogen receptor (ER) subtypes at transcript and protein level signifies negative influence of BPA on hepatic ERα/ERß homeostasis in this species. While at a lower dose it promotes Akt phosphorylation (activation), BPA at the higher dose attenuates ERK1/2 phosphorylation (activation), suggesting potential alteration in insulin sensitivity. Importantly, dose-dependent decrease in hepatic TNF-α, IL-1ß, iNOS (NOS2) expression and nitric oxide (NO) level corresponds well with progressive decline in p-NF-κB, p-p38 MAPK, albeit with differential sensitivity, in BPA-exposed groups. Collectively, BPA exposure has wide-spread negative influence on hematological, biochemical and hepatic events in this species.

Expression of nitric oxide synthase (NOS) in Anabas testudineus ovary and participation of nitric oxide-cyclic GMP cascade in maintenance of meiotic arrest.

Participation of cyclic nucleotide-mediated signaling in nitric oxide/soluble guanylate cyclase (NO/sGC) regulation of oocyte maturation (OM) in perch (Anabas testudineus) follicle-enclosed oocytes has been investigated. Congruent with sharp decline in follicular cyclic GMP (cGMP) level, nitric oxide synthase (NOS)-inhibitor (L-NAME) attenuates protein kinase A (PKA) phosphorylation but promotes p-ERK1/2 and p-p34Cdc2 (Thr-161) in maturing oocytes. Conversely, NO donor (SNP) prevents OM, potentially through elevated cGMP synthesis. Expression and localization of Nos2 and Nos3 immunoreactivity in perch ovary varied considerably at progressively higher stages of folliculogenesis. While sGC inhibitor (ODQ) alone could induce OM, 8-bromo-cGMP attenuates 17,20ß-P-induced OM indicating functional significance of NO/sGC/cGMP in perch ovary. Interestingly, high NO/cGMP inhibition of OM shows positive relation with elevated cAMP level. MIS induced OM is more susceptible to the oocyte-specific phosphodiesterase (PDE) 3 than PDE4 inhibition. Collectively, high NO/cGMP attenuation of OM potentially involves PDE3 inhibition, cAMP accumulation and PKA activation.

Nonylphenol attenuates SOCS3 expression and M1 polarization in lipopolysaccharide-treated rat splenic macrophages.

Endocrine disruptors interfere with normal sexual and reproductive development of numerous organisms. Widely used in several chemical and manufacturing industries, nonylphenol (NP), a potent xenoestrogen, has the potential to perturb immune system. Using rat splenic macrophages (SMΦ) as the model system, NP-modulation of lipopolysaccharide (LPS)-induced inflammatory response has been investigated. Our results demonstrate that NP (0.1-10 µM) attenuates catalase activity, reactive oxygen species (ROS) generation and nitric oxide (NO) synthesis in LPS-treated SMΦ in vitro. NP inhibition of LPS-induced nuclear factor kappa B (NF-κB) activation and pro-inflammatory cytokine gene expression corroborate well with attenuation of suppressor of cytokine signalling 3 (SOCS3). Besides, elevated expression of anti-inflammatory factors reveals inverse correlation with suppression of endotoxin-induced M1 polarization in NP pre-incubated cells. While LPS promotes, NP prevents ERK1/2 (extracellular-signa1-regulated kinase 1/2) phosphorylation and MEK-inhibitor abrogates SOCS3 expression and NO production suggesting involvement of ERK1/2 in NP inhibition of SOCS3 expression. Further, translational inhibitor cycloheximide prevents LPS-induced NF-κB activation indicating functional importance of de novo synthesis of SOCS3, at least in part, in toll-like receptor 4 (TLR4)-mediated inflammatory response. Collectively, present study provides evidence favouring participation of SOCS3 in NP modulation of inflammatory response in rat SMΦ.