Stability of housekeeping genes and expression of locally produced growth factors and hormone receptors in goat preantral follicles

Zygote ◽  
2010 ◽  
Vol 19 (1) ◽  
pp. 71-83 ◽  
Author(s):  
Isana M. A. Frota ◽  
Cintia C. F. Leitão ◽  
José J. N. Costa ◽  
Ivina R. Brito ◽  
Robert van den Hurk ◽  
...  
Keyword(s):  
Growth Factors ◽  
Hormone Receptors ◽  
Rna Extraction ◽  
Housekeeping Genes ◽  
Stable Gene ◽  
Mrna Levels ◽  
Preantral Follicles ◽  
The Stability ◽  
Suitable Reference

SummaryThe aim of the present study was to investigate the stability of six housekeeping genes, and the relative expression of growth factors (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 and KL) and hormone receptors (FSH, LH and GH) in goat preantral follicles. To evaluate to stability of housekeeping genes micro-dissected fresh follicles (150–200 μm) as well as follicles that have been in vitro cultured for 12 days were used. In addition, isolated fresh follicles were used to compare expression of various growth factors and hormone receptors before culture. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA. After amplification of cDNA by real-time PCR, the geNorm software program was used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), β-tubulin, β-actin, phosphoglycerokinase (PGK), 18S rRNA, ubiquitin (UBQ) and ribosomal protein 19 (RPL-19). In addition, follicular steady-state levels of mRNA from the various growth factors under study were compared. Results demonstrated that, in goat preantral follicles, UBQ and β-actin were the most suitable reference genes and thus could be used as parameters to normalize data from future in vitro studies. In contrast, 18S RNA appeared the least stable gene among the tested housekeeping genes. Analysis of mRNA for several hypophyseal hormone receptors in fresh preantral follicles showed significantly higher FSH-R mRNA levels than those of LH-R and GH-R, and no difference between GH-R and LH-R mRNA levels. In regard growth factor mRNA expression in goat preantral follicles, EGF mRNA levels appeared significantly lower than those of the other studied growth factors. Increasingly higher relative mRNA levels were observed for GDF-9, BMP-15, BMP-6, FGF-2, VEGF, Kl and IGF-1, successively. In conclusion, UBQ and β-actin are the most stable housekeeping genes in fresh and 12-days cultured caprine preantral follicles. Furthermore, in fresh follicles, high levels of FSH-R mRNA are detected while among eight growth factors, IGF-1 is the most highly expressed and EGF the weakest expressed compound.

Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro

2017 ◽  
Vol 29 (6) ◽  
pp. 1144 ◽  
Author(s):  
R. M. P. Rocha ◽  
L. F. Lima ◽  
I. R. Brito ◽  
G. M. Silva ◽  
H. H. V. Correia ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cytochrome P450 ◽  
Hormone Receptors ◽  
Mrna Levels ◽  
Follicular Growth ◽  
Steroid Secretion ◽  
Preantral Follicles ◽  
Treatment Groups ◽  
Cytochrome P450 Family

The aim of the present study was to evaluate the effect of anti-Müllerian hormone (AMH), with and without FSH, on the in vitro development of isolated caprine preantral follicles, as well as follicular steroid production and mRNA levels of AMH, hormone receptors (AMH and FSH), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP17 (cytochrome P450, family 17, subfamily A, polypeptide 1), HSD3B (3-beta-hydroxysteroid dehydrogenase) and Myc (myelocytomatosis oncogene). Isolated secondary follicles were cultured in minimum essential medium alpha (α-MEM+) alone or supplemented with 50 ng mL–1 AMH and/or 100 ng mL–1 FSH added sequentially on different days of culture. Follicles were cultured for a total of 18 days, with different media during the first (Days 0–9) and second (Days 10–18) halves of the culture period, resulting in six treatment groups, as follows: α-MEM+/α-MEM+, FSH/FSH, AMH/AMH, AMH+FSH/AMH+FSH, AMH/FSH, and FSH/AMH. Follicle development was evaluated on the basis of follicular growth, oocyte maturation and steroid secretion. There was a decrease in follicular growth rate in the AMH, AMH + FSH and AMH/FSH treatment groups compared with α-MEM+ and FSH treatment groups (P < 0.05). However, the different culture conditions had no effect on rates of meiotic resumption and steroid secretion (P > 0.05). Moreover, follicles cultured in the presence of FSH had lower levels of AMH receptor type II (AMHRII) mRNA compared with non-cultured control (freshly isolated follicles), and the AMH and AMH/FSH treatment groups. In conclusion, AMH reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.

Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella)

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 167-171 ◽  
Author(s):  
A.B. Brito ◽  
J.S. Lima ◽  
D.C. Brito ◽  
L.N. Santana ◽  
N.N. Costa ◽  
...  
Keyword(s):  
Reference Genes ◽  
Target Genes ◽  
Ovarian Tissue ◽  
Rna Extraction ◽  
Cebus Apella ◽  
Capuchin Monkeys ◽  
Stable Gene ◽  
Polymerase Chain ◽  
The Stability ◽  
Suitable Reference

SummaryThere is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

scholarly journals Role of Melatonin as a Survival Factor for In vitro Development of Sheep Preantral Follicles

2021 ◽  
Author(s):  
C. Chetan Kumar ◽  
B. Rambabu Naik ◽  
A.V.N. Siva Kumar ◽  
A. Ravi ◽  
L.S.S. Varaprasad Reddy ◽  
...  
Keyword(s):  
Growth Factors ◽  
Ovarian Function ◽  
Follicular Development ◽  
Oocyte Quality ◽  
Free Radical Scavenger ◽  
Radical Scavenger ◽  
Optimum Level ◽  
Preantral Follicles ◽  
Positive Effects

Background: Melatonin, a powerful free radical scavenger and broad-spectrum antioxidant may directly affect ovarian function by regulating folliculogenesis, maintenance of follicular integrity, oocyte quality and maturation capacity. Therefore, we aimed to study effects of melatonin and its interaction with growth factors in sheep preantral follicles. Methods: The influence of different concentrations of Melatonin (5-500 pM) on in vitro culture of preantral follicles (PFs’) isolated from sheep ovaries was studied. Experiments I and II were conducted to standardize the optimum concentration of Melatonin that supports better development of preantral follicles. Experiment III was conducted with the optimum level of Melatonin derived in the Experiments I and II to evaluate the effect of melatonin at 100pM in combination with various growth factors. Result: Overall follicular development was found to be the best in the PFs’ cultured in medium supplemented with 100pM of Melatonin. Melatonin supplementation showed positive effects on the preantral follicular development in combination with different growth factors.

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.

In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women

2020 ◽  
Vol 35 (12) ◽  
pp. 2793-2807
Author(s):  
P Asiabi ◽  
M M Dolmans ◽  
J Ambroise ◽  
A Camboni ◽  
C A Amorim
Keyword(s):  
Growth Factors ◽  
In Vitro Culture ◽  
Protein Detection ◽  
Basic Medium ◽  
Specific Marker ◽  
Mrna Levels ◽  
Theca Cells ◽  
Ovarian Cortex ◽  
Steroid Dehydrogenase

Abstract STUDY QUESTION Can human theca cells (TCs) be differentiated in vitro? SUMMARY ANSWER It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones. WHAT IS KNOWN ALREADY There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs. STUDY DESIGN, SIZE, DURATION After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53–74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line. PARTICIPANTS/MATERIALS, SETTING, METHODS To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Results obtained from qPCR showed a significant increase (P &lt; 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P &lt; 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P &lt; 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q &lt; 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q &lt; 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells. WIDER IMPLICATIONS OF THE FINDINGS This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests.

Effect of various growth factors on the in vitro development of goat preantral follicles

2006 ◽  
Vol 63 (1-2) ◽  
pp. 204-212 ◽  
Author(s):  
K. Rajarajan ◽  
B.S. Rao ◽  
R. Vagdevi ◽  
G. Tamilmani ◽  
G. Arunakumari ◽  
...  
Keyword(s):  
Growth Factors ◽  
In Vitro Development ◽  
Preantral Follicles ◽  
Vitro Development

scholarly journals Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Xu Zhao ◽  
Huanling Yang ◽  
Mingjie Chen ◽  
Xiaoxia Song ◽  
Changxia Yu ◽  
...  
Keyword(s):  
High Temperature ◽  
Real Time ◽  
Temperature Stress ◽  
Reference Gene ◽  
Reference Genes ◽  
Candidate Reference Gene ◽  
Housekeeping Genes ◽  
High Temperature Stress ◽  
Stable Gene ◽  
Mrna Levels

Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes,TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC,andH2A, were tested for their adequacy inLentinula edodes(L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia ofL. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm,TUBhad the lowest M values inL. edodesstrains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 wasTUB(0.030), and the best candidate reference gene in strain 18N44 wasUBI(0.047). In BestKeeper analysis, the standard deviation (SD) values ofUBC,TUA,H2A,EF1,ACT,18S, andGTPin strain 18 and those ofGADPHandGTPin strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, onlyUBIandTUBwere found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses,TUBwas the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction inL. edodesmycelia under high-temperature stress.

224. Platelet derived growth factors and receptors in the rat ovary contribute towards preantral follicle growth

2005 ◽  
Vol 17 (9) ◽  
pp. 87
Author(s):  
L. S. Sleer ◽  
C. C. Taylor
Keyword(s):  
Growth Factors ◽  
Preantral Follicle ◽  
Follicle Growth ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Follicle Diameter ◽  
The Family ◽  
Rat Ovary ◽  
In Cells

In this study, the family of platelet derived growth factors (PDGF) and receptors were identified and characterized in the rat ovary and a role in contributing towards growth of preantral follicles was revealed. Real-time polymerase chain reaction revealed the presence of mRNA for all platelet derived growth factors (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and receptors (PDGF-Rα and PDGF-Rβ). In situ hybridization identified oocytes of primordial/primary follicles and cells of the theca layer as a source of PDGF-B, PDGF-C and PDGF-D mRNA. Protein expression was explored through immunohistochemistry. In rats aged days 0 and 4, PDGF-Rα, PDGF-A and PDGF-C immunoreactivity was observed within oocyte clusters, and PDGF-Rβ and PDGF-B immunoreactivity in cells surrounding oocyte clusters. In primordial follicles, PDGF-Rβ and PDGF-C was observed in the oocyte, and PDGF-Rα and A in the either the oocyte or pregranulosa cells. In primary follicles, PDGF-A, PDGF-C, PDGF-Rα and PDGF-Rβ are expressed in the oocyte. PDGF-Rβ is also expressed in cells surrounding primordial and primary follicles, possibly the precursors to theca cells. In secondary and antral follicles, all four PDGF isoforms and both receptors are expressed in either theca or vascular cells of the theca layer, and PDGF-Rα and A are also expressed in some granulosa cells in rats aged day 20 and older. A role in preantral follicle growth was identified by in vitro culture of preantral follicles. Preantral follicles cultured in serum free medium increased in diameter by 11.0 ± 1.57% over 5 days. Addition of PDGF-AA, PDGF-AB or PDGF-BB to the medium resulted in increases in follicle diameter after 5 days of 18.32 ± 2.18%, 17.72 ± 2.3% and 17.6 ± 1.81%, respectively, representing a significant increase over control diameters. In summary, this study has identified and characterized the presence and localization of all members of the family of platelet derived growth factors and receptors in the rat ovary and revealed a role for these growth factors in positively influencing early follicle growth.

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