scholarly journals Identification of miRNAs associated with the follicular–luteal transition in the ruminant ovary

Reproduction ◽  
2012 ◽  
Vol 144 (2) ◽  
pp. 221-233 ◽  
Author(s):  
D McBride ◽  
W Carré ◽  
S D Sontakke ◽  
C O Hogg ◽  
A Law ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Expression Profiles ◽  
Oestrous Cycle ◽  
Corpora Lutea ◽  
Follicular Cells ◽  
Mirna Targets ◽  
Genome Wide ◽  
Ovulatory Follicles

Little is known about the involvement of microRNAs (miRNAs) in the follicular–luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0–5.5 mm), pre-ovulatory follicles (6.0–7.0 mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular–luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular–luteal transition.

scholarly journals Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice

Reproduction ◽  
2010 ◽  
Vol 139 (3) ◽  
pp. 587-598 ◽  
Author(s):  
Samu Myllymaa ◽  
Arja Pasternack ◽  
David G Mottershead ◽  
Matti Poutanen ◽  
Minna M Pulkki ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Microscopic Analysis ◽  
Corpora Lutea ◽  
Electron Microscopic ◽  
Oocyte Growth ◽  
Long Term Study ◽  
Ovarian Folliculogenesis

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain–Fc fusion protein (BMPR2ecd–Fc) is able to inhibit the actions of GDF9 and BMP15 in vitro. Here, we have produced bioactive BMPR2ecd–Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd–Fc during the postnatal days 4–12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd–Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4–28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd–Fc is a potent modulator of ovarian folliculogenesis in vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factors in vivo.

Inhibition of LH-stimulated cyclic AMP formation in pre-ovulatory rat granulosa cells by follicular fluid

1980 ◽  
Vol 95 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Knut Nordenström ◽  
Anita Sjögren ◽  
Lars Hamberger
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Female Rats ◽  
Bicarbonate Buffer ◽  
Ovulatory Follicles

Abstract. Immature female rats were injected sc with a single dose of PMSG to induce growth and maturation of ovarian follicles. In the morning of prooestrus the rats were given a single ip injection of LH (10 μg/rat) or 0.154 m NaCl, 2 h prior to sacrifice. Granulosa cells were isolated from the pre-ovulatory follicles and incubated in Krebs bicarbonate buffer, for 1 h with or without in vitro addition of various test substances. Following incubation the amounts of cAMP in tissue plus medium were determined. It was found that the isolated granulosa cells exposed to LH in vivo responded to the addition of LH in vitro with a production of high amounts of cAMP, i.e. these cells were not refractory to LH stimulation and in fact responded better than granulosa cells isolated from ovaries not exposed to LH in vivo. The addition to the incubation medium of follicular fluid (FFl) obtained from pre-ovulatory follicles decreased the effect of LH in vitro when added at a final concentration of 1% and completely abolished it at a concentration of 3%. Removal of steroids from the FFl did not influence the inhibitory effect and the addition of a phosphodiesterase inhibitor (IBMX) in vitro did not alter the results in principle. These results point to the existence of a factor in the FF1 which interacts with the sensitivity of the isolated preovulatory granulosa cells to repeated exposures to LH. Characterization of this factor is subject to further investigations.

Oestradiol synthesis by granulosa cells from immature rats treated with pregnant mare's serum gonadotrophin

1983 ◽  
Vol 104 (3) ◽  
pp. 372-380 ◽  
Author(s):  
Donald C. Johnson ◽  
Roger C. Hoversland
Keyword(s):  
Granulosa Cells ◽  
Steroid Synthesis ◽  
Bicarbonate Buffer ◽  
Immature Rats ◽  
Hypophysectomized Rats ◽  
System Inclusion ◽  
Ovulatory Follicles ◽  
Generating System

Abstract. Granulosa cells harvested from follicles in hypophysectomized or intact immature rats treated with 20 IU of pregnant mare's serum gonadotrophin (PMS) produced immunoreactive oestradiol (E2) when incubated in Krebs Ringer bicarbonate buffer containing an NADPH generating system; inclusion of steroid substrates in the medium increased the rate of synthesis. Further, tritiated E2 was synthesized when labelled progesterone was used as substrate. Granulosa cells removed from pre-ovulatory follicles on the morning of prooestrus in adult females also produced E2 in vitro. Although E2 synthesis was apparent by cells from immature hypophysectomized rats within 12 h of PMS treatment, it inceased greatly with longer in vivo exposure to the gonadotrophin. Production was linear with the number of cells incubated and with time, at least through the first 30 min; the production rate decreased slightly with longer incubations. Exposure of the cells in vivo to hCG or ovine LH, before incubation, destroyed most of their ability to synthesize E2 even if progesterone or pregnenolone was added to the medium, but conversion of testosterone to E2 was reduced by only about 50%. Inhibitors of steroid synthesis, i.e. 4-OH-androstenedione, SU-10603, cyanoketone, or aminoglutethimide, greatly reduced the amount of E2 synthesized by the cells. The results indicate that granulosa cells exposed in vivo to gonadotrophins can synthesize E2 without the addition of androgenic substrate provided that cofactors are supplied. This finding has important implications for the current 'two cell' theory for oestrogen production by the ovary. A deficiency in steroidogenic enzymes within the granulosa cell appears to be an inadequate basis for the theory. However, the total synthesis of E2 in vivo by granulosa cells has not been shown.

scholarly journals Response Gene to Complement 32 Expression Is Induced by the Luteinizing Hormone (LH) Surge and Regulated by LH-Induced Mediators in the Rodent Ovary

Endocrinology ◽  
2008 ◽  
Vol 149 (6) ◽  
pp. 3025-3036 ◽  
Author(s):  
Eun-Sil Park ◽  
Seungho Choi ◽  
Kenneth N. Muse ◽  
Thomas E. Curry ◽  
Misung Jo
Keyword(s):  
Granulosa Cells ◽  
Luciferase Reporter ◽  
Corpora Lutea ◽  
Functional Regression ◽  
Growth Factor Signaling ◽  
Response Gene ◽  
Specific Expression ◽  
Periovulatory Follicles

Response gene to complement 32 (Rgc32) has recently been suggested to be expressed in the ovary and regulated by RUNX1, a transcription factor in periovulatory follicles. In the present study, we determined the expression profile of the Rgc32 gene in the rodent ovary throughout the reproductive cycle and the regulatory mechanism(s) involved in Rgc32 expression during the periovulatory period. Northern blot and in situ hybridization analyses revealed the up-regulation of Rgc32 expression in periovulatory follicles. Rgc32 mRNA was also localized to newly forming corpora lutea (CL) and CL from previous estrous cycles. Further studies using hormonally induced luteal and luteolysis models revealed a transient increase in levels of Rgc32 mRNA at the time of functional regression of the CL. Next, the regulation of Rgc32 expression was investigated in vitro using rat preovulatory granulosa cells. The effect of human chorionic gonadotropin on Rgc32 expression was mimicked by forskolin, but not phorbol 12-myristate 13-acetate, and was mediated by the activation of progesterone receptors and the epidermal growth factor-signaling pathway. The mechanism by which RUNX1 regulates Rgc32 expression was investigated using chromatin immunoprecipitation and Rgc32 promoter-luciferase reporter assays. Data from these assays revealed direct binding of RUNX1 in the Rgc32 promoter region in vivo as well as the involvement of RUNX binding sites in the transactivation of the Rgc32 promoter in vitro. In summary, the present study demonstrated the spatial/temporal-specific expression of Rgc32 in the ovary, and provided evidence of LH-initiated and RUNX1-mediated expression of Rgc32 gene in luteinizing granulosa cells.

scholarly journals Genome-wide Analysis of Pre-mRNA Splicing

2004 ◽  
Vol 279 (50) ◽  
pp. 52437-52446 ◽  
Author(s):  
Aparna K. Sapra ◽  
Yoav Arava ◽  
Piyush Khandelia ◽  
Usha Vijayraghavan
Keyword(s):  
Splice Site ◽  
Dna Microarrays ◽  
Expression Profiles ◽  
Open Reading Frames ◽  
Specific Factor ◽  
Splicing Factors ◽  
Critical Function ◽  
Genome Wide

Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation. The spliceosome, a ribonucleoprotein performs this critical function; however, precise roles for many of its proteins remain unknown. Genome-wide consequences triggered by the loss of a specific factor can elucidate its function in splicing and its impact on other cellular processes. We have employed splicing-sensitive DNA microarrays, with yeast open reading frames and intron sequences, to detect changes in splicing efficiency and global expression. Comparison of expression profiles, for intron-containing transcripts, among mutants of two second-step factors, Prp17 and Prp22, reveals their unique and shared effects on global splicing. This analysis enabled the identification of substrates dependent on Prp17. We find a significant Prp17 role in splicing of introns which are longer than 200nts and note its dispensability when introns have a ≤13-nucleotide spacing between their branch point nucleotide and 3 ′ splice site.In vitrosplicing of substrates with varying branch nucleotide to 3 ′ splice site distances supports the differential Prp17 dependencies inferred from thein vivoanalysis. Furthermore, we tested the predicted dispensability of Prp17 for splicing short introns in the evolutionarily distant yeast,Schizosaccharomyces pombe, where the genome contains predominantly short introns. SpPrp17 was non-essential at all growth temperatures implying that functional evolution of splicing factors is integrated with genome evolution. Together our studies point to a role for budding yeast Prp17 in splicing of subsets of introns and have predictive value for deciphering the functions of splicing factors in gene expression and regulation in other eukaryotes.

scholarly journals The role of tyrosine kinase in gonadotropin-induced ovulation in the rat ovary

1998 ◽  
pp. 594-600 ◽  
Author(s):  
T Shimamoto ◽  
M Yamoto ◽  
R Nakano
Keyword(s):  
Tyrosine Kinase ◽  
Granulosa Cells ◽  
Kinase Inhibitor ◽  
Tissue Type ◽  
Corpora Lutea ◽  
Induced Ovulation ◽  
Rat Ovary ◽  
Ovulatory Process

OBJECTIVES: Our purpose was to elucidate the involvement of the tyrosine kinase pathway in gonadotropin-induced ovulation in the rat ovary. STUDY DESIGN: We investigated the effect of a tyrosine kinase inhibitor, tyrphostin, on the rat ovulatory process in vivo and in vitro. METHODS: In cultured rat granulosa cells, the effect of tyrphostin on LH-, dibutyryl cyclic AMP ((Bu)2cAMP)- or forskolin-stimulated tissue type plasminogen activator (tPA) activities was examined by using a fibrin autography technique. In an in vivo system, tyrphostin was injected into the bursal cavity of the ovary in pregnant mare serum gonadotropin-treated rats, just before human chorionic gonadotropin administration. After 24 h, the number of oocytes in the oviduct was counted and the tyrphostin-treated ovaries were examined histologically. RESULTS: Tyrphostin inhibited LH-stimulated tPA activity but did not affect (Bu)2cAMP- or forskolin-stimulated ones. In an in vivo study, tyrphostin suppressed oocyte release dose-dependently. Histological observations revealed that tyrphostin-treated ovaries contained many large unruptured follicles and a few corpora lutea. CONCLUSION: This study suggests that the suppressive effect of tyrphostin on ovulation may be partly due to tPA activity inhibition in the granulosa cells via the suppression of tyrosine kinase activity. Additionally, tyrosine kinase phosphorylation may be involved in gonadotropin-activated signaling systems in the rat ovulatory process.

Thyroid hormone as a regulator of basal and human chorionic gonadotrophin-stimulated steroidogenesis by cultured porcine theca and granulosa cells isolated at different stages of the follicular phase

1996 ◽  
Vol 8 (6) ◽  
pp. 961 ◽  
Author(s):  
EL Gregoraszczuk ◽  
M Skalka
Keyword(s):  
Thyroid Hormone ◽  
Granulosa Cells ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Follicular Cells ◽  
Steroid Secretion ◽  
Follicle Size ◽  
Androgen Secretion

To characterize thyroid hormone action in the ovary, the direct effect of triiodothyronine (T3) was investigated in vitro using a culture system of porcine theca cells (Tcs) and granulosa cells (Gcs) in mono- and co-culture (GT), the latter resembling follicles in vivo. The cells were cultured in the absence or presence of human chorionic gonadotrophin (hCG) with or without T3 (10(-7), 10(-9) or 10(-11) M). Follicular cells were obtained from follicles of different size (small, medium and large), and steroid secretion into the culture medium was detected by radioimmunoassay. T3 alone did not influence steroid secretion by Tcs and Gcs isolated from follicles that were small and medium in size. In preovulatory follicles, an increase in basal androgen secretion and a simultaneous decrease in oestradiol secretion were observed with Tcs and Gcs in both mono- and co-culture. T3 together with hCG decreased hCG-stimulated androgen secretion in Tcs isolated from medium-sized follicles and had a simultaneous stimulatory effect on hCG-stimulated oestradiol secretion by Gcs. In cultures of follicular cells obtained from large follicles, T3 decreased hCG-stimulated secretion of both androgen and oestrogen by Tcs and simultaneously stimulated oestradiol secretion in GT co-cultures. Thus, the interaction of T3 with gonadotrophin hormone modulated follicular steroidogenesis, depending on follicle size and cell type used in culture. The observed T3-induced increase in basal androgen secretion by Tcs could account for the atresia of follicles, since it is accompanied by a decrease in oestradiol secretion in GT co-culture. In its co-activity with hCG, an adequate level of T3 prevents excessive androgen production by Tcs, probably influencing aromatization processes in the follicle. An increase in hCG-stimulated oestradiol secretion in GT co-cultures is then observed. Further investigations are required to clarify whether this is linked with an effect on the aromatization processes occurring in the follicle.

Bleb formation in granulosa cells of rat pre-ovulatory follicles in vivo and in vitro

1981 ◽  
Vol 13 (4) ◽  
pp. 669-680 ◽  
Author(s):  
P. Bagavandoss ◽  
A.R. Midgley
Keyword(s):  
Granulosa Cells ◽  
Bleb Formation ◽  
Ovulatory Follicles

scholarly journals Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Shipra Kumari ◽  
Bashistha Kumar Kanth ◽  
Ju young Ahn ◽  
Jong Hwa Kim ◽  
Geung-Joo Lee
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Lilium Longiflorum ◽  
Biotic And Abiotic Stress ◽  
Biotic Stresses ◽  
Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

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