Cloning of carrier cells infected with oncolytic adenovirus driven by midkine promoter and biosafety studies.

BACKGROUND: A549 carrier cells infected with oncolytic adenovirus can induce complete tumor reduction of subcutaneous ovarian tumors but not intraperitoneal disseminated ovarian tumors. This appears to be a result of the insufficient antitumor effect of A549 carrier cells. Therefore, in the present study, we cloned a novel carrier cell with the aim of improving the antitumor effects. METHODS: Carrier cells infected with oncolytic adenovirus AdE3-midkine with a midkine promoter were cloned by limiting dilution. We examined the antitumor effects of these cells on subcutaneous and intraperitoneal OVHM ovarian tumors in a syngeneic mouse model. Biosafety tests were conducted in beagle dogs and rabbits. RESULTS: We cloned EHMK-51-35 carrier cells with 10-fold higher antitumor effects compared to A549 carrier cells in vitro. EHMK-51-35 carrier cells co-infected with AdE3-midkine and Ad-mGM-CSF induced a 100% complete tumor reduction in subcutaneous tumors and a 60% reduction of intraperitoneal disseminated tumors. Single-dose acute toxicity test on beagle dogs with EHMK-51-35 carrier cells co-infected with AdE3-midkine and Ad-cGM-CSF showed no serious side effects. Biologically active adenoviruses were not detected in the blood, saliva, feces, urine or whole organs. In a chronic toxicity test, VX2 tumors in rabbits were injected five times with EHMK-51-35 carrier cells infected with AdE3-midkine and these rabbits showed no serious side effects. CONCLUSIONS: Significant antitumor effects and safety of cloned EHMK-51-35 carrier cells were confirmed in intraperitoneal ovarian tumors and toxicity tests, respectively. These findings will be extended to preclinical efficacy studies using dogs and cats, with the aim of conducting human clinical trials on refractory solid tumors.

Preventive effect of oral mucosal epithelial cell sheets on intrauterine adhesions.

STUDY QUESTION: Can regenerative-medicine techniques using oral mucosal epithelial cell sheets (OMECS) provide a new treatment method for intrauterine adhesions (IUA) which cause female infertility? SUMMARY ANSWER: Transplantation of OMECS was confirmed to be effective in preventing IUA after endometrial damage in rats. WHAT IS KNOWN ALREADY: Uterine disorders such as IUA, commonly known as Asherman's syndrome, are one factor that can result in infertility. Clinical therapy for this kind of disease is targeted at the prevention of re-adhesion by surgical synechiotomy, administration of hormones after the operation, and the use of intrauterine devices. Recently, a new approach called 'cell-sheet engineering', which harvests confluent culture cells as a contiguous cell sheet having intact cell-cell junctions and an extracellular matrix, without having to use enzymatic treatment, has been developed for tissue regeneration. STUDY DESIGN, SIZE, DURATION: OMECS were prepared from rat oral mucosal tissues. An IUA model was made in rat uteri, and OMECS were transplanted into the model. Uteri transplanted with OMECS were compared with the non-transplanted control uteri by histological analysis at 1, 2 and 8 days after surgery (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS: Oral mucosal tissues were resected from neonatal rats, and oral mucosal epithelial cells were collected with enzymatic treatment. An isolated cell suspension was seeded on a temperature-responsive cell culture-insert and incubated. After being detached from the insert, a cell sheet was transplanted onto the endometrium defect. At 1, 2 and 8 days after surgery, uteri were resected and examined. MAIN RESULTS AND THE ROLE OF CHANCE: Histological examination of the non-treated specimens at 1, 2 and 8 days after surgery did not show any uterine cavities typically caused by IUA. In contrast, the histology of uteri transplanted with OMECS immediately after endometrial damage showed the presence of uterine cavities, and furthermore, stratified squamous epithelial cells on the luminal surface (n = 3). LIMITATIONS, REASONS FOR CAUTION: The results of this study are difficult to apply directly to humans, because the structure and function of rat uteri are different from those of human. WIDER IMPLICATIONS OF THE FINDINGS: Transplantation of OMECS offers a reliable method not only to protect the woman's fertility from intrauterine re-adhesion after synechiotomy for IUA or uterine lumen adhesion but also to prevent adhesion after any intrauterine surgery in clinical cases.

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering.

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor ß and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.

Novel method of gene transfer in birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms.

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

Phospholipase Czeta mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor.

This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Czeta (PLCzeta) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCzeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLCzeta cRNA at 60 microg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCzeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCzeta cRNA can induce development. In addition, RT-PCR revealed that PLCzeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCzeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.

Efficient intrahepatic tumor generation by cell sheet transplantation to fabricate orthotopic hepatocarcinoma-bearing model mice for drug testing.

Subcutaneous tumor-bearing mice are commonly used to evaluate antitumor activity in preclinical studies of anticancer drugs. However, these models often exhibit excessive antitumor responses to anticancer drug candidates. In this study, intrahepatic tumor-bearing mice as orthotopic tumor models were fabricated by transplanting hepatocarcinoma cell monolayers (sheets) to investigate differences in ectopic versus orthotopic antitumor response. Cell sheets, harvested from temperature-responsive cell culture dishes using thin gelatin gel supporters, were transferred onto mouse liver surfaces. Cell sheet transplantation drastically improved intrahepatic tumor formation compared with direct intrahepatic injection of dispersed cells. In particular, all cell sheet-transplanted mice formed well-developed tumors inside the liver following removal of the mesothelial membrane at the liver surface. Notably, these mice exhibited comparable life spans, indicating similar intrahepatic tumor development rates. Antitumor activity of doxorubicin (DOX) was examined using both subcutaneous and intrahepatic tumor-bearing mice. Although DOX administration yielded decreased subcutaneous tumor volumes, intrahepatic tumors exhibited no significant antitumor response. The results were considered to represent pharmacokinetic and histological structure differences between ectopic and orthotopic tumors, and partially supported the clinical uses of DOX. Therefore, cancer cell sheet transplantation constitutes a promising method to fabricate intrahepatic tumor-bearing mice for drug screening test in preclinical studies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1071-1079, 2019.

Suitable reference genes for the analysis of direct hyperplasia in mice.

The liver is capable of undergoing a proliferative growth, known as direct hyperplasia, in which the naïve liver increases in size due to stimulation with primary mitogens. To produce accurate gene expression data, housekeeping genes (HKGs) that are stably expressed need to be determined. In the present study, liver regeneration was promoted via the direct hyperplasia mode by inducing mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. Gene expression levels of nine commonly used HKGs were analyzed in the liver of different timing during the regeneration. The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, we identified that PPIA and RPL4 showed the most stable expression regardless of the status of the liver regeneration. In conclusion, the present study demonstrated that the use of PPIA and RPL4 were the most optimal in providing reliable normalization of gene expression when assessing liver regeneration attributed to direct hyperplasia.

Improved In Vivo Subcutaneous Tumor Generation by Cancer Cell Sheet Transplantation.

BACKGROUND/AIM: In vivo subcutaneous tumor models are generally prepared by the injection of a cancer cell suspension to evaluate the pharmaceutical effects on tumor tissues. However, dispersed cells show low biological activities because of enzyme-induced cell harvest treatment, thus limiting the formation of tumor tissues. In this study, a biologically active cancer cell monolayer (cell sheet) was used to improve the efficiency of subcutaneous tumor formation. MATERIALS AND METHODS: Mouse lung squamous cancer cells (KLN-205) were transplanted on the subcutis of immunocompetent and immunodeficient mice in the form of a dispersed cell suspension or cell sheet, and the tumor formation abilities were independently investigated with considering immunological effects. RESULTS: Mouse lung squamous cancer cells (KLN-205) scarcely formed malignant tumors on the mouse subcutis following injection of the cell suspension. On the other hand, cell transplantation in the cell sheet form successfully achieved effective tumor development due to only weak immunological reactions at the transplanted area. And thus, the cancer cells maintained their proliferative activity to form tumors. CONCLUSION: Transplantation of the cell sheet is effective to generate subcutaneous tumor-bearing mice, providing a useful alternative to the low tumor formation activities induced with the conventional injection method.

Endometrial regeneration using cell sheet transplantation techniques in rats facilitates successful fertilization and pregnancy.

OBJECTIVE: To regenerate functional endometrium tissue using "cell sheet" techniques as a regenerative medicine approach to address endometrial disorders causing female factor infertility. DESIGN: In vivo experimental study. SETTING: Preclinical surgical and biomedical research laboratories. ANIMAL(S): Green fluorescent protein (GFP) transgenic rats [SD-Tg (CAG-EGFP) rats] and nude rats (F344/NJcl-rnu/rnu). INTERVENTION(S): GFP-positive rat uterine-derived cells as cell sheets were transplanted into resected rat uterine endometrial sites. Transplanted cell sheet areas were then analyzed using macroscopic observations and histological analysis including immunohistochemistry. Subsequently, crossbreeding was performed to establish fertility and confirm pregnancy in the rat-regenerated uterus. MAIN OUTCOME MEASURE(S): Morphologic and biochemical markers of regenerated endometrium and establishment of pregnancy in otherwise sterile animals. RESULT(S): After cell sheet transplantation, regenerated endometrium was confirmed as GFP-positive tissue engraftment both visually and under histological analysis. After crossbreeding, GFP-positive tissue areas and living fetuses were observed in the transplantation group. CONCLUSION(S): Cell sheet transplantation can regenerate endometrial tissue with histological structure and physiological function supporting pregnancy similar to normal endometrial tissue. Translation of this endometrial cell sheet transplantation method to human patients with endometrial disorders could yield a novel therapy for uterine infertility.

Transplantation of cancerous cell sheets effectively generates tumour-bearing model mice.

Tumour-bearing mice were created by transplanting cancerous cell sheets onto the subcutaneous tissue of the dorsal region, using luciferase gene-transfected mammary gland adenocarcinoma cells, 4T1-luc2, to investigate the tumourigenicity of the cell sheet relative to a conventional injection of cell suspension. Contiguous breast cancerous cell sheets were harvested from temperature-responsive culture dishes by reducing the temperature from 37 °C to 20 °C; the sheets were then transplanted onto the dorsal side of the mouse subcutaneous tissue, using a chitin-based supporting membrane. Cell suspensions obtained by trypsin digestion were subcutaneously injected into the dorsal region of mice. The tumour growth of the transplanted cancer cells was evaluated by the tumour volume and by the bioluminescence from luciferase-gene transfected cancer cells, using an in vivo imaging system. The cell sheet method improved the 4 T1-luc2 engraftment efficiency in living mouse tissues at the initial stage by 13-fold compared with that from injecting cell suspensions. On day 14 after the transplantation, the tumour formation at the transplanted area of cell sheet-transplanted mice also accelerated, and the mean tumour volume became 1116 mm3 , which was 10 times larger than that in cell suspension-transplanted mice. The cell sheets engrafted on the recipient tissues efficiently due to the preserved extracellular matrix on their basal sides, such that cancer cells were supplied with sufficient oxygen and nutrients from the host tissues to develop tumour tissues. Therefore, cancerous cell sheet-based transplantation is a promising method for efficiently creating cancer-bearing mice. Copyright © 2013 John Wiley & Sons, Ltd.

Facile cell sheet manipulation and transplantation by using in situ gelation method.

Cell sheets harvested from temperature-responsive cell culture dishes (TRDs) has attracted considerable attention as effective tools for reconstructing the lost functions of tissues and organs in the regenerative medicine field. However, because of their thinness, handling problems sometimes arise when transferring cell sheets from a TRD to a target surface. In this study, we developed a facile cell transfer method referred to as in situ gelation by using both gelatin hydrogel and a support membrane. Gelation and low-temperature processes were simultaneously performed on TRD. Confluent cultured cells were efficiently harvested from TRD in less than 5 min by decreasing the incubation temperature to 20°C. Harvested cells were found to maintain their cell viability, extracellular matrix, and original shape, thus allowing transfer of the cells to another surface with a short incubation time at 37°C. This method is applicable for various cell types regardless of the formation of tight cell-cell junctions. In addition, because of the high flexibility of the gelatin-coated membrane, cells were efficiently transferred to the surface of a mouse subcutis and liver. When compared with conventional cell sheet manipulation methods, the interaction between the cell surface and membrane was reinforced by the uniformly formed gelatin gel layer without using a special device. Therefore, the in situ gelation method is a promising technique for cell sheet-based tissue engineering and regenerative medicine.

Cell shape regulation based on hepatocyte sheet engineering technologies.

The de novo engineering of a uniform hepatocyte sheet in vitro is considered as a novel approach for liver-directed therapeutics. Hepatocytes can be cultured on a temperature-responsive culture dishes coated with poly(N-isopropylacrylamide) (PIPAAm). Following multiple days of culturing, the hepatocytes can be easily harvested as a uniform sheet by decreasing temperature from 37°C to 20°C. By modifying the sheet harvesting protocol, we have noticed that two different forms of the hepatocyte sheets, "extended" and "shrinking," were obtained. This study describes the methods for harvesting the two different forms of sheets, and their cellular structure and hepatocyte-specific functions. To obtain an "extended sheet" form, a cluster of hepatocytes covered with a support membrane was harvested by the temperature reduction. For the "shrinking sheet" form, the hepatocyte sheet was floated after reducing the culture temperature, and the floating process allowed the sheet to shrink spontaneously. Histological analysis revealed that the hepatocytes in the extended sheet form were predominantly flat, whereas the shrinking sheet contained cuboidal shaped hepatocytes. The preservation of hepatocyte-specific ultrastructures was confirmed in both types of sheets. To investigate hepatocyte-specific functionality, the harvested hepatocyte sheets were recultured on Matrigel-coated dishes. Assessment of protein production levels and chemical metabolizing activities showed the similar functionalities for each form. In contrast, the recalculation of these values per sheet versus per square centimeter of sheet surface demonstrated that the function of the shrinking sheet was significantly higher than that of the extended sheets. This study demonstrated that the hepatocyte sheets created on the PIPAAm dish could spontaneously shrink in size, but retain their hepatocyte functionality. This type of hepatocyte sheet could be utilized for the engineering of liver tissue in limited areas that are unable to give adequate transplant space.

Engineering liver tissues under the kidney capsule site provides therapeutic effects to hemophilia B mice.

Recent advances in liver tissue engineering have encouraged further investigation into the evaluation of therapeutic benefits based on animal disease models. In the present study, liver tissues were engineered in coagulation factor IX knockout (FIX-KO) mice, a mouse model of hemophilia B, to determine if the tissue engineering approach would provide therapeutic benefits. Primary hepatocytes were isolated from the liver of wild-type mice and suspended in a mixture of culture medium and extracellular matrix components. The hepatocyte suspension was injected into the space under the bilateral kidney capsules of the FIX-KO mice to engineer liver tissues. The plasma FIX activities (FIX:C) of the untreated FIX-KO mice were undetectable at any time point. In contrast, the liver tissue engineered FIX-KO mice achieved 1.5-2.5% of plasma FIX activities (FIX:C) and this elevated FIX:C level persisted throughout the 90 day experimental period. Significant FIX mRNA expression levels were found in the engineered liver tissues at levels similar to the wild-type livers. The present study demonstrates that liver tissue engineering could provide therapeutic benefits in the treatment of hemophilia B.

Recombinant sendai virus-mediated gene transfer to mouse pancreatic stem cells.

Efficient gene transfer into stem cells is essential for the basic research and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (AdV) vectors, one of the most commonly used types of vectors, can mediate high, albeit transient, levels of expression of the transgene in pancreatic stem/progenitor cells. However, high multiplicity of infection (MOI) with AdV vectors can result in cellular toxicity. Therefore, AdV vectors have been of limited usefulness in clinical applications. In this study, we investigated the in vitro gene transfer efficiency of Sendai virus (SeV) vectors, a paramyxovirus vector that can efficiently introduce foreign genes without toxicity into several cell types, including pancreatic stem cells. The dose-dependent GFP expression of pancreatic stem cells transfected with SeV vectors after 48 h of culture at 37 degrees C was observed. The transfection of pancreatic stem cells with SeV vectors and AdV vectors results in equal expression of the transgene (GFP expression) in the cells after 48 h of culture at 37 degrees C. Although the transfection of pancreatic stem cells with AdV vectors at high MOIs was cytotoxic, transfection with SeV vectors at high MOIs was rarely cytotoxic. In addition, pancreatic stem cells transfected with SeV maintained their differentiation ability. These data suggest that SeV could provide advantages with respect to safety issues in gene-modified regenerative medicine.

Cell transplantation of adipose tissue-derived stem cells in combination with heparin attenuated acute liver failure in mice.

The effect of adipose tissue-derived stem cells (ASCs) in combination with heparin transplantation on acute liver failure mice with carbon tetrachloride (CCl(4)) injection was investigated. CCl(4) is a well-known hepatotoxin and induces hepatic necrosis. Heparin did not affect the viability of ASCs for at least 24 h. The injection of heparin into the caudal tail vein decreased slightly the activities of the alanine aminotransferase (ALT), asparate aminotransferase (AST), and lactate dehydrogenase (LDH) in plasma. In the transplantation of ASCs (1 x 10(6) cells) group, there was a trend toward decreased activities of all markers. However, four out of six mice died of the lung infarction. In the transplantation of ASCs in combination with heparin group, there was also a trend toward decreased activities of all markers. In addition, all mice survived for at least the duration of the study period. In conclusion, the transplantation of ASCs in combination with heparin was thus found to effectively treat acute liver failure.

Effects on coagulation factor production following primary hepatomitogen-induced direct hyperplasia.

AIM: To investigate the molecular mechanisms involved in coagulation factor expression and/or function during direct hyperplasia (DH)-mediated liver regeneration. METHODS: Direct hyperplasia-mediated liver regeneration was induced in female C57BL/6 mice by administering 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a representative hepatomitogen. Mice were weighed and sacrificed at various time points [Day 0 (D0: prior to injection), 3 h, D1, D2, D3, and D10] after TCPOBOP administration to obtain liver and blood samples. Using the RNA samples extracted from the liver, a comprehensive analysis was performed on the hepatic gene expression profiling of coagulation-related factors by real-time RT-PCR (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIIIbeta, plasminogen, antithrombin, protein C, protein S, ADAMTS13, and VWF). The corresponding plasma levels of coagulation factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIII, and VWF) were also analyzed and compared with their mRNA levels. RESULTS: Gavage administration of TCPOBOP (3 mg/kg body weight) resulted in a marked and gradual increase in the weight of the mouse livers relative to the total body weight to 220% by D10 relative to the D0 (control) ratios. At the peak of liver regeneration (D1 and D2), the gene expression levels for most of the coagulation-related factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, XI, XII, XIIIbeta, plasminogen, antithrombin, protein C, ADAMTS13, VWF) were found to be down-regulated in a time-dependent manner, and gradually recovered by D10 to the basal levels. Only mRNA levels of factor X and protein S failed to show any decrease during the regenerative phase. As for the plasma levels, 5 clotting factors (prothrombin, factors VIII, IX, XI, and XII) demonstrated a significant decrease (P<0.05) during the regeneration phase compared with D0. Among these 5 factors, factor IX and factor XI showed the most dramatic decline in their activities by about 50% at D2 compared to the basal levels, and these reductions in plasma activity for both factors were consistent with our RT-PCR findings. In contrast, the plasma activities of the other coagulation factors (fibrinogen, factors V, VII, XIII, and VWF) were not significantly reduced, despite the reduction in the liver mRNA levels. Unlike the other factors, FX showed a temporal increase in its plasma activity, with significant increases (P<0.05) detected at D1. CONCLUSION: Investigating the coagulation cascade protein profiles during liver regeneration by DH may help to better understand the basic biology of the liver under normal and pathological conditions.

Possible role of calcium on oocyte development after intracytoplasmic sperm injection in quail (Coturnix japonica).

Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development.

Fertilization and development of quail oocytes after intracytoplasmic sperm injection.

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.