Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes

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.

Repeated hyperstimulation affects the ultrastructure of mouse fallopian tube epithelium

“…In fact, while ROS production augmented after 4R of COH, the Oct4 transcripts started to decrease [ 49 ]. This is in agreement with our previous data, showing a progressive reduction of the protein OCT3/4 from 4 to 8R in mouse [ 18 ]. Moreover, oxidative damages were also detected in mouse ovaries.…”

“…Animals in which the early luteal phase of the estrous cycle was evaluated by examination of the vagina and vaginal smears (n = 4), and without any hyperstimulation, were used as Control (Ctr). Repetitive cycles of ovarian stimulation were performed according to previous studies [ 17 , 18 ]. Briefly, mice (ν = 11) were injected i.p.…”

“…with 5 IU of PMSG (pregnant mare serum gonadotropin) (Folligon, Milano, Italy) and after 48 h with 5 IU of hCG (human chorionic gonadotropin) (Corulon, Milano, Italy). The timing of four to eight repeated rounds (4R, 8R) of stimulation was selected according to [ 17 ] and [ 18 ]. In particular, the COH protocol (PMSG + hCG) was repeated four times in the mice of 4R group (n = 6) and eight times in those of 8R group (n = 5).…”

“…In our previous papers, we analyzed oocyte spindle morphology and the content of proteins involved in cell cycle control in ovaries and FTs retrieved from mice treated up to 8 times (8 Rounds, 8R) with a standardized protocol of gonadotropin [ 17 , 18 ]. Despite after 4R the only modification recorded was an increase in cyclin D1 content in FT, after 8R also p53, phospho-p53, phospho-AKT, GSK3B and OCT3/4 contents were significantly higher than in controls.…”

“…Despite after 4R the only modification recorded was an increase in cyclin D1 content in FT, after 8R also p53, phospho-p53, phospho-AKT, GSK3B and OCT3/4 contents were significantly higher than in controls. The over-expression of these proteins, all involved in the control of cell cycle progression, and the drastic decrease of oocyte number and spindle quality strongly supported an altered differentiation processes of FT epithelial cells and an impaired ovulation and oocytes spindle organization [ 17 , 18 , 19 ].…”