| Objective (1) To study the association of Foxo3a (Forkhead box group O)transcriptor with oocyte apoptosis in neonatal rat ovaries. (2) To investigatethe Foxo3a-regulated oocytes apoptosis pathways. To explore whether Bim,FasL(Fas ligand), p27KIP1, caspase-3 and caspase-8 take part in theFoxo3a-regulated oocytes apoptosis pathways or not.Materials and Methods (1) About 250g adult Sprague-Dawley rats were obtainedfrom Shantou University Medical College. Pregnant rats delivered pups at 19.5days of gestation. Female pups of 1,2,3, and 4 days old (5 rats per age group)were sacrificed by decapitation. The ovaries isolated were quickly fixed in10% formaldehyde for 1-1.5 hours. The ovaries were dehydrated through anincreased ethanol dilution and cleared with xylene. Then the ovaries wereparaffin-embedded, serially sectioned at 4μm and mounted in order onaminopropyltriethoxysilane-coated slides. (2) Ovarian sections weredeparaffinized in xylenes and hydrated through an ethanol series of 100%, 90%,80%, 70%, and 50%. Then sections were stained with hematoxylin-eosin. Afterclearing with fresh xylene, sections were mounted with Canada balsam forobservation and viewed by light microscopy. (3) We performed animmunohistochemical study (Avidin-Biotin Complex) to determine theexpression levels and loclizations of the proteins such as Foxo3a, Bim, Fasligand, caspase-3, caspase-8, and p27KIP1 at different developmental stagesin neonatal rat ovaries. Ovarian sections were deparaffinized and hydrated.A solution of 2% BSA (bovine serum albumin) in PBS (phosphate buffered saline)was used as a blocking agent prior to incubating sections with goat anti-ratagainst Foxo3a, FasL (1:25 dilution), rabbit anti-rat againstcaspase-3, Bim, caspase-8 (1:50 dilution) and mouse anti-rat againstp27KIP1(1:50 dilution) overnight at 4℃in humidified chamber. Then, theslides were incubated for 1h with biotinylated anti-rabbit IgG(1:100 dilution) for caspase-3, caspase-8, bim, biotinylated anti-goat IgG(1:100 dilution)for Foxo3a, FasL, and biotinylated anti-mouse IgG for p27KIP1(1:100 dilution).Finally, sections were incubated for 30 min in an Avidin-Biotin Complex(ABC)staining kit. Staining was performed with DAB (3,3-diaminobenzidinetetrahydrochloride, 0.5 mg/ml) in PBS. Sections were then lightlycounterstained with hematoxylin. Negative controls were incubated in thepresence of PBS insdead of a primary antibody. Sections were analyzed by lightmicroscopy. (4) The ovary apoptotic profiles were determined by TUNEL(terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)staining. Section was carried out using the TUNEL apoptosis detection kitfollowing the manufacturer's instructions. Tissue sections werepermeabilized by incubation with proteinase K for 15 min at 37℃, and thencounterstained with Nuclear Fast Red and analyzed by light microscopy. Theoocytes exhibiting blue staining were considered positive. (5)Immunofluorescene for Foxo3a or Bim staining and DAPI(4',6-Diamidino-2-phenylindole dihydrochloride) counterstaining wereperformed to determine whether Foxo3a positive oocytes were apoptotic ones.Ovarian sections were deparaffinized and hydrated. After blocking nonspecificprotein-binding sites with 2% BSA in PBS for 30 min at room temperature,sections were incubated with goat ployclonal antibody against Foxo3a or rabbitployclonal antibody against Bim (1:25 dilution) overnight at 4℃in humidifiedchamber. Sections were then incubated with anti-goat IgG conjugated with Alexa—555(1:50 dilution, red fluorescence) or anti-rabbit IgG conjugated withAlexa-488(1:50 dilution, green fluorescence) for 1h in a humidified chamberat room temperature. Nuclei were stained simultaneously with DAPI (bluefluorescence). Sections were mounted with the anti-fading agent DABCO andvisualized by fluorescence microscopy. For colocalization of Foxo3a or Bimand nuclear fluorescence signal, digital images were merged using SimplePCIanalysis software. (6) Double Labelings of immunofluorescene for Foxo3a orBim and TUNEL staining were performed to further determine whether Foxo3a positive oocytes were apoptotic ones. Ovarian sections were deparaffinizedand hydrated. Tissue sections were permeabilized by incubation withproteinase K for 8 min at room temperature. After blocking nonspecificprotein-binding sites with 2% BSA for 30 min at room temperature, sectionswere incubated with primary antibody against Foxo3a (1:50 dilution) for 2hat 37℃in humidified chamber, incubated with anti-rabbit IgG conjugated withCy3 (red fluorescence) for 1h at 37℃in humidified chamber. The sectionswere covered with terminal deoxynucleotidyl transferase (TdT) bufferconjugated with FITC (green fluorescence) and were incubated in a humidifiedchamber at 37℃for 1h. Nuclei were stained simultaneously with DAPI (bluefluorescence). Sections were mounted with anti-fading agent DABCO andvisualized by fluorescence microscopy. For colocalization of Foxo3a or Bimand nuclear fluorescence signal, digital images were merged by using SimplePCIanalysis software.Results (1) Kinetics of neonatal rat follicle development. Follicles werecounted and classified as unassembled, early primordial, primordial, anddeveloping follicles. HE-staining showed that sections of 1-day-old ratovaries mainly contained early primordial follicles (about 76.73%), andprimordial follicles dominate from 2-day-old to 4-day-old rat ovaries, andthe number of primordial follicles occupied about 70% in 4-day-old ratovaries. (2) TUNEL staining and immunohistochemical detections ofFoxo3a, Bim, p27KIP1, caspase—3, caspase-8 and FasL expression andlocalization in neonatal rat ovaries.①The majority of TUNEL-positiveoocytes were observed in some early primordial follicles and primordialfollicles. In 1,2,3 and 4-day-old rat ovaries, the percentages ofWUNEL-positive oocytes were 7.24%, 7.35%, 4.07%, 1.82%, respectively. Nostatistical differences were observed between 1 and 2-day-old rat ovaries(p＞0.05). There were statistical differences between the other consecutiveage groups (p＜0.05).②Active Foxo3a dominately expressed in the nuclei of some oocytes in early primordial follicles and primordial follicles of neonaterat ovaries. In 1,2,3 and 4-day-old rat ovaries, the rates of activeFoxo3a-positive oocytes were 6.71%, 7.61%, 3.62%, 1.82%, respectively.No statistical differences were observed between 1 and 2-day-old rat ovaries(p＞0.05). There were statistical differences between the other age groups(p＜0.05). Additionally, the positive percentages between Foxo3a and TUNELhad no statistical differences at the same age group (p＞0.05).③Bim mainlyexpressed in both the nuclei and cytoplasms of some oocytes in early primordialfollicles and primordial follicles of neonatal rat ovaries. In 1,2,3 and4-day-old rat ovaries, the rates of Bim-positive oocytes were about 4.76%, 6.47%, 3.29%, 1.76%, respectively. No statistical differences wereobserved between 1 and 2-day-old rat ovaries (p＞0.05). There were statisticaldifferences between the other consecutive age groups (p＜0.05). Additionally,the positive percentages between Foxo3a and TUNEL had no statisticaldifferences at the same age group except 1 day old(p＞0.05).④FasL was mainlydetected in the nuclei and cytoplasms of some oocytes in early primordialfollicles and primordial follicles of neonatal rat ovaries. The rates ofFasL-positive oocytes were about 2.83%, 4.28%, 2.57%, 0.99% in 1,2,3 and4-day-old rat ovaries, respectively. No statistical differences were observedbetween 1 and 2-day-old rat ovaries (p＞0.05). There were statisticaldifferences between the other consecutive age groups (p＜0.05). In addition,the positive percentages between Foxo3a and FasL had no statisticaldifferences at the same age group except 2 day old (p＞0.05).⑤p27KIP1 wasdetected mostly in the nuclei and cytoplasms of some oocytes in earlyprimordial follicles and primordial follicles of neonate rat ovaries. Therates of p27KIP1-positive oocytes were about 4.92%, 6.86%, 4.20%, 1.49% in 1,2,3 and 4-day-old rat ovaries, respectively. There are statisticaldifferences at the same age group (p＜0.05). In addition, the positivepercentages between Foxo3a and p27KIP1 had no statistical differences at thesame age groups (p＞0.05).⑥Caspase-8 expressed mainly in the nuclei and cytoplasms of some oocytes in early primordial follicles and primordialfollicles of neonatal rat ovaries. In 1,2,3 and 4-day-old rat ovaries, therates of caspase-8-positive oocytes were approximately 4.84%, 6.08%, 3.76%, 1.29%. Caspase-3 also expressed mainly in the nuclei and cytoplasms ofsome oocytes in early primordial follicles and primordial follicles ofneonatal rat ovaries. In 1,2,3 and 4-day-old rat ovaries, the rates ofcaspase-3-positive oocytes were approximately 6.92%, 10.05%, 5.30%, 1.87%. (3) Double Labelings of immunofluorescene for Foxo3a or Bim and TUNELstaining. The results showed that Foxo3a-positive oocytes counterstained byDAPI had the morphologic characteristics of apoptosis such as the condensednuclei and fragmented nature, and that the Foxo3a-positive oocyte was the veryTUNEL-positive ones. Moreover, Bim-positive oocytes showed also themorphologic characteristics of apoptosis and were also TUNEL-positive cells.Conclusions (1) Our primary results indicate that Foxo3a transcriptor maytake part in regulating the apoptosis of follicular oocytes in neonatal ratovaries, and (2)that Bim, as the target of Foxo3a, may also be invovled inthe pathway of oocyte apoptosis regulation. (3)In addition, FasL, p27KIP1,caspase-3 and caspase-8 may also play a role in regulating the apoptosisof follicular oocytes in neonatal rat ovaries.
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