Abstract
In this study, it was evaluated if increased rounds of gonadotropin stimulation could affect in mice: (i) expression levels of proteins regulating cell cycle and DNA repair in fallopian tubes and (ii) meiotic spindle morphology of ovulated oocytes. To this end, adult female mice were subjected or not (Control) to 6 or 8 rounds of gonadotropin stimulation. Ovulated oocytes were incubated with anti A/B tubulin to evaluate spindle morphology. Fallopian tubes were analyzed to detect Cyclin D1, phospho-p53/p53, phospho-AKT/AKT, phospho-GSK3B/GSK3B, SOX2, OCT3/4, phospho-B-catenin/B-catenin, phospho-CHK1 and phospho-H2A.X protein levels. After 6 rounds, Cyclin D1, p53 and phospho-p53 contents were higher than Control. After 8 rounds, the contents of phosphorylated AKT, GSK3B and p53 as well as of total p53, Cyclin D1 and OCT3/4 significantly increased in comparison with Control. Conversely, SOX2 and B-catenin were similarly expressed among all experimental groups. The finding that phospho-CHK1 and phospho-H2A.X protein levels were undetectable supported the absence of extensive DNA damage. Oocytes number and percentage of normal meiotic spindles drastically decreased from 6 rounds onward. Altogether, our results demonstrated that 6 and 8 cycles of gonadotropin stimulation reduce mouse reproductive performances by inducing over-expression and over-activation of proteins controlling cell cycle progression in fallopian tubes and by impairing oocyte spindle.
Introduction
Retrieval of human oocytes during assisted reproductive technology requires the use of stimulation protocols (Pacchiarotti et al. 2016, Alper & Fauser 2017). Among these, the mild ovarian stimulation (MOS) protocol (Fauser et al. 1999) became the preferable option for IVF treatments thanks to the reduction of patient discomfort, risk of developing ovarian hyperstimulation syndrome and costs, as well (Pennings & Ombelet 2007). However, the debate about mild and conventional ovarian stimulation (COS) protocols is nevertheless still controversial. In fact, several studies comparing ongoing pregnancy and live birth rates between MOS and COS protocols revealed that COS was more successful and gave better chance of positive outcome in comparison with MOS (Revelli et al. 2011, Crawford et al. 2016, Siristatidis et al. 2017).
Moreover, women frequently have to undergo repeated cycles of ovarian stimulation. The side effects of these treatments have been highlighted by Homburg and collaborators who found that pregnancy rate was hampered by increasing number of cycle attempts. Indeed, a drastic decline from 25% to 17% after 4 rounds (R) of stimulation, and to 11% after more than 12R was recorded (Homburg et al. 2009).
In recent years, there has been a great concern about the risk of developing reproductive cancers, i.e. breast, ovarian and endometrial cancer, following IVF treatments (Brinton et al. 2012). For what concerns ovarian cancer (OC), results on the association between repetitive treatments and the risk of developing OC are discrepant. Sanner and collaborators reported that the protocols utilizing gonadotropins were associated with increased risk of OC (Sanner et al. 2009). By contrast, other authors reported no elevated risk (Jensen et al. 2009, Gadducci et al. 2013), while Brinton and collaborators sustained a possible incidence in the development of borderline ovarian tumors (Brinton et al. 2012).
The etiological and clinical approach to OC deeply changed following the discovery that OC can also arise from the spread of high-grade intraepithelial serous carcinoma originated in fallopian tubes (FT) epithelium (Kurman & Shih 2010, Cibula et al. 2011, Kim et al. 2012, Hua et al. 2016). Indeed, the genetic expression profile of this kind of cancer is much more related to FT rather than ovarian epithelium (Marquez et al. 2005, Eckert et al. 2016).
Discrepancies in clinical data on women undergoing fertility treatments are mainly due to the presence of biases like parity/nulliparity, kind of treatment and, most of all, infertility (Jensen et al. 2009, Sanner et al. 2009, Gadducci et al. 2013). The use of an animal model, such as the Swiss CD1 mouse, avoids the biases mentioned before, highlighting the damages exclusively linked to the treatment.
In our previous study (Di Luigi et al. 2014), by using the gonadotropin full-stimulation protocol usually adopted for mice (Hogan et al. 1994, Van Blerkom & Davis 2001), we demonstrated that 4 rounds (4R) of gonadotropin stimulation induced a significant increase of Cyclin D1 content in the FT. On the other hand, the expression levels of p53, AKT and of malignant transformation markers such as B-catenin, OCT3/4 and SOX2 remained unchanged (Di Luigi et al. 2014). In addition, we observed that 4R of gonadotropin stimulation altered oocytes meiotic spindle (Di Luigi et al. 2014). Starting from these results, here, we evaluated if further increasing to 6 and to 8 the number of repeated stimulations could modulate the expression levels of above-mentioned proteins, as well as of GSK3B, CHK1 and H2A.X.
Materials and methods
Chemicals
All the chemicals were of the purest analytical grade and were purchased from Sigma Chemical Company, unless otherwise indicated. Mouse monoclonal p53 and OCT3/4, rabbit polyclonal Cyclin D1, phospho-B-catenin (Thr41/Ser45), B-catenin, phospho-AKT (Ser473), AKT, phospho-GSK3B (Ser9), GSK3B and SOX2 primary antibodies were purchased from Santa Cruz Biotechnology; mouse monoclonal phospho-p53 (Ser15), rabbit monoclonal phospho-CHK1 (Ser345) and phospho-H2A.X (Ser139) were purchased from Cell Signaling Technology. Specific secondary antibodies were purchased from Santa Cruz Biotechnology.
Collection of oocytes and fallopian tubes
Mus musculus Swiss CD1 adult female mice (2–3-month-old; Harlan Italy, Udine, Italy; N = 27) were housed in the animal facility under controlled temperature (21 ± 1°C) and light (12 h light/day) conditions, with free access to food and water. Animals in which early luteal phase of estrous cycle was confirmed by examination of the vaginal smears (Caligioni 2009, Byers et al. 2012, Cora et al. 2015) (N = 9) were used as Control (Ctr). Repetitive cycles of ovarian stimulation were performed according to the protocol utilized in our previous study (Di Luigi et al. 2014). Five IU of PMSG (Folligon, Milano, Italy) were injected in mice. After 48 h, 5 IU of hCG (Corulon, Milano, Italy) were injected in animals. Six and eight rounds (6R, 8R) of stimulation were performed with intervals of 1 week between each. Fallopian tubes (FT) were collected from Control (Ctr; N = 9) and hyperstimulated (N = 18; 9 for 6R, 9 for 8R) mice, snap-frozen and stored at −80°C for Western blotting analysis.
Naturally ovulated oocytes were collected from FT of Ctr mice. Oocytes ovulated from hyperstimulated mice were recovered 14 h after the last hCG injection. In total, almost 100 oocytes were fixed for spindle analysis.
All experimental procedures involving animals and their care were performed in conformity with national and international laws and policies (European Economic Community Council Directive 86/609, OJ 358, 1 Dec 12, 1987; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana n. 40, Feb 18, 1992; National Institutes of Health Guide for the Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985). The project was approved by the Italian Ministry of Health and the internal Committee of the University of L’Aquila. The method of killing consisted of an inhalant overdose of carbon dioxide (CO2, 10–30%), followed by cervical dislocation. All efforts were made to minimize suffering.
Western blotting
FT samples were resuspended in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% Igepal) containing protease inhibitors (1 mM phenylmethylsulphonylfluoride, 1 μg/mL leupeptin and 1 μg/mL aprotinin) and phosphatase inhibitor (1 mM sodium fluoride, 10 mM sodium pyrophosphate and 1 mM sodium orthovanadate) for 30 min. Lysates (80 μg/sample) were separated by electrophoresis and transferred to nitrocellulose membranes (Hybond C Extra, Amersham). Membranes were incubated overnight at 4°C with specific primary antibodies: phospho-CHK1 (1:1000), phospho-H2A.X (1:1000), phospho-p53 (1:1000), p53 (1:200), Cyclin D1 (1:200), phospho-B-catenin (1:200), B-catenin (1:200), phospho-AKT (1:200), AKT (1:200), phospho-GSK3B (1:200), GSK3B (1:200), OCT3/4 (1:200), SOX2 (1:200) and then for 1 h with a peroxidase-conjugated anti-mouse (1:5000) or anti-rabbit (1:10000) secondary antibody. After detection using a chemiluminescence reagent (ECL, Pierce), the nitrocellulose membranes were examined using the Alliance LD2-77WL imaging system (Uvitec, Cambridge, UK). Densitometric quantification was performed with the public domain software NIH image v.1.62 and standardized using actin (1:200) as a loading control.
Analysis of meiotic spindles
To detect meiotic spindle morphology, oocytes and chromosomes were labeled as previously described (Rossi et al. 2006, Di Luigi et al. 2014). Briefly, oocytes were incubated for 1 h at 37°C with anti-A/B tubulin primary antibody (1:100) and then with anti-mouse secondary antibody conjugated with fluorescein isothiocyanate (1:800). Chromosomes were labeled with Hoechst 33342 (1 µg/mL). The analysis was performed using a fluorescence microscope (40× objective; Axioplan 2; Zeiss) with digital images collected with Leica DFC350 FX camera interfaced with IM500 Leica software.
Statistical analysis
The experiments were replicated at least 3 times, and data were expressed as mean ± s.e.m. Differences between groups were analyzed for statistical significance using ANOVA with Tukey–Kramer multiple comparison test as post-test. Results were considered significantly different when P < 0.05.
Results
Analysis of proteins involved in the control of cell cycle and DNA repair in FT
Comparable levels of total AKT were found in Ctr and repeatedly stimulated mice (Fig. 1A, Ctr vs 6R vs 8R, P > 0.05). Conversely, phospho-AKT/AKT ratio significantly increased after 8R (+58%) when compared with Ctr and 6R (Fig. 1A; Ctr and 6R vs 8R, P < 0.05). Moreover, there was a noticeable increase in AKT2 isoform phosphorylation level (Fig. 1A).
Western blot analysis of proteins involved in cell cycle control in fallopian tubes (FT). Ctr, Control mice; 6R and 8R, mice undergoing 6 rounds and 8 rounds of gonadotropin stimulation. p-AKT/AKT (A); p-GSK3B/GSK3B (B); Cyclin D1 (C). Data are expressed as arbitrary units (a.u.) of phosphorylated/total protein ratio (A and B) and protein/actin (C), used as loading control. The results are presented as mean ± s.e.m. of at least three independent experiments. Different letters: P < 0.05.
Citation: Reproduction 155, 3; 10.1530/REP-17-0687
Total and phospho-kinase GSK3B contents were determined in Ctr and repeatedly stimulated mice. No variation of total GSK3B expression was found in all experimental groups (Fig. 1B; Ctr vs 6R vs 8R, P > 0.05). Conversely, phospho-GSK3B/GSK3B ratio increased significantly after 8R of stimulation (+67%; Fig. 1B; Ctr vs 6R, P > 0.05; Ctr, 6R vs 8R, P < 0.05).
A very low Cyclin D1 level was found in Ctr (Fig. 1C), while a +27% and a +31% increases were recorded after 6R and 8R, respectively (Fig. 1C; Ctr vs 6R, 8R, P < 0.05).
In contrast to the low level found in Ctr, a dose-dependent increase of total and phospho-p53 content occurred after 6R (+42%, +46%) and 8R (+67%, +63%) (Fig. 2A, B and C; 6R, 8R vs Ctr, P < 0.05).
Western blot analysis of p53 and p-p53 in fallopian tubes (FT). Ctr, Control mice; 6R and 8R, mice undergoing 6 rounds and 8 rounds of gonadotropin stimulation (A). Data are expressed as arbitrary units (a.u.) of p53/actin (B), used as loading control and p-p53/p53 (C). The results are presented as mean ± s.e.m. of at least three independent experiments. Different letters: P < 0.05.
Citation: Reproduction 155, 3; 10.1530/REP-17-0687
Prolonged treatments did not modulate total and phospho-B-catenin levels, while phospho-CHK1 and phospho-H2A.X remained undetectable (data not shown).
Expression levels of transcription factors SOX2 and OCT3/4 were reported in Fig. 3A and B. SOX2 was unaffected by treatments (Fig. 3A; Ctr vs 6R vs 8R, P > 0.05). By contrast, a +31% increase of OCT3/4 occurred after 8R (Fig. 3B; Ctr, 6R vs 8R, P < 0.05).
Western blot analysis of transcription factors in fallopian tubes (FT). Ctr, Control mice; 6R and 8R, mice undergoing 6 rounds and 8 rounds of gonadotropin stimulation. SOX2 (A); OCT3/4 (B). Data are expressed as arbitrary units (a.u.) of each protein/actin, used as loading control. The results are presented as mean ± s.e.m. of at least three independent experiments. Different letters: P < 0.05.
Citation: Reproduction 155, 3; 10.1530/REP-17-0687
Spindle analysis in ovulated oocytes
The percentage of Ctr oocytes with normal, focused spindles and chromosome aligned on metaphase II plate was 98% (Fig. 4A, B and C). The percentage of oocytes with disorganized and asymmetric spindles arose concomitantly with rounds of stimulation. After 8R, the number of recovered oocytes drastically dropped (Fig. 4; Ctr vs 6R, P < 0.05; Ctr vs 8R, P < 0.05; 6R vs 8R, P < 0.05).
Frequency of spindle defects in MII oocytes collected after 6 and 8 rounds (6R, 8R) of gonadotropin treatment. Spindles were labelled with anti A/B Tubulin antibody (A and D) and chromosomes with Hoechst 33342 (B and E; merge C and F). The different percentages of normal (C) and abnormal (F) configurations have been reported in the table. Data are expressed as mean ± s.e.m. of three independent experiments. Original magnification: ×40. Within each column, different superscripts indicate P < 0.05.
Citation: Reproduction 155, 3; 10.1530/REP-17-0687
Discussion
Results presented here demonstrate that in fallopian tubes (FT), the contents of phospho-AKT, phospho-GSK3B and phospho-p53, as well as of Cyclin D1, p53 and OCT3/4 increase together with the number of gonadotropin treatments.
Repetitive gonadotropin stimulation does not modulate total AKT expression, whilst kinase phosphorylation is enhanced after 8 rounds (8R). Interestingly, AKT2 is the isoform that is predominantly phosphorylated. Despite both AKT isoforms 1 and 2 sharing similar substrates (Cecconi et al. 2012), phospho-AKT2 seems to be more implicated in cancer cell invasiveness than phospho-AKT1 (Arboleda et al. 2003). To date, we do not know the reasons and consequences of AKT2 activation on FT epithelial cells functions. However, the fact that both AKT2 amplification and overexpression occur in OC (Bellacosa et al. 1995, Khabele et al. 2014) suggests that dysregulation of kinase activity might predispose to/take part in uncontrolled proliferation of tubal epithelial cells. After 8R, phospho-AKT is able to inactivate its substrate GSK3B, which is a regulator of Cyclin D1 phosphorylation and proteolytic turnover (Diehl et al. 1998). Since in FT the higher increase of Cyclin D1 content occurs after 8R, it could be possible that the high phospho-GSK3B level inhibits Cyclin D1 degradation (Diehl et al. 1998, Chang et al. 2003). Likely, the maximum increase of Cyclin D1 content recorded after 8R (+31% over Ctr) is insufficient to induce cancer.
It is of interest to note that elevated levels of gonadotropins are detected during post-menopause, when OC frequency rises (https://www.cancer.org/cancer/ovarian-cancer/causes-risks-prevention/risk-factors.html, Webb & Jordan 2017). Therefore, such physiological age-dependent modifications could contribute to determine conditions that, concomitantly with the presence of mutations/deletions of specific genes, facilitate OC onset (Corney et al. 2008, Bast et al. 2009, Kim et al. 2016).
The demonstration of stem cells in FT (Paik et al. 2012, Snegovskikh et al. 2014) prompted us to investigate the levels of expression of OCT3/4, B-catenin and SOX2, all key factors linked to stemness and usually overexpressed during malignant transformation in several adult tissues (Palma et al. 2008, Wang et al. 2014, Zeineddine et al. 2014, Hellner et al. 2016). In our previous study (Di Luigi et al. 2014), we found that 4R of gonadotropin stimulation did not modify the low expression levels of these proteins in FT. Conversely, a +31% increase of OCT3/4 content occurred after 8R, thereby supporting the existence of a relationship between repeated gonadotropin stimulation and overexpression of this transcription factor also in non-tumoral adult somatic cells. Our results support an interaction among OCT3/4, AKT phosphorylation and Cyclin D1. This observation is consistent with results obtained in embryonic stem cells (ESC) (Lin et al. 2012, Liu et al. 2017). In these cells, phospho-AKT and Cyclin D1 action can be correlated to the inhibition of OCT3/4 proteasomal degradation, thereby leading to the accumulation of this transcription factor (Lin et al. 2012, Liu et al. 2017). Based on the literature data and on our results, we can hypothesize that the significant increase of phospho-AKT, Cyclin D1 and OCT3/4 contents after 8R could be representative of altered differentiation processes of FT epithelial cells.
The detrimental effects of 6R and 8R on the production of good-quality oocytes are confirmed by reduction of ovulated oocytes and increased meiotic spindle abnormalities. Hence, excessive gonadotropin administrations are able to perturb follicle development and acquisition of oocyte developmental competence (Van Blerkom & Davis 2001, Di Luigi et al. 2014).
In conclusion, our study indicates that, in mouse, a number of cycles from 6 to 8 can induce a different modulation of AKT, GSK3B, Cyclin D1, p53 and OCT3/4, as well as an impairment of ovulation and oocytes spindle organization. It should be noted that 6R of gonadotropin stimulation seems to be the threshold number of cycles without irreversible consequences on oocytes quality and on the expression levels of the proteins here analyzed.
These observations could be of interest if translated from a mouse model to young healthy donors approaching uncontrolled and repetitive stimulations for oocyte donation. Indeed, the possibility that such protocols could increase the risk of developing gynecological cancers in adulthood cannot be ruled out and should be carefully considered.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research was supported by Ministero dell’Università e della Ricerca (RIA 2015-16) to S C and G M T.
Acknowledgements
Authors would like to thank Gianfranco Ciccone and Anna Rita Nardecchia of the animal staff of University of L’Aquila for providing animal care.
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