Lipid droplets, subspecies (Bos taurus indicus vs. Bos taurus taurus), and in vitro culture are known to influence cryopreservation of bovine embryos. Limited information is available regarding differences in membrane lipids in embryo, such as phosphatidylcholines (PC) and sphingomyelins (SM). The objective of the present study was to compare the profiles of several PC and SM species and relate this information to cytoplasmic lipid levels present in Nellore (B. taurus indicus) and Simmental (B. taurus taurus) blastocysts produced in vitro (IVP) or in vivo (ET). Simmental and IVP embryos had more cytoplasmic lipid content than Nellore and ET embryos (n = 30). Blastocysts were submitted to matrix-assisted laser desorption/ionization mass spectrometry. Differences in the PC profile were addressed by principal component analysis. The lipid species with PC (32:1) and PC (34:1) had higher ion abundances in Nellore embryos, whereas PC (34:2) was higher in Simmental embryos. IVP embryos had less abundant ions of PC (32:1), PC (34:2), and PC (36:5) compared to ET embryos. Moreover, ion abundance of PC (32:0) was higher in both Nellore and Simmental IVP embryos compared to ET embryos. Therefore, mass spectrometry profiles of PC and SM species significantly differ with regard to unsaturation level and carbon chain composition in bovine blastocysts due to subspecies and in vitro culture conditions. Because PC abundances of Nellore and Simmental embryos were distinct (34:1 vs. 34:2), as were those of IVP and ET embryos (32:0 vs. 36:5), they are potential markers of postcryopreservation embryonic survival.
To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0-3.0, 3.1-6.0, 6.1-8.0 and >or=8.1 mm. Cumulus-oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription-polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles>6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P<0.05) in oocytes from follicles>or=8.1 mm in diameter than in oocytes from follicles<6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.
Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.
Fibroblast growth factors (FGF) are involved in paracrine signaling between cell types in the ovarian follicle. FGF8, for example, is secreted by oocytes and controls cumulus cell metabolism. The closely related FGF18 is also expressed in oocytes in mice. The objective of this study was to assess the potential role of FGF18 in follicle growth in a monovulatory species, the cow. Messenger RNA encoding FGF18 was detected primarily in theca cells, and in contrast to the mouse, FGF18 was not detected in bovine oocytes. Addition of FGF18 protein to granulosa cell cultures inhibited estradiol and progesterone secretion as well as the abundance of mRNA encoding steroidogenic enzymes and the follicle-stimulating hormone receptor. In vivo, onset of atresia of the subordinate follicle was associated with increased thecal FGF18 mRNA levels and FGF18 protein in follicular fluid. In vitro, FGF18 altered cell cycle progression as measured by flow cytometry, resulting in increased numbers of dead cells (sub-G1 peak) and decreased cells in S phase. This was accompanied by decreased levels of mRNA encoding the cell cycle checkpoint regulator GADD45B. Collectively, these data point to a unique role for this FGF in signaling from theca cells to granulosa cells and suggest that FGF18 influences the process of atresia in ovarian follicles.
In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.
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