Expression of the bovine oestrogen receptor-β (bERβ) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERβ mRNA of second wave follicles after LH infusion into cows (original) (raw)
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Biology of …, 1995
The objective of the present study was to characterize expression of mRNAs encoding FSH and LH receptors during follicular development and at different stages of the first follicular wave in cattle. Following estrus, groups of heifers (3-5 per group) were ovariectomized on the day of initiation of the first follicular wave (as determined by ultrasonography; Day 0), or on Days 2, 4, 6, 8, or 10 after initiation of the first wave. FSH and LH receptor mRNAs were detected within follicles-4 mm and in some smaller follicles by in situ hybridization and were quantified by image analysis. FSH receptor mRNA was expressed in granulosa cells of all growing follicles, starting in some follicles with only one layer of granulosa cells. Irrespective of day of the follicular wave, the level of expression of FSH receptor mRNA in granulosa cells of healthy antral follicles ranging from 0.5 to 14 mm in diameter did not vary significantly with follicular size (r = 0.02, p > 0.10). Expression of LH receptor mRNA was first observed in theca interna cells of follicles shortly after antral formation. Irrespective of day of the follicular wave, the levels of LH receptor mRNA in theca interna cells of healthy antral follicles ranging from 0.5 to 14 mm increased with follicular size (r = 0.39, p < 0.01). In granulosa cells, LH receptor mRNA was expressed only in healthy follicles > 9 mm in diameter and was first observed in the dominant follicles collected on Day 4. Expression of mRNA for LH receptor, but not for FSH receptor, changed (p < 0.01) with the stage of the first follicular wave. LH receptor mRNA levels were highest in theca interna cells of dominant follicles collected on Day 4 and did not differ on other days. Only the healthy dominant follicles collected on Days 4, 6, and 8 expressed LH receptor mRNA in the granulosa cells. Levels of LH receptor mRNA in granulosa cells were similar on Day 4 and Day 6 but were reduced (p < 0.05) on Day 8. Atresia was associated with a loss of LH receptor mRNA in granulosa cells and with reduced expression of LH receptor mRNA in theca cells and FSH receptor mRNA in granulosa cells. In summary, acquisition of LH receptors in granulosa cells between Day 2 and Day 4 may be important in the establishment of follicular dominance during the first follicular wave.
Domestic Animal Endocrinology, 2016
Brain ribonuclease (BRB) is a member of the ribonuclease A superfamily that is constitutively expressed in a range of tissues, and is the functional homolog of human ribonuclease 1. This study was designed to characterize BRB gene expression in granulosa cells (GC) during development of bovine dominant ovarian follicles, and to determine the hormonal regulation of BRB in GC. Estrous cycles of Holstein cows (n = 18) were synchronized and cows were ovariectomized on either day 3 to 4 or day 5 to 6 post-ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GC were collected for RNA isolation and quantitative PCR. Follicles were categorized as small (1 to 5 mm; pooled per ovary), medium (5 to 8 mm; individually collected) or large (8.1 to 17 mm; individually collected) based on surface diameter. Estradiol (E 2) and progesterone (P 4) levels were measured by RIA in FFL. Abundance of BRB mRNA in GC was 8.6-to 11.8-fold greater (P < 0.05) in small (n = 31), medium (n = 66) and large (n = 33) subordinate E 2-inactive (FFL E 2 < P 4) follicles than in large (n = 16) dominant E 2-active (FFL E 2 > P 4) follicles. In the largest 4 follicles, GC BRB mRNA abundance was negatively correlated (P < 0.01) with FFL E 2 (r = −0.65) and E 2 /P 4 ratio (r = −0.46). In Exp. 2, GC from large (8 to 22 mm diameter) and small (1 to 5 mm diameter) follicles were treated with IGF1 (0 or 30 ng/mL), and/or tumor necrosis factor α (TNFα) (0 or 30 ng/mL); IGF1 increased (P < 0.05) BRB mRNA abundance and TNFα decreased (P < 0.001) the IGF1induced BRB mRNA abundance in large-follicle GC. In Exp. 3 to 6, E 2 , FSH, fibroblast growth factor 9 (FGF9), cortisol, wingless 3A (WNT3A), or Sonic hedgehog (SHH) did not affect (P > 0.10) abundance of BRB mRNA in GC; thyroxine and LH increased (P < 0.05) whereas prostaglandin E2 (PGE2) decreased (P < 0.05) BRB mRNA abundance in small-follicle GC. Treatment of small-follicle GC with recombinant human RNase1 increased (P < 0.05) GC numbers and estradiol production. In conclusion, BRB is a hormonally and developmentally regulated gene in bovine GC and may regulate estradiol production during follicular growth in cattle.
Molecular Reproduction and Development, 2008
Transcription factors inhibit or assist RNA polymerases in the initiation and maintenance of transcription; however, the cell specific expression and roles of transcription factors within bovine ovarian follicles during development are unknown. The aim of present study was to determine if the expression of transcription factors in theca and granulosa cells differ between the dominant and the largest subordinate follicles at different stages of the follicle wave. We used a bovine cDNA microarray to screen granulosa and theca cells from dominant and subordinate follicles for differential expression of genes coding for transcription factors. Expression was confirmed using reverse transcription polymerase chain reaction and differences in mRNA abundance further examined at Emergence, Selection and Dominance stages of the follicle wave. We have identified five genes encoding for transcription factors that have not been previously described in developing follicles with greater mRNA abundance in subordinate compared to dominant follicles. The genes (and their putative roles) are CEBP-b (responsible for luteinization), SRF (cell survival), FKHRL1 (stimulates apoptosis), NCOR1 (modulation of the actions of the oestradiol receptor) and Midnolin (control of development via regulation of mRNA transport in cells). Mol. Reprod. Dev. 75: 904-914, 2008.
Journal of Reproduction and Development, 2007
The aim of the present study was to examine the messenger RNA expressions of the endothelin and angiotensin systems during the periovulatory phase in gonadotrophin releasing hormone (GnRH)-treated cows. Ovaries were collected by transvaginal ovariectomy (n=5 cows/ group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (control, before LH surge), 3-5 h after GnRH (during LH surge), 10 h after GnRH; 20 h after GnRH, 25 h after GnRH (peri-ovulation), and early corpus luteum (CL) (Days 2-3). Expression of mRNA was investigated using quantitative real-time PCR. The expression of angiotensin converting enzyme (ACE) mRNA significantly decreased immediately after onset of the LH surge and remained at low levels. The levels of angiotensin II receptor type 1 (AT1R) and type 2 (AT2R) expression during the periovulatory period significantly decreased compared with other periods. The concentration of angiotensin II in follicular fluid began to increase 10 h after GnRH treatment and further increased as ovulation approached. The level of ET-1 mRNA significantly decreased 10 h after GnRH treatment compared with the levels before GnRH treatment and those of the early CL period. The expression of ETR-A and ETR-B mRNA during the periovulatory period were lower than in other periods. The expression of ECE-1 mRNA began to decrease in the LH surge period and significantly decrease in the periovulatory period compared with other periods. These results suggest that the vasoactive peptides angiotensin and endothelin may be associated with final maturation of follicles.
Biology of Reproduction, 2001
Ovarian growth and development are critically dependent upon the influence of endogenous estrogens, and both are highly regulated during the reproductive cycle. The observation that estrogen-receptor-␣-deficient mice still exhibit follicular growth and development, together with other evidence, suggests that responsiveness of the ovary to estradiol occurs predominantly through the second estrogen receptor, ER. We characterized the physiological regulation of ER expression in ovarian follicles during the follicular phase of sheep that were synchronized for estrus during the breeding season with intravaginal progesterone implants (controlled internal drug release [CIDR] device; InterAg, Hamilton, New Zealand). Ovaries were removed at times corresponding to the early (EF) and late follicular phases (LF) of the ovine estrous cycle (12 h [n ؍ 5] and 32 h [n ؍ 5] after CIDR device removal, respectively). Sections of ovary were then hybridized with a cRNA probe corresponding to the 5 region of ovine ER. ER mRNA expression within the granulosa layer of different size follicles (size classes: Յ3 mm, 3.1-4.0 mm, 4.1-5.0 mm, Ͼ5 mm) was quantified. ER mRNA expression varied both with follicle size (P Ͻ 0.01) and with cycle stage (P Ͻ 0.01). In EF ewes, the highest levels of ER mRNA expression were found in follicles Յ 3 mm in size. ER mRNA expression declined progressively thereafter among the different size classes with lowest levels expressed in Ͼ5-mm follicles. By contrast, expression of ER mRNA in the 3.1-to 4.0-mm follicles of LF group was significantly higher than in the Յ3-mm size follicles and declined thereafter progressively to the Ͼ5-mm size levels as in the EF group. Furthermore, expression of ER mRNA in Յ3-mm size follicles of LF group was significantly lower than the corresponding size class in the EF group. Lower expression of ER mRNA in Ͼ5-mm follicle is suggestive of a down-regulation by the local estrogen milieu.
Differential gene expression of granulosa cells after ovarian superstimulation in beef cattle
Reproduction, 2013
Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were u...
Journal of Ovarian Research, 2014
Background: Estradiol (E2) receptors mediate E2 effects on cell proliferation and apoptosis under normal and pathological conditions. However, the mechanisms involved in E2 signaling are not completely understood. The objectives in this study were to evaluate the expression of estrogen receptors (ESRs) during follicular selection in cattle, and the effect of intrafollicular injection of fulvestrant (an antagonist of ESRs) on follicular development and transcript abundance in granulosa cells. Methods: Granulosa cells were obtained from the two largest follicles around follicular deviation, after FSH treatment and after intrafollicular injection of fulvestrant. Ovarian follicular dynamics monitored by ultrasonography and quantitative real time PCR were used to validate the in vivo model and investigate the effects of FSH supplementation or ESR blockade on mRNA expression of estradiol-related genes. Results: ESR1 and ESR2 were expressed in granulosa cells of both dominant (F1) and subordinate (F2) follicles, but their transcripts levels were higher in F1 than F2 after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels for both ESR1 and ESR2 were not affected by fulvestrant injection. Analyses of FSH-regulated genes revealed that ESRs inhibition in the dominant follicle decreased the transcript levels of the GJA1 but not those of PRKAR2B, MRO or LRP11 genes. Conclusions: Our findings indicate that: both ESR1 and ESR2 are regulated during follicular deviation and dominance in cattle and in response to FSH treatment, and ESRs are required for normal gene expression and development of the dominant follicle. Furthermore, we have validated an in vivo model to study estrogen signaling during follicular development that allows paracrine signaling between different follicular cells in a physiological endocrine environment.
Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle
Biology of Reproduction, 1992
To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 aCi 5H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 (E,) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 ± 45.5 ng/ ml; 875.4 ± 68.2 pg E2/follicle) compared to subordinate follicles (40.6 ± 69.4 ng/ml; 99.4 ± 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 ± 118.1 pg E2/follicle) and 8 (1028.2 ± 118.1 pg E2/ follicle) compared to Day 12 (450.7 ± 118.1 pg E2/follicle). Concentrations of E, and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 ± 78.2 and 89.5 ± 15.7 ng/ml) compared to Days 8 (225.1 ± 78.6 and 5.9 ± 14.8 ng/ ml) and 12 (108.5 ± 78.6 and 13.0 ± 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 ± 1.4 to 32.5 ± 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of UI when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 'H-leucine into macromolecules and secreted high (90000-120000) and low (20 000-23000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12. Results indicate that evolution of the first-wave dominant follicle and the CL have important effects on plasma FSH and LH which regulate ovarian folliculogenesis and steroidogenesis.
Transcriptional effect of the LH surge in bovine granulosa cells during the peri-ovulation period
Reproduction (Cambridge, England), 2011
The LH surge induces a multitude of events that are essential for ovulation and corpus luteum formation. The transcriptional responses to the LH surge of preovulatory granulosa cells (GCs) are complex and still poorly understood. In this study, a genome-wide bovine oligo array was used to determine how the gene expression profile of GCs is modulated by the LH surge. GCs from three different stages were used to assess the short- and long-term effects of this hormone on follicle differentiation: 1) 2 h before induction of the LH surge, 2) 6 h and 3) 22 h after the LH surge. The results obtained were a list of differentially expressed transcripts for each GC group. To provide a comprehensive understanding of the processes at play, biological annotations were used to reveal the different functions of transcripts, confirming that the LH surge acts in a temporal manner. The pre-LH group is involved in typical tasks such as cell division, development, and proliferation, while the early res...
Reproduction, 2008
This study was designed to identify genes that regulate the transition from FSH-to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P%0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, b; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development.