Mitigation of premature ovarian failure by over-expression of lentivirus vector-mediated Wilms tumor-suppressor gene

Purpose: To investigate the effect of WT1(Wilms tumor-suppressor gene) overexpression on premature ovarian failure (POF)-mediated ovarian dysfunction. Methods: Three mice groups were used: control group (untreated mice), POF group (mice sterilized by intravenous injection of cyclophosphamide and busulfan), and POF-LV(lentiviral vector) GFP(green fluorescent protein) WT1 group (POF mice given intra-ovarian microinjection of LV GFP WT1, a WT1 overexpressing lentiviral vector, one week after sterilization). Real time-PCR was employed to analyze in vitro WT1 overexpression levels. Overall ovarian function was measured by hormonal assay, H & E staining, and immuno-histochemical techniques. Results: Overexpression of WT1 in mice models of POF alleviated ovarian granulosa cell (GC) damage, increased ovary weight, and significantly increased follicular number (p < 0.05). Radioimmunoassays revealed reduction in plasma estradiol (E2) and follicle-stimulating hormone (FSH, p < 0.05). However, results from immune-histochemical assays showed reduced Bax expression levels, and increased expression of Bcl-2 in WTI-overexpression mice, relative to POF mice. Conclusion: Overexpression of WT1 may stimulate repair of ovarian tissue while improving endocrine function by inhibiting ovarian cell apoptosis signaling pathway in POF mice.


INTRODUCTION
Chemotherapy-induced POF and infertility are areas of immense interest to clinicians.This is due to rising incidence of malignant tumors and chemotherapy-induced POF, as well as increasing population of relatively young patients with malignant ovarian tumors [1,2].Apart from POF and infertility, chemotherapy is accompanied by complications such as anorexia, amenorrhea, cardiovascular diseases and osteoporosis, all of which severely impact the life quality of young cancer survivors [3].Fertilityenhancing treatments are available for POF patients.These include in vitro fertilization and hormonal therapy [4].In contrast, there are limited options for patients with chemotherapyinduced POF.Thus, there is need for research aimed at developing treatment strategies for this group of POF patients.The WT1 gene is implicated in genito-urinary disorders [5,6].It is an important inhibitor of cellular apoptosis in male germline cells, and in the embryological development of the kidney [7].It has been demonstrated that granulosa cell (GC) survival is dependent on WT1 activation especially at the initial stages of development of the follicles [8].Moreover, it is involved in the control of the effectors of the apoptotic cascade [8].
Nevertheless, not much is known from direct in vivo investigations on the apoptosis-promoting or apoptosis-inhibitory effects of WTI protein, and its effect on the ovary is poorly understood.
In gene therapy, DNA-based RNAi can be stably introduced for once-treatment of shRNA genes from viral vectors [9].The transfer of genes by lentivirus vectors is more beneficial when compared with gene transfer by other carriers.Cells can be infected irrespective of whether they are fully differentiated or undergoing active division.Besides, lentiviruses are easily incorporated into host genome, and they do not elaborate gene products [10,11].Several studies have shown that POF is implicated in granulocyte apoptosis.The hypothesis that formed the plank of the present investigation was that lentivirus-mediated WT1 overexpression in GCs might induce transcription of genes necessary for conferring an advantage in cell survival.Thus, the effect of WTI overexpression on endocrine function and follicular development in POF mice was studied.The study was also carried out to investigate the link between the effects of WTI overexpression and the Bax and bcl-2 genes, which are integral components of cell life and death [12].

EXPERIMENTAL Animals and chemicals
Two month-old female C57BL/6 mice were used in this study.

Injection of lentivirus
The mice ovaries were injected with lentivirus via the subscapular approach by means of a 10-μl syringe (Nanofil Syringe, WPI).Slow injections were carried out, with needles kept in place for 5 min.Each ovary had a total injection of 5 μl, and each mouse received a total of 5 μl of lentivirus.

Hormone assays
On day 42 after cyclophosphamide and busulfan treatment, whole blood samples (1.0 -1.2 ml) were drawn (into anticoagulated containers) under anesthesia through the abdominal aorta.Following a 3-h incubation at 37 °C, plasma samples were obtained by centrifugation, and used for the assays of FSH and E2 (as indicators of ovarian senescence) with radioimmunoassay (RIA) techniques at the Beijing Sino-uk Institute of Biological Technology.

Ovarian morphologies and follicle enumeration
Ovarian tissue samples were fixed in paraformaldehyde and paraffinized after sequential dehydration in ethanol.Tissue sections were subjected to H & E staining in line with standard procedures, and the various follicular forms were enumerated under the light microscope.

Immunohistochemistry
Para-formaldehyde-fixed and paraffinized ovarian tissues were sectioned and microwaved after rinsing in phosphate buffer.The sections were subsequently incubated for 45 min at 37 ˚C with anti-WT1 (ab89901, Abcam), anti-Bax (2772T, CST) and 3498T (anti-Bcl-2).Following incubation with secondary antibody, visualization was enhanced by addition of ABC chromogenic reagent.The negative control was PBS.Using a light Olympus microscope, 5 views per section were assessed and used for quantification of immune-reactive cells.

Real-time PCR
In the determination of relative mRNA expression levels, mice ovarian tissue or granulosa cells were lysed with TRIzol Reagent (Beyotime Institute of Biotechnology, Shanghai, China).Total RNA was obtained as per manufacturer's protocol, and reverse-transcribed to cDNA.Then, qRT-PCR was done on Applied Biosystems 7500 with specific SYBR Premix Ex TaqTM, (primer sequences are depicted in Table 1).The qPCR and the 2 −ΔΔCT methods were used for determination of relative gene expressions.Mean fold changes were obtained from three independent experiments.

Statistical analysis
Each experiment was performed at least three times, and results are presented as mean ± standard error of the mean (SEM).The data were analyzed using GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA).Where applicable, differences were evaluated by Student's t test.Values of p < 0.05 were considered statistically significant.

Effect of LV-GFP-WT1 infection on basal WT1 transcript levels in GCs
Granular cells (GCs) are not susceptible to infection.Thus, to achieve more than 80 % infection efficiency, the MOI should be about 100.A 90 % lentiviral infection efficiency was successfully effected in GCs by LV-GFP-WT1 treatment, as confirmed by quantification of GFP expression 72 h after transfection (Figure 2 A).In addition, the negative control (NC) and blank control groups were established.There were significant increases in endogenous WT1 mRNA expressions in the WT1-overexpression group 72 h post-infection, when compared to the control group and NC group (p < 0.05, Figure 2 B).

Identification of mouse ovarian tissue transfected with LV-GFP-WT1
Fluorescence expression was observed under an inverted fluorescence microscope and the results are shown in Figure 3. Except for the control group, the POF group and the LV-GFP-WT1 group expressed green fluorescence.Ovarian granulosa cells and ovarian epithelial cells were expressed in green fluorescence.

WT1 was significantly overexpressed in POF-LV-GFP-WT1 groups
Immuno-histochemical staining was performed on ovarian tissues to localize WT1.As shown in Figure 4 A, WT1 was expressed specifically in the nucleus of GCs, a finding that is consistent with the results obtained by previous investigators.The POF group had significantlyattenuated WT1 levels, relative to the control and POF-LV-GFP-WT1 groups (Figure 4 B, p < 0.05).

WT1 overexpression increased normal follicular cells, endocrine function, and ovarian weight in POF mice
Mice ovaries in the control group contained a variety of mature and immature follicles at every developmental stage, as revealed by H & E staining (Figure 5 A).In contrast, ovaries of POF mice had fewer follicles at every developmental stage, and consisted mainly of interstitial cells.The ovaries of POF mice were generally smaller in size than those from control group.However, WTI overexpression in mice with POF was able to significantly reverse ovarian pathology and alleviate the degree of injury in ovarian GCs (Figure 5 A).Cell-counting of normal follicles revealed an absolute reduction in follicles in mice with POF (p < 0.05; Figure 5 B).The number of follicles in the POF-LV-GFP-WT1 group was higher than that in the POF group, but cell counts of normal follicles differed significantly between the POF-LV-GFP-WT1 group and the control group (p < 0.05).Ovary weight of mice in the POF and control groups differed significantly (Figure 5 C).However, the weight of ovarian tissue did not significantly differ between the POF and POF-LV-GFP-WT1 mice groups (p > 0.05).In the POF group, there were reduced blood levels of E2 but elevated levels of FSH, relative to the control group.Although E2 and FSH levels were also different between the POF-LV-GFP-WT1and control groups, mice in the POF-LV-GFP-WT1 group had elevated plasma E2 and reduced FSH levels, relative to the POF group (Figure 5 D and  E).

WT1 overexpression in GCs affected Bax and bcl-2 protein levels
The proportion of Bax-immunoreactive cells   c; and a´ b´ c´, respectively).B: Quantitation of Bax and bcl-2 immuno-reactive cells.Bax was significantly decreased in POF-LV-GFP-WT1 group, when compared with Bax expression in POF group.Bcl-2 was significantly increased in POF-LV-GFP-WT1 group, relative to its expression in POF group; *p < 0.05, relative to POF and control groups; #p < 0.05, compared to control and POF groups (percent of total cells) in POF-LV-GFP-WT1 group was significantly increased, when compared with the control group, but was lower than the value obtained in the POF group (p < 0.05; Figure 6 B).However, the percentage of bcl-2-immunoreactive cells in POF-LV-GFP-WT1 group was lower than that in the control group, but significantly higher than corresponding levels in the POF group (p < 0.05).

DISCUSSION
Premenopausal women subjected to chemotherapy treatment are at high risk of POF.Currently, there are some treatments for POF patients.However, there is still a need for improvement in the treatment strategies.The results obtained in the present study revealed that ovarian dysfunction in chemotherapyinduced ovarian injury could be mitigated by the overexpression of WT1 induced by intra-ovarian microinjection of lentiviral vectors in mouse ovaries.
Alkylating agents (e.g.cyclophosphamide and busulfan) are considered more gonadotoxic than other agents.It has been reported that about 4 out of every 10 women subjected to alkylating agent therapy experienced ovarian failure, in contrast to those who received alkaloid or platinum-based therapy [14].Alkylating agents exert their deleterious effects on cells through the induction of DNA cross-links [15].Cross-links in DNA influence mitochondrial dynamics and activate the process of apoptosis [16].Studies have shown that cyclophosphamide increases the levels of pro-apoptotic protein Bax [16].Therefore, cyclophosphamide significantly reduces GCs, compromises the development of follicles and reduces the population of oocytes [17,18].Agents with alkylating properties impair follicular growth and decrease the populations of all types of follicles [19].
Estrogen is secreted mainly by ovarian GCs.Chemotherapeutic drugs reduce the level of estradiol due to damage to GCs.The results of this study are consistent with the finding that cyclophosphamide induced apoptosis in GCs, resulting in the reduction of various types of follicles and impairment of ovarian endocrine function [20].
The fates of follicular cells are dictated by the relative expressions of tumor suppressors, survival factors and apoptotic proteins [21].It is known that WT1 is one of the zinc finger transcription that are strongly expressed in GCs [21].Studies by Kreidberg et al established for the first time that WT1 inhibits apoptosis associated with urogenital tract development [22].Studies on the effect of WT1 on GC programmed cell death are carried out in the absence of serum so as to provoke spontaneous apoptosis [23].Both Bax and bcl-2 proteins are primarily involved in the regulation of the secretion of pro-apoptotic factors in several stages of the apoptotic cascade [4].These proteins are in turn regulated by WT1, which also modulates several other pro-apoptotic genes in non-gonadal cells [16,24,25].
In the present study, LV-GFP-WT1 was applied to study the role of significantly overexpressed WT1 at the levels of gene transcription and protein expression.It was found that WT1 possesses the potential to repair ovarian injury and augment ovarian function.The POF mice which were modified to overexpress WT1 had a diminished level of Bax immune-reactive cells and elevated percentage of Bcl-2 immunoreactive cells, when compared to POF mice that did not have WT1 overexpression.Thus, WT1 may affect apoptosis by influencing the expressions of apoptotic signaling pathway factors, particularly Bax and Bcl-2.Although the exact cellular processes involved are yet to be delineated, the finding that WT1 influences the expression of factors in apoptotic signaling pathways is a novel information on the control of apoptosis in follicular development.Thus, the present study has identified a novel target molecule for the development of a new therapy for chemotherapy-induced POF.

Study limitations
The procedures used for delivering the lentivirus vectors into the ovaries involved tedious operations which may affect the safety of the vectors themselves.Further studies are necessary to deepen the current understanding of how WT1 influences the expression of factors involved in the apoptotic cascade.

CONCLUSION
It can be reasonably inferred that overexpression of WTI gene enhances recovery of ovarian tissue and stimulates release of estrogen via the activation of factors involved in the apoptotic signaling pathways that exist in ovarian tissue of POF mice models.Therefore, WT1 may serve as a potential novel marker for POF induced by cyclophosphamide and a new target for treatment.Future studies are required to investigate the specific mechanism underlying the effect of POF on WT1.

DECLARATIONS
NM_144783)] are shown in Figure 1.The lentivirus encoding the complete WT1 open reading frame (LV GFP WT1) and negative control virus were obtained from GeneChem Biotechnology (Shanghai, China).The concentration of the stock lentiviral suspension was 5×10 8 titer units (TU)/ml.

Figure 2 :Figure 3 :
Figure 2: Enhancement of WTI expression in GCs by lentiviral-mediated RNAi.A: C cells infected with lentivirus at an MOI of 100.(scale bars = 400, 200 and 100 μm for a, a´; b, b´; and c, c´, respectively.B: Relative expression of WT1 mRNA in GCs; *p < 0.05, vs control and negative control groups.(Con = control, Nc = negative control)

Figure 4 :
Figure 4: Immuno-histochemical detection of WT1 protein in the ovary.A: Expression of WT1 in ovarian granulosa cells (scale bars for a, b, c and a´, b´, and c´ are 400 and 200 μm, respectively.B: Quantitation of WT1 immuno-reactive cells.WT1 was significantly increased in POF-LV-GFP-WT1 group, when compared with the expression in POF group; *p < 0.05, compared to POF group; #p < 0.05, relative to control

Figure 5 :
Figure 5: Effect of WT1 overexpression on POF.A: WTI overexpression enhanced the population of normal follicles and decreased atretic follicles in POFinduced ovarian tissue pathology.B: Follicular number in POF-LV-GFP-WT1 group was significantly increased, relative to POF group.C: Ovarian tissue weight did not significantly differ between the POF-LV-GFP-WT1 and the POF group.D: E2 content of plasma was higher in POF-LV-GFP-WT1 group, relative to POF group.E: Plasma FSH were lower in the POF-LV-GFP-WT1 group, relative to the POF group.Values are mean ± SEM; *p < 0.05, compared to control and POF groups; #p < 0.05, compared to control

Figure 6 :
Figure 6: Immuno-histochemical localization of Bax and bcl-2 proteins in the ovary.A: Bax and Bcl-2 were expressed mainly in ovarian granulosa cells (scale bars: 400 and 200 μm for a, b, c; and a´ b´ c´, respectively).B: Quantitation of Bax and bcl-2 immuno-reactive cells.Bax was significantly decreased in POF-LV-GFP-WT1 group, when compared with Bax expression in POF group.Bcl-2 was significantly increased in POF-LV-GFP-WT1 group, relative to its expression in POF group; *p < 0.05, relative to POF and control groups; #p < 0.05, compared to control and POF groups

Table 1 :
Gene primer sequences used