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Conditional ablation of macrophages disrupts ovarian vasculature.

Turner EC, Hughes J, Wilson H, Clay M, Mylonas KJ, Kipari T, Duncan WC, Fraser HM - Reproduction (2011)

Bottom Line: As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16  h.These events were followed by necrosis and profound structural damage.These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Reproductive Sciences Unit, Queen's Institute of Medical Research, Centre for Reproductive Biology Obstetrics and Gynaecology, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK.

ABSTRACT
Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48  h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16  h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

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Micrographs of individual antral follicles illustrating different stages of follicle health and changes associated with treatment. (A) Healthy antral follicle with no pyknotic cells, (B) <10% pyknotic cells, (C) 10–50% pyknotic cells and (D) >50% pyknotic cells within the granulosa cell layer as observed 16 h post–DT. Black arrows indicate pyknotic cells. (E) Histogram showing percentages of antral follicles at different degrees of atresia in control ovaries and ovaries collected 2, 8 and 16 h post-DT. DT treatment adversely affected antral follicle health with a complete absence of pyknosis-free follicles by 16 h following treatment (P<0.05). Bar =50 μm.
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fig7: Micrographs of individual antral follicles illustrating different stages of follicle health and changes associated with treatment. (A) Healthy antral follicle with no pyknotic cells, (B) <10% pyknotic cells, (C) 10–50% pyknotic cells and (D) >50% pyknotic cells within the granulosa cell layer as observed 16 h post–DT. Black arrows indicate pyknotic cells. (E) Histogram showing percentages of antral follicles at different degrees of atresia in control ovaries and ovaries collected 2, 8 and 16 h post-DT. DT treatment adversely affected antral follicle health with a complete absence of pyknosis-free follicles by 16 h following treatment (P<0.05). Bar =50 μm.

Mentions: Using H&E-stained ovarian sections, antral follicles were examined under light microscopy to determine the presence of pyknotic cells within the granulosa cell layer (Fig. 7A–D). The DT treatment adversely affected antral follicle health increasing the proportion of follicles with pyknosis (χ2) (P<0.05). By 16 h following treatment, there was a complete absence of pyknosis free follicles, a reduction that was significant (P<0.05) (Fig. 7E). In addition, the presence of antral follicles containing more than 50% pyknotic cells within the granulosa cell layer was only observed at 16 h following DT treatment (Fig. 7D). These changes in follicle health were seen in increasing severity by 24 and 48 h following DT treatment (data not shown). The timing of this increase in granulosa cell pyknosis correlates with the first observation of thecal haemorrhage, indicating that changes in follicle health are likely to be secondary effects of hypoxia due to vascular disruption.


Conditional ablation of macrophages disrupts ovarian vasculature.

Turner EC, Hughes J, Wilson H, Clay M, Mylonas KJ, Kipari T, Duncan WC, Fraser HM - Reproduction (2011)

Micrographs of individual antral follicles illustrating different stages of follicle health and changes associated with treatment. (A) Healthy antral follicle with no pyknotic cells, (B) <10% pyknotic cells, (C) 10–50% pyknotic cells and (D) >50% pyknotic cells within the granulosa cell layer as observed 16 h post–DT. Black arrows indicate pyknotic cells. (E) Histogram showing percentages of antral follicles at different degrees of atresia in control ovaries and ovaries collected 2, 8 and 16 h post-DT. DT treatment adversely affected antral follicle health with a complete absence of pyknosis-free follicles by 16 h following treatment (P<0.05). Bar =50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101494&req=5

fig7: Micrographs of individual antral follicles illustrating different stages of follicle health and changes associated with treatment. (A) Healthy antral follicle with no pyknotic cells, (B) <10% pyknotic cells, (C) 10–50% pyknotic cells and (D) >50% pyknotic cells within the granulosa cell layer as observed 16 h post–DT. Black arrows indicate pyknotic cells. (E) Histogram showing percentages of antral follicles at different degrees of atresia in control ovaries and ovaries collected 2, 8 and 16 h post-DT. DT treatment adversely affected antral follicle health with a complete absence of pyknosis-free follicles by 16 h following treatment (P<0.05). Bar =50 μm.
Mentions: Using H&E-stained ovarian sections, antral follicles were examined under light microscopy to determine the presence of pyknotic cells within the granulosa cell layer (Fig. 7A–D). The DT treatment adversely affected antral follicle health increasing the proportion of follicles with pyknosis (χ2) (P<0.05). By 16 h following treatment, there was a complete absence of pyknosis free follicles, a reduction that was significant (P<0.05) (Fig. 7E). In addition, the presence of antral follicles containing more than 50% pyknotic cells within the granulosa cell layer was only observed at 16 h following DT treatment (Fig. 7D). These changes in follicle health were seen in increasing severity by 24 and 48 h following DT treatment (data not shown). The timing of this increase in granulosa cell pyknosis correlates with the first observation of thecal haemorrhage, indicating that changes in follicle health are likely to be secondary effects of hypoxia due to vascular disruption.

Bottom Line: As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16  h.These events were followed by necrosis and profound structural damage.These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Reproductive Sciences Unit, Queen's Institute of Medical Research, Centre for Reproductive Biology Obstetrics and Gynaecology, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK.

ABSTRACT
Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48  h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16  h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

Show MeSH
Related in: MedlinePlus