Kit Ligand Expression in granulosa cells during follicular culture and associated maturation rate of oocytes in buffalo (Bubalus bubalis)

2015 ◽  
Author(s):  
Atul Mahajan ◽  
Parveen Kumar ◽  
Sachin Atole ◽  
B P Brahmkshtri ◽  
K. R. Tajane ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Culture Media ◽  
Pcr Amplification ◽  
Bubalus Bubalis ◽  
Ovarian Follicles ◽  
Kit Ligand ◽  
Tissue Culture Media ◽  
Maturation Rate ◽  
Granulose Cells

The objectives of the study to associate the expression of Kit Ligand gene under different gonadotropins and growth factors supplementation with the maturation rate of oocytes in buffalo ovarian follicular cells during whole follicular culture at subsequent intervals of 24 hours. Intact ovarian follicles were dissected out of buffalo ovaries and cultured in Tissue Culture Media-199 supplemented with gonadotropins and growth factors for 24 hours. A semi-quantitative RT-PCR amplification was obtained in granulose cells of buffalo ovarian follicles and its expression was found to be restricted during initial stage of follicle culture. KL expression persistency in granulosa cells beyond 4 hours during follicular culture was found to be negatively correlated with maturation rate of oocytes. It was concluded that expression of Kit Ligand gene in granulose cells could be predictive role for assessing oocytes maturation rate and possibly the oocytes competence in buffalo.

scholarly journals Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 641-649 ◽  
Author(s):  
Inger B Carlsson ◽  
Mika P E Laitinen ◽  
Jennifer E Scott ◽  
Henna Louhio ◽  
Louiza Velentzis ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Culture Media ◽  
Follicular Development ◽  
Ovarian Follicles ◽  
Kit Ligand ◽  
Kit Receptor ◽  
Ovarian Follicular Development

The receptor tyrosine c-Kit and its cognate ligand, c-Kit ligand (KL, stem cell factor, SCF), are involved in ovarian follicular development in several animal species. We studied the expression of KL and c-Kit usingin situhybridization and immunohistochemistry in donated human ovarian cortical tissue. The KL transcripts were expressed in granulosa cells of primary follicles, whereas the expression of c-Kit was confined to the oocyte and granulosa cells in primary and secondary follicles. We employed an ovarian organ culture using firstly serum-containing and then serum-free medium to study the effects of KL and an anti-c-Kit antibody, ACK2, on the development and survival of ovarian folliclesin vitro. Culture of ovarian cortical slices for 7 days resulted in a 37% increase in the number of primary follicles and a 6% increase in secondary follicles. The proportion of viable follicles decreased in all cultures. The addition of KL (1, 10 and 100 ng/ml) into the culture media did not affect the developmental stages of the follicles or the proportion of atretic follicles. Inclusion of ACK2 (800 ng/ml) in the culture medium significantly increased the proportion of atretic follicles on days 7 (49 vs 28% in control cultures) and 14 (62 vs 38%) of culture. In conclusion, c-Kit and KL are expressed in human ovaries during follicular development. Blocking the c-Kit receptor induces follicular atresia. The KL/c-Kit signaling system is likely to control the survival of human ovarian follicles during early follicular development.

scholarly journals Serum deprivation affects secretory activity of cultured porcine ovarian follicles and granulosa cells and their response to hormones

2009 ◽  
Vol 54 (No. 10) ◽  
pp. 455-460
Author(s):  
A.V. Sirotkin
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Secretory Activity ◽  
Ovarian Follicles ◽  
Serum Deprivation ◽  
Ovarian Cells ◽  
Igf I ◽  
Granulose Cells ◽  
Vitro Model

The aim of the present study is to understand the hormonal mechanisms of the effect of malnutrition on ovarian follicle functions. For this purpose, we examined the effect of malnutrition/serum deprivation, addition of metabolic hormones and gonadotropin (IGF-I, leptin and FSH) and their combination on the release of progesterone (P<sub>4</sub>), testosterone (T), estradiol (E<sub>2</sub>) and insulin-like growth factor I (IGF-I) by cultured whole ovarian follicles and on P<sub>4</sub> and IGF-I output by cultured granulosa cells isolated from porcine ovaries. It was observed that in ovarian follicles cultured with nutrients/serum addition of IGF-I reduced release of P<sub>4</sub>, but not of T or E<sub>2</sub>. Exogenous leptin reduced output of E<sub>2</sub>, but not of P<sub>4</sub> or T, and increased IGF-I output. No significant effect of FSH on release of steroid hormones by isolated follicles was found. Serum deprivation did not affect release of P<sub>4</sub>, but reduced output of T and E<sub>2</sub>, and promoted IGF-I release by cultured ovarian follicles. Addition of hormones failed to prevent the effect of malnutrition on the secretory activity of cultured ovarian follicles. In cultured granulose cells, all the tested hormones promoted release of both P<sub>4</sub> and IGF-I. Food restriction/serum deprivation reduced both P<sub>4</sub> and IGF-I output. Additions of either IGF-I, leptin and FSH prevented the inhibitory action of malnutrition on both P<sub>4</sub> and IGF-I release. The present observations (1) confirm the involvement of the hormones IGF-I, leptin and FSH in the control of secretory activity of ovarian cells, (2) demonstrate, that both isolated ovarian granulosa cells and whole follicles cultured in the absence of serum nutrients could be an adequate in-vitro model for studying the effect of malnutrition on ovarian secretory functions, and (3) suggest, that malnutrition could affect ovarian functions through changes in the release of ovarian hormones.

scholarly journals Characteristics of Goat Ovarium Granulosa Cells With Extract Leaves ‎‎(Schleichera oleosa) at Different Passage Levels in Vitro

el–Hayah ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 28-38
Author(s):  
Lil Hanifah ◽  
Roihatul Muti’ah
Keyword(s):  
Granulosa Cells ◽  
Leaf Extract ◽  
Culture Media ◽  
Cell Protein ◽  
Cell Culture Media ◽  
Randomized Design ◽  
Schleichera Oleosa ◽  
Protein Membrane ◽  
Granulose Cells

S. oleosa belongs to the Sapindaceae family and has phytochemicals phenolic acid so that it has enormous benefits in the process antioxidant. The presence of antioxidant activity in cells can affect the defense of the cell protein membrane, so that the ability of cells to perform division is optimal. In vitro this is mostly done by adding hormones to cell culture media or natural compounds to increase cell proliferation. Every population of cell that has undergone division during the proliferation process to confluent is an indication that the cell is aging. This study was an experimental study was designed using a completely randomized design (CRD) with 5 treatments and 5 replications as follows: K0: granulose cells and culture media without treatment (0 µl), P1: granulose cells and culture media with 1% kesambi leaf extract. (30 µl), P2: granulose cells and culture media given kesambi leaf extract 1.5% (45 µl), P3: granulose cells and culture media given kesambi leaf extract 2% (60 µl) and P4: granulosa cells and The culture media was given 2.5% (75 µl) to the leaves extract., the characteristics of goat ovarian granulosa cells by giving kesambi (Scheichera oleosa) leaf extract at different passages were at the level of confluence showing the highest value in passage I, namely treatment P1. Meanwhile, the level of viability shows the highest value in section I, namely the P4 treatment

IGF-I Prevents Fas Ligand-Induced Apoptosis in Granulosa Cells of Buffalo (Bubalus bubalis) Ovarian Follicles In Vitro.

2011 ◽  
Vol 85 (Suppl_1) ◽  
pp. 657-657
Author(s):  
Ravindra P. Janivara ◽  
Sunil K. Patil ◽  
Jamuna V. Kolatalu ◽  
Dhanya Joseph ◽  
Raghavendra Subbarao ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Fas Ligand ◽  
Bubalus Bubalis ◽  
Ovarian Follicles ◽  
Igf I ◽  
Induced Apoptosis

Leptin Supplementation Stimulates Synergism with Growth Factors and Hormones to Express Its Receptor in Cultured Preantral Follicles of Sheep

2020 ◽  
Author(s):  
K. Sravani Pragna ◽  
V. Praveen Chakravarthi ◽  
Deepa Pathipati ◽  
B. Rambabu Naik ◽  
L.S.S. Varaprasad Reddy ◽  
...  
Keyword(s):  
Growth Factors ◽  
Leptin Receptor ◽  
Culture Media ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Ovarian Follicles ◽  
Antral Follicles ◽  
Receptor Mrna

Background: Leptin receptor is a transmembrane receptor that regulates reproduction at molecular level.Since for action of any hormone on target cell and to have local action on any tissue, expression of its own receptor is necessary and also it is not known whether such improvement in ovarian follicular development by Leptin is mediated through presence of its homologous receptors in the sheep ovaries. Therefore this study aimed on expression of Leptin receptor mRNA in cultured ovarian follicles of sheep by RT PCR.Methods: Leptin receptor mRNA expression in sheep was studied using qRT-PCR from: (i) In vivo grown preantral, early antral, antral, large antral follicles and cumulus oocyte complexes obtained from large antral follicles subjected to 24h of in vitro maturation and (ii) PFs’ exposed to three different culture media for 3min, two, four or six days and subsequently matured in vitro for 24h. Result: Leptin receptor was observed at all stages ovarian follicles in both cumulus cells and oocytes. Leptin supplementation along with other growth factors and hormones stimulated the expression of its receptor mRNA which is parallel to in vivo stages which could suggest synergistic action of growth factors and hormones with Leptin. 

scholarly journals Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation, granulosa cell proliferation, steroid hormone and PGE2 release in cultured rat ovarian follicles

Reproduction ◽  
2002 ◽  
pp. 891-898 ◽  
Author(s):  
PS Duggal ◽  
NK Ryan ◽  
KH Van der Hoek ◽  
LJ Ritter ◽  
DT Armstrong ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Food Intake ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Culture Media ◽  
Ovarian Follicles ◽  
Meiotic Maturation ◽  
Feed Restriction ◽  
Preovulatory Follicles ◽  
Igf I

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

scholarly journals Pengaruh Keberadaan Corpus Luteum terhadap Kualitas dan Tingkat Maturasi Oosit Domba Lokal Umur Pubertas Awal Secara In Vitro

2018 ◽  
Vol 18 (2) ◽  
pp. 83-89
Author(s):  
Rini Widyastuti ◽  
Mas Rizky Anggun Adipurna Syamsunarno ◽  
Alvin Yusuf ◽  
Muhammad Rosyid Ridhlo ◽  
Sigit Prastowo
Keyword(s):  
Corpus Luteum ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Polar Body ◽  
Early Puberty ◽  
Tissue Culture Media ◽  
Maturation Rate ◽  
Fetal Bovine

ABSTRAK. Penelitian ini bertujuan mengetahui pengaruh keberadaan corpus luteum (CL) pada ovarium domba umur pubertas awal terhadap kualitas oosit hasil koleksi dan tingkat maturasinya secara in vitro (IVM). Sebanyak 279 oosit digunakan, terbagi pada kelompok tanpa CL (CL-) sebanyak 143 dan dengan CL (CL+) sebanyak 136. Oosit dipilih berdasarkan homogenitas sitoplasma dan dikelompokkan sesuai jumlah lapisan sel kumulus yaitu grade 1 (4 lapis), grade 2 (3–4 lapis) dan grade 3 (0–2 lapis). Media IVM menggunakan Tissue Culture Media 199 ditambah antibiotik, Follicle Stimulating Hormone, dan 10% Fetal Bovine Serum. Maturasi dilakukan pada inkubator 38,5°C, 5% CO2 selama 24 jam. Pasca IVM, tingkat kematangan oosit dievaluasi berdasar kemunculan Polar Body I (PB I). Hasil menunjukkan bahwa keberadaan CL tidak berpengaruh terhadap kualitas oosit yang dikoleksi pada semua grade. Keberadaan CL berpengaruh pada tingkat kematangan oosit pada grade 1 sebesar 48,64% dibandingkan CL- sebesar 47,19% (p0,05). Berdasarkan hasil penelitian disimpulkan bahwa berpengaruh pada tingkat kematangan oosit setelah IVM. Hasil penelitian menggambarkan potensi penggunaan oosit ternak umur pubertas awal untuk digunakan lebih lanjut dalam program produksi embrio secara in vitro. (Effect of the presence of corpus luteum on oocytes quality and in vitro maturation rate of ewes at early puberty) ABSTRACT. This study aims to know effect of presence of corpus luteum (CL) to collected oocytes quality and its maturation rate post in vitro maturation (IVM), on local ewes ovary at early puberty. In total 279 oocytes were collected, 143 without CL (CL-) and 136 with CL present (CL+). Oocytes were selected according to sitoplasma hemogenity, divided into 3 grades according to cumulus cell (CC) layer namely Grade 1, 2 and 3 indicated by 4, 3–4, and 0–2 CC layers, respectively. The IVM media was Tissue Culture Media 199 supplemented with antibiotic, Follicel Stimulating Hormone, and 10% Fetal Bovine Serum following culture at 38.5°C and 5% CO2. Twenty four hours post IVM, oocytes were evaluated on the presence of Polar Body I. Result showed that oocytes quality were not different among group in all grades. The present of CL gives better maturation rate in grade 1 compared to CL- (48.64 vs 47.19%; p0.05). The present finding show that the presence of CL improves oocytes maturation rate post IVM. Moreover, this study shows the potency of using oocytes from ewes ovary at early puberty for further in vitro embryo production program

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

Eliminating Thrips in Microshoot Cultures

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 506d-506
Author(s):  
Robert R. Tripepi ◽  
Holly J. Schwager ◽  
Mary W. George ◽  
Joseph P. McCaffrey
Keyword(s):  
Betula Pendula ◽  
Culture Media ◽  
Dry Weight ◽  
Thrips Tabaci ◽  
Shoot Cultures ◽  
Onion Thrips ◽  
Ulmus Americana ◽  
Shoot Height ◽  
Tissue Culture Media ◽  
Plant Tissue Cultures

Two insecticides, acephate or azadirachtin, were added to tissue culture media to determine their effectiveness in controlling onion thrips (Thrips tabaci Lindeman.) and to determine if these insecticides could damage the plant shoot cultures. To test for insecticide phytotoxicity, microshoots from European birch (Betula pendula), American elm (Ulmus americana), `Pink Arola' chrysanthemum (Dendranthema grandiflora), `America' rhododendron (Rhododendron catawbiense), `Golden Emblem' rose (Rosa hybrida), and `Gala' apple (Malus domestica) were placed in 130-ml baby food jars containing 25 ml of medium supplemented with 6.5, 13, or 26 mg/l Orthene® (contained acephate) or 0.55, 1.1, or 2.2 ml/l Azatin® (contained azadirachtin). Control jars lacked insecticide. To test for thrips control, 13 mg/l Orthene® or 0.55 ml/l Azatin® was added to Murashige and Skoog medium, and 10 thrips were placed on `Gala' apple microshoots in each jar. Jars were sealed with plastic wrap. In both studies, microshoot dry weight and heights were determined. In the second study, the total number of thrips per jar was also determined 3 weeks after inoculation. Microshoots on Orthene®-treated media lacked phytotoxicity symptoms, regardless of the concentration used. In contrast, Azatin® hindered plant growth, decreasing shoot height or dry weight by up to 85% depending on the species. Both insecticides prevented thrips populations from increasing, since less than 10 thrips were found in jars with insecticide-treated medium. Control jars, however, contained an average of almost 70 thrips per jar. This study demonstrated that both Orthene® and Azatin® were effective for eradicating thrips from plant tissue cultures, but Orthene® should probably be used because Azatin® was phytotoxic to all species tested.

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