bluuebunny/crossref_metadata_2025 huggingface dataset:
Fertility control of mammal pests and the conservation of endangered marsupials
Authors: Sinclair A. R. E.
Published: November 2003

Populations are bounded by negative feedbacks operating through fertility or
mortality, termedpopulation regulation. If fertility is
artificially reduced, the average size of the population is also reduced, but
only under certain conditions. If (i) juvenile survival
or (ii) adult survival improve due to lower fertility,
or (iii) territoriality limits populations, the effects
of lower birth rate will not change population size unless such reduction
exceeds the effects of these processes.
Published data on population trends and birth rates have allowed a comparison
among species of instantaneous rates of change. The intrinsic rate of
increase, rm, and population
variability are both related to body size, because birth rates and
survivorship are also related to body size. These rates are trade-offs as
adaptations. Populations of species in exotic habitats may fluctuate more than
when they are in their indigenous habitats. Fertility control could reduce
such fluctations.
Marsupials have lower birth rates than eutherians, and so rely more on
survivorship, perhaps as an adaptation to unpredictable environments.
Compromising survival by either habitat change or increased predation will
affect marsupials more than eutherians. This explains why many marsupial
populations are declining towards extinction.
Neither human nor natural: ethics and feral animals
Authors: Singer Peter
Published: November 2003

There are three major ethical approaches to issues affecting nonhuman animals
and the natural environment: an anthropocentric ethic, an ethic of concern for
all sentient beings, and a biocentric approach. The ethic of concern for all
sentient beings is the most defensible basis for resolving conflicts between
the interests of humans and wild animals. There is no ethical basis for
discounting the suffering of an animal simply because that being is a member
of a different species. On the other hand, it is certainly true that human and
nonhuman animals differ in their capacities, and this does make a difference
to the ethics of what we may do to them, including rendering them infertile.
Since ethics is not a matter of adhering to absolute rules, but rather of
doing what will have best consequences, given the constraints under which we
act, the ethics of using a specific method of fertility control for feral
animals will depend on what other methods are being used, or will be used, if
the given method is not available. It will also depend on the consequences of
not using any method of controlling the population of the animals.
Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb
Authors: Armstrong D. T. , Kotaras P. J. , Earl C. R.
Published: November 2003

The use of juvenile donors in embryo-transfer (ET) programmes offers
considerable potential for accelerated genetic gain in domestic livestock
through reduced generation interval. The present paper reviews recent research
aimed at optimizing embryo production from oocytes collected from young calves
and lambs using in vitro methods of embryo production.
Emphasis is placed on criteria for donor selection, oocyte-collection methods,
and hormone-stimulation methods designed to produce maximum yields of viable
oocytes. In vitro fertilization (IVF) rates of calf and
lamb oocytes did not differ significantly whether matured
in vivo or in vitro, and rates of
development to blastocyst stages in culture were similar to those observed for
embryos derived from adult donors. Blastocysts produced by IVF of lamb and
calf oocytes established ET pregnancies at rates of 30–45%.
Pregnant recipients have reached full term and delivered normal offspring at
rates similar to those expected following ET of embryos produced
in vivo from superovulated donors. On the basis of
current follicle-stimulation protocols, on rates of blastocyst production in
vitro under optimal conditions, and on observed pregnancy rates from fresh
transfer of IVF embryos, 8–10 pregnancies may be expected per oocyte
collection from 10–12-week-old calves and from 6–8-week-old lambs.
Alternative strategies for the long-term preservation of spermatozoa
Authors: Holt W. V.
Published: November 2003

A number of perceived future requirements for stored germplasm in agriculture,
aquaculture, biotechnology and conservation are discussed in the present
review. In the light of these broad demands, it is apparent that current
approaches to gamete and embryo storage need considerable improvement, and
that novel approaches to the technologies of germplasm preservation should be
pursued if possible. The present article is presented in response to a request
for novel future research ideas in this area. Early literature on desiccation,
and later research into natural mechanisms of survival during desiccation, is
considered in relation to the development of freeze-drying and vitrification
methods. Developments in reproductive technologies themselves may mean that
freeze-dried spermatozoa could realistically be used for direct
intracytoplasmic microinjection of oocytes. Other radical methods that may
achieve germplasm preservation are harvesting testicular cells in culture,
cryopreserving immature or seasonally regressed testicular material, or a
combination of both. Evidence from non-mammalian testicular culture systems,
and recent success with the transmeiotic development of mouse
spermatidsin vitro, suggest that these approaches may
eventually become feasible.
Recent developments and potentialities for reducing embryo mortality in ruminants: the role of IFN-t and other cytokines in early pregnancy
Authors: Martal J. , ChÊne Nicole , Camous Sylvaine , Huynh L. , Lantier F. , Hermier Paloma , L'Haridon R. , Charpigny G. , Charlier Madia , Chaouat G.
Published: November 2003

This review considers the potential reduction of embryo mortality
in vitro and in vivo in ruminants.
Data on cytokines provided by different fields of reproductive immunology and
biology were collated. Because of the crucial importance of the local
interactions between the embryo and its dam, the expression of growth-factor
and cytokine genes was analysed in the embryo proper, trophoblast, oviduct and
endometrium by reverse transcriptase polymerase chain reaction in sheep and in
cattle during the pre- and periimplantation periods. Many deleterious
cytokines, such as tumour necrosis factor-α, intereron-γ (IFN-γ
), interleukin-2 (IL-2), and beneficial cytokines, such as transforming growth
factor-β, leukaemia inhibiting factor, colony-stimulating factor-1
(CSF-1), ganulocyte–macrophage CSF, IL-1, IL-3, IL-4, IL-6, IL-10 and
IFN-γ appeared to be involved in embryo survival in ruminants and other
species. Their administration is efficient in a murine experimental model
(CBA/J DBA/2) of embryonic and fetal mortality. For instance,
recombinant ovine IFN-τ (roIFN-τ ) injected at the moment of
implantation drastically reduces embryonic mortality in this model. In
ruminants, roIFN-τ and recombinant bovine IFN-τ are very efficient in
maintaining progesterone luteal secretion in cyclic animals. The involvement
of IFN-τ in the mechanisms of maternal pregnancy recognition are
particularly detailed in relation to inbition of 13,14
dihydro-15-keto-prostaglandin F2α (PGFM) pulses
and oxytocin uterine receptivity. A synthetic model of the anti-luteolytic
effects of IFN-τ on the endometrial cell is proposed. Finally, the
particular potential of serum pregnancy-specific proteins (PSPs: PSPB, PSP60,
pregnancy-associated glycoprotein) for monitoring embryo survival, with
examples given for cattle and sheep is underlined.
Do sperm cells age? A review of the physiological changes in sperm during storage at ambient temperature
Authors: Vishwanath R. , Shannon P.
Published: November 2003

In a liquid environment, at high dilutions, fertility of bull sperm is
maintained for 3–5 days when stored at ambient temperatures
(10–21°C), after which time it steadily declines at a rate of
3–6% per day. This decline in fertility occurs irrespective of
whether the sperm are stored at 5°C or at 15°C, but the rate is
greater once storage temperatures exceed 25°C. Sperm motility can be
maintained for extended periods in an environment where the extracellular
oxidative stress is minimized by reducing the oxygen tension, by addition of
antioxidants and chelating agents; however, this will not prevent a
significant drop in fertility after five days of storage at ambient
temperature. The requirement of energy by the sperm-motility apparatus demands
a high level of respiratory activity. This system is very active and the free
radicals produced in vivo during this process could lead to chromatin damage.
As no internal repair mechanism exists in sperm, an extraneous supply of
protectants, or an environment where damage is minimized, is essential to
maintain its fertilizing potential. The lack of extended storage potential of
sperm, even in the presence of antioxidants, seems to suggest that although
oocyte-penetrating ability of the sperm could still be intact, the high rate
of intracellular metabolic activity could lead to mitochondrial DNA damage and
chromosomal abnormalities that would compromise the viability of the resulting
conceptus.
Effect of undernutrition of ewes from the time of mating on fetal ovarian development in mid gestation
Authors: Borwick S. C. , Rhind S. M. , McMillen S. R. , Racey P. A.
Published: November 2003

Ewes were fed either 150% (High, H) or 50% (Low, L) of their
energy requirements for maintenance of liveweight during early gestation.
Effects of maternal nutrition on fetal ovarian size, histological structure
and steroidogenic capacity were studied at Day 47 and on ovarian size and
structure at Day 62 of gestation. At Day 47 of gestation, there were
significantly higher concentrations of oogonia in the ovaries of L fetuses
than H fetuses (105·9 v. 76·9 germ cells
mm-2; s.e. 4·94; P <
0·001). The capacity of the ovaries to secrete oestradiol
(pg/ovary/24 h) at Day 47 was not affected by treatment when they were
incubated either with (H, 773; L, 740; s.e. 179; not signicant, n.s.) or
without (H, 260; L, 290; s.e. 92·7; n.s.) ovine luteinizing hormone
(oLH). At Day 62 of gestation, the process of germ cell degeneration was less
advanced in L than H fetal ovaries, as indicated by higher oocyte
concentrations in the former (68·4 v. 48·6
germ cells mm-2; s.e. 3·85;
P < 0·01). There was a greater percentage of
meiotic cells in L ovaries (76·5 v. 18·6;
s.e. 5·82; P < 0·001). It is concluded
that undernutrition of the ewe from the time of mating signicantly retards
ovarian development in fetal ovaries
Triosephosphate metabolism by mature boar spermatozoa
Authors: Jones A. R.
Published: November 2003

Boar sperm rapidly interconverted dihydroxyacetone phosphate and
glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately
equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but
not glycerol or glycerol 3-phosphate. In the presence of
3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic
pathway, the triosephosphates were metabolized faster, produced less
fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but
not glycerol or glycerol 3-phosphate. This suggests that these cells may have
the capacity to convert glycolytic intermediates into a storage metabolite to
conserve carbon atoms for the eventual synthesis of lactate.
Effect of oestradiol on ovarian oxytocin secretion rate and luteolysis in the ewe after ovarian auto-transplantation
Authors: Al-Matubsi H. Y. , CSIRO J. Downing , Jenkin G. , Fairclough R. J.
Published: November 2003

The release of ovarian oxytocin and uterine prostaglandin
(PG)F2a in response to an oestradiol stimulus was
investigated. On Day 15 post-oestrus, ten ewes with ovarian auto-transplants
(n = 5 per group) received an intra-muscular
injection of either oestradiol benzoate (50 µg) or vehicle. Blood
samples were collected from the ovarian and jugular veins at 30 and 0 min
before, and at 15-min intervals up to 540 min after, injection. The secretion
rate of ovarian progesterone remained elevated in four of five treated ewes
and in all control ewes, indicating the presence of a functional corpus
luteum. Peripheral oestradiol concentrations were significantly
(P < 0·001) higher in treated than in control
ewes. The number of ewes that released pulses of ovarian oxytocin ≥240 min
following oestradiol benzoate injection was signicantly
(P < 0·05) greater than that in control ewes.
Mean amplitude and area under both ovarian-vein oxytocin and jugular-vein 15
keto-13,14 dihydro prostaglandin F2α (PGFM) pulses
were significantly increased in the treated ewes. These ndings demonstrate
that the administration of exogenous oestrogen provides a positive stimulus
for the release of ovarian oxytocin and uterine
PGF2α in the ovarian auto-transplanted ewe.
Nitric oxide mediates increased prostaglandin E production by oocyte - cumulus complexes in the non-insulin-dependent diabetic rat
Authors: Jawerbaum Alicia , Gonzalez Elida T. , Novaro Virginia , Faletti Alicia , Gimeno Martha A. F.
Published: November 2003

Previous work described an increase in prostaglandin E (PGE) production by
oocyte–cumulus complexes (OVA) obtained from non-insulin-dependent
diabetic rats. More recently, it has been found that in control OVA nitric
oxide (NO) mediates hCG-induced PGE secretion. To determine whether increases
in PGE secretion by diabetic OVA are mediated by NO, the present study has
evaluated the secretion of PGE by diabetic OVA, cultured in the absence or
presence of hCG, NO donors (sodium nitroprusside (NP) and
3-morpholino-sydnonimine-hydrochloride (SIN–1)), and a NO synthase
inhibitor (NG monomethyl-L-arginine; L-NMMA). hCG, NP
and SIN–1 increased PGE secretion by diabetic OVA. L-NMMA did not modify
basal secretion of PGE by control OVA but lowered PGE production in diabetic
OVA to control values. L-NMMA prevented the hCG-induced PGE accumulation in
control and diabetic OVA, and the quantities of PGE produced were similar to
those of control OVA but lower than in diabetic OVA incubated in the absence
of hCG. The effect of L-NMMA seems to be specific since
NG monomethyl-D-arginine had no effect. NO synthase
activity was higher in diabetic ovaries than in controls. The present results
suggest that NO mediates the increased PGE production by diabetic OVA,
probably a result of overproduction of NO.
In vitro maturation and fertilization, and subsequent development of buffalo (Bubalus bubalis) embryos: effects of oocyte quality and type of serum
Authors: Chauhan M. S. , Singla S. K. , Palta P. , Manik R. S. , Madan M. L.
Published: November 2003

In Experiment 1, to determine the developmental potential of buffalo oocytes
of different qualities, compact cumulus–oocyte complexes (COCs) with an
unexpanded cumulus mass, and with homogeneous ooplasm were classified as Grade
1 (with 5 layers of cumulus cells) and Grade 2 less than 4 layers of cumulus
cells). Grade-3 oocytes were either without cumulus cells or with expanded
cumulus mass, and with irregular ooplasm. The oocytes were matured for 24 h at
38·5°C, 5% CO2 in air in maturation
medium (10% fetal bovine serum (FBS) in TCM-199 supplemented with 5
µg mL-1 follicle stimulating hormone-P). The
nuclear maturation and cleavage rates, and the proportion of cleaved embryos
which developed to morula and blastocyst stage were in the order Grade
1>Grade 2>Grade 3 (P < 0·05). For
Experiment 2, the maturation medium consisted of TCM-199 supplemented with one
of the following sera at 10% concentration: (1)
buffalo oestrus serum (BOS), (2) superovulated buffalo
serum (SBS), (3) fetal bovine serum (FBS) and
(4) steer serum (SS). After
in vitro fertilization (IVF), the oocytes were
co-cultured with buffalo oviductal epithelial cells in TCM-199 containing the
respective sera at 10% concentration for the subsequent 9 days. The
extent of cumulus expansion and nuclear maturation were not different among
different groups. The cleavage rates were lower (P <
0·05) with FBS than with BOS, SBS and SS. The proportion of cleaved
embryos which developed to blastocyst stage was higher
(P < 0·05) with SBS than with BOS, FBS and SS.
Sheep oocyte activation after intracytoplasmic sperm injection (ICSI)
Authors: Gomez M. C. , Catt J. W. , Evans G. , Maxwell W. M. C.
Published: November 2003

The effect of calcium concentration on fertilization and activation was
examined in oocytes injected in vitro with sperm.
Oocytes were subjected to sperm injection, to sham injection or remained
uninjected, and were then cultured for 19 h in bicarbonate-buffered synthetic
oviduct fluid (BSOF) without calcium, or containing either calcium chloride or
calcium ionophore. There was no difference in fertilization rates after ICSI
when oocytes were cultured in vitro in media containing
calcium chloride or calcium ionophore but the rate was lower in calcium-free
media. There was also no difference in the fertilization rate after ICSI when
oocytes were culturedin vivocompared with that
observedin vitro in media containing calcium chloride or
calcium ionophore. In calcium chloride-treated oocytes, activation was induced
by mechanical injection, and in calcium ionophore-treated oocytes, by the
ionophore. In uninjected oocytes, calcium itself did not cause oocyte
activation. It is concluded that it is possible to induce activation by the
injection process, but that manipulation alone is inadequate to cause proper
oocyte activation unless calcium is also present. No difference in oocyte
activation between ICSI and sham injection was found, indicating that the
sperm may play no role in the early events of oocyte activation.
Genomic imprinting, development and disease—is pre-eclampsia caused by a maternally imprinted gene?
Authors: Graves Jennifer A. Marshall
Published: November 2003

Several genes in conserved clusters are expressed from only the maternal or
the paternal allele. The other allele has been genetically silenced
(‘imprinted’) by its passage through one sex. Many known imprinted
genes have effects on embryonic or trophoblast growth or fetal development,
and mutation or loss of the single active copy causes diseases such as
Prader–Willi, Angelmann and Beckwith–Wiederman syndromes.
Imprinted genes show an unusual mode of inheritance, since mutant genes have
an effect on the phenotype only if they come from the parent from which they
are expressed. This may explain some conditions which appear to be heritable
but show an inconsistant pattern in affected families. Of particular interest
is pre-eclampsia/eclampsia, the most serious complication of pregnancy,
which has some features suggesting that it results from fetal expression of
the mutant gene, but others which imply it results from maternal expression.
This could be resolved by proposing that the condition is due to mutation in a
paternally imprinted, maternally active gene which must be expressed by the
fetus in order to establish a normal placenta in the first pregnancy.
Maternal nicotine exposure during gestation and lactation interferes with alveolar development in the neonatal lung
Authors: Maritz G. S. , Dennis H.
Published: November 2003

The aim of this study was to investigate the effect of maternal nicotine
exposure during pregnancy and lactation on the development of the lungs of the
offspring as a gas- exchanger. Pregnant rats received nicotine (1 mg/kg
body mass day–1) subcutaneously during gestation
and lactation. Nicotine administration started one day after mating and lasted
until weaning on post natal Day 21. The offspring were exposed to nicotine
only via the placenta and the milk of the mother. The lung tissue of the
neonates was collected on post natal Days 14, 21, 35 and 42 and prepared for
morphometry. The results obtained show that maternal nicotine exposure
suppressed alveolarisation in the lungs of the offspring, which resulted in a
reduced internal surface area available for gas exchange. The radial alveolar
count as well as the number of capillaries in the septa were also
significantly lower than in the control animals. It is concluded that maternal
nicotine exposure had an adverse effect on the development of the gas exchange
region of the lungs of the offspring that persisted at least up to Day 42
after birth.
Advances and challenges with intersex disorders
Authors: Warne Garry L.
Published: November 2003

Many recent advances have come from basic research into the mechanisms of
sexual differentiation. These include the discovery of a number of genes
(SRY,
WT-1,SF-1,
SOX-9) and one locus (DSS)
involved in human gonadal differentiation, and understanding gained from
extensive studies of the androgen receptor and its gene. The challenge for the
clinician is to learn how best to use the new tools that have become available
as a result of these advances. At the same time, community-based organizations
whose members are adults with intersex disorders are issuing a challenge of a
different kind: that the medical profession should radically revise its
thinking on some of the most fundamental issues to do with treatment. The
opportunity to take a fresh look at our policies in collaboration with
well-informed patient groups is an advance as well as a challenge. Further
research on both the origins and outcomes of intersex conditions promises to
greatly improve the lives of affected people.
Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed
Authors: Wells David N. , Misica Pavla M. , Tervit H. Robin , Vivanco William H.
Published: November 2003

To preserve the female genetics of an endangered breed of cattle, adapted to
sub-Antarctic con-ditions, adult somatic cell nuclear transfer was used to
clone the last surviving Enderby Island cow from mural granulosa cells.
Embryos reconstructed with metaphase II cytoplasts and quiescent cells were
either activated and fused simultaneously (AFS) at 24 or 30 hours post
maturation (hpm) or alternatively, fused 4–6 h before activation at
26–30 hpm (FBA). A significantly higher proportion of fused embryos
developed in vitro to grade 1–3 blastocysts on Day
7 with FBA (39.82.8%) compared to AFS with activation either at 24 hpm
(10.63.9%, P<0.01) or at 30 hpm (18.64.1%, P<0.01).
Following the transfer of 74 embryos from the FBA treatment over two
experiments, survival rates on Days 30, 55, 85, 150 and 190 of pregnancy were
38%, 30%, 23%, 16% and 15%, respectively.
Of 22 embryos transferred in the first experiment, two calves were born alive
with one calf surviving. DNA analyses confirmed that the calves were
genetically identical to the Enderby Island cow. Additional pregnancies are
currently ongoing. These data show that embryo development is increased by
prolonged exposure of quiescent somatic cell nuclei to oocyte cytoplasm before
artificial activation, possibly facilitating nuclear reprogramming. The
successful demonstration of somatic cell nuclear transfer in animal
conservation extends the applications of the technology beyond the main
agri-cultural and biomedical interests.
Renal responses to angiotensin II receptor blockade in ventilated preterm newborn lambs
Authors: Smith Lynne M. , Ikegami Machiko , Calvario Glenda C. , Surdilla James , Ervin M. Gore
Published: November 2003

Renal and cardiovascular immaturity has been linked with poor outcomes in the
premature human newborn. Despite extensive study in the fetus, the
contribution of the renin–angiotensin system to renal and cardiovascular
function in the premature newborn has not been well characterized. To evaluate
the angiotensin II contribution to preterm newborn renal and cardiovascular
functions, preterm (120-day) and near-term (136-day) lambs were Caesarean
delivered and ventilated. One hour following delivery, animals were randomized
to receive angiotensin II receptor-blockade (saralasin; 20 g
kg–1 min–1) or saline
(CON). Prior to blockade, mean SEM values for urine flow
(UFlow), urinary sodium excretion
(UNaV), and fractional excretion of sodium (FENa) were
similar in all groups. Angiotensin II receptor-blockade decreased
Uflow, UNaV and FENa in the
120-day group with no changes in the 136-day animals. No changes in mean
arterial pressure, or plasma angiotensin II, aldosterone, and renin activity
levels were noted at either gestational age. Conclusions:
(1) angiotensin II contributes to the regulation of
renal function in 120-day preterm lambs without changing blood pressure and
(2) angiotensin II-mediated feedback inhibition of renin
release is uncoupled in preterm newborns.
Effect of serum on the in vitro maturation of canine oocytes
Authors: Otoi Takeshige , Fujii Maya , Ooka Aya , Tanaka Masaki , Suzuki Tatsuyuki
Published: November 2003

The meiotic competence of canine oocytes cultured for 72 h in medium
supplemented with three different concentrations (5, 10 and 20%) of
anoestrous, oestrous or metoestrous bitch serum, or with 0.3% bovine
serum albumin (BSA), was examined. The oestrous serum supplement had a
positive effect on the resumption of meiosis, compared with the other
supplements (P<0.05). The number of oocytes that
reached metaphase I (MI) and metaphase II (MII) was significantly higher
(P<0.05) with the oestrous serum supplement than with
the anoestrous serum supplement. There were no significant differences among
the three different concentrations in each serum type with respect to the
proportion of oocytes that completed meiosis (MI to MII). The number of
oocytes that resumed meiosis in the 10% oestrous serum supplement was
significantly higher (P<0.05) than those of each
concentration of the anoestrous and metoestrous serum supplements, and of the
0.3% BSA supplement. Moreover, a higher number of oocytes reached MII
in the presence of the 10% oestrous serum supplement than with the
10% anoestrous serum supplement. These results suggest that
supplementation of the culture medium with 10% oestrous serum is the
optimal treatment for in vitro maturation of canine
oocytes.
Role of the MAPK cascade in mammalian germ cells
Authors: Sun Q. Y. , Breitbart H. , Schatten H.
Published: November 2003

The mitogen-activated protein kinase (MAPK) cascade is one of the most
important of the intracellular signaling pathways that play a crucial role in
cell proliferation, cell differentiation and cell cycle regulation. Since the
first report in 1993 of MAPK’s involvement in the functional regulation
of mammalian oocytes, much work has been done on the role of the MAPK cascade
in germ cells in different species of mammals. This review describes the
possible involvement of the MOS/MEK/MAPK/RSK cascade in
spermatogenesis, sperm function, oocyte meiotic re-initiation, spindle
assembly, metaphase II arrest, pronuclear formation and the entry of first
mitosis, as well as the cross-talk of this cascade to maturation-promoting
factor (MPF) and other signal molecules in mammals.
Increased ovulation rate in Merino ewes immunized against small synthetic peptide fragments of the inhibin α subunit
Authors: Anderson S. T. , Bindon B. M. , Hillard M. A. , O'Shea T.
Published: November 2003

Four experiments were carried out in Merino ewes during a period of 4 years to
determine the long-term effects of immunization against different synthetic
peptides mimicking the amine terminal of the a subunit of porcine inhibin.
Peptides were conjugated to human serum albumin and 100–200 g emulsified
in Freund’s complete adjuvant for the primary immunization. Usually two
booster injections were given at monthly intervals with 50–100 g
conjugated peptide using either incomplete Freund’s adjuvant or
Montanide : Marcol. In some experiments a further immunization was carried in
the next year. Blood samples were taken 10 days after each immunization,
during the luteal phase, for estimation of gonadotrophin concentrations and
determination of inhibin antibody titres. One day after blood sampling
cloprostenol was used to induce luteolysis and laparoscopy was performed in
the subsequent oestrous cycle. Immunization of ewes with synthetic peptides
1–32, 1–26, 7–26 and 8–30 resulted in large increases
in the ovulation rate (OR). An approximately two-fold increase in OR was
observed following the first booster immunization with these peptides and a
three- to five-fold increase after the second booster immunization.
Immunization with these large peptides resulted in a sustained increase in OR
for a period of at least 1 year after the second booster immunization. Of the
shorter peptides, peptides 10–26 and 13–26 gave a reasonable
ovulatory response, although it was more difficult to obtain a response with
peptides 1–16, 8–22, 13–25, 8–19 and 10–19;
peptides 7–13 and 1–6 gave no response (but were examined for one
breeding season only). The smaller peptides led to lower inhibin antibody
titres that were not necessarily associated with increased
follicle-stimulating hormone (FSH) or OR. More intensive blood sampling in one
experiment showed that following primary immunization against peptide
1–32 there was a transient increase in plasma FSH, which did not lead to
an increased OR. Moreover, a prolonged period of raised FSH after the first
booster was significantly correlated with increased OR. In these animals
antibody titres were only slightly increased after primary immunization, but
after the first booster immunization higher titres were observed that were
significantly correlated with trough FSH values and the subsequent OR. These
results are interpreted as showing that (1) to obtain an
increase in OR peptides 1–32, 1–26 and 7–26 are suitable as
immunogens; (2) smaller peptides are less reliable,
often require multiple injections, and the response may be delayed; and
(3) an extended period of raised plasma FSH is needed to
give a large ovulatory response.
Molecular cloning of translocation breakpoints in a case of constitutional translocation t(11;22)(q23;q11) and preparation of probes for preimplantation genetic diagnosis
Authors: Fung Jingly , Munné Santiago , Garcia Jaqueline , Kim Ung-Jin , Weier Heinz-Ulli G.
Published: November 2003

In vitro fertilization (IVF) centres with
preimplantation genetic diagnosis (PGD) programmes are often confronted with
the problem of identifying chromosomal abnormalities in interphase cells
biopsied from preimplantation embryos of carriers of a reciprocal
translocation. The present authors have developed a DNA testing based approach
to analyse embryos from translocation carriers, and this report describes
breakpoint-spanning probes to detect abnormalities in cases of the most common
human translocation (i.e. the t(11;22)(q23;q11)). Screening a yeast artificial
chromosome (YAC) library for probes covering the respective breakpoint regions
in the patient lead to probes for the breakpoint on chromosome 11q23. The
physically mapped YAC and bacterial artificial chromosome (BAC) clones from
chromosome 22 were then integrated with the cytogenetic map, which allowed
localization of the breakpoint on chromosome 22q11 to an interval of less than
84 kb between markers D22S184 and KI457 and to prepare probes suitable for
interphase cell analysis. In summary, breakpoint localization could be
accomplished in about 4 weeks with additional time needed to optimize probes
for use in PGD.
Effects of calcium and bicarbonate on head-to-head agglutination in ejaculated boar spermatozoa
Authors: Harayama Hiroshi , Miyake Masashi , Shidara Osamu , Iwamoto Eiji , Kato Seishiro
Published: November 2003

The present study was conducted to reveal the effects of calcium and
bicarbonate on the occurrence of head-to-head agglutination in ejaculated boar
spermatozoa in vitro. Boar spermatozoa were washed and
incubated in a modified Krebs-Ringer bicarbonate (mKRB) in a 37˚C
CO2 incubator (5% CO2 in
air) for 1–5 h. Before and after the incubation, aliquots of each sperm
sample were fixed, smeared on glass slides, and stained with a
phosphate-buffered solution of Giemsa to assess the percentages of head-to-
head agglutinated spermatozoa. Before the incubation, only 5–12%
of the spermatozoa were agglutinated. After the 1-h incubation, however, the
percentage of head-to-head agglutinated spermatozoa rose to approximately
50%, followed by only minor increases thereafter. This rise was
dependent on the concentrations of calcium chloride contained in the mKRB and
was attenuated by the addition of 2 mM
[ethylene-bis(oxyethylenenitrilo)]tetra-acetic acid (EGTA) to the
medium. Moreover, the replacement of sodium bicarbonate with
2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (Hepes) in
the medium and treatment with ruthenium red, which have both been shown
previously to inhibit calcium uptake by boar spermatozoa, significantly
reduced the rise. Based on these findings, it was concluded that extracellular
calcium and bicarbonate are key factors regulating head-to-head agglutination
in boar spermatozoa. The possible relationship between agglutinability and the
fertilizing ability of boar spermatozoa is also discussed.
cAMP inhibits mitogen-activated protein (MAP) kinase activation and resumption of meiosis, but exerts no effects after spontaneous germinal vesicle breakdown (GVBD) in mouse oocytes
Authors: Sun Q. Y. , Lu Q. , Breitbart H. , Chen D. Y.
Published: November 2003

Various signaling molecules have been implicated in the oocyte G2/MII
transition, including protein kinase C (PKC), cAMP and mitogen-activated
protein (MAP) kinases. However, the cross-talk among these signaling pathways
has not been elucidated. The present study demonstrates that both germinal
vesicle break down (GVBD) and MAP kinase phosphorylation (activation) are
inhibited when intraoocyte cAMP is increased by treating the GV-intact oocytes
with dibutyryl cyclic AMP (dbcAMP), forskolin, or isobutylmethylxanthine
(IBMX). Okadaic acid, a specific inhibitor of protein phosphatase-1 and -2A,
completely overcame this effect. Calphostin C, a specific inhibitor of PKC,
accelerated both GVBD and MAP kinase phosphorylation, and this effect was
attenuated by increased intraoocyte cAMP, whereas PKC activation inhibited
these events. Once GVBD occurred, the progression of oocyte maturation and MAP
kinase phosphorylation were independent of cAMP. These results indicate that
an increase in intraoocyte cAMP, in synergy with PKC activation, initiates a
cascade of events resulting in inhibition of MAP kinase phosphorylation and
GVBD in the mouse oocyte.
Cryopreservation of macropodid spermatozoa: new insights from the cryomicroscope
Authors: Holt W. V. , Penfold L. M. , Johnston S. D. , Temple-Smith P. , Shaw J. , McCallum C. , Lindemans W. , Blyde D.
Published: November 2003

This study examined the effects of cooling and
cryopreservation upon macropod spermatozoa (eastern grey kangaroo,
Macropus giganteus and red-necked wallaby,
Macropus rufogriseus). Sperm survival during and after
freezing to –30˚C or –70˚C in minimum essential medium
(MEM) + 5, 10, 20 or 30% (v/v) glycerol, MEM + 10 or
20% (v/v) ethylene glycol and MEM containing a mixture of
7.5% (v/v) glycerol + 10% (v/v) dimethylsulphoxide
was examined by cryomicroscopy. The MEM/glycerol mixtures permitted better
post-thaw sperm recovery than the other cryoprotectants. After freezing to
–30˚C at 10˚C min –1 in 20%
glycerol, then rewarming at 20˚C min –1 ,
flagellar activity resumed in more than 50% of spermatozoa when the
temperature increased into the range 5–10˚C. However, as the
temperature increased, into the range 20–25˚C, motility declined
rapidly so that less than 5% motile cells were seen at 35˚C.
Spermatozoa in MEM without cryoprotectant were also examined by cryomicroscopy
to evaluate changes in flagellar configuration, swimming behaviour and
viability during cooling from 35˚C to approximately –7˚C, and
rewarming to 35˚C. Cooling from 35 to 28˚C induced kangaroo
spermatozoa to exhibit rigid principal-piece bending and non-linear motility,
which was reversed by further cooling and the spermatozoa resumed their normal
linear movement. Rewarming induced principal-piece bending in the range of
20–30˚C, but this effect was reversed by further warming. Although
red-necked wallaby spermatozoa showed these effects, they also exhibited a
tendency to form rosette-like clusters during rewarming, especially when the
temperature reached approximately 14˚C. The clusters were induced when
the flagellar end-pieces became anteriorly reflected, producing hook-like
flagellar conformations, which then became interlinked.
Partial purification and characterization of a phosphoprotein phosphatase from sperm plasma membrane
Authors: Barua Madhabi , Ghosh Anil K. , Majumder Gopal C.
Published: November 2003

A phosphoprotein phosphatase (PPase M-I) that
dephosphorylates serine and threonine residues of histones was isolated from
the goat cauda-epididymal sperm plasma membrane and partially characterized.
The PPase was solubilized from the sperm membrane by treating it with 0.1 N
NaOH at pH 11.4 and the solubilized enzyme was partially purified by
concanavalin A-sepharose affinity chromatography and high-performance liquid
chromatography (HPLC), revealing it to be a 520-kDa protein. The PPase gave a
single protein band in native polyacrylamide gel electrophoresis (PAGE), but
in the presence of SDS it resolved into multiple proteins (35–170 kDa)
showing that the isolated enzyme contained a few contaminating proteins. The
enzyme is a glycoprotein because it binds with high affinity to concanavalin
A. It was maximally active at pH 8.0 and its activity was not dependent on
bivalent metal ions. The enzyme is a specific phosphatase as it displayed
higher affinity for dephosphorylation of large molecular weight phosphate
esters. The PPase showed broad substrate specificity for the dephosphorylation
of a variety of proteins. The membrane-associated PPase was strongly
(70–80%) inhibited by detergents (0.5%) such as Nonidet
P-40, Lubrol PX, Triton X-100 and Tween-20. Pyrophosphate (5 mM) and
orthovanadate (400 M) had no significant effect on the activity of the
isolated PPase whereas polyamines such as spermine (10 mM) nd spermidine (10
mM) slightly inhibited (20%) the enzymatic activity. Inorganic
phosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolic
PPases, had no appreciable effect on the activity of PPase M-I, indicating
that the membrane-bound PPase is distinct from the cytosolic PPases. The
enzyme was radiolabelled when the intact spermatozoa were subjected to
lactoperoxidase-mediated radioiodination reaction. The results show that the
PPase M-I is an ecto-enzyme that may play an important role in sperm
physiology by causing the dephosphorylation of the sperm outer surface
phosphoproteins.
The ultrastructure and metabolism of ejaculated tammar wallaby sperm are impaired by swim-up procedures when compared with sperm from the cauda epididymidis
Authors: Murdoch R. N. , Jones R. C. , Wade M. , Lin M.
Published: November 2003

The metabolism, rate of intracellular
accumulation of sugars, motility and ultrastructure of ejaculated tammar sperm
were impaired by swim-up into artificial media, particularly when the cells
were subsequently exposed to N-acetyl-D-glucosamine (NAG). The inclusion of
hyaluronate, serum albumin, catalase or Desferal in swim-up media helped
prevent deterioration of sperm motility, but failed to prevent detrimental
NAG-induced metabolic and ultrastructural changes. However, the sperm were
unavoidably diluted during swim-up into artificial media and their behavioural
properties were modified by dilution. Thus, sperm collected from the cauda
epididymidis were immotile and their rate of oxygen uptake was low in
undiluted caudal epididymal semen (CES). Nevertheless, these sperm were
viable, and vigorous motility was induced by 5- to 50-fold dilution in
Krebs-Ringer phosphate (KRP). Sperm respiration also dramatically increased
with moderate dilution (5- or 15-fold) in KRP, but decreased again at higher
rates (50-fold). This suggested that motility and the metabolic properties of
tammar sperm are modified both by dilution and on leaving the suppressing
conditions of the epididymis. Diluted tammar epididymal sperm also displayed a
Pasteur effect, but rapidly lost capacity for motility in an oxygen-depleted
atmosphere. It was concluded hat swim-up procedures compromise ejaculated
tammar sperm by promoting dilution-induced changes. This may alter the
permeability of the membrane with loss of the enzymes that process the ammonia
generated during the metabolism of NAG in seminal plasma. Subsequent exposure
to NAG further promotes ultrastructural damage culminating in loss of
viability.
Endothelial Cells Require STAT3 for Protection against Endotoxin-induced Inf lammation
Authors: Kano Arihiro , Wolfgang Michael J. , Gao Qian , Jacoby Joerg , Chai Gui-Xuan , Hansen William , Iwamoto Yoshiki , Pober Jordan S. , Flavell Richard A. , Fu Xin-Yuan
Published: November 2003

Endothelial cells (ECs) are believed to be an important component in the protection from lipopolysaccharide (LPS)-induced endotoxic shock. However, the cellular and molecular mechanism is not well defined. Here, we report that signal transducer and activator of transcription (STAT) 3 is an essential regulator of the antiinflammatory function of ECs in systemic immunity. Because STAT3 deficiency results in early embryonic lethality, we have generated mice with a conditional STAT3 deletion in endothelium (STAT3E−/−). STAT3E−/− mice are healthy and fertile, and isolated ECs initiate normal tube formation in vitro. Conditional endothelial but not organ-specific (i.e., hepatocyte or cardiomyocyte) STAT3 knockout mice show an increased susceptibility to lethality after LPS challenge. The LPS response in STAT3E−/− mice shows exaggerated inflammation and leukocyte infiltration in multiple organs combined with elevated activity of serum alanine aminotransferase and aspartate aminotransferase, indicating organ damage. Concomitantly, proinflammatory cytokines are produced at an exaggerated level and for a prolonged period. This defect cannot be explained by lack of antiinflammatory cytokines, such as interleukin 10 and transforming growth factor β. Instead, we have shown that a soluble activity derived from endothelia and dependent on STAT3 is critical for suppression of interferon γ. These data define STAT3 signaling within endothelia as a critical antiinflammatory mediator and provide new insight to the protective function of ECs in inflammation.
Blockade of Inflammation and Airway Hyperresponsiveness in Immune-sensitized Mice by Dominant-Negative Phosphoinositide 3-Kinase–TAT
Authors: Myou Shigeharu , Leff Alan R. , Myo Saori , Boetticher Evan , Tong Jiankun , Meliton Angelo Y. , Liu Jie , Munoz Nilda M. , Zhu Xiangdong
Published: November 2003

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Δp85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85α, which was fused to HIV-TAT (TAT-Δp85). Intraperitoneal administration of TAT-Δp85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3–10 mg/kg of TAT-Δp85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Δp85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.
Tumor Rejection by Modulation of Tumor Stromal Fibroblasts
Authors: Schüler Thomas , Körnig Sandra , Blankenstein Thomas
Published: November 2003

Interleukin (IL)-4–secreting tumors are rejected in mice, an effect that is thought to be immune mediated. However, solid tumors are embedded in a stroma that often contains tumor-promoting fibroblasts, a cell population whose function is also affected by IL-4. Here we show that IL-4–secreting tumors grew undiminished in IL-4 receptor (R)–deficient (IL-4R−/−) mice. In IL-4R+/+ mice they were long-term suppressed in the absence of T cells but complete rejection required T cells, compatible with the assumption that hematopoietic cells needed to respond to IL-4. Surprisingly, bone marrow (BM) chimeric mice revealed that IL-4R expression exclusively on non-BM–derived cells was sufficient for tumor rejection. Fibroblasts in the tumor stroma were identified as a target cell type for IL-4 because they accumulated in IL-4–secreting tumors and displayed an activated phenotype. Additionally, coinjection of IL-4R+/+ but not IL-4R−/− fibroblasts was sufficient for the rejection of IL-4–secreting tumors in IL-4R−/− mice. Our data demonstrate a novel mechanism by which IL-4 contributes to tumor rejection and show that the targeted modulation of tumor-associated fibroblasts can be sufficient for tumor rejection.
Vav1/2/3-null Mice Define an Essential Role for Vav Family Proteins in Lymphocyte Development and Activation but a Differential Requirement in MAPK Signaling in T and B Cells
Authors: Fujikawa Keiko , Miletic Ana V. , Alt Frederick W. , Faccio Roberta , Brown Tracie , Hoog Jeremy , Fredericks Jessica , Nishi Shinzo , Mildiner Shirly , Moores Sheri L. , Brugge Joan , Rosen Fred S. , Swat Wojciech
Published: November 2003

The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late “transitional” stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)–induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho–guanine nucleotide exchange factors.
Computer‐assisted audit tools and techniques: analysis and perspectives
Authors: Braun Robert L. , Davis Harold E.
Published: November 2003

In light of the increasing demand on auditors to make the audit more effective and efficient, this paper presents a brief summary of the most prominent computer assisted audit tools and techniques (CAATTs), which auditors can use to increase audit efficiency and effectiveness. Additionally, the results of a survey of governmental auditors which inquired about their perceptions of a specific type of CAATTs (generalized audit software proxied by the use of Audit Control Language or ACL) are presented. Results show that auditors seemed to perceive the potential benefits associated with ACL; however, they displayed a lower confidence in their technical abilities in using the application. Also, the auditors surveyed expressed a desire to increase their skills through increased ACL training. Taken together, these results give audit decision makers evidence that additional technical training is needed and desired by auditors.