178 EXPRESSION OF mRNA ENCODING LUTEINIZING HORMONE RECEPTOR AND MEVALONATE KINASE AROUND FOLLICLE DEVIATION IN NELORE HEIFERS (BOS INDICUS)

2013 ◽  
Vol 25 (1) ◽  
pp. 238
Author(s):  
A. C. Souza Castilho ◽  
R. L. Ereno ◽  
M. Fernandes Machado ◽  
R. A. Satrapa ◽  
M. F. Gouveia Nogueira ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Luteinizing Hormone Receptor ◽  
Mrna Abundance ◽  
Mevalonate Kinase ◽  
Dominant Follicle ◽  
Significant Difference

Luteinizing hormone (LH) plays a key role in controlling physiological processes in the ovary, and the expression of LHR by bovine granulosa cells is crucial to the follicular transition from FSH to LH dependency. There are controversies about the time at which follicles acquire LHR in granulosa cells. In Nelore breed (Bos indicus), the morphological divergence occurs, on average, 2.5 days after ovulation when the diameter of the dominant follicle is ~6.0 mm. In our previous work with semiquantitative PCR, the mRNA expression of LHR isoforms was detected more clearly after deviation (Day 3). The LHR mRNA binding protein, mevalonate kinase (MVK), is responsible for the down-regulation of LHR mRNA, thereby controlling the steady-state of LHR mRNA expression. In rats, there is an inverse correlation between the mRNA expression of LHR and MVK in luteal cells; however, there is no evidence about MVK expression in the bovine antral follicle. To gain insight about the involvement of the LHR/MVK system in the control of follicle deviation, we assessed the mRNA expression of LHR and MVK in granulosa cells from dominant and subordinate follicles close to deviation in Nelore heifers. Animals (n = 10) were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (before deviation; n = 3), 2.5 (around deviation; n = 4), and 3 (post-deviation; n = 3) days after ovulation. Granulosa cells were harvested from the 2 largest follicles and submitted to total RNA extraction and reverse transcribed with oligo-dT. The mRNA abundance of LHR and MVK was measured by real-time RT-PCR using the Sybr Green system with bovine-specific primers and normalized by the expression of endogenous gene, cyclophilin A (PPIA), using the ΔΔct method corrected by Pffafl’s equation. Dominant and subordinate follicles were considered those expressing the greatest and second greatest abundance of aromatase mRNA (CYP19) in granulosa cells within each heifer. Effects of the day and follicle status on the mRNA abundance of LHR and MVK were tested by ANOVA and the mean values compared by paired t-test or Tukey test (P < 0.05 indicated significant difference). The LHR mRNA was detected at the predicted time of follicle deviation in Nelore heifers (Day 2.5) and was higher in dominant follicle on Day 3 (32.8 ± 12.6) compared with Day 2.5 (3.2 ± 0.9). The second largest follicle (subordinate follicles) had lower mRNA abundance of LHR when compared with future dominant follicles (largest follicles) on days 2.5 (0.8 ± 0.4 v. 3.2 ± 0.9) and 3 (1.9 ± 0.8 v. 32.8 ± 12.6). In contrast to the mRNA expression of LHR, MVK mRNA was more expressed in the subordinate follicles than in the largest follicles at Days 2.5 (3.1 ± 0.9 v. 0.9 ± 0.3) and 3 (2.6 ± 0.6 v. 0.9 ± 0.1) after ovulation, suggesting that it may be necessary to decrease the MVK expression in future dominant follicles to increase their LHR expression and follow up to ovulation. Supported by FAPESP.

230 EXPRESSION OF mRNA ENCODING FGF10 AND COGNATE RECEPTORS (FGFR1B AND FGFR2B) AROUND FOLLICLE DEVIATION IN NELORE HEIFERS

2010 ◽  
Vol 22 (1) ◽  
pp. 273
Author(s):  
A. C. S. Castilho ◽  
M. F. Machado ◽  
D. M. Guerra ◽  
R. Ereno ◽  
C. M. Barros ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Mrna Abundance ◽  
Morphological Divergence ◽  
Dominant Follicle ◽  
Ultrasound Monitoring ◽  
Follicle Diameter ◽  
Follicle Selection

A member of the FGF7 subfamily, FGF10 acts via FGFR2B and FGFR1B. In bovine antral follicles, FGF-10 was detected in oocytes and theca cells (TC). Levels of mRNA were negatively correlated with intrafollicular concentrations of estradiol, and FGF10 inhibited estradiol production from granulosa cells (GC). In Nellore (Bos indicus), morphological divergence occurs on average 2.5 days after ovulation, when dominant follicle diameter is around 6.0 mm. To gain insight into the involvement of the FGF10 system in the control of follicle selection, we assessed mRNA expression of FGF10 in TC and of FGFR1B and FGFR2B in GC from dominant and subordinate follicles around deviation in Nellore heifers. Thirteen Nellore heifers were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (n =4), 2.5 (n = 5), and 3 (n = 4) days after ovulation. Granulosa cells and TC were separated from the 2 largest follicles and submitted to total RNA extraction. mRNA abundance of CYP19 (aromatase), FGF10, FGFR1B, and FGFR2B was measured by real-time RT-PCR and normalized by the expression of cyclophilin A (CYCA) and GAPDH, for TC and GC, respectively. Dominant and subordinate follicles were considered those expressing the greatest and second-greatest abundance of CYP19 mRNA in GC within each heifer. Effects of follicle status and day on CYP19, FGF10, FGFR2B, and FGFR1B mRNA abundance were tested by ANOVA. On Day 2, FGFR2B mRNA abundance was greater in GC of subordinate follicles compared with dominant follicles (P = 0.006), and that of FGF10 in TC tended to exhibit the same pattern (P = 0.06). Follicle diameter was not different between dominant and subordinate follicles on Day 2 (5.5 ± 0 v. 5.12 ± 0.3 cm). On Day 2.5, FGF10 expression was greater in TC from subordinate follicles (P = 0.01), and FGFR2B expression in GC was no longer different between dominant and subordinate follicles. Follicle diameter was greater in dominant follicles on Day 2.5 (6.7 ± 0.2 v. 5.8 ± 0.3 cm; P = 0.04). On Day 3, no differences were observed between dominant and subordinate follicles for any of the genes assessed. mRNA expression of FGFR1B in GC did not change with follicle status or day. In conclusion, expression of FGF10 and FGFR2B was decreased in dominant follicles around morphological divergence, suggesting their involvement in the mechanisms controlling dominant follicle selection. As FGF10 inhibits estradiol production of GC, we propose that FGF10 and FGFR2B are suppressed in the dominant follicle to allow acquisition of full steroidogenic capacity. This research was supported by FAPESP.

176 GENE EXPRESSION OF LUTEINIZING HORMONE RECEPTOR (LHR) ISOFORMS IN GRANULOSA CELLS OF FOLLICLES FROM NELLORE HEIFERS BEFORE, DURING, AND AFTER FOLLICULAR DEVIATION

2009 ◽  
Vol 21 (1) ◽  
pp. 187 ◽  
Author(s):  
C. M. Barros ◽  
R. L. Ereno ◽  
M. F. Machado ◽  
J. Buratini ◽  
M. F. Pegorer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Rna Extraction ◽  
Follicular Development ◽  
Sao Paulo ◽  
Luteinizing Hormone Receptor ◽  
São Paulo ◽  
Dominant Follicle ◽  
The Future ◽  
Group 2

During bovine follicular development, there is a phase known as follicular deviation in which the future dominant follicle grows faster than the other follicles and acquires LH receptors (LHR). In Nellore breed, deviation occurs 2.5 days after ovulation, and at this time, the dominant follicle has in average a diameter of 6.0 mm. Some authors believe that LHRs are present in the future dominant follicle before deviation and are essential for this process. However, others are convinced that LHRs are present only during or after follicular deviation. The aim of the present experiment was to evaluate the expression of 4 LHR isoforms (M1 to M4) in granulosa cells of follicles from Nellore heifers before, during, and after follicular deviation. At a random stage of the estrous cycle (D0), Nellore heifers (n = 21) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB, i.m., Estrogin®, Farmavet, Sao Paulo, Brazil). Eight days later (D8) PGF2α was administered (150 μg d-cloprostenol, i.m., Prolise®, ARSA S.R.L., Buenos Aires, Argentina), and the device was removed. Twenty-four hours after device removal, cows were treated with EB (1.0 mg, i.m.), and from this point in time, the growth of the dominant follicle growth was observed by ultrasonography (US, Aloka 900, Tokyo, Japan) every 12 h. The animals were allocated in 3 groups: Group 2 (G2, 2 days after ovulation, n = 7), Group 2.5 (G2.5, 2.5 days after ovulation, n = 7), and Group 3 (G3, 3 days after ovulation, n = 7), and were slaughtered 2, 2.5, and 3 days after ovulation, respectively, in order to remove the ovaries. The granulosa cells, obtained from ovarian follicles, were separated for total RNA extraction, and the gene expression of LHR isoforms was measured by semiquantitative RT-PCR. Since LHR expression was not detected in Group 2 (follicles with 4.5 to 6.7 mm), comparisons were performed between groups G2.5 and G3 by ANOVA. The LHR expression was detected only in 2 samples of Group G2 (7.0-mm follicles) and was significantly higher in Group G3 (63.6%; follicles from 8 to 14 mm, P < 0.05). In all samples that expressed LHR, the 4 isoforms were present. It is concluded that LHR expression is present in granulosa cells of follicles from Nellore heifers after follicular deviation. Support and fellowship from FAPESP (Sao Paulo, Brazil).We are grateful to Tecnopec (Sao Paulo, Brazil) for providing intravaginal devices used in the experiment.

scholarly journals Association of luteinizing hormone receptor gene expression with cell cycle progression in granulosa cells

2009 ◽  
Vol 296 (6) ◽  
pp. E1392-E1399 ◽  
Author(s):  
Jennifer D. Cannon ◽  
Srinivas V. Seekallu ◽  
Catherine A. VandeVoort ◽  
Charles L. Chaffin
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Luteinizing Hormone ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Hormone Receptor ◽  
Cell Cycle Progression ◽  
Luteinizing Hormone Receptor ◽  
Cycle Progression ◽  
Immature Rats

During hormonally induced ovarian follicle growth, granulosa cell proliferation increases and returns to baseline prior to the administration of an ovulatory stimulus. Several key genes appear to follow a similar pattern, including the luteinizing hormone receptor (LHCGR), suggesting an association between cell cycle progression and gene expression. The expression of LHCGR mRNA in granulosa cells isolated from immature rats and treated in culture with FSH increased in a time-dependent manner, whereas administration of the cell cycle inhibitor mimosine completely suppressed expression. Although forskolin was able to induce luteinization in cells treated with mimosine, human chorionic gonadotropin had no effect, indicating the functional loss of LHCGR. The effects of mimosine on cell cycle progression and LHCGR mRNA expression were reversible within 24 h of mimosine removal. Cell cycle inhibition did not alter the stability of LHCGR mRNA, indicating that the primary effect was at the transcriptional level. To determine whether the relationship between LHCGR expression and cell cycle were relevant in vivo, immature rats were given a bolus of PMSG, followed by a second injection of either saline or PMSG 24 h later to augment levels of proliferation. The expression of LHCGR mRNA was elevated in the ovaries of animals receiving a supplement of PMSG. Mimosine also blocked cell cycle progression and LHCGR mRNA expression in macaque granulosa cells isolated following controlled ovarian stimulation cycles and in two different mouse Leydig tumor lines. These data collectively indicate that LHCGR mRNA is expressed as a function of the passage of cells across the G1-S phase boundary.

Neurotensin: A Neuropeptide induced by hCG in the Human and Rat Ovary during the Periovulatory Period

2021 ◽  
Author(s):  
Linah Al-Alem ◽  
Muraly Puttabyatappa ◽  
Ketan Shrestha ◽  
Yohan Choi ◽  
Kathy Rosewell ◽  
...  
Keyword(s):  
Cell Migration ◽  
Growth Factor ◽  
Mrna Expression ◽  
Vascular Permeability ◽  
Signaling Pathway ◽  
Granulosa Cells ◽  
Transvaginal Ultrasound ◽  
Dominant Follicle ◽  
Rat Ovary ◽  
Pka Pathway

Abstract Neurotensin (NTS) is a tridecapeptide that was first characterized as a neurotransmitter in neuronal cells. The present study examined ovarian NTS expression across the periovulatory period in the human and the rat. Women were recruited into this study and monitored by transvaginal ultrasound. The dominant follicle was surgically excised prior to the LH surge (preovulatory phase) or women were given 250 μg hCG and dominant follicles collected 12-18 h after hCG (early ovulatory), 18-34 h (late ovulatory) and 44-70 h (postovulatory). NTS mRNA was massively induced during the early and late ovulatory stage in granulosa cells (15,000 fold) and theca cells (700 fold). In the rat, hCG also induced Nts mRNA expression in intact ovaries and isolated granulosa cells. In cultured granulosa-lutein cells (GLC) from IVF patients, NTS expression was induced 6 h after hCG treatment whereas in cultured rat granulosa cells NTS increased 4 h after hCG treatment. Cells treated with hCG signaling pathway inhibitors revealed that NTS expression is partially regulated in the human and rat GC by the epidermal-like growth factor (EGF) pathway. Human GLC and rat granulosa cells also showed that Nts was regulated by the PKA pathway along with input from the PI3K and MAPK pathways. The predominate NTS receptor present in human and rat granulosa cells was SORT1, whereas NTSR1 and NTSR2 expression was very low. Based on NTS actions in other systems, we speculate that NTS may regulate crucial aspects of ovulation such as vascular permeability, inflammation, and cell migration.

347 COULD THE DIFFERENTIAL EXPRESSION OF LUTEINIZING HORMONE RECEPTOR ISOFORMS EXPLAIN THE VARIABILITY IN SUPEROVULATORY RESPONSES IN CATTLE?

2015 ◽  
Vol 27 (1) ◽  
pp. 261
Author(s):  
S. Wohlres-Viana ◽  
E. K. N. Arashiro ◽  
J. G. V. Grazia ◽  
L. S. A. Camargo ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Bos Indicus ◽  
Luteinizing Hormone Receptor ◽  
Poor Responders ◽  
Embryo Production ◽  
Lh Receptor ◽  
Follicular Wave ◽  
Follicular Aspiration ◽  
Receptor Isoforms

Embryo production in vivo is highly variable among donors. The Gir breed (Bos indicus) is well known to show a low embryo production after superovulation (2.5 to 3.5 viable embryos per flush), and a high variance in superovulatory responses, which makes this breed an interesting model to study this trait. The aim of this study was to evaluate the expression pattern of LHR isoforms in Gir heifers previously characterised as good (10.3 ± 1.2 embryos/flush, N = 5) or poor (1.1 ± 0.3 embryos/flush, N = 5) responders to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system (Arashiro et al. 2012 Reprod. Fertil. Dev. 24, 175) was used to collect granulosa cells (GC) from 8-mm follicles growing in either a synchronized but not stimulated follicular wave (FW) or in the fourth day of superovulation (SOV), induced with 200 UI of FSHp (Pluset, Serono). The recovered follicular fluid was centrifuged and the cells were washed with NaCl 0.9% saline and kept in RNA Later (Ambion, Austin, TX, USA). Total RNA extraction was performed using the commercial RNeasy Micro Kit (Qiagen, Valencia, CA, USA). The RNA samples were quantified and reverse transcribed using the commercial Superscript III kit (Invitrogen, Carlsbad, CA, USA). Complementary DNA samples were amplified through real-time PCR, using a LH receptor primer – not selective for LHR isoforms (total LHR) – and 4 sets of isoform selective primers (S1, S10, S10+11, and S11). All samples were previously tested for theca cell contamination through detection of CYP17A1 gene, and those showing contamination were excluded. The β-actin gene was used as endogenous control. Analyses were performed using the REST software and the expression values are shown as mean ± s.e.m. For comparisons between good and poor responders, the first was set as 1.00. For comparisons between FW and SOV, FW was set as 1.00. In the good responder group, there was no difference (P > 0.05) in total LHR expression among GC samples from FW and SOV. However, the S10+11 isoform was down-regulated (0.4 ± 0.1; P < 0.01) after SOV. In the poor responders group, total LHR expression was down-regulated (0.2 ± 0.1; P < 0.01) after SOV, but there was no difference in the expression of isoforms (P > 0.05). Contrasting the response groups (good and poor), total LHR (15.1 ± 7.6; P < 0.001), and the isoforms S10 (5.7 ± 2.7; P < 0.01), S10+11 (1.9 ± 0.6; P < 0.01), and S11 (5.1 ± 2.5; P < 0.01) were up-regulated in FW of poor responders, but there was no difference (P > 0.05) in any LHR form during SOV. We concluded that 1) LHR expression is different between heifers characterised as good or poor responders to superovulation; 2) superovulation modulates the LHR expression and reduces the original differences observed in unstimulated cycles; 3) diminished expression of total LHR, but not in the isoforms, in poor responders heifers could suggest a reduction in the expression of full-length LHR, with possible consequences to ovulatory capability after superovulation.Financial support was provided by CNPq Project 477701 and Fapemig PPM 0067/11.

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