Atretic Antral Follicles Research Articles

Folliculogenesis and steroidogenesis alterations after chronic exposure to a human-relevant mixture of environmental toxicants spare the ovarian reserve in the rabbit model

BackgroundIndustrial progress has led to the omnipresence of chemicals in the environment of the general population, including reproductive-aged and pregnant women. The reproductive function of females is a well-known target of endocrine-disrupting chemicals. This function holds biological processes that are decisive for the fertility of women themselves and for the health of future generations. However, insufficient research has evaluated the risk of combined mixtures on this function. This study aimed to assess the direct impacts of a realistic exposure to eight combined environmental toxicants on the critical process of folliculogenesis.MethodsFemale rabbits were exposed daily and orally to either a mixture of eight environmental toxicants (F group) or the solvent mixture (NE group, control) from 2 to 19 weeks of age. The doses were computed from previous toxicokinetic data to reproduce steady-state serum concentrations in rabbits in the range of those encountered in pregnant women. Ovarian function was evaluated through macroscopic and histological analysis of the ovaries, serum hormonal assays and analysis of the expression of steroidogenic enzymes. Cellular dynamics in the ovary were further investigated with Ki67 staining and TUNEL assays.ResultsF rabbits grew similarly as NE rabbits but exhibited higher total and high-density lipoprotein (HDL) cholesterol levels in adulthood. They also presented a significantly elevated serum testosterone concentrations, while estradiol, progesterone, AMH and DHEA levels remained unaffected. The measurement of gonadotropins, androstenedione, pregnenolone and estrone levels yielded values below the limit of quantification. Among the 7 steroidogenic enzymes tested, an isolated higher expression of Cyp19a1 was measured in F rabbits ovaries. Those ovaries presented a significantly greater density/number of antral and atretic follicles and larger antral follicles without any changes in cellular proliferation or DNA fragmentation. No difference was found regarding the count of other follicle stages notably the primordial stage, the corpora lutea or AMH serum levels.ConclusionFolliculogenesis and steroidogenesis seem to be subtly altered by exposure to a human-like mixture of environmental toxicants. The antral follicle growth appears promoted by the mixture of chemicals both in their number and size, potentially explaining the increase in atretic antral follicles. Reassuringly, the ovarian reserve estimated through primordial follicles number/density and AMH is spared from any alteration. The consequences of these changes on fertility and progeny health have yet to be investigated.

Participation of LIF in follicular development and steroidogenesis in rat ovaries.

Leukaemia inhibitory factor (LIF) is a cytokine belonging to the IL-6 family that is important at the reproductive level in the uterine implantation process. However, there is very little evidence regarding its effect at the ovarian level. The aim of this work was to study the local involvement of the LIF/LIFRβ system in follicular development and steroidogenesis in rat ovaries. To carry out this research, LIF/LIFR/GP130 transcript and protein levels were measured in fertile and sub-fertile rat ovaries, and in vitro experiments were performed to assess STAT3 activation. Then, in in vivo experiments, LIF was administered chronically and locally for 28 days to the ovaries of rats by means of an osmotic minipump to enable us to evaluate the effect on folliculogenesis and steroidogenesis. It was determined by q-PCR and WB that LIF and its receptors are present in fertile and sub-fertile ovaries, and that LIF varies during the oestrous cycle, being higher during the oestrus and meta/dioestrus stages. In addition to this, it was found that LIF can activate STAT3 pathways and cause pSTAT3 formation. It was also observed that LIF decreases the number and size of preantral and antral follicles without altering the number of atretic antral follicles, and can increase the number of corpora lutea, with a notable increase in the levels of progesterone. It is therefore possible to infer that LIF exerts an important effect in vivo on folliculogenesis, ovulation and steroidogenesis, specifically the synthesis of progesterone.

Effects of myo-inositol plus folic acid on ovarian morphology and oocyte quality in PCOS mouse model.

Although the role of myo-inositol (MYO) in promoting the oocyte quality of PCOS patients has been documented in human studies; the cellular effects of this supplement on oocytes have not been directly examined due to ethical limitations. In the first phase of this study, MYO dosimetry was carried out simultaneously with the PCOS model development. An effective dose was obtained following the assessment of fasting insulin and testosterone levels using ELISA and ovarian morphology appraisal by histopathology. In the second phase, following the continuous administration of the effective dose of MYO and dehydroepiandrosterone (DHEA), cellular evaluation was performed. The quality of oocytes from superovulation was analyzed by examining maturity and normal morphology percentage using a stereomicroscope, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels using fluorometry, and ATP count evaluation using ELISA. The results revealed that, among the four different MYO concentrations, the 0.36 mg/g dose compared with the DHEA group reduced testosterone levels and large atretic antral follicles (LAtAnF) diameter. This dose also increased the corpus luteum count and the granulosa:theca (G/T)layer thickness ratio in antral follicles. Furthermore, this dose increased mature oocytes and normal morphology percentage, ATP count, and GSH levels; however, it decreased ROS levels in mature oocytes. Our findings provide the grounds for further cellular and molecular studies on the PCOS mouse model, suggesting that the improvement in mitochondrial function and its antioxidant properties is probably one of the mechanisms by which MYO increases oocyte quality.

RF10 | PMON241 Temporal Changes in Phenotype and Gut microbiota in PCOS Mouse Model Induced by Prenatal Androgen Exposure.

Abstract PCOS is a complex multigenic disorder with strong epigenetic and environmental influence. Previous reports have suggested that fetal over-exposure to androgens contributes to the development of PCOS after birth. On the other hands, recent studies on both human and rodent models of PCOS have demonstrated the relationship between PCOS and gut microbiome in adulthood. Furthermore, gut microbiome in obese adolescent with PCOS are different from obese adolescent without PCOS. However, the mechanism has not been revealed and it is unclear which events appear first, PCOS phenotypes or gut microbiome. We wondered if prenatal androgen exposure leads gut microbial dysbiosis early in life and is associated with the development of PCOS in later life. To test this hypothesis, we examined the temporal changes in the phenotypes of PCOS and gut microbiome using prenatally androgenized (PNA) model mice, an well-established model of PCOS. PNA model was generated by subcutaneously injecting pregnant dams with dehydroepiandrosterone (DHT) on days 16, 17, and 18 of gestation. Phenotypes and gut microbiome activity were compared between PCOS model mice (n=12/group) and control mice (n=10/group) at each developmental stage of 4 weeks (prepuberty), 6 weeks (puberty), 8 weeks (adolescent), 12 weeks (young adulthood), and 16 weeks (adulthood), respectively. The determinants for PCOS development are onset of puberty, estrous cycle, morphology of ovaries, serum testosterone levels, body weight, the size of parametrial adipocytes, and insulin resistance. For evaluation of gut microbiome, next generation sequencing and bioinformatics analysis of 16S rRNA genes were performed on obtained DNA from mouse fecal samples. PNA groups resulted in delayed puberty onset, disrupted estrous cycle, and increased testosterone levels from 6 weeks. Increased atretic antral follicles were observed in PNA groups at 6, 12, and 16 weeks. Additionally, PNA groups showed increased body weight, hypertrophy of parametrial adipocytes, and insulin resistant from 12 weeks. As for gut microbiome, PNA exhibited altered alpha-diversity from 8 weeks and beta-diversity at 8 weeks. Composition of gut microbiome was already altered from 4 weeks. At phylum level, Firmicutes phylum are significantly increased in PNA groups at 4 and 8, and decreased at 16 weeks. Actinobacteria phylum showed significant decrease at 6 and 8 weeks in PNA groups. At genus level, relative abundance of several bacterial taxa differed significantly between control and PNA groups; Allobaculum, Adlercreutzia which produce equol, Roseburia which produce butyric acid, and Sutterella were significantly decreased in PNA groups at multiple stages of development. In conclusion, our findings suggest that the alteration of gut microbiome appears simultaneously or even earlier than the presence of PCOS phenotypes, and that normalizing microbiome could improve pathologic condition of PCOS. In addition, early intervention of gut microbiome might indicate preventive care for women at high-risk of developing PCOS. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Comparative effects of garlic (Allium sativum) powder and atorvastatin in female reproductive system of hypercholesterolemic rats: A histological and biochemical evaluation

Background and aims: The abnormal increase in blood cholesterol can cause many problems. Statins have a cholesterol-lowering effect, but they also have adverse effects. Garlic prevents the formation of cholesterol due to its antibiotic properties. This study aimed to investigate the comparative effect of garlic powder and atorvastatin on hypercholesterolemia-induced reproductive failure in female rats. Methods: In the present experimental study, 48 adult female Wistar rats were divided into eight groups (n=6), including control, atorvastatin (10 mg/kg/d; orally), atorvastatin (20 mg/kg/d; orally), garlic powder (100 mg/kg/d; orally), hypercholesterolemia (1.5 mg/kg/d of cholesterol; orally), hypercholesterolemia + atorvastatin (10 mg/kg/d), hypercholesterolemia + atorvastatin (20 mg/kg/d), and hypercholesterolemia + garlic powder. After 30 days, rats were euthanized and blood samples were obtained from their heart for serological assessments. The right ovary was transferred to 10% formalin for histological analyses, and the left ovary was transferred to a −80°C freezer for evaluation of oxidative stress markers. Data were statistically analyzed by ANOVA and Tukey’s test using SPSS version 24.0 (P<0.05). Results: The number of healthy primordial, primary, secondary, and antral follicles, catalase activity, total antioxidant capacity (TAOC) as well as estrogen and progesterone levels were lower in hypercholesterolemic rats compared to controls (P<0.001). Additionally, the number of the atretic primary, secondary, and antral follicles and malondialdehyde (MDA) levels were higher in hypercholesterolemic rats (P<0.001). However, garlic powder and atorvastatin 10 improved alterations in the mentioned parameters (P=0.99). Conclusion: The results showed that hypercholesterolemia could have adverse effects on rat ovaries. However, the garlic powder improves ovarian toxicity in hypercholesterolemia rats better than atorvastatin.

Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle.

Mammalian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation, and release of the eggs, i.e., ovulation. The steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered membrane progesterone receptor α/β hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation of Abhd2 caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries of Abhd2 knockout (KO) females resemble polycystic ovary morphology (PCOM) with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowed Abhd2 KO females to ovulate a significantly increased number of mature and fertile eggs in comparison with their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in non-genomic steroid regulation of the female reproductive cycle.

P–632 Examination of temporal changes in phenotype and gut microbiome during the process of growth in polycystic ovary syndrome (PCOS) model induced by prenatal androgen exposure

Abstract Study question From when do abnormality in gut microbiome and phenotypes of PCOS appear during the process of growth? Summary answer Reproductive phenotypes of PCOS appear from 6 weeks and metabolic phenotypes from 12 weeks onward. Alteration in gut microbiome appears as early as 4 weeks. What is known already The etiology of PCOS remains largely unknown, however PCOS is considered as a complex multigenic disorder with strong epigenetic and environmental influence. Previous studies have suggested that fetal over-exposure to androgens could be the main factor of the development of PCOS after birth. On the other hands, recent studies on both human and PCOS rodent models have demonstrated the association between PCOS and alteration of gut microbiome in adulthood. Furthermore, it was recently reported that gut microbiome in obese adolescent with PCOS is different from obese adolescent without PCOS. Study design, size, duration A rodent PCOS model induced by prenatal dehydroepiandrosterone (DHT) exposure was applied to this study. Phenotypes and gut microbiome were compared between PCOS model mice (n = 12/group) and control mice (n = 10/group) at each stage of growth; 4 weeks (prepuberty), 6 weeks (puberty), 8 weeks (adolescent), 12 weeks (young adult), and 16 weeks (adult). The determinants for PCOS phenotypes are onset of puberty, estrous cycle, morphology of ovaries, serum testosterone level, body weight, and insulin resistance. Participants/materials, setting, methods Pregnant dams were subcutaneously injected on days of 16, 17, and 18 of gestation with either sesame oil for control groups or sesame oil containing 250µg of DHT for prenatal DHT groups. The evaluation of PCOS phenotypes and gut microbiome in female offspring were performed at each stage of growth. For examination of gut microbiota, next generation sequencing and bioinformatics analysis of 16S rRNA genes were performed on DNA extracted from mouse fecal samples. Main results and the role of chance Prenatal DHT mice exhibited delayed puberty onset, disrupted estrous cycle, and significantly increased testosterone levels from 6 weeks onward. Significantly increased atretic antral follicles were observed in prenatal DHT mice at 6, 12, and 16 weeks. Prenatal DHT mice showed significantly decreased body weight at 4, 6, 8 weeks and increased body weight from 12 weeks onward. As for gut microbiome, alpha-diversity was significantly different between control and prenatal DHT mice from 8 weeks onward and beta-diversity was significantly different at 6 and 8 weeks. Altered composition of gut microbiota was observed as early as 4 weeks. At phylum level, Firmicutes are significantly increased in prenatal DHT mice at 4 and 8 weeks and decreased at 16 weeks. Actinobacteria phylum showed significant decrease at 6 and 8 weeks in prenatal DHT mice. At genus level, relative abundance of several bacterial taxa significantly differed between control and prenatal DHT mice; some taxa, such as Allobaculum, Adlercreutzia, Bilophila, Clostridium, Gemella, Gemmiger, Roseburia, Ruminococcus, Staphylococcus, and Sutterella, exhibited constant increase or decrease in prenatal DHT mice during the process of growth. Interestingly, Roseburia was never detected in prenatal DHT mice, while approximately half of control mice harbored Roseburia at 12 and 16 weeks. Limitations, reasons for caution It is not clearly determined whether alteration in gut microbiome is cause or result of PCOS development, although the changes in gut microbiome seemed to precede the appearance of typical PCOS phenotypes in the present study. Mouse model does not completely recapitulate human PCOS. Wider implications of the findings: Our findings suggest that prenatal androgen exposure causes alteration of gut microbiome from pre-puberty onward, even before PCOS phenotypes become apparent. Intervention for girls at risk of PCOS with pre/pro-biotics may prevent them from developing PCOS in future. Trial registration number Not applicable

Upregulation of Aryl Hydrocarbon Receptor in Granulosa Cells by Endoplasmic Reticulum Stress Contributes to the PCOS Pathophysiology

Studies have demonstrated that endocrine disrupting chemicals (EDC) are involved in the pathophysiology of PCOS, and aryl hydrocarbon receptor (AHR) mediates the cellular effect of EDC by inducing xenobiotic metabolizing enzymes including cytochrome P450 1B1 (CYP1B1). However, recent studies suggest the novel role of AHR in various diseases, including obesity and cancer progression, independent from the EDC metabolism. We previously demonstrated that endoplasmic reticulum (ER) stress, a newly recognized local factor, contributes to PCOS pathology by affecting diverse functions of granulosa cells. We hypothesized that ER stress induces the expression of AHR and activates its downstream signaling in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. At first, we determined the upregulation of AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) in the granulosa cells of PCOS patients and model mice by immunohistochemical staining and qPCR. We examined CYP1B1 as a representative AHR target gene. Treatment of cultured human granulosa-lutein cells (GLCs) with tunicamycin (ER stress inducer) upregulated the expression of AHR, ARNT and CYP1B1. Knockdown of AHR decreased the tunicamycin-induced expression and activity of CYP1B1, suggesting the intermediary role of AHR in upregulation of AHR activity by ER stress. To confirm the role of AHR in vivo, we administered the AHR antagonist CH223191 to PCOS model mice. The administration of the antagonist restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, this study indicates that AHR and downstream signaling are activated by ER stress in GLCs of PCOS. Moreover, downregulation of local AHR expression and activation restores a normal reproductive phenotype in a PCOS mouse model. Our findings demonstrate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Ufmylation regulates granulosa cell apoptosis via ER stress but not oxidative stress during goat follicular atresia

Follicular atresia is primarily caused by granulosa cell (GC) apoptosis, although the mechanisms are largely unknown. Ufmylation is a recently identified ubiquitin-like post-translational modifier that plays an important role in cell proliferation and apoptosis. The purpose of this study was to investigate the effects of Ufmylation on GC apoptosis during goat follicular atresia. Ubiquitin-fold modifier 1 (UFM1) and its target DDRGK domain containing 1 (DDRGK1) proteins were identified in granulosa cells (GCs) isolated from all stages of preantral follicles and from healthy (HF), early atretic (EF) and progressed atretic (PF) antral follicles. The expression levels were higher in GCs derived from antral atretic follicles than healthy follicles. Although the viability of GCs was not affected after overexpression of UFM1, siRNA-mediated UFM1 silencing significantly inhibited GC proliferation and induced apoptosis. Notably, components of the ufmylation pathway were significantly upregulated in GCs induced by the ER stress agent tunicamycin (Tm) and thapsigargin (Tg), but not affected by oxidative stress inducer H2O2. Furthermore, UFM1 silencing markedly increased the apoptosis of GCs upon Tg treatment by stimulating the ER stress-related gene expression. Our results provide evidence that UFM1 and its target DDRGK1 are expressed in the goat GCs during follicular development and atresia, and ufmylation may play an important role in the prevention of ER stress but not oxidative stress-induced GCs apoptosis.

Early Exposure to High-Sucrose Diet Leads to Deteriorated Ovarian Health.

BackgroundThe metabolic syndrome (MetS) is correlated with disorders of the reproductive system, such as the polycystic ovary syndrome (PCOS). While consumption of a diet rich in carbohydrates is linked to the development of MetS, it is still unclear if this diet leads to ovarian dysfunction and PCOS.ObjectivesWe investigated the influence of a high-sucrose diet (HSD) on the ovarian milieu of Wistar rats and studied the correlation between high consumption of sugary drinks and the prevalence of PCOS in women.MethodsWistar rats were given a standard laboratory diet (CTR, 10% sucrose, n = 8) or HSD (HSD, 25% sucrose, n = 8) from postnatal day 21 to 120. Animals were evaluated weekly to calculate food intake, feed efficiency and weight gain. Both onset of puberty and estrous cycle were monitored. Metabolic serum biochemistry, organ morphometry and ovarian histology were performed upon euthanasia. In parallel, a fixed-effects multiple linear regression analysis was performed using data from Brazilian states (459 state-year observations) to test the correlation between the consumption of sugar-sweetened beverages (surrogate for HSD intake) and the prevalence of PCOS (surrogate for ovarian dysfunction).ResultsHSD animals showed increased adipose tissue accumulation, hyperglycaemia and insulin resistance when compared to CTR. Interestingly HSD rats also entered puberty earlier than CTR. Moreover, ovaries from HSD animals had an increased number of atretic antral follicles and cystic follicles, which were correlated with the hypertrophy of periovarian adipocytes. Finally, there was a positive correlation between the intake of sugary drinks and prevalence of PCOS in women of reproductive age.ConclusionsHSD ingestion leads to ovarian dysfunction in rats and could be correlated with PCOS in women, suggesting these alterations could lead to public health issues. Therefore, we reinforce the deleterious impact of HSD to the ovarian system and suggest that the reduction of added sugars intake could be beneficial to ovarian health.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = −6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

Induction of aryl hydrocarbon receptor in granulosa cells by endoplasmic reticulum stress contributes to pathology of polycystic ovary syndrome.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously showed that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

D-ribose-L-cysteine modulates lead acetate-induced hematobiochemical alterations, hormonal imbalance, and ovarian toxicity in adult female Wistar rats

Lead is a common environmental toxicant associated greatly with hematological and hormonal imbalance, biochemical alterations, and reproductive abnormalities. This study was conducted to evaluate the effects of D-ribose-L-cysteine (DRLC) on hematobiochemical and reproductive toxicity associated with lead acetate exposure in adult female Wistar rats. Thirty-two adult female Wistar rats (165 ± 20 g) were divided into four groups (n = 8). Group A received normal saline as placebo; Group B received 100 mg/kg BW of lead acetate only; Group C received 100 mg/kg BW of lead acetate and 10 mg/kg BW DRLC (low dose); Group D received 100 mg/kg BW of lead acetate and 30 mg/kg BW of DRLC (high dose). All administration was done via oral gavage for 42 days, thereafter animals were sacrificed; serum was obtained from the blood collected for analysis, ovaries, and uterus was harvested for analysis. The lead acetate only group showed a significant difference in hematological indices relative to control. Additionally, there was a significant decrease in body weight, sodium dismutase, catalase, reduced glutathione, progesterone with a corresponding increase in ovarian weight, MDA, FSH, and LH among the lead acetate only group relative to the control. Histological observation showed atretic antral follicles, with detached granulosa cells, pyknotic nuclei in the granulosa wall in the ovaries of the lead-exposed only group compared to the control. Co-administration of DRLC and lead attenuate the toxicity of lead exposure by restoring the hematological values, biochemical parameters, hormone profile, and morphology of the ovary. Exposure to lead acetate causes deleterious toxicity to hematological and reproductive functions which were ameliorated DRLC supplementation through its antioxidant mechanisms.

Characteristics of Circular RNA Expression Profiles of Porcine Granulosa Cells in Healthy and Atretic Antral Follicles.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions—circ_CBFA2T2 and circ_KIF16B—were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes—the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

Characteristics of circular RNA expression profiles of porcine granulosa cells in healthy and atretic antral follicles

Characteristics of circular RNA expression profiles of porcine granulosa cells in healthy and atretic antral follicles

PTEN and FOXO3 expression in the prenatal and postnatal human ovary

The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

Androgens Increase Accumulation of Advanced Glycation End Products in Granulosa Cells by Activating ER Stress in PCOS

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism, and we previously found that androgens activate endoplasmic reticulum (ER) stress in granulosa cells from patients with PCOS. In addition, recent studies demonstrated the accumulation of advanced glycation end products (AGEs) in granulosa cells from PCOS patients, which contribute to the pathology. Therefore, we hypothesized that androgens upregulate the receptor for AGEs (RAGE) expression in granulosa cells by activating ER stress, thereby increasing the accumulation of AGEs in these cells and contributing to the pathology. In the present study, we show that testosterone increases RAGE expression and AGE accumulation in cultured human granulosa-lutein cells (GLCs), and this is reduced by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Knockdown of the transcription factor C/EBP homologous protein (CHOP), an unfolded protein response factor activated by ER stress, inhibits testosterone-induced RAGE expression and AGE accumulation. The expression of RAGE and the accumulation of AGEs are upregulated in granulosa cells from PCOS patients and dehydroepiandrosterone-induced PCOS mice. Administration of the RAGE inhibitor FPS-ZM1 or TUDCA to PCOS mice reduces RAGE expression and AGE accumulation in granulosa cells, improves their estrous cycle, and reduces the number of atretic antral follicles. In summary, our findings indicate that hyperandrogenism in PCOS increases the expression of RAGE and accumulation of AGEs in the ovary by activating ER stress, and that targeting the AGE-RAGE system, either by using a RAGE inhibitor or a clinically available ER stress inhibitor, may represent a novel approach to PCOS therapy.

The potential biological functions of circular RNAs during the initiation of atresia in pig follicles

The specific expression profile and function of circular RNAs (circRNAs) in mammalian ovarian follicles, especially during the atresia process, are unclear. In this study, genome-wide deep circRNA sequencing was applied to screen circRNAs in healthy and early atretic antral follicles in pig ovaries. A total of 40,567 distinct circRNAs were identified in follicles, among which 197 circRNAs (108 upregulated and 89 downregulated) were significantly shifted during the early atresia process. Most differentially expressed circRNAs (DECs) lacked protein-coding potential. Annotation analysis of the DECs revealed 162 known host genes, or noncoding RNAs, and 10 intergenic regions. The key pathways in which these host genes are involved include the focal adhesion-PI3K-Akt-mTOR signaling pathway, vascular endothelial growth factor A (VEGFA)–vascular endothelial growth factor receptor 2 signaling pathway and transforming growth factor-beta signaling pathway. Further comparison analysis between host genes of DECs and the differentially expressed linear messenger RNA transcripts revealed the cotranscription of circRNAs and their linear mRNAs in inhibin beta units (INHBA and INHBB), glutathione S-transferase (GSTA1), and VEGFA. In addition, we predicted 196 pairs of potential circRNA-micro RNA (miRNA) interactions among 77 DECs and 101 porcine miRNAs. We have identified 16 functional miRNAs by comparing the 101 miRNAs to the functional miRNAs reported in mammal ovarian follicle atresia and granulosa cell apoptosis studies. Our study adds new knowledge to circRNA distribution profiles in pig ovarian follicles, offers a valuable reference for transcriptomic profiles in the initiation of follicular atresia, highlights warranted circRNAs for further functional investigation, and provides possible biomarkers for ovarian dysfunctions.

Roles of extracellular matrix in follicular development

The cellular biology and changes in the extracellular matrix of ovarian follicles during their development are reviewed. During growth of the bovine ovarian follicle the follicular basal lamina doubles 19 times in surface area. It changes in composition, having collagen IV α1–26 and laminin α1, ß2 and γl at the primordial stage, and collagen IV α1 and α2, reduced amounts of α3–α5, and a higher content of laminin α1, ß2 and γl at the antral stage. In atretic antral follicles laminin α2 was also detected. The follicular epithelium also changes from one layer to many layers during follicular growth. It is clear that not all granulosal cells have equal potential to divide, and we have evidence that the granulosal cells arise from a population of stem cells. This finding has important ramifications and supports the concept that different follicular growth factors can act on different subsets of granulosal cells. In antral follicles, the replication of cells occurs in the middle layers of the membrana granulosa, with older granulosal cells towards the antrum and towards the basal lamina. The basal cells in the membrana granulosa have also been observed to vary in shape between follicles. In smaller antral follicles, they were either columnar or rounded, and in follicles >5 mm the cells were all rounded. The reasons for these changes in matrix and cell shapes are discussed in relation to follicular development.