Showing papers in "Journal of Reproduction and Development in 1993"
TL;DR: It is concluded that Hokkaido brown bears exhibit obligate delayed implantation and that this period of delayed implanted implantation continues at least until late November.
Abstract: Ovaries having corpora lutea and uteri of 6 female Hokkaido brown bears (Ursus arctos yesoensis), which were legally killed by hunters from October to December in Hokkaido, Japan, were examined macroscopically and histologically. Four of the 6 female bears had unimplanted embryos in the uterine lumen during the period of delayed implantation between October 19 and November 20. Ovaries of bears which had unimplanted embryos contained the corpora lutea with extensive vacuolation or numerous granules in the cytoplasm of luteal cells. These bears had well-developed uterine glands which had tall epithelial cells with vacuoles in the cytoplasm of glandular cells and coagula in the glandular lumina. It is concluded that Hokkaido brown bears exhibit obligate delayed implantation and that this period of delayed implantation continues at least until late November.
51 citations
TL;DR: An optimum time for an intrauterine insemination with frozen-thawed semen appears to be different in the use of MAP sponge (range: 36 to 60h) and CIDR® (48h), and a significant difference in the lambing rate was found among the sheep farms conducting the present study.
Abstract: During the non-breeding season (April to July), 187 ewes treated wth either an intravaginal sponge impregnated with medroxyprogesterone acetate (MAP) or a controlled internal drug release (CIDR®) device for 9 days and an injection of pregnant mare serum gonadotropin (PMSG) at one day before the cessation of progestogen treatment were inseminated into the uterus by laparoscopy with frozen-thawed semen at fixed-time basis (36, 48 or 60h after the removal of sponge or device). Insemination was also performed at 18 h after estrus detection as a control. The time to the onset of estrus after CIDR® treatment was significantly (P<0.05) earlier (mean: 24.9h) than that in ewes treated with MAP sponge (mean: 30.0h). The percentages of ewes lambing and lambs born per ewe lambing for inseminations at 36, 48 and 60 h were 66.7% and 1.69; 52.0% and 1.31; 46.2% and 1.75 for MAP-treated ewes, and 33.3% and 1.50; 73.9% and 1.53; 40.9% and 1.56 for CIDR®-treated ewes. For control ewes, they were 59.1% and 1.62; and 47.6% and 1.90 for MAP and CIDR® treatments, respectively. There was no significant differences in the lambing rates and prolificacy between MAP and CIDR® treatments and among the insemination times. An optimum time for an intrauterine insemination with frozen-thawed semen appears to be different in the use of MAP sponge (range: 36 to 60h) and CIDR® (48h). A significant difference in the lambing rate was found among the sheep farms conducting the present study.
23 citations
TL;DR: Results indicate that during 2 to 3 h of incubation with oocytes in the caffeine-containing medium, the spermatozoa become highly fertile, and that a high concentration of cyclic AMP in sperm might not have any direct effects on the ability of sperm to penetrate oocytes.
Abstract: Experiments were conducted to assess the effects of caffeine on the ability of frozen-thawed boar epididymal spermatozoa to penetrate oocytes that had been matured in culture. The spermatozoa were preincubated in modified TCM199 medium and subsequently incubated for 2 h in either a fertilization medium with 2 mM caffeine or in a fertilization medium without caffeine. Then a sample of the spermatozoa in the caffeine-containing medium was introduced into another caffeine-containing medium that contained matured oocytes, while another sample was introduced into a medium with matured oocytes without caffeine. This procedure was repeated for the spermatozoa that had been incubated in the caffeine-free medium. The two sets of manipulations resulted in higher rates of penetration of oocytes inseminated in the caffeine-containing medium than of oocytes in the caffeine-free medium. Furthermore, when oocytes with attaching spermatozoa were washed and transferred to caffeine-free medium after 1, 2 and 3 h of incubation in the presence of caffeine, penetration rates were 14%, 76% and 94%, respectively. Addition of caffeine to the fertilization medium caused as much as a 1.4-fold elevation in levels of cyclic AMP in sperm, the effect being maximal within about 0.5 h of incubation. These results indicate that during 2 to 3 h of incubation with oocytes in the caffeine-containing medium, the spermatozoa become highly fertile, and that a high concentration of cyclic AMP in sperm might not have any direct effects on the ability of sperm to penetrate oocytes.
23 citations
TL;DR: The results suggested that expression of the transgenes were probably higher in organs with exocrine functions, including the brain, and that hGH was produced in the brain.
Abstract: We have generated 2 transgenic mouse lineages expressing a mWAP/hGH fusion gene where 5' sequence from the mouse whey acidic protein (mWAP) gene is linked to the coding region for human growth hormone (hGH). In the present study, we investigated whether hGH was synthesized in the mammary gland of the transgenic mice and secreted into milk, and whether physiological functions of the transgenic mice were affected by the expression of the transgenes or not. Milk taken from female mice of the transgenic lineages contained about 4.77±0.1 μg/ml (n=4) hGH in average. The hGH was also detected in plasma of the transgenic mice expressing the transgene. The plasma hGH was probably derived from several organs, including submandibular glands, pancreases and tongues as well as mammary glands. The results suggested that expression of the transgenes were probably higher in organs with exocrine functions. In addition, hGH was produced in the brain. Transgenic females derived from one of the founder males showed the sterility accompanied by obesity, insulinemia and hyperglycemia. Histological examination confirmed that the number of azocarmine-positive cells which represent GH secreting-cells in the anterior hypophysis was dramatically reduced in the sterile transgenic females compared to the control mice. The ovary showed signs of pronounced disorders accompanied by failure of follicular growth and by inability of the corpus luteum formation.
22 citations
TL;DR: It is shown that a delay of embryo development had already appeared before male and female genomes fused (pre-syngamy), and the incidence of triploidy was higher in F1 eggs, in both the pronuclear and mitotic stages.
Abstract: Superovulated eggs in (BALB/c×C57BL/6) F1 and ICR (outbred in a closed colony) female mice were fertilized in vitro with spermatozoa obtained from caudal epididymides of ICR males. Air-dried chromosome preparations were made from colcemid-primed 1-cell eggs and stained by the C-banding method. The fertilization rate was lower in F1 eggs than in ICR eggs (88 vs. 92%, P<0.02). The incidence of metaphase ("syngamy") eggs was higher in F1 eggs, and the incidence of pronuclear and late prometaphase ("pre-syngamy") eggs was higher in ICR eggs (P<0.001, P<0.005), showing delayed progress of the first cleavage in the ICR eggs. Developmental rates from 2-cell to 4-cell stage and from morula to blastocyst stage were significantly lower in ICR eggs (P<0.001) than in F1 eggs. These results show that a delay of embryo development had already appeared before male and female genomes fused (pre-syngamy). The incidence of triploidy, which might be caused by dispermy, was higher in F1 eggs, in both the pronuclear and mitotic stages (P<0.001). A little aneuploidy and structural aberration of chromosomes occurred in both F1 and ICR eggs, and no significant difference was observed between F1 and ICR eggs. The sex ratio at the first cleavage stage in both F1 and ICR eggs showed no significant deviation from equality.
21 citations
TL;DR: Bovine male-specific repetitive DNA's were cloned for the sexing of bovine embryos by means of the polymerase chain reaction (PCR) and a combination of 2 pairs of primers gave discrete PCR products allowing discrimination between male and female DNA.
Abstract: Bovine male-specific repetitive DNA's were cloned for the sexing of bovine embryos by means of the polymerase chain reaction (PCR). The cloning method used was PERT (phenol emulsion reassociation technique), in which most male DNA's were absorbed by an excess amount of female DNA's to enrich Y-specific DNA's. Among 20 clones examined, one clone was male-specific. The others were gender-neutral; three of these were used as probes for internal controls. The male-specific clone was used as a probe to isolate other male-specific clones from a bovine male genomic DNA library. A clone thus obtained contained at least 3 male-specific EcoRI fragments. The original and the secondarily cloned DNA's were all sequenced. They were repetitive in nature. Oligonucleotides (20 mers) were synthesized on the basis of both the male-specific and gender-neutral DNA sequences as primers for PCR. A combination of 2 pairs of primers, i.e., male-specific and gender-neutral, gave discrete PCR products allowing discrimination between male and female DNA. The detection limit of the template DNA was 10 pg, equivalent to the DNA from 3 cells.
20 citations
TL;DR: The method appears to be an efficient and reliable one to determine the sex of bovine preimplantation embryos.
Abstract: We have previously cloned and characterized both bovine male-specific and genderneutral DNA's. Primers for the polymerase chain reaction (PCR) were synthesized on the basis of these DNA's to give differential PCR products between males and females. Serial dilution of bovine somatic cells of both sexes revealed that 10 cells were sufficient to discriminate males from females under our conditions. The usefulness of our primers to sex bovine embryos was confirmed by using purified control DNA's, cytogenetic analysis of demi-embryos, coincidence of a pair of asymmetrical biopsy samples and embryo transfer. All the embryos used here were produced by in vitro fertilization and culture methods. A successful determination of sex was made for 83 out of 84 embryos examined (98.8%). The sex ratio (54.2% male) did not differ significantly from the expected 1:1 ratio. The viability of biopsied embryos (86.3%) after 24 h culture in vitro was not significantly different from that of intact embryos (92.3%). Also, the mean numbers of cells of the biopsied (153.1, n=42) and intact (170.7, n=39) embryos were not significantly different. Out of seven recipients received the embryos with predicted sex, three went to term, producing one male and 2 female calves in accordance with the sex prediction before transfer. In conclusion, our method appears to be an efficient and reliable one to determine the sex of bovine preimplantation embryos.
19 citations
TL;DR: Analysis of newly synthesized proteins of the reconstituted embryos showed that 68-70 kDa heat shock proteins, initial markers of embryonic genome activation, were synthesized at the late 2-cell stage.
Abstract: Thymocytes from fetal, newborn, and adult mice were transferred into enucleated oocytes by standard micromanipulation. The developmental ability of oocytes receiving a thymocyte was examined in vitro. Most of the reconstituted eggs were successfully activated and formed a pronucleus-like structure. The percentage of reconstituted eggs developing to the 2-cell stage was high, 75-84%, but development of embryos to more advanced stages was limited. Only 8 and 1% of the reconstituted eggs receiving a thymocyte from fetal and newborn mice developed to the blastocyst stage, respectively. None developed to the 8-cell stage when a thymocyte from adult mice was transferred. Analysis of newly synthesized proteins of the reconstituted embryos showed that 68-70 kDa heat shock proteins, initial markers of embryonic genome activation, were synthesized at the late 2-cell stage.
19 citations
TL;DR: The effects of culture time for oocyte maturation and the injection conditions of capacitated sperm (with/without manipulation medium) on the subsequent morphological changes in male and female nuclei and the development of embryos derived from microfertilization of bovine oocytes were investigated and it was suggested that the injected manipulation medium was harmful to the ooplasm and affected theDevelopment of embryos.
Abstract: The effects of culture time for oocyte maturation and the injection conditions of capacitated sperm (with/without manipulation medium) on the subsequent morphological changes in male and female nuclei and the development of embryos derived from microfertilization of bovine oocytes were investigated. The rates of cleavage and development to blastocyst stage increased with the extension of maturation time of oocytes from 18h to 25h.The best results (2-cell: 71.4%, blastocysts: 11.4%) were obtained from the oocytes cultured for 25 h. Microinjection of sperm into the oocytes without manipulation medium, resulted in higher (P<0.05) developmental rates (2-cell: 28.8%, blastocysts: 9.6%) than those (19.5% and 4.9%) from sperm with manipulation medium. This result suggested that the injected manipulation medium was harmful to the ooplasm and affected the development of embryos. The development of embryos derived from microfertilization up to 3-to 7-cell stage and over 8-cell stage was delayed for 4 h and 8 h than those from in-vitro morphological observation of the male and female nuclei after the microinjection, the formation of male pronucleus was slower than that of female pronucleus.
18 citations
TL;DR: The results indicate that even a low insemination dose (0.05 ml) can result in similar rates of lambing compared with 0.1 or 0.2 ml of inSEmination dose per uterus in Suffolk ewes treated with either P sponge or CIDR® during the non-breeding season.
Abstract: During the non-breeding season, a total of 176 Suffolk ewes at 3 sheep farms were treated with either a self-made, progesterone-impregnated intravaginal sponge (P sponge) or controlled internal drug release (CIDR®) for 9 days and an intramuscular injection of pregnant mare's serum gonadotropin (PMSG) 1 day before the cessation of progesterone treatment. At 44-52 h after treatment, 174 ewes were inseminated with 0.2, 0.1 or 0.05 ml of frozen-thawed ram semen per uterine horn with the aid of a laparoscope. There was no significant difference in lambing rate between P sponge (54.2%) and CIDR®(61.5%). The insemination dose did not also affect the lambing rates in both P sponge-treated (51.7, 66.7 and 44.4% for 0.2, 0.1 and 0.05 ml, respectively) and CIDR®-treated (58.1, 59.4 and 67.9%, respectively) ewes. Prolificacy was not significantly different between progesterone treatments nor among insemination doses. Lambing rates and prolificacy at the 3 sheep farms were not also significantly different. These results indicate that even a low insemination dose (0.05 ml) can result in similar rates of lambing compared with 0.1 or 0.2 ml of insemination dose per uterus in Suffolk ewes treated with either P sponge or CIDR® during the non-breeding season.
18 citations
TL;DR: It is concluded that the culture systems examined are competent to support development of porcine zygotes to blastocysts and evaluate in vitro fertilized porcINE embryos.
Abstract: To identify an in vitro culture procedure which can be used to assess porcine in vitro fertilized embryos, three culture systems were examined. A total of 103 in vivo fertilized zygotes were co-cultured in modified BMOC-3 medium with granulosa cells, or in modified BMOC-2 with oviduct cells, and 35 were cultured in Whitten's medium supplemented with 1.5% BSA. After 8 days in culture, 66.0 and 73.6% of the co-cultured zygotes, and 51.4% of the zygotes cultured in the modified Whitten's medium developed to blastocyst stage. In contrast, only 20.5% (18/88) and 1.4% (9/627) of in vitro fertilized zygotes derived from in vivo and in vitro matured oocytes developed to blastocysts in the co-culture with modified BMOC-3 and granulosa cells. It is concluded that the culture systems examined are competent to support development of porcine zygotes to blastocysts and evaluate in vitro fertilized porcine embryos.
TL;DR: The vomeronasal organ of the rat fetuses at 10, 11, 12, 13, 14, 16 and 18 days of gestation was studied by light and scanning electron microscopy and the findings were similar to those reported in other rodents such as mice and golden hamsters, however in the hamster the vomer onasal duct, cartilage and gland, and in the mouse the vOMS nerve appeared earlier respectively than those in the rat.
Abstract: The vomeronasal organ of the rat fetuses at 10, 11, 12, 13, 14, 16 and 18 days of gestation was studied by light and scanning electron microscopy. The primordium of the vomeronasal organ was observed first on 11 days of gestation as a thickening of the ectodermal layer on the mediorostral wall of the olfactory pit, whereas the olfactory placode appeared on the 10th day. The ectodermal thickening, which was identified as the vomeronasal placode, was situated on the separate geometric territory lying apart from the olfactory epithelium, moreover the vomeronasal placode appeared one day later than the olfactory placode did. The vomeronasal nerves and intraepithelial capillaries were observed on 13 days of gestation and immature vomeronasal cartilage cells accumulated around the vomeronasal duct on 14-days. The vomeronasal complex appeared 1 or 2 days later than the structures in the olfactory organ. These findings were similar to those reported in other rodents such as mice and golden hamsters, however in the hamster the vomeronasal duct, cartilage and gland, and in the mouse the vomeronasal nerve, appeared earlier respectively than those in the rat.
TL;DR: In this article, the efficacy of 30% polyvinylpyrrolidone solution (PVP; molecular weight 40, 000) as a solvent for follicle stimulating hormone (FSH) injection for superovulating beef cattle and to compare its efficacy in inducing superovulation with descending doses of FSH dissolved in saline.
Abstract: . This study was designed to establish the efficacy of 30% polyvinylpyrrolidone solution (PVP; molecular weight 40, 000) as a solvent for follicle stimulating hormone (FSH) as a single intramuscular (i.m.) injection for superovulating beef cattle and to compare its efficacy in inducing superovulation with descending doses of FSH dissolved in saline. In experiment 1, 16 Japanese Black cows were randomly alloted to 2 treatment groups. Each of the 8 cows in the PVP group or saline group were given a single i.m. injection with 30 mg of FSH dissolved in either 10 ml of PVP or 10 ml of saline, respectively. In Experiment 2, the efficacy of a single FSH injection was compared between 15 Japanese Black cows and 12 heifers. In experiment 1, a larger number of ova and embryos was observed with FSH dissolved in PVP than in saline (9.8±2.4 versus 0). In experiment 2, the average number of embryos and transferable embryos in the control group (12.0±8.4 and 6.0±4.7) and experimental cows (10.4±7.6 and 5.2±2.2) were significantly higher (P<0.01) than in heifers (2.8±3.1 and 1.1±1.9).9).
TL;DR: Bovine cumulus-oocyte complexes were inseminated in fertilization medium and the proportions of oocytes developed to the morula and blastocyst stages, respectively, were significantly (P<0.01) higher in culture medium without than with glucose regardless of the presence of glucose during fertilization.
Abstract: Bovine cumulus-oocyte complexes were inseminated in fertilization medium, mBO medium including caffeine (5 mM), heparin (10 μg/ml) and bovine serum albumin (BSA; 10 mg/ml) with or without glucose (13.9 mM). Oocytes were freed from cumulus cells 8 h post-insemination and transferred into culture medium, mTCM-199 supplemented with BSA (3 mg/ml) and with or without glucose (5.56 mM). When oocytes were examined 24 and 72 h post-insemination, high proportions of oocytes were penetrated (88-92%) and cleaved to more than the 2-cell stage (68-71%), respectively, regardless of the presence or absence of glucose during fertilization and/or further culture without cumulus cells. At 144 and 192 h post-insemination, however, the proportions of oocytes developed to the morula and blastocyst stages, respectively, were significantly (P<0.01) higher in culture medium without than with glucose regardless of the presence of glucose during fertilization. In addition, significantly (P<0.05) higher proportions of oocytes developed to the morula (23 vs. 40%) and blastocyst (5 vs. 12%) stages when they were cultured in glucose-free medium after fertilization in medium without than with glucose. When glucose was added in culture medium 120 or 144 h post-insemination, 23 to 24% of inseminated oocytes developed to the early or expanded blastocyst stage 192 h post-insemination.
TL;DR: The results suggest that the low developmental ability in Ham's F-10 may be mainly due to the deleterious effect of heavy metal ions and hypoxanthine.
Abstract: To elucidate what kind of medium would be desirable for mammalian embryo culture, we evaluated the effect of heavy metal ions on mouse in vitro embryonic development. Pronuclear stage embryos recovered from ICR mice were cultured in Ham's F-10 and α-MEM and the developmental ability was compared between them. The major difference of these 2 media is that only Ham's F-10 contains heavy metal ions such as Zn2+, Fe2+ and Cu2+, and hypoxanthine. When pronuclear stage embryos were cultured in α-MEM, the rates of embryos reaching the 4-cell, blastocyst and hatched blastocyst stages were 96.5%, 75.4% and 66.7%, respectively. These values were significantly (P<0.01) higher than the rates of embryos cultured in Ham's F-10; 61.8%, 5.5% and 3.6% respectively. The deletion of CuSO4, ZnSO4, FeSO4 and hypoxanthine from Ham's F-10 significantly increased the rates of embryos reaching the 4-cell, blastocyst and hatched blastocyst stages to the extent comparable to those in α-MEM. In contrast, the addition of all or one of CuSO4, ZnSO4, FeSO4 and/or hypoxanthine to α-MEM significantly decreased the in vitro embryonic development. The strongest inhibition was observed when all of them were added. The developmental ability in α-MEM to which all of them were added was as low as that in Ham's F-10. These results suggest that the low developmental ability in Ham's F-10 may be mainly due to the deleterious effect of heavy metal ions and hypoxanthine. The toxic effect of heavy metal ions and hypoxanthine might be interpreted as the damage on embryos by an increased generation of oxygen radicals and the medium without constituents which may enhance the production of oxygen radicals seems to be desirable for the culture of mammalian embryos.
TL;DR: The processes of postnatal differentiation of seminiferous tubules from primitive sex cords; inhibition of germ cells entering mitosis, proliferation and distribution of the Sertoli cells, colonization of the gonocyte and elongation of the tubules, were defective in the hgn/hgn testis and might result in the loss of normal spermatogonia, leading failure of sperMatogenesis in the adult.
Abstract: In order to determine the initial alterations in histopathological and hormonal milieu in the male hypogonadism mutant rats (gene symbol: hgn, a single autosomal recessive trait), postnatal morphological development of the affected testis, and testicular testosterone (T) contents, plasma concentrations of T, follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL) were examined in the hgn/hgn and phenotypically normal male littermates (+/?) on days 0 to 21 after birth. The processes of postnatal differentiation of seminiferous tubules from primitive sex cords; inhibition of germ cells entering mitosis, proliferation and distribution of the Sertoli cells, colonization of the gonocyte and elongation of the tubules, were defective in the hgn/hgn testis. These histopathological alterations were present at birth. These defects might result in the loss of normal spermatogonia, leading failure of spermatogenesis in the adult. Severe progressive degenerations were observed in the tubular diameter and cells within the tubules on day 7 and afterwards, and the affected status seen in the adult was achieved by day 18. There have been marked elevations in the plasma FSH and LH levels from early postnatal age in the hgn/hgn male rat, while testicular and plasma T contents and PRL values were almost comparable between hgn/hgn and +/?.
TL;DR: The results suggest that the LH surge is induced by a surge release of Gn RH from the hypothalamus but ends despite a continued supply of GnRH.
Abstract: Neurosecretion of hypothalamic GnRH was monitored during the LH surge in ovariectomized goats given estradiol using microdialysis sampling. The microdialysis probe with a polycarbonate dialysis membrane was stereotaxically implanted in the median eminence, and was perfused with the artificial cerebrospinal fluid at a flow rate of 5ƒÊl/min under conscious and unrestrained conditions. The LH surge was induced by means of an intravenous infusion of estradiol (3 ƒÊg/h, i.v.) for 16 h and hourly fractions of microdialysis perfusate were collected for GnRH measurement. The perfusate GnRH showed a marked increase to peak levels of 25.3•}6.9 pg/ml over basal levels of 2.6•}1.1 pg/ml at the onset of the LH surge, and then gradually decreased so that the GnRH surge lasted longer than the LH surge. These results suggest that the LH surge is induced by a surge release of GnRH from the hypothalamus but ends despite a continued supply of GnRH.
TL;DR: The proposed vitrification procedure can be useful in the cryopreservation of mouse preimplantation embryos at 1-cell and morula stages and for blastocyst stage survival was significantly lower than that in control embryos.
Abstract: The aim of the work is to determine the susceptibility of mouse preimplantation embryos to vitrification at different developmental stages. The experiment was carried out in embryos at 1-cell, morula and blastocyst stages. As a vitrification solution, a mixture of 2.5 M dimethylsulphoxide + 2.5 M propylene glycol (DP), DP + 1.0 M sucrose (DPS) or DP + 0.16 M raffinose (DPR), was used. The in vitro survival, evaluated by the development into a late blastocyst stage in vitro, of control and vitrified mouse embryos was examined. A high survival rate (80%) after vitrification was obtained for 1-cell embryos after a 15 sec-exposure to DPS. High survival rates for morulae (91-96%) and relatively low survival rates for blastocysts (30-55%) were observed after vitrification in either DPS or DPR after short exposures (within 30 sec). In vivo survival rate for each embryos vitrified in DPS by a short exposure (within 30 sec) at 1-cell, morula or blastocyst stage was 56, 53 or 40%. Though the rates for 1-cell and molura stages were comparable with those in control embryos (55 and 58%), the rate for blastocyst stage was significantly lower (P<0.05) than that in control embryos (56%). Thus, the proposed vitrification procedure can be useful in the cryopreservation of mouse preimplantation embryos at 1-cell and morula stages.
TL;DR: The objective of the present investigation was to purify the early pregnancy factor (EPF) from swine pregnancy serum using diafiltration, ion-exchange chromatography, HPLC-gel permeation, and affinity chromatography to find the major active form of the swine EPF in this gestation period.
Abstract: The objective of the present investigation was to purify the early pregnancy factor (EPF) from swine pregnancy serum using diafiltration, ion-exchange chromatography, HPLC-gel permeation, and affinity chromatography. EPF activity was determined by using RIT after the pregnancy serum obtained from a mature swine 25 days after mating was divided into 2 parts (below M.W. 100KD and above M.W. 100KD) by the diafiltration. Using RIT, EPF activity was found in the fraction below M.W. 100KD. When this fraction was applied onto the DEAE-Shepharose column, the EPF activity was only found in the unadsorbed fraction. Next, this fraction was applied onto the CM-Sepharose column and was divided into 3 parts. A fraction which was eluted by 50 mM ammonium acetate buffer containing 50 mM NaCl had the EPF activity and the other fractions did not have this activity. The fraction which was eluted with 50 mM NaCl was analysed by SDS-PAGE and contained 6 bands between M.W. 30KD and 20KD. When the pattern of this SDS-PAGE was compared with the bands obtained from non-pregnant swine serum (day 0 of cycle, estrus), the 3 bands were not confirmed in the non-pregnancy serum. The molecular weight of these bands were 21KD, 23.7KD, and 26KD, respectively. On the HPLC analysis, EPF activity was found between M.W. 14 and 21KD. Therefore, it was thought that the swine EPF active material of M.W. 21KD might be the major active form of the swine EPF in this gestation period. In addition, the affinity gel which was coupled with Rabbit anti nonpregnancy swine whole serum lgG was effective for the purification of EPF.
TL;DR: The present result suggests that vole spermatozoa have a high ability to penetrate zona pellucida of heterologous eggs.
Abstract: Penetration of the zona pellucida of heterologous eggs by spermatozoa of Japanese field vole, Microtus montebelli, was examined in vitro. Spermatozoa from the vole epididymis were more active and survived longer in mKRB medium than did mouse spermatozoa. When vole spermatozoa were used for insemination, they passed through the zona pellucida of mouse and hamster eggs at a high rate. Although many spermatozoa invaded the perivitelline space of such eggs, penetration of the vitelline membrane was not confirmed. The present result suggests that vole spermatozoa have a high ability to penetrate zona pellucida of heterologous eggs.
TL;DR: Results indicate that capacitated mouse spermatozoa stored for 24 h at 24C can fertilize ova that subsequently develop normally through pre- and post-implantation stages.
Abstract: Capacitated mouse spermatozoa were stored for 24 to 72 h under the following conditions: 4C in air, 24C in air, 24C in 5% CO2 in air, or 37C in 5% CO2 in air. None or few of the oocytes inseminated with the capacitated spermatozoa stored for 24 h at 37C in 5% CO2 in air or 4C in air were fertilized. Fertilization rates of oocytes inseminated with capacitated spermatozoa stored for 24 h at 24C in 5% CO22 in air and 24C in air were 46% and 37%, respectively. When capacitated spermatozoa were stored at high concentrations, over 2×104 cells/μl, 87% of the oocytes were fertilized. Seventy-one percent of the fertilized eggs developed to term after transfer into the oviduct of recipients. These results indicate that capacitated mouse spermatozoa stored for 24 h at 24C can fertilize ova that subsequently develop normally through pre-and post-implantation stages.
TL;DR: A simplified procedure for bisection of bovine morulae and blastocysts involving a fine microsurgical blade and Dulbecco's phosphatebuffered saline (PBS) supplemented with 0.3 M sucrose and 0.1 mM ethylene diamine tetra acetic acid (EDTA) was employed to examine the developmental ability of the bisected demi-embryos as discussed by the authors.
Abstract: A simplified procedure for bisection of bovine morulae and blastocysts involving a fine microsurgical blade and Dulbecco's phosphate-buffered saline (PBS) supplemented with 0.3 M sucrose and 0.1 mM ethylene diamine tetra acetic acid (EDTA) was employed to examine the developmental ability of the bisected demi-embryos. This procedure did not require a holding pipet to restrain the embryo and only a single micromanipulator was needed. Bisected embryos were transferred to recipient heifers either as pairs of demi-embryos or as single demi-embryo to compare maintenance of pregnancy and fetal survival as to the incidence of identical twins. Blastocyst formation rate was significantly (P<0.05) higher (78.3% vs 60.5%) in embryos bisected in medium containing sucrose as compared to controls when early or mid-blastocyst stage embryos were bisected. Bilateral transfer of bovine demi-embryos generated more fetuses per original embryo than unilateral transfer of a single demi-embryo (105% vs 93%). Embryos producing identical twin pregnancies from sets of demi-embryos (bisected-twins) represented 30.7% of the total while 39.8% of embryos produced one pregnancy and the remaining 29.5% failed to result in pregnancy. The results show that bovine embryos can be bisected by a simplified method and lead to normal pregnancies with a high incidence of identical twins.
TL;DR: Cloned the Ren-1C gene from the C57BL/6 mouse and determined its genomic structure and characterized the structure and expression of the renin gene in embryonic stem D-3 cells and in its parental mouse strains, 129/Sv mouse.
Abstract: Renin is the rate-limiting enzyme in the renin-angiotensin system, the only known biochemical cascade that produces the potent octapeptide effector molecule angiotensin II that controls, to a large degree, cardiovascular homeostasis and reproductive function in mammals. In the present study, we have cloned the Ren-1C gene from the C57BL/6 mouse and determined its genomic structure. In order to provide the molecular basis for the application of the gene targeting method to the renin gene, we also characterized the structure and expression of the renin gene in embryonic stem D-3 (ES-D3) cells and in its parental mouse strains, 129/Sv mouse. The Ren-1C gene is 12 kilobases long and is composed of 9 exons interrupted by 8 introns. Renin mRNA was undetectable in ES-D3 cells but was found to be highly expressed in the submandibular gland of the 129/Sv mouse, in addition to the kidney. Southern blot analysis reveals that ES-D3 cells carry the additional duplicated renin gene, Ren-2, consistent with the classification of the 129/Sv mouse as a two-renin gene strain.
TL;DR: It is suggested that PMSG promotes early oocyte growth and zona pellucida formation of oocytes in neonatal mice, although it has a little effect on the proliferation of granulosa cells.
Abstract: Ovaries from newborn female mice were cultured for 4 or 8 days in Eagle's minimum essential medium supplemented with pregnant mare's serum gonadotrophin (PMSG) at a concentra-tion of 0, 2, 10 or 50IU/ml. At the beginning of culture, most oocytes in the ovaries were smaller than 20μm in diameter and no zona pellucida was formed around them. During 4 days of culture, promotion of oocyte growth by PMSG (10 and 50IU/ml) was observed. After 8 days, proportions of oocytes with a continuous zona pellucida were significantly higher in PMSG-supplementation groups. In the cultured ovaries, however, the multilayered granulosa cells, that could be often seen around growing oocytes in 8-day-old mouse ovaries, were not observed at all irrespective of the presence of PMSG. These results suggest that PMSG promotes early oocyte growth and zona pellucida formation of oocytes in neonatal mice, although it has a little effect on the proliferation of granulosa cells.
TL;DR: Among correlations between reproductive parameters and urinary component levels, litter size on the day of parturition and total pup weight on day 12 of lactation had the positive and the negative correlations with urinary levels of lactic acid and taurine, respectively.
Abstract: Urinary component level was examined by proton nuclear magnetic resonance (1H-NMR) spectroscopy 2-3 days before mating, on day 17-19 of pregnancy and on day 9-11 of lactation in SHN female mice. Urinary levels of taurine and betaine, both of which were found in milk, continued to decline through pregnancy and lactation. Creatinine and creatine decreased and increased, respectively, in the middle of lactation. Allantoin also increased at the end of pregnancy. There was little difference in the levels of urea and lactic acid at any stage examined. Concurrent pregnancy affected little any component level in the middle of lactation. Among correlations between reproductive parameters and urinary component levels, litter size on the day of parturition and total pup weight on day 12 of lactation had the positive and the negative correlations with urinary levels of lactic acid and taurine, respectively. The significance of these findings was considered and the possibility that the measurement of urinary components by 1H-NMR is a useful method to estimate the metabolic change of small experimental animals during reproduction was provided.
TL;DR: Determination of EPF in the serum is proved to be useful for monitoring the viability of the transferred embryo and indicates the occurrence of embryonic death.
Abstract: The viability of embryos transferred to 37 cows were monitored by detecting early pregnancy factor (EPF) in the bovine serum Embryos were transferred nonsurgically to the uterine horn ipsilateral to the ovary bearing the corpus luteum on day 7 to day 11 (day 0=day of estrus) The transferred embryos included 33 in vitro fertilized embryos; 3 fresh embryos, 24 freezed embryos, 6 freezed, thawed and cultured embryos, and 4 in vitro fertilized embryos which were freezed and thawed Blood samples were collected weekly after the embryo transfer for the EPF assay and progesterone determination Total pregnancy rate on day 42 to 43 obtained by ultrasonographic examination was 189% Percentage of cows with viable embryo on 7 and 28 days after the transfer was 595% and 189%, respectively This discrepancy indicated the occurrence of embryonic death All 7 cows in which EPF was detected on 28 days after embryo transfer were diagnosed with ultrasonographic examination as pregnant on 35 days after the transfer Determination of EPF in the serum is proved to be useful for monitoring the viability of the transferred embryo
TL;DR: The results suggest that the oviduct may have embryo growth promoting effect besides low oxygen tension, and that not only the normality of structural components but the inherent character of the oVIDuct may play an important role in the promotion of embryo development.
Abstract: To elucidate whether or not the promoting effects on embryo development is derived from the characteristic factors which the oviduct possesses, we cultured mouse embryos in the oviduct, the ureter and the vein, all of which have "ductal structures", and we compared the culture efficacy among 3 organs. Furthermore, we measured oxygen tensions within the 3 organs and performed an ultrastructural analysis of the three ductal organs after the culture. Embryos cultured in the oviduct developed to blastocysts at a significantly higher rate (13.5%) than those in the ureter (1.7%) and the vein (0%). The oxygen tensions of the inner lumen of all the ductal organs were relatively low, 36-45 mmHg, and no remarkable difference was observed among the 3 organs. These results suggest that the oviduct may have embryo growth promoting effect besides low oxygen tension. Furthermore, the ultrastructural results suggest that not only the normality of structural components but the inherent character of the oviduct may play an important role in the promotion of embryo development.