Gonadotropin-Releasing Hormone Antagonist: RESULTS
Prepubertal rats were superovulated with eCG (control group, C) and treated with LA (LA group; 1 ^g rat day during 48 h), or Ant (Ant group; 10 ^g rat day during 48 h), or both (LA + Ant group). When the GnRH-agonist, LA, was injected alone, the ovarian weight decreased by 0.5-fold, whereas injection of GnRH-Ant increased ovarian weight by 0.4-fold compared with controls. Coinjection of both GnRH substances did not show differences from that of controls (Table 1).
Histological ovarian slides were stained with hematox-ylin-eosin to determine the number of different follicle stages and the percentage of apoptotic cells (Fig. 1). Table 1 shows that injection of LA significantly increased the number of PFs and atretic follicles (ATFs).
However, this treatment decreased the number of EAFs and POFs. The coinjection of GnRH-Ant interfered with the inhibitory effect of LA. Thus, the results show no change in the number of PFs, EAFs, and ATFs after cotreatment with the GnRH analogs. However, in this group, an increase in the number of POFs compared with that of the control group was observed. Surprisingly, the treatment with GnRH-Ant alone did significantly increase the number of POFs compared with that of the control group. canadian neighbor pharmacy online
Morphological and Biochemical Studies on Ovarian Apoptosis
To elucidate whether the changes observed in ovarian follicles content were mediated by a consequence of programmed cell death, the percentage of apoptotic cells in each follicular stage was determined (Table 2). LA increased the percentage of apoptotic cells in PFs, EAFs, and POFs. By contrast, GnRH-Ant treatment prevented the effect observed with LA. Unexpectedly, treatment with GnRH-Ant alone produced a decrease in the percentage of apoptotic cells in EAFs and POFs compared with that of the control group (Table 2).
These results were partially confirmed by DNA fragmentation analysis. Preovulatory follicles cultured in serum-free medium showed spontaneous onset of apoptotic DNA fragmentation (Fig. 2, lane 2). An increase in the spontaneous onset of apoptotic DNA fragmentation was observed in follicles obtained from LA-treated rats, with a 2.6-fold increase compared with that of controls (Fig. 2, lane 3). However, the treatment of superovulated rats with the GnRH-ant, Antide (5 ^g/injection every 12 h), did not change the level of ovarian DNA fragmentation (Fig. 2, lane 4). On the other hand, coincubation with the GnRH-ant reverted the stimulation of apoptosis by LA, because quantitative estimation of DNA cleavage from ovarian follicles revealed no changes in DNA fragmentation after in vivo LA + Ant treatment when compared with control cultured follicles (Fig. 2, lane 5). DNA fragmentation was minimal in freshly isolated preovulatory follicles, showing an increase in those follicles obtained from LA-treated rats (data not shown).
Protein Levels of FAS and FASL in Antral Follicles
Studies from a number of laboratories have suggested that FAS and FASL may be central to the regulation of cell death in the ovary. In addition, it was described that GnRH controls FAS and FASL expression in other cell types. Therefore, we decided to evaluate the protein expression of these cytokines. The follicular contents of FASL and FAS were evaluated by Western blotting (Fig. 3, A and B). No changes in the levels of FASL and FAS were observed when we compared fresh preovulatory follicles obtained from controls with those from the LA, Ant, or LA + Ant groups.
Effect of Analogs of GnRH on Subcellular Distribution of BAX and Cytochrome C
In previous works from our laboratory we showed that the apoptotic action of GnRH-a was correlated with an imbalance in the ratio of antiapoptotic:proapoptotic proteins (BCL-Xl:BCL-Xs), reducing the stability of the BCL-XL protein. In this study we have determined the in vivo effect of GnRH analogs on the subcellular distribution of BAX and cytochrome C. Mitochondrial and cytosolic fractions were prepared from isolated preovulatory follicles from the control, LA, Ant, and LA + Ant groups, and the levels of these proteins were determined by Western blot analysis. Mitochondrial preparation efficiency (80%) was estimated by the presence of P450scc enzyme, known to be specific for the mitochondrial membrane (Fig. 4C).
In the control group, BAX protein was mainly detected in the mitochondria (Fig. 4A). Similar results were observed in the LA group. Conversely, BAX protein was retained in cytosol when the animals were treated only with Ant. This effect was partially blocked when LA was coinjected with Ant. The analysis of cytochrome C distribution showed that this protein is localized mainly in mitochondria of samples belonging to animals injected with Ant or LA + Ant (Fig. 4B). We also noted that, despite the mitochondrial localization of BAX, the distribution of cytochrome C was similar in both fractions in the control group (Fig. 4B).
FIG. 1. Representative photomicrographs of ovarian sections from gonadotropin-stimulated rats treated with vehicle (control) or GnRH analogs (LA, 0.5 ^g/rat; Ant, 10 ^g/rat) during 2 days. A-D) Sections stained with hematoxylin-eosin. Note the presence of numerous PFs in the LA group (B), and POFs in the Ant group (C) and the LA + Ant group (D), in comparison with control group (A). EAF; early antral follicle, Oo; oocyte, Gc; granulosa cells, Tc; theca cells. Magnification X100.
TABLE 1. Effects of GnRH analogs on the ovarian response to gonadotropins.
a Data are expressed as mean ± SEM.
b-d Values with different superscripts are significantly different (P < 0.05).
FIG. 2. In vivo effect of treatment with GnRH analogs on ovarian inter-nucleosomal DNA fragmentation. Upper) Representative agarose gel showing DNA fragmentation and quantitative estimation of DNA cleavage. Prepubertal rats were superovulated with eCG and were injected every 12 h for 48 h with LA (0.5 |xg/rat), Ant (10 |xg/rat), or both. Control animals were treated with vehicle alone. Three hours after the last injection, preovulatory follicles were isolated and cultured for 24 h in serum-free medium. Four micrograms of follicular DNA extracted from each culture was analyzed by ethidium bromide staining, as described in Materials and Methods. Low-molecular-weight DNA (
TABLE 2. In vivo effect of GnRH analog treatment on the percentage of apoptotic ovarian cells.3
|Control||8.4 ± 0.67b||10.5 ± 0.71 b||11.3 ± 0.52b|
|LA||43.6 ± 5.41c||31.2 ± 2.42c||27.3 ± 2.53c|
|Ant||6.3 ± 0.53b||5.7 ± 0.60d||4.6 ± 0.74d|
|LA+Ant||3.4 ± 0.64b||4.9 ± 0.80d||5.4 ± 0.84d|
a Five randomly-selected fields from each ovarian section were analyzed (6 sections/ovary, 6-8 ovaries/group). Data are expressed as mean ± SEM. b-d Values with different letters in each follicular stage are significantly different (P < 0.05).
FIG. 3. Effect of GnRH analogs on FAS/FASL protein content in preovulatory follicles. A) Upper panel: representative immunoblot of FASL protein content in preovulatory follicles from control rats, and those treated with LA, Ant, or both. After homogenization, proteins were extracted and subjected to 15% SDS-PAGE, and transferred onto nitrocellulose membranes. FASL protein was visualized by using an anti-FASL antibody. Lower panel: Densitometric quantification of FAS content. Bars represent mean ± SEM of four experiments. B) Upper panel: representative immunoblot of FAS protein content in preovulatory follicles from control rats, and those treated with LA, Ant, or both. FAS protein was visualized by using an anti-FAS antibody. Lower panel: densitometric quantification of FAS content. Bars represent mean ± SEM of four experiments.
FIG. 4. GnRH analog-induced alteration in subcellular localization of BAX and cytochrome C. Cytosolic and mitochondrial fractions were prepared from isolated preovulatory follicles from prepubertal superovulated rats and treated with vehicle alone (control group, C) or with LA (1 rat day during 48 h; LA group), Ant (10 ^g rat day during 48 h; Ant group), or both LA and Ant (LA + Ant group). A) Upper panel: representative immunoblot of BAX in cytosolic and mitochondrial fractions. Fractions were analyzed by Western blot analysis by using primary antibody against BAX as described in Materials and Methods. B) Lower panel: densito-metric quantification of BAX content. Bars represent mean ± SEM of three experiments. *P < 0.05 comparing cytosol vs. mitochondria fraction contents in each treatment. B) Upper panel: representative immunoblot of cytochrome C in cytosolic and mitochondrial fractions. Fractions were analyzed by Western blot analysis using primary antibody against cytochrome C as described in Materials and Methods. Lower panel: densito-metric quantification of cytochrome C content. Bars represent mean ± SEM of three experiments. *P < 0.05 comparing cytosol vs. mitochondria fraction contents in each treatment. C) Representative immunoblot of P450scc in cytosolic and mitochondrial fractions. Fractions were analyzed by Western blot analysis using primary antibody against P450scc as described in Materials and Methods.