Motoaki Umezu

Rapid detection of male-specific DNA sequence in bovine embryos using fluorescence in situ hybridization

An accurate, reliable, and quick (less than an hour) method for determining the sex of bovine embryos was developed using a fluorescence in situ hybridization (FISH), with a probe designed from a bovine Y chromosome specific DNA (BC1.2). First, to improve a protocol of FISH and evaluate an accuracy of the method, lymphocyte nuclei prepared from three bulls, two cows, and one freemartin were tested. We found that 5 min was enough for hybridization. The washing solution adequate for posthybridization was 0.5× SSC at 72°C for 5 min. The whole procedure for FISH can be accomplished in less than an hour. A male‐specific signal was detected, on average, as 97, 0.5, and 83%, respectively, of lymphocytes in males, females, and a freemartin. Using the rapid FISH protocol developed, 28 embryos were divided. According to the presence of the digoxigenin signal, 16 embryos (57.1%) were predicted as male, and 12 embryos (42.9%), predicted as female. Mol. Reprod. Dev. 51:390–394, 1998.

Effect of hyaluronic acid on the development of porcine 1-cell embryos produced by a conventional or new in vitro maturation/fertilization system.

In pigs, it is difficult to produce normal fertilized embryos from immature oocytes in vitro. However, a new maturation/fertilization system in which the percentage of normal fertilized embryos is comparatively high has been developed recently. In the present study, porcine 1-cell embryos were produced both by a conventional and a new system and then cultured in NCSU-23 supplemented with hyaluronic acid at various concentrations. In the conventional system, the percentage of oocytes with monospermic penetration and 1 male pronucleus and 1 female pronucleus was only 6%. At 144 h after insemination, the percentage (5%) of embryos developing to the blastocyst stage in medium supplemented with 0.5 mg/mL hyaluronic acid was significantly (P<0.05) higher than that (2%) in medium without hyaluronic acid. When oocytes were matured and inseminated using the new system, monospermic penetration and the formation of 1 male and 1 female pronucleus were observed in 69% of the penetrated oocytes. However, blastocyst formation (8 to 14%) at 144 h after insemination was not affected by the concentration (0 to 1.0 mg/mL) of hyaluronic acid. These results indicate that the effect of hyaluronic acid on the development of in vitro-produced porcine embryos varies with the conditions of oocyte maturation and fertilization.

Microtubule and microfilament dynamics in rat embryos during the two‐cell block in vitro

Developmental block of rat embryo is induced by phosphate at the late two-cell stage. The present study uses immunocytochemistry and laser scanning confocal microscopy to examine microtubule and microfilament dynamics in blocked and nonblocked two-cell-stage rat embryos. Thin fibrous microtubules were distributed homogeneously in the cytoplasm in nonblocked embryos during the interphase of the two-cell stage and then translocated into mitotic spindles at the M-phase. In embryos blocked at the two-cell stage, much thicker fibrous microtubules were formed and distributed as rude meshwork structures in the cytoplasm. Microfilaments were distributed adjacent to nuclei and along the inside of the plasma membrane in nonblocked embryos during the two-cell stage, at M-phase, and at cleavage to the four-cell stage. In embryos blocked at the two-cell stage, however, microfilaments formed granules and dispersed in the cytoplasm. The distribution of microtubules and microfilaments changed relative to the occurrence of two-cell block. In summary, these results indicate that both microtubules and microfilaments are closely involved in the developmental block in two-cell rat embryos.

Endocrine profiles and embryo quality in superovulated Japanese Black cattle

The relationships between plasma concentrations of progesterone (P4), estradiol-17beta (E2) and lutenizing hormone (LH) and embryo yield and quality were examined in 40 superovulated Japanese Black cattle. The results indicated that the ovarian function, especially the function of corpus luteum on the first treatment day, is an important factor for reliable superovulation in cattle. The levels of plasma E2 and LH at estrus were related to embryo yield and quality.

Characteristics of Infertility and the Improvement of Fertility by Thyroxine Treatment in Adult Male Hypothyroid rdw Rats

Abstract We previously reported that rdw rats were infertile in both sexes. The present study was conducted to determine whether hypothyroidism in adult male rdw rats induced infertility by impairing sexual behavior or testicular function, whether the infertility could be reversed by thyroxine (T4) treatment, and whether the mutant could be produced by infertile rdw rats via in vitro fertilization. The sexual behavior was analyzed by pairing with normal female rats. The fertility of epididymal sperm was determined by in vitro fertilization. The results indicated that the infertility resulted from both defective sexual behavior and testicular function. No untreated rdw rats mated. The weights of epididymides were significantly low, whereas those of testes were not different from those of untreated normal rats. Epididymal sperm with cytoplasmic droplets were observed at a significantly high frequency. No fertilization was detected either in vivo or in vitro. Thyroxine treatment markedly increased serum T4 levels and the weights of both epididymides and testes. Partial reversion of the impaired sexual behavior was observed, and the percentage of epididymal sperm with cytoplasmic droplets was markedly decreased after T4 treatment. Fertility of epididymal sperm was completely reversed when determined both in vivo and in vitro, and homozygous embryos developed to term after transfer without loss of viability.

Serum inhibin, FSH, LH and testosterone levels and testicular inhibin content in beef bulls from birth to puberty

Developmental changes in testicular inhibin content and serum inhibin levels were observed with serum levels of gonadotrophins and testosterone in beef bulls up to 8 months of age. Twenty-two beef bulls (8 Japanese Black and 14 Japanese Shorthorn) were assigned to monthly groups from 1 to 8 months old (n=2 for 1 and 8 months, and n=3 for 2–7 months). Inhibin activity in the testicular extracts was determined using an in vitro rat pituitary cell culture system. Serum levels of inhibin and other hormones were measured by radioimmunoassays. The concentrations of inhibin in the testis (U/g) were high during the first 3 months (P < 0.05). They decreased temporarily between 4 and 6 months and then increased (P < 0.05). Serum inhibin levels were high during the first 4 months and decreased after 5 months (P < 0.05). Serum FSH levels were higher at 2 months than at any other month of age (P < 0.05). Serum testosterone levels remained low during the first 3 months, increased at 4 months and continued to rise thereafter. A significant positive relationship was found between serum FSH and serum inhibin levels (r=0.068; P < 0.001), whereas a negative relationship was found between serum testosterone and serum inhibin levels (r = −0.58; P < 0.01). During the first 5 months of age, the concentrations of inhibin in the testis (U/g) correlated positively with serum FSH levels (r=0.69; P < 0.01), while they correlated negatively with serum testosterone levels (r=−0.85; P <0.001). These results indicate that the immature bovine testis produces and secretes high levels of inhibin, and that serum inhibin levels reflect this high inhibin production in the testis before the onset of puberty. After the onset of puberty, inhibin production and inhibin release into the circulation are no longer parallel, which is probably due to the formation of the blood-testis barrier. It is suggested that FSH is the principal regulator of inhibin production in the testis during the first 5 months of age in bulls. After the onset of puberty, testosterone might play a dominant role in suppression of FSH from the pituitary.

Fluorescence in situ hybridization with Y chromosome-specific probe in decondensed bovine spermatozoa.

This study was carried out to demonstrate bovine Y chromosome-bearing spermatozoa by rapid fluorescence in situ hybridization (FISH), using a digoxigenin (Dig)-labeled DNA probe specific to bovine Y chromosome. Before the FISH procedure, sperm heads were treated for decondensation with dithiothreitol (DTT) and glutathione (GSH) with or without heparin supplementation. Concentrations of either above 2 mM DTT or above 100 mM GSH induced swelling of the sperm head, which resulted in sufficient detection of the Y chromosome signal in sperm nuclei by rapid FISH (49.8 to 53.4%). When FISH was used with 2 mM DTT or 100 mM GSH on specimens from 7 sires, the rate of detection of the Y chromosome signal varied among sires (5.4 to 49.6%), especially that of the GSH treatment. Supplementation of GSH with heparin (100 U/mL), however, could induce reliable, repeatable detection of the Y chromosome signal in sperm nuclei of all the 7 sires (48.4 to 50.3%). These results show that in bovine spermatozoa decondensed with GSH and heparin, rapid FISH can detect Y chromosome-bearing spermatozoa.

Plasma endocrine profiles and total cholesterol levels in superovulated cows

Abstract Changes in plasma follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17β (E2) and progesterone levels between superovulatory hormone injection and flushing were monitored. FSH level at gonadotropin injection was significantly correlated with the peak level (r = 0.73) and the total level between 30 h after cloprostenol injection and first service (r = 0.76). There was a significant relationship between the parameters of the FSH level and the number of transferable embryos. Early or delayed preovulatory LH surge was observed in 33.3% of the cows ( 6 18 ; Group I). Three cows (16.7%) had low basal FSH levels of less than 5 ng/ml between the gonadotropin injection and the preovulatory LH surge (Group II). The remaining nine cows (50.0%) made up Group III. The numbers of recovered and transferable embryos in each group were 4.5 and 0.5 (Group I); 4 and 3 (Group II); and 8 and 4 (Group III), respectively. A significant difference in the number of transferable embryos was observed between the groups (P The number of recovered embryos in the cows with a total cholesterol level of less than 130 mg/dl was significantly less than those in the cows with a total cholesterol level of 130 mg/dl and higher (4:11, P Our study indicated that early or delayed preovula tory LH surge, low FSH, and low total cholesterol levels adversely affect superovulation results.

Seasonal changes in inhibin activity in seminal plasma and serum concentrations of FSH, LH and testosterone in the male goat ( Capra hircus )

Inhibin activity in goat seminal plasma was measured by in vitro assay throughout successive 9 months and its relationship with the serum FSH, LH and testosterone concentrations was investigated. Total inhibin activity (TIA) in seminal plasma gradually increased from spring to summer, reduced in autumn (P<0.05) and recovered toward winter (P<0.05). Serum FSH and LH reached a peak in mid-summer (P<0.01) and returned to the low levels in autumn. Serum testosterone also increased in mid-summer and kept the high levels until the early winter (P<0.05). Some positive correlation was found in monthly levels between seminal TIA and serum FSH (r=0.305; P<0.05). Results suggest that the summer increase of inhibin activity in seminal plasma relates with the mid-summer rise of serum FSH levels in the male goat.

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.