The in vitro development of mammalian ovarian follicles still represents a challenge. Results achieved in the past several years have clarified some important aspects concerning the regulation of follicle and oocyte development, but the complexity of the process leading in vivo to the production of a fertilizable egg makes quite difficult to obtain large numbers of fully-grown mature germ cells starting from in vitro cultured follicles. The main problem is that culture systems currently utilized do not assure satisfactorily a co-ordinate development of both the somatic and germinal components of the ovarian follicle. An ideal culture media might be specially designed by the addition of specific components in correct ratio, in the attempt to reflect the dynamic changes which characterize follicle development. This is important, especially in the light of the fact that the period of culture could vary considerably, depending on the timing of follicle growth in vivo in the various species, being also influenced by the age of donors, methods and aims of culture. Despite this, in vitro technology represents not only an important tool to understand regulative processes underlying follicle development, but also a future option for the preservation of fertility. The present paper reviews current knowledge on advances and problems relevant to the culture of primordial and preantral mammalian follicles.

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Inactivation of the X-linked gene in the female embryo is one of the major events during mammalian early embryogenesis. Before this inactivation, enzyme activity encoded by the X-linked gene is different between male and female embryos. In the present study, we demonstrated a possibility that there may be a different response to specific culture conditions in vitro between male and female early embryo. Glucose-6-phosphate dehydrogenase (G6PD) is one of the X-linked enzymes and its activity is semi-quantitated by brilliant cresyl blue (BCB). In experiment 1, the relationship between semi-quantitated G6PD activity and the sex ratio of the embryos was examined. In experiment 2, the relationship between semi-quantitated G6PD activity and the developmental competence of embryos was examined. No relationship was found between G6PD activity and sex in 8-cell, 16-cell and blastocyst stage embryos. However, G6PD activity was high in female embryos and low in male embryos at the morula stage. When morula stage embryos were categorized by BCB and cultured under high oxygen tension (5% CO₂, 95% air), the developmental competence of embryos having high G6PD activity was higher than those having low enzyme activity. However, when the categorized morula stage embryos were cultured under low oxygen tension (5% CO₂, 5% O₂, 90% N₂), there was no difference in the developmental rates among the categories. These results indicate that G6PD activity is high in female morula stage embryos compared with that in male embryos, and that the activity of this enzyme strongly affects the developmental competence of morula stage embryo when they are cultured under high oxygen tension.

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This experiment was carried out to collect fetal blood and fluid via catheters fitted to elucidate interaction between the mother and her fetus during a comparatively long term. Fifteen cows in late gestation were used for this study. Under regional anesthesia, an incision was made in the left paralumbar fossa in the standing position. A catheter for fetal blood was inserted into a vein of the fetus and catheters for fetal fluid were inserted into amniotic and allantoic sacs respectively. Four types of catheters were used for collecting fetal blood. The best of these catheters for fetal vein was the angiographic catheter covered with spring tube. Fetal blood samples were able to be collected for more than seven days constantly from four fetuses until parturition. Fetal fluid could be collected daily from three fetuses until parturition perfectly. Parturition occurred at the end of a normal gestation period. The concentration of fetal and maternal cortisol decreased to the basal level within 24 hours (P<0.05) after operation. The changes in fetal and maternal cortisol during periparturition were the same as previously reported. These results suggested that this method would be beneficial for fetal cannulation and the sample collected by this method was useful for endocrine study during the perinatal period in cattle.

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Rat cauda epididymal spermatozoa treated with α-chlorohydrin at concentrations of 0, 0.01, 0.1, and 1.0 under conditions that support in vitro fertilization were used to evaluate the effects of acrosomal status and sperm motility in predicting their fertilizing capacity. Acrosomal status was assessed by fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) lectin assay in combination with supravital stain using calcein acetoxy methyl ester (CAM) and ethidium homodimer-1 (EthD-1) at 1, 3, and 5 h of incubation. Sperm motility was also examined with a computer assisted sperm analysis (CASA) system at the same time points as acrosomal status. The movement of spermatozoa treated with α-chlorohydrin at 0.01 mM was similar to the control spermatozoa over 5 h of incubation. The 0.1 mM treated spermatozoa showed progressive movement at 1 h of incubation, and low vigorous movement was seen after 3 h of incubation. The 1.0 mM treated spermatozoa showed low progression and low vigorous movement over 5 h of incubation. The FITC-ConA patterns of rat spermatozoa undergoing acrosome reaction were marked by the appearance of bright green fluorescence from the acrosomal region on their head, and were clearly distinguished from the degenerative acrosome loss in dead sperm. A time-related increase in the percentage of live acrosome lost sperm was observed in the control and 0.01 mM α-chlorohydrin treated groups, but a low percentage and no time-related increase in live acrosome lost sperm without degenerative acrosome loss in dead sperm were observed in the 0.1 and 1.0 mM treated groups. On the basis of these results, rat spermatozoa labeled with FITC-ConA combined with CAM and EthD-1 can have their acrosomal status clearly distinguished. The α-chlorohydrin treated spermatozoa were suppressed not only vigorous movement but also acrosome reaction, and the results suggested that the present staining procedure for acrosomal status can be a useful indicator for predicting spermatozoal fertilizing capacity.

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As a prerequisite for the production of cloned goats by nuclear transfer from the cultured somatic cells, we established cloned fibroblast cell lines, CPF-1 and other related CPF subclones from the placenta of the Shiba goat. CPF-1 cells ceased to proliferate at and about the 51st population doubling level (PDL) when the morphological signs of senescence, such as an unusual increase in cell volume, became obvious. The mean surface area of a cell at 51PDL was about 3 times greater than that of an early-passage cell (3PDL) (p<0.001). When the CPF-1 cells at 14PDL were subjected to chromosomal analysis, 49.3% of the cells turned out to be of normal diploid chromosomal constitution (2n=60). All the chromosomes were of acrocentric types. Part of the reason for the rather high incidence of aneuploidy might be due to the well-known technical difficulty in preparing good chromosomal spreads in the caprine cells. We analyzed the rates of telomere length shortening in the CPF-1 cells, according to the number of passages and culture periods. The telomere length in the late-passage cells became approximately 1/2.5 of that of the early-passage cells (p<0.05). The estimated mean rate of telomere shortening in the CPF-1 cells was approximately 260 bases per PDL and faster than the values (50-200 bases/PDL) so far reported in the literature for other types of cells. Furthermore, in the CPF-1 cells the replicative capacity was exhausted when the mean telomere length reached approximately 7.5 kb which is significantly longer than the values generally obtained in other cultured cell lines. The reasons for the observations have not yet been clarified.

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We previously reported that thyroxine (T4) treatment significantly improved follicular development and increased the number of eggs ovulated in immature hypothyroid infertile rdw rats in the presence of gonadotrophins. The present study was conducted to determine whether (1) infertile rdw rats would respond to repeated stimulation with gonadotrophins, and whether (2) T4 treatment would increase the total number of ovulated eggs in an individual rat induced to ovulate up to three consecutive times at intervals of about 30 days. eCG was repeatedly given from the immature stage on postnatal day 28 and then at day of metestrus in the mature stage at intervals of about 30 days, followed by hCG 54 h later in rdw and normal rats, both of which were also given T4 (T4-rdw and T4-Normal), and in normal rats without T4 treatment (Normal). The results indicate that there were no significant differences in the weights of ovaries at any age tested among T4-rdw, T4-Normal and Normal. The number of eggs ovulated in the first treatment was significantly higher than in subsequent treatments in the same group (P<0.05). No significant differences were observed in the number of eggs ovulated in each corresponding treatment among the three groups, although more eggs (85.0 ± 4.6) were collected after the first treatment in T4-rdw than in the other groups, while fewer eggs (4.0 ± 2.7) were collected in the third treatment in T4-Normal than in the other groups. There were no significant differences in the percentages of degenerative or fertilized eggs in the same treatment among groups or among subsequent treatments in the same groups. However, the total number of eggs collected in the first three treatments in T4-rdw was significantly more than in Normal (P<0.05). T4 treatment also increased the total number of eggs ovulated in first three treatments in normal rats compared to those in Normal although no significant difference was observed between the two groups. In conclusion, the results of the present study indicate that infertile rdw rats responded well to repeated stimulation with gonadotrophins, and that T4 treatment increased the total number of ovulated eggs in an individual rat treated three times with gonadotrophins.

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The present study was conducted to investigate fertility of estrus-induced ewes during the non-breeding season, artificially inseminated with frozen semen imported from New Zealand. A total of 122 Suffolk and Suffolk-crossed ewes in three different sheep farms (T, S, M) were treated with an intravaginal progesterone release device (CIDR-G) for 12 days, and an intramuscular injection of 500 IU equine chorionic gonadotropin one day before CIDR removal. Effects of intrauterine insemination doses (0.2 and 0.4 ml per head) and two rams on fertility were compared at Farm T using 75 ewes. At the other two farms (S and M), 0.1 to 0.2 ml doses were inseminated into the uteri on the fixed-time basis. The lambing rates of ewes inseminated with 0.2 and 0.4 ml doses were 68.4 and 59.5%, and the lambing rates of ewes inseminated with the two rams were 61.3 and 65.9%. No significant differences were found between any of the factors. The lambing rates on Farms S and M were 54.2% and 47.8%, respectively. Overall, there was no significant difference in the lambing rates (58.3% and 59.5%) and prolificacy (2.03 and 1.61) between the two rams. These results indicate that the fertility of ewes inseminated during the non-breeding season with frozen-thawed semen imported from New Zealand is acceptable and that new blood lines of Suffolk sires and dams can be produced on Japanese sheep farms.

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Levels of pituitary thyrotropin messenger RNA and circulating hormones during molting induced by feed and water deprivation were determined by reverse-transcription polymerase chain reaction and radioimmunoassays. Plasma levels of luteinizing hormone and estradiol decreased from the next day (Day 1), and progesterone from Day 3, of deprivation treatment, and were associated with the cessation of egg laying on Day 4. The plasma thyroxine level of experimental hens increased on Day 5 and remained high until Day 17, whereas the plasma concentration of triiodothyronine was lower than the control level throughout the experimental period. The messenger RNA level of the pituitary thyrotropin β subunit of experimental hens decreased markedly at first, but then increased showing a peak on Day 3 followed by a gradual decrease until Day 17. The serum prolactin level decreased on Day 3 and remained low until Day 17. These results suggest that (1) a decline in hormones associated with egg laying is followed by activation of the pituitary-thyroid axis, (2) thyrotropin triggers the increase in thyroxine but is not necessarily responsible for the maintenance of the high thyroxine level, and (3) prolactin might be involved in molting induced by feed and water deprivation.

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The present study was conducted to determine the effect of a low dose of human chorionic gonadotropin (hCG) on the induction of fertile estrus in Shiba goats pretreated intravaginally with progesterone during the early postpartum period. Nursing Shiba goats (n=13) around 3 weeks after the parturition were pretreated with a controlled internal drug release dispenser (CIDR) containing 0.3 g of progesterone for 7 day, and 60 μg of cloprostenol 24 hr before CIDR removal. Goats in Group I (n=6) received subcutaneous injections of a low dose of hCG (1 IU/kg) after the removal of CIDR at 12-h intervals until the occurrence of estrus. Goats in Group II (n=7, control) received subcutaneous injections of saline instead of hCG. The rate of estrus induction in Group I (83.3%) tended to be higher than that in Group II (42.9%), although the difference was not significant (P=0.14). Time from the removal of CIDR to the beginning of estrus in Group I was significantly shorter than that in Group II (P<0.05). A prominent peak of estradiol-17 β concentration in plasma was observed with a maximum value at 30 h after the removal of CIDR in Group I, while two relatively small rises were detected with a maximum value at 48 h in Group II. The pregnancy rate in Group I (83.3%) was significantly higher (P<0.05) than in Group II (28.6%). These results suggest that the injections of low dose hCG after the treatment with CIDR promote follicular maturation and fertile estrus induction in the goats during the early postpartum nursing period.

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The present study was performed to establish a rat cell line from blastocysts and to examine the developmental ability of nuclear transfer embryos reconstituted from the established cells and cumulus cells with enucleated mature oocytes. No colonies of embryo-derived cells were observed when morulae were cultured. In contrast, 89% and 40% of the attached embryos formed colonies when blastocysts and hatched blastocysts were cultured, respectively. When the colonies were subcultured in the absence of feeder cells, a cell line with round cell morphology was obtained. This cell morphology was stable up to at least passage 23. The percentage of these embryo-derived cells at G0/G1 was increased when cells were cultured in medium with 0.5% FCS for 48 and 72 h. Ninety-three percent of fresh, non-cultured cumulus cells were at G0/G1. The percentages of reconstructed embryos survival (44-48%), and developed to the 2-cell stage (35%), were significantly higher when the nuclei of cumulus cells were used than when the nuclei of blastocyst-derived cells were used (29% and 3%). The results of the present study indicate that a rat cell line can be established from blastocysts and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cell nuclei into enucleated oocytes have poorer development potential than those obtained using cumulus cell nuclei. No reconstructed embryos developed beyond the 2-cell stage in vitro.

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To elucidate abnormality of acrosome formation in germ cells of the B10.BR-Ydel (Ydel) mutant mouse, male mouse lines were produced by crossing the Ydel males with females from a transgenic mouse line, Acr-EGFP, carrying a transgene encoding a fusion protein of mouse pre-proacrosin and jellyfish enhanced green fluorescence protein (EGFP) under the control of the promoter of the mouse acrosin gene. Morphological analysis indicated that green fluorescence due to EGFP was non-uniformly localized in the acrosomal cap regions of cauda epididymal sperm in Ydel/Acr-EGFP mouse sperm. When spermatogenic cells at twelve stages of the seminiferous epithelial cycle were examined, no significant difference of the acrosome formation was observed between the wild-type and Ydel/Acr-EGFP mice, except that small, round vesicles without green fluorescence were frequently found in the acrosome of elongating spermatids at stage IX and later stages only in the mutant mouse. Some fluorescence-less vesicles were eliminated from the acrosome of elongating spermatids during late spermiogenesis. However, most of the fluorescence-less vesicles were retained in the acrosome. These results suggest that the occurrence of the small, round vesicles within the acrosome may correlate with the production of abnormal sperm with flat heads in the Ydel mutant mouse.

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Development of convenient techniques to diagnose pregnancy status is important for efficient animal production. Considering that pregnancy is an exciting biological event in the maternal body, the metabolism of amino acids during this period might be different from other periods in life. Pregnant animals would then be distinguished from non-pregnant ones, if relative differences in plasma amino acid concentrations could be identified. In the present investigation, a linear discriminant analysis was performed to characterize specific and possibly unique aspects of plasma amino acid concentrations in pregnant animals. Pregnant group (PG, n=5) and non-pregnant group (NPG, n=5) of Shiba goats were subjected to this study at the Experimental Station for Bio-Animal Science, The University of Tokyo. On approximately 50^th day after mating (day 0 = day of estrus), the animals' pregnancy status was diagnosed by using an ultrasonography. In addition to body weight (BW) measurements, blood samples, from which plasma amino acid concentrations were determined, were obtained every week via the jugular vein from two groups of animals. Because variations of plasma amino acid concentrations (Ile, Leu, Glu and Tau) and BW in PG among pregnant days after mating were high (P<0.01), they were adjusted to the average days passed after mating (adjusted to 70^th, 77^th, 84^th, 91^st, 98^th, 105^th and 112^th days). An effect of pregnant status was significant only for Glu (P<0.01). Correlations between BW and plasma amino acid concentrations of PG and NPG were low, suggesting that concentrations of these plasma amino acids were not affected directly by BW gain. Groups of PG and NPG were discriminated significantly using a linear discriminant model with plasma amino acid concentrations in the middle period of pregnancy (P<0.05). These results suggest that the pregnancy status can be evaluated easily and at low cost by using mathematical models and suitable weighted variables of biological factors such as plasma amino acid concentrations.

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A cloned full-length cDNA encoding bovine luteinizing hormone (LH) receptor was expressed in COS-7 cells and the binding activity of the protein to human chorionic gonadotropin (hCG) was examined. Molecular cloning of the bovine LH receptor cDNA was carried out by reverse transcription-polymerase chain reaction using total RNA extracted from a bovine corpus luteum. Three overlapping partial fragments of the receptor cDNA were amplified, sequenced, and engineered to generate the entire cDNA coding sequence and it was subcloned into a mammalian expression vector. Sequence analysis showed a 2,103-nucleotide open reading frame encoding the bovine LH receptor, predicting a putative 26-amino acid signal peptide, and a 675-amino acid mature receptor protein that is 94, 88, 86 and 85% identical to the porcine, human, rat and mouse homologs, respectively. Scatchard analysis of the intact COS-7 cells transfected with the bovine LH receptor cDNA showed high affinity and low capacity of [¹²⁵I]-hCG binding sites on the cells. These results suggest that the protein expressed from the cloned bovine LH receptor cDNA is located on the cell surface with its binding activity to LH/hCG.

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