225 FOLLICULAR DIAMETER, OVULATION RATE, AND LH RECEPTOR GENE EXPRESSION IN NELLORE COWS

2010 ◽  
Vol 22 (1) ◽  
pp. 270 ◽  
Author(s):  
R. A. L. Simões ◽  
R. A. Satrapa ◽  
F. S. Rosa ◽  
M. Piagentini ◽  
A. C. S. Castilho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Internal Control ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Sao Paulo ◽  
Ovulation Rate ◽  
São Paulo ◽  
Lh Receptor ◽  
Theca Cells

The aim of the present experiment was to verify the relationship among follicular diameter, ovulation rate, and gene expression of LH receptor (LHR) isoforms in order to know whether these aspects could or could not influence ovulation rates in Nellore cows. In Experiment 1, at a random stage of the estrous cycle (Day 0), Nellore cows (n = 53) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, São Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB; i.m. Estrogin®; Farmavet, São Paulo, Brazil). On Day 8, PGF2 (150 μg d-cloprostenol; Prolise® ARSA S.R.L., Buenos Aires, Argentina) was administered i.m. and the device was removed. Twenty-four hours after device removal, cows were treated i.m. with EB (1.0 mg) and, 48 h afterwards, ovulation was determined by ultrasonography (US; Aloka 900, Tokyo, Japan). Three days after ovulation, follicular growth was observed daily by US and cows were randomly allocated into 3 groups according to follicular diameter (mm) [G1 (7.0-8.0), G2 (8.1-9.0), and G3 (9.1-10.0)] to receive 6.25 mg of LH (i.m. Lutropin®-V, Bioniche, Belleville, Ontario, Canada), which corresponds to twice the minimum ovulatory dose (3.12 mg) as determined in a preliminary experiment. The results were analyzed by logistic regression (PROC GEN MOD, SAS Institute, Cary, NC). The ovulation rates were 9 (2/21), 36 (8/22), and 90% (9/10) for G1, G2, and G3, respectively. There were significant differences when comparing G1 v. G3 (P < 0.01), G2 v. G3 (P < 0.02), and G1 v. G2 (P < 0.03). In Experiment 2, granulosa and theca cells from Nellore cows were recovered from follicles obtained in a local abattoir and submitted to total RNA extraction and expression of LHR isoforms (LHR-B3, LHR-B4, LHR-B5, and LHR-B6) by semiquantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPD) as the internal control. Follicles were dissected, measured with a paquimeter, and allocated in 3 groups according to follicular diameter (mm): A (8.0-9.0), B (9.1-10.0), and C (10.1-11.0). Considering that follicles measured with paquimeter are on average 1.0 mm larger than those measured by US, Groups A, B, and C correspond to Groups G1, G2, and G3 (Experiment 1). In order to select only nonatretic (healthy) follicles, the E2/P4 >1.0 ratio was used. Therefore, from a total of 400 ovaries, only 5, 4, and 4 granulosa (n = 13) and 7, 8, and 8 theca samples (n = 23) from Groups A, B, and C, respectively, were obtained. The data were analyzed by ANOVA and Pearson’s correlation. There were no significant differences in total LHR expression (LHR-B3 + LHR-B4 + LHR-B5 + LHR-B6) in theca cells from Groups A, B, and C. However, in granulosa cells, follicles from Group A had lower LHR expression (16.5; mRNA LHR/mRNA GAPD) compared with Group C (37.6; P < 0.05). There was a positive correlation between expression of LHR-B5 and LHR-B6 isoforms and an increase in follicular diameter. In conclusion, these preliminary results indicate that ovulatory capacity in Nellore cattle is related to an increase in follicular diameter and LHR expression in granulosa cells. R. A. L. Simões, R. A. Satrapa, and A. C. S. Castilho are recipients of fellowship and funding from FAPESP (São Paulo, Brazil).

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.

115 FOLLICULAR GROWTH AND BLOOD FLOW OF THE DOMINANT FOLLICLE IN CROSSBRED RECIPIENTS TREATED WITH eCG

2014 ◽  
Vol 26 (1) ◽  
pp. 171
Author(s):  
M. P. Palhao ◽  
N. S. Junior ◽  
C. R. B. Guimarães ◽  
C. A. C. Fernandes ◽  
M. E. O. Ferreira ◽  
...  
Keyword(s):  
Blood Flow ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Sao Paulo ◽  
Ovulation Rate ◽  
São Paulo ◽  
Follicular Growth ◽  
Dominant Follicle ◽  
Follicle Diameter ◽  
Transfer Protocol

This study aimed to explore changes in follicle diameter and blood flow of the dominant follicle (DF), in ovulation and embryo transfer rates, after inclusion of eCG in a protocol for timed embryo transfer. The effect presence or absence of a corpus luteum (CL) at the start of treatment was also included. Crossbred heifers (n = 116, Bos taurus × Bos indicus), with (n = 61) or without (n = 55) CL, were included in the same hormone protocol: Day 0 (D0), insertion of progesterone (P4) device (1.0 g, Sincrogest®, Ouro Fino, São Paulo, Brazil) and 2 mg of oestradiol benzoato (EB, Sincrodiol®, Ouro Fino); D8, removal of P4 device and injection of sodium Cloprostenol (0.250 mg mL–1, Sincrocio®, Ouro Fino). On D8, the animals with and without CL – at the beginning of the protocol – were equally divided into 2 groups (G): G1 – injection of 300 IU (2.0 mL) of eCG (n = 56; Synchro eCG®, Ouro Fino); G2 – 2.0 mL of saline (n = 60). The ovulations were synchronized with 1 mg of EB on D9. From D8 to D11, the diameter of the DF and blood flow in its wall were recorded daily (M5 ultrasound with colour Doppler technology, 7.5-MHz linear array, DPS medical equipment, São Paulo, Brazil). Approximately 100 frames in colour-flow mode, containing entire cross-sections of the DF, were recorded during each examination. The area of the follicular wall with coloured pixels was measured with ImageJ software (Image Processing and Analysis in Java) from the frame with the largest blood flow signal. Before embryo transfer, all heifers were evaluated, and those with good-quality CL received frozen/thawed embryos (ethylene glycol 1.5 mol). Follicle diameter and blood flow area were compared between groups with or without CL before timed embryo transfer protocol and between eCG treatments. The PROC GLM procedure of SAS (version 9.0) and the t-test were used to assess the differences between means. Pregnancy diagnosis was performed on D35. Embryo transfer (ET) rate of the recipients and pregnancy rate were compared between CL or eCG treatments by the chi-squared test. Ovarian status, before hormone protocol, did not change (P > 0.05) the follicular growth of the DF. However, ovulation rate (78.8 v. 65.4%, P < 0.05) and ET rate (78.7 v. 65.4%, P < 0.05) were higher in animals with CL on D0. From D8 to D10, the inclusion of eCG did not affect (P > 0.05) follicular growth and blood flow of the DF. The time effect (P < 0.0001) for follicular blood flow had shown an increase in area of blood flow 24 h after implant removal (7.7 ± 0.7,b 10.2 ± 0.7,a and 12.3 ± 1.0a mm2, for Days 8, 9, and 10, respectively). The eCG did not affect (P > 0.05) the ovulation rate (71.4 and 73.3%, respectively, eCG and no eCG), however, approached an increased (P < 0.06) ET rate (78.8 v. 66.7%). The overall pregnancy rate (51.2%, 43/84) was not affected (P > 0.05) by evaluated variables. In summary, the addition of 300 IU of eCG on D8 of the timed embryo transfer protocol did not change the development of DF but increased the ET rate of the recipients. Biotran, FAPEMIG (project number APQ-1454-12), and CnPQ are acknowledged.

198 PROGESTERONE, ESTROGEN (ER-α AND ER-β), AND OXYTOCIN RECEPTOR GENE EXPRESSION IN CANINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 248
Author(s):  
A. A. P. Derussi ◽  
A. C. S. Castilho ◽  
R. W. A. Souza ◽  
R. Volpato ◽  
C. R. F. Guaitolini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Hormone Receptors ◽  
Target Genes ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Amplification Efficiency ◽  
Mrna Levels ◽  
Lh Surge ◽  
Total Rna

The aim of this study was to compare the mRNA levels of hormone receptor for progesterone (PR), oestrogen α (ER-α), oestrogen β (ER-β), and oxytocin (OTR) in canine morulae and blastocysts. Ten healthy mature bitches were inseminated based on monitoring vaginal cytology and progesterone concentration. The first insemination was performed on Day 2 after the preovulatory LH surge (progesterone 4 ng mL–1), and the second was performed 48 h later. All females were submitted to ovariohysterectomy (OVH), and the oviduct as well the uterurs were flushed with PBS solution to obtain the embryos. The females were divided into two groups: Group A (n = 5), morulae were collected 8 days after the LH surge and Group B (n = 5), blastocysts were collected 12 days after the LH surge. The pools (n = 10) of embryos (5 embryos/pool) were stored in RNAlater® (Ambion, Life Technologies, USA) at –80°C. The samples were analysed together. The RNA later was removed used PBS calcium free and the total RNA extraction was performed using the Qiagen RNeasy micro-kit (Hildesheim, Germany). Before reverse-transcription (RT) reaction, the total RNA was treated with DNase I Amplification Grade (Invitrogen Life Technologies, Carlsbad, CA, USA). The gene expression of target genes was assessed by real-time RT-qPCR, using SuperScript III for RT and power SYBR Green PCR Master Mix (Applied Biosystems, USA) for cDNA for PCR. The primers for target genes were designed using the software Primer Express® (Applied Biosystems, USA). The gene expression of target genes was normalized by HPRT gene and the relative abundance of mRNA was determined by the ΔΔct method corrected by amplification efficiency using Pffafl’s equation. The means of mRNA relative abundance were compared by t-test. The PR mRNA expression only in blastocysts is similar to the results obtained by Hou et al. (1997) in rat embryos. It is believed that the absence of PR in the early stages of cleavage is due to the indirect action of progesterone by growth factors produced by the maternal reproductive tract (2). Apparently, ER-β action does not occur in the embryo canine phases analysed; however, the action of ER-α seems related to the deployment signal as seen by Hou et al. (1996) in rats. Similarly to findings in the literature, OTR expression decreased in canine embryonic development. This receptor was produced by blastocysts while present in the uterus, which may represent an incidental mechanism to the embryo control of endometrial receptivity, such as also to prevent the development of endometrial luteolytic mechanism. The variation in hormone receptors gene expression in canine embryos can be influencing the transition from morula to blastocyst. In addition, a hormonal influence on these structures can occur in different ways.

scholarly journals LH-Receptor Gene Expression in Human Granulosa and Cumulus Cells from Antral and Preovulatory Follicles

2012 ◽  
Vol 97 (8) ◽  
pp. E1524-E1531 ◽  
Author(s):  
Janni Vikkelsø Jeppesen ◽  
Stine Gry Kristensen ◽  
Maria Eilsø Nielsen ◽  
Peter Humaidan ◽  
Maria Dal Canto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Receptor Gene ◽  
Cumulus Cells ◽  
Lh Receptor ◽  
Preovulatory Follicles ◽  
Receptor Gene Expression

Control of low density lipoprotein receptor gene expression in steroidogenic cells

1989 ◽  
Vol 67 (8) ◽  
pp. 968-973 ◽  
Author(s):  
Koichiro Takagi ◽  
Jerome F. Strauss III
Keyword(s):  
Gene Expression ◽  
Cyclic Amp ◽  
Granulosa Cells ◽  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Steroidogenic Cells ◽  
Receptor Gene Expression

Low density lipoprotein (LDL)-carried cholesterol is a primary substrate for steroid hormone synthesis by luteinized human granulosa cells. Chorionic gonadotropin and 8-bromo-cAMP both increase LDL receptor levels in granulosa cells by stimulating accumulation of the receptor mRNA. LDL and 25-hydroxycholesterol reduce LDL receptor expression, but this suppressive effect is partially overcome by 8-bromo-cAMP. Using fusion gene constructs containing the LDL receptor gene promoter transfected into JEG-3 cells, a cyclic AMP responsive enhancer could not be identified in the LDL receptor gene upstream promoter in transfection studies. We suggest that the LDL receptor gene in human steroidogenic cells is under negative control by a sterol effector, but that a cyclic AMP triggered process overcomes, to some extent, the sterol-mediated suppression. The detailed mechanisms by which sterol and cyclic AMP modulate LDL receptor gene expression remain to be elucidated.Key words: low density lipoproteins, low density lipoprotein receptors, cholesterol, steroidogenesis, gonadotropins.

scholarly journals LH receptor gene expression in cumulus cells in women entering an ART program

2012 ◽  
Vol 29 (5) ◽  
pp. 409-416 ◽  
Author(s):  
Myrto Papamentzelopoulou ◽  
Despina Mavrogianni ◽  
George A. Partsinevelos ◽  
Spyros Marinopoulos ◽  
Vasiliki Dinopoulou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Receptor Gene ◽  
Cumulus Cells ◽  
Lh Receptor ◽  
Art Program ◽  
Receptor Gene Expression

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

2012 ◽  
Vol 77 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Renato A.L. Simões ◽  
Rafael A. Satrapa ◽  
Fernanda S. Rosa ◽  
Marcelo Piagentini ◽  
Anthony C.S. Castilho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovulation Rate ◽  
Lh Receptor ◽  
Follicle Diameter

scholarly journals Differential gene expression of granulosa cells after ovarian superstimulation in beef cattle

Reproduction ◽  
2013 ◽  
Vol 146 (2) ◽  
pp. 181-191 ◽  
Author(s):  
F C F Dias ◽  
M I R Khan ◽  
M A Sirard ◽  
G P Adams ◽  
J Singh
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Stress Response ◽  
Granulosa Cells ◽  
Rna Extraction ◽  
Oxidative Stress Response ◽  
Endoplasmic Reticulum Stress Response ◽  
Control Group ◽  
Matrix Remodeling ◽  
Upregulated Genes

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 upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.

scholarly journals Comparison of Q223R leptin receptor polymorphism to the leptin gene expression in Greek young volunteers

2021 ◽  
Vol 8 (4) ◽  
pp. 301-310
Author(s):  
Panagiotis Halvatsiotis ◽  
◽  
Argyris Siatelis ◽  
Panagiotis Koulouvaris ◽  
Anthimia Batrinou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Leptin Receptor ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Genomic Region ◽  
Small Scale ◽  
Coding Region ◽  
Gender And Age ◽  
Study Results ◽  
Pcr Rflp

<abstract><sec> <title>Objective</title> <p>The objective of the present study was to identify the leptin gene expression and the leptin receptor polymorphisms in blood samples and to correlate gene expression values with anthropometric characteristics.</p> </sec><sec> <title>Methods</title> <p>Blood from 140 Greek young volunteers was subjected to polymerase chain reaction–restricted fragment length polymorphism (PCR–RFLP), for the genomic region of Q223R polymorphism at codon 223 in the leptin receptor gene (<italic>LEPR</italic>) coding region. RNA extraction, cDNA synthesis and Quantitative Real-Time PCR was performed for assessing the expression of the leptin gene (<italic>LEP</italic>).</p> </sec><sec> <title>Results</title> <p>Leptin gene was identified in all tested specimens and the gene was expressed in 88.9% of all volunteers with BMI &lt; 25. In addition, it was observed that gene expression is affected by various external factors, such as Body Mass Index (BMI), eating behavior, gender and age. It was also shown that as for the Q223R polymorphism (A to G) allele G occurs with a frequency of 100% in men with BMI &gt; 30 and 75.9% in men and 88.9% in women with BMI 25–30. Volunteers with BMI 25–30 who were homozygous on the G allele were 50% and 77.8% in men and women respectively. All subjects with a BMI &gt; 30 were homozygous on the G allele at 100%.</p> </sec><sec> <title>Conclusions</title> <p>In this small-scale study, results have shown that the leptin gene expression correlates with BMI and that the allele G in Q223R polymorphism is linked to overweight individuals.</p> </sec></abstract>

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