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.

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)

scholarly journals The activin-follistatin system and in vitro early follicle development in goats

2006 ◽  
Vol 189 (1) ◽  
pp. 113-125 ◽  
Author(s):  
J R V Silva ◽  
T Tharasanit ◽  
M A M Taverne ◽  
G C van der Weijden ◽  
R R Santos ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Activin A ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Follicle Development ◽  
Cortical Tissue ◽  
Primary Follicle ◽  
Growth And Survival ◽  
Primordial Follicles

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

132. ACTIVIN A HAS A STIMULATORY EFFECT IN VITRO ON EARLY FOLLICLE DEVELOPMENT IN RAT OVARIES

2010 ◽  
Vol 22 (9) ◽  
pp. 50
Author(s):  
D. A. Cossigny ◽  
J. K. Findlay ◽  
A. E. Drummond
Keyword(s):  
Treatment Group ◽  
Real Time ◽  
Granulosa Cells ◽  
Combined Treatment ◽  
Activin A ◽  
Tunel Staining ◽  
Follicle Development ◽  
Primordial Follicles ◽  
Preantral Follicles

Activins are dimers of inhibin β subunits and are growth and differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily (1). Both βA and βB subunits are highly expressed in rat granulosa cells, while theca cells express little or no β subunit mRNAs (2). Oocytes lack expression of either subunit (3, 4). Activin is suggested to facilitate the responsiveness of granulosa cells to FSH (5). We hypothesized that activin, with or without FSH, could enhance the transition from the primordial to later preantral stages of follicle development. In two independent experiments, day 4 rat ovaries (n = 3 from different rats per treatment) were randomly assigned and cultured (6, 7) for 10 days in DMEM/Hams F-12 media with either no additives, FSH (100 ng/mL), activin A (50 ng/mL), or both. Day 4 fresh ovaries were also used as controls. Media and treatments were refreshed every alternate day. Ovaries were fixed andsectioned, or placed into Ultraspec for RNA extraction and real-time PCR analysis. Follicle numbers were counted as described previously (7). The proportion of atretic follicles (TUNEL staining) was determined in 3 randomly selected sections per ovary. Primordial follicles in all treatment groups were approximately 20% of those in Day 4 fresh ovaries. Primary follicles increased significantly (P < 0.05) only in the combined treatment group, where preantral follicles increased significantly (P < 0.0001) only when treated with activin A alone. Activin A alone decreased the proportion of atretic follicles in the primary and preantral classes, where the combined treatment increased the proportion of atretic preantral follicles. Real-time analysis revealed that expression levels of follistatin, FSH receptor and activin βA and βB subunits were all expressed at significantly higher levels in the Activin A-only treated group (P < 0.05). In summary, there was no effect on primordial follicle activation by any treatment. Activin alone had a stimulatory effect in vitro on subsequent folliculogenesis, but in the presence of FSH its effect was counteracted shown by an increase in atresia. Reasons for an increase in atretic preantral follicles in the combined treatment group are unclear. These studies support a stimulatory role for activin A in early follicle development and confirm the in vivo effects of activin on folliculogenesis (4). NHMRC program grant # 494802 and Fellowship (# 441101) provided financial support. (1) Vale W et al. 1986. Nature 321: 776–779.(2) Meunier H et al. 1988. Proc Natl Acad Sci USA 85: 547–251.(3) Roberts V et al. 1993. Journal of Clinical Endocrinology & Metabolism 7: 1402–1410.(4) Sidis Y et al. 1998. Biology of Reproduction 59(4): 807–812.(5) Drummond A et al. 2002. Endocrinology 143 (4): 1423–1433.(6) Nilsson E et al. 2001. Molecular and Cellular Endocrinology 182 (2): 145–155.(7) Rosairo D et al. 2008. Reproduction 136: 799–809.

scholarly journals Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers

2003 ◽  
Vol 177 (1) ◽  
pp. 45-55 ◽  
Author(s):  
TM Lovell ◽  
RT Gladwell ◽  
NP Groome ◽  
PG Knight
Keyword(s):  
Functional Role ◽  
Ovarian Follicle ◽  
Fold Increase ◽  
Activin A ◽  
Inhibin B ◽  
Follicle Development ◽  
Inhibin A ◽  
Ovarian Follicle Development ◽  
Tissue Extracts

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 mm but increased ~50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (< or =9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (approximately 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 mm; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (approximately 6-fold higher in F4; P<0.0001); levels then fell approximately 2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose approximately 3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell approximately 4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.

Effects of follicular fluids on the growth of porcine preantral follicle and oocyte

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 239-247 ◽  
Author(s):  
T. Metoki ◽  
H. Iwata ◽  
M. Itoh ◽  
M. Kasai ◽  
A. Takajyo ◽  
...  
Keyword(s):  
Calf Serum ◽  
Follicle Development ◽  
Preantral Follicle ◽  
Promoting Effect ◽  
Oocyte Growth ◽  
Preantral Follicles ◽  
Molecular Weights ◽  
Heat Treated ◽  
Growth Promoting ◽  
Follicular Fluids

SummaryWe examined the effect of supplementing the culture medium with follicular fluid (FF) on the growth of porcine preantral follicles and oocytes. Firstly, preantral follicles were retrieved from ovaries and then FF was collected from all antral follicles that were 2–7 mm in diameter (AFF), which included large follicles of 4–7 mm in diameter (LFF) and small follicles of 2–3 mm in diameter (SFF). When preantral follicles with a diameter of 250 μm were cultured in medium containing AFF, the growth of follicles and oocytes was greater than when follicles were cultured in medium containing fetal calf serum (FCS). When this growth-promoting effect in AFF was compared for LFF and SFF, the LFF were shown to be significantly more effective than SFF. This LFF effect was lost, however, when the concentration of LFF in the medium was decreased from 5% to 0.5% or when LFF were heat treated (60 °C for 30 min) or trypsin was added. In contrast, a decrease in SFF concentration from 5% to 0.5% and heat treatment of the SFF enhanced preantral follicle growth. Furthermore, proteins obtained from LFF that had molecular weights greater than 10 kDa (LFF > 10 kDa) had similar, but relatively reduced, growth-promoting properties. The remaining three LFF protein fractions (<10 kDa or <100 kDa or >100 kDa), however, did not have these growth-promoting properties. In conclusion, the supplementation of medium with LFF, rather than serum, enhanced preantral follicle and oocyte growth. Factors that enhanced follicle development in LFF and factors that suppressed follicle development in SFF were proteins and these LFF factors ranged in size from 10 kDa to over 100 kDa.

Morphogenesis of the silkworm egg

Development ◽  
1976 ◽  
Vol 36 (1) ◽  
pp. 13-18
Author(s):  
J. M. Legay
Keyword(s):  
Ovarian Follicle ◽  
Temporal Organization ◽  
Width Ratio ◽  
Follicle Development ◽  
Ovarian Follicle Development ◽  
Length Width ◽  
Form Stability

Ovarian follicle development, which accompanies morphogenesis of the silkworm egg has three distinct phases: spheric, ellipsoidal and flattened-ellipsoid. Transitions between phases are rapid and form-stability (characterized by length/width ratio) is preserved from the beginning of the ellipsoidal phase. The geometric stability of the follicle-oocyte-ovariole system, the polarity of the egg and the determinism in form changes reveal strikingly coordinated spatial and temporal organization.

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