The homeobox genes expressed in highly invasive murine hemochorial placentae and in non-invasive caprine epitheliochorial/syndesmochorial placentae were surveyed by cloning and sequencing RT-PCR products generated by using sets of degenerate primers. All HOX9 paralogues, i. e., HOXA-9 - HOXD-9, were expressed both in the murine and in the caprine placenta. Among the non-clustered HOX genes, the placental expression of EMX2 and ATBF1 was newly demonstrated in the present study. In the murine placenta, Hoxa-10 was the dominant homeobox gene expressed. In the intercotyledon region of the caprine placenta, HOXA-10 was expressed at a low level, while HOXB-7 transcript levels were high. By contrast, no Hoxb-7 expression was found in the murine placenta. The high HOXB-7 mRNA levels in the caprine placenta might represent the expression in the allantois which enormously expands in the caprine epitheliochorial/syndesmochorial placenta. Despite the difference in the pattern of HOXA-10 and HOXB-7 expression, the homeobox genes expressed in two morphologically and functionally different types of placentae were surprisingly similar with each other. In the mouse plcenta, the expression levels of Hoxa-9, Emx2, Msx2 and ATBF1 did not show any consistent changes during the pregnancy period examined. Hoxb-9 expression levels exhibited a decline between Day 13 and Day 15 of pregnancy, while the expression levels of Hoxc-9 and Hoxd-9 gradually increased from Day 13 to Day 19. The Hoxc-9 expression level reached to the maximum level on Day 19. The functional significance of these homeobox genes remains to be clarified.
A new inbred strain SD/gShi male rat with small testes was studied from the reproductive, histopathological and endocrinological viewpoints from 3 to 35 weeks of age. SD/gShi males showed only a slight increase in sperm production at puberty, in contrast to a rapid increase in normal SD males. At 6 weeks of age, spermiation became detectable in both SD/gShi and normal SD males, however, the numbers of spermatocytes and round spermatids per Sertoli cell in SD/gShi males were less than those in normal SD males. Moreover, the number of round spermatids per Sertoli cell did not increase in SD/gShi males at puberty, while they did in normal SD males. Histological analysis of germ cell numbers in SD/gShi males indicated that the lower numbers of spermatocytes and spermatids are caused by suppression of germ cell proliferation at the step from the spermatogonium to the preleptotene spermatocyte. Adult SD/gShi males showed normal fertilization ability, however, they had only 10-20% of sperm production in normal adult SD males and showed depressed sperm motion. Plasma total testosterone in SD/gShi males was lower for all ages, though testis testosterone concentration was normal. Plasma gonadotropin concentrations, especially FSH, remained higher than in normal SD males, suggesting that the spermatogenic defects are not caused by gonadotropin deficiency. These results indicate that the SD/gShi male is characterized by spermatogenic defects at the preleptotene spermatocyte step which occur from the first spermatogenic wave. The SD/gShi male rat can serve as a useful model for studying spermatogenesis in rats.
This study was conducted to examine the possibility of selection of transgenic bovine embryos by use of polymerase chain reaction (PCR) following microinjection of exogenous DNA before embryos were transferred to recipient cattle. DNA of bovine α-lactalbumin (α-LA) gene was microinjected into pronuclei of bovine embryos derived from in vitro maturation and fertilization. They were then cultured for 7 to 8 days to the blastocyst stage. To examine persistence of injected DNA during embryo development, embryos were analyzed by PCR at 0, 1, 3, 5 and 7 days following DNA microinjection. To distinguish the injected α-LA from the endogenous gene, gene-gene junction regions of head-to-tail concatemers of the injected DNA, which were formed when linearized DNA were injected into cell nuclei, were amplified by PCR. Injected DNA persisted at high rates (72 to 80%) until 5 days following DNA injection, but the detection rate at the blastocyst stage (20%) was lower than earlier stage (P<0.05), indicating that injected DNA into bovine embryos drastically decreased at the blastocyst stage. To determine if the PCR signals reflect transgenic status, biopsy samples of DNA-injected blastocysts were analyzed. Total of 1, 583 embryos were microinjected, and 124 (8.9%) developed to blastocysts. Twelve (11%) out of 109 samples biopsied from microinjected blastocysts were positive by PCR analysis. Eight PCR-positive embryos were transferred to 8 recipients. Also, 31 negative embryos were transferred to recipient heifers. Four out of 8 recipients that received PCR-positive embryos were pregnant and fetuses and placentae were recovered at 60 to 75 days of pregnancy. One of the samples, of which only a placenta was recovered, was found to be transgenic by both PCR analysis and Southern hybridization. The other samples had no injected DNA. On the other hand, 14 fetuses with placentae were recovered from recipients that received PCR-negative embryos. None of these samples were found to carry injected DNA. Our system could not detect single, or head-to-head- or tail-to-tail-joining transgenes; however, the results described here imply selective transfer of potential transgenic embryos to recipient heifers by the PCR selection.
To elucidate the beneficial effects of follicular fluid (FF) added to maturation medium on nuclear maturation and postfertilization development of bovine oocytes in vitro, we attempted to separate largely bovine FF (bFF) collected from small follicles into heparin-binding and -nonbinding fractions (HBF and HNF, respectively) by heparin affinity chromatography. Each fraction was dialyzed (molecular weight cut off ？？1, 000 Da) and lyophilized, then dissolved in a half original bFF volume of modified synthetic oviduct fluid (mSOF). Experiment 1: Cumulus-oocyte-complexes (COCs) were matured in mSOF in the presence or absence (control) of whole bFF (wFF), HBF or HNF at a concentration of 10% (v/v). After in vitro fertilization, oocytes were freed from COCs and then cultured in mSOF with fetal calf serum until Day 8. Nuclear maturation rates were not different among wFF, HBF, HNF and control (69.8, 75.6, 63.6 and 72.9%, respectively). However, HBF favored postfertilization development, especially to the blastocyst stage (27.2%) compared with the control (19.0%), whereas HNF showed inhibitory effects, although wFF had no effect. Experiment 2: To determine whether the effects of HBF and HNF would become more evident by high-dose supplementation, HBF or HNF was added to in vitro maturation (IVM) medium at concentrations of 10, 20 or 40% (v/v). HBF at all concentrations examined resulted in significantly higher (p<0.05) cleavage rates (80.5-86.2%) and blastocyst yield (31.1-44.2%) than the control(cleavage: 70.1%, blastocyst: 22.4%), whereas high dosages of HNF (？？20%) markedly inhibited embryonic development. These results indicated that heparin-binding fraction of bFF when added to the IVM medium was highly effective for enhancement of developmental competence of bovine oocytes in vitro.
The testes of bulls in an artificial breeding unit were examined with low frequency (1.6 and 2.25 MHz) transducers to determine if acoustical impedance could be used to detect bull differences in testicular tissue. The initial studies accompanied measurement of the linear dimensions and circumference of the testes with simple calipers and tapes to estimate size. Simple instruments proved to provide testicular size information that was more accurate and more easily obtained than by ultrasonography. Subsequent studies dealt with spermatogenic elements. Internal characteristics of the testes of 10 normal young bulls measured by ultrasonography were similar and were not significantly correlated with semen characteristics. Ultrasonograms on the testes of 68 bulls 20 to 144 months old differed, particularly associated with age. There was more dense tissue distributed in the testicular parenchyma of older bulls. Twenty-six bulls that had preslaughter ultrasonic analysis of the testes were classified into two groups after slaughter on the basis of normal or high proportions of fibrous testicular tissue. An independent analysis of the ultrasonograms concurred 81% of the time with the postmortem findings. While ultrasonography has greatly aided the understanding of many events in reproductive development and sexual function, it appears that its capabilities of providing unique information on internal testicular characteristics may be limited to special cases, such as old bulls when semen quality is marginal.
The Cre-loxP site-specific recombination system is useful for tracing the fate of cells via transient expression of the Cre recombinase gene directly introduced into the cells with a vector carrying loxP sites. In this study, we used this system to examine whether cell lineage-specific expression of a reporter gene occurs in murine preimplantation embryos. An expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken β-actin promoter (CAG), a portion of the rabbit β-globin gene (including the 2nd intron, 3rd exon and 3' noncoding region), loxP-flanked DNA sequence [containing enhanced green fluorescent protein (EGFP) cDNA and chloramphenicol acetyltransferase gene (CAT)], and lacZ gene encoding E. coli β-galactosidase (β-gal), was constructed. When pCETZ-17 DNA was microinjected into the pronuclei of fertilized eggs, 30.0% (6/20) of the surviving 2-cell embryos exhibited distinct fluorescence in both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for β-gal. When both plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, all (12/12) embryos exhibited low or no fluorescence, but 66.7% (8/12) exhibited positive staining for β-gal activity in one or both blastomeres. This indicates that transient expression of Cre removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only 2-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing 2-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 2 days up to 8-cell stage. When the developing 8-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for β-gal activity was observed in all (7/7) embryos. These findings indicate that this Cre-loxP system, which is based on transient expression of the Cre gene directly introduced into nuclei of embryonic cells by microinjection, is useful for tracing cell lineage as well as for expressing a gene of interest in a lineage-specific manner in early embryogenesis
To directly examine the cellular roles of the retinoblastoma gene product (pRb), we constructed an effective antisense Rb RNA vector that expressed an antisense Rb-1 sequence directed toward the 3' untranslated region of Rb-1 mRNA. In this study, we introduced the antisense Rb vector into mouse fibroblast SV-T2 cells expressing large amounts of SV40 LT, which inhibits the biological functions of pRb through direct binding. Only a small number of drug-resistant colonies were obtained after drug selection. Co-transfection of SV-T2 cells with pRb expression vectors and the antisense Rb vector resulted in an apparent increase in the number of drug-resistant colonies, suggesting that a decrease in cellular pRb content induced cell death in the cells. The cell death in SV-T2 cells was also abrogated by simultaneous transfection with antisense p53 RNA expression vector. These results indicate that a decrease in the amount of biologically active pRb by both the effects of the transfected antisense Rb RNA and the large amount of endogenous SV40 LT induces cell death in SV-T2 cells through a p53-dependent pathway. The antisense Rb and p53 vectors used in this study are useful to examine the biological functions of these genes in established cell lines.
This study was carried out to simplify the previous method for the mucus penetration test in Japanese beef cattle. Frozen-thawed semen was brought into contact with frozen-thawed estrous cervical mucus loaded in a rectangular capillary tube under the following conditions for placement of the tube and temperature of incubation: 1) horizontal/38 C (the previous method, Horizontal 38 C), 2) upright/38 C (Upright 38 C), or 3) upright/room temperature (Upright RT). The previous method included fixing the capillary tube with a mixture of vaseline and paraffin and a cover of semen with paraffin oil and a cover slip on a glass slide. This procedure was simplified by use of a small vial, in which semen was placed and covered with paraffin oil in Upright 38 C and Upright RT. Mucus penetration, expressed as the distance travelled by the sperm cell that penetrated the mucus the farthest in 10 min, was similar to Horizontal 38 C and Upright 38 C but was significantly lower with Upright RT. Intra-assay and inter-assay coefficients of variation in Upright 38 C were below 5%. Thus this study simplified the technique of the mucus penetration test using Upright 38 C
We have previously demonstrated that the gonadal function of sika deer (Cervus nippon) can be successfully monitored by using fecal steroid analysis. In this study we examined some technical aspects of applying this method to the estimation of the reproductive status of wild sika deer. Enzymeimmunoassays (EIA) for fecal progesterone and testosterone were established, and the fecal concentrations of these steroids obtained were compared with those measured by conventional radioimmunoassays (RIA). The patterns of fecal progesterone during the estrous cycle and the annual fecal testosterone profile in a male deer measured by EIA were virtually identical with those measured by RIA. Then the effect of environmental temperature on the fecal progesterone concentration was examined by monitoring the change in fecal progesterone at 4 C and 20 C up to 144 h after defecation. Fecal progesterone levels increased markedly at 20 C (but not at 4 C) with the degree of increase being variable in each sample. This increase was inhibited by adding ethanol, antibiotics or silica gel. It therefore appears that the increase was caused by the action of intestinal microorganisms, which converted conjugated steroids to unconjugated forms. These results suggest that wild sika deer fecal samples should be collected in winter time when the environmental temperature is low for steroid analysis in field conditions. The EIAs appeared more feasible means for field-endocrinological studies than RIA considering their simpler and less time-consuming procedures.