Motozumi Matsui

Other transgenic mutation assays: A new transgenic mouse mutagenesis test system using Spi− and 6-thioguanine selections

A new transgenic mouse mutagenesis test system has been developed for the efficient detection of point mutations and deletion mutations in vivo. The mice carry lambda EG10 DNA as a transgene. When the rescued phages are infected into Escherichia coli YG6020‐expressing Cre recombinase, the phage DNA is converted into plasmid pYG142 carrying the chloramphenicol‐resistance gene and the gpt gene of E. coli. The gpt mutants can be positively detected as colonies arising on plates containing chlaramphenicol and 6‐thioguanine. The EG10 DNA carries a chi site along with the red and gam genes so that the wild‐type phages display Spi+ (sensitive to P2 interference) phenotype. Mutant phages lacking both red and gam genes can be positively detected as plaques that grow in P2 lysogens of E. coli. These mutant phages are called lambda Spi−. The spontaneous gpt mutation frequencies of five independent transgenic lines were 1.7 to 3.3 × 10−5 in bone marrow. When the mice were treated with ethylnitrosourea (single i.p. treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four‐ to sevenfold over the background in bone marrow. The average rescue efficiencies were more than 200,000 chloramphenicol‐resistant colonies per 7.5 μg bone marrow DNA per packaging reaction. In contrast to gpt mutation frequencies, spontaneous Spi− mutation frequencies were 1.4 × 10−6 and 1.1 × 10−6 in bone marrow and sperm, respectively. No spontaneous Spi− mutants have been detected so far in spleen, although 930,000 phages rescued from untreated mice were screened. In gamma‐ray‐treated animals, however, induction of Spi− mutations was clearly observed in spleen, at frequencies of 1.4 × 10−5 (5 Gy), 1.2 × 10−5 (10 Gy), and 2.0 × 10−5 (50 Gy). These results suggest that the new transgenic mouse “gpt delta” could be useful for the efficient detection of point mutations and deletion mutations in vivo.

Spectra of gpt mutations in ethylnitrosourea-treated and untreated transgenic mice.

We have established a new transgenic mouse mutagenicity assay for the efficient detection of point mutations and deletions in vivo (Nohmi et al. [1996] Env. Mol. Mutagen. 28:465–470). In this assay, the gpt gene of Escherichia coli is used as a reporter for the detection of point mutations. Treatment of mice with ethylnitrosourea (ENU, 150 mg/kg) enhances by several‐fold the mutant frequency of gpt in bone marrow. Here, we report the mutation spectra of the gpt gene recovered from bone marrow of ENU‐treated and untreated transgenic mice. In the gpt mutants rescued from ENU‐treated mice, more than 90% of the mutations were base change mutations; the predominant types were A:T to T:A transversions and G:C to A:T transitions. On the contrary, in the mutants rescued from untreated mice, 54% were base substitutions and the remainders were short deletions and insertions. Among untreated mice, the most frequently observed base substitution was G:C to A:T transitions (7/14 mutants). Three of these occurred at 5′‐CpG‐3′ sites. Interestingly, the mutation spectra of the gpt gene were different from those of the gpt gene in ENU‐treated and untreated E.coli, whereas they were similar to those of the lacZ and lacI genes in ENU‐treated and untreated other transgenic mice or cultured mammalian cells. We also report the establishment of homozygous transgenic mice that have transgene λEG10 DNA in both chromosome 17 of C57BL/6J mouse. Environ. Mol. Mutagen. 34:1–8, 1999

Effect of local neutralization of basic fibroblast growth factor or vascular endothelial growth factor by a specific antibody on the development of the corpus luteum in the cow

Active angiogenesis and progesterone (P) synthesis occur in parallel during development of the corpus luteum (CL). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis and P synthesis in vitro. The aim of the present study was to investigate the impact of bFGF or VEGF on the CL development in the cow by using a specific antibody against bFGF or VEGF. bFGF antibody, VEGF antibody, or saline as a control (n = 4 cows/treatment) were injected directly into the CL immediately after ovulation (Day 1), and the treatment was continued for 3 times/day over 7 days. Luteal biopsies were applied on Day 8 of the estrous cycle to determine the expression of genes associated with P synthesis and angiogenesis. Intraluteal injections with the bFGF antibody or the VEGF antibody markedly decreased the CL volume, plasma P concentration and StAR mRNA expression. bFGF antibody treatment decreased the mRNA expression of bFGF, FGF receptor‐1, VEGF120, and angiopoietin (ANPT)‐1, and increased ANPT‐2/ANPT‐1 ratio. However, VEGF antibody treatment decreased ANPT‐2 mRNA expression and ANPT‐2/ANPT‐1 ratio. These results indicate that local neutralization of bFGF or VEGF changes genes regulating angiogenesis and P synthesis, and remarkably suppresses the CL size and P secretion during the development of CL in the cow, supporting the concept that bFGF and VEGF control the CL formation and function. Mol. Reprod. Dev. 75: 1449–1456, 2008.

Prostaglandin F2α increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis

Prostaglandin F(2)(alpha) (PGF(2)(alpha)) released from the uterus causes alterations in luteal blood flow, reduces progesterone secretion, and induces luteolysis in the bovine corpus luteum (CL). We have recently discovered that luteal blood flow in the periphery of the mature CL acutely increases coincidently with pulsatile increases in a metabolite of PGF(2)(alpha) (PGFM). In this study, we characterized changes in regional luteal blood flow together with regional alterations in endothelial nitric oxide synthase (eNOS) expression during spontaneous luteolysis and in response to PGF(2)(alpha). Smooth muscle actin-positive blood vessels larger than 20 microm were observed mainly in the periphery of mature CL. PGF(2)(alpha) receptor was localized to luteal cells and large blood vessels in the periphery of mid-CL. PGF(2)(alpha) acutely stimulated eNOS expression in the periphery but not in the center of mature CL. Injection of the NO donor S-nitroso-N-acetylpenicillamine into CL induced an acute increase in luteal blood flow and shortened the estrous cycle. In contrast, injection of the NOS inhibitor l-NAME into CL completely suppressed the acute increase in luteal blood flow induced by PGF(2)(alpha) and delayed the onset of luteolysis. In conclusion, PGF(2)(alpha) has a site-restricted action depending on not only luteal phase but also the region in the CL. PGF(2)(alpha) stimulates eNOS expression, vasodilation of blood vessels, and increased luteal blood flow in periphery of mature CL. Furthermore, the increased blood flow is mediated by NO, suggesting that the acute increase in peripheral blood flow to CL is one of the first physiological indicators of NO action in response to PGF(2)(alpha).

Stimulation of the development of bovine embryos by insulin and insulin-like growth factor-I (IGF-I) is mediated through the IGF-I receptor

To study the effects of insulin and insulin-like growth factor-I (IGF-I) on the development of bovine embryos, fertilized bovine embryos in vitro were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid (mSOF) supplemented with 1 mg/ml polyvinyl alcohol. Dose-response studies showed that insulin (0.5 to 10 microg/ml) and IGF-I (2 to 200 ng/ml) stimulated the development of bovine embryos to the morula stage 5 d after in vitro fertilization. The addition of 0.5 microg/ml insulin or 2 ng/ml IGF-I to the mSOF had beneficial effects on embryonic development to the morula stage in the presence of amino acids, but insulin and IGF-I did not affect the development of bovine embryos to the morula stage in the absence of amino acids. The antiIGF-I receptor antibody (alphaIR-3) completely blocked the stimulation of development to the morula stage by insulin and IGF-I. These findings suggest that the stimulation of embryonic development by insulin and IGF-I is mediated through the IGF-I receptor.

Evaluation of ovarian blood flow by colour Doppler ultrasound: Practical use for reproductive management in the cow

Transrectal real-time ultrasonography (US) has been developed as a research and practical tool in bovine reproduction. Non-invasive US observations have made it possible to provide real-time and serial analyses of ovarian morphological changes and fetal development and have generated new information on reproductive physiology during the bovine oestrous cycle and pregnancy. This has greatly contributed to an understanding of the real-time dynamics of follicular development. US has also allowed for more accurate diagnosis compared with rectal palpation in reproductive management in cattle. Practical applications of US include early diagnosis of pregnancy, identification of twin fetuses, detection of ovarian and uterine pathologies and determination of fetal sex. In recent years, local blood flow has been analysed in individual ovarian follicles and the corpus luteum (CL) in the cow using colour Doppler US. From these observations, it has been found that (1) the blood supply to follicles is closely related to follicular growth, atresia and ovulation, (2) the blood supply to the CL increases in parallel with its growth, and (3) there is an acute increase in blood flow in the mature CL prior to luteal regression. Colour Doppler US may provide an estimate of the physiological status of follicles and corpora lutea. For example, images of blood flow can be used to assess the thickness of the follicular wall and provide a differential diagnosis of follicular and luteal cysts. Assessment of the area of blood flow in the CL using colour Doppler imaging may offer a useful adjunct in estimating CL function, which could be applied to the diagnosis of non-pregnancy and fetal loss. The number of small follicles which have blood flow at the start of gonadotrophin treatment may be a useful index to predict the superovulatory response. With improvements in portability and cost-effectiveness, the evaluation of ovarian blood flow by colour Doppler US is likely to become widely used as a diagnostic tool for monitoring ovarian function in dairy cattle.

Involvement of angiopoietin-tie system in bovine follicular development and atresia : messenger RNA expression in theca interna and effect on steroid secretion

Abstract Angiogenesis is involved in the local mechanisms that regulate follicular development and ovulation. Recently, the angiopoietin (ANPT)-Tie system has been shown to be required to regulate angiogenesis and blood vessel regression. Expression of the ANPT-Tie system in the cyclic ovary suggests that the relative changes in the expression of ANPT-1 and ANPT-2 influence the stability of ovarian blood vessels. In this study, we investigated 1) the mRNA expression for ANPT-1, ANPT-2, and endothelial cell-specific receptors Tie1 and Tie2 in the theca interna (TI) of the bovine developing, mature, and atretic follicles by using a semiquantitative reverse transcription polymerase chain reaction assay and 2) the effect of ANPT on the secretion of steroid hormones from bovine preovulatory follicles in vitro using a microdialysis system (MDS) implanted in the thecal layer. Bovine follicles were classified as developing, mature, and atretic according to size, follicular fluid content of estradiol (E2) and progesterone (P4), and characteristics of granulosa cells (GCs). Both ANPT and Tie mRNA were expressed in the TI, whereas GCs expressed ANPT mRNA only. The expression of ANPT-2 mRNA was decreased in the mature follicles. This decrease resulted in a decrease in the ANPT-2:ANPT-1 ratio (an index of instability of blood vessels), indicating that the blood vessels became more stable or mature. The early atretic follicles showed a higher ANPT-2:ANPT-1 ratio and higher Tie2 mRNA expression than did other follicles at healthy or later atretic stages. This finding may imply that blood vessels become unstable at the initial stage of follicular atresia. In both mid and late atretic follicles, Tie2 mRNA expression dramatically decreased, indicating a disruption of the ANPT-Tie system. In the MDS experiment, an infusion of ANPT-1 or ANPT-2 increased P4 release, whereas both ANPTs inhibited the release of androstenedione. ANPT-1 also increased E2 release. These results showed that the mRNA expression for ANPT-1, ANPT-2, Tie1, and Tie2 changes during follicular development, maturation, and atresia in bovine follicles and that ANPTs affect steroidogenesis in the preovulatory follicle. The results suggest that the ANPT-Tie system is involved the structural (angiogenesis) and secretory changes that occur during follicular development and atresia.

Real-Time Relationships in Intraluteal Release among Prostaglandin F2α, Endothelin-1, and Angiotensin II During Spontaneous Luteolysis in the Cow

Abstract It is well known that prostaglandin F2α (PGF2α) is a physiological luteolysine, and that its pulsatile release from the endometrium is a luteolytic signal in many species. There is now clear evidence that the vasoactive peptides endothelin-1 (ET-1) and angiotensin II (Ang II) interact with PGF2α in the luteolytic cascade during PGF2α-induced luteolysis in the cow. Thus, we investigated the local secretion of PGF2α, ET-1, and Ang II in the corpus luteum (CL) and their real-time relationships during spontaneous luteolysis in the cow. For this purpose, an in vivo microdialysis system (MDS) implanted in the CL was utilized to observe local secretion changes within the CL microenvironment. Each CL of cyclic Holstein cows (n = 6) was surgically implanted with MDS capillary membranes (18 lines/6 cows) on Day 15 (estrus = Day 0) of the estrous cycle. The concentrations of PGF2α, ET-1, Ang II, and progesterone (P) in the MDS samples were determined by enzyme immunoassays. The intraluteal PGF2α secretion slightly increased from 12 h after the onset of luteolysis (0 h) and drastically increased (by about 300%) from 24 h. Intraluteal ET-1 secretion increased from 12 h. Intraluteal Ang II secretion was elevated from 0 h and was maintained at high levels (about 180%) toward estrus. In each MDS lines (in the same microenvironment) within the regressing CL, the local releasing profiles of PGF2α, ET-1, and Ang II CL positively correlated with each other (P < 0.05) at high proportions in 18 MDS lines (PGF2α vs. ET-1, 44.4%; PGF2α vs. Ang II, 55.6%; ET-1 vs. Ang II, 38.9%). In contrast, there was no clear relationship among these substances released into different MDS lines implanted in the same CL (with different microenvironments). In conclusion, we propose that the increase of PGF2α, ET-1, and Ang II within the CL during luteolysis is a common phenomenon for both PGF2α-induced and spontaneous luteolysis. Moreover, this study illustrated the in vivo relationships in intraluteal release among PGF2α, ET-1, and Ang II during spontaneous luteolysis in the cow. The data suggest that these vasoactive substances may interact with each other in a local positive feedback manner to activate their secretion in the regressing CL, thus accelerating and completing luteolysis.

Possible involvement of IFNT in lymphangiogenesis in the corpus luteum during the maternal recognition period in the cow

The corpus luteum (CL), which secretes large amounts of progesterone and is thus essential for establishing pregnancy, contains various types of immune cells that may play essential roles in CL function by generating immune responses. The lymphatic system is the second circulation system and is necessary for immune function, but the lymphatic system of the bovine CL has not been characterized in detail. We collected bovine CLs on days 12 and 16 of the estrous cycle (C12 and C16) and days 16 and 40 of early pregnancy (P16 and P40). Lymphatic endothelial hyaluronan receptor 1 (LYVE1) protein was detected in the CL by immunohistochemistry and western blotting and increased at P40 compared with C16. The mRNA expression levels of lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGFC), VEGFD, and their common receptor VEGFR3, as well as the lymphatic endothelial cell (LyEC) marker podoplanin, increased in P16 and P40 CLs. Thus, it is suggested that the lymphatic system of the bovine CL reconstitutes during early pregnancy. Interferon tau (IFNT) from the conceptus in the uterus is a candidate for activating luteal lymphangiogenesis during the maternal recognition period (MRP). We found that treatment of LyECs isolated from internal iliac lymphatic vessels with IFNT stimulated LyEC proliferation and significantly increased mRNA expression of VEGFC and IFN-stimulated gene 15. Moreover, both IFNT and VEGFC induced LyECs to form capillary-like tubes in vitro. In conclusion, it is suggested that new lymphangiogenesis in the bovine CL begins during the MRP and that IFNT may mediate this novel phenomenon.

Lipopolysaccharide in ovarian follicular fluid influences the steroid production in large follicles of dairy cows

In postpartum dairy cows, various inflammatory diseases depress reproductive performance. Lipopolysaccharide (LPS) derived from infections of the uterus or mammary gland with Gram-negative bacteria was shown to suppress steroid production in the granulosa cells of follicles in vitro. The aim of the study was to investigate the relationship between LPS in ovarian follicular fluid and steroidogenesis by the theca and granulosa cells of the large follicles in vivo. Bovine ovaries were collected from a slaughterhouse, and the largest (F1) and the second largest (F2) follicles were used (>8 mm in diameter, n=38). LPS concentration in the follicular fluid was measured using quantitative kinetic assay. Follicular steroidogenesis was evaluated by measuring the estradiol (E2) and progesterone (P4) concentration in follicular fluid and by analysing transcription levels of steroidogenesis-related genes in theca and granulosa cells. LPS concentration detected in follicular fluid ranged from 0.2 to 2.0 EU/mL. In follicles with a high level of LPS (>0.5 EU/mL, n=15), the concentration of E2 was lower and that of P4 was higher when compared to those in follicles with a low level of LPS (<0.5 EU/mL, n=23), which was observed both in F1 and F2 follicles. Furthermore, in follicles with a high level of LPS, transcripts of steroidogenic enzymes such as CYP17 and P450arom were lower. In those follicles, the expression of caspase-3 was high, suggesting an association with follicular atresia. These findings indicate that LPS present in follicular fluid may cause ovarian dysfunction by inhibiting follicular activity.