Changes in cortical endoplasmic reticulum clusters in the fertilized mouse oocyte†.

Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum (ER) specializations (cortical ER clusters), which are thought to be essential for egg activation. In examination of cortical ER clusters, we observed that they were tethered to previously unreported fenestrae within the cortical actin layer. Furthermore, studies demonstrated that sperm preferentially bind to the plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global change in volume and shape that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males. In addition to these global changes, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, and then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection.

DOT1L Methyltransferase Regulates Calcium Influx in Erythroid Progenitor Cells in Response to Erythropoietin.

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca2+) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1lKO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1lKO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca2+ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca2+ influx by TRPC2, Dot1lKO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca2+ influx. Such heightened Ca2+ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.

Sperm-oocyte signaling: the role of IZUMO1R and CD9 in PTK2B activation and actin remodeling at the sperm binding site†.

Sperm-oocyte binding initiates an outside-in signaling event in the mouse oocyte that triggers recruitment and activation of the cytosolic protein kinase PTK2B in the cortex underlying the bound sperm. While not involved in gamete fusion, PTK2B activity promotes actin remodeling events important during sperm incorporation. However, the mechanism by which sperm-oocyte binding activates PTK2B is unknown, and the present study examined the possibility that sperm interaction with specific oocyte surface proteins plays an important role in PTK2B activation. Imaging studies revealed that as IZUMO1R and CD9 became concentrated at the sperm binding site, activated (phosphorylated) PTK2B accumulated in the cortex underlying the sperm head and in microvilli partially encircling the sperm head. In order to determine whether IZUMO1R and/or CD9 played a significant role in PTK2B recruitment and activation at the sperm binding site, the ability of oocytes null for Izumo1r or Cd9, to initiate an increase in PTK2B content and activation was tested. The results revealed that IZUMO1R played a minor role in PTK2B activation and had no effect on actin remodeling; however, CD9 played a very significant role in PTK2B activation and subsequent actin remodeling at the sperm binding site. These findings suggest the possibility that interaction of sperm surface proteins with CD9 or CD9-associated oocyte proteins triggers PTK2B activation at the sperm binding site.

Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm - oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline - rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm - oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm-oocyte binding sites that exhibited this actin response was reduced in pyk2-/- oocytes and the pyk2-/- oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm-oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm - binding sites under conditions where sperm - oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm - oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm.

Manipulation-free cultures of human iPSC-derived cardiomyocytes offer a novel screening method for cardiotoxicity.

Induced pluripotent stem cell (iPSC)-based cardiac regenerative medicine requires the efficient generation, structural soundness and proper functioning of mature cardiomyocytes, derived from the patient's somatic cells. The most important functional property of cardiomyocytes is the ability to contract. Currently available methods routinely used to test and quantify cardiomyocyte function involve techniques that are labor-intensive, invasive, require sophisticated instruments or can adversely affect cell vitality. We recently developed optical flow imaging method analyses and quantified cardiomyocyte contractile kinetics from video microscopic recordings without compromising cell quality. Specifically, our automated particle image velocimetry (PIV) analysis of phase-contrast video images captured at a high frame rate yields statistical measures characterizing the beating frequency, amplitude, average waveform and beat-to-beat variations. Thus, it can be a powerful assessment tool to monitor cardiomyocyte quality and maturity. Here we demonstrate the ability of our analysis to characterize the chronotropic responses of human iPSC-derived cardiomyocytes to a panel of ion channel modulators and also to doxorubicin, a chemotherapy agent with known cardiotoxic side effects. We conclude that the PIV-derived beat patterns can identify the elongation or shortening of specific phases in the contractility cycle, and the obtained chronotropic responses are in accord with known clinical outcomes. Hence, this system can serve as a powerful tool to screen the new and currently available pharmacological compounds for cardiotoxic effects.

Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway.

Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase-protein kinase B/AKT-mammalian target of rapamycin (PI3K/AKT-mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K-AKT-mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids.

Role of focal adhesion kinase in oocyte-follicle communication.

Germ cells require communication with associated somatic cells for normal gametogenesis, as exemplified by an oocyte that interacts with granulosa cells via paracrine factors as well as gap junctions located at sites of contact between these two cell types. The objective of the present study was to define the mechanisms by which cell-cell contact with the oocyte is controlled and to determine the extent that the oocyte actively participates in this association. Proline-rich tyrosine kinase 2 (PTK2), a focal adhesion kinase, was found to be activated at sites of contact between the oocyte and trans-zonal cell processes from the surrounding granulosa cells. In order to determine the functional significance of oocyte-derived PTK2 signaling in oocyte-follicle communication, an oocyte-specific Ptk2 knockout was produced through a breeding strategy pairing a floxed Ptk2-CAT-eGFP mouse with the Zp3-Cre line. Since Ptk2-null mice never develop to birth, this represents the first opportunity to define the role of PTK2 in oocyte-follicle communication. Ablation of Ptk2 within the developing oocyte resulted in lower fertility with reduced numbers of pups, lower rates of blastocyst formation, and reduced cell numbers per blastocyst. Follicles containing Ptk2-null oocytes exhibited reduced oocyte diameter, reduced numbers of connexin 37 and 43 foci at the oocyte surface, and impaired dye coupling between oocyte and granulosa cells. These findings are consistent with a model in which PTK2 plays a critical role in establishing or maintaining oocyte-granulosa cell contacts that are essential for gap junction-mediated communication between granulosa cells and the oocyte.

PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone.

PTK2b function during fertilization of the mouse oocyte.

Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

Post-ovulatory aging of oocytes disrupts kinase signaling pathways and lysosome biogenesis.

Post-ovulatory aging of oocytes results in the progressive loss of fertilization and developmental competence. This degradation of oocyte quality has been the object of numerous investigations, primarily focused on individual signaling pathways which provide limited insight into the status of global signaling events. The purpose of the present investigation was to comprehensively assess broad patterns of signaling pathway activity during in vitro aging as an initial step in defining control points that can be targeted to prevent the reduction in oocyte quality during prolonged culture. An antibody microarray-based phospho-proteome analysis performed on oocytes before and after eight hours of culture revealed significant changes in the abundance or activation state of 43 proteins that function in a wide variety of protein kinase-mediated signaling pathways. Several of the most significantly affected kinases were studied by Western blot and confocal immunofluorescence to corroborate the array results. Prolonged culture resulted in global changes in the abundance and activity of protein kinases that regulate the response to calcium, stress, and cell-cycle control. Examination of intracellular structures revealed a previously unrecognized increase in the abundance of large autophogagic lysosomes, which correlates with changes in protein kinase pathways. These results provide insight into the stresses experienced by oocytes during culture and the diversity of responses that results from them. The observed increase in autophagy-related activity, together with the disruptions in calcium signaling, cell-cycle, and stress-response pathways, have the potential to negatively impact oocyte quality by interfering with the normal sequence of biochemical changes that constitute egg activation following fertilization.

SRC-family tyrosine kinases in oogenesis, oocyte maturation and fertilization: an evolutionary perspective.

The oocyte is a highly specialized cell poised to respond to fertilization with a unique set of actions needed to recognize and incorporate a single sperm, complete meiosis, reprogram maternal and paternal genomes and assemble them into a unique zygotic genome, and finally initiate the mitotic cell cycle. Oocytes accomplish this diverse series of events through an array of signal transduction pathway components that include a characteristic collection of protein tyrosine kinases. The src-family protein kinases (SFKs) figure importantly in this signaling array and oocytes characteristically express certain SFKs at high levels to provide for the unique actions that the oocyte must perform. The SFKs typically exhibit a distinct pattern of subcellular localization in oocytes and perform critical functions in different subcellular compartments at different steps during oocyte maturation and fertilization. While many aspects of SFK signaling are conserved among oocytes from different species, significant differences exist in the extent to which src-family-mediated pathways are used by oocytes from species that fertilize externally vs those which are fertilized internally. The observation that several oocyte functions which require SFK signaling appear to represent common points of failure during assisted reproductive techniques in humans, highlights the importance of these signaling pathways for human reproductive health.

Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

Role of FYN kinase in spermatogenesis: defects characteristic of Fyn-null sperm in mice.

FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation was slightly modified in Fyn-null sperm, with reduced abundance of several minor phosphoproteins. These findings are consistent with a model in which FYN kinase plays an important role in proper shaping of the head and acrosome within the testis and possibly an additional role in the sperm acrosome reaction, events required for development of full fertilizing capacity in sperm.

The autoimmune regulator prevents premature reproductive senescence in female mice.

Loss-of-function mutations in the autoimmune regulator (AIRE) gene are responsible for autoimmune polyglandular syndrome type 1 (APS-1), which commonly manifests as infertility in women. AIRE is a transcriptional regulator that promotes expression of tissue-restricted antigens in the thymus, including antigens specific to the ovary. Thymic expression of ovarian genes under AIRE's control may be critical for preventing ovarian autoimmune disease. Because mice lacking Aire are an important APS-1 model, we examined the reproductive properties of female Aire-deficient (Aire(-/-)) mice. Female Aire(-/-) mice on the BALB/c background were examined for reproductive parameters, including fertility, litter sizes, and ovarian follicular reserves. Although delayed puberty was observed in Aire(-/-) mice, all mice entered puberty and exhibited mating behavior. Only 50% of Aire(-/-) females gave an initial litter, and only 16% were able to produce two litters. Ovarian histopathologic examination revealed that 83% of previously bred females lost all ovarian follicular reserves. Among virgin females, follicular depletion was observed in 25% by 8 wk, and by 20 wk, 50%-60% of mice lost all follicles. This was associated with elevated serum follicle-stimulating hormone level and ovarian infiltration of proliferating CD3+ T lymphocytes. Ovulation rates of 6-wk-old Aire(-/-) mice were reduced by 22%, but this difference was not statistically significant. Finally, transplantation experiments revealed that follicular loss depended on factors extrinsic to the ovary. These results suggest that immune-mediated ovarian follicular depletion is a mechanism of infertility in Aire(-/-) mice. The results have important implications in the pathogenesis of ovarian autoimmune disease in women.

Signaling Proteins Recruited to the Sperm Binding Site: Role of ß-Catenin and Rho A.

Sperm interaction with the oocyte plasma membrane triggers a localized response in the mouse oocyte that leads to remodeling of oocyte surface as well as the underlying cortical actin layer. The recent demonstration that PTK2B is recruited and activated at the sperm binding site raised the possibility that multiple signaling events may be activated during this stage of fertilization. The present study demonstrated that ß-catenin and Rho A were recruited to the cortex underlying bound/fused sperm. To determine whether sperm-oocyte contact was sufficient to initiate ß-catenin recruitment, Cd9-null, and PTK2b-null oocytes were tested for the ability to recruit ß-catenin to sperm binding sites. Both Cd9 and Ptk2b ablation reduced ß-catenin recruitment raising the possibility that PTK2B may act downstream of CD9 in the response to sperm binding/fusion. Further immunofluorescence study revealed that ß-catenin co-localized with f-actin in the interstitial regions between actin layer fenestrae. Rho A, in contrast, was arranged underneath the actin layer in both the fenestra and the interstitial regions suggesting that they may play different roles in the oocyte.

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes.

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.

Protein tyrosine kinase signaling during oocyte maturation and fertilization.

The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans.

ERß regulated ovarian kisspeptin plays an important role in oocyte maturation.

Kisspeptin (KISS1) signaling in the hypothalamic-pituitary (H-P) axis plays an essential role in regulating gonadotropin secretion. KISS1 and KISS1 receptor (KISS1R) are also expressed in the ovary; however, the role of intraovarian KISS1 signaling remains unclear. Granulosa cell (GC)-specific expression of KISS1, and oocyte-specific expression of KISS1R indicate that GC-derived KISS1 may act on oocytes. Expression of KISS1 in GCs is induced by gonadotropins but it is absent in estrogen receptor ß knockout (Erßnull) rat ovaries. We also observed that gonadotropin stimulation failed to induce maturation of Erßnull oocytes. Interestingly, KISS1 treatment of cumulus oocyte complexes (COCs) isolated from antral follicles promotes in vitro maturation of oocytes. Treatment of oocytes with KISS1 induced intracellular Ca2+ release, and increased activation of MAP kinase ERK1/2. KISS1 treatment also induced the expression of oocyte genes that are crucial for differentiation of GCs, and maturation of oocytes. Our findings suggest that ovarian KISS1-signaling plays an important role in gonadotropin induced follicle development and oocyte maturation.

Functions of Fyn kinase in the completion of meiosis in mouse oocytes.

Oocyte maturation invokes complex signaling pathways to achieve cytoplasmic and nuclear competencies for fertilization and development. The Src-family kinases FYN, YES and SRC are expressed in mammalian oocytes but their function during oocyte maturation remains an open question. Using chemical inhibitor, siRNA knockdown, and gene deletion strategies the function of Src-family kinases was evaluated in mouse oocytes during maturation under in vivo and in vitro conditions. Suppression of Src-family as a group with SKI606 greatly reduced meiotic cell cycle progression to metaphase-II. Knockdown of FYN kinase expression after injection of FYN siRNA resulted in an approximately 50% reduction in progression to metaphase-II similar to what was observed in oocytes isolated from FYN (-/-) mice matured in vitro. Meiotic cell cycle impairment due to a Fyn kinase deficiency was also evident during oocyte maturation in vivo since ovulated cumulus oocyte complexes collected from FYN (-/-) mice included immature metaphase-I oocytes (18%). Commonalities in meiotic spindle and chromosome alignment defects under these experimental conditions demonstrate a significant role for Fyn kinase activity in meiotic maturation.

Role of Fyn kinase in oocyte developmental potential.

Fyn kinase is highly expressed in oocytes, with inhibitor and dominant-negative studies suggesting a role in the signal transduction events during egg activation. The purpose of the present investigation was to test the hypothesis that Fyn is required for calcium signalling, meiosis resumption and pronuclear congression using the Fyn-knockout mouse as a model. Accelerated breeding studies revealed that Fyn-null females produced smaller litter sizes at longer intervals and exhibited a rapid decline in pup production with increasing age. Fyn-null females produced a similar number of oocytes, but the frequency of immature oocytes and mature oocytes with spindle chromosome abnormalities was significantly higher than in controls. Fertilised Fyn-null oocytes frequently (24%) failed to undergo pronuclear congression and remained at the one-cell stage. Stimulation with gonadotropins increased the number of oocytes ovulated, but did not overcome the above defects. Fyn-null oocytes overexpressed Yes kinase in an apparent effort to compensate for the loss of Fyn, yet still exhibited an altered pattern of protein tyrosine phosphorylation. In summary, Fyn-null female mice exhibit reduced fertility that appears to result from actin cytoskeletal defects rather than calcium signalling. These defects cause developmental arrest during oocyte maturation and pronuclear congression.