Background: Young cancer patients may occasionally face infertility and premature gonadal failure. Apart from its direct effect on follicles and oocytes, chemotherapy may induce ovarian toxicity via an impact on the entire ovary. The role of doxorubicin in potential ovarian failure remains obscure. Our intention was to elucidate doxorubicin-related toxicity within ovaries. Methods: Female mice were injected intraperitoneally with 7.5 or 10 mg/kg doxorubicin and their ovaries were visualized in vivo by high resolution MRI, one day and one month following treatment. Ovaries of other treated mice were excised and weighed at the same post-treatment intervals. Ovarian histological sections were stained for TUNEL or active caspase-3 and follicles were counted and categorized. Ovulation rates were evaluated in superovulated female mice treated with doxorubicin. Results: A single injection of doxorubicin resulted in a major reduction in both ovarian size and weight that lasted even one month post treatment. A dramatic reduction in ovulation rate was observed one week after treatment, followed by a partial recovery at one month. Histological examination revealed positive staining of TUNEL and active caspase-3. We observed a significant reduction in the population of secondary and primordial follicles one month following treatment. Conclusions: Our results may imply a mechanism of chemotherapy-induced ovarian toxicity, manifested by reduced ovulation and accompanied by a reduction in ovarian size, caused probably by an acute insult to the ovary.

Fyn, a member of the Src family kinases (SFKs) has a pivotal role in cell adhesion, proliferation, migration and survival, and its overexpression is associated with several types of cancer. microRNAs (miRNAs) play a major role in post-transcriptional repression of proteins expression. In light of the significant functions of Fyn, together with studies demonstrating miR-125a as a tumor suppressing miRNA that is down-regulated in several cancer cell types and based on our bioinformatics studies, we chose to examine Fyn's post-transcription regulation by miR-125a-3p, in a model of HEK 293T cell line. We show that Fyn expression can be dramatically reduced by elevated levels of miR-125a-3p, after which the activity of Fyn-downstream-proteins such as FAK, paxillin and Akt, proteins known to be overexpressed in various tumors, is also reduced. On a broader level, we showed that miR-125a-3p causes an arrest of the cell cycle at the G2/M stage and decreases cell viability and migration probably in a Fyn-directed manner. The results are reinforced by control experiments conducted using Fyn siRNA and anti-miR-125a-3p as well as by numerous cancer cell lines showing a significant down-regulation of Fyn after mir-125a-3p overexpression. Collectively, we conclude that miR-125a-3p has an important role in the regulation of Fyn expression and of its signaling pathway, implying it has a therapeutic potential in overexpressed Fyn-related diseases.

The earliest visible indications for the transition to embryos in mammalian eggs, known as egg activation, are cortical granules exocytosis (CGE) and resumption of meiosis (RM); these events are triggered by the fertilizing spermatozoon through a series of Ca²⁺ transients. The pathways, within the egg, leading to the intracellular Ca²⁺ release and to the downstream cellular events, are currently under intensive investigation. The involvement of Src family kinases (SFKs) in Ca²⁺ release at fertilization is well supported in marine invertebrate eggs but not in mammalian eggs. In a previous study we have shown the expression and localization of Fyn, the first SFK member demonstrated in the mammalian egg. The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs. All three kinases examined: Fyn, c-Src and c-Yes are distributed throughout the egg cytoplasm. However, Fyn and c-Yes tend to concentrate at the egg cortex, though only Fyn is localized to the spindle as well. The different localizations of the various SFKs imply the possibility of their different functions within the egg. To examine whether SFKs participate in the signal transduction pathways during egg activation, we employed selective inhibitors of the SFKs activity ((PP2 and SU6656). The results demonstrate that RM, which is triggered by Ca²⁺ elevation, is an SFK-dependent process, while CGE, triggered by either Ca²⁺ elevation or protein kinase C (PKC), is not. The possible involvement of SFKs in the signal transduction pathways that lead from the sperm–egg fusion site downstream of the Ca²⁺ release remains unclear. Abstract The earliest visible indications for the transition to embryos in mammalian eggs, known as egg activation, are cortical granules exocytosis (CGE) and resumption of meiosis (RM); these events are triggered by the fertilizing spermatozoon through a series of Ca²⁺ transients. The pathways, within the egg, leading to the intracellular Ca²⁺ release and to the downstream cellular events, are currently under intensive investigation. The involvement of Src family kinases (SFKs) in Ca²⁺ release at fertilization is well supported in marine invertebrate eggs but not in mammalian eggs. In a previous study we have shown the expression and localization of Fyn, the first SFK member demonstrated in the mammalian egg. The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs. All three kinases examined: Fyn, c-Src and c-Yes are distributed throughout the egg cytoplasm. However, Fyn and c-Yes tend to concentrate at the egg cortex, though only Fyn is localized to the spindle as well. The different localizations of the various SFKs imply the possibility of their different functions within the egg. To examine whether SFKs participate in the signal transduction pathways during egg activation, we employed selective inhibitors of the SFKs activity ((PP2 and SU6656). The results demonstrate that RM, which is triggered by Ca²⁺ elevation, is an SFK-dependent process, while CGE, triggered by either Ca²⁺ elevation or protein kinase C (PKC), is not. The possible involvement of SFKs in the signal transduction pathways that lead from the sperm–egg fusion site downstream of the Ca²⁺ release remains unclear.

Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca²⁺]_i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca²⁺]_i transient. Strontium chloride (SrCl₂) binds to and activates the Ca²⁺-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca²⁺]_i oscillations. Although SrCl₂ has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca²⁺]_i oscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl₂ (2, 4 or 6 mM) on [Ca²⁺]_i, by monitoring [Ca²⁺]_i oscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl₂ was optimal for inducing the first [Ca²⁺]_i transient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca²⁺]_i oscillations were lower and longer in SrCl₂-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl₂, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca²⁺]_i oscillations (8, 15 or 60 min in SrCl₂ respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl₂ caused a delay in CM. Our results demonstrate that SrCl₂ is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca²⁺]_i oscillations, CGE and CM.

The process of resumption of the first meiotic division (RMI) in mammalian oocytes includes germinal vesicle breakdown (GVBD), spindle formation during first metaphase (MI), segregation of homologous chromosomes, extrusion of the first polar body (PBI) and an arrest at metaphase of the second meiotic division (MII). Previous studies suggest a role for Fyn, a non-receptor Src family tyrosine kinase, in the exit from MII arrest. In the current study we characterized the involvement of Fyn in RMI. Western blot analysis demonstrated a significant, proteasome independent, degradation of Fyn during GVBD. Immunostaining of fixed oocytes and confocal imaging of live oocytes microinjected with Fyn complementary RNA (cRNA) demonstrated Fyn localization to the oocyte cortex and to the spindle poles. Fyn was recruited during telophase to the cortical area surrounding the midzone of the spindle and was then translocated to the contractile ring during extrusion of PBI. GVBD, exit from MI and PBI extrusion were inhibited in oocytes exposed to the chemical inhibitor SU6656 or microinjected with dominant negative Fyn cRNA. None of the microinjected oocytes showed misaligned or lagging chromosomes during chromosomes segregation and the spindle migration and anchoring were not affected. However, the extruded PBI was of large size. Altogether, a role for Fyn in regulating several key pathways during the first meiotic division in mammalian oocytes is suggested, particularly at the GV and metaphase checkpoints and in signaling the ingression of the cleavage furrow.

Prior to fertilization, the spindle of vertebrate eggs must remain stable and well organized during the second meiotic meta-phase arrest (MII). In a previous study we have determined that the completion of meiosis is a Src family kinase (SFK)-dependent event. In the current study we have used the SFK inhibitors, SU6656 and PP2, and demonstrated that inhibition of SFKs caused the formation of a disorganized spindle. The observation that proper organization of an MII spindle is an SFK-dependent process, combined with our previous finding that Fyn kinase is localized at the microtubules (MTs), prompted us to examine the potential role of Fyn in MT signaling. Our results show an association between Fyn and tubulin, the ability of Fyn to phosphorylate tubulin in vitro and stimulation of meiosis completion by injection of a constitutively active form of Fyn (CAF). We suggested that SFKs mediate significant functions during the organization of the MII spindle. In view of CAF injection experiments, and of the pronounced concentration of Fyn kinase at the spindle, we propose that Fyn may play an important role in some aspects of the spindle functions, possibly those involving the MTs.