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An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats.

Salvetti NR, Panzani CG, Gimeno EJ, Neme LG, Alfaro NS, Ortega HH - Reprod. Biol. Endocrinol. (2009)

Bottom Line: We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w.These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ciencias Morfológicas, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Argentina. salvetti@fcv.unl.edu.ar

ABSTRACT

Background: Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.

Methods: We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.

Results: The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.

Conclusion: These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

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Localization of caspase-3, Ki-67 and PCNA by immunohistochemistry and in situ apoptosis by TUNEL. Positive staining is shown as brown colouring of the cytoplasm/nucleus of the cells. Figures A, D, G and J correspond to healthy tertiary follicles; B, E, H and K correspond to atretic follicles and C, F, I and L correspond to cystic follicles. (A-C) caspase-3 immunolocalization, (D-F) TUNEL, (G-I) Ki-67 immunolocalization and (J-L) PCNA immunolocalization. G: Granulosa, TI: Theca Interna, TE: Theca Externa. Bars = 20 μm.
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Figure 1: Localization of caspase-3, Ki-67 and PCNA by immunohistochemistry and in situ apoptosis by TUNEL. Positive staining is shown as brown colouring of the cytoplasm/nucleus of the cells. Figures A, D, G and J correspond to healthy tertiary follicles; B, E, H and K correspond to atretic follicles and C, F, I and L correspond to cystic follicles. (A-C) caspase-3 immunolocalization, (D-F) TUNEL, (G-I) Ki-67 immunolocalization and (J-L) PCNA immunolocalization. G: Granulosa, TI: Theca Interna, TE: Theca Externa. Bars = 20 μm.

Mentions: The proliferation index was evaluated by PCNA and Ki-67. Only nuclear staining was found with both cellular proliferation markers while the quantity of PCNA-positive cells was higher than that of Ki-67, possibly due to their higher sensitivity. Both antibodies showed similar patterns in the different follicular categories. Granulosa cells of tertiary follicles from the control group (Figures 1G and 1J) showed a higher proliferation index in relation to tertiary and cystic follicles from the treated group [See Additional file 1]. In theca interna cystic follicles presented lower proliferation index when compared with tertiary follicles in both groups [See Additional file 2] (Figures 1I and 1L). Cystic and atretic follicles showed minor proliferation in theca interna layer (Figures 1H, I, K and 1L). The theca externa also had reduced proliferation index in atretic follicles of both groups and in cystic follicles [See Additional file 3] (Figures 1H, I, K and 1L).


An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats.

Salvetti NR, Panzani CG, Gimeno EJ, Neme LG, Alfaro NS, Ortega HH - Reprod. Biol. Endocrinol. (2009)

Localization of caspase-3, Ki-67 and PCNA by immunohistochemistry and in situ apoptosis by TUNEL. Positive staining is shown as brown colouring of the cytoplasm/nucleus of the cells. Figures A, D, G and J correspond to healthy tertiary follicles; B, E, H and K correspond to atretic follicles and C, F, I and L correspond to cystic follicles. (A-C) caspase-3 immunolocalization, (D-F) TUNEL, (G-I) Ki-67 immunolocalization and (J-L) PCNA immunolocalization. G: Granulosa, TI: Theca Interna, TE: Theca Externa. Bars = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713246&req=5

Figure 1: Localization of caspase-3, Ki-67 and PCNA by immunohistochemistry and in situ apoptosis by TUNEL. Positive staining is shown as brown colouring of the cytoplasm/nucleus of the cells. Figures A, D, G and J correspond to healthy tertiary follicles; B, E, H and K correspond to atretic follicles and C, F, I and L correspond to cystic follicles. (A-C) caspase-3 immunolocalization, (D-F) TUNEL, (G-I) Ki-67 immunolocalization and (J-L) PCNA immunolocalization. G: Granulosa, TI: Theca Interna, TE: Theca Externa. Bars = 20 μm.
Mentions: The proliferation index was evaluated by PCNA and Ki-67. Only nuclear staining was found with both cellular proliferation markers while the quantity of PCNA-positive cells was higher than that of Ki-67, possibly due to their higher sensitivity. Both antibodies showed similar patterns in the different follicular categories. Granulosa cells of tertiary follicles from the control group (Figures 1G and 1J) showed a higher proliferation index in relation to tertiary and cystic follicles from the treated group [See Additional file 1]. In theca interna cystic follicles presented lower proliferation index when compared with tertiary follicles in both groups [See Additional file 2] (Figures 1I and 1L). Cystic and atretic follicles showed minor proliferation in theca interna layer (Figures 1H, I, K and 1L). The theca externa also had reduced proliferation index in atretic follicles of both groups and in cystic follicles [See Additional file 3] (Figures 1H, I, K and 1L).

Bottom Line: We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w.These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ciencias Morfológicas, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Argentina. salvetti@fcv.unl.edu.ar

ABSTRACT

Background: Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.

Methods: We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.

Results: The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.

Conclusion: These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

Show MeSH
Related in: MedlinePlus