We investigated the relationship between the morphology of oocytes collected from small antral follicles and their developmental capacity. Immature oocytes were classified into seven groups and cultured in vitro for maturation (IVM), fertilization (IVF) and development to blastocysts (IVC). After IVF, sperm penetration and normal fertilization rates were higher in the oocytes whose cytoplasm appeared brown. The rate of polyspermy was highest in the oocytes whose cytoplasm was black. After IVC, the rates of cleavage and of development to the blastocyst stage were also higher in the brown oocytes. Although the oocytes with dark clusters in a pale cytoplasm showed lower cleavage rates, cleaved zygotes had high developmental rates the same as the oocytes with a brown cytoplasm. Transmission electron microscopy showed that the oocytes with a pale or black cytoplasm had organelles arranged differently from other oocytes before IVM. Most of the oocytes with a brown and homogeneous cytoplasm or small diameter had the characteristics of immature cytoplasm (large clusters of cortical granules) even after IVM. On the other hand, the brown oocytes with a dark zone at the periphery or with dark clusters showed the same arrangement of organelles as in vivo matured oocytes. The oocytes with a pale or black cytoplasm appeared to be degenerating and/or ageing. In conclusion, a dark ooplasm indicates an accumulation of lipids and good developmental potential, while a light-coloured ooplasm indicates a low density of organelles and poor developmental potential. A black ooplasm indicates ageing and low developmental potential.

SummaryWe examined the relationship among morphological appearance (six groups) of bovine oocytes, ATP content and maturational/developmental ability. Oocytes with a brown ooplasm (with or without a dark region) had intermediate levels of ATP at the germinal vesicle (GV) stage and showed higher rates of first polar body (PB) extrusion than the other groups. Oocytes with a low level of ATP (oocytes with a pale ooplasm without dark clusters) and oocytes with a high level of ATP (oocytes with a black ooplasm) showed lower rates of PB extrusion. During in vitro maturation, ATP levels in oocytes decreased at around GV breakdown and increased toward metaphase II (MII). MII oocytes having a brown ooplasm with a dark region, which had good developmental capacity, had a relatively high level of ATP. MII oocytes with a brown or pale ooplasm without dark clusters, which had poor developmental capacity, had low ATP levels. MII oocytes with a black ooplasm, which had poor developmental capacity, had an unusually high level of ATP. These results suggest that the morphological appearance of bovine oocytes is closely related to their ATP levels and that cytoplasmic morphology will give an advantage for the selection of oocytes with a high maturational and developmental ability.

ABSTRACT. A 10-year-old Miniature Schnauzer with bilateral cryptorchidism and male external genitalia was referred with a history of abdominal enlargement. Upon exploratory laparotomy, two tumors and a connecting structure similar to fluid-filled uterus were recognized. After cytological and bacterial examinations of the fluid and histological examination, this dog was diagnosed with bilateral Sertoli cell tumor with hydrometra. The karyotype of this dog was 78, XY and the sry gene was detected positive by PCR. We diagnosed this dog as a case of persistent Müllerian duct syndrome (PMDS), which is male pseudohermaphroditism. This is the first report regarding the incidence of PMDS in Miniature Schnauzers in Japan, and it suggests the involvement of a gene carrier. A 10-year-old male Miniature Schnauzer was referred to Tottori University Animal Teaching Hospital with a history of abdominal enlargement. Physical examination revealed bilateral cryptorchidism with male external genitalia and non-pruritic dorsal lumbar alopecia. On palpation, a large mass was felt in the abdomen. The general condition seemed to be normal, and visible feminization was not recognized.Full CBC and serum biochemistry tests were performed. Slight elevations were observed in the levels of alanine amino transferase (182 U/l), alkaline phosphatase (1,363 U/ l), and total cholesterol (259 mg/dl). The concentrations of serum estradiol and testosterone were 103 pg/ml (reference range of normal male: < 15 pg/ml) and 246 ng/dl (reference range of normal male: 100-300 ng/dl), respectively. Abdominal radiographs detected large masses compressing the normal intestinal canal. Abdominal ultrasonography revealed two masses with mixed echogenecity. Additionally, hypoechogenecity tubular cysts cranial to the bladder were observed, which appeared to be fluid-filled like pyometra or hydrometra.Although the tubular cysts were unidentifiable, the abovementioned findings suggested the possibility of testicular tumors; thus, exploratory laparotomy was performed to explore the possibility of resecting the masses and cysts. A large and a small mass, which were speculated to represent the testicles, were located at the center of the abdomen and attached to the structure like the uterus which was inflated with fluid material. This uterus-like structure had two horns that caudally joined into the body of the uterus, the vagina terminated cranially into the prostate gland. Due to the growing mass, obvious structures like the fallopian duct or the testicular canal were not recognized. After the vagina was ligated, both the tumor and the uterus were extracted (Fig 1). Cytodiagnosis revealed intact erythrocyte and keratinizing epitheliums without pathogenic microorganisms. To identify the presence of aerobic bacteria, culture was performed and any multiplication of bacteria were not observed. The walls of both the uterine horns were thin. Histological examination confirmed bilateral Sertoli cell tumor and normal uterus; no ovarian tissue was found. The postoperative gen...

Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.

The objective of this research was to clarify the aging-related changes in in vitro-matured bovine oocytes. Firstly, we examined the fertilization and embryonic development of bovine oocytes after 22 and 30–34 h of in vitro maturation (IVM). The oocytes after 30–34 h of IVM (penetrated by sperm at around 40 h after starting IVM) showed a lower developmental rate to blastocysts (P<0.01), although normal fertilization rates were similar regardless of IVM duration. In the next experiment, reactive oxygen species (ROS), mitochondrial activity and ATP content in oocytes after 20, 30 and 40 h of IVM were examined. The lowest level of ROS was found in the group subjected to 30 h of IVM. The mitochondrial activity and ATP content in the group subjected to 40 h of IVM were higher than in the group subjected to 20 h of IVM (P<0.01), and those in the group subjected to 30 h of IVM showed intermediate values. Thereafter, the mitochondrial activities at 3 days after in vitro fertilization in embryos derived from the oocytes subjected to 22 and 34 h of IVM were evaluated. In the group subjected to 34 h of IVM, high-polarized mitochondria were frequently observed at the periphery of blastomeres. The present results suggest that high mitochondrial activity observed in oocytes after prolonged IVM culture and localization of high-polarized mitochondria at the periphery of blastomeres during early embryonic development may be associated with the low developmental competence in aged bovine oocytes.