ABSTRACT
This study evaluated the effects of frutalin (0.6, 6.0 or 60.0⯵g/mL) and doxorubicin (0.3⯵g/mL) on survival, growth and ultrastructure of in-vitro cultured goat secondary follicles. The effects of these substances on the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 were also investigated. Results showed that, after 6â¯days of culture, frutalin or doxorubicin reduced the percentage of normal follicles (Pâ¯<â¯0.05), but doxorubicin had higher toxicity than frutalin. Except for follicles cultured with 0.6⯵g/mL frutalin, follicular growth rate was reduced after culture with doxorubicin or frutalin (Pâ¯<â¯0.05). The presence doxorubicin or 60.0⯵g/mL frutalin increased the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 (Pâ¯<â¯0.05). Higher mRNA levels for Casp3, Casp6 and Bax were found in follicles cultured with doxorubicin, but higher levels of Bcl2 mRNA were found in follicles cultured with frutalin (Pâ¯<â¯0.05). In conclusion, frutalin has lower toxic effects than doxorubicin on secondary follicles cultured in vitro.
Subject(s)
Antineoplastic Agents/pharmacology , Doxorubicin/pharmacology , Galectins/pharmacology , Goats , Ovarian Follicle/drug effects , Animals , Antineoplastic Agents/administration & dosage , Dose-Response Relationship, Drug , Female , Galectins/administration & dosage , Gene Expression Regulation/drug effects , Ovarian Follicle/ultrastructure , RNA, Messenger/genetics , Tissue Culture TechniquesABSTRACT
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.
Subject(s)
Dexamethasone/pharmacology , Gene Expression Regulation/drug effects , Ovarian Follicle/metabolism , Tissue Culture Techniques/veterinary , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Apoptosis , Cattle , Cell Survival , Female , Ovarian Follicle/ultrastructure , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
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.
Subject(s)
Gonadotropin-Releasing Hormone/pharmacology , Luteinizing Hormone/metabolism , Ovarian Follicle/drug effects , RNA, Messenger/metabolism , Receptors, Tumor Necrosis Factor, Type II/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Cattle , Cells, Cultured , Cumulus Cells/metabolism , Cumulus Cells/ultrastructure , Female , Gene Expression , Luteinizing Hormone/pharmacology , Oocytes/metabolism , Oocytes/ultrastructure , Ovarian Follicle/cytology , Ovarian Follicle/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type II/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
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-1ß 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-1ß (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-1ß, 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-1ß (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-1ß promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.
Subject(s)
Cattle/physiology , Interleukin-1/analysis , Interleukin-1beta/pharmacology , Ovarian Follicle/chemistry , Ovarian Follicle/physiology , RNA, Messenger/analysis , Animals , Blotting, Western , Female , Granulosa Cells/chemistry , Immunohistochemistry/veterinary , Interleukin 1 Receptor Antagonist Protein/analysis , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin-1/genetics , Interleukin-1beta/analysis , Interleukin-1beta/genetics , Oocytes/chemistry , Ovarian Follicle/drug effects , Real-Time Polymerase Chain Reaction/veterinary , Receptors, Interleukin-1 Type II/analysis , Receptors, Interleukin-1 Type II/genetics , Theca Cells/chemistryABSTRACT
The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (â¼0.2â mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200â µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1â µg/mL PHA (96.43%); 10â µg/mL PHA (84.85%); 50â µg/mL PHA (85.29%); 100â µg/mL PHA (88.57%), and 200â µg/mL PHA (87.50)], but the presence of 10â µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10â µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10â µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.
Subject(s)
Follicle Stimulating Hormone/metabolism , Mitogens/pharmacology , Ovarian Follicle/drug effects , Phytohemagglutinins/pharmacology , Animals , Female , Follicle Stimulating Hormone/genetics , Goats , In Vitro Techniques , Ovarian Follicle/growth & development , Ovarian Follicle/ultrastructure , Proliferating Cell Nuclear Antigen/metabolism , RNA, Messenger/metabolismABSTRACT
The objective of the present study was to determine the effects of bone morphogenetic protein (BMP)-15 and FSH on the growth, viability, and expression of mRNA for FSH (FSH-R) and BMP-15 (BMPR-IB and BMPR-II) receptors in cultured bovine secondary follicles. Secondary follicles were microdissected and cultured for 12 days in minimum essential medium-α alone or supplemented with BMP-15, sequential FSH, both BMP-15 and FSH, or BMP-15 from days 0 to 6, and FSH from days 7 to 12. Thereafter, the effect of these treatments on the follicular volume, viability, and antrum formation and the levels of mRNA for BMPR-IB, BMPR-II, and FSH-R were assessed. Compared with day 0, the follicles cultured with FSH or BMP-15, or both, had a significant and progressive increase in volume (P < 0.05). However, the follicles cultured for 12 days with both BMP-15 and FSH had the greatest volume and a greater rate of antrum formation than those in control medium, but results similar to those cultured with FSH (days 0 to 12) or BMP-15 (days 0 to 6) and FSH (days 7 to 12). Together with their accelerating effect on in vitro follicle growth, the combination of FSH and BMP-15 induced ultrastructural changes in the cultured follicles and increased atresia. However, adding either BMP-15 or FSH to the culture medium, not only promoted follicular growth and follicular antrum formation, but also maintained follicular viability during culture. Except for follicles cultured in minimal essential medium-α, the levels of mRNA for BMPR-IB were reduced, and the levels of mRNA for FSH-R were significantly greater in follicles cultured in medium supplemented with BMP-15. In conclusion, all in vitro follicle treatments supported growth of bovine preantral follicles; however, adding both BMP-15 and FSH to the culture medium (minimal essential medium-α) for 12 days provided the greatest stimulation. Furthermore, the viability and ultrastructural integrity of cultured follicles were only maintained when only BMP-15 or FSH was added to the culture medium.
Subject(s)
Bone Morphogenetic Protein 15/pharmacology , Cattle , Follicle Stimulating Hormone/pharmacology , Follicular Atresia/drug effects , Ovarian Follicle/drug effects , Animals , Bone Morphogenetic Protein Receptors/genetics , Bone Morphogenetic Protein Receptors/metabolism , Female , Gene Expression Regulation , Ovarian Follicle/growth & development , Ovarian Follicle/ultrastructure , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, FSH/genetics , Receptors, FSH/metabolismABSTRACT
The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.
Subject(s)
Animals , Female , Follicle Stimulating Hormone/metabolism , Mitogens/pharmacology , Ovarian Follicle/drug effects , Phytohemagglutinins/pharmacology , Follicle Stimulating Hormone/genetics , Goats , In Vitro Techniques , Ovarian Follicle/growth & development , Ovarian Follicle/ultrastructure , Proliferating Cell Nuclear Antigen/metabolism , RNA, Messenger/metabolismABSTRACT
The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⻹) from Days 0 to 6 and 500 ng mL⻹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⻹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⻹ bovine serum albumin, 10 µg mL⻹ insulin, 5.5 µg mL⻹ transferrin, 5 ng mL⻹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⻹ ascorbic acid (α-MEMâº). Comparisons of uncultured (0.2 mm) and α-MEM⺠cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.
Subject(s)
Follicle Stimulating Hormone/metabolism , Gene Expression Regulation, Developmental , Growth Differentiation Factor 9/metabolism , In Vitro Oocyte Maturation Techniques/veterinary , Oogenesis , Ovarian Follicle/metabolism , RNA, Messenger/metabolism , Abattoirs , Animals , Cattle , Cell Survival , Female , Follicular Fluid/enzymology , Follicular Fluid/metabolism , Glucuronosyltransferase/antagonists & inhibitors , Glucuronosyltransferase/biosynthesis , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Hyaluronan Synthases , Isoenzymes/antagonists & inhibitors , Isoenzymes/biosynthesis , Isoenzymes/genetics , Isoenzymes/metabolism , Oocytes/cytology , Oocytes/enzymology , Oocytes/metabolism , Ovarian Follicle/cytology , Ovarian Follicle/growth & development , Proliferating Cell Nuclear Antigen/biosynthesis , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Proteoglycans/antagonists & inhibitors , Proteoglycans/biosynthesis , Proteoglycans/genetics , Proteoglycans/metabolism , Receptors, FSH/antagonists & inhibitors , Receptors, FSH/biosynthesis , Receptors, FSH/genetics , Receptors, FSH/metabolism , Tissue Culture Techniques/veterinaryABSTRACT
This study investigated the stability of housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, ß-tubulin, ß-actin, phosphoglycerate kinase (PGK), 18S rRNA, ubiquitin and ribosomal protein 19) and the levels of mRNA for bone morphogenetic protein-2 (BMP-2), -4 (BMP-4), -6 (BMP-6), -7 (BMP-7) and -15 (BMP-15), their receptors (BMPR-IA, -IB and -II) and Similar to Mothers Against Decapentaplegic (SMADs) (-1, -5 and -8) in goat follicles of 0.2, 0.5 and 1.0mm, as well as in secondary follicles before and after culture for 18 days. ß-tubulin and PGK were the most stable housekeeping genes and the levels of mRNA for BMP-2 in follicles of 0.2mm were higher than in follicles of 0.5 and 1.0mm. For BMP-4, -6 and -7, the highest levels of mRNA were found in follicles of 1.0mm. The expression of BMPR-IB was higher in follicles of 0.2mm, whereas the levels of BMPR-II were higher in follicles of 0.5mm. The levels of mRNA for SMAD-5 were higher in follicles of 0.2mm, whereas SMAD-8 had higher levels in 0.5-mm follicles. After culture, follicles showed increased levels of mRNA for BMP-2 and reduced mRNA for BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, ß-tubulin and PGK are the most stable reference genes, and BMPs, their receptors and SMADs have variable levels of mRNA in the follicular size classes analysed.