Clinically relevant animal models of human cancer are important for studies of cancer biology, invasion and metastasis, and for investigating new forms of prognostic diagnosis and therapy. An ovarian tumor line (RMG-1: human clear cell carcinoma of the ovary) previously grown subcutaneously was implanted orthotopically as intact tissue into the ovarian capsule of 22 nude mice. The tumors showed progressive growth at the orthotopic site in all animals. Tumor-associated serum galactosyltransferase (GAT) tended to be positive in all nude -mice. The tumors invaded or metastasized to the contralateral ovary, retroperitoneum, mesentery and peritoneum, and omentum, and metastasized to the subcutaneous tissue, lymph nodes and distant organs including the liver, kidney, pancreas, and diaphragm. In striking contrast, subcutaneous transplantation of this tumor resulted in growth in only 2 of 5 animals with local lymph node and kidney involvement but no retroperitoneal or peritoneal involvement. These findings suggest that orthotopic implantation provides a suitable micro-environment in which ovarian cancer can express its intrinsic clinically-relevant properties. This approach is relevant to the clinical features of ovarian cancer and is thought to be a useful model for studies of therapy for this cancer.

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A personal computer-controlled micromanipulation system was developed for automatic injection of spermatozoa into the perivitelline space of mouse ova. A pair of three-dimensional hydraulic micromanipulators driven by pulse motors was used for this automatic system. The pulse signals that regulate the motors are initiated by the computer program, and these signals cause the micromanipulator to move the microtool precisely. The computer program was designed to perform the most effective movements of the sperm injection needle used during manual micromanipulation. Prior to the manipulation, the computer locates the tip of the injection needle and the end of the egg-holding pipette in the microscope field using image processing. The trajectory of the injection needle is determined according to these initial positions. Using this robotic system, subzonal insemination with a single mouse spermatozoon was attempted in a total of 143 ova. The sperm insertion was successfully completed in all cases without damaging any of the ova. Spermatozoa treated with ionophore A23187 and those without the treatment were used. The fertilization rate (68.8%) of the ova inseminated with treated sperm was significantly higher than that (37.5%) obtained with the nontreated sperm (P less than 0.05). These findings suggest the feasibility and potential for further applications of a robotic microinsemination system and, in addition, that a higher fertility rate in the subzonal insemination of mouse ova can be achieved with the ionophore treatment of spermatozoa.

The effect of calcium concentration in culture medium on the fertilization of subzonally microinseminated mouse oocytes was examined. Oocytes were injected with a single spermatozoon so that the sperm head was forced to adhere onto the ooplasmic membrane with a micromanipulation technique. For the inseminations, epididymal spermatozoa preincubated in culture medium and those treated with ionophore A23187 were used. Inseminated oocytes were cultured using media with three different calcium concentrations of 1.71, 3.42, and 5.13 mM; 40.0%, 71.6%, and 47.9% of oocytes microinjected with preincubated sperm were fertilized after incubation with those media, respectively. When the oocytes inseminated with ionophore-treated sperm were incubated in media containing 1.71 and 3.42 mM calcium, their fertilization rates were 58.2% and 87.5%. Thus fertility of subzonally microinseminated oocytes was obviously enhanced when cultured in medium with 3.42 mM of calcium, irrespective of being inseminated with preincubated sperm (P < 0.01) or with ionophore-treated sperm (P < 0.005). Some of the microinseminations with preincubated sperm were performed without sperm adhered to the oolemma. In these cases, the incidence of fertilization was not improved by incubating the inseminated oocytes in medium containing 3.42 mM calcium (32.6%) as compared to those incubated in medium with 1.71 mM calcium (28.3%). These results suggest that the concentration of extracelluar calcium exerts an important effect on the progress of fertilization events subsequent to sperm adherence onto the ooplasmic membrane. Almost 80% of the zygotes fertilized via incubation in medium with 3.42 mM of calcium developed into blastocysts after culturing in vitro.

Abstract.Primers possessing base sequences complementary to the 3512-3534th base (Y1