SH2 domain-mediated activation of an SRC family kinase is not required to initiate Ca2+ release at fertilization in mouse eggs

SRC family kinases (SFKs) function in initiating Ca2+ release at fertilization in several species in the vertebrate evolutionary line, but whether they play a similar role in mammalian fertilization has been uncertain. We investigated this question by first determining which SFK proteins are expressed in mouse eggs, and then measuring Ca2+ release at fertilization in the presence of dominant negative inhibitors. FYN and YES proteins were found in mouse eggs, but other SFKs were not detected; based on this, we injected mouse eggs with a mixture of FYN and YES Src homology 2 (SH2) domains. These SH2 domains were effective inhibitors of Ca2+ release at fertilization in starfish eggs, but did not inhibit Ca2+ release at fertilization in mouse eggs. Thus the mechanism by which sperm initiate Ca2+ release in mouse eggs does not depend on SH2 domain-mediated activation of an SFK. We also tested the small molecule SFK inhibitor SU6656, and found that it became compartmentalized in the egg cytoplasm, thus suggesting caution in the use of this inhibitor. Our findings indicate that although the initiation of Ca2+ release at fertilization of mammalian eggs occurs by a pathway that has many similarities to that in evolutionarily earlier animal groups, the requirement for SH2 domain-mediated activation of an SFK is not conserved.     

NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary

Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.     

Evidence that activation of Src family kinase is not required for fertilization-associated [Ca2+]i oscillations in mouse eggs

Recent evidence in marine invertebrate, frog, and zebrafish eggs suggests the involvement of a Src family kinase (SFK) in fertilization-induced Ca2+ release. In the present study, we have investigated whether activation of an SFK is required for initiation of intracellular Ca2+ ([Ca2+]i) oscillations in mouse fertilization. We detected a Hck-like protein and tyrosine-phosphorylated proteins in soluble and insoluble sperm fractions, respectively. However, the presence of these proteins did not correspond to the active fractions of porcine sperm extracts (pSE). Moreover, [Ca2+]i oscillations induced by pSE in mouse eggs were unaltered by pre-incubation of pSE with specific SFK inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]-pyrimidine (PP2) or lavendustin A, despite the fact that the inhibitors were shown to be active both in vivo and in vitro. Another SFK inhibitor, peptide A, blocked oscillations when incubated with pSE prior to injection into eggs, but this inhibition required more than ten times the concentration reportedly required to inhibit SFK activity. In addition, pre-injection or pre-incubation of eggs with these inhibitors did not affect the ability of pSE to trigger [Ca2+]i oscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]i release. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]i oscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations.     

Role of facilitative glucose uptake in the glucose-inorganic phosphate-mediated retardation and inhibition of development in different strains of mouse embryos

Mouse embryos from different strains develop differently in vitro depending on the composition of the culture medium, and in particular on the presence or absence of glucose and inorganic phosphate. Glucose is both stimulatory and inhibitory in certain conditions. Glucose uptake by cells can be passive, down concentration gradients, or active, through sodium driven pumps, or can occur through facilitative transport. This study investigated the effects of inhibition of facilitative glucose transport on the glucose-inorganic phosphate-mediated blocks in development in three different strains of mouse embryo, CF-1, CD-1 and an F2 hybrid. Development of CF-1 and CD-1 embryos is blocked in medium containing glucose and inorganic phosphate but not in medium containing glucose alone, and F2 embryos are not affected. Inhibition of facilitated glucose transport to the eight-cell-morula stage in CF-1 and CD-1 embryos resulted in development in medium containing both glucose and inorganic phosphate, indicating that the prevention of facilitative glucose uptake can overcome the developmental block. Removal of inhibition before the eight-cell-morula stage resulted in total arrest of CF-1 embryos and minimum development of CD-1 embryos. F2 embryos are not affected by inorganic phosphate and glucose and showed no response to the transporter inhibitor at any stage. These data support the contention that facilitated glucose transport is active in embryos, is phosphate-dependent and that its inhibition can overcome the glucose-inorganic phosphate-mediated developmental blocks in mouse embryos.     

Phosphatidylinositol 3-kinase signaling in mammalian preimplantation embryo development

The development of the preimplantation mammalian embryo is an autopoietic process; once initiated development proceeds without an absolute requirement for external information or growth cues. This developmental autonomy is partly explained by the generation of autocrine trophic ligands that are released and act back on the embryo via specific receptors. Several embryotrophic ligands cause receptor-dependent activation of 1-o-phosphatidylinositol 3-kinase. This enzyme phosphorylates phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate. Genetic or pharmacological ablation of this enzyme activity disrupts normal development of preimplantation embryos. Phosphatidylinositol-3,4,5-trisphosphate is a membrane lipid that acts as a docking site for a wide range of proteins possessing the pleckstrin homology (PH) domain. Such proteins are important regulators of cell survival, proliferation, and differentiation. RAC-alpha serine/threonine protein kinase is an important PH domain protein and its activity is required for normal preimplantation embryo development and survival. The activity of a range of PH domain proteins is also implicated in the normal development of the embryo. This review critically examines the evidence for the activation of 1-o-phosphatidylinositol 3-kinase in the generation of pleiotypic trophic response to embryotrophins in the autopoietic development of the preimplantation embryo.     

Delayed and stage specific phosphorylation of H2AX during preimplantation development of gamma-irradiated mouse embryos

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated in the serine 139 residue at the damage site. The phosphorylated H2AX, designated as gamma-H2AX, is visible as nuclear foci in the irradiated cells which are thought to serve as a platform for the assembly of proteins involved in checkpoint response and DNA repair. It is known that early stage mammalian embryos are highly sensitive to radiation but the mechanism of radiosensitivity is not well understood. Thus, we investigated the damage response of the preimplantation stage development by analyzing focus formation of gamma-H2AX in mouse embryos gamma-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy gamma-irradiation is hindered up to the two cell stage of development. When left in utero for another 24-64 h, however, these irradiated embryos showed delayed phosphorylation of H2AX. In contrast, phosphorylation of H2AX was readily induced by radiation in post-compaction stage embryos. It is possible that phosphorylation of H2AX is inefficient in early stage embryos. It is also possible that the phosphorylated H2AX exists in the dispersed chromatin structure of early stage embryonic pronuclei, so that it cannot readily be detected by conventional immunostaining method. In either case, this phenomenon is likely to correlate with the lack of cell cycle arrest, apoptosis and high radiosensitivity of these developmental stages.     

Role of androgens and fibroblast growth factors in prostatic development

This review focuses on the role of androgens and fibroblast growth factors (FGFs) in regulating the growth and development of the prostate. It is known that androgens and mesenchymal-epithelial interactions are required for the formation and growth of the prostate, but little is known of the molecular mediators regulating prostatic organogenesis. Paracrine signalling from the mesenchyme to the epithelium is a key element of prostatic development and the action of androgens in mesenchymal cells is essential for prostatic development. This finding has led to the hypothesis that androgens regulate the expression of paracrine-acting growth factors. Although several families of growth factors play a role in regulating prostatic growth, the FGF family contains members that have been studied most comprehensively in regard to prostatic growth and branching morphogenesis. The role of FGFs in prostatic development is described in detail, since two members of the FGF family function as mesenchymal paracrine-acting factors in the prostate. It has been shown that FGF7 and FGF10 play important roles during prostatic development yet they do not appear to be regulated directly by androgens. Current models propose that growth factor expression (including FGF7 and 10) is regulated directly by androgens. However, it is possible that androgen regulation is indirect and a model outlining indirect androgen regulation of growth factors is proposed.     

Expression profile of protein kinase C isozymes in preimplantation mouse development

In the preimplantation mouse embryo, the protein kinase C (PKC) family has been implicated in regulation of egg activation, progression of meiotic and mitotic cell cycles, embryo compaction, and blastulation, but the involvement of the individual isozymes is largely unknown. Here, using semiquantitative immunocytochemistry and confocal microscopy we analyze the relative amount and subcellular distribution of ten isozymes of PKC (alpha, betaI, betaII, gamma, delta, epsilon, eta, theta, zeta, iota/lambda) and a PKC-anchoring protein, receptor for activated C-kinase 1 (RACK1). Our results show that all of these isoforms of PKC are present between the two-cell and blastocyst stages of mouse preimplantation development, and that each has a distinct, dynamic pattern and level of expression. The data suggest that different complements of the isozymes are involved in various steps of preimplantation development, and will serve as a framework for further functional studies of the individual isozymes. In particular, there was a transient increase in the nuclear concentration of several isozymes at the early four-cell stage, suggesting that some of the PKC isozymes might be involved in regulation of nuclear organization and function in the early mouse embryo.     

Role of gap junctions during early embryo development

Gap junctional communication plays a central role in the maintenance of cellular homeostasis by allowing the passage of small molecules between adjacent cells. Gap junctions are composed of a family of proteins termed connexins. During preimplantation development several connexin proteins are expressed and assembled into gap junctions in the plasma membrane at compaction but the functional significance of connexin diversity remains controversial. Although, many of the connexin genes have been disrupted using homologous recombination in embryonic stem cells to obtain unique phenotypes, none of these studies has demonstrated a specific role for connexins during preimplantation development in the null mutants. This review surveys evidence for the involvement of gap junctional communication during embryo development highlighting discrepancies in the literature. Although some evidence suggests that gap junctions may be dispensable during preimplantation development this is difficult to envisage particularly for the process of cavitation and the maintenance of homeostasis between the differentiated trophectoderm cells and the pluripotent inner cell mass cells of the blastocyst.     

Endocytosis in the mouse oocyte and its contribution to cAMP signaling during meiotic arrest

Mammalian oocytes are arrested at prophase I of meiosis until a preovulatory surge of LH stimulates them to resume meiosis. Prior to the LH surge, high levels of cAMP within the oocyte maintain meiotic arrest; this cAMP is generated in the oocyte through the activity of the constitutively active, G(s)-coupled receptor, G-protein-coupled receptor 3 (GPR3) or GPR12. Activated GPRs are typically targeted for desensitization through receptor-mediated endocytosis, but a continuously high level of cAMP is needed for meiotic arrest. The aim of this study was to examine whether receptor-mediated endocytosis occurs in the mouse oocyte and whether this could affect the maintenance of meiotic arrest. We found that constitutive endocytosis occurs in the mouse oocyte. Inhibitors of receptor-mediated endocytosis, monodansylcadaverine and dynasore, inhibited the formation of early endosomes and completely inhibited spontaneous meiotic resumption. A red fluorescent protein-tagged GPR3 localized in the plasma membrane and within early endosomes in the oocyte, demonstrating that GPR3 is endocytosed. However, overexpression of G-protein receptor kinase 2 and β-arrestin-2 had only a modest effect on stimulating meiotic resumption, suggesting that these proteins do not play a major role in GPR3 endocytosis. Inhibition of endocytosis elevated cAMP levels within oocytes, suggesting that there is an accumulation of GPR3 at the plasma membrane. These results show that endocytosis occurs in the oocyte, leading to a decrease in cAMP production, and suggest that there is a balance between cAMP production and degradation in the arrested oocyte that maintains cAMP levels at an appropriate level during the maintenance of meiotic arrest.     

Role of nutrition in the development of functional shifts in the thyroid

The authors studied the effect of the diets differing in macroelements and trace elements (excess silicon in association with moderate deficiency of iodine and cobalt) on thyroid function in normal people living in the endemic regions with regard to goiter. The shifts in thyroid function were found to correlate with the increased content of silicon and some other trace elements in the diets. The silicon subregion is marked by the incidence of dysthyrosis of the thyroid gland.     

In vitro follicular development of cryopreserved mouse ovarian tissue

In a previous report, we showed that follicles isolated from frozen/thawed mouse ovarian tissues reached the mature follicle stage on the 12th day of culture. However, the developmental ability was lower than that of fresh ovarian tissue. The purpose of this study was to define a culture system with some technical modification for preantral follicles isolated from frozen/thawed ovarian tissue and to confirm cell injury. Ovaries obtained from three-week-old female mice were cryopreserved by the rapid freezing method. Preantral follicles isolated from frozen/thawed ovarian tissues were cultured for 12-16 days. The follicles were then stimulated with human chorionic gonadotropin. In vitro fertilization was performed on the released cumulus-oocyte complexes (COCs). Preantral follicle viability was assessed by supravital staining using Hoechst 33258. Using this stain cell death was found in part of the granulosa cells of a follicle obtained from frozen/thawed ovarian tissue. On the 14th and 16th days of culture, the diameters of follicles isolated from frozen/thawed ovaries were larger than on the 12th day of culture. The released COCs were fertilized and developed to the blastocyst stage in 15.8% (12/76) of the oocytes taken from the fresh group, and in 0% (0/73), 2.9% (2/69) and 19.1% (22/115) of the oocytes taken from the frozen/thawed group that had been cultured for 12, 14 and 16 days respectively. The preantral follicles isolated from frozen/thawed mouse ovarian tissues developed slowly compared with the freshly prepared preantral follicles. During prolonged culture from 12 to 16 days, these follicles obtained the potential to fertilize and develop to the blastocyst stage.     

IGFs对小鼠乳腺发育及细胞凋亡的影响

为揭示IGFs在乳腺发育和乳腺上皮细胞凋亡中的作用，运用荧光显微镜及光镜技术，采用小鼠乳腺组织培养及小鼠乳腺上皮细胞培养方法，对IGFs的作用进行了研究。结果表明，IGFs可促进小鼠乳腺导管的生长发育，并且IGF—I促小鼠乳腺导管生长发育的功能强于IGF—II；IGFs抑制泌乳期小鼠乳腺上皮细胞凋亡，并且IGF—I抑制凋亡的作用强于IGF—II。     

Postnatal testicular development in mouse species with different levels of sperm competition

Postcopulatory sexual selection leads to an increase in sperm numbers which is partly the result of an increase in relative testes mass and could also be the consequence of changes in testis architecture or function. Very little is known regarding developmental changes during the first spermatogenic wave that may lead to enhanced spermatogenic efficiency and increased sperm production. We examined testicular development after birth in four mouse species with different sperm competition levels to assess changes in testicular architecture and function. Differences in relative testes mass between species appeared soon after birth and were exacerbated thereafter. The volume of testes occupied by seminiferous tubules differed between species postnatally and were associated with sperm competition levels. Finally, changes over time in the proportions of tubules with different germ cell types were also associated with sperm competition levels, with the time taken for the transition between various cell stages being negatively associated with levels of sperm competition. We conclude that postnatal testis development differs between closely related species with different sperm competition levels influencing testis architecture and the rate of progression of spermatogenesis, leading to differences in testis function at reproductive maturity.     

PTK+PRK治疗LASIK术中角膜瓣碎裂

目的研究激光辅助原位角膜磨镶术(LASIK)手术过程中,角膜瓣碎裂的处理及临床结果与疗效。方法对开展LASIK手术过程中,发生角膜瓣碎裂者行PTK(激光治疗性角膜切除术) PRK(激光屈光性角膜切削术),包括视力、屈光状态及术后Haze(混浊度)的情况,随访1周、1月、3月、6月、1年。结果自2000年5月～2004年5月共进行LASIK手术7 858眼,发生角膜瓣碎裂11例14眼,对其中7例8眼行PTK PRK。术后1周最佳矫正视力均大于0.6,Haze 0级;术后1月视力均大于1.0,Haze大于1级;术后3月、1年视力均大于1.0,Haze0级。结论PTK PRK治疗LASIK术中角膜瓣碎裂安全有效。