270. TGF-β and ovarian follicle development

2008 ◽  
Vol 20 (9) ◽  
pp. 70
Author(s):  
D. A. Rosairo ◽  
J. K. Findlay ◽  
A. E. Drummond
Keyword(s):  
Ovarian Follicle ◽  
Combined Treatment ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Preantral Follicles ◽  
Ovarian Follicle Development ◽  
Apoptosis Detection ◽  
Rat Ovary ◽  
Tgf Β1

The role TGF-β plays in ovarian follicular growth and differentiation was investigated using a ‘physiological' culture system. TGF- β ligand and receptors are present in the rat ovary from 4 days after birth. Therefore we established organ cultures with these ovaries in order to assess the potential impact of TGF- β1 on follicle growth and transition from the primordial through to the primary and preantral stages of development. Whole ovaries were isolated and cultured for 10 days on floating filters with the addition of supplemented DMEM/Hams F-12 media and either FSH (100ng/mL), TGF- β1 (10ng/mL), or a combination of the two. Media as well as treatments were refreshed every second day. At the end of the culture period, ovaries were fixed in 10% formalin, embedded in paraffin and sectioned at 5µm. Sections were used for morphological assessment and ovarian follicle counting with three serial sections mounted/slide and every alternate slide used for counting of primordial, primary and preantral follicles. An evaluation of atresia by the detection of apoptotic cells was undertaken using terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick-end labelling (TUNEL) via the ApopTag® Peroxidase in situ apoptosis detection kit. Results gathered from this study show preantral follicle numbers declined significantly when treated with the combination of FSH and TGF- β1, consistent with our morphological appraisal of atresia where the combined treatment appeared to produce more apoptotic follicles than healthy follicles, suggesting an increase in atretic primary and preantral follicles. These preliminary findings suggest an inhibitory role for TGF- β1 in the presence of FSH, resulting in fewer follicles making the transition from the primary to the preantral stage. Further studies are required to test the effects of other TGF-β superfamily members on follicle transition in vitro. Supported by the NHMRC of Australia (Regkeys 241000, 441101, 465415, 198705)

123. ACTIVIN A AND OVARIAN FOLLICLE DEVELOPMENT

2009 ◽  
Vol 21 (9) ◽  
pp. 42
Author(s):  
D. A. Cossigny (Rosairo) ◽  
J. K. Findlay ◽  
A. E. Drummond
Keyword(s):  
Ovarian Follicle ◽  
Combined Treatment ◽  
Rna Extraction ◽  
Activin A ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Follicle Development ◽  
Preantral Follicle ◽  
Primary Follicle ◽  
Preantral Follicles ◽  
Ovarian Follicle Development

A significant developmental stage in ovarian folliculogenesis is the acquisition of gonadotropin sensitivity by ovarian follicles. Activin has previously been suggested to be involved in the responsiveness of granulosa cells to FSH (1). Therefore, the role of activin was investigated using a ‘physiological’ culture system to determine if pathways exist to transduce activin signals within the postnatal rat ovary. Organ cultures with day 4 whole ovaries were employed in order to assess the potential impact of Activin A on follicle growth and transition from the primordial through to the primary and later preantral stages of development. Ovaries were isolated and cultured for 10 days with the addition of supplemented DMEM/Hams F-12 media (2)and either FSH (100ng/ml), Activin A (50ng/ml), or a combination of the two. Media and treatments were refreshed every alternate day. At the end of the culture period, ovaries were fixed and sectioned, or placed immediately into Ultraspec for RNA extraction for future real-time PCR. Sections were used for morphological assessment and ovarian follicle counting of primordial, primary and preantral follicles. An evaluation of atresia by the detection of apoptotic cells was undertaken using terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick-end labeling (TUNEL). Primary follicle numbers increased significantly (P<0.05) in the combined treatment group whereas, preantral follicle numbers increased significantly (P<0.0001) when treated with Activin A alone. This is consistent with a morphological appraisal of atresia where a decrease in atresia was found in primordial and primary follicles, supporting the primary follicle development data and Activin A treatment alone resulted in more healthy primary and preantral follicles than atretic ones. Therefore, a stimulatory role for Activin A both in the presence of FSH (primary follicle development) or alone (preantral follicle development) has resulted in more follicles making the transition from the primordial to primary stages, as well as to the later preantral stages.

scholarly journals Comparison of Apoptosis Detection Markers Combined with Macrophage Immunostaining to Study Phagocytosis of Apoptotic Cells in Situ

2006 ◽  
Vol 1 ◽  
pp. 117727190600100 ◽  
Author(s):  
Dorien M. Schrijvers ◽  
Guido R.Y. De Meyer ◽  
Mark M. Kockx ◽  
Arnold G. Herman ◽  
Wim Martinet
Keyword(s):  
Lupus Erythematosus ◽  
Caspase 3 ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Immunological Responses ◽  
Apoptosis Detection ◽  
Apoptosis Markers ◽  
Suitable Marker ◽  
Parp 1

Efficient phagocytosis of cells undergoing apoptosis by macrophages is important to prevent immunological responses and development of chronic inflammatory disorders such as systemic lupus erythematosus, cystic fibrosis and atherosclerosis. To study phagocytosis of apoptotic cells (AC) by macrophages in tissue, we validated different apoptosis markers (DNA fragmentation, caspase-3 activation and cleavage of its substrate poly(ADP-ribose)polymerase-1) in combination with macrophage immunostaining. Human tonsils were used as a model because they show a high apoptosis frequency under physiological conditions as well as efficient phagocytosis of AC by macrophages. On the other hand, advanced human atherosclerotic plaques were examined since plaques show severely impaired phagocytosis of AC. Our results demonstrate that the presence of non-phagocytized terminal deoxynucleotidyl transferase end labelling (TUNEL)-positive AC represents a suitable marker of poor phagocytosis by macrophages in situ. Other markers for apoptosis, such as cleavage of caspase-3 or PARP-1, should not be used to assess phagocytosis efficiency, because activation of the caspase cascade and cleavage of their substrates can occur in AC when they have not yet been phagocytized by macrophages.

Culture of goat preantral follicles in situ associated with mesenchymal stem cell from bone marrow

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Camila Arrivabene Neves ◽  
Lucilene dos Santos Silva ◽  
Camila Ernanda Sousa de Carvalho ◽  
Marina Silva Carvalho ◽  
José Lindenberg Rocha Sarmento ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Culture Medium ◽  
Reduced Glutathione ◽  
Follicular Growth ◽  
Morphological Classification ◽  
Nitrite Concentration ◽  
Preantral Follicles ◽  
Rate Of Activation

SummaryThis study aims to develop an in vitro co-culture system of in situ goat preantral follicles with bone marrow-derived mesenchymal stem cells (BM-MSC), evaluating the influence of these cells on follicular growth, rate of activation and morphologically normal follicles. Fragments of ovarian cortex were cultured for 1 or 7 days in the presence of BM-MSC (BM-MSC+) and absence of BM-MSC (BM-MSC−). Histological sections of the fragments were analysed and data were obtained regarding morphological classification, survival rate of morphologically normal follicles and rate of follicular activation. Culture medium on days 1 and 7 was also sampled for nitrite concentration and reduced glutathione activity. There was a reduction (P < 0.05) in the percentage of morphologically normal follicles in the BM-MSC+ compared with the fresh control only on the seventh day of culture. When comparing treatments, on the seventh day of culture, a higher rate of morphologically normal preantral follicles was observed in BM-MSC+ (P < 0.05). In both treatments, primordial and developing follicle rates were similar to the fresh control (P > 0.05). When comparing treatments with each other, as well as with the fresh control, no differences were observed in follicular diameter (P > 0.05) or nitrite concentration (P > 0.05). The concentration of reduced glutathione was lower on the seventh day of co-culture in both treatments (P < 0.05). In conclusion, co-culture had no influence on follicular or oocyte development. However, it was critical to maintain the survival of preantral follicles during 7 days of culture.

scholarly journals Interpenetrating fibrin–alginate matrices for in vitro ovarian follicle development

Biomaterials ◽  
2009 ◽  
Vol 30 (29) ◽  
pp. 5476-5485 ◽  
Author(s):  
Ariella Shikanov ◽  
Min Xu ◽  
Teresa K. Woodruff ◽  
Lonnie D. Shea
Keyword(s):  
Ovarian Follicle ◽  
Follicle Development ◽  
Ovarian Follicle Development ◽  
Alginate Matrices

scholarly journals Involvement of miRNAs in equine follicle development

Reproduction ◽  
2013 ◽  
Vol 146 (3) ◽  
pp. 273-282 ◽  
Author(s):  
S N Schauer ◽  
S D Sontakke ◽  
E D Watson ◽  
C L Esteves ◽  
F X Donadeu
Keyword(s):  
Cell Survival ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Ovarian Follicle ◽  
Follicle Development ◽  
Previous Evidence ◽  
Ovarian Follicle Development ◽  
Fluid Levels ◽  
Follicle Selection

Previous evidence fromin vitrostudies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P≤0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels ofCYP19A1andLHCGR(P<0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets,PTEN,RASA1, andSMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.

Localization of ovine follistatin and α and βA inhibin mRNA in the sheep ovary during the oestrous cycle

1994 ◽  
Vol 12 (2) ◽  
pp. 181-193 ◽  
Author(s):  
D J Tisdall ◽  
N Hudson ◽  
P Smith ◽  
K P McNatty
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Indirect Evidence ◽  
Oestrous Cycle ◽  
Corpora Lutea ◽  
Α Subunit ◽  
Ovarian Follicle Development ◽  
Antral Follicles

ABSTRACT The sites of follistatin and α and βA inhibin gene expression were examined by in situ hybridization in sheep ovaries during the early and mid-luteal phases (days 3 and 10) of the oestrous cycle and a prostaglandin F2α (PGF2α)-induced follicular phase. Follistatin mRNA was detected in the granulosa cells of preantral, antral and early atretic follicles at all stages of the oestrous cycle, and in the corpora lutea at the early and mid-luteal stages of the cycle. However, only low levels of expression of follistatin were observed in the presumptive preovulatory follicle at 56 h after treatment with PGF2α. Both α and βA inhibin were shown to be expressed in ovaries at all stages of the oestrous cycle. In situ hybridization localized α subunit mRNA to the granulosa cells of most, but not all, healthy antral follicles, and to no other ovarian cell type. In contrast, expression of the βA subunit was confined to a few medium-to-large healthy antral follicles. In antral follicles expressing βA inhibin, mRNAs for α inhibin and follistatin were always detected, but the converse was not true. Unlike follistatin, no α and βA inhibin expression was seen in preantral follicles, developing corpora lutea, or follicles undergoing atresia. These results show that, in the adult sheep ovary, follistatin gene expression is a constitutive event in all growing follicles from the early preantral stage, and also provide indirect evidence of the involvement of follistatin, but not inhibin or activin, in the early stages of ovarian follicle development in sheep.

scholarly journals Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stromal Cells Prevent TGF-β1-Induced Myofibroblast Generation but Are Not Synergistic when Combined: Morphological in vitro Analysis

2018 ◽  
Vol 206 (6) ◽  
pp. 283-295 ◽  
Author(s):  
Flaminia Chellini ◽  
Alessia Tani ◽  
Larissa Vallone ◽  
Daniele Nosi ◽  
Paola Pavan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transforming Growth Factor ◽  
Synergistic Effects ◽  
Platelet Rich Plasma ◽  
Therapeutic Option ◽  
Smooth Muscle Actin ◽  
Combined Treatment ◽  
Biologically Active ◽  
Tgf Β1

The persistence of activated myofibroblasts is a hallmark of fibrosis of many organs. Thus, the modulation of the generation/functionality of these cells may represent a strategical anti-fibrotic therapeutic option. Bone marrow-derived mesenchymal stromal cell (MSC)-based therapy has shown promising clues, but some criticisms still limit the clinical use of these cells, including the need to avoid xenogeneic compound contamination for ex vivo cell amplification and the identification of appropriate growth factors acting as a pre-conditioning agent and/or cell delivery vehicle during transplantation, thus enabling the improvement of cell survival in the host tissue microenvironment. Many studies have demonstrated the ability of platelet-rich plasma (PRP), a source of many biologically active molecules, to positively influence MSC proliferation, survival, and functionality, as well as its anti-fibrotic potential. Here we investigated the effects of PRP, murine and human bone marrow-derived MSCs, and of the combined treatment PRP/MSCs on in vitro differentiation of murine NIH/3T3 and human HDFα fibroblasts to myofibroblasts induced by transforming growth factor (TGF)-β1, a well-known pro-fibrotic agent. The myofibroblastic phenotype was evaluated morphologically (cell shape and actin cytoskeleton assembly) and immunocytochemically (vinculin-rich focal adhesion clustering, α-smooth muscle actin and type-1 collagen expression). We found that PRP and MSCs, both as single treatments and in combination, were able to prevent the TGF-β1-induced fibroblast-myofibroblast transition. Unexpectedly, the combination PRP/MSCs had no synergistic effects. In conclusion, within the limitations related to an in vitro experimentation, our study may contribute to providing an experimental background for supporting the anti-fibrotic potential of the combination PRP/MSCs which, once translated “from bench to bedside,” could potentially offer advantages over the single treatments.

scholarly journals Correction to: Synergy of Paracrine Signaling During Early-Stage Mouse Ovarian Follicle Development In Vitro

2018 ◽  
Vol 11 (6) ◽  
pp. 537-537
Author(s):  
Hong Zhou ◽  
Joseph T. Decker ◽  
Melissa M. Lemke ◽  
Claire E. Tomaszewski ◽  
Lonnie D. Shea ◽  
...  
Keyword(s):  
Early Stage ◽  
Ovarian Follicle ◽  
Follicle Development ◽  
Paracrine Signaling ◽  
Ovarian Follicle Development ◽  
Development In Vitro
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