Normal Pups Research Articles

Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

358 MICROINSEMINATION USING MALE GERM CELLS FROM EPIDIDYMIDES AND TESTES STORED IN FREEZERS WITHOUT CRYOPROTECTANT

Cryopreservation of male germ cells is a strategy for the conservation of species and strains valuable to biomedical researchers. However, to minimize damage that may occur during freezing and thawing, complex cryopreservation protocols that have been optimized for the stage and species of the male germ cell are usually employed. This study was undertaken to see whether mouse male germ cells could be safely cryopreserved for later use by freezing the whole epididymides and testes without cryoprotectant. Furthermore, we examined whether frozen male germ cells maintained their fertilization ability after international transportation on dry ice. Epididymides and testes were collected from sexually mature male ICR and C57BL/6Cr mice and placed in polypropylene cryotubes. The cryotubes were frozen at -80�C with or without a freezing container, or were plunged directly into liquid nitrogen (LN2). They were stored at -80�C or in LN2 from between one week and one year. Epididymides and testes were thawed by placing the cryotubes in a water bath at room temperature. B6D2F1 and C57BL/6Cr oocytes were microinseminated with either epididymal and testicular spermatozoa or round spermatids. After embryo transfer into pseudopregnant females, normal pups were obtained irrespective of the method of cryopreservation and cell type used. However, their birth rates (2-33%) were lower than those of our conventional microinsemination using fresh sperm or spermatids (20-60%). For transportation experiments, testes were collected from C57BL/6J mice and placed in a cryotube. The cryotubes were frozen at -80�C in a freezing container. On the day of transportation, the cryotubes were placed in a polystyrene foam case filled with dry ice and were transported from Harwell (UK) to Tsukuba (Japan) by air and land. After three days, the samples were delivered to the recipient facility and were stored at -80�C until use (about 1 month). After thawing and collection of spermatogenic cells, C57BL/6J oocytes were microinseminated with either testicular spermatozoa or elongated spermatids. After embryo transfer, 24 (34% per transfer) and 8 (16%) offspring, respectively, were obtained from the two groups. These results indicate that mouse male germ cells retain their nuclear integrity even after freezing epididymides or testes in freezers without cryoprotectant. Since this cryopreservation technique is very simple and allows storage at -80�C for at least several months, it may enable transportation of mouse male germ cells internationally on dry ice, even when the senders are not specialized in cryopreservation.

322 DIFFERENTIAL DEVELOPMENT OF RABBIT EMBRYOS FOLLOWING MICROINSEMINATION USING SPERM AND SPERMATIDS

Microinsemination is a technique that delivers male germ cells directly into the ooplasm. The efficiency of fertilization and subsequent embryo development after microinsemination varies with species and the male germ cells used. This study examined the developmental ability of rabbit embryos in vitro and in vivo following microinsemination using haploid male germ cells at different stages. First, we injected rabbit spermatozoa, elongated spermatids, and round spermatids into mouse oocytes to assess their oocyte-activating capacity. Mouse oocytes are a good experimental model for assessing the oocyte-activating capacity of male germ cells from different species. The majority of mouse oocytes were activated irrespective of the stage of rabbit male germ cells injected (77, 61, and 73% for spermatozoa, elongated spermatids, and round spermatids, respectively). By contrast, these male germ cells activated homologous rabbit oocytes at rates of 100, 59, and 29%, respectively. After 120 h in culture, 69, 55, and 13% of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts, respectively. The rate of embryo development into blastocysts following round spermatid injection was significantly improved when oocytes were activated by an electric pulse shortly before microinsemination. The total number of cells was counted in embryos that reached the morula/blastocyst stages in culture using nuclear-staining with propidium iodide. The average cell number of embryos derived from elongated (89 ± 41; mean ± SD) or round spermatid (98 ± 34) injection was significantly lower than that of control embryos (in vivo fertilization) (211 ± 44) (P < 0.01). After 24 h in culture, some four- to eight-cell-stage embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from embryos involving sperm (4 offspring/16 transfers; 25%) and elongated spermatid (3/26; 12%) injection, but none from those involving round spermatid injection (0/68). These findings indicate that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. Immaturity of the nuclear genome or difficulty in coordinating the behavior of the male and female chromosomes might compromise embryo development.

P.8.004 Effects of hyperhomocysteinemia on the system of haemostasis under stress

Various dgrees of folate deficiency were produced inrats during gestation and lactation to evaluate maternal folate status effects during both the fetal and neonatal periods. Female rats were fed folate-deficient diets with 1% succinylsulfathiazole for 0, 5 and 9 wk before breeding and until 2 wk postpartum; others were fed a control diet. The most severely deficient group had significantly lower wt gain during gestation, with fetal resorption and no live pups being born. The moderately deficient group, with reduced plasma and red blood cell (RBC) folate concentrations prior to breeding, had impaired gestational wt gain, and, at gestational d 21, their rat fetuses weighed less than controls and had lower folate levels in blood, liver and placenta. The low birthweight pups in this group, at d 14 of age, had poor folate status with depressed brain DNA concentrations. The marginally deficient group produced normal birthweight pups, but growth was impaired at d 14, associated with poor folate status. Milk folate was significantly lower and urinary formiminoglutamic acid (FIGLU) was elevated in folatedeficient dams. These experimental results produced statistically significant correlation coefficients between folate status in rat dams and growth, hematology and folate stauus in their pups, even when folate status was marginal.

176 EFFECT OF HYPERTHERMIA ON DEVELOPMENTAL COMPETENCE OF GERMINAL-STAGE OOCYTES: IN VIVO AND EX VIVO STUDIES IN MICE

Mammalian oocytes are susceptible to thermal stress at various stages of follicular development. The present study was performed to determine the effects of maternal hyperthermia on germinal vesicle (GV)-stage oocytes in mice. Evaluation parameters included (a) the oocyte's ability to become fertilized, (b) ex vivo embryonic development, (c) pregnancy outcome, and (d) quality of offspring. Female mice (CB6F1) were synchronized with pregnant mare serum gonadotropin (PMSG) followed by hCG 48 h later. One hour after hCG administration, control mice were kept under normo-thermal conditions (22�C, 45% RH), whereas the treated mice were exposed to thermal stress (40�C, 70% RH) for 1.5 to 2 h to induce a rise of 1.8�C in rectal temperature. After a 1-h recovery period, both treated and control mice were paired with stud males overnight. In Exp. 1, mated mice were sacrificed 20 h after hCG administration and embryos were recovered and cultured in vitro. The number of putative zygotes recovered was counted, and cleavage and blastocyst-formation rates were recorded on Days 1 and 5 post-fertilization, respectively. Examination of oocyte developmental stage during hyperthermia induction revealed that oocytes were at the GV stage. The percentage of putative zygotes that cleaved into the two-cell stage was higher (P < 0.05) in the control (91.2 � 1.3) than in the treated group (84.5 � 1.7). In addition, blastocyst-formation rate was higher in the control (P < 0.05; 84.3 � 1.8%) than in the treated group (57 � 2.6%). In Exp. 2, females were left with the stud males until litter delivery. The date of delivery and the number of pups were documented. Eight-week-old offspring were submitted to behavioral (locomotor activity and episodic memory) tests. The one-way ANOVA procedure of JAMPIN (SAS Institute, Inc., Cary, NC, USA, 2004) was used for statistical analysis. The percentage of females that gave birth during the first and second estrous cycles after hyperthermia induction was 59% and 71% for treated and control groups, respectively. The average number of pups in the first estrous cycle was lower (P < 0.05) for the treated group, and only became equal to that in the control group in the third estrous cycle. Behavioral tests did not reveal any differences between pups from the two experimental groups. In summary, GV-stage oocytes from stimulated ovaries are highly sensitive to thermal stress, since induction of maternal hyperthermia during this stage disrupts oocyte developmental competence both in vivo and ex vivo. However, oocytes that survive thermal stress exhibit normal developmental potential, since heat-stressed mice produced normal pups, at least as reflected by offspring behavioral tests. Thus, strategies designed to protect the ovarian pool of oocytes from thermal stress are likely to improve fertility in heat-stressed females.

ALTERATION IN POSTNATAL DEVELOPMENT OF MASSETER INNERVATION IN HYPOTHYROID RATS

The thyroid hormones have profound effects on the development of neuromuscular system. These hormones exert their influence on both muscle fibers and related motoneurons during development. Masseter is one of the most important muscles for mastication in mammals. Thyroid hormone deficiency 3 weeks after birth, the period in which an alteration from sucking to biting occurs, could influence the developmental pattern of the muscle. Horseradish peroxidase was injected into the masseter (0.5-5 µlit, 40%) of normal and prenatal hypothyroid pups at 1,7,15 and 23 postnatal days. After 24-48 h, the 50µm brainstem sections were processed for TMB histochemical procedure. Labeled motoneurons in Mo5 were counted, measured and on the basis of their tracing intensity classified into heavy (H), intermediate (I) and light (L) groups. In each age group motoneurons were divideded into small, intermediate and large populations. In 1-day old pups, there was no significant difference between the labeling intensity and size of motoneurons in normal and hypothyroid rats. From the day 7, a significant decrease in the number of H motoneurons was observed (P

Pharmacological inhibition of phosphodiesterase 4 triggers ovulation in follicle-stimulating hormone-primed rats.

Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides to render them biologically inactive. As such, these enzymes are critical regulators of signal transduction pathways that use cyclic nucleotides as second messengers. PDE4 is one such member that has been identified in ovarian tissue and purported to have a role in the regulation of gonadotropin action. In the present study, selective PDE4 inhibitors enhanced intracellular signaling in a human LH receptor-expressing granulosa cell line. In vivo, PDE4 inhibition in FSH-primed rats resulted in ovulation, indicating that the PDE4 inhibitors can substitute for LH and human chorionic gonadotropin (hCG) in this process. Moreover, when coadministered with a subeffective dose of hCG, PDE4 inhibitors acted synergistically to enhance the ovulation response. Inhibitors of PDE3 or PDE5 had no ovulatory effect under similar conditions. Oocytes that were ovulated after PDE4 inhibition could be fertilized in vitro at a rate similar to that of oocytes from hCG-induced ovulation. Moreover, such oocytes were fully capable of being fertilized in vivo and developing into normal live pups. These results indicate that small molecule PDE4 inhibitors may be orally active alternatives to hCG as part of a fertility treatment regimen.

Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic–ischemic injury

Severe perinatal hypoxia–ischemia is an important cause of brain injury in both full-term and premature newborns, with a high risk of future behavioral and neurological deficits. The most commonly used animal model of neonatal hypoxia–ischemia is the unilateral ligation of the common carotid artery followed by exposure to hypoxia in 7-day-old rats. In spite of the wide use of this model, lot of contradictions and discrepancies exist between the results obtained by different laboratories regarding behavioral deficits and there are no data regarding the possible delay of the appearance of neurological reflexes and the time-course of reflex performances following neonatal hypoxic–ischemic injury in rats. In the present study we showed that neonatal hypoxia–ischemia retarded the development of somatic growth and several neurological reflexes (ear twitch, grasping, gait and negative geotaxis). Hypoxic animals also displayed retarded performance in righting, geotaxis and gait reflexes. Although hypoxic pups performed worse in most tests for motor coordination, they reached normal levels by 5 weeks of age except in the footfault test. In the open-field, hypoxic animals were generally more active, except at 3 weeks, when activity of normal pups increased enormously as well. Brain areas were significantly reduced in hypoxic animals, but no close correlation was found with behavioral deficits.

Increased incidence of spontaneous malformations in pups and increased litter size from pinealectomized dams.

The aim of the present study was to clarify whether maternal pinealectomy increases: (i) the incidence of spontaneous malformations in offspring; and (ii) litter size. More than 30 female rats in each of the control groups (normal and sham-pinealectomized) and the pinealectomized group were mated repeatedly with normal male rats and pups were autopsied mostly before weaning. No malformations were seen in offspring from the normal and sham-pinealectomized groups (n = 350 and n = 736, respectively). In contrast, in offspring from pinealectomized mothers (n = 1123), spontaneous malformations were found in five (taillessness in three and unilateral hydronephrosis or large renal cyst in the other two) or maybe six (unilateral renal hypoplasia in another) pups. This increased incidence of malformations in the latter group was statistically significant (P < 0.034 or 0.017 (Fisher's exact test), respectively). The frequency of still-born cases was not higher in pups born from pinealectomized mothers. The mean litter size was larger in the pinealectomized group compared with the control groups (P < 0.005-0.001, Student's t-test) at the first delivery (at approximately 100 days of age), but was not different at later deliveries at older ages. Our results suggest that the maternal pineal hormone suppresses: (i) the incidence of spontaneous malformations in offspring until mothers reach an old age; and (ii) litter size during the reproductively maturational phase of life.

The effects of pregnancy malnutrition on the development of insulin resistance in rat offspring

To investigate the effects of pregnancy malnutrition on the occurrence of insulin resistance (IR) in rat offspring during adult stage and to find out the relationship between TNF-alpha and IR; and to find out a reasonable early nutritional intervention measure for the prevention of IR, through giving different diets to offspring. An IUGR model was built by maternal nutrition restriction. 80 newborn IUGR female pups were randomly divided into 4 groups, the mother rats were given the following diet respectively for 3 weeks after delivery, pups were fed by mother milk: (1) The IUGR (intrauterine growth retardation) rat model was used and the animals were divided into: IUGR control group (group S/N) fed with normal diet, (2) IUGR high-caloric diet group (group A), (3) IUGR high-protein and high-caloric diet group (group B) and (4) IUGR high-protein isocaloric diet group (group C). Each group had 20 pups and another 20 normal female pups were fed with normal diet as the normal control group (group C/N). All pups were weaned at the 4th week of age and fed with normal diet till the end of the experiment. At the 12th week (adulthood) and 48th week (senility) of life, body weight and length, the fasting blood glucose, insulin concentration, TNF-alpha of adipose tissue and body weight were measured. Body mass index (BMI), ISI (insulin sensitive index), IRI (insulin resistant index) and HBCI (beta cell insulin excretion index) and their correlation to TNF-alpha were calculated. At 12th week and 48th week of life, the insulin sensitivity of IUGR model group was significantly lower than group C/N, although there was no significant difference of body weight between these two groups. TNF-alpha was negatively correlated with ISI, positively correlated with IRI and no relation to HBCI. Group A and B was fatter and developed more severe IR. There were no significant differences in ISI, IRI, HBCI and TNF-alpha between group C and group C/N. IUGR offspring of pregnancy malnutrition mother rats showed IR at the age of 12th week. TNF-alpha was closely related to the occurrence of IR in IUGR pups. IUGR pups fed with high caloric diet or high protein and caloric diet at the early postnatal period amplified the metabolic abnormality. The high protein isocaloric diet is effective early nutritional intervention measure for the prevention of occurrence of IR at adulthood.

Prolonged interval between parturition of normal live pups in a bitch

On day 64 after artificial insemination, a six-year-old primiparous briard bitch whelped three live pups between 05.00 and 08.00. It was presented at 11.00 on the same day with failure to complete parturition. On ultrasound examination, a normal live fetus was observed and the bitch was treated with oxytocin three times during the day (1.0, 2.0 and 2.0 iu intramuscularly), with no effect. The following day, a higher dose of oxytocin (5.0 iu) was administered intramuscularly at 11.00, after a uterine ultrasound examination confirmed viability of the fetus. At 18.00 of the same day, the bitch whelped the fourth normal live pup, 37 hours after initiation of parturition and 34 hours after expulsion of the last fetus. Effectiveness of oxytocin and normal versus prolonged parturition due to uterine inertia are discussed.

Microinsemination with first-wave round spermatids from immature male mice.

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.

221.Effects of exogenous gonadotrophin stimulation on ovarian tissue grafts in the mouse

Ovarian tissue grafts commonly contain only limited numbers of follicles. The functional life span and ability to retrieve as many mature oocytes as possible from ovarian grafts is important when grafting is used to restore fertility. This study aimed to determine whether ovarian grafts responded to exogenous hormones in a similar manner to that of in situ ovaries. Ovaries of C57BlxCBA F1 mice were cut in half and grafted to one of three different graft sites in females of the same F1 line; bursal capsule (BC, n = 12), kidney capsule (KC, n = 6), subcutaneous tissue (SC, n = 24). Three weeks after grafting, half of the graft recipients in each group were treated with 5IU PMSG followed by 5IU hCG 48 hours later. Oocytes were collected directly from the grafted ovaries 10 hours after the hCG injection and fertilized in vitro. Oocytes from the ovaries of superovulated normal mice (n = 4) of the same hybrid strain were used as controls. Two-cell embryos were transferred to pseudopregnant recipients and collected at day 15 of gestation or the animals were allowed to go to term. Mature fertilisable MII oocytes were retrieved from stimulated grafts from all graft sites, however, the number (BC 9, KC 5, SC 2 oocytes per ovary) and proportion of two-cell embryos in each grafted group (BC 52%, KC 32%, SC 32%) was significantly (P &lt; 0.05) lower than in the in vivo matured control (16 oocytes, 85% two-cell). The fetal and placental weights of fetuses produced from graft-derived oocytes were not significantly different to the control group. Phenotypically normal pups were born in each of the graft and control groups. In conclusion, ovarian grafts treated with exogenous gonadotrophins produce significantly fewer mature oocytes and two cell embryos compared to in situ ovaries. Work supported by ARC and NIH RFA.

Effect of transient embryonic in vivo exposure to the endocrine disruptor methoxychlor on embryonic and postnatal testis development.

The current study was designed to examine the effects of a transient embryonic exposure to the pesticide methoxychlor, an endocrine disruptor, on in vivo rat testis development and function. Gestating female rats were transiently administered methoxychlor (MXC) from embryonic day 7 (E7; EO = plug date) through E15. Embryonic testes were collected at E16 and postnatal (PO = day of birth) testes at P4, P10, P17-20, and P60. Seminiferous cords formed in testes from MXC exposed males. However, at E16, there was a decrease in the area of cords and an increase in interstitial area in MXC exposed testes when compared with controls. At all postnatal ages collected, there did not appear to be differences in seminiferous cord/tubule area, interstitial area, or number of seminiferous cords/tubules between untreated controls and males exposed to MXC. Exposure to the endocrine disruptor also had no effect on the postnatal organ weights of a variety of different organs, nor were testosterone levels altered. Interestingly, there were reductions in the number of germ cells in testes from MXC-exposed males at P17-P20 when compared with untreated controls. Furthermore, there was a twofold increase in apoptotic cells in tubules from pubertal P17-P20-MXC exposed males when compared with untreated controls. Testes were collected from adult P60 males to determine if early embryonic and postnatal alterations in germ cell numbers or testis cellular composition had compromised spermatogenesis. In adult P60 MXC exposed testes there were no gross morphological changes in testis structure or cellular composition over that of controls. However, there was an increase in apoptotic cell number in elongating spermatids in MXC exposed testes. Four P60 males that were exposed to MXC during gestation and 4 control males were bred with unexposed females to determine their ability to produce offspring. All MXC exposed males were capable of impregnating females and had normal litter size and pup weights. Combined observations demonstrated that exposure to MXC during gestation at a critical stage of testis development (ie, sex determination) affects embryonic testis cellular composition, germ cell numbers, and germ cell survival. While alterations in these parameters does not affect the ability of males to produce offspring, there appears to be a reduced spermatogenic capacity associated with MXC treatment. Therefore, transient embryonic exposure to an endocrine disruptor (methoxychlor) during gestation can influence the germline and fertility in adult males.

Role of Hypoxia during Normal Retinal Vessel Development and in Experimental Retinopathy of Prematurity

To test the hypothesis that retinal hypoxia is present during vascular development in normal rat pups and in a newborn rat model of retinopathy of prematurity (ROP). Preretinal vitreous PO(2) measurements were made during room air breathing using (19)F magnetic resonance spectroscopy (MRS) and a perfluoro-15-crown-5-ether droplet in normal adult and newborn (postnatal day [P]1-P20) rats, and in newborn rats exposed first to 14 days of variable oxygen (before NV) and six additional days in room air after variable oxygen exposure (during NV). After each experiment, blood gas values were measured, and retinas were isolated. Retinas were adenosine diphosphatase (ADPase) stained, and flatmounted to determine peripheral avascular extent and NV incidence and severity. In the vascularized rat retina, no significant difference (P > 0.05) was found between the droplet-derived preretinal vitreous oxygen tension (24 +/- 2 mm Hg, mean +/- SEM, n = 18) and previously reported electrode-measured oxygen tension (22 +/- 1 mm Hg). Only during normal retinal vessel growth (P1-P10) and before the appearance of NV was evidence for retinal hypoxia found at the border of the vascular and avascular retina. However, the mean PO(2) (range, 24-28 mm Hg) over the vascular retina was not different (P > 0.05) between any of the newborn rat groups in this study. (19)F MRS of a perfluorocarbon droplet provides an accurate measure of preretinal vitreous oxygen tension in rats. These data support an important role of physiologic hypoxia in normal retinal circulatory development and raises the possibility that, in experimental ROP, retinal hypoxia is a necessary but not sufficient condition for the development of retinal NV.

Central blockade of oxytocin receptors during late gestation disrupts systemic release of oxytocin during suckling in rats.

There is evidence that the central oxytocin system is activated and undergoes reorganization before parturition. The present study was designed to determine the effects of central oxytocin receptor blockade during late gestation on parturition, pup growth, and oxytocin release during suckling. Female Sprague-Dawley rats were implanted on gestation day 12-14 with Alzet osmotic minipumps containing an oxytocin receptor antagonist (d(CH2)5, Tyr(Me)(2), Orn(8)-vasotocin; OT-X) or artificial cerebrospinal fluid (VEH), which was infused into the third cerebral ventricle. Pumps were removed within 24 h of parturition. Daily maternal body weight and food intake were monitored during gestation and lactation. The length of gestation, duration of parturition, pup number, litter weight and interbirth interval were recorded. Subsequently, pup number and litter weights were recorded daily until lactation day 10 or 11, when maternal and pup behaviour, and plasma oxytocin concentration before and during suckling were measured. Central oxytocin blockade had no effect on the timing of parturition, maternal behaviour, litter size, still births, or litter weights at birth. However, beginning on day 3 of lactation, average weights of litters of OT-X females were significantly lower than litters of VEH-treated females. Furthermore, while basal plasma oxytocin concentrations, oxytocin increases in response to suckling and dam/pup interactions did not differ between groups, a significant delay in suckling-induced systemic oxytocin release was observed in OT-X females. Finally, OT-X dams weighed less than VEH dams during the postpartum observation period, although food intakes were similar. These data suggest that central actions of oxytocin during late gestation are necessary for the normal timing of systemic release of oxytocin during suckling, normal pup weight gain, and maintenance of maternal body weight.

Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning.

Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 x 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 +/- 2.1%) was obtained with (B6 x 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 x JF1)F1 and (JF1 x 129)F1 genotypes, whereas no pups were born from cells with the (B6 x JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.

Passive and adaptive immunity alters enterocyte-specific gene regulation in developing mice

Background: Breast-feeding decreases the incidence and seventy of many disorders. It is thought that passive immunity (PI) & the infants own adaptive immunity (AI) play an important role in this process, however, the underlying mechanism of how they protect against these various disorders is poorly understood Objective: We hypothesized that the influence of PI & AI on the regulation of enterocyte specific genes that code for the transport of immunoglobulins, such as plgR & FcI~n, & nutrient processing, such as Ghit5, SGLT1, Lactase & IBAT play a major role in the nrevention of disease. To test the hypothesis, we developed a mouse model where normal'& immunodeficient pups were born & nursed by immunodeficient or normal dams Design/Methods: Mice were immunodeficient, RAG-1 knockouts (ID), or wild type, C57BL6 mice (normal). RNA was isolated from the proximal & distal small intestine & liver throughout weaning. RNA protection assays were then performed with FcRn, plgR, Glut5, Lactase, IBAT & SGLT 1 riboprobes Results: All transcripts studied were developmentally regulated around the time of weaning, & the transcripts in the proximal small intestine had the highest level of expression of the three tissues studied. In normal pups reared in the absence of ~ , the most significant changes from those reared in the presence of P1 were seen in the expression of plgR, which was increased almost 2fold, & a significant decrease in Lactase during the weaning process. When ID pups reared in the presence of PI were compared to normal pups reared in the presence of P1, there were significant decreases in the expression of plgR & Lactase, and also significant increases in expression levels of FcRn, Gfut5 & IBAT during weaning. When ID pups reared in the absence of PI were compared to normal pups reared in the presence of PI, there was a significant decline in expression of plgR & Lactase, & also a significant increase in the expression of IBAT. The expression levels of SGLT1 were not changed significantly by any of the experimental conditions Conclusions: This murine model allows us to simultaneously evaluate the role of passive & adaptive immunity in controlling the expression of enterocytespecific transcripts and altering the various physiologic functions during breast-feeding Finally, we demonstrate that both adaptive & passive immunity influence the developmental expression of genes that may be involved in the protective effect of breast-feeding.

Milk intake and survival in newborn cannabinoid CB 1 receptor knockout mice: evidence for a “CB 3” receptor

Cannabinoids, whether plant-derived, synthetic or endogenous, have been shown to stimulate appetite in the adult organism. We have reported previously that cannabinoid receptors play a critical role during the early suckling period: The selective cannabinoid CB 1 receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide (SR141617A) permanently prevented milk ingestion in a dose-dependent manner, when administered to (Sabra, albino) mouse pups, within 1 day of birth. As a consequence, these pups died within the first week of life. We now generalize this finding to a different strain of mice (C57BL/6). Further, we show that cannabinoid CB 1 receptor blockade (20 mg/kg SR141716A) must occur within 24 h after birth as injection of SR141716A into 2- or 5-day-old pups had a much smaller effect or no effect at all, respectively. Cannabinoid CB 1 receptor knockout mice did not ingest milk on the first day of life, similarly to SR141716A-treated normal pups, as measured by the appearance of “milkbands”. However, the knockout pups started to display milkbands from day 2 of life. Survival rates of cannabinoid CB 1 receptor knockout mice were affected significantly, but to a lesser extent than normal pups, by the administration of SR141716A. Daily administration of the endocannabinoid 2-arachidonoyl glycerol, or the synthetic agonists ( R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2, 5 mg/kg) or (−)- cis-3-[2-Hydroxy4-(1,1-dimethylheptyl) phenyl]- trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940, 5 or 20 mg/kg) did not promote survival or weight gain in CB 1 −/− pups. Our data support previous evidence for a critical role of cannabinoid CB 1 receptors for the initiation of suckling. Further, the present observations support the existence of an unknown cannabinoid receptor, with partial control over milk ingestion in newborns. Our data also suggest that the CB 1 −/− neonates possess a compensatory mechanism which helps them overcome the lack of cannabinoid CB 1 receptors.