Disruption of the temporally regulated cloaca endodermal ß-catenin signaling causes anorectal malformations.

The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. ß-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of ß-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the ß-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (Shh(CreERT2)). Both ß-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the ß-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the ß-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The Shh(CreERT2/+); ß-catenin(flox(ex3)/+); BmprIA(flox/-) mutants displayed partial restoration of URS elongation compared with the ß-catenin GOF mutants. These results indicate that some ARM phenotypes in the ß-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal ß-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.

The influence of reduced glutathione in fertilization medium on the fertility of in vitro-matured C57BL/6 mouse oocytes.

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo-matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.

ß-Catenin signaling regulates Foxa2 expression during endometrial hyperplasia formation.

The Wnt/ß-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated ß-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/ß-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of ß-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that ß-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that ß-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that ß-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of ß-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia.

Combination medium of cryoprotective agents containing L-glutamine and methyl-{beta}-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm.

Recently, a vast number of genetically-engineered mice have been created in various laboratories worldwide, all of which need to be effectively archived. The cryopreservation of mouse sperm provides a simple and economical means of storing the mice in mouse resource facilities. The current protocol for sperm cryopreservation using 18% raffinose pentahydrate and 3% skim milk (R18S3) has been adopted in most laboratories. In general, we can attain relatively high fertilization rates for frozen/thawed sperm in many inbred and F1 hybrid strains. However, the sperm of C57BL/6J mice shows an extremely low fertility rate after freezing and thawing (0-20%). In this study, we attempted to improve the low fertility of frozen/thawed C57BL/6J mouse sperm. Our results showed that a combination of R18S3 containing l-glutamine and methyl-beta-cyclodextrin (MBCD) in a preincubation medium dramatically increased the rate of fertilization (69.2 +/- 12.2%). Furthermore, the developmental potencies of two-cell embryos produced by frozen/thawed sperm to live young were normal (fresh: 46.0 +/- 8.2%, frozen/thawed: 51.5 +/- 11.1%). In summary, we conclude that a new method of sperm cryopreservation and in vitro fertilization using modified R18S3 with l-glutamine and MBCD in a preincubation medium yields a high fertilization rate for frozen/thawed C57BL/6J strain sperm. Furthermore, the new method provides a reliable archiving and reproducing system for genetically-engineered mice using sperm cryopreservation.

Aurora A overexpression induces cellular senescence in mammary gland hyperplastic tumors developed in p53-deficient mice.

Aurora A mitotic kinase is frequently overexpressed in various human cancers and is widely considered to be an oncoprotein. However, the cellular contexts in which Aurora A induces malignancy in vivo are still unclear. We previously reported a mouse model in which overexpression of human Aurora A in the mammary gland leads to small hyperplastic changes but not malignancy because of the induction of p53-dependent apoptosis. To study the additional factors required for Aurora A-associated tumorigenesis, we generated a new Aurora A overexpression mouse model that lacks p53. We present evidence here that Aurora A overexpression in primary mouse embryonic fibroblasts (MEFs) that lack p53 overrides postmitotic checkpoint and leads to the formation of multinucleated polyploid cells. Induction of Aurora A overexpression in the mammary glands of p53-deficient mice resulted in development of precancerous lesions that were histologically similar to atypical ductal hyperplasia in human mammary tissue and showed increased cellular senescence and p16 expression. We further observed DNA damage in p53-deficient primary MEFs after Aurora A overexpression. Our results suggest that Aurora A overexpression in mammary glands is insufficient for the development of malignant tumors in p53-deficient mice because of the induction of cellular senescence. Both p53 and p16 are critical in preventing mammary gland tumorigenesis in the Aurora A overexpression mouse model.

Offspring derived from oocytes injected with rat sperm, frozen or freeze-dried without cryoprotection.

Sperm preservation is a valuable technique for maintaining genetic resources in biomedical research. In the present study, 10mM Tris-HCl and 1mM EDTA (TE buffer; a simple solution without cryoprotection), was used to freeze or freeze-dry rat sperm. The results were compared with rat sperm frozen using a solution containing Equex STM and egg yolk. Sperm from Wistar and Sprague-Dawley (SD) rats were evaluated by injecting them individually into oocytes derived from the same strain. Of the oocytes that survived after sperm injection, more than 94% were fertilized in all treatments of both strains. In the Wistar rat, 27, 20, 43, and 30% of 2-cell embryos developed to blastocysts, and 35, 9, 11, and 14% of 2-cell embryos developed to offspring from oocytes injected with fresh, frozen (Equex STM/egg yolk), frozen (TE buffer), and freeze-dried sperm, respectively. Using the analagous sources of sperm in the SD rat, 45, 14, 27, and 7% of 2-cell embryos developed to blastocysts, and 22, 0, 14, and 4% of 2-cell embryos developed to offspring. These results demonstrated that rat sperm could be frozen or freeze-dried using TE buffer. We concluded that this simple preservation method, in which cryoprotection was not required, allowed sperm to be preserved efficiently with maintenance of their fertilizing ability.

Molecular genetic cascades for external genitalia formation: an emerging organogenesis program.

External genitalia are anatomical structures located at the posterior embryonic region as part of several urogenital/reproductive organs. The embryonic anlage of the external genitalia, the genital tubercle (GT) develops as a bud-shaped structure with an initial urethral plate and later urethra. Embryonic external genitalia are considered to be one of the appendages. Recent experiments suggest that essential regulatory genes possess similar functions for the outgrowth regulation of the GT and limb appendages. The transient embryonic epithelia located in the distal GT are called the distal urethral epithelium (DUE) regulating, at least in part, the (distal) GT development. This review covers the available data about early patterning of GT and discusses the molecular developmental similarities and points of divergence between the different appendages. Development of the male and female external genitalia is also reviewed.

Developmental biology meets with reproductive engineering; interdisciplinary science area as a breakthrough.

It has been postulated many times that different scientific topics and strategies often encounter each other, which create cutting edge research field resulting in further significant progresses of science. This was also a lesson bestowed by Prof. Raymond Wegmann where he created innovative research field for biology, molecular biology and biochemistry-biophysics. Progresses of developmental biology were boosted by molecular biology and reproductive engineering where ES cells and embryonic manipulation are necessary. There are no questions about the utility of their technologies. Reviews on their contributions with respect to the condition of genome manipulation are addressed.

Cryopreservation and transport of mouse spermatozoa at -79 degrees C.

In the present study, 2 experiments were carried out. In experiment 1, mouse spermatozoa were frozen and stored in an ultra-low temperature freezer maintained at -79 degrees C, from 1 week to 8 months. In vitro fertilization rates of the frozen-thawed sperm after 1 week and 4 months of storage were high at 71 and 71%, respectively. These values did not differ significantly from the value (73%) of the control stored at -196 degrees C. In contrast, the 8-month storage rate was significantly lower at 51%. In experiment 2, frozen spermatozoa were transported in a Styrofoam box packed in dry ice from Hokkaido to Tokyo. In vitro fertilization rate of frozen-thawed sperm after transport at -79 degrees C was high at 88%, which was not significantly different from that (84%) of the transported control at -190 degrees C. After transferring two-cell embryos derived from frozen spermatozoa to recipients, 37-62% of the embryos developed into offspring in both experiments. These results indicate that mouse spermatozoa can survive cryopreservation in an ultra-low temperature freezer (-79 degrees C) for up to 4 months and transport at -79 degrees C.

Cryopreservation of mouse spermatozoa.

Recently, it has become possible to freeze a large number of mouse spermatozoa immediately after collection from the epididymides of a small number of males. The cryopreservation of spermatozoa is simpler, less time-consuming, and less costly than that of embryos for maintaining various strains of mice with induced mutations. This chapter attempts to provide a realistic overview of the cryopreservation of mouse spermatozoa and to describe a detailed procedure for mouse sperm freezing.

Production of chimeric mice from cryopreserved blastocysts.

Mouse blastocysts derived from in vivo fertilization were cryopreserved by a simple vitrification method. Most of the cryopreserved blastocysts survived while demonstrating a normal morphology after warming (88.1%). The rate of cryopreserved blastocysts which developed to term as normal young was 56.8%, and chimeric mice were also produced from cryopreserved blastocysts injected with ES cells. As a result, 3.4% of the injected embryos developed to term as germ line chimeric mice.

Positive effect of partial zona-pellucida dissection on the in vitro fertilizing capacity of cryopreserved C57BL/6J transgenic mouse spermatozoa of low motility.

Although cryopreservation of mouse spermatozoa has recently become available for use, as yet there are considerable differences in fertilization efficiency of cryopreserved spermatozoa among various mouse strains. In this study, oocytes subjected to partial dissection of the zona pellucida (PZD) were inseminated with frozen-thawed C57BL/6J mouse spermatozoa. At 30, 60, 120, and 240 min after insemination, the oocytes were washed in human tubal fluid medium and cultured for 3-6 h. The fertilization rates of the PZD oocytes in each group at 6-7 h after insemination were significantly higher than that of the zona-intact control (73-88% vs 12%, respectively) (p < 0.01); but the incidence of polyspermy was nevertheless quite low (1.3-2.4%). The development rates of the monospermic oocytes to the morula and early blastocyst stages were in the 87-92% range, with 31-40% of those developing into offspring after embryo transfer. When the cryopreserved spermatozoa of C57BL/6J transgenic mice were used to fertilize PZD oocytes, the fertilization rates were as high as (73-76%) those of the PZD oocytes inseminated with the cryopreserved C57BL/6J spermatozoa, with 30-31% of the morulae and early blastocysts derived from the monospermatic oocytes developing into offspring. These results indicate that PZD of oocytes may provide an alternative when the fertilizing capacity of mouse spermatozoa has been compromised by cryopreservation.

Simple and efficient vitrification procedure for cryopreservation of mouse embryos.

Mouse pronuclear oocytes and 2-cell embryos derived from in vitro fertilization were cryopreserved by a novel simple vitrification procedure. Most cryopreserved oocytes/embryos were morphologically normal after warming, and 89-92% of them developed to the blastocyst stage during the culture. Moreover, the rate of morphologically normal pronuclear oocytes after being repeatedly cooled and warmed three times was as high as that of oocytes cooled and warmed only once, and 85% of them developed to the blastocyst stage. In addition, 43-57% of the cryopreserved oocytes/embryos transferred to recipients had developed normally to live fetuses observed on day 18.5 of pregnancy.

Cryopreservation of wild mouse spermatozoa.

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats.

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T3 injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about approximately 35-40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly approximately 25-32 and approximately 29-41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by approximately 72-81 and approximately 51-70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels.

Onset of paternal gene activation in early mouse embryos fertilized with transgenic mouse sperm.

We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken beta-actin promoter-driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1- to 2-cell stage. RNA transcripts of the luciferase gene were first detected in the 1-cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon-count imaging, functional luciferase was identified at the 2-cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1-cell embryos, although paternally derived protein is not synthesized until the 2-cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1-cell stage.