Oocytes In Ovarian Follicles Research Articles

Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice.

In oocyte biology, the zona pellucida has long been known to operate three extracellular functions downstream of the secretory pathway, namely, encasing the oocytes in ovarian follicles, mediating sperm-oocyte interaction, and preventing premature embryo contact with oviductal epithelium. The present study uncovers a fourth function that is fundamentally distinct from the other three, being critical for embryonic cell survival in mice. Intriguingly, the three proteins of the mouse zona pellucida (ZP1, ZP2, ZP3) were found abundantly present also inside the embryo 4 days after fertilization, as shown by mass spectrometry, immunoblotting, and immunofluorescence. Contrary to current understanding of the roles of ZP proteins, ZP3 was associated more with the cytoskeleton than with secretory vesicles in the subcortical region of metaphase II oocytes and zygotes, and was excluded from regions of cell-cell contact in cleavage-stage embryos. Trim-away-mediated knockdown of ZP3 in fertilized oocytes hampered the first zygotic cleavage, while ZP3 overexpression supported blastocyst formation. Transcriptome analysis of ZP3-knockdown embryos pointed at defects of cytoplasmic translation in the context of embryonic genome activation. This conclusion was supported by reduced protein synthesis in the ZP3-knockdown and by the lack of cleavage arrest when Trim-away was postponed from the one-cell to the late two-cell stage. These data place constraints on the notion that zona proteins only operate in the extracellular space, revealing also a role during the oocyte-to-embryo transition. Ultimately, these data recruit ZP3 into the family of maternal factors that contribute to developmental competence of mouse oocytes.

O-238 Critical requirement of intracellular zona pellucida proteins for mouse embryo survival

Abstract Study question Are the intracellular deposits of zona pellucida (ZP) proteins relevant to embryonic development? Summary answer Contrary to longstanding views, ZPs are not only oocyte-specific proteins with extracellular tasks, but also have intracellular tasks that are essential for embryo survival. What is known already Mutation studies always defined the ZP functions as extracellular, namely: to encase oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, ZP mutations cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryo and oviductal epithelium. However, when these limitations were obviated in mice by monospermic fertilization in vitro and blastocyst transfer to uterus, the concepti of ZP2-null and ZP3-null oocytes were still unviable (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed. Study design, size, duration After monospermic fertilization, wild-type mouse oocytes were depleted of intracellular ZP proteins using the ‘Trim-away’ method (PMID 29153837). ZP1-3 were targeted by prevalidated antibodies (producing a dominant band in Western blot) and committed for proteasomal degradation by the ubiquitine ligase Trim21. Antibodies were either folded or denatured (heat-inactivated), thereby allowing for efficient or abolished depletion, respectively (negative control). The two groups were examined for development and efficiency of ‘Trim-away’ at degrading the ZPs. Participants/materials, setting, methods Mature B6C3F1 female mice were stimulated with gonadotropins (eCG, hCG) and mated to CD1 stud males to collect fertilized superovulated oocytes. These were microinjected with a cocktail of mCherry-Trim21 mRNA and anti-ZP antibody, followed by culture in KSOM(aa) medium for 96 hours. Successful depletion of ZP proteins was measured by mCherry fluorescence, ZP Western blotting and mass spectrometry. Developmental rates were compared between the groups of the folded vs. denatured antibodies. Main results and the role of chance Fertilized oocytes depleted of ZP3 did not cleave and remained arrested at 1-cell stage (0 blastocysts / 300 oocytes; 7 replicates). Oocytes depleted of ZP2 cleaved once or twice, and remained arrested at 2- or 4-cell stage (0 blastocysts/ 100 oocytes; 3 replicates). These failures stood in contrast to the blastocyst progression of oocytes depleted of ZP1 (42 blastocysts / 100 oocytes; 3 replicates) and negative controls injected with either of the denatured antibodies (>50% blastocysts) (p < 0.01; chi test). Molecular efficacy of ‘Trim-away’ was manifest from the decline of mCherry fluorescence, and from the reduction of the ZP signal intensity in Western blot and mass spectrometry; whereas the negative controls remained bright-fluorescent and retained the dominant band in Western blot. Inspection of publicly available datasets from two-hybrid and affinity capture screens reveals that ZP3 has more than 20 interaction partners in cell lines. Collectively, this information supports that ZP proteins are required for developmental competence not only outside but also inside of oocytes, where the ZPs might interact with more proteins than just ZP1, ZP2 and ZP3. Our study provides novel insight into the pathogenic mechanism of ZP gene mutations, adding a novel intracellular route to the longstanding extracellular roles. Limitations, reasons for caution Although there is sufficient evidence for expanding on the tasks of ZPs in mice, human oocytes contain ZP4 in addition to ZP1-3. While the novel tasks in mice rely on stable protein deposits that persist from oocyte to blastocyst, it is not known if the human counterparts persist that long. Wider implications of the findings ZPs may qualify as ‘moonlighting proteins’ that enrich - but at the same time also complicate - the determination of primary infertility phenotype from genotype. Trial registration number not applicable

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

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.

Previtellogenic and vitellogenic oocytes in ovarian follicles of cultured siberian sturgeon Acipenser baerii (Chondrostei, Acipenseriformes).

Previtellogenic and vitellogenic oocytes in ovarian follicles from cultured Siberian sturgeon Acipenser baerii were examined. In previtellogenic oocytes, granular and homogeneous zones in the cytoplasm (the ooplasm) are distinguished. Material of nuclear origin, rough endoplasmic reticulum, Golgi complexes, complexes of mitochondria with cement and round bodies are numerous in the granular ooplasm. In vitellogenic oocytes, the ooplasm comprises three zones: perinuclear area, endoplasm and periplasm. The endoplasm contains yolk platelets, lipid droplets, and aggregations of mitochondria and granules immersed in amorphous material. In the nucleoplasm, lampbrush chromosomes, nucleoli, and two types of nuclear bodies are present. The first type of nuclear bodies is initially composed of fibrillar threads only. Their ultrastructure subsequently changes and they contain threads and medium electron dense material. The second type of nuclear bodies is only composed of electron dense particles. All nuclear bodies impregnate with silver, stain with propidium iodide, and are DAPI-negative. Their possible role is discussed. All oocytes are surrounded by follicular cells and a basal lamina which is covered by thecal cells. Egg envelopes are not present in previtellogenic oocytes. In vitellogenic oocytes, the plasma membrane (the oolemma) is covered by three envelopes: vitelline envelope, chorion, and extrachorion. Vitelline envelope comprises four sublayers: filamentous layer, trabecular layer 2 (t2), homogeneous layer, and trabecular layer 1 (t1). In the chorion, porous layer 1 and porous layer 2 are distinguished in most voluminous examined oocytes. Three micropylar cells that are necessary for the formation of micropyles are present between follicular cells at the animal hemisphere. J. Morphol. 278:50-61, 2017. ©© 2016 Wiley Periodicals,Inc.

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.

Concentrations of bone morphogenetic protein-15 (BMP-15) and growth differentiation factor-9 (GDF-9) in follicular cysts, mono - and polyoocyte follicles in gilts

Abstract The objective of the study was to determine the concentration of BMP-15 and GDF-9 in the fluid of follicular cysts and ovarian follicles, and to compare their concentrations in mono- and polyoocyte follicles in gilts. The study involved two experiments conducted on the ovaries collected post-slaughter from gilts (7-8 months old). The first experiment covered 31 follicular single cyst gilts (15-25 mm in diameter) and 41 gilts without cysts. Follicular fluid from follicles of 8-10 mm in diameter (n=41) and 5-8 mm in diameter (n=41), and cystic fluid (n=31) were collected for analysis. The second experiment involved collecting follicular fluid from poly- (n=19) and monooocyte (n=22) follicles. The concentration of BMP-15 and GDF-9 was then determined in the samples using specimen-specific ELISA kits. The differences in the concentration of these factors were calculated by means of analysis of variance and a posthoc test. Duncan’s multiple range test was used to verify the significance of differences at P<0.05 and P<0.01. In addition, correlations between the factors were calculated. BMP-15 and GDF-9 levels in the cystic fluid were significantly higher than those in the follicular fluid (P<0.01). However, no differences were observed between various size follicles or between mono- and polyoocyte follicles. BMP-15 and GDF-9 concentrations were found to be positively correlated (P<0.01). Differences in BMP-15 and GDF-9 concentrations in ovarian follicles and follicular cysts, as evidenced by our study, indicate that these factors may be related to folliculogenesis disorders in gilts. What is more, the number of oocytes in ovarian follicles does not influence the intrafollicular concentration of BMP-15 and GDF-9.

Aromatase-null mice expressing enhanced green fluorescent protein in germ cells provide a model system to assess estrogen-dependent ovulatory responses

Enhanced green fluorescent protein (EGFP) has provided us with valuable approaches for tracking living cells. We established a novel line of transgenic mice, which express EGFP in the testis and ovary. Histological analysis demonstrated that spermatids in the testis and oocytes in ovarian follicles beyond preantral stages were positive for EGFP. By exploiting these features, we evaluated ovulatory responses of aromatase-gene (Cyp19a) knockout mouse expressing the EGFP transgene, which is totally anovulatory due to 17β-estradiol (E2) deficiency. Ovulation in the knockout mice was induced by sequential injections of E2 on days 1, 4 and 5, pregnant mare serum gonadotropin on day 4 and human chorionic gonadotropin on day 6. Fluorescent oocytes were readily detectable at 15 h after the last gonadotropin injection in the oviduct under a fluorescence stereomicroscope, even when only one oocyte was present. However, when E2 supplementation on day 4 or day 5 in the regimen was omitted, no ovulated oocytes were detected, indicating that exogenous E2 supplementation at the time of gonadotropin stimulation is necessary to induce ovulation in aromatase-gene knockout mice. Our results further demonstrated that the current mouse line can provide an alternative tool to study germ cell biology, including oogenesis, ovulation and senescence.

Oocyte-derived R-spondin2 promotes ovarian follicle development.

R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.

The Expression Patterns of Reg IV Gene in Normal Rat Reproduction System

Reg IV, the latest member of the regenerating gene family, has been documented in different tissues of human and rat, such as the colon, small intestine, stomach, and pancreas. Expression of Reg IV gene in distinct cell types has been correlated with its various functions in regeneration, cell growth and survival, proliferation and differentiation, cell adhesion, and resistance to apoptosis. However, there was no evidence to show whether the Reg IV protein is present in the reproductive system of normal rat. The aim of this study was to reveal the expression patterns of Reg IV in rat ovary and uterus. The expression of Reg IV was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at mRNA and protein levels, respectively. The localization of Reg IV protein within rat ovary and uterus was investigated by immunohistochemistry (IHC). Our results showed that the expression of Reg IV in ovary was significantly higher than that in the uterus. The strong immunoreactive signals of Reg IV was observed in granulosa cells and oocytes of ovarian follicles, corpus luteum, and interstitial cells in rat ovary; only weak signals were detected in luminal and gland epithelium of rat endometrium. These findings first demonstrate the expression of Reg IV in ovary and uterus of the healthy rat at both mRNA and protein levels. It provides an evidence of Reg IV expression in rat reproductive system, which may help elucidate a potential role in cell growth and proliferation of reproductive system.

Passage of 17 kDa calmodulin through gap junctions of three vertebrate species

Gap junctions of some vertebrates are capable of passing the elongate molecule, calmodulin, with a molecular weight 8–17 times greater than the previously recognized size limits. Fluorescently labeled calmodulin (FCaM) (17.34 kDa) microinjected into oocytes of ovarian follicles from an amphibian, Xenopus laevis, and from two species of teleost fish, Danio rerio (Zebrafish) and Oryzias latipes (Medaka), is shown to transit their gap junctions and enter the surrounding epithelial cells. Passage of FCaM was terminated when follicles were first treated with 1 mM octanol, a molecule known to down-regulate gap junctions. There was no FCaM detected in the surrounding medium, nor did epithelial cells become fluorescent when follicles were incubated in medium containing dye. Calmodulin is well known to modulate many cytoplasmic reactions; thus, its passage through gap junctions opens possibilities of additional means by which cells may be supplied with this signaling molecule, and by which their supply may be regulated.

Reduced GLUT1 expression in Igf1-/- null oocytes and follicles.

Granulosa cells provide nutritional and trophic support for growing oocytes in ovarian follicles. The granulosa cells closest to the oocyte produce abundant insulin-like growth factor 1 (IGF1) and the IGF1 receptor is highly expressed by oocytes, suggesting that granulosa-derived IGF1 may have trophic effects on oocyte growth and development. To investigate this possibility, in the present study we used in situ hybridization and immunohistochemistry to examine glucose transporter (GLUTs 1, 3 & 4) expression in follicles from pre-pubertal Igf1-/- and littermate wildtype (wt) mice. Pre-pubertal mice were used for this study because the Igf1 null mice do not mature sexually. GLUT1 mRNA and immunoreactivity were most abundant in oocytes and in granulosa cells immediately surrounding the oocyte. Expression of this transporter was significantly reduced in Igf1 null oocytes and granulosa cells and was restored by exogenous IGF1 treatment to wt levels. These effects on GLUT expression were significant at both the mRNA and immunoreactive protein levels. Oestrogen treatment significantly increased GLUT1 levels in oocytes and granulosa from both wt and Igf1-/-, although the latter were still significantly lower than wt. Oocyte glycogen stores, determined by PAS staining, did not appear different in Igf1-/- and wt mice.GLUT3 was expressed in thecal cells surrounding growing follicles and was not appreciably different in Igf1 null compared with wt ovaries. GLUT4 expression was not detected in the prepubertal mouse ovary. Together with observations from previous studies showing that ovulation is blocked in Igf1 null mice, the present data suggest that IGF1's augmentation of granulosal and oocyte GLUT1 expression may be essential for oocyte maturation and successful ovulation.

Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation.

The zona pellucida (ZP), a glycoprotein layer that encloses the mammalian oocyte, is formed during follicular development in the ovary, persists at the time of fertilization within the oviduct, and then surrounds the embryo until implantation in the uterus. Although the structure and chemical properties of the ZP have been extensively studied, the precise site of origin of the ZP remains a matter of controversy. Moreover, the mechanism of synthesis and secretion of the ZP constituents is not fully elucidated. We have recently developed monoclonal antibodies (MAbs) against oviductal ZP of the golden hamster. We have used one of these MAbs (an immunoglobulin G) and the protein A-gold technique to study the localization of the corresponding antigenic sites, and we report here their distribution in the oviduct and within the cumulus oophorus complex of the superovulated hamster. In the oviductal epithelium, immunolabeling was observed in non-ciliated secretory cells in structures involved in protein secretion. In the cumulus masses collected from the oviduct, the sites of immunoreactivity were localized exclusively in the ZP encompassing the oocyte. Gold particles were evenly distributed throughout the entire thickness of the ZP. Treatment of the cumulus masses with hyaluronidase prior to preparation of isolated oocytes for immunocytochemistry did not affect this uniformity. The ZP of the preovulatory oocytes in ovarian follicles was not labeled. Our study provides immunocytochemical evidence for the secretion of an oviductal antigen that becomes intimately associated with the ZP of the oocytes during their passage through the oviduct.

Occurrence or Morphologically Changed Oocytes in Ovarian Follicles of Cows During the Reproductive Cycle

Occurrence or Morphologically Changed Oocytes in Ovarian Follicles of Cows During the Reproductive Cycle