•TNF-α system members are expressed at different follicular stages in bovine ovaries.•TNF-α reduces follicular survival in cultured bovine ovarian tissue.•TNF-α increases the total number of ...apoptotic cells in cultured follicles.•Dexamethasone improves ultrastructure of bovine follicle cultured in vitro.
This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM+ supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM+). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue.
During the last 10 to 15 yr, in vitro research to predict antral follicle growth and oocyte maturation has delivered interesting advances in the knowledge of processes regulating follicle growth and ...developmental competence of oocytes. This review discusses the contribution of cumulus and mural granulosa cells in the process of oocyte maturation and cumulus expansion in cumulus–oocyte complexes (COCs) from follicles of different sizes and shows that differences in gene expression in oocytes, granulosa, and theca cells of small and large follicles impact the success of in vitro blastocyst development. In addition, the molecular mechanisms by which COC metabolism and antioxidant defense provide oocyte competence are highlighted. Furthermore, new insights and perspectives on molecular and cellular regulation of in vitro oocyte maturation are emphasized.
•Role of hormones and growth factors on maturation of different-sized oocytes is discussed.•Maturation stage of granulosa cells is important for oocyte competence.•Cumulus–oocyte complex metabolism and antioxidant defense provide oocyte competence.•Perspectives are given on molecular and cellular regulation for in vitro oocyte maturation.
The peroxisome proliferator-activated receptor gamma (PPARG, also called NR1C3) is a nuclear receptor of the peroxisome proliferator-activated receptor family (PPAR). PPARs are involved in the ...regulation of apoptosis, cell cycle, estradiol and progesterone synthesis, and metabolism. However, the role of PPARs and their regulation during follicular development and ovulation in monovular species remain poorly understood. In this study, a well-established intrafollicular injection model was used to investigate if the PPARG participates in the regulation of dominant follicle development and ovulation in cattle. Findings from this study revealed that the relative mRNA abundance of PPARG was similar between dominant and subordinate follicles around follicle deviation, decreased after the LH surge, and increased before ovulation. In addition, a quadratic correlation was found between PPARG mRNA levels in granulosa cells and progesterone concentration in the follicular fluid. Intrafollicular injection of 50 μM Troglitazone (TGZ; a PPARG agonist) inhibited follicular growth and decreased CYP19A1 mRNA abundance in granulosa cells. These findings indicate that PPARG is involved in the regulation of steroidogenesis, follicle growth and ovulation in cattle.
•Activation of PPARG by synthetic agonist inhibit follicular growth in cattle.•Activation of PPARG decrease CYP19A1 mRNA abundance in granulosa cells.•mRNA abundance of PPARG decrease after the LH surge, and increase before ovulation.
This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro ...influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.
•The proteins by TNF-α and its receptors (TNFR1 and TNFR2), are present in antral follicles compartments.•GnRH treatment increases TNF-α mRNA level in granulosa cells of preovulatory follicles in vivo.•In vitro, expression of TNF-α mRNA in cumulus cells is also upregulated by gonadotropins.•TNF-α maintains ultrastructure and regulates gene expression during COC culture.
This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows ...and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM+ supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.
•Interleukin (IL) 1 system members are expressed in bovine follicles at different stages.•Follicular growth is followed by an increase in expression of interleukin 1 system.•Follicular wall is the main site of production of IL-1α, IL-1β, IL-1RA, and IL-1RI.•IL-1β promotes activation of bovine primordial follicles in vitro.
The purpose of this study was to determine the effects of in vivo GnRH treatment on mRNA expression of IL-1β system in bovine granulosa cells from preovulatory follicles and to analyze the in vitro ...effect of gonadotropins on the IL-1β system gene expression in cumulus cells. Moreover, the additive effect of IL-1β on cumulus expansion, gene expression and ultrastructural integrity of COCs cultured in vitro was evaluated. In vivo studies in granulosa cells demonstrated that IL-1RA mRNA levels were increased after 24h of GnRH treatment. Similarly, the presence of gonadotropins increased the levels of mRNAs for IL-1R1 and IL-1RA in cumulus cells after 24h of culture in vitro. The presence of IL-1β in culture medium did not influence cumulus expansion, but it increased the expression of mRNAs for IL-1β, IL-1RA, HAS2 and iNOS after COC culture. In addition, the ultrastructure of COCs cultured in vitro in the presence of IL-1β was maintained. In conclusion, IL-1RA mRNA is upregulated in granulosa cells at later stages of the ovulatory process in cattle. In cumulus cells, mRNA expression of IL-1RI and IL-1RA is induced by gonadotropins and IL-1β maintains the ultrastructure of COC cultured in vitro.
•IL-1RA mRNA is upregulated in bovine granulosa cells after in vivo GnRH treatment.•mRNA expression of IL-1RI and IL-1RA is induced by gonadotropins in cultured cumulus cells.•IL-1β increases iNOS mRNA in cultured cumulus cells in a time-dependent manner.•IL-1β maintains the ultrastructure of COC cultured in vitro.
Contents
The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We ...hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR‐1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH‐induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.
Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that ...they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2-6-nucleotide patch size) pathways need AP endonuclease to generate a 3' hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.
The spin-orbit coupling relating the electron spin and momentum allows for spin generation, detection and manipulation. It thus fulfils the three basic functions of the spin field-effect transistor. ...However, the spin Hall effect in bulk germanium is too weak to produce spin currents, whereas large Rashba effect at Ge(111) surfaces covered with heavy metals could generate spin-polarized currents. The Rashba spin splitting can actually be as large as hundreds of meV. Here we show a giant spin-to-charge conversion in metallic states at the Fe/Ge(111) interface due to the Rashba coupling. We generate very large charge currents by direct spin pumping into the interface states from 20 K to room temperature. The presence of these metallic states at the Fe/Ge(111) interface is demonstrated by first-principles electronic structure calculations. By this, we demonstrate how to take advantage of the spin-orbit coupling for the development of the spin field-effect transistor.
PDF