bluuebunny/crossref_metadata_2025 huggingface dataset:
The metabolism of glutamine by the preimplantation sheep conceptus and its interaction with glucose
Authors: Wales RG , Du ZF
Published: October 2005

The metabolism of glutamine and glucose, separately and in combination, by the sheep conceptus recovered on Days 2, 6, 13, 15, 17, and 19 of pregnancy was assessed over 2.5 h. At Day 2, the production of CO2 from glutamine was similar to that from glucose, with additive effects seen when both substrates were present. Between Day 2 and Day 6, there was a three-fold increase in glucose oxidation but no change in the oxidation of glutamine. From Day 13 to Day 19, the oxidation of glutamine was relatively high in embryonic tissue, low in trophoblastic tissue and intermediate in the yolk sac but in all tissues decreased as development progressed. Over this latter period the oxidation of glutamine was reduced to approximately 50% by the addition of glucose to the medium but glucose oxidation was unaffected by the addition of glutamine. At the early stages of development, the incorporation of substrate carbon from glutamine was less than that from glucose but in each case, incorporation into the acid-insoluble macromolecular fraction increased 2-3 times between Day 2 and Day 6. Incorporation of glutamine into the Day-17 and Day-19 conceptus was also measured; embryonic tissue exhibited the highest rate of incorporation and trophoblastic tissue the lowest. Incorporation was lower on Day 19 than on Day 17 and the proportion of carbon isolated in the acid-insoluble fraction represented 20% of the total incorporated. At no time did the addition of glucose to the medium alter incorporation of glutamine into either embryonic tissue or extraembryonic membranes.
Removal of the zona pellucida and parthenogenetic activation affect rates of survival of ultrarapidly frozen mouse oocytes
Authors: Lai AC , Ryan JP , Saunders DM
Published: October 2005

Survival rates on thawing were assessed for ultrarapidly frozen mouse oocytes following the removal of zonae pellucidae using an acid Tyrode's solution, pronase or a mechanical dissection technique. Significantly higher rates of survival were observed for zona-free oocytes than for zona-intact control oocytes (303/684, 44% v. 130/498, 26%; P < 0.001). The rates of survival observed for pronuclear stage embryos (72-76%) were much greater than those observed for oocytes and were not influenced by zona removal techniques. Parthenogenetic activation of oocytes by exposure to a 7% (v/v) ethanol solution was also shown to increase survival rates of ultrarapidly frozen oocytes (155/185, 84% v. 19/102, 19%) indicating that fusion of sperm to the plasma membrane or formation of a male pronucleus are not directly responsible for the increased survival rates of pronuclear stage embryos compared with oocytes. These data support the hypothesis that increased survival of ultrarapidly frozen pronuclear stage embryos is the result of changes to the plasma membrane and/or to the zona pellucida that occur following fertilization.
Localization of neutral endopeptidase in the ovine uterus and conceptus during the oestrous cycle and early pregnancy
Authors: Riley SC , Wong E , Findlay JK , Salamonsen LA
Published: October 2005

Neutral endopeptidase (NEP; EC 3.4.24.11), an enzyme which metabolizes several peptides (including oxytocin and endothelins) implicated in the control of uterine function, was found to be localized in the ovine uterus throughout the oestrous cycle and in the uterus and conceptus during early pregnancy, using immunohistochemical techniques. Positive NEP immunoreactivity was found in the endometrium principally in stromal cells, in the vasculature in endothelial and vascular smooth muscle cells, and also weakly in some glandular epithelial cells. In a layer of stromal fibroblasts several cells in thickness underlying the luminal epithelium, staining was much weaker than that in the deeper stromal cells throughout the period examined. NEP staining was also present in smooth muscle cells of the myometrium at all times, and was most intense in the layer of cells adjacent to the endometrium. In the conceptus, NEP immunohistochemical staining was found in uninucleate cells, but not in binucleate trophoblast cells, in epithelial cells of the allantois and amnion, and in the heart and brain of the Day-20 embryo. In ovariectomized ewes treated with oestrogen or progesterone separately or remaining untreated, immunohistochemical staining of NEP was stronger when compared with intact ewes, in caruncular and intercaruncular stroma and epithelia, in glands, in the vasculature and in myometrium. The staining was less intense in all cell types in ewes receiving oestrogen plus progesterone. The expression of NEP and its specific regionalization within the uterus indicate a mechanism by which the availability of biologically important peptides involved in the regulation of the oestrous cycle and implantation, including oxytocin and endothelin, can be controlled by regulation of their metabolism.
Characterization of glucose transport in preimplantation mouse embryos
Authors: Gardner HG , Kaye PL
Published: October 2005

Membrane transport of glucose divorced from metabolism, was analysed in 2-cell embryos, morulae and blastocysts in the preimplantation mouse. A non-metabolizable radiolabelled analogue, 3,0 methyl D-glucose (3OMG) was used, and glucose was used as well in morulae and blastocysts; incubation times were < or = 5 min. Uptake occurred by combination of a non-saturable process, resistant to cytochalasin-B, and a facilitated process exhibiting classic Michaelis-Menten kinetics. The rate constant for the non-saturable component increased from 1.22 +/- 0.12 pL embryo-1 min-1 in 2-cell embryos to 2.08 +/- 0.44 pL embryo-1 min-1 in blastocysts, determined using 3OMG. The Km values of the saturable component for 3OMG at 22 degrees C were relatively constant at about 6.5 mM in 2-cell embryos, morulae and blastocysts. At 37 degrees C, the Km increased from 6 mM in 2-cell embryos to 17 mM in blastocysts. Vmax increased about five-fold during development from the 2-cell stage to the morula stage and about three-fold during development to the blastocyst. The Km values for glucose in morulae and blastocysts were constant at about 1.3 mM at 37 degrees C. Uptake of 3OMG in blastocysts was inhibited by glucose and stimulated by incubation in glucose-free medium. There was no kinetic evidence for the participation of multiple saturable components in uptake by blastocysts or morulae. This supports the observation that the glucose transporter GLUT2, which is first expressed at the 8-cell stage to supplement GLUT1 expressed in the oocyte, does not contribute to the uptake of environmental glucose and is, therefore, probably restricted in expression to abcoelic membrane areas of the trophectoderm. Together with the known values of glucose in uterine fluid, the kinetic data indicate that most glucose enters the trophectoderm by this GLUT1 at a rate which directly reflects the external glucose concentrations. The activity increased on a cellular basis as development proceeded, suggesting increased activity to meet the increasing metabolic requirements of the blastocyst for glucose.
Comparative response of the immature and mature ovine fetus to corticotrophin-releasing hormone (CRH)
Authors: McFarlane AC , Cooper EJ , Fernley RT , Watabe T , Wintour EM
Published: October 2005

The aim of this study was to address the possibility that the low concentrations of adrenocorticotrophin (ACTH) seen in the ovine fetus between 90 and 120 days of gestation could be attributed to an alteration in the sensitivity or responsiveness of the fetal pituitary to corticotrophin-releasing hormone (CRH), a key regulator of ACTH secretion. Chronically cannulated ovine fetuses at Days 104-108 (n = 11, representing fetuses from this 90-120-day period) and Days 138-142 (n = 6) of pregnancy received graded doses of ovine CRH (0.8, 1.6, 3.8 and 7.6 micrograms h-1 for 60 min each, given consecutively and in ascending order) or isotonic saline (n = 4 at both age groups studied). Arterial blood samples were taken concurrently for analysis of plasma immunoreactive CRH, ACTH and cortisol throughout the infusion to assess the pituitary-adrenal response. Regression lines describing the relationship between log.PCRH and log.PACTH were calculated for both age group studied. A significant (P < 0.001) rightward shift in the log.PCRH/log.PACTH regression line for the Day 104-108 group was found, suggesting that the ovine fetus at this age is less sensitive or responsive to exogenous oCRH than the mature Day 138-142 fetus. This decreased responsiveness could explain the low concentrations of endogenous ACTH seen during the 90-120-day period.
Effect of protein kinase C modulation on gonadotrophin-induced granulosa cell steroidogenesis
Authors: He H , Herington AC , Roupas P
Published: October 2005

The effect of protein kinase C (PKC) modulation on gonadotrophin-induced ovarian granulosa cell differentiation was investigated by using an activator of PKC, phorbol 12-myristate 13-acetate (PMA) and inhibitors of PKC, sphingosine (SPH) and staurosporine (ST). The effects of PMA (at doses which activate PKC (10 ng mL-1), and down-regulate PKC (1000 ng mL-1)), sphingosine (25 microM) and staurosporine (10(-10)-10(-7) M) on gonadotrophin-induced granulosa cell differentiation were studied by the determination of steroidogenesis and cAMP accumulation in immature rat ovarian granulosa cells treated with or without pregnant mare serum gonadotrophin (100 mU mL-1). PMA (10 ng mL-1) inhibited gonadotrophin-induced granulosa cell steroidogenesis and cAMP accumulation. PMA (1000 ng mL-1)-induced down-regulation of PKC did not affect gonadotrophin-induced steroidogenesis. The inhibitory effect of PMA (10 ng mL-1) on gonadotrophin-induced granulosa cell steroidogenesis was not present in PKC-down-regulated cells. These data indicate that PKC activation by PMA inhibits gonadotrophin-induced steroidogenesis. SPH also inhibited gonadotrophin-induced steroidogenesis and cAMP accumulation. This effect of SPH was not affected by PMA-induced PKC down-regulation, indicating that this action of SPH does not require PKC or is mediated via a phorbol ester-insensitive PKC isoform. ST induced steroidogenesis in the absence of gonadotrophin, but was not synergistic with gonadotrophin. PMA-induced down-regulation of PKC abolished the effect of ST, suggesting that the action of ST requires PKC. The data suggest that ST and PMA, which antagonize each other in gonadotrophin-induced steroidogenesis, act via a PKC-mediated mechanism whereas the cAMP-associated actions of gonadotrophins and SPH are not dependent on PKC.
Calicin in human sperm fertilizing zona-free hamster eggs in vitro
Authors: Paranko J , Salonen I
Published: October 2005

Calicin, a basic cytoskeletal protein has been proposed to be involved in the formation and maintenance of the highly regular organization of the postacrosomal perinuclear theca, the calyx of mammalian spermatozoa. The fate of human sperm calicin in the zona-free hamster egg fertilized in vitro has been analysed at the light and electron microscope level using polyclonal mouse anti-calicin antibodies. Calicin was localized in the postacrosomal dense lamina of the capacitated acrosome-reacted spermatozoa attached on the surface of the egg as well as in the spermatozoa at an early stage of ooplasmic incorporation. As determined by the aid of the DNA-specific fluorescent dye 4,6-diamidino-2-phenylindole (DAPI), the pattern of staining with anti-calicin changed from a funnel-shape into a ring soon after the onset of the nuclear decondensation. Later, no anti-calicin labelling could be detected around the more decondensed sperm nuclei. Because the residual, ring-like accumulation of calicin was associated with the least decondensed chromatin, it appears that the degradation of calicin-containing perinuclear theca is intimately involved with the posteriorly advancing nuclear disintegration. The ring formation also suggests that the calyx of human spermatozoa is not structurally homogeneous at least in terms of sensitivity to ooplasmic degradation. Calicin appears to be unaffected by lytic enzymes of the acrosome. The present study further shows that DAPI can be effectively used to analyse sperm nuclear decondensation in vitro.
Comparative intracytoplasmic sperm injection (ICSI) in human and domestic species
Authors: Catt JW , Rhodes SL
Published: October 2005

The current clinical use of intracytoplasmic sperm injection (ICSI) for the alleviation of male factor infertility has prompted a re-investigation of sperm injection techniques in a number of animal species. This report examines sperm injection of in vitro matured oocytes in the major domestic species and compares the results with the human. Ovine, bovine and porcine oocytes can undergo fertilization and at least limited development without exogenous activation either prior to or subsequent to injection. Porcine is temperature sensitive during fertilization and the early stages of embryo development. The oocytes of all three domestic species, particularly ovine, have a tendency to activate after the injection procedure regardless of the presence or absence of sperm. The implications for early development studies and the practical use of direct sperm injection for domestic species are discussed.
Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI)
Authors: Flaherty SP , Payne D , Swann NJ , Matthews CD
Published: October 2005

The assessment of fertilization is an important part of intracytoplasmic sperm injection (ICSI) and oocytes are routinely examined about 17 h after injection using Nomarski differential interference contrast optics. However, it is not possible to conclusively determine the aetiology of fertilization anomalies in this manner, so cytological studies were undertaken to determine the causes of failed and abnormal fertilization after ICSI. Oocytes which exhibited no evidence of fertilization, one pronucleus (PN) or 3 PN were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify PN, metaphase chromosomes, sperm heads and polar bodies. A total of 428 unfertilized oocytes were examined from 170 ICSI cycles. Overall, 82% of these unfertilized oocytes were still at metaphase II (non-activated) while the remaining 18% were activated and had 1 PN and two polar bodies. The majority (71%) of the metaphase II oocytes contained a swollen sperm head, which indicates that the spermatozoon was correctly injected but the oocyte did not activate and complete its second meiotic division. The swollen sperm head was located among the metaphase chromosomes in 4.3% of these oocytes, while in some cases (6.6%), the sperm chromosomes had undergone premature chromosome condensation (PCC). Other aetiologies of failed fertilization in these metaphase oocytes were ejection of the spermatozoon from the oocyte (19%) and complete failure of sperm head decondensation (10%). A similar pattern of anomalies was found in 1 PN oocytes, although the ratios were different (swollen sperm head, 51%; ejection of the spermatozoon, 19%; undecondensed sperm head, 30%). Seventy abnormally fertilized oocytes were also examined, of which 63 had 3 PN and a single polar body, indicating that the unextruded second polar body developed into the third PN. In conclusion, the present study demonstrates that the principal cause of fertilization failure after ICSI is failure of oocyte activation and not ejection of the spermatozoon from the oocyte. It is also apparent that further studies are needed to elucidate the mechanisms that control oocyte activation and sperm head decondensation in injected oocytes.
Factors affecting success with intracytoplasmic sperm injection
Authors: Tucker MJ , Morton PC , Wright G , Ingargiola PE , Jones AE , Sweitzer CL
Published: October 2005

In this study, 141 couples underwent 163 cycles of in vitro fertilization (IVF) and embryo transfer in which the eggs were inseminated by intracytoplasmic sperm injection (ICSI). Overall, 41% of the injected eggs were normally fertilized and 81% of the resulting embryos were suitable for cryopreservation (91 embryos) or uterine transfer. From 153 fresh embryo transfers, 45 ongoing or delivered pregnancies (27.6% per cycle) were achieved, and of the 507 embryos transferred, 54 successfully implanted giving an implantation rate per embryo of 10.7%. Five additional pregnancies did not yield a viable fetus or underwent a spontaneous abortion, giving a miscarriage rate of 10% (5/50). Increased maternal age or a prior diagnosis of failed fertilization after conventional IVF had a significantly negative impact on success. Sperm from the testis and epididymis, those retrieved by electro-ejaculation, and completely immotile ejaculated sperm all gave rise to pregnancies. ICSI reinsemination was used with limited success to rescue failed fertilization cycles, although the implantation rate per embryo was poor (5%). ICSI has greatly improved the ability to use IVF for treating couples with a poor fertilization potential.
The use of intracytoplasmic sperm injection for the treatment of severe and extreme male infertility
Authors: Bourne H , Richings N , Harari O , Watkins W , Speirs AL , Johnston WI , Baker HW
Published: October 2005

The outcome of treatment by intracytoplasmic sperm injection (ICSI) is described for patients with severe male infertility. In 296 consecutive cycles, a normal fertilization rate of 69% was achieved with 288 cycles (97%) resulting in embryos suitable for transfer. A total of 32 clinical pregnancies were achieved from the transfer of fresh embryos (clinical pregnancy rate of 12% per transfer) and an additional 44 clinical pregnancies were obtained after the transfer of frozen-thawed embryos (clinical pregnancy rate of 16% per transfer). Overall, 57 of the 76 pregnancies were ongoing or delivered. An analysis of outcome in 5 male factor subgroups revealed no significant differences in pregnancy and implantation rates between the categories. However, the fertilization rate was significantly lower in patients with oligoasthenoteratozoospermia and significantly higher in those patients for whom epididymal sperm were used for insemination. The treatment of patients with extreme male infertility is also described; normal fertilization and embryo development were obtained using ICSI in patients with mosaic Klinefelter's syndrome, severe sperm autoimmunity, round-headed acrosomeless sperm (globozoospermia), completely immotile sperm selected by hypo-osmotic swelling and sperm isolated from testicular biopsies. Three ongoing pregnancies were obtained from 6 patients for whom testicular sperm were used. These results demonstrate the value of ICSI in the management of severe male infertility, however, the treatment of some types of extreme male infertility using ICSI may be limited.
Clinical results from intracytoplasmic sperm injection at monash IVF
Authors: McLachlan RI , Fuscaldo G , Rho H , Poulos C , Dalrymple J , Jackson P , Holden CA
Published: October 2005

The impact of a modification of the intracytoplasmic sperm injection (ICSI) technique on fertilization and pregnancy rates was examined in a retrospective analysis of 171 consecutive ICSI treatment cycles (156 patients). Patients were selected for ICSI on the basis of severe oligoasthenozoospermia (65 patients) or following conventional in vitro fertilization (IVF) with failed or poor fertilization (70 patients). Seven patients in which epididymal or testicular sperm was used, 10 patients with sperm antibodies and 4 patients with retrograde ejaculation or who required electro-ejaculation were also treated with ICSI. In the first 105 cycles (102 patients), single sperm, rendered immotile, were injected into the ooplasm of 979 metaphase II (M II) oocytes using an established technique (Method 1). In the following 66 cycles (513 M II oocytes injected), the ICSI procedure was modified by increased aspiration of the oolemma to ensure the intracytoplasmic deposition of sperm (Method 2). The patient groups did not differ between the two injection procedures. The normal (two pronuclear) fertilization rate increased significantly (P < 0.001) from 34.3% with Method 1 to 73.1% with Method 2, with no difference in the oocyte degeneration rate (4.3% v. 4.5% respectively). The incidence of failed fertilization was significantly (P < 0.01) reduced from 17.1% (18 cycles) to 1.6% (1 cycle) with the change in technique. As a consequence of the increased fertilization rates with Method 2, more embryos were available for assessment and transfer, and a pregnancy rate per oocyte retrieval of 21.2% was obtained for Method 2. Fertilization, embryo transfer and pregnancies were obtained in all patient groups treated with ICSI.(ABSTRACT TRUNCATED AT 250 WORDS)
Fertilizing capacity of epididymal and testicular sperm using intracytoplasmic sperm injection (ICSI)
Authors: Silber SJ , Devroey P , Tournaye H , Van Steirteghem AC
Published: October 2005

For men with uncorrectable obstructive azoospermia, their only hope of fathering a child is microsurgical epididymal sperm aspiration (MESA) combined with in vitro fertilization (IVF). In 1988, proximal epididymal sperm were demonstrated to have better motility than senescent sperm in the distal epididymis, and it was thought that retrieval of motile sperm from the proximal epididymis would yield reliable fertilization and pregnancy rates after conventional IVF. However, the results to date have been poor, and although a minority of patients achieved good fertilization rates with IVF, the vast majority (81%) had consistently poor or no fertilization and the pregnancy rate averaged only 9%. Recently, intracytoplasmic sperm injection (ICSI) has been successfully used to achieve fertilization and pregnancies for patients with extreme oligoasthenozoospermia. ICSI has therefore been applied to cases of obstructive azoospermia and, in this report, 67 MESA-IVF cases are compared with 72 MESA-ICSI cases. The principle that motile sperm from the proximal segments of the epididymis should be used for ICSI was followed, although in the most severe cases in which there was an absence of the epididymis (or absence of sperm in the epididymis), testicular sperm were obtained from macerated testicular biopsies. These sperm only exhibited a weak, twitching motion. In 72 consecutive MESA cases, ICSI resulted in fertilization and normal embryos for transfer in 90% of the cases, with an overall fertilization rate of 46%, a cleavage rate of 68%, and ongoing or delivered pregnancy rates of 46% per transfer and 42% per cycle. The pregnancy and take-home baby rates increased from 9% and 4.5% with IVF to 53% and 42% with ICSI. There were no differences between the results for fresh epididymal, frozen epididymal or testicular sperm, and the number of eggs collected did not affect the outcome. The results were also unaffected by the aetiology of the obstruction such as congenital absence of the vas deferens or failed vasoepididymostomy. The only significant factor which affected the pregnancy rate was female age. It is concluded that although complex mechanisms involving epididymal transport may be beneficial for conventional fertilization of human oocytes (in vivo or in vitro), none of these mechanisms are required for fertilization after ICSI. Given the excellent results with epididymal and testicular sperm, ICSI is obligatory for all future MESA patients. Finally, the use of ICSI with testicular sperm from men with non-obstructive azoospermia is also discussed.
Fetal plasma insulin-like growth factor-binding protein-3 concentrations are elevated following bilateral nephrectomy in fetal sheep
Authors: Beanland C , Browne C , Young R , Owens J , Walton P , Thorburn G
Published: October 2005

Insulin-like growth factors mediate many of the effects of growth hormone and are important in the regulation of growth, especially in the fetus where growth is less dependent on circulating growth hormone. In the ovine fetus, insulin-like growth factor-I (IGF-I) is bound mainly to the low molecular weight insulin-like growth factor-binding proteins (IGFBP), IGFBP-1 and IGFBP-2, with little binding to IGFBP-3 until near term at 147 days gestation. To determine if there was any difference in plasma IGF-I and IGFBP-3 concentrations in growth-retarded fetal sheep with altered renal status, concentrations were measured by specific radioimmunoassay from bilaterally nephrectomized fetal sheep between Days 113 and 135 gestation. Plasma IGFBP-3 concentrations were significantly (P < 0.001) increased in bilaterally nephrectomized fetuses (4.19 +/- 0.19 micrograms mL-1, n = 7) compared with control fetuses (2.33 +/- 0.10 micrograms mL-1, n = 7). There was no change in plasma IGFBP-3 concentration with gestational age in either experimental group. Maternal plasma IGFBP-3 concentrations did not differ between the bilateral nephrectomy group (3.11 +/- 0.09 micrograms mL-1, n = 7) and the control group (3.25 +/- 0.11 micrograms mL-1, n = 7) and showed no change within groups over the experimental period. Total plasma IGF-I concentrations in bilaterally nephrectomized fetuses and ewes were similar to those in control fetuses and ewes. The results indicate that the profile of IGF binding in fetal plasma is altered in the anephric fetal sheep. In nephrectomized fetal sheep, increased IGFBP-3 concentrations, and therefore increased IGF-binding capacity in fetal plasma, may have contributed to a decrease in free IGF in plasma and decreased IGF-I bioactivity. This would provide a possible mechanism for the growth retardation reported in bilaterally nephrectomized fetal sheep.
Regulation of placental metabolism by glucose supply
Authors: Hay WW
Published: October 2005

Glucose is supplied to the placenta and fetus from the maternal plasma according to concentration-dependent mechanisms exhibiting saturation kinetics that are mediated by facilitative transporter proteins on both the maternal-facing microvillus and fetal-facing basal trophoblast membranes. Placental glucose transport to the fetus requires a net maternal-to-fetal plasma glucose concentration gradient that is determined by placental as well as fetal glucose consumption. Fetal plasma glucose concentration, independent of maternal glucose concentration, regulates the partition of placental glucose uptake into transfer to the fetus and consumption by the placenta. Placental transport capacity increases with advancing gestation, probably by an increased number of transporter proteins as surface area increases. Placental glucose consumption contributes to most or all of placental lactate and fructose production and other less well defined non-oxidative pathways of carbon metabolism. Placental glucose consumption accounts for at least 50% of placental oxygen consumption which remains independent of short-term or long-term changes in placental glucose supply, thus requiring varying amounts of other carbon substrates. Placental glucose supply, therefore, plays a key role in regulating placental glucose metabolism and placental carbon balance, and interacts reciprocally with other carbon substrates to maintain placental oxidative metabolism.
Characterization of 11 beta-hydroxysteroid dehydrogenase activity in fetal and adult ovine tissues
Authors: Kim EK , Wood CE , Keller-Wood M
Published: October 2005

11 Beta-hydroxysteroid dehydrogenase (11 beta-HSD) converts 11-hydroxycorticosteroids to 11-oxocorticosteroids, thereby influencing the availability of bioactive cortisol or corticosterone in target tissues. The activity of this enzyme was investigated in sheep by: (1) measuring relative 11 beta-HSD activities in kidney, liver and placenta, and in various areas of the brain (hypothalamus, hippocampus, and brainstem); (2) characterizing the optimum pH of activities in the tested tissues; (3) investigating the possible effect of gonadal steroids on 11 beta-HSD activity in adult hypothalamus and kidney; and (4) investigating possible developmental changes in activities in the tested tissues. The optimum pH in liver and placenta was pH 9-10, whereas the optimum pH in kidney was pH 7-8. In tissues from adult ewes, 11 beta-HSD activity was highest in liver (84.6 +/- 3.8%) and kidney (49.8 +/- 11.6%), lower but measurable in pituitary (38.8 +/- 3.7%), and near the limit of detection in hypothalamus and hippocampus (2.7 +/- 0.9% and 3.2 +/- 0.8% respectively). Liver, kidney and pituitary from late-gestation fetal sheep contained activities which were similar to those in the adult (76.9 +/- 4.5%, 66.0 +/- 6.7% and 26.3 +/- 3.0% respectively). Activity in the pituitary was not related to fetal gestational age. Placenta also contained measureable 11 beta-HSD activity (21.4 +/- 4.7%). However, no activity was detected in hypothalamus (-1.7 +/- 0.2%), hippocampus (-0.2 +/- 0.6%) or brainstem (-1.0 +/- 0.6%) in late-gestation fetal or neonatal sheep. Enzyme activities in kidney and hypothalamus did not change significantly when the circulating concentrations of ovarian steroids were altered over a 1-3-week period. It is concluded that the ovine kidney, liver and placenta, but not hypothalamus or cerebral cortex, contain 11 beta-HSD activity. In addition, there is no change in 11 beta-HSD activity between late-gestation fetal life and adult life, and the relative activities are not altered by the ovarian steroid milieu.
Acute hypoxia induces elevation of ornithine decarboxylase activity in neonatal rat brain slices
Authors: Longo LD , Packianathan S
Published: October 2005

Recent studies in vivo have demonstrated that ornithine decarboxylase (ODC) activity in the fetal rat brain is elevated 4-5-fold by acute maternal hypoxia. This hypoxic-associated increase is seen in the rat brain in both the newborn and the adult. Because of the intimate involvement of ODC in transcription and translation, as well as in growth and development, it is imperative that the manner in which hypoxia affects the regulation of this enzyme be better understood. In order to achieve this, a brain preparation in vitro was required to eliminate the confounding effects of the dam on the fetal and newborn brain ODC activity in vivo. Therefore, brain slices from 3-4-day-old (P-3) newborn rats were utilized to test the hypothesis that ODC activity increases in response to hypoxia in vitro. Cerebral slices from the P-3 rat pups were allowed to equilibrate and recover in artificial cerebrospinal fluid (ACSF) continuously bubbled with a mixture of 95% O2 and 5% CO2 for 1 h before beginning hypoxic exposures. Higher basal ODC activities were obtained by treating the slices with 0.03% fetal bovine serum (FBS) and 0.003% bovine serum albumin (BSA), rather than with ACSF alone. Hypoxia was induced in the slices by replacing the gas with 40%, 21%, 10%, or 5% O2, all with 5% CO2 and balance N2. With FBS and BSA treatment, ODC activity was maintained at about 0.15-0.11 nM CO2 mg-1 protein h-1 throughout the experiment, which was 2-3-fold higher than that without FBS and BSA. ODC activity increased significantly and peaked between 1 h and 2 h after initiation of hypoxia. For instance, with 21% O2, ODC activity increased approximately 1.5-fold at 1 h and approximately 2-fold at 2 h. These studies demonstrate that: (1) the hypoxic-induced increases observed in vivo in the fetal and newborn rat brain ODC activity can be approximated in a newborn rat brain slice preparation in vitro; (2) newborn rat brain slice preparations may provide an alternative to methods in vivo or cell culture methods for studying the regulation of acute hypoxic-induced enzymes; and (3) high, stable baseline ODC activities in brain slices suggest that the cells in the slice are capable of active metabolism if FBS and BSA are available to mimic conditions in vivo.
Role of prostaglandin I2 and prostaglandin E2 in the initiation of nonshivering thermogenesis during the simulation of birth in utero
Authors: Ball KT , Takeuchi M , Yoneyama Y , Power GG
Published: October 2005

Because maximal nonshivering thermogenesis can commence only after occlusion of the umbilical cord, circulating stimulators and inhibitors were hypothesized to alter brown fat activity in the perinatal period. The roles of prostaglandin I2 (PGI2) and PGE2 in the initiation of nonshivering thermogenesis at birth were investigated. Indomethacin (45 mg bolus, 3 mg h-1 thereafter) was infused into 10 near-term fetal sheep to decrease prostanoid synthesis; 6 age-matched fetuses were infused with saline as controls. Sixteen hours later, birth was simulated in utero by sequentially cooling the fetus, ventilating its lungs with oxygen and occluding the umbilical cord. In the control fetuses, the plasma concentrations of PGI2 and PGE2 and free fatty acids, an index of nonshivering thermogenesis, were unaffected by cooling. Ventilation caused the concentration of PGI2 to increase 108% (P < 0.001) and that of PGE2 to decrease 26% (P < 0.05), while fatty acid concentrations increased 100% (P < 0.05). After cord occlusion, PGI2 concentrations remained elevated whereas PGE2 concentrations decreased a further 46% (P < 0.01), and fatty acid concentrations increased a further 100% (P < 0.05). In the indomethacin-treated fetuses, PGI2 and PGE2 concentrations decreased to 20% of the preinfusion values (P < 0.001) and did not change during the experiment. Cooling initiated a 300% increase in fatty acid concentrations (P < 0.05) and ventilation and cord occlusion induced no further significant changes. Thus, prostanoid concentrations follow changes in nonshivering thermogenic activity and support a regulatory role for PGI2 and PGE2 in the initiation of thermogenesis. Before birth, high concentrations of PGE2 favour suppression of thermogenesis, and after birth this inhibition is removed and there is stimulation by PGI2.
Sustained alterations in postnatal respiratory function following sub-optimal intrauterine conditions
Authors: Harding R
Published: October 2005

This paper reviews recent evidence from epidemiological, follow-up and experimental studies that sub-optimal conditions during gestation can cause alterations in respiratory function that persist during postnatal life. Several studies indicate that placental insufficiency, which can be associated with fetal substrate deprivation, hypoxia and low birthweight, may be followed by evidence of respiratory compromise in later life. Similarly, it is becoming evident that maternal smoking affects fetal lung development and that the effects can persist into postnatal life. A reduced period of fetal development, due to preterm birth, may be associated with prolonged postnatal respiratory consequences which are independent of factors operating during the early postnatal period. Disorders of pregnancy that compress the fetal lungs, or that cause the abolition of fetal breathing movements, commonly lead to lung hypoplasia. We have been interested in the prenatal causes and postnatal effects of fetal lung hypoplasia and have used an ovine model of lung hypoplasia induced by prolonged removal of amniotic fluid. This leads to a reduction in the expansion of the fetal lungs which appears to be a common underlying cause of fetal lung hypoplasia. Studies of lung function in lambs chronically exposed as fetuses to a lack of amniotic fluid showed that, although lung hypoplasia was apparently present throughout the 28-day postnatal study period, major alterations in respiratory function were attributable to changes in chest wall compliance. Thus, it is apparent that sub-optimal intrauterine conditions can have lasting effects on the structure and function of respiratory organs. Available evidence indicates that the degree to which these organs can recover postnatally may be restricted.
Ultrastructural and functional features of the developing mammalian heart: a brief overview
Authors: Smolich JJ
Published: October 2005

The heart undergoes marked ultrastructural alterations during fetal and postnatal development. Early in fetal development, cardiac myocytes contain abundant pools of glycogen, scattered mitochondria and sparse, peripheral myofibrils. Transverse tubules are absent, and sarcoplasmic reticulum and intercalated discs are poorly developed. During late fetal and early postnatal development, myofibrils extend into the myocyte interior and attain a mature appearance, and the glycogen pools are reduced in size. In addition, transverse tubules develop and the morphological appearance of the sarcoplasmic reticulum and intercalated disc becomes increasingly complex. Experimental studies in sheep, corroborated by clinical studies in humans, also point to marked functional changes during development. In the fetus, the right ventricle is the dominant pumping chamber because right ventricular output exceeds left ventricular output, while pulmonary arterial and aortic pressures are similar. This functional difference is reflected in myocardial blood flow patterns, with blood flow to the right ventricle exceeding that to the left ventricle. The ventricular outputs equalize after birth, but a functional left ventricular dominance rapidly emerges following a postnatal increase in systemic vascular resistance and a decrease in pulmonary vascular resistance. This postnatal switchover in functional dominance is accompanied by a corresponding alteration in the relative level of ventricular myocardial blood flows. Consistent with right ventricular dominance in utero, myocytes in the right ventricle of the fetal sheep are larger and contain more myofibrillar material than those in the left ventricle. Left ventricular myocytes become larger than right ventricular myocytes after birth, but this adaptation to altered postnatal haemodynamics requires some weeks to become fully established.
Baroreflex and chemoreflex control of fetal hormone secretion
Authors: Wood CE
Published: October 2005

In recent years, the expanding literature in fetal cardiovascular physiology and endocrinology has improved our understanding of the afferent mechanisms involved in the reflex hormonal and neural responses to cardiovascular distresses, such as hypotension, hypoxia, hypercapnia, and asphyxia. For some endocrine systems, the relative roles of peripheral and central baroreceptors and chemoreceptors are well understood. The best example of such a system is AVP, summarized in Fig. 6. Published experiments have demonstrated that AVP secretion is influenced by arterial baroreceptors, but not by arterial chemoreceptors. On the other hand, there is most likely a chemosensitive area within the central nervous system controlling AVP secretion which responds to hypoxia as well as hypercapnia. Cardiac receptors are less important in the control of AVP secretion in fetal life than in postnatal life. Our understanding of the control of other hormonal systems, including renin and ACTH, is less complete. The challenge for the future in this field will be to fill in the gaps in our knowledge of these fetal reflexes, and to provide a functional understanding of the mechanisms underlying the quantitative differences between fetus and adult. For example, does the rearrangement of the circulation at birth 'activate' the cardiac receptors by changing intracardiac pressures, or are the neuronal pathways poorly developed in the fetus compared with the postnatal animal? Also, what is the mechanism of the chemoreflex control of AVP which is independent of peripheral chemoreceptor integrity in the fetus? The answers to these questions and others will significantly improve our understanding of fetal cardiovascular and endocrine physiology. These answers will also provide information which will prove to be of practical importance in a modern age of obstetrics, paediatrics, and surgery, where the fetus will increasingly be treated as a young patient.
Postnatal renal adaptation in preterm and term lambs
Authors: Berry LM , Ikegami M , Woods E , Ervin MG
Published: October 2005

The present experiments determined if increases in renal reabsorptive capacity during the transition from fetal to neonatal life are gestation dependent. Renal function was studied in chronically-catheterized fetal lambs (133 +/- 1 days; term, 145-150 days). Additionally, renal function was studied in anaesthetized, ventilated, caesarean-delivered preterm lambs (109-139 days gestation) and term lambs (148 days gestation), and in 2-day-old spontaneously-delivered term lambs. Newborns < or = 120 days old received surfactant to facilitate ventilation and maintenance of physiologic blood gases. Two hours after caesarian delivery, urine osmolality, urine flow, glomerular filtration rate (GFR), and fractional sodium excretion (FENa) values were similar for all gestations. Relative to fetal values, caesarean-delivered newborn renal values included lower urine flow rates (0.20 +/- 0.03 v. 0.05 +/- 0.01 mL min-1 kg-1), higher urine osmolalities (118 +/- 15 v. 422 +/- 16 mOsmol kg-1 H2O), and no differences in GFR or FENa. Relative to caesarean-delivered newborns, 2-day newborn renal function included higher values for GFR (0.7 +/- 0.1 v. 3.0 +/- 0.1 mL min-1 kg-1) and urine osmolality (724 +/- 32 mosmol kg-1 H2O), and lower FENa (7.0 +/- 1.5 v. 0.2 +/- 0.02%), and urine flow (0.005 +/- 0.003 mL min-1 kg-1). The 132- and 139-day animals were ventilated for 5 h and 10 h respectively; the only functional change at 10 h was a decrease in FENa (7.0 +/- 1.5 v. 2.8 +/- 0.1%). It is concluded that: (1) relative to fetal animals, renal adaptive responses in anaesthetized, ventilated newborns begin within 2 h following caesarian delivery; (2) initial adaptive responses are not gestation dependent after 109 days; and (3) the combined effects of ventilation and/or anaesthesia delay postnatal renal adaptations for at least 10 h after birth.
Studies on glucocorticoid hormone actions in the regulation of human amnion PGHS
Authors: Zakar T , Olson DM
Published: October 2005

The human amnion is an excellent model in which to study the regulation of the expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) mRNA by glucocorticoids, as glucocorticoids can both stimulate and inhibit PGHS activity and can have their stimulatory effect reversed to an inhibitory one. Our studies suggest the presence of a glucocorticoid-induced inhibitory protein which affects the post-transcriptional activity of PGHS-2 mRNA. Glucocorticoid-induced expression of PGHS-2 mRNA may be dependent on protein phosphorylation.
Regulation of the switch from myometrial contractures to contractions in late pregnancy: studies in the pregnant sheep and monkey
Authors: Nathanielsz PW , Giussani DA , Mecenas CA , Wu W , Winter JA , Garcia-Villar R , Baguma-Nibasheka M , Honnebier MB , McDonald TJ
Published: October 2005

Myometrial contractility occurs throughout pregnancy and characteristic patterns of myometrial activity exist according to the endocrine status and the relationship to parturition. These characteristic patterns differ between species, yet certain common features can be observed. Throughout pregnancy, myometrial activity is of the contractures type, long-lasting, low-amplitude epochs of activity switching to contraction-type activity at term. This switch from contractures to contractions tends to occur at night and is related to alteration in maternal plasma oestrogen concentrations, and maternal oxytocin function. Studies in several animal species support the hypothesis that maternal oestrogen prepares the myometrium for a periodic signal that causes the switch from contractures to contractions. Several lines of evidence implicate oxytocin in the switch. These studies show that the detailed preparation for parturition takes longer than previously considered and is brought about by a carefully regulated sequence of events in which oestrogen production by the placenta plays a central role.
Myometrial transcriptional regulation of the gap junction gene, connexin-43
Authors: Lefebvre DL , Piersanti M , Bai XH , Chen ZQ , Lye SJ
Published: October 2005

The mechanisms that enable the myometrium to switch from a state of relative quiescence during pregnancy to a muscle that is spontaneously active, very responsive to endogenous uterotonins and exhibits a high degree of cell-cell coordination are poorly understood. It is hypothesized that this switch or 'activation' of the myometrium results from the coordinated expression of a cassette of 'contraction-associated proteins'. The molecular mechanisms that regulate the expression of one of these, namely the myometrial gap junction protein connexin-43 (Cx-43), have been analysed. Myometrial Cx-43 expression is significantly increased during labour, associated with an increase in plasma oestrogen:progesterone, and positively regulated by oestrogen in non-pregnant rats. The genomic structure of the murine Cx-43 gene and the sequence of its 5' flanking sequence are reported here. This region functions as a promoter and contains several putative cis-acting elements which may be important in the regulation of Cx-43 transcription. Among these elements are several half-palindromic sequences that may function as oestrogen response elements and several AP-1 sites that may bind the transcription factors Fos and Jun. Oestrogen treatment of cells transiently transfected with a plasmid containing the Cx-43 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, increased CAT activity indicating that the murine Cx-43 gene is oestrogen responsive. In addition, treatment of rats with oestrogen significantly increased mRNA encoding c-fos and c-jun in the myometrium and this occurred before any increase in Cx-43 mRNA. These data suggest that oestrogen may increase transcription of the Cx-43 gene through direct mechanisms (via the putative oestrogen response elements) or indirect mechanisms (by increased expression of c-fos and c-jun acting via the putative AP-1 sites). Since oestrogen may be an important modulator of myometrial activation, these mechanisms may be critical to the processes leading to increased synthesis of gap junctions at term and, hence, to the onset of labour.
Fetal growth retardation and increased placental weight in the spontaneously hypertensive rat
Authors: Johnston BM
Published: October 2005

Epidemiological studies have linked low birth weight and increased placental weight with increased risk of hypertension in adult life. It has been proposed that the cardiovascular changes which lead to hypertension are initiated in utero by processes associated with intrauterine growth retardation. The alternative possibility, that hypertension may result from genetic influences which also determine fetal and placental size, has had less support because birth weight is not determined genetically in humans. However, in the spontaneously hypertensive rat (SHR) essential hypertension is known to be transmitted genetically. Fetal and placental weights were, therefore, measured at Day 20 gestation in SHRs and compared with those in the normotensive Wistar Kyoto (WKY) control strain. Fetal weight (1.93 +/- 0.04 g) was significantly (P < 0.001) reduced in SHRs compared with WKY fetuses (2.23 +/- 0.01 g) but placental weight was heavier (P < 0.001) in SHRs (0.347 +/- 0.005 g) than in WKY rats (0.300 +/- 0.006 g) although litter size was not different. As expected, maternal blood pressure recorded under 1% halothane anaesthesia was higher (126 +/- 2.7 mm Hg) in SHR than WKY rats (100 +/- 2.1 mm Hg; 1 mm Hg = 133 Pa). In addition the concentration of maternal blood glucose in SHR was significantly (P < 0.001) higher (4.8 +/- 0.32 mM v. 3.7 +/- 0.11 mM) and the concentration of plasma insulin was significantly (P < 0.05) lower in SHRs (18.8 +/- 3.0 ng mL-1) than in WKY dams (29.4 +/- 3.1 ng mL-1). Thus, the data support human population studies which show an association between adult hypertension and a reduced fetal:placental weight ratio at birth. However, because hypertension in the SHR is genetically determined, these data suggest that fetal growth retardation and increased placental weight may also be determined genetically.