RESUMEN
Most of follicles undergo a degenerative process called follicular atresia. This process directly affects the egg production of laying hens and is regulated by external and internal factors. External factors primarily include nutrition and environmental factors. In follicular atresia, internal factors are predominantly regulated at 3 levels; organic, cellular and molecular levels. At the organic level, the hypothalamic-pituitary-ovary (HPO) axis plays an essential role in controlling follicular development. At the cellular level, gonadotropins and cytokines, as well as estrogens, bind to their receptors and activate different signaling pathways, thereby suppressing follicular atresia. By contrast, oxidative stress induces follicular atresia by increasing ROS levels. At the molecular level, granulosa cell (GC) apoptosis is not the only factor triggering follicular atresia. Autophagy is also known to give rise to atresia. Epigenetics also plays a pivotal role in regulating gene expression in processes that seem to be related to follicular atresia, such as apoptosis, autophagy, proliferation, and steroidogenesis. Among these processes, the miRNA regulation mechanism is well-studied. The current review focuses on factors that regulate follicular atresia at organic, cellular and molecular levels and evaluates the interaction network among these levels. Additionally, this review summarizes atretic follicle characteristics, in vitro modeling methods, and factors preventing follicular atresia in laying hens.
Asunto(s)
Pollos , Atresia Folicular , Animales , Atresia Folicular/fisiología , Femenino , Pollos/genética , Pollos/fisiología , Folículo Ovárico/fisiología , Enfermedades de las Aves de Corral/genéticaRESUMEN
The proliferation and death of granulosa cells (GCs) in poultry play a decisive role in follicular fate and egg production. The follicular fluid (FF) contains a variety of nutrients and genetic substances to ensure the communication between follicular cells. Exosomes, as a new intercellular communication, could carry and transport the proteins, RNA, and lipids to react on GCs, which had been found in FF of various domestic animals. Whether exosomes of FF in poultry play a similar role is unclear. In this study, geese, a poultry with low egg production, were chosen, and the effect of FF exosomes on the proliferation and death of GCs was investigated. Firstly, there were not only a large number of healthy small yellow follicles (HSYFs) but also some atresia small yellow follicles (ASYFs) in the egg-laying stage. Also, the GC layers of ASYFs became loose interconnections, inward detachment, and diminished survival rate than that of HSYFs. Besides, compared to HSYFs, the contents of E2, P4, and the mRNA expression levels of ferroptosis-related genes GPX4, FPN1, and FTH1 were significantly decreased, while COX2, NCOA4, VDAC3 mRNA were significantly increased, and the structure of mitochondrial cristae disappeared and the outer membrane broke in the GC layers of ASYFs. Moreover, the ROS, MDA, and oxidation levels in the GC layers of ASYFs were significantly higher than those of HSYFs. All these hinted that ferroptosis might result in a large number of GCs death and involvement in follicle atresia. Secondly, FF exosomes were isolated from HSYFs and ASYFs, respectively, and identified by TEM, NTA, and detection of exosome marker proteins. Also, we found the exosomes were phagocytic by GCs by tracking CM-Dil. Moreover, the addition of ASYF-FF exosomes significantly elevated the MDA content, Fe2+ levels, and the mitochondrial membrane potential (MMP) in GCs, thus significantly inhibiting the proliferation of GCs, which was restored by the ferroptosis inhibitor ferrostatin-1. Thirdly, the proteomic sequencing was performed between FF-derived exosomes of HSYFs and ASYFs. We obtained 1615 differentially expressed proteins, which were mainly enriched in the protein transport and ferroptosis pathways. Among them, HMOX1 was enriched in the ferroptosis pathway based on differential protein-protein interaction network analysis. Finally, the role of HMOX1 in regulating ferroptosis in GCs was further explored. The highly expressed HMOX1 was observed in the exosomes of ASYF-FF than that in HSYF-FF. Overexpression of HMOX1 increased ATG5, LC3II, and NCOA4 expression and reduced the expression of FTH1, GPX4, PCBP2, FPN1 in the ferroptosis pathway, also promoted intracellular Fe2+ accumulation and MDA surge, which drove ferroptosis in GCs. The effects of HMOX1 on ferroptosis could be blocked by its inhibitor Znpp. Taken together, the important protein HMOX1 was identified in FF, which could be delivered to GCs via exosomes, triggering ferroptosis and thus determining the fate of follicles.
Asunto(s)
Exosomas , Ferroptosis , Atresia Folicular , Líquido Folicular , Gansos , Células de la Granulosa , Hemo-Oxigenasa 1 , Animales , Ferroptosis/fisiología , Femenino , Exosomas/metabolismo , Células de la Granulosa/fisiología , Células de la Granulosa/metabolismo , Atresia Folicular/fisiología , Líquido Folicular/metabolismo , Gansos/fisiología , Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/genética , Proteínas Aviares/metabolismo , Proteínas Aviares/genéticaRESUMEN
In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.
Asunto(s)
Autofagia , Atresia Folicular , Células de la Granulosa , Femenino , Animales , Atresia Folicular/fisiología , Ovinos/fisiología , Autofagia/fisiología , Células de la Granulosa/fisiología , Ovario , Folículo Ovárico/fisiología , ApoptosisRESUMEN
BACKGROUND: The mammalian ovary is a unique organ that displays a distinctive feature of cyclic changes throughout the entire reproductive period. The estrous/menstrual cycles are associated with drastic functional and morphological rearrangements of ovarian tissue, including follicular development and degeneration, and the formation and subsequent atrophy of the corpus luteum. The flawless execution of these reiterative processes is impossible without the involvement of programmed cell death (PCD). MAIN TEXT: PCD is crucial for efficient and careful clearance of excessive, depleted, or obsolete ovarian structures for ovarian cycling. Moreover, PCD facilitates selection of high-quality oocytes and formation of the ovarian reserve during embryonic and juvenile development. Disruption of PCD regulation can heavily impact the ovarian functions and is associated with various pathologies, from a moderate decrease in fertility to severe hormonal disturbance, complete loss of reproductive function, and tumorigenesis. This comprehensive review aims to provide updated information on the role of PCD in various processes occurring in normal and pathologic ovaries. Three major events of PCD in the ovary-progenitor germ cell depletion, follicular atresia, and corpus luteum degradation-are described, alongside the detailed information on molecular regulation of these processes, highlighting the contribution of apoptosis, autophagy, necroptosis, and ferroptosis. Ultimately, the current knowledge of PCD aberrations associated with pathologies, such as polycystic ovarian syndrome, premature ovarian insufficiency, and tumors of ovarian origin, is outlined. CONCLUSION: PCD is an essential element in ovarian development, functions and pathologies. A thorough understanding of molecular mechanisms regulating PCD events is required for future advances in the diagnosis and management of various disorders of the ovary and the female reproductive system in general.
Asunto(s)
Atresia Folicular , Ovario , Animales , Femenino , Humanos , Ovario/fisiología , Atresia Folicular/fisiología , Apoptosis/genética , Muerte Celular/fisiología , Oocitos/metabolismo , MamíferosRESUMEN
Follicle-stimulating hormone (FSH), luteinizing hormone (LH), miR-23a, apoptosis signal-regulating kinase 1(ASK1)/c-Jun N-terminal kinase (JNK), autophagy and apoptosis play crucial roles in follicular development. However, their role in yak granulosa cells (GCs) remains unknown. Therefore, we examined the effect of miR-23a, ASK1, FSH, and LH on apoptosis, autophagy, and the release and reception of some steroid hormones in these cells. Our results showed that miR-23a overexpression significantly increased the abundance of Beclin1, the LC3II/I ratio, and the number of Ad-mRFP-GFP-LC3-labeled autophagosomes, and decreased p62 abundance. Additionally, Bax abundance and the number of terminal deoxynucleotidyl transferase deoxynucleotide triphosphate nick end labeling-positive cells were reduced, while Bcl2 expression was increased. Overexpression of miR-23a also significantly increased the abundance of estradiol receptor α (ER-α) and ß (ER-ß) and the concentrations of estradiol (E2), progesterone (P4) in yak GCs. Here, treating yak GCs with miR-23a decreased ASK1 expression, which regulates ASK1/JNK-mediated apoptosis, autophagy, E2 and P4 levels, and ER-α/ß abundance. In contrast, treatment of yak GCs with FSH (10 µg/mL) and LH (100 µg/mL) increased miR-23a abundance, regulating the subsequent effect on ASK1/JNK-mediated apoptosis, autophagy, ER-α/ß abundance, and E2 and P4 concentrations. In conclusion, miR-23a enhances autophagy in yak GCs, attenuates apoptosis, and increases ER-α/ß abundance and E2 and P4 concentrations by downregulating ASK1. Additionally, FSH and LH can regulate these effects of miR-23a by altering its expression. These results provide important insights that can inform the development of strategies to reduce abnormal follicular atresia and improve the reproductive rate of yaks.
Asunto(s)
Hormona Luteinizante , MicroARNs , Animales , Bovinos , Femenino , Apoptosis , Autofagia , Estradiol/metabolismo , Hormona Folículo Estimulante/farmacología , Atresia Folicular/fisiología , Células de la Granulosa/metabolismo , Hormona Luteinizante/farmacología , Hormona Luteinizante/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Progesterona/metabolismoRESUMEN
In brief: Genistein contributes to granulosa cell (GC) survival by two routes: one is that genistein induced p-AMPK and inhibited p-mTOR, which induces LC3 activation and autophagy; the other is that genistein inhibited caspase-3 and its cleavage, which induces PARP1 activation and PARylation. Abstract: Genistein is an isoflavone which is beneficial for health, but little is known regarding its function on granulosa cell fate during follicular atresia. In the present study, we established an in vitro model of porcine follicular granulosa cell apoptosis by serum deprivation and showed that treatments with 1 µM and 10 µM genistein significantly reduced the apoptotic rate of granulosa cells compared to the blank control (P < 0.05). These results suggest that genistein at micromolar levels alleviates serum deprivation-induced granulosa cell apoptosis, and the ameliorative effect of genistein on granulosa cell apoptosis is likely to be able to inhibit nutrient depletion-induced follicular atresia. Further experimental results revealed that the expression of the autophagic marker protein LC3II in 100 nM-10 µM genistein treatment increased in a dose-dependent manner and was higher than the control (P < 0.05). Genistein also dose dependently promoted the phosphorylation of AMPK (adenosine 5'-monophosphate-activated protein kinase) in granulosa cells. Poly(ADP-ribose) (pADPr) formation in genistein-treated groups was also notably higher than in the controls (P < 0.05). Collectively, genistein alleviates serum deprivation-induced granulosa cells in vitro through enhancing autophagy, which involving AMPK activation and PARylation signaling. However, further study should be carried out to investigate the role of the aforementioned signaling on this process.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Genisteína , Femenino , Animales , Porcinos , Genisteína/farmacología , Genisteína/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Atresia Folicular/fisiología , Células de la Granulosa/metabolismo , ApoptosisRESUMEN
The majority of activated ovarian follicles undergo atresia during reproductive life in mammals, and only a small number of follicles are ovulated. Though hormone treatment has been widely used to promote folliculogenesis, the molecular mechanism behind follicle selection and atresia remains under debate due to inconsistency among investigation models. Using a high-throughput molecular pathology strategy, we depicted a transcriptional atlas of mouse follicular granulosa cells (GCs) under physiological condition and obtained molecular signatures in healthy and atresia GCs during development. Functional results revealed hypoxia-inducible factor 1 (HIF1) as a major effector downstream of follicle-stimulating hormone (FSH), and HIF1 activation is essential for follicle growth. Energy shortage leads to prevalent AMP-activated protein kinase (AMPK) activation and drives follicular atresia. FSHR-mTOR-HIF1 signaling helps follicles escape from the atresia fate, while energy stress persists. Our work provides a comprehensive understanding of the molecular network behind follicle selection and atresia under physiological condition.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Células de la Granulosa , Animales , Femenino , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Atresia Folicular/fisiología , Células de la Granulosa/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Mamíferos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Ovarian aging is the main reason of female reproductive problems. Excessive oxidative stress can induce ovarian senescence and follicular atresia, thereby reducing the reproductive performance. Follicles were divided into five groups for in vitro culture based on the duration of stimulation with tert-butyl hydroperoxide (t-BHP)-control group and groups 1 h, 2 h, 6 h, and 12 h. The results revealed that the ratio of progesterone (P4) to estradiol (E2) was increased after 24 and 36 h of follicle culture, shifting follicles toward atresia (P < 0.05). Stimulated by 200 µM t-BHP, follicles showed progressive aging phenotype. Senescence-associated ß-galactosidase staining (SA-ß-Gal) showed a significant increase in the number of positive cells (P < 0.05). Reactive oxygen species were also significantly upregulated (P < 0.05). t-BHP treatment for 6 h induced significant increases in Caspase 3, P53, and Foxo1 mRNA and protein levels (P < 0.05) and significant decreases in SOD mRNA and protein levels (P < 0.05). Transcriptome sequencing analysis of the follicles showed that the aged and treatment groups were clustered together in hierarchical clustering. Correlation analysis indicated significant changes at the transcriptome level in the treatment groups versus the control group. The common differentially expressed genes in the treatment groups were enriched in three growth-factor signaling pathways associated with cell proliferation and apoptosis (P53, mTOR, and MAPK). In conclusion, induction of follicular senescence by treatment with 200 µM t-BHP for 6 h is an effective in vitro model to simulate ovarian senescence in sows.
Asunto(s)
Atresia Folicular , Proteína p53 Supresora de Tumor , Femenino , Animales , Porcinos , terc-Butilhidroperóxido/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Atresia Folicular/fisiología , Folículo Ovárico/metabolismo , ARN Mensajero/metabolismoRESUMEN
Although silica nanoparticles (SNPs) are generally thought to be biocompatible and safe, the adverse effects of SNPs were also reported in previous studies. SNPs cause follicular atresia via the induction of ovarian granulosa cell apoptosis. However, the mechanisms for this phenomenon are not well understood. This study focuses on exploring the relationship between autophagy and apoptosis induced by SNPs in ovarian granulosa cells. Our results showed that 25.0 mg/kg body weight (b.w.)/intratracheal instillation of 110 nm in diameter spherical Stöber SNPs caused ovarian granulosa cell apoptosis in follicles in vivo. We also found that SNPs mainly internalized into the lumens of the lysosomes in primary cultured ovarian granulosa cells in vitro. SNPs induced cytotoxicity via a decrease in viability and an increase in apoptosis in a dose-dependent manner. SNPs increased BECLIN-1 and LC3-II levels, leading to the activation of autophagy and increased P62 level, resulting in the blockage of autophagic flux. SNPs increased the BAX/BCL-2 ratio and cleaved the caspase-3 level, resulting in the activation of the mitochondrial-mediated caspase-dependent apoptotic signaling pathway. SNPs enlarged the LysoTracker Red-positive compartments, decreased the CTSD level, and increased the acidity of lysosomes, leading to lysosomal impairment. Our results reveal that SNPs cause autophagy dysfunction via lysosomal impairment, resulting in follicular atresia via the enhancement of apoptosis in ovarian granulosa cells.
Asunto(s)
Atresia Folicular , Nanopartículas , Femenino , Humanos , Atresia Folicular/fisiología , Células de la Granulosa/metabolismo , Apoptosis , Autofagia/fisiologíaRESUMEN
Amphetamine derivatives negatively impact serotonin (5-HT) production, which triggers apoptosis in different tissues, depending on the receptor they bind. 5-HT in the ovary stimulates estradiol secretion, a survival factor of granulosa cells. The effect of amphetamine derivatives on the serotonergic system of the ovary and follicular development is unknown. Therefore, in this study, we investigated the effects of p-chloroamphetamine (pCA), derived from amphetamines, on estradiol production, follicular development, apoptosis of granulosa cells, and serotonin 5-HT7 receptor (R5-HT7) expression. Female rats (30 days old) were injected with 10 mg/kg of pCA intraperitoneally and were euthanized 48 or 120 h after treatment. The concentration of 5-HT in the hypothalamus decreased at 48 and 120 h after treatment and in the ovary at 120 h. The serum concentration of estradiol decreased at all times studied. Follicular atresia, TUNEL-positive (apoptotic) granulosa cells and Bax expression were elevated by pCA, but none of these effects was associated with R5-HT7 expression. These results suggest that pCA induces the dysregulation of the serotonergic system in the hypothalamus and the ovary, negatively impacting estradiol production and follicular development.
Asunto(s)
Atresia Folicular , Serotonina , Anfetamina , Animales , Apoptosis , Estradiol/metabolismo , Femenino , Atresia Folicular/fisiología , Células de la Granulosa/metabolismo , Ratas , p-Cloroanfetamina/farmacologíaRESUMEN
Epidemiological investigations and animal studies demonstrate a significantly positive relationship between polycyclic aromatic hydrocarbons (PAHs) exposure and reproductive disorders. However, few researches are focused on the reproductive toxicity of low-molecular-weight PAHs (number of benzene ring ≤ 3) which occupy a large part of PAHs. Phenanthrene (Phe), a typical low-molecular-weight PAH, is one of the most abundant PAHs detected in foods. In the present study, oral treatment with Phe at a human exposure related level during gestation (60 µg/kg body weight every three days, six times in total) induced reproductive disorders in F1 adult female mice: the number of antral follicles (an immature stage of follicular development) were significantly increased, while the maturation of oocytes was inhibited and aggravated follicular atresia was observed; the serum levels of luteinizing hormone (LH), testosterone and estradiol were significantly reduced; the receptor of follicle-stimulating hormone (FSHR) and aromatase in the ovary were significantly upregulated; transcriptome analysis demonstrated that the phosphatidylinositol 3-kinase and protein kinase B (PI3K/Akt) signal pathway was upregulated, and the calcium signal pathway was disturbed, which probably accounts for the exacerbated atresia of the growing follicles and the excessive consumption of follicles. The reproductive toxicity of low-molecular-weight PAHs could not be neglected.
Asunto(s)
Atresia Folicular , Fenantrenos , Animales , Estradiol/metabolismo , Femenino , Hormona Folículo Estimulante/metabolismo , Atresia Folicular/fisiología , Hormona Luteinizante/metabolismo , Ratones , Folículo Ovárico , Fenantrenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Ovarian granulosa cells (GC) play an essential role in the development and atresia of follicles. Emerging studies suggest that non-coding RNAs are involved in the regulation of GC apoptosis. Here, we aimed to analyze the function of ssc-circINHA-001, coded by the first exon of the inhibin subunit α gene (INHA), in resisting GC apoptosis and follicular atresia by enhancing the expression of the inhibin subunit ß A (INHBA) through a cluster of miRNAs. A higher expression of ssc-circINHA-001 in healthy follicles compared to early atretic follicles was detected by qRT-PCR. Its circular structure was confirmed by RNase R treatment and reversed PCR. The function of ssc-circINHA-001 in GC resistance to apoptosis was detected by in vitro transfection of its si-RNA. Furthermore, the dual-luciferase reporter assay suggested that ssc-circINHA-001 adsorbed three miRNAs, termed miR-214-5p, miR-7144-3p, and miR-9830-5p, which share the common target INHBA. A low expression of ssc-circINHA-001 increased the levels of the free miRNAs, inhibited INHBA expression, and thus raised GCs apoptosis through a shift from the secretion of activin to that of inhibin. Our study demonstrated the existence of a circRNA-microRNAs-INHBA regulatory axis in follicular GC apoptosis and provides insight into the relationship between circRNA function and its coding gene in inhibin/activin balance and ovarian physiological functions.
Asunto(s)
Activinas/genética , Apoptosis , Atresia Folicular/metabolismo , Células de la Granulosa/metabolismo , Inhibinas/genética , MicroARNs/genética , ARN Circular/metabolismo , Animales , Femenino , Atresia Folicular/fisiología , Regulación de la Expresión Génica , Células de la Granulosa/fisiología , Inhibinas/metabolismo , MicroARNs/metabolismo , Ovario/metabolismo , Ovario/fisiología , Sus scrofa/genética , Sus scrofa/metabolismo , Sus scrofa/fisiologíaRESUMEN
Increased follicular atresia occurs with aging and results in reduced fecundity in laying chickens. Therefore, relieving follicular atresia of aging poultry is a crucial measure to maintain sustained high laying performance. As an antiaging agent, metformin was reported to play important roles in preventing aging in diverse animals. In this study, the physiological state of the prehierarchical follicles in the peak-laying hens (D280) and aged hens (D580) was compared, followed with exploration for the possible capacity of metformin in delaying atresia of the prehierarchical follicles in the aged D580 hens. Results showed that the capacity of yolk deposition within follicles declined with aging, and the point of endoplasmic reticulum- (ER-) mitochondrion contact decreased in the ultrastructure of the follicular cells. Meanwhile, the expression of apoptosis signaling genes was increased in the atretic small white follicles. Subsequently, the H2O2-induced follicular atresia model was established to evaluate the enhancing capacity of metformin on yolk deposition and inhibition of apoptosis in the atretic small white follicles. Metformin inhibited apoptosis through regulating cooperation of the mitochondrion-associated ER membranes and the insulin (PI3K/AKT) signaling pathway. Furthermore, metformin regulated calcium ion homeostasis to relieve ER-stress and inhibited release of mitochondrion apoptosis factors (BAD and caspase). Additionally, metformin activated PI3K/AKT that suppressed activation of BAD (downstream of the insulin signaling pathway) in the atretic follicles. Further, serum estrogen level and liver estrogen receptor-α expression were increased after dietary metformin supplementation in D580 hens. These results indicated that administration of dietary metformin activated the PI3K/AKT and calcium signaling pathway and enhanced yolk deposition to prevent chicken follicular atresia.
Asunto(s)
Envejecimiento/fisiología , Señalización del Calcio/efectos de los fármacos , Atresia Folicular/efectos de los fármacos , Metformina/farmacología , Animales , Caspasas/metabolismo , Pollos/metabolismo , Femenino , Atresia Folicular/fisiología , Células de la Granulosa/metabolismo , Peróxido de Hidrógeno/metabolismo , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The chemokine receptor 2 (CCR2) was first described as a chemotactic factor involved in immune responses, but it also plays an essential function in several biological processes. The chemokine (C-C motif) ligand 2 (CCL2) binds to CCR2 triggering G protein-coupled receptor (GPCR) signaling in leukocytes, including activation of PI3K/Akt/mTOR, a key pathway that is also related to follicular activation and survival. However, the potential role of CCR2 in ovarian follicular physiology remain unexplored. Thus, we investigated the role of CCR2 on follicular growth during adult life and aging. Ovaries and oocytes were collected from wild type (WT) mice at 1.5 months old (mo), and CCR2 expression was observed predominantly in oocytes included in growing follicles, as well as after ovulation. Follicle populations were assessed in WT and CCR2-/- mice at 1.5 mo, and CCR2-/- mice had more primordial and less primary and secondary follicles, while there were no differences in antral follicle numbers. Pro-apoptotic genes Bax and Casp3 were downregulated, while anti-apoptotic Bcl2 was upregulated in CCR2-/- mice. To further characterize the role of CCR2 in ovarian aging, follicle populations were assessed in WT and CCR2-/- mice at 1.5, 2.5, 6, 10, and 12 mo. A larger ovarian follicular reserve at 1.5-6 mo was observed in CCR2-/- mice. Finally, CCR2-/- aged mice (6-12 mo) ovulated more oocytes than WT mice. Altogether, these data suggest that CCR2 plays an important role in the regulation of murine folliculogenesis, potentially affecting the reproductive lifespan.
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Fertilidad/fisiología , Atresia Folicular/fisiología , Oogénesis/fisiología , Folículo Ovárico/crecimiento & desarrollo , Receptores CCR2/deficiencia , Animales , Apoptosis/fisiología , Caspasa 3/metabolismo , Femenino , Ratones , Ratones Noqueados , Modelos Animales , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores CCR2/genética , Factores de TiempoRESUMEN
The present study describes in detail the morphological characteristics of the process of ovarian follicular atresia in Redbelly tilapia (Coptodon zillii) during the nonbreeding season using light and electron microscopy and immunohistochemistry. The follicular regression process was initiated with shrinkage and disintegration of the nuclear membrane of oocytes resulting in dispersing of chromatin within the ooplasm, followed by marked hyperplasia and hypertrophy of follicular and granulosa cells, which exhibited a strong phagocytic activity to engulf the liquefied yolk particles. Rodlet cells and granulocytes were recorded on the follicular wall and invaded the regressed follicles. Rodlet cells expressed a strong immunoreactivity to matrix metalloperoxidase (MMP-9) and α-smooth muscle actin, while neutrophils expressed a strong reactivity to Myeloperoxidase-3 (MPO). In the advanced stage of follicular atresia, the yolk was almost phagocytized and resorbed and the regressed follicle lost its integrity and appeared to be formed of a cellular mass of phagocytic cells. Transmission electron microscopy revealed the presence of neutrophils, eosinophils, and dendritic cells within the atretic follicle in between these phagocytic cells. Moreover, numerous lysosomes, granules, and phagosomes were observed within the cytoplasm of both phagocytic cells and granulocytes. Telocytes were also demonstrated within the highly thickened richly vascularized theca layer during the late stages of follicular atresia. Immunohistochemical staining for caspase-3 established the participation of apoptosis in the advanced stages of follicular regression. Immune cells, rodlet cells, and telocytes in combination with follicular cells play an essential role in follicular atresia. In conclusion, the present study provides a new evidence on the role of both somatic and immune cells in the phenomenon of ovarian follicular atresia in Redbelly tilapia (Coptodon zillii) during the nonbreeding season.
Asunto(s)
Peces/anatomía & histología , Atresia Folicular/fisiología , Folículo Ovárico/citología , Ovario/citología , Animales , Apoptosis , Caspasa 3 , Células Dendríticas/citología , Femenino , Células de la Granulosa , Inmunohistoquímica , Metaloproteinasa 9 de la Matriz/metabolismo , Neutrófilos , Oocitos , Ovario/metabolismo , Peroxidasa/metabolismo , Telocitos/citologíaRESUMEN
The goldfish is a model organism showing great potential for research, particularly in comparative endocrinology concerning the neuroendocrine signalling and regulation of vertebrate reproduction. Furthermore, this teleost is increasingly stressed as a relevant alternative to more common fish model organisms, namely zebrafish. However, quality descriptions and illustrations of the complete goldfish gonadal histology are surprisingly scarce, but needed, to support research using this fish. Therefore, the main aim of this work is to describe in detail and adequately illustrate the goldfish oogenesis, from oogonia to late maturation, by applying routine stains (haematoxylin-eosin) and special procedures (periodic acid-Schiff and Goldner's trichrome). We hypothesized that the combined strategies would enable not only to observe the most general features but also to perceive some poorly described details of oocytes better. We describe the details of the following maturation stages: oogonia proliferation, chromatin-nucleolus, primary growth (one nucleolus step, multiple nucleoli step, perinucleolar step, cortical alveoli step) and secondary growth (early secondary growth step, late secondary growth step). Additionally, we report aspects of early and late follicular atresia. The study allowed comparisons with other species and showed that the Goldner's trichrome has the best discriminative power and should be the preferred stain, despite more time-consuming.
Asunto(s)
Carpa Dorada/anatomía & histología , Folículo Ovárico/crecimiento & desarrollo , Animales , Nucléolo Celular/fisiología , Cromatina/fisiología , Femenino , Atresia Folicular/fisiología , Carpa Dorada/crecimiento & desarrollo , Carpa Dorada/fisiología , Modelos Animales , Oocitos/crecimiento & desarrollo , Oocitos/fisiología , Oogénesis/fisiología , Oogonios/crecimiento & desarrollo , Oogonios/fisiología , Folículo Ovárico/fisiología , Coloración y EtiquetadoRESUMEN
The greater amberjack Seriola dumerili is a new aquaculture fish that may display reproductive dysfunctions. During extensive follicular atresia, which is a common reproductive dysfunction in females during vitellogenesis, part of the reabsorbed yolk returns to the liver to be metabolized and recycled. Melanomacrophage centres (MMCs) are aggregates of macrophage-like cells that play a role in the destruction, detoxification and recycling of endogenous and exogenous materials, and have been associated with systemic stress. Wild and captive-reared greater amberjack were sampled in the Mediterranean Sea during two different phases of the reproductive cycle. The liver of reproductively dysfunctional captive-reared females sampled during the spawning season showed a high density of both MMCs and apoptotic cells. A weak liver anti-cytochrome P450 monooxygenase 1A immunoreactivity was observed, suggesting that the examined fish were not exposed to environmental pollutants. We propose that the observed increase in MMCs and apoptosis in captive-reared fish was related to the hepatic overload associated to the metabolism of yolk proteins reabsorbed during extensive follicular atresia. Since follicular atresia is a frequent physiological and pathological event in teleosts, we suggest that the reproductive state should be always assessed when MMCs are used as markers of exposure to stress or pollutants.
Asunto(s)
Enfermedades de los Peces/patología , Peces , Atresia Folicular/fisiología , Hepatopatías/veterinaria , Macrófagos/patología , Animales , Acuicultura , Biomarcadores/análisis , Italia , Hepatopatías/patología , Estrés FisiológicoRESUMEN
Follicular atresia is the mechanism by which the oocyte contents are degraded during oogenesis in response to stress conditions, allowing the energetic resources stored in the developing oocytes to be reallocated to optimize female fitness. Autophagy is a conserved intracellular degradation pathway where double-membrane vesicles are formed around target organelles leading to their degradation after lysosome fusion. The autophagy-related protein 8 (ATG8) is conjugated to the autophagic membrane and has a key role in the elongation and closure of the autophagosome. Here we identified one single isoform of ATG8 in the genome of the insect vector of Chagas Disease Rhodnius prolixus (RpATG8) and found that it is highly expressed in the ovary during vitellogenesis. Accordingly, autophagosomes were detected in the vitellogenic oocytes, as seen by immunoblotting and electron microscopy. To test if autophagosomes were important for follicular atresia, we silenced RpATG8 and elicited atresia in vitellogenic females by Zymosan-A injections. We found that silenced females were still able to trigger the same levels of follicle atresia, and that their atretic oocytes presented a characteristic morphology, with accumulated brown aggregates. Regardless of the difference in morphology, RpATG8-silenced atretic oocytes presented the same levels of protein, TAG and PolyP, as detected in control atretic oocytes, as well as the same levels of acidification of the yolk organelles. Because follicular atresia has the ultimate goal of restoring female fitness, we tested if RpATG8-silenced atresia would result in female physiology and behavior changes. Under insectarium conditions, we found that atresia-induced control and RpATG8-silenced females present no changes in blood meal digestion, survival, oviposition, TAG content in the fat body, haemolymph amino acid levels and overall locomotor activity. Altogether, we found that autophagosomes are formed during oogenesis and that the silencing of RpATG8 impairs autophagosome biogenesis in the oocytes. Nevertheless, regarding major macromolecule degradation and adaptations to the fitness costs imposed by triggering an immune response, we found that autophagic organelles are not essential for follicle atresia in R. prolixus.
Asunto(s)
Autofagosomas , Atresia Folicular/fisiología , Silenciador del Gen , Proteínas de Insectos/metabolismo , Rhodnius/fisiología , Animales , Femenino , Atresia Folicular/genética , Proteínas de Insectos/genética , Oocitos , Ovulación/fisiología , Rhodnius/genética , VitelogénesisRESUMEN
In mammals, apoptosis has been accepted as the type of programmed cell death (PCD) that occurs in ovarian follicles undergoing atresia. Results of recent studies, however, indicate autophagy may be an alternative mechanism involved in follicle depletion through independent or tandem actions with apoptosis. Western blotting and immunofluorescence procedures were used in the present study to investigate the abundances of LC3B protein in freshly collected granulosa cells (GCs), cumulus cells (CCs), and oocytes to evaluate whether autophagy is an important process of antral follicle atresia in sexually mature sows. Furthermore, apoptosis was analyzed using annexin V and TUNEL assays in the same cellular cohorts to evaluate the correlation between the two processes. Immunostaining results indicate autophagy was induced in the majority of GCs, CCs, and oocytes from early and advanced stage atretic follicles. The quantitative results of western blot analysis indicate there is a progressive increase (Pâ¯<⯠0.05) in abundance of autophagy-related protein (LC3B-II) in these cells compared with cells in non-atretic follicles. Furthermore, there is confirmation that apoptosis occurs in the GCs of atretic follicles, thus indicating that in pigs apoptosis and autophagy are processes in GCs that regulate PCD and as a consequence antral follicle depletion. There was a greater abundance of LC3B-II in CCs and oocytes of atretic follicles, while apoptosis was not detected. It, therefore, is suggested that in these cells the two processes function independently, with autophagy having a cytoprotective rather than PCD mechanism of action.
Asunto(s)
Autofagia/fisiología , Células del Cúmulo/fisiología , Atresia Folicular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Oocitos/fisiología , Porcinos , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
SummaryPrevious studies have established a model of atresia in preovulatory follicles after stimulation of immature rats with equine chorionic gonadotropin (eCG). This gonadotropin recruits a follicular pool and the deprivation of preovulatory luteinizing hormone (LH) surge induces the atresia in preovulatory follicles. The present study investigated the occurrence of ovulation and provided some morphological features of granulosa cell (GC) apoptosis of atretic follicles at 0, 48, 72 and 120 h after eCG stimulation. Histological sections of ovaries from untreated animals (0 h) showed primordial, primary, secondary and early antral follicles. After 48 h ovaries showed large antral follicles. Preovulatory follicles were observed at 72 h, and two out of five rats displayed cumulus-oocyte complexes (COCs) in the oviducts. All animals exhibited corpora lutea after 120 h. We observed increased estradiol (E2) levels 48 h after eCG treatment that might trigger an endogenous preovulatory gonadotropin surge. Higher progesterone (P4) level, which is the hallmark of a functional corpus luteum, was observed at 120 h. Atresia in secondary and antral follicles was observed by pyknotic granulosa cell nuclei in histology and positive immunolabelling for cleaved caspase 3. We also observed macrophages in secondary and antral follicles in atresia. Transmission electron microscopy revealed GCs with compacted chromatin against the nuclear envelope, nuclear fragmentation, cell shrinkage and fragmentation. No preovulatory follicles showed apoptosis of GCs. In conclusion, our results suggested the occurrence of an endogenous gonadotropin surge, promoting ovulation and preventing atresia of preovulatory follicles.