bluuebunny/crossref_metadata_2025 huggingface dataset:
Embryo development and placentome formation during early pregnancy in red deer
Authors: McMahon C. D. , Fisher M. W. , Mockett B. G. , Littlejohn R. P.
Published: November 2003

Early embryo development and placentome formation were assessed in red deer
between Days 27 and 55 of gestation. Uteri were collected from 12 pregnant
hinds in which mating was observed following a synchronized oestrus, and the
tissues retained for measurements and histological processing for light
microscopy. Twelve embryos were recovered with mean embryo weights increasing
from 0·02 ± 0·01 g at Day 27 to 7·56 ±
1·39 g at Day 55 of gestation. Similarly, crown-rump lengths increased
from 5·7 ± 0·7 mm to 55·3 ± 5·9 mm
over this period. The trophoblast had extended throughout both uterine horns
and gastrulation was completed by Day 27. Limb buds were apparent by Day 34,
and by Day 48 the phalanges had separated into hooves and dew claws. Plaques
were evident on the trophoblast at Day 34 and, by Day 41, placentomes had
formed adjacent to the embryo. These placentomes grew in size as pregnancy
advanced; by Day 55 most caruncles had formed placentomes. It is therefore
conrmed that placentome formation occurs at about the sixth week of gestation.
These results indicate that embryo growth and placentome formation in red deer
are generally typical of that observed in other
ruminants.Extra keyword: caruncle.
Oestrogen regulation of insulin-like growth factor binding protein-3 (IGFBP-3) and expression of IGFBP-3 messenger RNA in the ovine endometrium
Authors: Peterson A. J. , Ledgard A. M. , Hodgkinson S. C.
Published: November 2003

Transcriptional regulation of insulin-like growth factor binding protein-3
(IGFBP-3) by oestrogen was investigated by Northern blot hybridization of
endometrial tissue mRNA from anoestrous ewes treated with oestradiol-17β
at either 0, 40 or 80 µg per day for 7 days. The 2.6 kb IGFBP-3
transcript, seen in control animals, was virtually undetectable in treated
animals. This suppressive effect was reflected in Western ligand blots of the
uterine luminal fluid (ULF) proteins where the concentration of IGFBP-3 was
significantly decreased with increasing oestrogen treatments. IGFBP-2 levels
were increased with oestradiol treatment but no significant effect was seen on
the other minor IGFBP’s present in the ULF. Northern analysis also
showed that the IGFBP-3 transcript was present from days 12 to 16 of the
oestrous cycle but was either absent or very weak on days 0 (oestrus) and 9 of
cycling ewes. In situ hybridization of endometrial
tissue sections localized the IGFBP-3 mRNA to the luminal epithelial cell
layer, areas of the stromal tissue and in some glandular epithelial cells.
Oestradiol treatment of ewes down-regulated expression of IGFBP-3 in the
endometrium; therefore, the low levels of IGFBP-3 in ULF during the early part
of the oestrous cycle is possibly due to elevated levels of plasma oestradiol
around oestrus.
Ultrastructural features of bovine cumulus-corona cells surrounding oocytes, zygotes and early embryos
Authors: Familiari Giuseppe , Verlengia Cristina , Nottola Stefania A. , Tripodi Alessandro , Hyttel Poul , Macchiarelli Guido , Motta Pietro M.
Published: November 2003

Integrated transmission and scanning electron microscopic (TEM and SEM)
techniques have provided the first detailed description of the ultrastructural
features of the bovine cumulus-corona (CC) cells surrounding oocytes at the
time of final maturation, zygotes and early cleaving embryos (2/4 to
6/8 blas-tomeres). TEM revealed the presence of rough endoplasmic
reticulum and Golgi complexes in the cyto-plasm of CC cells surrounding
immature, mature and fertilized eggs, and also revealed an increasing amount
of smooth endoplasmic reticulation membranes, lipid droplets and mitochondria
with villiform and/or tubular cristae in the cytoplasm of CC cells during
maturation and fertilization of the oocyte. In addition, a loss of
cell-to-cell junctions between CC cells was evident. TEM also demonstrated
that a few residual CC cells were still associated with early embryos and that
these cells showed rather degenerative or apoptotic patterns, the latter
pattern also observed on cells associated with fertilized eggs. SEM revealed
that the complex of CC cells of immature oocytes was compact with narrow
intercellular spaces, which progres-sively enlarged in size around mature
oocytes. This phenomenon is mostly due to the production of abun-dant
extracellular matrix. Immature CC cell complexes possessed characteristic long
and filiform microvilli whereas the surface of CC cells surrounding mature
oocytes showed numerous blebs and occasional large cytoplasmic protrusions as
well as microvilli. Zygotes and early embryos were covered with a few
polyhe-dral CC cells possessing scarce and short microvilli and a large amount
of pleomorphic blebs. This study demonstrated a precocious luteinization
occurring in bovine CC cells at ovulation until zygote segmenta-tion, and this
process was associated with a progressive apoptotic mechanism that ended in
the complete denudation of the zona pellucida covering the early embryo. The
presence of CC cells around the maturing oocyte and fertilized egg could have
important functions related to the microenvironmental requirements of ovum
maturation as well as facilitating activities related to fertilization.
Differential expression of activin subunits, activin receptors and follistatin genes in bovine oocytes and preimplantation embryos
Authors: Yoshioka K. , Takata M. , Taniguchi T. , Yamanaka H. , Sekikawa K.
Published: November 2003

The presence of inhibin subunits (α, βA and βB), activin binding
protein follistatin and activin receptors (ActR-I, ActR-IB, ActR-II and
ActR-IIB) mRNA transcripts in bovine oocytes and preimplantation embryos was
examined by reverse transcription-polymerase chain reaction. Immature and
matured oocytes, zygotes, 2-cell, 3–4-cell, 5–8-cell,
9–16-cell embryos, morulae, blastocysts and hatched blastocysts were
produced by in vitro maturation, fertilization and
culture for this study. Activin βA subunit and follistatin transcripts
were detected in all of the stages tested. However, α subunit
transcripts were detected only in oocytes (immature and matured), blastocysts
and hatched blastocysts, and no βB subunit transcripts were found in any
of the stages. Whereas ActR-I transcripts were detectable in all stages,
ActR-IB transcripts were not detectable in oocytes or embryos except in
matured oocytes. Activin type II receptors (ActR-II and ActR-IIB) transcripts
were detectable in oocytes and embryos except for ActR-IIB, which was not
found in the 5–8-cell stage embryos. The results suggest that bovine
embryos may produce activin A (homodimer of the βA subunits) at least from
zygotes to morulae. Furthermore, bovine oocytes and embryos express the
activin receptors mainly by the combination of ActR-I and either ActR-II or
ActR-IIB.
Obesity: genes, glands or gluttony?
Authors: Chisholm D. J. , Samaras K. , Markovic T. , Carey D. , Lapsys N. , Campbell L. V.
Published: November 2003

Distribution as well as amount of fat has health implications; central
abdominal fat seems to be the major contributor to insulin resistance and risk
of diabetes, hypertension and cardiovascular disease. Physical activity and
diet affect overall adiposity; moreover, exercise specifically reduces
visceral fat. The sexes differ in fat distribution; in particular,
pre-menopausal women, despite greater overall adiposity, have much less
visceral fat than men. There is a strong genetic determination of overall
obesity and central abdominal adiposity. Genes regulating obesity (e.g.
Ob) could modulate appetite, satiety, metabolic rate or
physical activity. Moderate obesity probably results from interaction between
genetic predisposition and an environment of abundant calories and reduced
physical activity. Single gene mutations are being identified in a few
morbidly obese people; however, the common genetic predisposition for obesity
may relate to more subtle variations in regulatory controls.
Diet and exercise are effective for some, but the response is often
disappointing. Definition of pathways controlling appetite, metabolic rate and
lipid metabolism may generate improved pharmacological compounds. Education
and availability of lower-energy foods may help, but more radical approaches
may be needed, such as environmental restructuring to increase physical
activity. The problem is great, but failure will mean intolerably increased
health costs.
Are sperm counts really falling?
Authors: Kretser D. M. de
Published: November 2003

Semen quality is said to have been declining over the past 50 years. The
biological significance of these changes is emphasized by a concomitant
increase in the incidence of genitourinary abnormalities such as testicular
cancer and cryptorchidism. The increase in regional frequency of testicular
abnormalities over a relatively short period of time may be due to local
environmental factors, including the comparatively recent fashion for wearing
tight-fitting underwear. Data also indicates that prenatal exposure to
environmental agents can affect the development of the male genital tract.
From the reproductive point of view, an increased environmental impact on the
human male gonad is of concern and merits the development of sensitive
techniques that can detect deleterious agents.
Epidemiological approaches to infertility
Authors: Lumley Judith
Published: November 2003

There are a number of reasons why epidemiological approaches to infertility
have not made a major contribution to research in Australia. They include the
success of generic treatments, the high public profile of infertility and the
consequent polarization of discussion over treatment versus prevention, some
reluctance to draw attention to possible aetiologic factors which may be
perceived negatively in public debates, and the lack of graduate training in
reproductive and perinatal epidemiology. Voluntary infertility is now common
for most of the fertile life span in developed countries, and intended family
size is small. Many important conditions cannot be diagnosed without the use
of invasive procedures or complex investigations, and the more widespread use
of less invasive procedures has shown other conditions to be relatively common
in healthy populations. If epidemiological approaches are to make a greater
contribution towards an increased understanding and control of infertility,
research should focus on retrospective and prospective cohort studies of the
incidence and prevalence of infertility, nested case-control studies of
occupational and environmental exposures, and an extension of the developing
use of record-linkage across routinely collected data systems and registers.
Uterine nitric oxide and prostaglandin E during embryonic implantation in non-insulin-dependent diabetic rats
Authors: Novaro V. , Jawerbaum A. , Faletti A. , Gimeno M. A. F. , González E. T.
Published: November 2003

In the process of embryo implantation in the rat, both nitric oxide and
prostaglandins act as vascular and myometrial regulators. The aim of the
present work was to evaluate the effect of diabetes on the synthesis of both
agents during embryo implantation. In diabetic rats, uterine activity of the
enzyme nitric oxide synthase and prostaglandin E production were increased
during peri-implantation compared to the control group
(P < 0·05 and P <
0·001, respectively). Both parameters showed a prolonged increase in
temporal profile during peri-implantation days. Local production of nitric
oxide and prostaglandin E in the implantation sites was higher in diabetic
rats (P < 0·05), but the intersite : site
ratio was similar to that of the control group. On the other hand, the
implantation rate and the timing of the beginning of this process were not
altered in the diabetic group. These results suggest that the vasoactive
modulators of the implantation process, nitric oxide and prostaglandins, are
increased in this diabetic pathology, and that this increase is probably
functioning as a compensatory mechanism, so as to allow an unaltered rate of
embryo implantation in this model.
Extra keyword: diabetes mellitus.
Glucocorticoid activity in the fetal spontaneously hypertensive rat
Authors: Lewis R. M. , Batchelor D. C. , Cockrem J. F. , Johnston B. M. , Bassett N. S. , Skinner S. J. M.
Published: November 2003

Fetal exposure to high concentrations of corticosteroids in the rat is
associated with elevated blood pressure in postnatal life. In this study we
have investigated indicators of corticosteroid activity in fetal spontaneously
hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in order to determine
whether fetal corticosteroid exposure is increased in the SHR. Placental
11β-hydroxysteroid dehydrogenase (11β-HSD) activity, which prevents
maternal steroids from crossing the placenta, was not impaired in the SHR.
Concentrations of amniotic fluid corticosterone were significantly decreased
in the SHR compared with the WKY at fetal Day 20, but were not significantly
different on fetal Days 16 or 22. This suggests that rather than increased
exposure to corticosteroids in the SHR fetus corticosteroid exposure may be
reduced. Expression of lung surfactant protein A (Sp-A), a gene induced in
late gestation by corticosteroids, was decreased in the SHR. In addition,
differences in amniotic fluid electrolyte concentrations were observed which
may reflect delayed renal maturation in the fetal SHR. These data suggest that
the SHR fetus is exposed to low concentrations of corticosteroids and that the
late gestation rise in fetal corticosteroid may be delayed in the SHR.
Potential benefits of cell cloning for human medicine
Authors: Trounson A. , Pera M.
Published: November 2003

The successful cloning of a mammal from an adult somatic cell nucleus opens
new avenues for major advances in reproductive medicine, biotechnology and
cellular-based transplantation therapies for degenerative diseases. At the
same time, this breakthrough has generated much heated discussion concerning
the ethics of cloning. Twinning is a form of cloning, and there are instances
in clinical assisted reproduction in which the deliberate formation of twins
by embryo dissection would seem ethically acceptable. Nuclear transfer
technology might facilitate the derivation of human embryonic stem cells,
capable of differentiation into a wide variety of somatic cell lineages.
Directed differentiation of human embryonic stem cells into specific cell
types in vitro could provide a universal source of cells
for transplantation therapy. The potential benefits of therapeutics based on
cloning technologies are considerable, and hasty legislation to ban all such
procedures could block progress in critical arenas of biomedical research
Cystic fibrosis and reproduction
Authors: Phillipson Greg
Published: November 2003

Cystic fibrosis (CF) is the most common autosomal recessive disease. CF and
congenital bilateral absence of the vas deferens (CBAVD) share a genetic and
embryological background. Since the 1960s, medical therapy to reduce the
progressive obstructive lung disease and nutritional deficiencies has resulted
in most CF patients reaching adulthood. With the improved life expectancy of
CF patients, new issues in reproductive health and pregnancy management have
arisen. Puberty is delayed, with menarche often occurring eighteen months
later than the average. Almost all men with CF are azoospermic. In both CF and
CBAVD, the vas deferens is absent and the seminal vesicles are often
hypoplastic. Many women with CF are subfertile, and if pregnancy is achieved
there is an observed increase in maternal morbidity and mortality. The
understanding of the molecular basis of CF and CBAVD has evolved, with the
identification of hundreds of CF gene mutations and discovery of an associated
intron polymorphism of the CF gene. The concept of severe and mild mutations
has been introduced to explain the severe and mild phenotype variations such
as the pancreatic insufficient and pancreatic sufficient patient. This paper
reviews the above issues to assist with the management of infertile couples
with CF or CBAVD.
The effects of intrafetal ACTH administration on the outcome of pregnancy in the mare
Authors: Ousey J. C. , Rossdale P. D. , Dudan F. E. , Fowden A. L.
Published: November 2003

Enhanced adrenocortical activity in the fetus is related to the onset of
parturition in many species. The aim of this study was to determine the effect
of injection of fetal ACTH on gestational length and fetal viability in the
horse. Pony mares (n=23) were studied from 300
days gestation. Seven control mares (Group 1) received three consecutive
intrafetal injections of sterile water, while fetuses of a further 16 mares
received Depot ACTH1–24. These mares were either
allowed to foal spontaneously (Group 2, n=4) or
delivery was induced within 3 days of the last fetal injection (Group 3,
n=7); 5 mares aborted within 48 h of intrafetal
ACTH injection. Maternal plasma progestagen concentrations increased
significantly (P<0.05) in Groups 2 and 3 mares following intrafetal ACTH
injection, compared with Group 1 mares. All Group 1 mares delivered live foals
at a median gestational age of 327 days (interquartile range, 323–334),
except one that aborted 16 days after the last fetal injection. Gestational
length was significantly (P<0.05) shortened in Group 2 mares (median 314
[312–314]) days compared with controls. Group 1 and Group 2
foals showed normal postnatal adaptive responses and endocrine (plasma
cortisol/progestagens) patterns. Group 3 mares delivered live foals at 306
(306–308) days gestation except one that aborted. Their foals were less
mature compared with Groups 1 and 2 foals. Eight ACTH-injected fetuses were
meconium-stained at delivery, including four that were aborted. Eight mares
had thickened placentae, including three that aborted. The data show that both
precocious maturation of the equine fetus and a significant reduction in
gestational length compared with controls may be achieved in pony mares by
intrafetal ACTH injections. This is probably mediated via adrenal regulation
of fetal maturation and production of maternal progestagens.
Improved fertility through superoxide removal by Toki-shakuyaku-san in mice - a preliminary study
Authors: Ota H. , Igarashi S. , Tanaka T.
Published: November 2003

The effect of a superoxide dismutase inhibitor on pregnancy rate and fetal
survival, and the effect of concomitant treatment with Toki-shakuyaku-san, was
studied. Twelve week-old IV CS female mice were given diethyldithiocarbamate
(DEDC; 0.9, 9, 45, and 225 mg kg–1)
intraperitioneally, from Day 3 to Day 5 of pregnancy, every 12 h, for a total
of six times. Two additional groups received both 45 mg
kg–1 DEDC and Toki-shakuyaku-san (50 or 200
mg/100 mL), by water bottle, from 12 h before to 12 h after DEDC
administration. For each group, the pregnancy rate, the number of surviving
fetuses, and the number of resorbed fetuses was calculated. Pregnancy rates
were 95.5, 81.8, 54.5 and 0%, respectively, in the DEDC groups and were
100% in the control group. The fetal resorption rate was 0.9 in the
control group, and 1.0, 1.7, and 2.3, respectively, in the 0.9, 9, 45 mg
kg–1 DEDC groups. Administration of 200 mg
Toki-shakuyaku-san significantly (P<0.05) improved
the pregnancy rate to 81.8%, and improved the degree of reduced
intercellular interactions and insufficient decidual reactions found on Day 5
in the endometrium of the DEDC groups. Toki-shakuyaku-san reduces the adverse
effects of excess superoxides on the endometrium during implantation.
Expression of Bcl-2 and 3-ß hydroxysteroid dehydrogenase protein during oocyte and follicle development in fetal and post-natal pig ovaries
Authors: Garrett Wesley M. , Guthrie H. David
Published: November 2003

The fetal and post-natal development of the pig ovary involves both
proliferation and apoptotic loss of germ cells, follicle formation and growth,
and the initiation of oocyte meiotic maturation. The present study measured
the expression of the proto-oncogene Bcl-2 immunohistochemically on paraffin
sections of pig ovaries to determine its relationship with folliculogenesis on
Days 50 and 80 post coitum (p.c.) and on Days 1, 21, and 56 post partum
(p.p.). The expression of the steroidogenic enzyme 3β-hydroxy-steroid
dehydrogenase (3βHSD) was used to determine the lineages of the cells
forming the ovarian follicles, and the expression of the cell
proliferation-associated nuclear antigen Ki-67 was used to determine germ cell
proliferation and the initiation of follicle growth. Expression of Ki-67
showed that many oogonia were proliferating on Days 50 and 80 p.c. Granulosa
cells were more proliferative on Day 56 p.p. than at any other stage; Ki-67
was expressed in 70% of growing follicles and granulosa cells had a
3% mean staining index per section. Less than 4% of germ cells
and follicles had morphological signs of degeneration during the period of the
study. Bcl-2 was most abundant on Days 21 p.p. and 56 p.p.; staining was
localized to stromal cells among follicles and in small clusters in the
cortical–medullary junction (CMJ). 3βHSD staining on Day 50 p.c. was
seen in cords of stromal cells within the medulla of the ovary, and in the
stromal cells investing the oogonial nests. On Days 80 p.c., 1 p.p., 21 p.p.,
and 56 p.p., 3βHSD was expressed in the granulosa cells of primary or
primordial follicles at the CMJ. Production of Bcl-2 by somatic cells may
support germ cell and preantral follicle survival.
Determinants of the annual pattern of reproduction in mature male Merino and Suffolk sheep: modification of endogenous rhythms by photoperiod
Authors: Martin Graeme B. , Tjondronegoro Soedjiharti , Boukhliq Rachid , Blackberry Margaret A. , Briegel Jan R. , Blache Dominique , Fisher James A. , Adams Norman R.
Published: November 2003

In mediterranean environments, pastures are
very poor during the autumn and consequently small ruminants, such as sheep,
would have been losing body mass for many months so, during mating, gamete
production would be depressed in both sexes. Effectively, the nutritive
requirements for a photo-period-driven, annual reproductive cycle are out of
phase with seasonal changes in food availability. The problem could be
overcome through more flexible timing of reproduction, perhaps explaining
variations in seasonality between breeds that originate from differing
latitudes. To study these concepts and the mechanisms involved, the endogenous
rhythms and responses to photoperiod were compared in rams of
‘mediterranean origin’ (Merino) and ‘temperate origin’
(Suffolk). Groups of 16 rams of each breed were given a constant food supply
and subjected to 16 months of constant equinoctial photoperiod (12L : 12D) or
simulated ‘mediterranean’ changes in daylength (from 14L : 10D to
14D : 10L). With nutritional and photoperiodic inputs held constant, Merino
and Suffolk rams showed similar endogenous rhythms in reproductive activity.
Under constant nutritional inputs and a mediterranean photoperiodic cycle, the
endogenous rhythms were modified differently in the two breeds, with the
Merinos starting and finishing their seasons about 2 months earlier than the
Suffolks. These observations partially explain the patterns observed in rams
kept under field conditions. It is now necessary to test whether the rhythms
of reproduction in these breeds are also modified by changes in nutrition and
social cues.
Secretory proteins from the female reproductive tract of the brushtail possum Trichosurus vulpecula: binding to sperm and effects on sperm survival in vitro
Authors: Sidhu K. S. , Mate K. E. , Molinia F. C. , Glazier A. M. , Rodger J. C.
Published: November 2003

Previous studies have demonstrated that
co-culture of brushtail possum epididymal spermatozoa with oviduct epithelial
cell monolayers prolongs sperm survival and results in the re-orientation of
the sperm head and tail to the T-shape (thumbtack) configuration.
Transformation of sperm to thumbtack orientation is believed to be associated
with marsupial sperm capacitation. Here we report that incubation in
oviduct-conditioned media also significantly prolongs sperm survival and
results in the transformation of sperm to the thumbtack orientation. The major
objective of the current study was to examine the proteins present in the
conditioned media, to determine whether any of these proteins specifically
bound to sperm and the relationship between these proteins and sperm survival
and thumbtack orientation. Co-culturing brushtail possum sperm with
biotin-labeled proteins in conditioned media (CM) from ampulla, isthmus and
uterine explants demonstrated strong binding of two proteins of molecular mass
230 and 61 kD and weak binding of nine proteins of molecular mass 200, 180,
120, 140, 55, 52, 48, 34, 30 kD to sperm within 30 min. The binding of the
61-kD protein from the conditioned media appeared specific as increasing
concentrations of non-labeled oviduct proteins, but not serum proteins,
inhibited the binding of labeled proteins. The binding of oviduct and uterine
proteins in the conditioned media significantly prolonged sperm survival and
percentage motility and also transformed a large number of sperm to a
thumbtack orientation. The implication of binding of these proteins is
discussed in the context of sperm survival and capacitation in this species.
Role of developmental factors in the switch from pyruvate to glucose as the major exogenous energy substrate in the preimplantation mouse embryo
Authors: Martin Karen L. , Leese Henry J.
Published: November 2003

Preimplantation mouse embryos, cultured in vitro and
those freshly flushed from the reproductive tract, exhibit a switch in energy
substrate preference, from pyruvate during the early preimplantation stages,
to glucose at the blastocyst stage. Although the biochemical basis of this
phenomenon is quite well characterized, its timing and possible association
with developmental factors have not been considered. We have therefore
examined the role of five developmental factors in determining the timing of
the switch, namely: (1) embryo age (in hours post hCG);
(2) developmental stage; (3)
cytokinesis; (4) cell number; and
(5) activation of the embryonic genome. One-cell
embryos, which develop more slowly than 2-cell embryos
in vitro, were used to investigate the role of embryo
age and developmental stage. Cytochalasin D, which inhibits cytokinesis and
delays the timing of compaction and cavitation, was used to investigate the
role of cell division and developmental stage. Finally, transcription of the
embryonic genome was examined with the inhibitor, α-amanitin. Pyruvate
and glucose consumption by single embryos were measured using a non-invasive
ultramicrofluorometric technique. The results showed that the timing of the
switch in energy substrate preference is precisely regulated in the mouse
preimplantation embryo. Activation of the embryonic genome is a prerequisite
for the switch and its timing is closely associated with developmental stage,
specifically compaction and/or cavitation. Cell number, cytokinesis and
embryo age appeared to be unrelated to the timing of the switch. These
conclusions may well be extrapolated to other species, since an increase in
net glucose uptake, if not always at the expense of pyruvate, is a feature of
preimplantation embryo metabolism in all mammals studied.
Low-dose flunarizine does not affect short-term fetal circulatory responses to acute asphyxia in sheep near term
Authors: Garnier Yves , Berger Richard , Pfeiffer Doris , Jensen Arne
Published: November 2003

Asphyxia is one of the major causes of perinatal brain damage and neuronal
cell loss, which may result in psychomotor deficits during later development.
It has been shown previously that the immature brain can be protected from
ischemic injury by flunarizine, a class IV calcium antagonist. However,
cardiovascular side-effects of flunarizine, when applied at the dosages used
in those studies, have been reported. Recently, the present authors were able
to demonstrate that even by injecting flunarizine at a far lower dosage (1 mg
kg) estimated bodyweight) neuronal cell damage, caused by occlusion of both
carotid arteries for 30 min, can be reduced in fetal sheep near term. The aim
of the present study was, therefore, to examine whether low-dose flunarizine
affects fetal cardiovascular responses to acute asphyxia in sheep near term.
Ten fetal sheep were chronically instrumented at a mean gestational age of 132
1 days (term is at 147 days). Fetuses from the study group received a bolus
injection of flunarizine (1 mg kg–1 estimated
fetal weight) 60 min before asphyxia, whereas the solvent was administered to
the fetuses from the control group. Organ blood flows, physiological variables
and plasma concentrations of catecholamines were measured before, during and
after a single occlusion of uterine blood flow for 2 min (i.e. at 0, 1, 2, 3,
4, and 30 min). Before asphyxia, the distribution of combined ventricular
output and physiological variables, as well as concentrations of
catecholamines, in fetuses from the control group were in the normal range for
chronically prepared fetal sheep near term. During acute asphyxia there was a
redistribution of cardiac output towards the central organs accompanied by a
pronounced bradycardia and a rapid increase in arterial blood pressure. After
asphyxia circulatory centralization did not resolve quite as rapidly as it
developed, but was almost completely recovered at 30 min after the insult.
There were nearly no differences in the time course of physiological and
cardiovascular variables measured before, during and after acute intrauterine
asphyxia between the control and study groups. From the present study it was
concluded that low-dose flunarizine does not affect short-term fetal
circulatory responses to acute asphyxia in sheep near term.
Cytogenetic analysis of human preimplantation embryos following developmental arrest in vitro
Authors: Almeida Paula A. , Bolton Virginia N.
Published: November 2003

The relationship between chromosomal abnormalities in the human
preimplantation embryo and developmental arrest in vitro
was investigated. Cytogenetic analysis of 171 embryos that had arrested
between the pronucleate and the 8-cell stages demonstrated that the overall
incidence of chromosomal abnormality among these embryos was 63.4%. Of
the embryos that arrested at the pronucleate stage (n
= 48), 47.9% were chromosomally abnormal, compared with
59.5% of those that arrested between the 2- and 4-cell stages
(n = 50), and 82.8% of those arrested
between the 5- and 8-cell stage (n = 73). The
rate of abnormality in embryos with poor morphology (irregular shaped
blastomeres and considerable extracellular fragmentation) was significantly
higher (86.8%; n = 33) than those with
good morphology (60%; n = 51;
P<0.005). These results suggest that there is an
association between chromosomal abnormality, developmental arrest
in vitro, and poor morphology.
Strategies for activating nuclear transfer oocytes
Authors: Macháty Zoltán , Prather Randall S.
Published: November 2003

The technique of nuclear transfer can have enormous applications in the fields
of agriculture and biomedicine. This is especially true if a cell line that
has been transformed can be used as a source of nuclei for the nuclear
transfer. One major aspect of the nuclear transfer procedures is that of
oocyte activation. Without oocyte activation the transferred nucleus would
never progress to the first interphase. It is therefore of utmost importance
that the oocyte be activated in a fashion that is as normal as fertilization.
The inability to obtain development after artificial activation of pig oocytes
has been a limiting factor in the application of the nuclear transfer
technology. Recently, a number of techniques have been developed that result
in blastocyst stage embryos after oocyte maturation
in vitro and artificial activation. The theories behind
normal oocyte activation are reviewed as well as a number of methods of
artificial oocyte activation. It is anticipated that such a review will
provide the basis for the development of additional methods that are as
efficient, or more efficient, at activating the unfertilized oocyte.
Impact of cloning on cattle breeding systems
Authors: McClintock A. E.
Published: November 2003

The concept of clone-family testing is compared with existing progeny testing
systems. The critical factors that will decide how cloning is utilized are the
potential size of cloned families, and the cost per embryo (or per calf born).
If family sizes of 100 000 become routinely achievable (cheaply), then clone
testing becomes viable. In rough figures, cloned embryos costing $30
with a 50% calving rate would be attractive to farmers and would be
cheap enough that farmers would buy more (crossbred) embryos in order to breed
further replacement cows. At $300 per embryo, farmers would be more
inclined to buy a number of cloned pure-bred female embryos and then to use
conventional artificial insemination to breed further replacements from these
superior cows. At $3000 per embryo, farmers would probably only be
interested in very small numbers of cloned animals, most of which would be
males. The relative importance of adult versus fetal cloning is discussed. The
need for gene banks to preserve genetic variation is emphasized; both gametes
and somatic tissue cultures should be considered.
Genetically identical primate modelling systems for HIV vaccines
Authors: Kent Stephen J. , Lewis Ian M.
Published: November 2003

There is an urgent need for a safe and effective vaccine to prevent human
immunodeficiency virus (HIV) infection. Several HIV vaccine candidates have
shown promise, but many concerns regarding the safety and efficacy of current
vaccines remain. A major hindrance in HIV vaccine development is a poor
understanding of precisely what functions HIV vaccines are required to perform
in order to protect humans from HIV-1. Only higher primates (i.e. macaques,
chimpanzees and humans) are susceptible to HIV-1 or the closely related virus
‘simian immunodeficiency virus’. These species are outbred and
there are remarkable genetic differences in both the immune responses to
vaccines and their susceptibility to infection. The development of genetically
identical macaques would be a major step towards dissecting what immune
responses are required to protect from HIV infection. For example, live
attenuated HIV-1 vaccines are likely to be highly efficacious, but will induce
disease in a substantial proportion of recipients. Defining why a live
attenuated vaccine is effective should allow safer vaccines to be developed,
retaining only the immunologic properties of an effective vaccine. The
reduction in ‘background genetic noise’ obtained by studying
genetically identical primates would provide concise answers to critical HIV
vaccine issues, by studying a minimal number of animals. Such an approach
could potentially be employed in other diseases where non-human primates are
the only available model. Small studies can be performed where identical twins
are generated by embryo bisection; however, larger studies where multiple
immune parameters are simultaneously evaluated would be facilitated by cloning
technology. Despite the technical difficulties to be overcome, the potential
gains in human health from the development of genetically identical non-human
primates are worthy of careful consideration.
Gestational changes in fetal renal and hepatic angiotensinogen mRNA and protein
Authors: Zhang David Y. , Lumbers Eugenie R. , Wu June J.
Published: November 2003

The aim of the study was to determine the amount of angiotensinogen expression
and its protein product in fetal sheep liver and kidney in the last third of
gestation. Angiotensinogen mRNA was measured by RNase protection assay and its
protein levels were measured by radioimmunoassay. Levels were measured at 80,
95, 111, 125 and 139 days. Angiotensinogen mRNA was present in all fetal liver
and kidney samples tested. The ratio of hepatic angiotensinogen mRNA/18 S
rRNA increased by 100% (P<0.001) and
angiotensinogen levels increased by 33%
(P<0.001) in fetal sheep from 80 to 139 d. Over the
same period the ratio of renal angiotensinogen mRNA/18 S rRNA increased by
170% (P<0.001) and renal angiotensinogen
protein increased by 41% (P<0.001). The levels
of angiotensinogen mRNA and its protein in the adult kidney were less than in
kidneys of 139 d old fetuses (P<0.01). There was a
direct relationship between levels of angiotensinogen mRNA and its protein in
the liver (r = 0.53,
P<0.01, n = 25) and in
the kidney (r = 0.75,
P<0.0001, n = 24). These
findings demonstrate that there is a significant increase in both hepatic and
renal angiotensinogen gene expression in the last third of gestation in the
fetal sheep and that this increase is associated with an increase of
angiotensinogen levels in both tissues. This increase in angiotensinogen in
late gestation could influence the activity of both the intrarenal and
circulating renin angiotensin systems.
The production of transgenic domestic livestock: successes, failures and the need for nuclear transfer
Authors: Ward Kevin A. , Brown Bruce W.
Published: November 2003

The direct transfer of recombinant DNA to embryos is conceptually a powerful
method for the manipulation of the genetic potential of domestic animals, but
in practice the technology has yet to fulfil its promise. In this paper, two
examples are given of research utilising direct genetic manipulation, both of
which are aimed at increasing aspects of productivity in sheep. The first of
these involves the modification of the growth hormone status of sheep by the
use of the ovine growth hormone gene, the regulation of which has been altered
by the use of an ovine metallothionein promoter. While there has been a large
amount of research already conducted in this area by many groups, our recent
results provide one of the first demonstrations that this approach can
increase the growth rates of sheep while maintaining the animals in good
health. The second project involves the modification of intermediary
metabolism in sheep by the introduction of the cysteine biosynthetic pathway.
The results to date demonstrate that it is possible to change intermediary
metabolism in animals using our approach but that there are species-specific
requirements that must be satisfied in order to make the approach a practical
method for improving animal productivity.
Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm
Authors: Nichols Jennifer , Smith Austin , Buehr Mia
Published: November 2003

In this study we have compared the in vitro
differentiation potential of epiblast tissue from mouse and rat embryos.
Epiblasts were isolated from egg cylinder stage embryos by microdissection and
placed in culture. Rat cultures were distinguished by the copious production
of parietal endoderm cells. Mouse epiblasts, in contrast, did not produce
parietal endoderm. This difference in capacity to regenerate extraembryonic
endoderm marks a surprising distinction in development of the pluripotential
lineage between these two closely related rodents.
Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro
Authors: Pelton T. A. , Bettess M. D. , Lake J. , Rathjen J. , Rathjen P. D.
Published: November 2003

Early mammalian embryogenesis is characterised by the coordinated
proliferation, differentiation, migration and apoptosis of a pluripotent cell
pool that is able to give rise to extraembryonic lineages and all the cell
types of the embryo proper. These cells retain pluripotent differentiation
capability, defined in this paper as the ability to form all cell types of the
embryo and adult, until differentiation into the three embryonic germ layers
at gastrulation. Our understanding of pluripotent cell biology and molecular
regulation has been hampered by the difficulties associated with experimental
manipulation of these cells in vivo. However, a more
detailed understanding of pluripotent cell behaviour is emerging from the
application of molecular technologies to early mouse embryogenesis. The
construction of mouse mutants by gene targeting, mapping of gene expression
in vivo, and modelling of cell decisions
in vitro are providing insight into the cellular origin,
identity and action of key developmental regulators, and the nature of
pluripotent cells themselves. In this review we discuss the properties of
early embryonic pluripotent cells in vitro and
in vivo, focusing on progression from inner cell mass
(ICM) cells in the blastocyst to the onset of gastrulation.
Laboratory production of buffalo (Bubalus bubalis) embryos
Authors: Palta P. , Chauhan M. S.
Published: November 2003

There is an increasing interest in large-scale in vitro
production (IVP) of buffalo embryos through in vitro
maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes for faster
multiplication of superior germplasm. The recovery of total and usable quality
oocytes from slaughterhouse ovaries is low in this species. The nuclear
maturation rates of buffalo oocytes matured in the presence of follicular
fluid or serum and hormones like luteinizing hormone, follicle-stimulating
hormone and oestradiol vary from 70 to 80% and are comparable to those
reported for cattle oocytes. However, with fertilization rates of
40–55%, and the yield of blastocysts at around
10–15%, the efficiency of IVP is much lower than that in cattle.
The in vitro sperm preparation procedures and the
systems employed for performing IVF and culture of zygotes up to blastocyst
stage are suboptimal and need substantial improvements. The quality and
viability of blastocysts produced need to be checked by cell count, and after
transfer to synchronized recipients, for development of quality control
standards.
Maintenance of pluripotential embryonic stem cells by stem cell selection
Authors: Mountford Peter , Nichols Jennifer , Zevnik Branko , O'Brien Carmel , Smith Austin
Published: November 2003

As gastrulation proceeds, pluripotential stem cells with the capacity to
contribute to all primary germ layers disappear from the mammalian embryo. The
extinction of pluripotency also occurs during the formation of embryoid bodies
from embryonic stem (ES) cells. In this report we show that if the initial
differentiated progeny are removed from ES cell aggregates, further
differentiation does not proceed and the stem cell population persists and
expands. Significantly, the presence of even minor populations of
differentiated cells lead to the complete loss of stem cells from the
cultures. This finding implies that the normal elimination of pluripotent
cells is dictated by inductive signals provided by differentiated progeny. We
have exploited this observation to develop a strategy for the isolation of
pluripotential cells. This approach, termed stem cell selection, may have
widespread applicability to the derivation and propagation of stem cells.
Reprogramming cattle somatic cells by isolated nuclear injection
Authors: Trounson Alan , Lacham-Kaplan Orly , Diamente Maria , Gougoulidis Tiki
Published: November 2003

The development of a somatic cell nuclear transfer procedure for the
production of blastocyst stage cattle embryos is described. Bovine fetal
fibroblasts were used for fusion experiments with surgically enucleated
oocytes (cytoplasts) following the establishment of optimal parameters for
electrofusion from isofusion contours. Fusion rates were increased by
decreasing size of the cytoplasts used but cleavage was decreased by
decreasing size of the cytoplast used (quarter, half and whole cytoplasts).
The use of double cytoplasts did not improve cleavage, and development to
blastocysts could not be achieved. In a comparison of electrofusion of
fibroblasts with cytoplasts in the subzonal perivitelline space with
intracytoplasmic injection of nuclei and parthenogenetically activated
oocytes, 2%, 14% and 24% developed to blastocysts
respectively. In the group injected with isolated nuclei, the passage number
(4 to 9) had no apparent influence on developmental competence to blastocysts.
The embryos produced by nuclear injection of somatic cell nuclei showed the
normal pattern of cell surface appearance of TEC-3 and TEC-4 stage-specific
epitopes during development, as seen in fertilized oocytes. We conclude that
the nuclear injection of somatic cell nuclei is a relatively efficient way to
clone bovine embryos.
Comparison of the merits of measuring equine chorionic gonadotrophin (eCG) and blood and faecal concentrations of oestrone sulphate for determining the pregnancy status of miniature horses
Authors: Henderson K. , Stevens S. , Bailey C. , Hall G. , Stewart J. , Wards R.
Published: November 2003

The relative merits of measuring blood concentrations of equine chorionic
gonadotrophin (eCG, previously known as pregnant mare serum gonadotrophin
(PMSG)), or oestrone sulphate (OS), or faecal OS concentrations for
determining pregnancy status in miniature horses were investigated. Pregnant
mares between 40 and 140 days after mating had serum eCG concentrations >1
I.U. mL–1, with the highest concentrations
occurring between days 50 and 120. However, eCG measurements were susceptible
to returning a ‘false positive’ diagnosis of pregnancy. Plasma OS
concentrations ranged from 0.1 to 3.6 ng mL–1 in
non-pregnant mares, whereas pregnant mares beyond 100 days post-mating all had
plasma OS concentrations >30 ng mL–1. Faecal OS
concentrations ranged from 4 to 89 ng g–1 in
non-pregnant mares. For faecal samples collected from pregnant mares 150 days
or more after mating, 97% of samples had OS concentrations >85 ng
g–1, the value 3 standard deviations above the
mean non-pregnant value. None had values below 67 ng
g–1, the value 2 standard deviations above the
mean non-pregnant value. These results show that measurement of eCG is
suitable for determining pregnancy status in miniature mares between 40 and
100 days post-mating. However, mares returning a ‘pregnant’
diagnosis should undergo a blood OS test 100 or more days after mating to
eliminate the possibility of a ‘false positive’ diagnosis.
Measuring blood OS is recommended as the method of choice for determining
pregnancy status in miniature mares 100 or more days after mating. Faecal OS
measurements provide a non-invasive alternative to blood OS testing from 150
days post-mating. However, the discrimination between ‘pregnant’
and ‘non-pregnant’ levels of OS is not as great in faeces as it is
in blood.
Developmental changes in plasma angiotensin-converting enzyme concentration in fetal and neonatal lambs
Authors: Forhead Alison J. , Melvin Robert , Balouzet Vanessa , Fowden Abigail L.
Published: November 2003

Relationships between plasma angiotensin-converting enzyme (ACE) and cortisol,
and blood pressure were examined in chronically catheterized ewes and their
fetuses during late gestation (111–141 days, term 145 2 days). Plasma
ACE was also measured in non-pregnant adult ewes and in lambs over the first 5
weeks of life. In fetuses near term (136–141 days), plasma ACE was
greater than in those studied earlier in gestation; overall, plasma ACE
correlated with gestational age (r = 0.72). The
ontogenic rise in plasma ACE was associated with prepartum increases in plasma
cortisol (r = 0.67) and blood pressure
(r = 0.66). No relationship was observed between
plasma ACE and partial pressure of oxygen in utero. Peak
plasma ACE concentration observed in fetuses near term was maintained in
newborn lambs for 3 days after birth. By 2 weeks of postnatal age, plasma ACE
had decreased to the value seen in non-pregnant adult ewes. Maternal plasma
ACE was similar at all gestational ages studied, and was lower than that
observed in non-pregnant ewes. Therefore, in the sheep fetus, plasma ACE
increased towards term in association with the prepartum cortisol surge.
Developmental changes in ACE activity may be partly responsible for the
ontogenic rise in fetal blood pressure.