Adenylate cyclase (AC) activity in mouse oocytes immediately after ovulation was electron histochemically detected by the method of Wagner et al., and compared among the 3 age groups of 30-day-old, 60- to 90-day-old and 180- to 210-day-old animals. AC activity was detected as lead granules, high in electron density on the cytoplasmic membrane. The activity was found in all the oocytes, but the intensity was highest in 30-day-old mice and lowest in 180- to 210-day-old ones with statistically significant differences.

View full abstract

The localization of β_A subunit of inhibin/activin in rat testis was studied by means of immunohistochemistry using antibody raised against synthetic polypeptide. The intense reaction with anti-β_A antibody was shown in the cytoplasm but not in the nucleus and the plasma membrane of spermatocytes. The reaction was also found in the cytoplasm of spermatids, through the immunostaining pattern was different between the two cells; the granule-like one in spermatocytes and diffused one in spermatids, respectively. Furthermore, the plasma membrane became positive in spermatids, suggesting that β_A subunit may be produced in spermatocytes and secreted from spermatids. The expression of β_A subunit in spermatogenic cells is thought to be stage-specific, because the reaction was not found in spermatogonia and spermatozoa. Although both Sertoli and Leydig cells are reported to produce β_A subunit of inhibin/activin, no reaction was observed in these two cells. This suggests that the antibody used in this study may recognize the epitopes different from those detected by previously-used immunological probes.

View full abstract

The present study was carried out to examine effects of storage conditions of bovine ovaries and/or oocytes on the success rate of in vitro fertilization (IVF) and subsequent in vitroculture (IVC) of the zygotes. Ovaries obtained from a slaughter house were stored in Ringer solution at 39C (group 1), 20C (group 2), or 4C (group 3). In another group, immature oocytes were isolated from ovaries and stored in TCM-199 medium at 39C (group 4). The entire collection procedures of the ovaries took approximately 3 h at the slaughter house from the time of slaughter. The ovaries and/or the oocytes were stored under the conditions described above for 5 h before the experiment. In the control group, oocytes were isolated from the ovaries and cultured immediately to induce maturation. Following IVF and IVC, none of the zygotes developed to morula stage in group 1 and 3. In group 2, some zygotes developed to morula or blastocyst stage (11%, 7% respectively), but the rates were significantly lower than in the control group (32%, 15%). Group 4 gave high success rates (38%, 13%) similar to those attained in the control gorup.

View full abstract

Single blastomeres were isolated from 2-cell mouse embryos and analysed for their sex by using partially deleted Y-chromosome as a marker. Sex identification of 83% of the embryos was achieved with conventionally Giemsa-stained chromosome preparations. The other half-embryos were cultured individually or recombined with another half-embryo of the same sex, and were transferred to pseudopregnant recipient females. Six percent of the half and 18% of the recombined embryos developed into live fetuses. The sex of these fetuses was identical with that determined prior to embryo transfer. Sex analysis in this way was accurate and early enough to allow utilization of sex-known embryos for development and sex differentiation studies at the cleavage stage.

View full abstract

Investigations on the pre-parturition, immediately after (during) parturition and post-parturition blood levels of 13, 14-dihydro-15-keto-PGF_2α (PGFM) and sex-steroid hormones of 5 thoroughbred mares were carried out for a period of 12 months (May, 1981-April, 1985). PGFM and hormonal levels were determined with radioimmunoassays. PGFM and cortisol concentrations on pre-parturition day (prePD) 1 and 0 were much higher than on prePD 2, and returned to pre-parturition basal values on following parturition, respectively. Progesterone, estrone and estradiol-17β gradually decreased from prePD 7 to parturition day. Estradiol-17β was consistently low compared with estrone. In view of the above findings, PGF_2α probably influences parturition inpregnant mares.

View full abstract

Peroxidase-conjugated lectins were used to examine histochemical changes in glycoconju-gates on the cell-surface of mouse embryos in the course of blastocyst formation. The binding of PNA, GS-l, DBA, SBA, WGA and FBP was observed in all the blastomeres of embryos at the stages of 8-cell to blastocyst, but LPA binding was not. The UEA-l binding was found in the blastomeres of uncompacted 8-16 cell embryos, in flattend blastomeres of compacted 8-16 cell embryos, and in trophoblast cells of blastocysts, but not in round blastomeres of compacted 8-16 cell embryos nor in inner-cell-mass cells of blastocysts. The binding of GS-ll, BPA, MPA or Con A was found in all the blastomeres of uncompacted and compacted 8-cell embryos, in flattened blastomeres of compacted 16-cell embryos, but not in round blastomeres of compacted 16-cell embryos. In blastocysts, the binding of BPA, MPA or Con A was found in trophoblast cells only, though that of GS-ll was not in trophoblast cells and inner-cell-mass cells. These findings imply that loss of the binding of UEA-l, GS-ll, BPA, MPA or Con A in round blastomeres of compacted embryos must be a participating factor in blastocyst formation.

View full abstract

The sex of embryos was determined by the polymerase chain reaction (PCR) method using the DNA fragment of a sex-related gene or Y-chromosome specific nucleotide sequence. By using the DNA fragment of the human sex determinant (SRY), cells of male humans, rabbits, rats, mice and goats can be distinguished from the female cells of each animal. By PCR using DNA of the mouse sex determinant (Sry), male cells of mice and rats were selected from the cell population. The sex of cattle cells and goat cells could be determined by PCR using BOV97M (cattle Y-specific repeated DNA sequence). The DNA preparation of rabbit or cattle embryos was amplified by the primer of either SRY or BOV97M, and the embryonic sex was successfully determined. The sex of the embryos predicted using the H-Y antibody or sex chromosome was confirmed by PCR analysis for biopsy of the trophoblastic cells. The bovine growth hormone gene (bGH) was used as an internal standard of cattle-specific DNA fragment. High efficiency and accuracy for embryo sexing was attained from biopsied embryos. Three of the 8 (38%) biopsied embryos transferred into the recipient resulted in pregnancy, and the sex of 2 calves born was the same as expected.

View full abstract

Southern blot analysis of the porcine pituitary glycoprotein common α-subunit gene was performed using porcine genomic DNA from 2 different sources. The BamHI fragments of the common α-subunit gene showed a difference in size between the DNA of Landrace-Yorkshire breed and the other sample purchased from Clontech, whereas the EcoRI-fragments were identical. The results indicate that restriction fragment length polymorphism exists in the porcine common α-subunit gene.

View full abstract

We have found previously that there are 2 kinds of 20α-hydroxysteroid dehydrogenase (20α-HSD) (EC 1.1.1.149) molecule (HSD1 and HSD2) in the mature rat ovary. In the present study, each enzyme activity was measured during pseudopregnancy (PSP) in the rat. Ovarian cytosol was collected in early (day 5), mid (day 9) or late (day 15) PSP. HSD1 activity was low in early and mid PSP and increased markedly at the end of PSP, while HSD2 activity was lower than HSD1 activity in early PSP and decreased as PSP advanced. These results suggest that the regulatory mechanism of the enzyme activity varies between HSD1 and HSD2, and that HSD1 mainly contributes to the increase in 20α-dihydroprogesterone secretion at the end of PSP. Interestingly, when ovaries at early and mid-term PSP were used, the total 20α-HSD activity in the cytosol fraction was increased markedly (163-296%) by passage through a DEAF column. This phenomenon was not observed with ovarian cytosol obtained at late PSP. Thus, we conclude that the ovary contains 2 different kinds of 20α-HSD molecule whose activities seem to be regulated separatedly, and that the HSD1 molecule is responsible for termination of the luteal phase.

View full abstract

Primers possessing base sequences complementary to the 3512-3534th base (Y1.1) and 78-100th base (Y1.2) of a specific repeat base sequence (DYZ1) on the long of the human Y chromosome were prepared. Using these primers, 1μg of cellular DNA from humans, cattle, pigs, rats and mice (both sexes) and testis DNA from salmon and herring were amplified by Polymerase Chain Reaction. In the case of human cellular DNA, the appearance of amplified DNA fragments (154 bp) following electrophoresis, and 55C and 65C hybridization, was noted only for male DNA. In the case of bovine, swine, rat and mouse DNA, such amplified DNA fragments were seen in both male and female DNA after 55C and 65C hybridization. The DNA fragments were also detected in salmon and herring DNA after 55C hybridization. These results suggest that the DYZ1 region is present in the DNA from many mammalian speces and some fishes but also on an autosome in various mammals with the exception of humans.

View full abstract

This study was conducted to examine the possibility of nuclear transplantation using the bovine oocytes matured in vitro. Sixteen-cell stage embryos (fresh, frozen or embryos developed from the oocytes matured and fertilized in vitro) were designated to donor cells. The follicular oocytes matured in vitro were used as enucleated recipient oocytes. The enucleated oocytes were fused with a blastomere from donor embryos by electrofusion. Most recipient oocytes (325/417, 78%) fused with donor cells. The reconstituted eggs developed to 2-cell stage when cultured on a layer of cumulus cells (187, 58%). Out of the 325 reconstituted eggs, 35(11%) developed into morulae and 9 (3%) into blastocysts stage respectively. Eighteen morula or blastocyst stage embryos were transferred nonsurgically to 9 recipient cows into 1-3 embryos per recipient. Two recipients were confirmed pregnant. One of recipients produced a live offspring resulting from a fresh donor blastomere. The present study showed that in vitro matured oocytes can be used as recipient cytoplasm.

View full abstract

Ejaculated canine spermatozoa were incubated at a concentration of 0.6-4.0×10⁸/ml, using a modified Krebs-Ringer bicarbonate solution (TYH) at 37C under 5% CO₂ in air. Motility, aggregation, viability, hyperactivated movement and acrosome reaction of spermatozoa were examined at 0, 1, 2, 3, 4, 5 and 6 h of incubation. Motility and viability decreased slightly and aggregation increased at 1h of incubation, then did not significantly change until 6h of incubation (about 3.4, 65% and 75%, respectively). The percentage of spermatozoa showed hyperactivated movement and acrosome reaction increased remarkably at 3 h (53.9%) and 4h (68.0%) of incubation, respectively. The 4 h-incubated spermatozoa were inseminated at a concentration of 0.5 or 1.0×10⁶/ml to the oocytes incubated in TYH (bovine serum albumin (BSA) -) with 10% fetal bovine serum for 48 h or 72 h. The spermatozoa penetrated into the zona pellucida at 1h after insemination. At 2 h after insemination, the spermatozoa penetrated into the cytoplasm (34.0%), then the percentage increased significantly to 63.4% at 4h after insemination. These results suggest that ejaculated canine spermatozoa are capacitated by 4h incubation in TYH.

View full abstract

Feasibility of utilizing follicular oocytes derived from gonadotropin treated prepubertal gilts and matured in vitro in nuclear transplantation of porcine embryos as the recipient cytoplasts was examined. After culture of 806 oocytes collected from gilts which had received an injection of 1500 i.u. PMSG followed by 750 i.u. hCG 72 h later, 488 (60.5%) matured normally, whilst fewer oocytes matured (144/308, 46.8%, P<0.05) when gilts were treated with 2000 i.u. PMSG. The proportion of oocytes activated after electric stimulation by double pulses of 150 V/mm D.C. for 30 or 60, usec were 62.9% (22/35) and 73.2% (30/41), respectively. Of 433 matured oocytes 342 (79.0%) were successfully manipulated to remove about 1/3 of cytoplasm adjacent to the polar body, with 68 of 77 (88.3%) being confirmed after acridine orange staining to be successfully enucleated. Attempts of electrofusion of the enucleated oocytes with 6 to 8 cell stage blastomeres resulted in 82.5% (66/80) successful fusion. Sixteen (45.7%) of the 35 nuclear transplanted embryos cleaved to 2 to 4 cell stage in culture with modified Kreb's Ringer Bicarbonate solution supplemented with 10% sheep serum.

View full abstract

Spontaneous arrest of cleavage takes place at the 2-cell stage (the 2-cell block) in vitro, in some mouse strains (the "blocking strains"), whereas in others, no such arrest has been recorded (the "non-blocking strains"). We found that activin A releases the 2-cell block, and stimulates the early embryogenesis in mice of the blocking strains (Lu et al. 1990). In this paper, presence of activin/inhibin subunits in zygotes, 2-cell embryos, and oviductal epithelial cells was demonstrated immunohistoche-mically, using the blocking (CD-1 and DBA/2NJcl) and the non-blocking (C57BL/6NCrj) strains of mice. Strong immunoreactivity for the β_A-, and β_B-subunits of activins/inhibins was detected, in the cytoplasm of 1-cell and 2-cell embryos of the 3 strains. Staining intensities with the β_A-subunit was higher during the 2-cell stage than during the 1-cell stage in the blocking strains. In the non-blocking strains, no such increase was observed. In contrast with the case of β_A-subunit, immunoreactivity of the embryos to the β_B-subunit decreased in the blocking strains, while in the non-blocking strain, it stayed at the same level between the two stages. Possible role played by activin AB in the early embryogenesis, was discussed. The immunohistochemically detected activin/inhibin subunits in the zona, probably represented those transferred passively from the oviductal fluid.

View full abstract

Investigation of regulation of estradiol production by porcine granulosa cells is hindered by lack of a culture system that consistently produces estradiol for extended periods. Granulosa cells that are characterized by their strong attachment to each other (TB-cells) were compared to cells that lack such attachments (WA-cells) for their responses to FSH, LH, and EGF in androstenedione supplemented medium. TB-cells were separated from WA-cells by filtration and were predominantly aggregates that had a smooth contour on one side of the aggregate. After enzymatic disruption, TB-cells had greater estradiol production than WA-cells when cultured initially, and they maintained this advantage through day 8 when stimulated with FSH or LH. Progesterone production was greater for TB- than WA-cells during initial culture, but by day 8 FSH and LH stimulated progesterone production was similar for these two subpopulations. EGF increased DNA concentrations and decreased production of estradiol and progesterone by similar amounts for each cell type. When TB-and WA-cells were cultured together, estradiol, but not progesterone, production was reduced disproportionately. This indicates that WA-cells or some component of these pools of cells interact with TB-cells to reduce estradiol production. We conclude that TB-cells are primarily from the mural layers within the follicle and are more differentiated at time of collection. The means through which TB-cells sustain FSH stimulated estradiol production in culture and why WA-cells fail to achieve this level of production are unknown.

View full abstract