Follicle Oocyte Research Articles

Prediction of major microRNAs in follicular fluid regulating porcine oocyte development

The aim of the present study was to identify key microRNAs (miRNAs) in porcine follicular fluid (FF) that regulate oocyte growth. miRNAs contained in FF were determined by small RNA-seq of exosome RNA. Upstream regulator miRNA was determined by ingenuity pathway analysis using differentially expressed genes in granulosa cells (GCs) between small follicles (1-2 mm in diameter) and large follicles (3-5 mm), and between follicles containing oocytes of high developmental ability and follicles containing oocytes of low developmental ability. The candidate miRNAs overlapping among the three miRNAs group were determined. Lastly, the effect of supplementation with FF, exosome-depleted FFs, or each miRNA on in vitro oocyte growth was examined. The miRNAs determined were miR-17, -27, -92a, and -145. These miRNAs were found in the spent culture medium of oocytes and granulosa cells complexes and serum by small RNA sequencing. Culturing of oocytes and granulosa cells complexes collected from porcine early antral follicles (0.5-0.7 mm in diameter) with FF for 14 days improved oocyte growth; depletion of exosomes from the FFs neutralized the beneficial effect observed. miR-92a mimic increased the antrum formation and diameter, together with acetylated levels of H4K12 in oocytes. In addition, supplementation of miRNA mimics miR-17b, -92a, and -145b improved the rate of chromatin configuration, and miR-17b and -92a mimics improved the developmental ability of oocytes to the blastocyst stage. miR-17, -92a, and -145 are major miRNA candidates in follicular fluids regulating oocyte growth.

Canine oviductal exosomes improve oocyte development via EGFR/MAPK signaling pathway.

Oviduct cells produce a favorable environment for the development of gametes by generating multiple growth factors. Particularly, in canine species, immature oocytes undergo serial maturation processes in the oviduct, while the other mammals already possess matured oocytes in ovulatory follicles. However, little is known about the potential effect exhibited by the components released from canine oviduct cells (OCs) for modulating the biological function of oocytes. Recently, exosomes are regarded as promising extracellular vesicles because they represent considerable data for molecular cargo. Therefore, we first investigated the effect of canine oviductal exosomes (OC-Exo) on oocyte development via EGFR/MAPK pathway. Our results showed that OC-Exo labeled with PHK67 are successfully incorporated with cumulus cells and oocytes during IVM. Also, OC-Exo markedly increased the proportion of cumulus-oocyte complexes (COCs) exhibiting cumulus expansion as well as cumulus cell proliferation and maturation rate of oocytes (P < 0.05). Furthermore, gene expression patterns related with EGFR/MAPK pathway including EGFR, PKA, TACE/ADAM17, MAPK1/3, MAPK14, PTGS2, TNFAIP6, GDF9, and BMP15 were positively modified in COCs cultured with OC-Exo (P < 0.05). In addition, OC-Exo significantly up-regulated the protein expression levels of p-EGFR, p-MAPK1/3, GDF9 and BMP15 in COCs (P < 0.05). Consequently, the current study provides a model for understanding the roles of OC-Exo as bioactive molecules for canine oocyte maturation via EGFR/MAPK pathway, which would open a new avenue for the application of exosomes to improve assisted reproductive technology in mammals, including humans.

The PARP inhibitor, olaparib, depletes the ovarian reserve in mice: implications for fertility preservation.

What is the impact of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, alone or in combination with chemotherapy on the ovary in mice? Olaparib treatment, when administered alone, depletes primordial follicle oocytes, but olaparib does not exacerbate chemotherapy-mediated ovarian follicle loss in mice. The ovary contains a finite number of oocytes stored within primordial follicles, which give rise to all mature ovulatory oocytes. Unfortunately, they are highly sensitive to exogenous DNA damaging insults, such as cytotoxic cancer treatments. Members of the PARP family of enzymes are central to the repair of single-strand DNA breaks. PARP inhibitors have shown promising clinical efficacy in reducing tumour burden, by blocking DNA repair capacity. Olaparib is a PARP1/2 inhibitor recently FDA-approved for treatment of BRCA1 and BRCA2 mutation carriers with metastatic breast cancer. It is currently being investigated as an adjunct to standard treatment at an earlier stage, potentially curable, BRCA1- and BRCA2-associated breast cancer which affects reproductive age women. Despite this, there is no preclinical or clinical information regarding the potential impacts of olaparib on the ovary or on female fertility. Unfortunately, it may be many years before clinical data on fertility outcomes for women treated with PARP inhibitors becomes available, highlighting the importance of rigorous preclinical research using animal models to establish the potential for new cancer therapies to affect the ovary in humans. We aimed to comprehensively determine the impact of olaparib alone, or following chemotherapy, on the ovary in mice. On Day 0, mice (n = 5/treatment group) were administered a single intraperitoneal dose of cyclophosphamide (75 mg/kg/body weight), doxorubicin (10 mg/kg), carboplatin (80 mg/kg), paclitaxel (7.5 mg/kg) or vehicle control. From Days 1 to 28, mice were administered subcutaneous olaparib (50 mg/kg) or vehicle control. This regimen is proven to reduce tumour burden in preclinical mouse studies and is also physiologically relevant for women. Adult female wild-type C57BL6/J mice at peak fertility (8 weeks) were administered a single intraperitoneal dose of chemotherapy, or vehicle, then either subcutaneous olaparib or vehicle for 28 days. Vaginal smears were performed on each animal for 14 consecutive days from Days 15 to 28 to monitor oestrous cycling. At 24 h after final treatment, ovaries were harvested for follicle enumeration and immunohistochemical analysis of primordial follicle remnants (FOXL2 expressing granulosa cells), DNA damage (γH2AX) and analysis of apoptosis by TUNEL assay. Serum was collected to measure circulating anti-Müllerian hormone (AMH) concentrations by ELISA. Olaparib significantly depleted primordial follicles by 36% compared to the control (P < 0.05) but had no impact on other follicle classes, serum AMH, corpora lutea number (indicative of ovulation) or oestrous cycling. Primordial follicle remnants were rarely detected in control ovaries but were significantly elevated in ovaries from mice treated with olaparib alone (P < 0.05). Similarly, DNA damage denoted by γH2AX foci was completely undetectable in primordial follicles of control animals but was observed in ∼10% of surviving primordial follicle oocytes in mice treated with olaparib alone. These observations suggest that functional PARPs are essential for primordial follicle oocyte maintenance and survival. Olaparib did not exacerbate chemotherapy-mediated follicle depletion in the wild-type mouse ovary. N/A. This study was performed in mice, so the findings may not translate to women and further studies utilizing human ovarian tissue and sera samples should be performed in the future. Only one long-term time point was analysed, therefore olaparib-mediated follicle damage should be assessed at more immediate time points in the future to support our mechanistic findings. Olaparib dramatically depleted primordial follicles and this could be attributed to loss of intrinsic PARP-mediated DNA repair mechanisms. Importantly, diminished ovarian reserve can result in premature ovarian insufficiency and infertility. Notably, the extent of follicle depletion might be enhanced in BRCA1 and BRCA2 mutation carriers, and this is the subject of current investigations. Together, our data suggest that fertility preservation options should be considered for young women prior to olaparib treatment, and that human studies of this issue should be prioritized. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. This work was supported by funding from the National Health and Medical Research Council (NHMRC); (K.J.H. #1050130) (A.L.W. #1120300). K.A.P. is a National Breast Cancer Foundation Fellow (Australia-PRAC-17-004). K.A.P. is the Breast Cancer Trials (Australia) Study Chair for the OlympiA clinical trial sponsored by AstraZeneca, the manufacturer of olaparib. All other authors declare no competing financial or other interests.

Unraveling the mechanisms of chemotherapy-induced damage to human primordial follicle reserve: road to developing therapeutics for fertility preservation and reversing ovarian aging.

Among the investigated mechanisms of chemotherapy-induced damage to human primordial follicle reserve are induction of DNA double-strand breaks (DSBs) and resultant apoptotic death, stromal-microvascular damage and follicle activation. Accumulating basic and translational evidence suggests that acute exposure to gonadotoxic chemotherapeutics, such as cyclophosphamide or doxorubicin, induces DNA DSBs and triggers apoptotic death of primordial follicle oocytes within 12-24 h, resulting in the massive loss of ovarian reserve. Evidence also indicates that chemotherapeutic agents can cause microvascular and stromal damage, induce hypoxia and indirectly affect ovarian reserve. While it is possible that the acute reduction of the primordial follicle reserve by massive apoptotic losses may result in delayed activation of some primordial follicles, this is unlikely to be a predominant mechanism of loss in humans. Here, we review these mechanisms of chemotherapy-induced ovarian reserve depletion and the potential reasons for the discrepancies among the studies. Based on the current literature, we propose an integrated hypothesis that explains both the acute and delayed chemotherapy-induced loss of primordial follicle reserve in the human ovary.

Ultrastructural Characterization of Porcine Growing and In Vitro Matured Oocytes.

Simple SummaryDuring oocyte growth and maturation, the organelle’s morphology of porcine oocytes changed and populated different compartments depending on the differentiation status. Changes in ultrastructural or subcellular level of porcine oocytes during oogenesis/folliculogenesis were observed, potentially leading to future mitochondrion replacement therapies of oocytes.This study aimed to investigate ultrastructural changes of growing porcine oocytes and in vitro maturated oocytes. Light microscopy was used to characterize and localize the primordial, primary, secondary, and tertiary follicles. During oocyte growth and maturation, the morphology of mitochondria was roundish or ovoid in shape depending on the differentiation state, whereas their mean diameters oscillated between 0.5 and 0.7 µm, respectively, from primary and secondary follicles. Hooded mitochondria were found in the growing oocytes of the tertiary follicles. In addition to the pleomorphism of mitochondria, changes in the appearance of lipid droplets were also observed, along with the alignment of a single layer of cortical granules beneath the oolemma. In conclusion, our study is apparently the first report to portray morphological alterations of mitochondria that possess the hooded structure during the growth phase of porcine oocytes. The spatiotemporal and intrinsic changes during oogenesis/folliculogenesis are phenomena at the ultrastructural or subcellular level of porcine oocytes, highlighting an in-depth understanding of oocyte biology and impetus for future studies on practical mitochondrion replacement therapies for oocytes.

Shift of the Vegetal Pole Area of Full-Grown Oocytes Toward the Ovulatory Site of the Ovary in the Medaka Fish, Oryzias latipes (Beloniformes: Adrianichthyidae).

This study presents novel findings on the dynamics of growing oocytes in the ovary of the medaka fish, Oryzias latipes. In the ovary of mature females, all follicles are anchored tightly to the abdominal ovarian rete via fibrous follicular stalks on the follicle surface. The follicular stalks lie at the end of the follicle opposite the site of its attachment to the ovarian wall. Various lengths of the follicular stalks reflect the spatial arrangement of follicles in the ovary. Herein, a line that connects the follicular stalk to the opposite side of the follicle toward or attaching to the ovarian wall is called the follicle axis. The animal-vegetal axis in late stage III oocytes is already recognizable as a line that connects the center of the oocyte nucleus and the vegetal pole area; this is ascertainable by the morphological landmark of a compact distribution of granulosa cells or rudimentary attachment filaments. In growing oocytes later than stage V, the beginning of vitellogenesis, the tufted attachment filaments are located on a discrete region of the vegetal pole area. Our observations reveal that during growth, oocytes are arranged randomly between the animal-vegetal axis and the follicle axis, whereas the vegetal pole area of full-grown oocytes in preovulatory follicles turns close to the inner surface of the ovarian wall, from which mature oocytes subsequently ovulate into the ovarian lumen. It is suggested that in the O. latipes ovary, mature oocytes always transpose the vegetal pole area to the ovulatory site of the ovarian wall prior to ovulation. The expulsion of mature oocytes from the vegetal pole appears to be the regular mode of ovulation in O. latipes.

Molecular cloning, characterization and expression profiles of a SoxB2 gene related to gonadal development in mud crab (Scylla paramamosain)

ABSTRACT The molecular mechanisms underlying sex differentiation and gonad development remain elusive in crustaceans. Here, we report the cloning and characterization of a Sry-related high-mobility group family B2 gene (SpSoxB2) from an economic crustacean species, the mud crab Scylla paramamosain. The SpSoxB2 encodes a putative HMG-box protein (317 amino acids) clustered with invertebrate Sox14 homologues in the SoxB2 subgroup, but shared low similarity with SpSox14. SpSoxB2 was expressed in the hepatopancreas, thoracic ganglion, and gonads (relatively higher expression in testis than ovary). In hepatopancreas, SpSoxB2 expression was not significantly affected by sex or gonadal development stages. In testis, it was demonstrated that the SpSoxB2 expression level increased from Stage I to Stage III and then decreased afterwards. During ovarian development, the SpSoxB2 expression level continually increased from Stage I to Stage V. Furthermore, fluorescence in-situ hybridization showed that SpSoxB2 transcripts were present in both somatic and developing germ cells in the gonads. In testis, SpSoxB2 transcripts were strongly localized in spermatocytes and spermatids. In ovary, SpSoxB2 transcripts were detected in follicle cells, oogonia and oocytes, with the strongest signal in follicle cells. Our results demonstrated that the SpSoxB2 gene may play an important role during gonadal development in mud crab.

Calorie restriction during gestation affects ovarian reserve in offspring in the mouse.

The aim of this study was to investigate the effect of calorie restriction (CR) during pregnancy in mice on metabolism and ovarian function in the offspring. Pregnant female mice were divided into two groups, a control group and a CR group (n=7 in each). Mice in the CR group were fed 50% of the amount consumed by control females from Day 10 of gestation until delivery. After weaning, the offspring received diet ad libitum until 3 months of age, when ovaries were collected. Ovaries were serially cut and every sixth section was used for follicle counting. Female offspring from CR dams tended to have increased bodyweight compared with offspring from control females (P=0.08). Interestingly, fewer primordial follicles (60% reduction; P=0.001), transitional follicles (P=0.0006) and total follicles (P=0.006) were observed in offspring from CR mothers. The number of primary, secondary and tertiary follicles did not differ between the groups (P>0.05). The CR offspring had fewer DNA double-strand breaks in primary follicle oocytes (P=0.03). In summary, CR during the second half of gestation decreased primordial ovarian follicle reserve in female offspring. These findings suggest that undernutrition during the second half of gestation may decrease the reproductive lifespan of female offspring.

127 Chemokine receptor 2 (CCR2) is expressed in growing oocytes, and its deficiency affects follicular activation and long-term female fertility in mice

Chemokine receptor 2 (CCR2) has important functions in several biological processes, including activation of PI3K/Akt/mTOR signalling, a key pathway in follicular activation. However, there is no report about the role of CCR2 in ovarian follicular physiology. The objectives of this study were (1) to immunolocalize CCR2 in mice ovaries and (2) to evaluate the effects of CCR2 deficiency on follicular growth during adult life and aging. A total of 74 C57Bl/6 (wild type, WT) and 68 B6.129S4-Ccr2tm1Ifc/J (CCR2−/− (knockout)) female mice were used. For objective 1, ovaries were collected from WT mice at 1.5 months old (m.o.), fixed in 4% PFA, embedded in paraffin, and used for immunoperoxidase staining with an anti-CCR2 antibody [EPR19698] (ab222496; 1:200) and using anti-rabbit IgG (Ab6721, 1:100) as a secondary antibody. Also, MII oocytes from oviducts of superovulated WT mice were processed with the same primary antibody for immunofluorescence. For objective 2, body and ovarian weight were evaluated. Follicle populations were assessed in WT and CCR2−/− mice at 1.5, 2.5, 6, 10, and 12m.o., by serial sectioning; the total follicle population was counted in every third section in the whole ovary. Additionally, ovarian total RNA isolation was performed from WT and CCR2−/−. Real-time PCR was used to evaluate differential gene expression according to standard protocols, using primers for Bax, Casp3, Bcl2, Fshr, and B-actin (endogenous control). The data were analysed using the GraphPad Prism Software, using t-test, and a P-value of 0.05 was considered as significant. Localization of CCR2 was observed exclusively on the membrane and cytoplasm of growing oocytes in primary, secondary, antral, and atretic follicles, as well as on ovulated MII oocytes membrane and cytoplasm. Body and ovarian weight were similar between WT and CCR2−/− mice. At 1.5m.o., CCR2−/− mice had more primordial follicles and fewer primary and secondary follicles compared with WT (P&amp;lt;0.05), whereas there was no difference in the antral follicle populations. Follicular activation (primordial to primary transition) and atresia rates were decreased in CCR2−/− (P&amp;lt;0.05) at 1.5m.o. A downregulation of pro-apoptotic genes (Bax and Casp3) was observed on CCR2−/− (P&amp;lt;0.05), while anti-apoptotic Bcl2 was upregulated (P&amp;lt;0.05) compared with WT in 1.5-m.o. animals. A larger ovarian follicular reserve at 1.5, 2.5, and 6m.o., but not at 10 or 12m.o., was observed in CCR2−/− mice. At 6 to 12m.o., CCR2−/− ovulated more (P&amp;lt;0.05) MII oocytes than WT. Altogether, these data may suggest that CCR2 plays an important role in the regulation of ovarian follicular reserve mobilization, potentially affecting reproductive lifespan. Financial support was provided by Fapemig and CNPq.

128 Study of preantral ovarian follicular population in fetal alpaca (Vicugna pacos) ovaries

In mammals, folliculogenesis begins at the fetal stage and is a complex, dynamic process that involves follicular quiescence, activation, growth, follicular migration, and cell interactions. At birth, the preantral ovarian follicular population, drastically reduced, constitutes &amp;gt;90% of all ovarian follicles, representing the ovarian reserve that will be used throughout the reproductive life. In alpacas, changes in follicular wave growth patterns and ovulation are different from other species; thus, it is important to know the ovarian reserve at fetal stage, as a starting point for future studies. Therefore, the aim of the present study was to establish the morphological characterisation and estimate the population of alpaca preantral follicles in the fetal stage. Ovaries from alpacas (n=5) in the fetal stage (fetus during the last third of gestation) were collected at a local slaughterhouse. Whole ovaries were individually fixed in 4% paraformaldehyde phosphate-buffered saline overnight at room temperature for routine histology. Ovaries were dehydrated in alcohol, cleared with xylene, and embedded in paraffin, and all tissue was serially sectioned at 7μm with a rotating microtome (Leica). The histological sections were mounted and stained with periodic acid-Schiff and hematoxylin. Preantral follicles were classified according to their developmental stage: primordial (one layer of flattened granulosa cells surrounding the oocyte), primary follicle transition (flattened cuboidal granulosa cells surrounding the oocyte), primary (single layer of cuboidal granulosa cells around the oocyte), or secondary (oocyte surrounded by more than one complete layer of cuboidal granulosa cells). We estimated the number of preantral follicles by counting all follicles in each histological section. Only follicles in which the oocyte nucleus was visible were counted. In addition, for each follicle category (n=40 per group), oocyte and follicle diameters were measured using an ocular micrometer. The variable means were compared using unpaired Student's t-test analysis, with significance set at P ≤ 0.05. Estimation of preantral follicular population (mean±standard deviation) was 80 516±14 575 in the ovaries of alpaca fetuses. Most of the follicles found belong to the primordial (49.2%) or primary follicle transition (39.2%) categories, followed by primary (10.8%) and secondary (0.8%) stages. Follicle and oocyte diameters of primordial (33.3±7.2; 21.5±4.6μm) and primary follicle transition stages (36.7±3.0; 23.4±2.6μm) were significantly (P&amp;lt;0.05) smaller than those of primary-stage follicles (77.9±15.8; 50.02±11.1μm). Finally, preantral follicles classified with normal morphological integrity appearance for each developmental stage were 98.2% (primordial), 96.7% (primary follicle transition), 91.5% (primary), and 88.1% (secondary), respectively. In conclusion, this study shows for the first time an estimation of the population of preantral follicles in alpaca fetal ovaries and establishes follicle and oocyte diameters and their normal morphological integrity.

12 Assessment of oocyte and embryo production in senescent beef cows

Fertility associated with age has been poorly documented in cattle. There is evidence that more than 50% of cows became infertile (failure to bear a calf for two successive years) at the age of 15. The causes of infertility in cows have been attributed to poor oocyte quality and/or follicle depletion. The present study aimed to compare the rates of oocyte and invitro embryo production of fertile cows older than 15 years. Sixteen Nellore cows (Bos taurus indicus) 200.3±4.9 months old were submitted to two ovum pickup series (OPU1 and OPU2) at a 2-year (2017 and 2019) interval. Before the OPU1 of each series, the cows were prepared with an IM injection of 2mg estradiol benzoate and 0.15mg prostaglandin F2α analogue and a progesterone-releasing intravaginal device (1g). After five days, the progesterone-releasing intravaginal device was withdrawn and the OPU1 was performed. The second OPU (OPU2) was performed 5 days later to aspirate only the growing follicles. The number and morphologic quality of recovered cumulus-oocyte complexes were registered. After IVM (tissue culture medium 199) and fertilization (Tyrode's solution) of cumulus-oocyte complexes, the presumptive zygotes were cultured in synthetic oviductal fluid with amino acids to assess developmental rates to blastocyst. Data were analysed by chi-squared and Wilcoxon test. The number of oocytes recovered at OPU1 was higher in 2107 than in 2019 (27.8±6.6 vs. 10.8±4.2; P&amp;lt;0.01), whereas in OPU2 the production of oocytes was not different (14.7±3.3 vs. 10.9±3.1). In OPU1, eight cows in 2017 and four in 2019 produced more than 20 oocytes, compared with only one cow in OPU2 of both years (P=0.02). Although there was no significant difference in the proportion of viable oocytes in OPU1 (83.6% and 87.2%) and OPU2 (85.5 and 68.0%), more viable oocytes were produced at OPU1 of 2017 (23.2±5.8) compared with OPU1 of 2019 (9.4±4.1), OPU2 of 2017 (12.6±2.7) and OPU2 of 2019 (7.4±2.4), respectively (P&amp;lt;0.05). There was no significant difference in the proportion of cleaved oocytes in OPU1 (74.2 and 75.1%) and OPU2 (68.9 and 79.8%). Two cows in 2017 (12.5%) and five in 2019 (31.2%) did not produce embryos in both OPUs. Cows produced more embryos (blastocysts) in OPU1 at 2017 (7.3±1.6) than 2019 (2.8±0.6; P&amp;lt;0.05), whereas in OPU2 there was no difference (4.1±0.8 and 3.1±0.8). In conclusion, senescent fertile cows decreases the number of oocytes and embryos as they approach 20 years of age; however, the quality of oocytes does not appear to be compromised. Financial support was provided by FAPESP (proc. 2018/07450-5).

Abundances of autophagy-related protein LC3B in granulosa cells, cumulus cells, and oocytes during atresia of pig antral follicles

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 (P < 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.

NMN does not protect the ovarian reserve from cancer treatments.

Primordial follicle oocytes are extremely vulnerable to DNA damage caused by exogenous agents, such as those commonly used to treat cancer. Consequently, female cancer patients often have diminished ovarian reserve, which if severe enough, can cause premature ovarian failure and early menopause. Advances in cancer therapies have resulted in significantly improved cancer survival rates; therefore, it is becoming increasingly important to devise strategies to protect the ovarian reserve from cancer treatments, to avoid loss of fertility and endocrine dysfunction. In this study, we aimed to determine whether supplementation with nicotinamide mononucleotide (NMN) could preserve the ovarian reserve following exposure to DNA-damaging cancer treatments. Adult female mice (n = 5-6/group) received saline or NMN (500 mg/kg/day) for 8 days. Mice were left untreated or exposed to γ-irradiation (0.1 Gy) or cyclophosphamide (150 mg/kg) on day 7 and ovaries and serum collected for analysis on day 12. We report that γ-irradiation treatment significantly reduced the number of primordial follicles, but supplementation with NMN did not prevent the observed follicle loss. Similarly, cyclophosphamide treatment significantly reduced primordial follicle numbers, but these losses were not prevented by NMN supplementation. In conclusion, depletion of the ovarian reserve following γ-irradiation or cyclophosphamide was not protected by NMN supplementation under the conditions employed in this study.

The effect of tamoxifen on IGF signaling pathway in the mouse ovary

Tamoxifen (TAM) is one of selective estrogen receptor modulators used in breast cancer treatment and prevention. The objective of this study was to determine whether or not insulin-like growth factor-I (IGF-1) and its receptor (IGF-1R), has any role in the effect mechanism of TAM on the ovary. Experimentally, animals were divided into three groups as control group (n= 20), low dose TAM treatment group (0.5 mg/mouse/day, n= 20) and high dose TAM treatment group (1.5 mg/mouse/day, n= 20). TAM was injected 0.5 and 1.5 mg/mouse/day for 5 days. Ovarian sections were used to examine the general structure by trichrome staining method and to determine IGF-1 and IGF-1R expressions by immunohistochemical staining method. After the experiment, the presence of atretic follicles and small cystic structures in the TAM-treated animals was determined. Also, antral follicles and the corpus luteum were much less in the high dose TAM group than in the control. TAM did not change the expression of IGF-1 in granulosa cells, but increased the expression of IGF-1R. In TAM groups, IGF-1 and IGF-1R expression were increased in oocytes of follicles and in interstitial cells depending on TAM doses. However, while IGF-1 expression was unchanged in the corpus luteum, decreased in treatment group. TAM generally stimulated IGF-1 and IGF-1R expression in a dose-dependent manner. The results suggest that IGF-1 signaling pathway is involved in the mechanism of action of TAM on the ovary. We may assert that it may be useful to use IGF-1 signaling pathway regulators to adjust the effects of TAM on the ovary.

Ovarian Activity and the Size of Oocytes in Ovarian Follicles in Sexually Mature Gilts in Individual Seasons

Abstract The aim of this study was to evaluate ovarian activity and the size of oocytes in ovarian follicles in sexually mature Landrace-Yorkshire gilts in relation to the individual seasons of the year. The study was carried out on 240 gilts slaughtered at an abattoir during the four yearly seasons. The size and weight of the ovaries, the number of follicles and corpora lutea (CL) according to individual size categories were evaluated. The oocytes were aspirated from follicles and their sizes were measured. Our evaluation of the size of the ovaries showed that they were the largest in autumn, when their mean length reached 25.8 ± 3.4 mm, while in winter their mean length was 24.2 ± 2.9 mm. The smallest weight of the ovaries was determined in autumn (mean 5.7 ± 1.4 g) and the highest in spring (mean 6.2 ± 2.2 g). The largest number of follicles in the ovaries of the gilts was recorded during the autumn months, with a predominance of follicles up to 3 mm (mean number 17.9 ± 7.5). The largest number of corpora lutea was observed in spring (mean number 12.1 ± 2.6) and the smallest in winter (mean number 6.1 ± 1.1). The oocytes from follicles of up to 3 mm size, were the smallest in spring (mean size 16.99 × 103 ± 3.42 × 103 µm2) and the largest in winter (mean size 18.90 × 103 ± 2.99 × 103 µm2). In total, the largest oocytes were aspirated from 4—6 mm follicles in autumn (mean size 19.60 × 103 ± 5.37 × 103 ± µm2). The values recorded indicated that the seasons affected the ovarian activity and the growth of oocytes in gilts.

Chemotherapy causes primordial follicle death in the human ovary via multiple apoptotic pathways and not by “burn out”

It has been proposed that gonadotoxic chemotherapy results in the “burn out” of primordial follicle reserve by activating PI3K/PTEN/Akt signaling pathway. Others have challenged this concept and put forward DNA damage and apoptosis as the main mechanism of follicle loss. We conducted this study to answer this controversy and conclusively determine the mechanism of chemotherapy-induced damage to ovarian reserve in women. Ovarian cortical pieces from organ donors aged ≤32 years were xenografted subcutaneously to SCID mice (n=12 mice/ tissue from 4 donors each). After 10 days, the mice were given an injection of cyclophosphamide (75mg/kg) or the vehicle. The tissues were recovered 12 hours later. The recovered xenografts were assessed for apoptosis by anti-caspase-3 (AC3) and DNA double strand breaks by γ-H2AX immunostaining as well as follicle growth initiation rate (FGIR) by primary/primordial follicle ratios. Single primordial follicle oocytes were laser captured for RNA sequencing and single cell quantitative real time PCR (qRT-PCR) to determine the signaling pathways activated in response to chemotherapy exposure. Immunofluorescence microscopy and image analysis were used to assess phosphorylation of PI3K/PTEN/Akt pathway and Bad-Bcl2 co-localization. There was no difference in the primordial follicle growth initiation rate (FGIR) between the vehicle and the cyclophosphamide-treated xenografts (p=0.35). However, cyclophosphamide caused a significant increase in the percentage of apoptotic (65.02±3.55% vs. 28.27±5.77%; p=0.001) and DNA-damaged (82± 2.7% vs. 23.82±8.66%;p=0.0001) primordial follicles . Ingenuity Pathway Analysis (IPA) of the RNA sequencing data from laser captured primordial follicles showed significant decrease in the expression of PECAM (p=0.00009), IKBKE (P=0.0001), and ANGPT1 (p=0.003) after cyclophosphamide treatment. These are positive regulators of the Akt pathway and hence results indicate that cyclophosphamide suppresses rather than activating Akt. In addition, IPA predicted that suppression of these pathways favor apoptosis in single primordial follicles. By qRT-PCR analyses, there was no change in the expression of Akt (p=0.9). Further, cyclophosphamide treatment did change the expression of the phospho-Akt (P=0.14), phospho-Foxo3a (p=0.17), or phospho-rpS6 (0.48) compared to the vehicle. By qRT-PCR, chemotherapy increased the expression of the anti-apoptotic Bcl2 (p=0.0003) accompanied by enhanced colocalization of the pro-apoptotic BAD-Bcl2 complex (p=0.006) in the primordial follicles, confirming that cyclophosphamide induces follicle death via apoptosis. This single cell transcriptomic and immunohistochemical analysis of human primordial and primary follicles prove that gonadotoxic chemotherapy agents do not cause follicle activation; they rather create a pro-apoptotic state resulting in massive loss of ovarian reserve. Future research on pharmacological fertility preservation should target preventing DNA damage and apoptosis rather than follicle activation.

Doxorubicin obliterates mouse ovarian reserve through both primordial follicle atresia and overactivation

Ovarian toxicity and infertility are major side effects of cancer therapy in young female cancer patients. We and others have previously demonstrated that doxorubicin (DOX), one of the most widely used chemotherapeutic chemicals, has a dose-dependent toxicity on growing follicles. However, it is not fully understood if the primordial follicles are the direct or indirect target of DOX. Using both prepubertal and young adult female mouse models, we comprehensively investigated the effect of DOX on all developmental stages of follicles, determined the impact of DOX on primordial follicle survival, activation, and development, as well as compared the impact of age on DOX-induced ovarian toxicity. Twenty-one-day-old CD-1 female mice were intraperitoneally injected with PBS or clinically relevant dose of DOX at 10 mg/kg once. Results indicated that DOX primarily damaged granulosa cells in growing follicles and oocytes in primordial follicles and DOX-induced growing follicle apoptosis was associated with the primordial follicle overactivation. Using the 5-day-old female mice with a more uniform primordial follicle population, our data revealed that DOX also directly promoted primordial follicle death and the DNA damage-TAp63α-C-CASP3 pathway was involved in DOX-induced primordial follicle oocyte apoptosis. Compared to 21-day- and 8-week-old female mice that were treated with the same dose of DOX, the 5-day-old mice had the most severe primordial follicle loss as well as the least degree of primordial follicle overactivation. Taken together, these results demonstrate that DOX obliterates mouse ovarian reserve through both primordial follicle atresia and overactivation and the DOX-induced ovarian toxicity is age dependent.

Ovarian cholesterol efflux: ATP-binding cassette transporters and follicular fluid HDL regulate cholesterol content in mouse oocytes†

High density lipoproteins (HDL) take up cholesterol from peripheral tissues via ABC transporters and deliver it to the liver via scavenger receptor class B type I (SR-B1). HDL are the main lipoproteins present in follicular fluid (FF). They are thought to derive from plasma, but their origin is still controversial. SR-B1 knock-out (KO) mice have provided important evidence linking HDL metabolism and female fertility. These mice have cholesterol-rich circulating HDL and female infertility that can be restored by treating mice with the cholesterol-lowering drug probucol. Ovulated oocytes from SR-B1 KO females are dysfunctional and show excess cholesterol. The mechanisms explaining the contribution of FF HDL to oocyte cholesterol homeostasis are unknown. Here, using quantitation of filipin fluorescence we show that in SR-B1 KO ovaries, cholesterol excess is first observed in immature oocytes in antral follicles. By performing cross-transplant experiments between WT and apolipoprotein A-I deficient (ApoA-I KO) mice, which lack the main protein component of HDL, we provide evidence supporting the plasmatic origin of FF HDL. Also, we demonstrate that probucol treatment in SR-B1 KO females results in lowering of cholesterol content in their oocytes. Incubation of oocytes from SR-B1 KO mice with purified WT HDL reduces their cholesterol content, suggesting that HDL promote efflux of excess cholesterol from oocytes. In agreement with this hypothesis, we identified ABC transporters in oocytes and observed that ABCA1 KO oocytes have excess cholesterol and lower viability than WT oocytes.

The use of pituitary adenylate cyclase-activating polypeptide in the pre-maturation system improves in vitro developmental competence from small follicles of porcine oocytes

ObjectiveWe investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles.MethodsWe divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (Pre-SF[−]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF).ResultsCleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(−)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(−)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05).ConclusionThe PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

Ovarian and follicular variables used to determine ewes with different FecGE genotypes

Based on ovarian and follicular variables, there was determination of ewes with different FecGE genotypes. Based on the FecGE genotype, 65 Santa Inês ewes were assigned to three experimental groups: homozygous wild-type (n = 25; FecG+/+), mutant heterozygous (n = 27; FecG+/E) and mutant homozygous (n = 13; FecGE/E). The ewe’s ovaries were weighed and measured, then the follicles (oocyte, nucleus and nucleolus) were histologically evaluated for morphometry and morphology. Morphologically normal follicles, in the primordial and transitional stages, explained 70.18% of the variability morphological characteristics between mutant and wild-type ewes. Conducting the morphometric evaluation resulted in a more precise determination of the genotype groups when there was assessment of the primordial and secondary follicular developmental stages. The diameter of the oocyte and the oocyte nucleus of the primordial follicles explained 36.76% of the variability in follicular morphology between ewes with the mutation and those with the wildtype group. Similarly, the core diameter of oocytes in secondary follicles explained 10.63% of the variability in follicular morphology among FecGE/E, FecG+/E and FecG+/+ ewes. Thus, morphologically normal follicles in the primordial and transitional stages of development are the variables that allow for a more precise differentiation of Santa Inês ewes with the FecGE mutation. These variables may be evaluated to make more efficient the adoption of biotechniques that when conducted there is utilisation of follicles in the initial developmental stages as a physiological basis for classifying whether specific follicles are useful when conducting the techniques.