The Placenta plays a pivotal role in the maternofetal exchange of substances. In the chorioallantoic placentas of rodents, maternal and fetal bloods come close in the labyrinth. Hemotrichorial placenta is formed in most of rodents including rat, mouse, and hamster. Three trophoblast layers composed of a single cytotrophoblast layer and two syncytiotrophoblast layers and endothelium of fetal capillaries lie between maternal and fetal circulations in the labyrinthine wall. Many gap junctions are present between syncytiotrophoblast layers. The double-syncytiotrophoblast layers connected by gap junctions serve as a structural basis of the placental barrier as well as a site of specific transfer of various substances. In the guinea pig placenta, only a single syncytiotrophoblast layer is formed, thereby it is classified as a hemomonochorial placenta. Endotheliochorial placenta was reported in the kangaroo rat. The ultrastructural features of the labyrinthine wall are described and discussed in relation to specific transplacental transfer of substances, especially glucose.

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The developmental ability of reconstituted embryos that received nuclei from 2-, 4- and 8-cell embryos at M phase of the cell cycle was examined in vitro and in vivo. After fusion of M phase nuclei with enucleated oocytes, the reconstituted oocytes were artificially activated and cultured with cytochalasin B to achieve formation of two “pronuclei-like nuclei” (PN-like nuclei). To obtain embryos with a diploid set of chromosomes, nuclei from each reconstructed embryo were transferred individually into separate enucleated fertilized 1-cell embryos. These nuclear transplants developed to multicellular stages at higher rates and the number of reconstituted embryos doubled. Of the embryos reconstituted with 2-, 4-, and 8-cell nuclei at M phase, 26 (23/88), 40 (32/80) and 4% (5/135) developed to blastocysts, respectively. Live young were produced from blastocysts obtained from reconstituted embryos that received 2- and 4-cell nuclei at M phase, after transfer into recipient females.

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In the present study, we investigated changes in plasma concentrations of inhibin, estradiol-17β, progesterone and FSH in the golden hamster (Mesocricetus auratus) superovulated by equine chorionic gonadotropin (eCG) treatment. In addition, the hamsters were injected at various times with human chorionic gonadotropin (hCG) to determine the follicular development after treatment with eCG. After treatment with 30 IU eCG at 1100 h on day 1 of the estrous cycle (day 1=day of ovulation), superovulation (36.5 ± 1.1 v.s. 13.5 ± 0.4 in controls treated with saline) occurred on the expected next day 1. Plasma concentrations of inhibin and estradiol-17β increased and plasma concentrations of FSH decreased in the animals given 30 IU eCG. Plasma concentrations of progesterone also increased after eCG treatment; however, they decreased to control levels within 48 h after eCG injection. The number of follicles capable of ovulating after hCG administration was dramatically increased within 36 h after eCG injection (43.4 ± 3.4). Thereafter, the number of follicles capable of ovulating decreased to about a half of the peak value during the next 6 h period, then increased gradually to the numbers of ovulations observed on the next day 1. Plasma inhibin and estradiol-17β were augmented and plasma concentrations of FSH were suppressed in the animals in which follicular development was stimulated, though there is a difference in the changing pattern between plasma inhibin and estradiol-17β. The present results also suggest that plasma concentrations of inhibin may be an indicator for the number of developing follicles and estradiol-17β may be an indicator for follicular maturation.

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The mouse myogenin gene carrying the LacZ gene coding for β-galactosidase was microinjected into fertilized eggs of dystrophic (dy/dy) mice. Concentrations of the LacZ gene varied between the fast and slow regions of skeletal muscle. The slow or gastrocnemius muscle region, exhibited unusually high levels of the LacZ gene compared to the levels found in the fast muscle region which is also known as the extensor digitorum longus muscle (EDL). In addition, morphological studies indicated that a maturational defect was present in the dystrophic dy/dy EDL muscle.

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The aromatic hydrocarbon receptor (AHR) mediates the toxicity of several halogenated aromatic hydrocarbons, including 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In order to better understand the systemic endocrine disruption induced by AHR agonists, we examined the regulation of AHR by exposure to TCDD during gestation and lactation. Pregnant rats were given an oral dose of 1 μg TCDD/kg body weight or vehicle control on day 15 of gestation, and female pups sacrificed on post-natal day 21. Total RNA from ovaries, uteri, and hypothalami was immobilized on a nylon membrane and probed with a murine AHR cDNA. The gel mobility-shift assay was used to asses changes in the DNA-binding activity of AHR in vitro. Ovarian AHR mRNA and DNA-binding capability were decreased 2.1- and 1.7-fold, respectively; while in the uterus, there was a 1.3- and 1.5-fold increase in message and DNA binding. Hypothalamic mRNA for AHR showed no change, while there was a 1.7-fold increase in DNA-binding activity. Our results show that in-utero and lactational exposure to TCDD alters the expression of AHR in a tissue-specific fashion, and that this altered regulation may either be a part or a cause of the general endocrine disruption observed in these animals.

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This study was undertaken to determine the relationship between the first cleavage division of in vitro produced (IVP) bovine embryos and the following few cleavage divisions in culture. The “early-”, “intermediate-“ and “late-” cleaving embryos presented at 22, 26 and 30 h post insemination (hpi) were cultured separately until they were fixed and stained with Hoechst 33342 at the end of each culture period (30, 48, 72, 96 and 120 hpi). Distributions of embryos that underwent the first cleavage at 22, 26 and 30 hpi were 21.5, 34.7 and 10.0%, respectively. No significant difference among the mean number of cells in the 3 groups was observed until 48 hpi. At 72 hpi, the number of cells in the early-cleaving embryos (10.8 ± 0.5) was higher than those in the intermediate- and late-cleaving embryos (8.7 ± 0.4 and 6.5 ± 0.6, respectively). Higher numbers of cells were also observed in the early-cleaving embryos (18.0 ± 1.5) at 120 hpi, compared with those in the intermediate- and late-cleaving embryos (12.5 ± 0.8 and 11.4 ± 1.5, respectively). More than 80% of the early- and intermediate-cleaving embryos had completed the second cleavage division at 48 hpi, whereas that of the late-cleaving embryos was lower (56.5%). No difference in the proportion of >8-cell embryos among the 3 groups was observed at 48 hpi (22.9 vs 24.7 vs 26.1%). However, the completion of the third cleavage in the early-cleaving embryos was higher than in the intermediate- and late-cleaving embryos at 72 hpi (65.4 vs 49.4 vs 25.0%), and from there onwards (96 hpi; 81.1 vs 63.2 vs 45.5%, 120 hpi; 83.6 vs 62.2 vs 65.0%). The proportion of >16-cell embryos in the early-cleaving embryos was also higher than that of the intermediate- and late-cleaving embryos between 96 hpi (18.9 vs 2.9 vs 0.0%) and 120 hpi (49.1 vs 22.0 vs 25.0%). Our results suggest that the development of IVP bovine embryos is possibly controlled as early as at the 2-cell stage, and the differences between the early and late cleaving embryos are associated with 1) the developmental delay or arrest of embryos during the second cleavage, and 2) the lengthening of the third cleavage, which seems to be related to the subsequent developmental ability of the embryos.

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Intraperitoneal administration of all-trans retinoic acid (RA) to pregnant mice on 8.5 or 10.5 days post coitum (dpc) resulted in malformed fetuses in all litters in a dose-dependent and developmental stage-specific manner. The pregnant mice were injected with RA dissolved in dimethyl sulfoxide, at a dose of 25 or 50 mg/kg of body weight on 8.5 or 10.5 dpc. In the 8.5 dpc-50 mg/kg treatment group, all fetuses had tail anomalies and vertebral malformations, and some fetuses showed craniofacial anomalies such as exencephaly, spina bifida, micrognathia and agenesis of auricles. Excessive cell death was seen in the tail bud 24 h after RA injection. In the 8.5 dpc-25 mg/kg treatment group, however, fetuses showed palliation of these defects. In the 10.5 dpc-50 mg/kg treatment group, limb disorders were induced, but the tail showed normal morphology. The present results indicate that RA-inducible malformation in mouse embryos is dose-dependent and developmental stage-specific, and these teratogenicity may correlate with excessive cell death. RA is considered to affect rapidly growing parts of embryonic tissues and causes disorders in normal pattern formation of embryonic tissues.

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Mastomys (Praomys coucha) is a small murine rodent native in Africa. The present study was undertaken to investigate the developmental time course of mastomys embryos in vivo and the rate of development in vitro. Mastomys embryos developing in vivo exhibited a long 2-cell stage, which lasted from late Day 1 through Day 3. At Day 4, most embryos developed to the 8-cell or morula stages and at Day 5, all embryos reached the blastocyst stage. The attachment of embryos to the endometrium began at Day 5, as revealed by histological examinations. The development in vitro was investigated in different media with three inbred strains and an F1 hybrid. When 1-cell embryos from inbred mastomys were cultured in Whitten's medium (WM), none of them (n=61) developed beyond the 2-cell stage. Embryos of the same strains collected at Day 2 (2-cells) reached the blastocyst stage (46%, 31/67) in the same medium. Meanwhile, 1-cell embryos from F1 hybrid mastomys developed to the blastocyst stage in WM (13%, 13/71), indicating the presence of a strong heterosis effect. CZB medium, which lacks glucose and contains glutamine and EDTA, supported the development of 1-cell inbred embryos to the blastocyst stage (24%, 17/70). Supplementation of glutamine, but not EDTA, to WM stimulated the development of 1-cells to the blastocyst stage (22%, 11/51). These findings indicate that the development of mastomys embryos in vitro is both strain and medium dependent and that glutamine may play a crucial role in early development of mastomys embryos.

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Pig immature oocytes were cultured for 24, 36 and 48 h in serum-free maturation medium with or without 0.57 mM cysteine. The addition of cysteine to the medium was associated with increased glutathione synthesis by oocytes but did not promote nor inhibit nuclear maturation, sperm penetration in vitro, and decondensation of sperm nuclei in penetrated oocytes. The incidence of activation of penetrated oocytes 14 h after insemination in vitro was lower in those cultured both in the presence and absence of cysteine for 24 h than 36 and 48 h. The lower ability of oocytes cultured for 24 h to be activated was not improved by neither the addition of cysteine (0.57 mM) in fertilization medium nor prolonged culture time after insemination. However, male pronuclear formation in activated oocytes after sperm penetration at any stages of maturation was largely accelerated when cysteine was added to maturation medium. An increased concentration of glutathione may induce full decondensation of sperm nuclei in immature pig oocytes penetrated in vitro ensuring transformation of the decondensed sperm nuclei to male pronuclei only in synchronization with oocyte activation.

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The production of transgenic animals with YAC constructs is required for overcoming position effects on transgene expression. The preparation of YAC DNAs for microinjection is a critical factor in the production of transgenic animals carrying large YAC DNA inserts. We report a method for the preparation of 210-400 kb intact YAC DNAs for microinjection. We screened four YAC clones containing the α-lactalbumin gene, glutathione S-transferase alpha class gene, hepatocyte growth factor gene and casein gene family from a human YAC library, and analyzed the location of the genes in the YAC inserts to determine which fragmentation vectors should be used. YAC clones were transferred from their hosts to the CGY2516 yeast strain and fragmented to appropriate lengths to allow YAC copy number amplification. The YAC DNA was prepared and concentrated by filter centrifugation. Two-five ng/μl of intact purified YAC DNA was obtained. The YAC DNA was microinjected into rat embryos and transgenic rats were produced as efficiently as with conventional plasmid constructs.

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Monozygotic mouse twins were produced by micromanipulation of 2-cell stage embryos and factors affecting the embryonic development were examined. In experiment 1, 2-cell embryos obtained from superovulated CD-1 strain or F1 (C57BL/6 × CBA) females mated with CD-1 strain males were separated into a single blastomere. Pairs of twin blastomeres were cultured with or without supplementation of growth factors, a mixture of IGF-1, TGF-β and EGF for 3 days and twin blastocysts developed were transferred to recipient females. Of 174 pairs of F1 embryos, 88 to 100% developed to twin blastocysts. In contrast, development of CD-1 embryos (835 pairs) was significantly lower (25 to 36%). Of 63 F1 twin blastocysts transferred, 7 to 29% developed to twin fetuses, whereas only 0 to 8% of 62 pairs of CD-1 embryos developed to twins. In experiment 2, one nucleus of a 2-cell stage F1 embryo was electrically fused with an enucleated recipient 2-cell embryo. The 2-cell embryo from which a nucleus was removed was cultured together with the reconstituted embryo as a monozygotic twin pairs. Of 24 and 28 twin pairs cultured with or without growth factors, 92 and 86% developed to twin blastocysts, respectively. After transfer of 13 twin blastocysts to recipients, 3 and 2 twin fetuses were obtained. These findings indicated that monozygotic twin mice can be produced more efficiently from F1 embryos than from CD-1 embryos. Methods of removal of zona pellucida and addition of growth factors to the culture medium had no effect on the efficiency of production of monozygotic twins.

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Seasonally or postpartum anestrous Suffolk ewes were divided into the following 4 groups: Groups 1 (barren, n=5), 2 (postpartum/non-lactating, n=5) and 3 (postpartum/lactating, n=5) ewes were fed 4 mg melatonin daily for 60 days from 26 June (Day 1), and control ewes in Group 4 (postpartum/lactating, n=3) were fed vehicle pellets not containing melatonin. The plasma concentration of progesterone was monitored to assess the resumption of ovulatory cyclicity. The ewes were teased by a vasectomized ram daily on Day 4 onward, and introduced to fertile rams on Day 41. The resumption of ovulatory cyclicity in postpartum ewes fed melatonin were significantly earlier than that in postpartum ewes in the control (p<0.05). However, there was no difference in the timing of the onset of ovulatory cyclicity and return to estrus between non-lactating and lactating ewes treated with melatonin. The interval from the start of melatonin feeding to the onset of ovulatory cyclicity in postpartum ewes was longer than that in barren ewes (p<0.05). These results suggest that the physiological status in early postpartum ewes results in a delay of the reproductive response to melatonin administration mimicking the short-day pattern, but that the lactational suppression of gonadal function could be overcome by the melatonin treatment.

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Sequential sampling of hypophyseal portal blood was performed in the Shiba goat. The technique for portal blood collection originally developed for sheep was adapted with some modification. Portal blood was withdrawn by lesioning a part of the pituitary portal vessels via a collection apparatus placed beforehand at the rostral surface of the anterior pituitary gland. In four ovariectomized goats, portal blood was collected successively at 5 min intervals for several hours to examine the neurosecretory dynamics of hypothalamic GnRH. A pulsatile pattern of GnRH discharge into the pituitary portal circulation was clearly observed. Mean ± SD inter-pulse intervals in each of the 4 animals were 29.0 ± 2.0 min, 38.8 ± 2.2 min, 29.3 ± 1.7 min and 23.9 ± 2.1 min. Each GnRH pulse was associated with an LH pulse, and temporal correlation between the GnRH and LH pulses was consistent despite differing pulse frequencies among individual animals. Thus the technique for continuous pituitary portal blood collection from conscious goat has become available.

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