Neurotensin induces sustainable activation of the ErbB-ERK1/2 pathway, which is required for developmental competence of oocytes in mice.

Purpose: LH induces the expression of EGF-like factors and their shedding enzyme (ADAM17) in granulosa cells (GCs), which is essential for ovulation via activation of the ErbB-ERK1/2 pathway in cumulus cells (CCs). Neurotensin (NTS) is reported as a novel regulator of ovulation, whereas the NTS-induced maturation mechanism in oocytes remains unclear. In this study, we focused on the role of NTS in the expression of EGF-like factors and ErbBs, and ADAM17 activity, during oocyte maturation and ovulation in mice. Methods: The expression and localization in GC and CC were examined. Next, hCG and NTS receptor 1 antagonist (SR) were injected into eCG-primed mice, and the effects of SR on ERK1/2 phosphorylation were investigated. Finally, we explored the effects of SR on the expression of EGF-like factors and ErbBs, and ADAM17 activity in GC and CC. Results: NTS was significantly upregulated in GC and CC following hCG injection. SR injection suppressed oocyte maturation and ERK1/2 phosphorylation. SR also downregulated part of the expression of EGF-like factors and their receptors, and ADAM17 activity. Conclusions: NTS induces oocyte maturation through the sustainable activation of the ERK1/2 signaling pathway by upregulating part of the EGF-like factor-induced pathway during oocyte maturation in mice.

Pre-culture with transferrin-Fe3+ before in vitro maturation improves the developmental competence of porcine oocytes matured in vitro.

Purpose: Since the developmental competence of oocytes cultured after in vitro maturation (IVM) is low, it is necessary to improve the IVM method for efficient offspring production. In this study, we revealed that transferrin (TF)-Fe3+ was accumulated in follicular fluid with increasing the follicular diameter, and that TF receptor (TFR1) was localized in granulosa cells of pig. Thus, we hypothesized that TF-Fe3+ would be a factor in the induction of developmental competence of porcine oocytes. Methods: To mimic the follicular development environment, cumulus-oocyte complexes (COCs) were cultured in pre-IVM medium (low dose of FSH) without or with Holo-TF (monoferric or diferric TF) or Apo-TF (non-iron bond TF). After pre-IVM without or with Holo-TF, COCs were cultured in IVM medium (high dose of FSH and EGF) without or with Holo-TF. Results: Cultivation with Holo-TF increased the expression of follicular development maker (Cyp19a1 and Ccnd2), E2 production, and proliferative activity of cumulus cells, whereas cultivation with Apo-TF did not show these positive effects. The treatment with Holo-TF during pre-IVM, but not during IVM, dramatically induced oocyte maturation with increasing the blastocyst rate. Conclusion: We succeeded in showing for the first time that the cultivation with Holo-TF in pre-IVM can produce embryos in pig with high efficiency.

Anti-microbial resistance of Salmonella isolates from raw meat-based dog food in Japan.

BACKGROUND: Salmonella contamination of raw meat-based diets (RMBDs) for pets poses a major public health concern but has not been investigated in Japan. OBJECTIVE: To investigate Salmonella contamination in RMBDs for dogs marketed in Japan and the anti-microbial resistance profiles of the Salmonella isolates. METHODS: Sixty commercial RMBD samples were collected in the Okayama and Osaka Prefectures, Japan, between December 2016 and March 2017. The obtained Salmonella isolates were serotyped, their anti-microbial resistance patterns were determined, and the anti-microbial-resistant isolates were screened for the presence of resistance genes by polymerase chain reaction. RESULTS: Salmonella enterica subsp. enterica was detected in seven of the 60 RMBD samples. Among them, five isolates were identified as S. Infantis (n = 3), S. Typhimurium (n = 1) and S. Schwarzengrund (n = 1), while the serotypes of two isolates were unable to be identified. All isolates were susceptible to ampicillin, cefazolin, cefotaxime and gentamycin. Two isolates were resistant to more than one anti-microbial agent; one of the S. Infantis isolates was resistant to streptomycin, kanamycin, tetracycline and trimethoprim, while the S. Typhimurium isolate was resistant to nalidixic acid, ciprofloxacin and chloramphenicol. The S. Schwarzengrund isolate was resistant to tetracycline. Additionally, the S. Typhimurium isolate harboured the anti-microbial resistance gene gyrA with a mutation corresponding to Ser-83→Phe amino acid substitution. CONCLUSION: The study findings suggest that RMBDs for dogs marketed in Japan can be a potential source of Salmonella infection for dogs and humans including infections caused by quinolone-resistant isolates.

LH Induces De Novo Cholesterol Biosynthesis via SREBP Activation in Granulosa Cells During Ovulation in Female Mice.

In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulate cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared with the control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum; however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.

Cortisol induces follicle regression, while FSH prevents cortisol-induced follicle regression in pigs.

During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus-oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18ß-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.

Iron deficiency induces female infertile in order to failure of follicular development in mice.

Iron is important for many cellular functions, including ATP synthesis and cell proliferation. Insufficient of iron in the diet causes iron deficiency anemia (IDA), which often occurs in people living in the world. Since 50% of women with IDA show amenorrhea, the relationship of between iron deficiency and reproductive function was assessed using mice fed a low Fe diet (LFD). The estrous cycle in the LFD mice was blocked at diestrus, which impair follicle development, and fertility. Further, even LFD mice were injected with exogenous pregnant mare serum gonadotropin (PMSG), follicular development was ceased at the secondary follicle stage, and preovulatory follicles were not observed. Amount of ATP decreased in the ovary of the LFD mice, and expression of follicle development markers (Fshr, Cyp19a1, Ccnd2) and estradiol-17ß (E2) was low level compared to levels mice fed a normal diet. Feeding a normal diet with sufficient iron to the LFD mice for an additional 3 weeks completely reversed absence the effects of iron insufficient on the estrous cycle and infertility. Thus, iron restriction depresses ovary functions, especially follicular development from secondary follicle to antral follicles and infertility. These effects are fully reversible by supplementation of a normal diet containing iron.

The Novel Pig In Vitro Maturation System to Improve Developmental Competence of Oocytes Derived from Atretic Non-Vascularized Follicle.

The in vitro maturation (IVM) technique is beneficial for producing animal offspring, but the blastocyst rate is low after IVM. In this technique, cumulus-oocyte complexes (COCs) are collected from medium size follicles. The follicles are ultimately selected as large dominant follicles or atretic follicles; therefore it is possible that the COCs collected using IVM are contaminated by follicles that will develop into large follicles and induce atresia. In the dominant follicles, estradiol-17beta and progesterone induce the differentiation of follicular somatic cells which exhibit the ability to respond to ovulation during follicular development. Thus, we hypothesized that changes in the hormonal condition of healthy follicles are essential for oocyte maturation during IVM. In this study, we performed a comparative analysis of the steroid hormone concentrations in non-vascularized follicles (NVFs) and vascularized follicles (VFs). The estradiol-17beta concentration increased in medium VFs, whereas the level was low in NVFs of the same size. The progesterone concentration increased with large follicular size in VFs, but the level remained low in follicles of any size among NVFs. To improve the oocyte quality derived from NVFs, NVF COCs were cultured with FSH alone or FSH under theVF hormonal conditions. Cultivation under the VF hormonal conditions dramatically improved the proliferation and survival of cumulus cells, meiotic maturation of oocytes, cumulus expansion, and blastocyst rate following in vitro fertilization. Thus, the cultivation of NVF COCs under VF hormonal conditions improves the developmental potential to the blastocyst stage by NVF oocytes.

Protein kinase C (PKC) increases TACE/ADAM17 enzyme activity in porcine ovarian somatic cells, which is essential for granulosa cell luteinization and oocyte maturation.

During in vitro maturation of porcine cumulus cell-oocyte complexes and in vitro luteinization of porcine granulosa cells, FSH induces the expression of the protease TNFα-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) and the epidermal growth factor (EGF)-like factors, which activate the EGF receptor (EGFR)-MAPK3/1 pathway in both cumulus and granulosa cells. FSH is known to activate not only protein kinase A and p38MAPK pathways in both cell types but also activates protein kinase C (PKC). Because PKC-induced association of cellular-Sarcoma (c-Src) and TACE/ADAM17 is required for TACE/ADAM17 enzyme activation in some cancer cells, we hypothesized that PKC and c-Src impact TACE/ADAM17-mediated activation of EGFR signaling pathway in porcine granulosa and cumulus cells. When granulosa cells or cumulus cell-oocyte complexes were cultured with FSH, PKC activity and c-Src phosphorylation increased and were associated with increased TACE/ADAM17 enzyme activity. The PKC inhibitor calphostin C (CalC) and the c-Src inhibitor (4 amino 5 (4 chlorophenyl) 7 (t butyl)pyrazolo[3,4 d]pyrimidine [PP2]) suppressed TACE/ADAM17 enzyme activity, whereas these inhibitors did not affect Tace/Adam17 mRNA expression. Immunoprecipitation analysis showed that FSH mediated the association of c-Src with TACE/ADAM17 via a PKC-dependent mechanism. Either CalC or PP2 suppressed EGFR downstream signaling pathway (MAPK3/1) in these ovarian cell types and reduced cumulus expansion, meiotic maturation of oocytes, and progesterone production. The negative effects were overcome by the addition of amphiregulin. Collectively, these results indicate that activation of TACE/ADAM17 via a PKC-induced c-Src-dependent manner mediates proteolytic activation of the EGF-like factors that are involved in the induction of granulosa cell differentiation, cumulus expansion, and meiotic maturation of porcine oocytes in vitro.

Etodolac, a selective cyclooxygenase-2 inhibitor, induces upregulation of E-cadherin and has antitumor effect on human bladder cancer cells in vitro and in vivo.

OBJECTIVES: Cyclooxygenase-2 (COX-2) is highly expressed in several human cancers, including bladder cancer. Thus, a selective COX-2 inhibitor could be useful as an antitumor agent for a range of cancers. In the present study, we investigated the antitumor effect and E-cadherin induction of etodolac, a highly selective COX-2 inhibitor, on human bladder cancer cells in vitro and in vivo. METHODS: We examined the cytotoxicity of etodolac against three human bladder cancer cell lines, T24, 5637, and KK47, and performed quantitative reverse transcriptase-polymerase chain reaction to measure the mRNA expression of COX-2, and E-cadherin. RESULTS: Etodolac showed significant cytotoxicity only to T24 cells, which expressed the greatest level of COX-2 mRNA and the lowest level of E-cadherin mRNA among the three cell lines. Etodolac also increased the E-cadherin mRNA expression in T24 cells in vitro. We also found that etodolac suppressed tumor growth and induced E-cadherin expression and cell apoptosis in a T24 tumor xenograft mouse model. CONCLUSIONS: Etodolac exhibited antitumor activity and induced E-cadherin expression in bladder cancer cells and might be useful for the clinical treatment and prevention of bladder cancer, especially in poorly differentiated bladder cancer with high COX-2 and low E-cadherin expression.

SCCmec typing and detection of VISA-related genes in methicillin-resistant Staphylococcus aureus clinical strains from Kobe University Hospital, Japan.

A total of 50 clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) were collected from Kobe University Hospital in 2003. Molecular typing of SCCmec was performed by multiplex polymerase chain reaction (PCR) and the presence of six genes (vraR, vraG, vraA, vraF, fruA, and fruB) associated with vancomycin (VCM) resistance was examined by simple PCR analysis. Out of 50 MRSA strains isolated 47 strains contained Type II SCCmec and the remaining contained Type IV SCCmec. Thirty seven strains contained pUB110 plasmid. VraA was present in 69% of the strains, vraF in 10%, vraG in 53%, and vraR in 16%. Noteworthy, strains without pUB110 contained vraR in relatively higher frequency (31%) compared with strains with pUB110 (11%).