The male-enhanced antigen (Mea) gene, the product of which was reactive with the H-Y antibody, was isolated from a bovine testicular cDNA library, and its expression in bovine early embryos was analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Analyses using diluted mRNA from blastocyst embryos suggested that it could detect the Mea transcripts from just a few blastocyst cells. Expression of the Mea gene was not detected at the 2-cell stage, but it was detected in 8-cell embryos to hatched blastocysts. When the sex of one half of a bisected blastocyst was determined by the usual PCR using bovine Y-chromosome specific primers and the other half by RT-PCR, all blastocysts having Y-chromosome sequences were found to express the Mea gene. Most embryos without Y specific sequences did not express the Mea gene, but a small fraction of them unexpectedly did. These findings indicate that the Mea gene could be useful for sexing early embryos by RT-PCR and especially to identify female embryos.
Supplementing Whitten's medium with both activin A and superoxide dismutase (SOD), either of which singly has a beneficial effect, can enhance synergistically the development of 1-cell mouse embryos to the blastocyst stage in vitro. To release the 2-cell block and support embryonic development up to blastocysts, the lowest concentrations were 1.0 ng/ml and 2.5 ng/ml of activin A in CD-1 and DBA/2NJcl mice, respectively, and 300IU/ml of SOD in both strains. The percent-age of cultured embryos developing from the pronuclear stage to the 3-or 4-cell stage was signifi-cantly higher in media containing both supplements at the concentrations mentioned above than in media supplemented with either activin A or SOD. Pronuclear embryos developed to either moru-lae or blastocysts in the activin A plus SOD group at significantly higher proportions (p<0.01) than those in groups treated with activin A or SOD separately. The synergism between the 2 supple-ments for releasing 2-cell block indicates that the modes of action are different for activin A and SOD.
We have already reported that the block to development in cultured 1-cell rat embryos was overcome using HECM-1, a protein-free chemically defined medium without glucose nor phosphate. In addition, we showed the beneficial effect of low oxygen tension in rat embryos. In this study, in order to clarify the reason why in vitro developmental block of rat embryos is over-come in HECM-1, we examined the effects of phosphate, glucose and L-cysteine on the in Vitro development of rat embryos. Furthermore, we examined their effects on the production of H2O2 in embryos by the fluorimetric method. Addition of KH2PO4 even at only 1 μM completely inhibited the development of rat (Wistar) embryos either from the 1-cell stage or the 2-cell stage. Addition of glucose at the concentration of 1000 μg/ml significantly decreased the blastulation rate. The pro-duction of H2O2 in embryos cultured under 5% O2 was significantly (P<0.01) lower than that of embryos cultured under 20% O2 in HECM-1. H2O2 production in embryos cultured in mKRB was significantly (P<0.01) higher than that in embryos cultured in HECM-1, irrespective of the oxygen tension. Addition of neither phosphate nor glucose to HECM-1 affected the H2O2 production at any concentration examined. L-cysteine significantly decreased the H2O2 production in embryos in mKRB, but developmental block could not be overcome by addition of L-cysteine to mKRB. These results revealed that glucose and phosphate independently inhibited the development of rat em-bryos in vitro, and suggest that the mechanism of in vitro developmental block of rat embryos could not be explained simply in terms of oxygen toxicity.
Experiments were conducted to find optimal conditions for obtaining high survival of expanded mouse blastocysts after vitrification in glycerol-based solutions. The solutions were GFS20, GFS30, GFS40 and GFS50, which contained 20%, 30%, 40% and 50% glycerol, respectively, diluted in PB1 medium containing 30% Ficoll + 0.5 M sucrose. The toxicity tests of the solutions and glycerol showed that longer exposure to a higher concentration of glycerol is more injurious to the embryos. For vitrification, expanded blastocysts were exposed to the GFS solution at 20 or 25 C for various periods; they were then vitrified in liquid nitrogen, and were warmed rapidly. When the embryos were directly exposed to GFS40 for 2 min at 25 C, 68% of them re-expanded during 48 h of post-warming culture. The re-expansion rates decreased, when exposure time was shortened or extended, when the exposure temperature was lowered (20 C), or when embryos were vitrified in GFS20, GFS30, and GFS50. When embryos had been pretreated in a dilute (10-20%) ethylene glycol solution for 5 min, followed by short exposure (0.5 min) to GFS40 at 25 C, the post-vitrifica-tion survival rate increased to 82-87%; furthermore, the rate reached 92% when GFS40 was re-placed by GFS50. Expanded blastocysts cryopreserved in GFS50 had the ability to develop into live young. The findings showed that glycerol-based solutions are as effective as ethylene glycol-based solutions, although the optimal solution needs a higher concentration of glycerol, probably because glycerol permeates the embryos more slowly than ethylene glycol.
Equine chorionic gonadotropin (eCG) is a member of the glycoprotein family, which includes lutropin (LH), follitropin (FSH), and thyrotropin (TSH), all consisting of two noncovalently linked a-and β-subunits. In the present study, we determined the full length of the equine α-subunit cDNA sequence. mRNA was extracted from equine placenta on day 70 of gestation. The nucleotide sequence of the eCG a-subunit cDNA was amplified by PCR using mixed primers designed on the basis of the nucleotide sequence of human, rat and mouse a-subunits. Analysis of the nucleotide sequence revealed that the a-subunit amplified contained 387 bp which included the signal peptide (24 amino acids containing 4 amino acids expected) and the first 3 amino acid residues. When the expression of both subunit mRNAs in the equine placenta was compared by Northern blotting, the expression of the β-subunit mRNA was relatively greater than that of the a-subunit.
The present study demonstrated the microvasculature of bovine mature follicles and microvascular changes with ovulation. The microvasculature of bovine mature follicles consisted of 2 capillary plexuses, the inner and outer. The inner capillary plexus consisted of a thick, dense sinusoidal capillary layer and a rough layer of arterioles and venules. Their plexus was character-ized as an independent microcircular unit receiving only one afferent vessel. Microvascular changes with ovulation appeared in the inner capillary plexus. In preovulatory follicles, inner sinusoidal capillaries gave rise to topographical increases in permeability. These phenomena may play an important role in causing the radical increase of follicular fluid pressure and cumulus oophorus discharge. In postovulatory follicles, the inner capillary plexus protruded into the follicular antrum and underwent remarkable angiogenesis to rapidly form the blood vascular bed of the corpus luteum.
To clarify the effect of triploidy on the development of bovine embryos, one or 2 sperm were injected into mature bovine oocytes and subsequent development and chromosome composi-tion were investigated. The cleavage rates of oocytes injected with one or 2 sperm using oocytes matured in vitro for 24 h were 36.3% (69/190) and 39.0% (53/136), respectively. Most of the embryos injected with 2 sperm had arrested development before the 8-cell stage and none devel-oped to the morula stage, although three (1.6%) embryos injected with one sperm developed to the blastocyst stage. The incidence of triploid embryos derived from 2-sperm injection was 12.9%, and the incidences of diploid and haploid embryos were 48.4% and 12.9%, respectively. About half (44.9%) of the uncleaved oocytes injected with one sperm and one-third (32.1%) of those injected with 2 sperm stopped developing at the second anaphase just after activation by ionophore treat-ment. Three pronuclei (10.7%) and one-cell metaphase (28.6%) with chromosome sets spread in 3 directions were observed only in uncleaved oocytes after 2-sperm injection. These facts suggested that triploidy is not the sole cause of arrested development before the 8-cell stage in cleaved embryos, although 2-sperm penetration and pronuclei formation strongly influenced oocyte cleav-age.
Mouse embryos were biopsied by squeezing, and viability was investigated. The biopsy method includes the following procedures: holding the embryo and partially dissecting the zona pellucida, squeezing a single blastomere from the embryo, sucking the blastomere into a blunt micropipette, and expelling the blastomere from the blunt micropipette. Mean cell numbers for 4-cell biopsied blastocysts (24.7 ± 5.2) were lower than in controls (38.5 ± 6.1) (P<0.0001). Preimplan-tation rates of development to blastocyst for embryos biopsied at the 4-cell and morula stages were 93% and 95%, respectively. In vivo survival rates to full term were similar for embryos biopsied at the 4-cell and morula stages (38% and 45%) and control embryos (46% and 47%, respectively). These results indicate that the squeeze method does not affect in vitro or in vivo viability of biopsied mouse embryos. This procedure may be useful for biopsy of preimplantation embryos for the purpose of sexing or diagnosing defective genes in humans and domestic animals.
Mastomys [Praomys(mastomys) coucha] of both sexes have morphologically functional prostates. To evaluate endocrine control of the prostate and other genital organs in mastomys, TAP-144-SR, a biodegradable sustained-release formulation of a potent Gonadotrophin-releaseing hormone (GnRH) agonist (leuprolide acetate), which is able to induce desensitization of the pitu-itary-gonadal axis, was used in this study. Three weeks after a single sc injection of TAP-144-SR (5 mg/kg), the mastomys were autopsied. In males, administration of TAP-144-SR resulted in decreases in the weights of testes, prostates and seminal vesicles in association with a marked reduction in serum testosterone levels. In females, TAP-144-SR treatment caused remarkable ovarian and uterine regression and reductions in serum levels of estradiol-17β and progesterone. However, the weights of female prostates and serum levels of testosterone remained unchanged by TAP-144-SR. These results show that the structure and function of male accessory sex organs and the uterus are mainly controlled by the gonads, which are regulated by gonadotrophins, but that those of the female prostate are less influenced by gonadal control.
The number of follicles more than 4 mm in size in the ovaries of 2 Japanese Black cows was determined by ultrasonography from 14 to 147 days after parturition. We found that the number of ultrasonographically identified follicles (uiFOL) with a diameter of 4 mm or more mark-edly decreased at about 92 days postpartum. We measured the number of uiFOL in 7 cows from 14 to 98 days postpartum and conducted superovulatory treatment with follicle stimulating hor-mone (FSH-P) at 95-98 days postpartum. The number of uiFOL decreased similarly at about 92 days after parturition, and that of recovered and transferable embryos was 17.7 ± 5.3 and 11.9 ± 4.4, respectively. The results suggest that it is preferable to start superovulatory treatment in the period when the number of uiFOL decreases. It is also important to examine whether these results can be applied to other periods excepting the period (92-98 days postpartum) in which the uiFOL de-creases in number. We measured both the number of uiFOL at the beginning of superovulatory treatment and that of recovered transferable embryos in 10 cows at more than 120 days postpartum. As a result, the coefficiency of correlation (r) obtained was 0.319, indicating that there was no correlation between the number of uiFOL and that of transferable embryos. This suggested that the number of uiFOL at the time of superovulatory treatment did not influence the yield of transferable embryos, except for the period (92-98 days postpartum) in which the uiFOL decreases in number.
Real-time ultrasound was successfully applied to predict bovine fetal sex in early preg-nancy. Ultrasound examinations were conducted in 68 cows through 117 trials between days 35 to 93 of pregnancy (day 0 = day of last artificial insemination). Regardless of fetal sex, the genital tubercle was readily recognized as a hyper-echogenic bilobar structure, and each lobe was elon-gated and oval shaped. The tubercle was first identified between the hind limbs on day 45 and then moved towards the umbilical cord in the male, while it moved towards the tail in the female. Thus sex of the fetus was diagnosed by ultrasonography in 90 cases. The diagnosis of sex was not possible in 27 cases: 18 cases with small fetuses before day 52, 2 cases with twin pregnancy, 2 cases with metroptosis after day 80, and 5 cases with maternal obesity. Accuracy of the ultrasonic diagnosis of fetal sex was 93.5% (43/46) for males and 90.9% (40/44) for females. The average umbilical-genital distance in frontal planes was significantly different between male and female fetuses by day 56. In conclusion, ultrasonic examination of the relative location of the genital tubercle was a reliable technique for determining bovine fetal sex between days 56 to 80 of preg-nancy.
The epidermal growth factor (EGF) concentration and its binding characteristics in bo-vine corpus luteum (CL) during the estrous cycle (early: Days 3-5, mid-: 8-12, late stage: 15-18) were determined. The EGF concentrations in corpus luteum tissue, evaluated by a radioreceptor assay using human placenta, were 3.0 ± 0.7 ng/g in early luteal phase, 43.9 ± 2.2 ng/g in mid-luteal phase and 18.6 ± 2.7 ng/g in late luteal phase. Specific EGF receptors were present in bovine corpus luteum of all luteal phases. TGFα, but not IGF-I, FGF and NGF competitively displaced EGF binding. The binding affinity of luteal EGF receptors differed between the luteal stages and decreased significantly from early (Kd=0.8 ± 0.04 nM) to mid-luteal stage (Kd=1.6 ± 0.34 nM, P<0.05). The binding capacity of luteal EGF receptor at different luteal stages were similar (early stage; Bmax=35.1 ± 5.7 fmol/mg protein, mid-luteal stage; Bmax=46.3 ± 4.0 fmol/mg protein, late stage; Bmax=35.1 ± 5.7 fmol/mg protein, all values mean ± SEM). These results suggest that EGF plays a physiological role for the regulation of luteal function during the estrous cycle.
The present study investigated effects of β-merchaptoethanol (β-ME) on the development of bovine embryos derived from In vitro matured and fertilized (IVM-IVF) oocytes. IVM oocytes were inseminated with frozen-thawed bovine sperm, and at 24, 48 and 72 h after insemination, 2-, 8-and 16-cell embryos, respectively, were transferred to TCM-199 medium containing 5% fetal bovine serum and 0, 5, 10, 20, 50 or 100 μm β-ME without a cumulus cell monolayer or with a cumulus cell monolayer but without addition of β-ME as a control. All embryos were subsequently incubated such that the total duration of culture was 8 days. The addition of low concentrations ( 5 or 10μM) of β-ME significantly enhanced the development of all the embryos to the blastocyst stage. High concentrations (20, 50 or 100μM) of β-ME were effective only for the development of 8-and 16-cell embryos, but not of 2-cell embryos, to the blastocyst stage. Compared with embryos in the co-culture system (control), 2-cell embryos that were cultured with β-ME but without a cumulus cell monolayer showed significantly lower rates of development to the blastocyst stage. However, even though without a cumulus cell monolayer, 8-cell embryos showed the same rates of develop-ment to the blastocyst stage as controls when low concentrations of β-ME (5 or 10μM) were added to the medium. Furthermore, significantly higher rates of development of embryos to the blasto-cyst stage were obtained when 16-cell embryos were cultured in medium supplemented with 20μM β-ME than that of controls. Twenty blastocysts developed from 16 cell IVM-IVF embryos in the medium containing 20μM β-ME were transferred to 20 recipients, and 11 of them became pregnant, resulting in 10 calves. These results indicate that β-ME is effective for blastocyst development of embryos that are cultured without a cumulus cell monolayer, and the effect is remarkable for embryos of advanced stages.
To improve chimera production efficiency, we examined whether frozen-thawed embry-onic stem (ES) cells would be useful in a coculture method. A3-1 ES cells were electroporated with the targeting vector pE 10.29NEO-TK. Exon 2 of the endothelin-1 gene in the ES cells was disrupted by homologous recombination. The homologous recombinant was cocultured at a density of 5.5 × 105 cells/ml with zona-free 8-cell to morula stage embryos in a coculture medium for 3-3.5 h. Cocultured embryos were transferred into Whitten's medium and cultured overnight. Morula and blastocyst stage embryos were transferred into the recipients on day 2.5 of pseudopregnancy. The development rate to blastocyst after coculture and subsequent overnight cultivation was 62%. As a result of embryo transfer, four male chimeras were obtained. Two of these were identified as germ-line chimeras by a progeny test. These results indicate that frozen-thawed ES cells can be used in coculture to produce chimeras and contribute a germ-line.