bluuebunny/crossref_metadata_2025 huggingface dataset:
Maternal constraint influences muscle fibre development in fetal lambs
Authors: McCoard S. A. , Peterson S. W. , McNabb W. C. , Harris P. M. , McCutcheon S. N.
Published: November 2003

The objective was to examine myogenesis in two situations expected to be
characterized by maternal constraint: (i) in fetuses due
to be born in spring (n = 10) or autumn
(n = 10); and (ii) in
single (n = 16) and twin (n
= 20) fetal lambs. Maternal constraint operating through limitation of
placental size, as measured by placentome weight per fetus, was evident in
each study. Although a lower placental weight did not inßuence body and
muscle weights of fetuses due to be born in the spring or autumn, twins had
lower body and muscle weights than singles. Fibre number and average bre
cross-sectional (CS) area were differentially affected by season and fetal
number. The differences in muscle bre morphology between spring- and
autumn-born fetuses suggest that muscle bre development was inßuenced by
maternal constraint in the absence of an effect on fetal weight. The
differences in muscle bre number and CS area in particular muscles from twin
and single fetuses suggest that more severe maternal constraint,
reßected in a lower placental size per fetus, not only inßuences
fetal weight but can also affect muscle development.
Energy balance and ovulation: small cages versus natural habitats
Authors: Bronson F. H.
Published: November 2003

In the laboratory, ovulation is suppressed when a mammal is in negative energy
balance whether that state is caused by inadequate food intake, excessive
locomotor activity or heavy thermoregulatory costs. In this paper, knowledge
generated in the laboratory about the link between ovulation and energy
balance is examined in relation to the kinds of energetic challenges mammals
actually face in natural habitats. When viewed in that context, several
conclusions can be drawn. First, females ovulate whenever extant energetic
conditions permit unless the process is blocked by non-metabolic stress,
social cues or a predictive seasonal cue such as photoperiod. In the latter
case, most mammals show at least a seasonal tendency in their reproduction and
the majority do not use a predictive cue; they reproduce opportunistically in
relation to seasonal variation in the energetic characteristics of their
environment. Second, the widely held assumption that a female’s fat
reserves must exceed a critical level in order that she may ovulate finds no
support in the literature dealing with natural populations. Third, the
surprisingly rapid responsiveness of the gonadotrophin releasing hormone
(GnRH) pulse generator to energetic manipulation probably reflects the study
of animals that are in a pure survival mode. Fourth, the complexity of the
energetic challenges mammals face in the wild suggests that there are probably
multiple metabolic and neural pathways coupling ovulation to energy balance
and that these pathways are probably characterized by considerable overlap and
redundancy. Thus, fifth, to develop a more realistic overview of these
pathways there is a need for experimental designs that present mammals with
the kinds of complex challenges they actually face in the wild habitats in
which they evolved.
Construction and immunological assessment of Salmonella typhimurium expressing fox sperm LDH-C4
Authors: Bird Peter , Hayes Christine , Jersey James de , Bradley Mark
Published: November 2003

This study examined immune responses of foxes to oral doses of recombinant
Salmonella typhimurium expressing fox sperm-specific
lactate dehydrogenase (fLDH). The cDNA for fLDH was cloned into the expression
plasmid pKK233· 2 (pKKfLDH).
Salmonella typhimuriumaroA–
(SL3261) was transformed with either the pKK233·2 plasmid alone (SpKK)
or the pKKfLDH construct (SpKfLDH). The fLDH expressed by SpKfLDH retained
enzymatic activity and was recognized by human LDH-C4-specific antibody. Male
European red foxes (Vulpes vulpes) were given an initial
oral dose of 1 × 1011 cfu of either SpKK (control,
n = 3) or SpKfLDH (test, n
= 6), followed four weeks later with a further dose of 1 ×
1011 cfu. Antibodies to Salmonella
lipopolysaccharide (LPS-04) and fLDH were measured in plasma and saliva for
eight consecutive weeks after the initial doses. Both LPS-04 IgG- and
IgA-specific antibodies as well as fLDH-specific IgG antibodies were detected
in plasma and saliva. However, there was a marked fLDH-specific IgA response
in saliva consistent with induction of the common mucosal immune system. The
antibody measurements demonstrated the feasibility of using recombinant
Salmonella as an oral vaccine to elicit gamete
antigen-specific mucosal immune responses in foxes.
Influence of early post partum ovulation on the re-establishment of pregnancy in multiparous and primiparous dairy cattle
Authors: Smith M. C. A. , Wallace J. M.
Published: November 2003

To investigate the influence of early ovulation after calving on subsequent
fertility, milk progesterone concentrations were monitored twice weekly in
multiparous (n = 87) and primiparous
(n = 60) Holstein/Friesian dairy cattle.
Luteal activity before 21 days post partum was defined as ‘early’.
In multiparous cows, calving-to-conception interval (CCI) was longer, services
per cow were more numerous and conception rate to all inseminations was lower
with early luteal activity (n = 41,
16·8±0·39 days post partum) than with later luteal
activity (n = 46, 37·8±2·09
days post partum); multiparous cows with early luteal activity required more
exogenous fertility treatments and were more likely to fail to conceive by 100
days post partum. In contrast, in primiparous cows, no significant
relationships were detected between early (n =
32) or later (n = 28) luteal activity and the
CCI, number of services per cow or conception rates to all inseminations.
However, primiparous cows that ovulated before 21 days post partum had fewer
normal progesterone profiles. Early ovulation after calving was associated
with reduced pregnancy rates and prolonged CCIs in multiparous but not in
primiparous dairy cows.
Extra keyword: luteal function.
Fetal growth and development following temporary exposure of Day 3 ovine embryos to an advanced uterine environment
Authors: Sinclair K. D. , Dunne L. D. , Maxfield E. K. , Maltin C. A. , Young L. E. , Wilmut I. , Robinson J. J. , Broadbent P. J.
Published: November 2003

The effect of exposing Day 3 ovine embryos to an advanced uterine environment
for a period of 3 days on subsequent fetal growth and development between Day
35 and Day 135 of gestation was studied. Day 3 embryos were recovered from
superovulated donor ewes and transferred to synchronous final or asynchronous
temporary recipients for 3 days. Embryos were recovered from these temporary
recipients and transferred to Day 6 final recipients. Gravid uteri were
recovered, weighed and dissected on Days 35, 45, 60, 90, 110, 125 and 135 of
gestation. Fetal weight and length data were analysed by fitting non-linear
Gompertz models of the form loge y = a –
be–ct, where y is fetal size
and t is time from conception. Various terms including treatment, gestational
age, embryo stage at transfer and fetal sex were fitted to this model. Fetal
development was assessed by relating organ weight to fetal bodyweight using
the linear allometric equation loge y =
loge a + b loge x, where y is
organ weight and x is fetal weight. Temporary exposure of Day 3 embryos to an
advanced uterine environment did not increase the rate of embryo development
and had no effect on fetal growth and development between Days 35 and 135 of
gestation in this study. A single Gompertz model (loge y
= 10.134 – 17.047e –0.01733t)
explained 99.8% of the variation in fetal weight. Of terms fitted to
this model only gestational age and fetal sex influenced fetal weight, with
male fetuses being 5% heavier (P<0.05) than
female fetuses. Fetal development was also unaffected by experimental
treatment in this study. Allometric coefficients established for various fetal
components agreed well with those from previously published studies.
The proteolysis of insulin-like growth factor binding proteins in ovine uterine luminal fluid
Authors: Peterson A. J. , Ledgard A. M. , Hodgkinson S. C.
Published: November 2003

During days 12–15 after oestrus (day 0), the uterine luminal fluid (ULF)
of both pregnant and non-pregnant ewes contains only two prominent
insulin-like growth factor binding proteins (IGFBPs) of 16–18 kDa and
22–24 kDa which preferentially bind IGF-2. Immunoblotting with an
IGFBP-3 antibody revealed these to be proteolytic fragments of IGFBP-3. In
contrast, the ULF from anoestrus and ovariec-tomized ewes contained intact
IGFBP-3 (40–44 kDa) and IGFBP-2 (34 kDa). Co-incubation of ULF from an
anoestrus ewe with that from a day 12 cycling ewe cleaved the IGFBP-3 present
into the two lower molec-ular weight IGFBPs characteristic of ewes in the late
luteal phase of the oestrous cycle. The variation in pro-teolytic activity
both during the year and during the cycle suggested an influence of
progesterone. Supplementation of progesterone to long-term ovariectomized ewes
via a CIDR-G™ breeding device for 5, 10 or 15 days induced marked
proteolytic activity in all 10-day treated sheep. The ULF from the 15-day
treated ewes showed reduced activity and could inhibit the activity present in
10-day ULF, suggesting the induction of an inhibitor after prolonged exposure
to progesterone treatment. A possible role of IGFBP-3 proteolysis in the ovine
ULF may be to selectively increase the bioavailability of IGF-1 in the uterine
microenvironment, which may be crucial for the rapid elongation of trophoblast
that begins during days 12–15 after mating.
Pre- and post-natal growth of the human ductus epididymidis. A morphometric study
Authors: Miguel María P. De , Mariño José M. , Martínez-García Francisco , Nistal Manuel , Paniagua Ricardo , Regadera Javier
Published: November 2003

A histometric study of the development of the human epididymis from the fetal
period to adulthood has been carried out in males without testicular or
related pathology, distributed into the following groups: (I) fetuses (between
the 28th and 37th week of pregnancy); (II) newborns (1–30 days of age);
(III) infants (2–4 months of age); (IV) infants (5–12 months of
age); (V) infants (1–4 years of age); (VI) children (5–14 years
[prepubertal]); and (VII) adults (15–60 years of age). For
each age group and each epididymal portion (efferent ducts, caput, corpus and
cauda epididymidis) the parameters measured were (1)
total surface (epithelium + muscular layer + lumen);
(2) the surface occupied by the lumen;
(3) the surface occupied by the muscular layer;
(4) total diameter of the duct;
(5) total diameter of the lumen; and
(6) the height of the epithelium. The results of the
present study revealed that the development of the efferent ducts and ductus
epididymidis follows a biphasic pattern. A progressive development occurs from
the fetal period to infants 2–4-months of age. However, this development
is transient and regresses during infancy (groups IV and V). At childhood
(group VI), a definitive development is initiated and completed at puberty
(group VII). These changes seem to be related to the androgen-dependence of
the epididymis, the different stages of testicular maturation, and the
steroidogenic activity of Leydig cells.
Ontogeny of pro-opiomelanocortin (POMC) gene expression and translated products adrenocorticotrophin (ACTH) and α-melanocyte stimulating hormone (α-MSH) in the ovine fetal pituitary gland
Authors: Hagan D. M. , Brooks A. N.
Published: November 2003

This study sought to determine the ontogeny of pro-opiomelanocortin
(POMC) mRNA by in situ
hybridization, and the expression of α–melanocyte stimulating
hormone (α-MSH) and adrenocorticotrophin (ACTH) by immunohistochemistry,
in the ovine fetal pituitary gland. Detection of POMC
mRNA, and of ACTH and α-MSH immunoreactivity was first noted at Day 40
of gestation in the pars intermedia (PI) and the pars distalis (PD). After Day
70, α-MSH immunoreactivity was confined to the PI whereas
POMC mRNA and ACTH immunostaining remained in both the
PI and the PD. Increases (P < 0.05) of
POMC mRNA expression were observed in the PI between
Days 50–70 and levels then remained constant to Day 141 (term, 145
days). In the basal region of the PD, POMC mRNA
expression was high at Day 40, declined (P < 0.01) by
Day 50 and then increased progressively to Day 141 of gestation. The
proportion of ACTH-immunopositive cells in the PD also fell from 14% at
Day 40 to 11.4% at Day 70 and then increased to 15.3% by Day
141. In contrast, neither the level of POMC mRNA
expression nor the percentage of corticotrophs changed in the region of the PD
immediately adjacent to the PI. These data provide evidence for differential
processing of POMC in a tissue-specific manner during
early fetal pituitary development.
Short Communication: Cloning of the red kangaroo (Macropus rufus) follicle stimulating hormone beta subunit
Authors: Belov Katherine , Harrison Gavan A. , Cooper Desmond W.
Published: November 2003

The cDNA encoding the follicle stimulating hormone beta subunit (FSH-β)
was isolated from a red kangaroo pituitary cDNA library by using a porcine
probe and the nucleotide sequence for the coding region was determined. The
highest degree of deduced amino acid sequence identity (91%) was
observed between the red kangaroo and another marsupial, the brushtail possum
(Trichosurus vulpecula), followed by eutherian species
(76%, 75% and 74%, respectively, for pig, mouse and
sheep). Based on the deduced red kangaroo FSH-β amino acid sequence,
putative antigenic sites have been identified that may prove useful for
studying the hormonal control of reproduction in marsupials.
Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses
Authors: Grupen Christopher G. , Verma Paul J. , Du Zhong Tao , McIlfatrick Stephen M. , Ashman Rodney J. , Nottle Mark B.
Published: November 2003

The current protocols used to activate pig nuclear transfer embryos are less
efficient than those used for other species. To address this problem, the
effect of multiple sets of electrical pulses on the parthenogenetic
development of in vivo- and
in vitro-derived porcine oocytes was examined. Each set
of pulses consisted of two 1.5 kV cm–1 DC pulses
of 60 s duration each, administered 1 s apart. For
in vivo-derived oocytes, application of a second set of
pulses 30 min after the first set increased the proportion of oocytes that
developed to the blastocyst stage compared with a single treatment (51
v. 34%). Application of a third set of pulses 30
min after the second set reduced the rate of blastocyst formation compared
with two sets of pulses. In contrast, the rate of blastocyst formation was
greater with one set of pulses compared with two sets for in vitro matured
oocytes (31 v. 16%). Additional sets of
electrical pulses did not affect the number of cells in blastocysts obtained
from either group of oocytes compared with a single treatment. In summary, the
study demonstrates that the application of a second set of activating pulses
30 min after the first set is beneficial to
in vivo-derived oocytes, but detrimental to
in vitro matured oocytes, in terms of their ability to
develop parthenogenetically to the blastocyst stage.
Embryonic and somatic cell cloning
Authors: Wilmut Ian , Young Lorraine , Campbell Keith H.S.
Published: November 2003

Revolutionary opportunities in biology, medicine and agriculture arise from
the observation that offspring are obtained after nuclear transfer if somatic
donor cells are induced to become quiescent. Exploitation of many of these
opportunities will depend upon optimizing procedures for nuclear transfer.
This may come about through an understanding of the means by which factors in
the oocyte cytoplasm act upon the DNA of the transferred nucleus to regulate
gene expression. Similarly, research will extend the procedure to other
species. This technology may be used for embryo production, the introduction
of genetic change and the derivation of cells needed to treat human diseases.
Groups of genetically identical animals will be used in research to control
genetic variation and to allow transfer of cells between individuals. In
agriculture, production of a small number of clones will separate genetic and
environmental effects, whereas production of larger numbers of offspring will
disseminate genetic improvement from nucleus herds. Precise genetic
modification will be achieved by site specific recombination in the donor
cells before nuclear transfer. In all mammals it will become possible to
define the role of any gene product and to analyse the mechanisms that
regulate gene expression. Medical uses of these techniques will include the
production of proteins needed to treat disease and the supply of organs such
as hearts, livers and kidneys from pigs. As genome mapping projects identify
loci associated with traits of commercial importance in agriculture then gene
targeting will be used to study this effect. Finally, cells capable of
differentiation into any of the tissues of a patient will provide treatment
for diseases reflecting damage to a specific cell population that neither
repairs nor replaces itself.
Influence of epigenetic changes during oocyte growth on nuclear reprogramming after nuclear transfer
Authors: Kono Tomohiro
Published: November 2003

Genomic imprinting is the epigenetic mechanism that distinguishes whether the
loci that are inherited from the maternal or paternal genome lead to
parent-specific gene expression. The mechanism also regulates development in
mammalian embryos. Genomic imprinting is established after implantation
according to the specific markers that are imposed on the genome during
gametogenesis; the allele-specific gene expression is then maintained
throughout embryogenesis. The genomic imprinting markers are erased and
renewed on an own-sex basis only in cells that differentiate into germline
cells. This report shows that the epigenetic modifications that occur during
oogenesis perform the crucial function of establishing the allele-specific
expression of imprinted genes, and also suggests that the epigenetic DNA
modification is related to the reprogramming and aberrant development seen in
manipulated embryos.
Recycling bovine embryos for nuclear transfer
Authors: Peura Teija T. , Trounson Alan O.
Published: November 2003

The terms recycling and serial transfer refer to the use of cells from nuclear
transfer embryos as a source of donor nuclei for a subsequent round of nuclear
transfer. This approach has two benefits: improved reprogramming of the donor
nucleus and an increase in absolute numbers of identical offspring. The
beneficial effect of sequential exposure of donor nuclei to the early
cytoplasm has been proven in several species, including amphibia, mice and
cattle. Transferable embryos and live offspring have also been obtained from
cattle and goat embryos produced by three and five rounds of nuclear transfer
respectively. Although the limits to recycling have not been determined,
embryonic development has been obtained from up to 10 cycles of nuclear
transfer. Our results do not indicate differences in the embryonic
developmental competence between clones from three generations in bovine
embryo recycling. Although one live offspring from the second generation and
pregnancies from third generation clones have been obtained, overall lower
pregnancy rates and higher fetal losses than from first generation clones have
been observed. Application of a novel vitrification method to nuclear transfer
recycling removes many of the practical limitations of the technology.
Development of the membrana granulosa of bovine antral follicles: structure, location of mitosis and pyknosis, and immunolocalization of involucrin and vimentin
Authors: van Wezel I. L. , Rodgers R. J. , Krupa M.
Published: November 2003

The membrana granulosa of the ovarian follicle is termed the ‘follicular
epithelium’, yet there have been no studies considering its epithelial
nature and how it changes during follicular development. Therefore, these
issues were investigated using histology (n = 45
ovaries), considering its structure and the location of proliferating and
dying cells, and drawing analogies with other epithelia. Additionally,
differences between the layers of granulosa cells were demonstrated by
immunohistochemistry (n =7 ovaries). The
structure of the membrana granulosa differed between follicles. Six arbitrary
classifications were designed based on these structures, 80 follicles were
allocated (n = 13 ovaries) to these classes and
the follicular diameters were then measured. For the first time, differences
in membrana granulosa structure were shown to correspond to follicle size.
Follicles in classes 1–3, where basal granulosa cells were columnar with
nuclei positioned basally in the cell, were all ≤3 mm in diameter. All
follicles larger than 3 mm had either columnar basal cells with nuclei
positioned centrally (class 4), or had rounded basal cells (class 5), and all
follicles >5 mm had only rounded basal cells. In all these classes, cells
in the middle zone were rounded; cells aligning the antrum were often
flattened. Irrespective of follicle class, cell proliferation and cell death
were shown to be predominantly in the middle portions, rather than the most
antral or most basal portions, of the membrana granulosa of healthy and
atretic follicles. Involucrin, a marker of keratinocyte differentiation, was
localized to the suprabasal region of the membrana granulosa of healthy
follicles, particularly in the second and third cellular layers in from the
follicular basal lamina. Conversely, the staining intensity for the
intermediate filament protein vimentin was lowest in this region, and greatest
in the more antral and basal regions. In atretic follicles, there was
widespread staining for involucrin and vimentin throughout the membrana
granulosa. In conclusion, the membrana granulosa is highly structured, and
alters with follicular development. Layers in the membrana granulosa can
differ in terms of cell shape, and differ in proliferation and gene
expression. In the light of the current work, and an associated study, it is
proposed that proliferation occurs in the middle layers, and that granulosa
cells then progress basally or antrally, the latter undergoing terminal
differentiation.
Sperm antibodies in rat models of male hormonal contraception and vasectomy
Authors: Veräjänkorva Esko , Martikainen Mika , Saraste Antti , Sundström Jari , Pöllänen Pasi
Published: November 2003

The presence of sperm antibodies correlates with nearly every pathological
condition of the male reproductive tract. In the seasonal breeder, mink, a
decrease in gonadotrophin secretion and testicular regression also induces
sperm antibodies. Because the Sertoli cells and the principal cells of the
epididymis (i.e. the cells mainly responsible for protection of germ cells
from autoimmune destruction) are dependent on androgens, and because the
androgen concentration decreases in both the testis and epididymis during male
hormonal contraception, the presence of IgG class sperm antibodies in serum
was studied in rats during the suppression and recovery phases of testosterone
contraception and after vasectomy. Five-centimetre long testosterone implants
were placed under the dorsal skin of rats under pentobarbitone anaesthesia.
The control rats received empty implants. All implants were left in the rats
for 27 or 53 days. The total number of testicular antigens detected by sera
from the vasectomized rats increased significantly until 66 days
post-operation, and then decreased to the levels of intact rats. The number of
testicular antigens detected by sera from rats receiving contraceptive doses
of testosterone did not increase before the testosterone capsules were
removed, but at 40 days post removal of the silastic capsules, the number of
antigens detected by the sera was significantly higher than in intact rats and
at 77 days post removal of the silastic capsules, the number of antigens
detected by the sera was significantly higher than at 27 days after starting
testosterone administration. No significant changes in the number of antigens
detected by the sera could be observed after the implanting of empty capsules
or after their removal. Vasectomy mostly induced antibodies against testicular
antigens in the molecular ratio ranges of 70–82, 25–33 and
21–24.5 kD. Antibodies against antigens in these molecular ratio ranges
were not significantly induced during or after treatment with contraceptive
doses of testosterone. Cell nuclei with apoptotic morphology could be observed
in the seminiferous tubules of the vasectomized rats, but DNA
in situ 3′-end labelling of testes could not
confirm any differences between the testes of vasectomized and sham-operated
rats or between testosterone-treated and empty implant-treated rats. CD3
+ T cells could not be observed in the testes of
any of the treatment groups. These results suggest that the immunological
conditions remain stable in the testes after vasectomy and during testosterone
treatment, but that the animals are more prone to develop autoantibodies after
vasectomy and during recovery from treatment with exogenous testosterone.
Differentiation of human pluripotent teratocarcinoma stem cells induced by bone morphogenetic protein-2
Authors: Pera Martin F. , Herszfeld Daniella
Published: November 2003

Pluripotent human teratocarcinoma stem cells cultured
in vitro provide a resource for the study of early
embryonic development in man, as well as a means for discovery of novel
factors controlling cell differentiation and commitment. We previously
reported that the human teratocarcinoma stem cell line GCT 27X-1 could be
induced to differentiate into an endodermal progenitor cell by treatment with
high doses of retinoic acid. A search for polypeptide inducers of
differentiation in this system has identified bone morphogenetic protein-2
(BMP-2) as a potent inducer of differentiation. In cell line GCT 27X-1,
treatment with BMP-2 reduces proliferation, induces morphological changes
similar to obtained following treatment with retinoic acid, and causes a
decrease in the expression of transcripts for the stem cell markers CD30 and
Oct-4. Preliminary immunochemical studies indicate that the differentiated
cells produced by BMP-2 are endodermal precursors with a pattern of marker
expression similar to that found in retinoic acid treated cells. Models of
endoderm differentiation in humans will be useful for identifying the
molecules which mediate cell interactions in development, and in achieving
directed differentiation of cells for use in transplantation.
Successful development in vitro of hamster 8-cell embryos to ‘zona-escaped’ and attached blastocysts: assessment of quality and trophoblast outgrowth
Authors: Mishra Archana , Seshagiri P. B.
Published: November 2003

The peri-implantation development involves zona escape (hatching) of
blastocysts and their attachment and proliferation. These events are difficult
to studyin vivo, so in this study hamster 8-cell embryos
were cultured through the hatched and attached blastocyst stages using
different formulations of hamster embryo culture medium (HECM)-2.
Supplementation of succinate, amino acids, vitamins (inositol, pantothenate,
choline chloride) and bovine serum albumin (BSA) to HECM-2 supported
100% development of ‘zona-escaped’ blastocysts. In this
medium (designated as hatching, i.e. HECM-2h) all blastocysts invariably
deflated and escaped from focally lysed zonae, which underwent complete
dissolution. In their presence, pre-morula stage embryos also escaped from
zonae. Omission of BSA from HECM-2h failed to support zona escape whereas that
of vitamins reduced zona escape (34.0% 7.0). Blastocysts with the
potential to undergo zona escape in HECM-2h were of high quality as they had a
higher mean cell number (MCN) than the MCN of those developing in BSA-free
HECM-2h (35.2 1.6 v. 24.3 1.1). Cell allocation (i.e. trophectoderm to inner
cell mass ratio) in blastocysts remained unaltered in both media (2.6 0.2 v.
2.7 0.2). Supplementation of 10% bovine fetal serum (BFS) to HECM-2h
was detrimental to the development of blastocysts (22.3% 7.4) and none
of them underwent zona escape. Interestingly, BFS was required either as a
supplement to the medium or as a coating on dishes for azonal blastocysts to
attach (≥70%) and exhibit trophoblast (TB) outgrowth (30.3 ×
103 2.9 × 103
µm2 at 48 h). These results show that HECM-2h
supports maximal development of zona-escaped blastocysts with the potential to
attach and exhibit TB outgrowth, and there is a developmental stage-specific
requirement for serum during peri-attachment in hamster development.
Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle
Authors: Wei Hong , Fukui Yutaka
Published: November 2003

This study investigated the effects of the bull, sperm type (dead, immotile or
motile) and sperm pretreatment (i.e. mechanical (tail-cutting or tail-scoring)
or chemical (heparin, heparin + caffeine, calcium ionophore A23187 or
dithiothreitol)) on male pronuclear formation after intracytoplasmic sperm
injection (ICSI) in cattle. Three experiments were conducted. In Experiment 1,
spermatozoa from three bulls (A, B and C) were used for both ICSI and
in vitro fertilization (IVF). The results were that
sperm from bull B yielded a higher penetration/male pronuclear formation
rate than that of bull C when used for IVF (89.6%
v. 25.6%, P<0.01).
However, when injected into oocytes by ICSI, sperm from bull C had a higher
male pronuclear formation rate than that of bull B (34.6%
v. 16.1%, P<0.05). The
effects of sperm type and mechanical pretreatment were examined in Experiment
2. No significant difference was found in the male pronuclear formation rate
when the three types of sperm were injected into oocytes. Tail-scored sperm
achieved a higher male pronuclear rate than that of non-mechanically treated
ones (38.2% v. 13.2%,
P<0.005). In Experiment 3, chemical pretreatments
were tested and compared. Higher male pronuclear rates, compared with the
control, were obtained when sperm were pretreated with heparin +
caffeine, calcium ionophore A23187 and dithiothreitol (48.2%,
62.5% and 64.5% v. 25.0%,
P<0.05, 0.005 and 0.005, respectively). These results
indicate that (1) there is a bull variation in male
pronuclear formation with ICSI, and the male pronuclear rate by ICSI is not
coincident with the results by IVF, (2) immobilization
of a spermatozoon by tail-scoring before ICSI can improve the formation of the
male pronucleus, and (3) an appropriate chemical
pretreatment of spermatozoa is necessary to achieve a higher rate of male
pronuclear formation.
Current and future prospects for xenotransplantation
Authors: French Andrew J. , Greenstein Julia L. , Loveland Bruce E. , Mountford Peter S.
Published: November 2003

The transplantation of organs and tissues between animal species, orxenotransplantation, is the focus of a growing field of research, owingprimarily to the increasing shortage of allogeneic donor organs. The pigstands out as the most suitable donor animal for humans; however, xenografts(e.g. pig organs) used for human transplantation are normally destroyed by thehost within minutes by hyperacute xenograft rejection. An improvedunderstanding of the immune recognition and rejection of xenografts hasresulted in new therapies that can partially overcome hyperacute rejection(HAR), delayed xenograft rejection (DXR) or acute vascular xenograftrejection. Strategies to diminish immunogenicity following xenotransplantationcan be divided into two approaches: those directed at the recipient (e.g.antibodies or complement depletion or inhibition and tolerance induction) andthose directed at the donor (e.g. transgenic modifications to express humancomplement-regulatory proteins or removal or displacement of αGalepitopes). DXR is likely to be controlled by transgenic inhibition ofendothelial cell activation (e.g. inhibition of NF-κB). Transgenic pigsrequired for xenotransplantation will soon be generated at a greaterefficiency and precision using nuclear transfer and cloning when compared topronuclear injection. Of greater significance is that nuclear transfer offersthe ability to target gene insertion selectively to specific gene loci and todelete specific genes in the pig. Experimental pig-to-primate organxenotransplantation is currently under way, and results show increasedtransplant function from minutes to days and weeks. The final therapeuticregimen that allows survival of a discordant xenograft is likely to involve acombination of ‘modified’ functional genes in the donor organ, thedevelopment of immunological tolerance to pig antigens and administration ofnovel therapeutic agents, including immunosuppressants, that can controlnatural killer (NK) cell and monocyte mediated responses.
Is nitric oxide an autocrine modulator of bovine granulosa cell function?
Authors: Basini G. , Baratta M. , Ponderato N. , Bussolati S. , Tamanini C.
Published: November 2003

Nitric oxide (NO) is an important intra- and intercellular messenger
controlling many biological processes. It is synthesized by NO synthases,
which have been found also in granulosa cells. The present study examined
whether NO is present in bovine follicular fluid and is produced by granulosa
cells in culture. Secondly, it aimed to determine if NO affects the main
parameters of granulosa cell function. The NO donor
S-nitroso-L-acetyl-penicillamine (10 –3 , 10
–4 , 10 –5 М) was
used to evaluate whether NO might influence steroidogenesis, proliferation and
apoptosis in bovine granulosa cells collected from follicles divided according
to their size in small (<5 mm) and large (>8 mm). The data demonstrate
the presence of NO in follicular fluid and its production by granulosa cells
in culture: the most active cells in producing NO are those from the small
follicles, as confirmed by the NO levels in follicular fluid. This study also
shows that NO donor significantly (P<0.001) inhibits
progesterone (P4) and oestradiol 17β (E2) production by the granulosa
cells from both kinds of follicle; moreover, the highest concentration of NO
donor significantly (P<0.001) inhibits DNA
fragmentation in all the cells whereas the lowest concentration stimulates
(P<0.001) cellular apoptosis only in granulosa cells
from large follicles. NO donor does not seem to modify cell proliferation.
Taken together these data lead point to NO as a local modulator of granulosa
cell function.
Non-invasive diagnosis of pregnancy in wild black rhinoceros (Diceros bicornis minor) by faecal steroid analysis
Authors: Garnier J. N. , Green D. I. , Pickard A. R. , Shaw H. J. , Holt W. V.
Published: November 2003

The objective of this study was to determine whether faecal progestagen
measurement could be used to diagnose pregnancy in wild black rhinoceros cows.
Immunoreactive 20α-progestagens were measured in faecal samples
collected regularly (one or two times times per week) from pregnant and
non-pregnant wild black rhinoceros females (n =6)
in Zimbabwe. Fresh dung piles deposited by the study animals were serially
sampled during prolonged periods of tracking with local game scouts. Samples
were stored frozen, and dried prior to methanol extraction. Immunoreactivity
in faecal extracts was measured with a 20α-dihydroprogesterone enzyme
immunoassay and was shown to reflect circulating progesterone concentrations.
Mean concentrations of faecal 20α-progestagens during each month of
gestation were significantly higher than faecal concentrations in non-pregnant
animals (P<0.05), except during the second month of
gestation. Faecal 20α-progestagens remained 5–10 times higher than
concentrations in non-pregnant animals from the 4th to 15th month of
gestation. It was concluded that regular non-invasive reproductive monitoring
of black rhinoceros in the wild was possible and that pregnancy could be
accurately diagnosed from the measurement of 20α-progestagens in faecal
samples. The use of this technique in wild black rhinoceros populations will
offer new perspectives for in situ management of this
endangered species.
Expression of polo-like kinase (PLK) in the mouse placenta and ovary
Authors: Takai Noriyuki , Yoshimatsu Jun , Nishida Yoshihiro , Miyakawa Isao , Hamanaka Ryoji , Narahara Hisashi
Published: November 2003

The polo-like kinase (PLK) is a mammalian serine/threonine kinase involved
in cell cycle regulation. Much evidence for the role of PLK in the cell cycle
has come from studies of cultured cells; however, little is known about its
function or even expression in vivo . The present study
examined the features of PLK expression in the mouse placenta and ovary.
Immunohistochemical studies showed that PLK is highly expressed in the
basement membrane of the endometrial gland, in some endothelial cells, in
endometrium after embryo implantation, in trophoblastic tissue invading the
decidua, in the ovarian stroma and in some lutein bodies. In contrast, PLK was
not detectable by immunohistochemistry in endometrial stroma before
decidualization, in decidua, in trophoblastic tissue not invading the decidua
or in ovarian follicles. PLK expression seemed to be correlated with the
expression of proliferation cellular nuclear antigen (PCNA) in many placental
and ovarian cells, reflecting a role in cellular proliferation. Nevertheless,
in ovarian stroma and lutein bodies where PCNA was not expressed, PLK was
strongly expressed. This finding indicates that PLK may have some post mitotic
functions in certain specialized cell types.
Matrix metalloproteinases and their endogenous inhibitors during the implantation period in the rat uterus
Authors: Hurst Peter R. , Palmay Rochelle D.
Published: November 2003

Rats were studied to determine if matrix metalloproteinases (MMPs) and tissue
inhibitors of MMPs (TIMPs) were detectable in developing embryonic and uterine
tissues during the implantation period; that is, Days 6–8 of pregnancy.
Tissue extracts were studied by zymography, reverse zymography and Western
blotting. Immunohistochemistry procedures using antibodies against MMPs and
TIMPs were applied to tissue sections that passed through implantation sites.
The MMP inhibitor, doxycycline, was injected intraluminally into uterine horns
of pseudopregnant animals. MMP-2 and -9, and TIMP-2 were detected in gelatin
and reverse zymographs, respectively, from extracts of implantation sites on
each day studied. Western blots defined bands corresponding to MMP-1, but
casein zymographs did not consistently show evidence of extractable MMP-1 or
-3. In tissue sections the primary decidual tissue area stained consistently
for TIMP-2 and on Days 7 and 8 this area also had positive staining for MMP-1
in close proximity to the implanting embryo. On Day 8 positive staining in the
outer stromal tissue was detected using an antibody against MMP-9, and another
MMP (possibly MMP-3) was localized exclusively in ectoplacental
cone/trophoblastic cells. Intraluminal injection of doxycycline did not
significantly prevent the growth of the uterine horns following surgical
induction of pseudopregnancy on Day 5. The strong localizationof TIMP-2 in the
primary decidual tissue is similar to that reported previously for TIMP-3,
indicating that these inhibitors have a role in decidualization, including the
regulation of trophoblastic invasion.
Involvement of serotoninergic pathways in the control of luteinizing hormone secretion in red deer hinds
Authors: Villa-Diaz Luis G. , Barrell Graham K.
Published: November 2003

Two experiments were conducted to determine whether serotoninergic pathways,
which are implicated in the neuroendocrine regulation of luteininzing hormone
(LH) secretion in domestic animals, have a similar action in red deer hinds.
In the non-breeding season (August), ovariectomized (n
=5) and ovariectomized-thyroidectomized (n
=5) hinds received a vehicle solution followed 4 h later by either
serotonin (66 µg kg–1 i.v.) every 10 min for
a further 4 h or the serotonin antagonist, cyproheptadine (3 mg
kg–1 i.v.) as a single injection. This procedure
was repeated in the breeding season (June). In the non-breeding season
serotonin was without effect, but cyproheptadine reduced LH pulse frequency
and amplitude in ovariectomized-thyroidectomized hinds
(P<0.01). During the breeding season, serotonin
reduced LH pulse amplitude in ovariectomized hinds
(P<0.05) and cyproheptadine reduced LH pulse
frequency in both ovariectomized and ovariectomized–thyroidectomized
hinds (P<0.05 and P<0.01,
respectively). On each occasion, cyproheptadine increased
(P<0.01) plasma prolactin concentration, whereas
serotonin had no effect. These results indicate a stimulatory role for
serotoninergic neurons on the hypothalamic GnRH pulse generator mechanism of
red deer hinds during the breeding season. In a second experiment, the LH
response to GnRH (5 µg i.v.) was examined in ovariectomized hinds
(n = 5) following administration of a serotonin
infusion (6.6 µg kg–1
min–1 i.v. for 15 min), cyproheptadine (3 mg
kg–1 i.v. as a single dose) or vehicle, in the
breeding season (July) after induction of halothane anaesthesia and in the
non-breeding season (December) without anaesthesia. Halothane anaesthesia
eliminated endogenous pulses of LH. In comparison with the vehicle-treated
controls, the response of plasma LH to exogenous GnRH was not altered by
serotonin or cyproheptadine in either season, which shows that serotonin has
no effect on LH release at the pituitary gland level in these animals. It was
concluded that in the regulation of LH release in red deer hinds,
serotoninergic pathways have a stimulatory role operating at the hypothalamic
level.
Diminished levels of prostaglandin E in type I diabetic oocyte - cumulus complexes. Influence of nitric oxide and superoxide dismutase
Authors: Carolina Pustrovh , Jawerbaum Alicia , Debora Sinner , Christian Perotti , Gimeno Martha A. F. , Gonzalez Elida T.
Published: November 2003

In the present work the prostaglandin E (PGE) production by ovulated, immature
and in vitromatured oocyte–cumulus complexes (OCC)
was evaluated in a rat model of type I diabetes induced by streptozotocin (60
mg kg–1). A diminished number of ovulated OCC were
found in the type I diabetic rat. In contrast to the increment in PGE
generation found previously in OCC and embryos from type II diabetic rats, it
was found that PGE production by type I diabetic OCC was diminished in
comparison with the controls. Nitric oxide synthase (NOS) activity is enhanced
in proestrous ovaries from type I diabetic rats, but cGMP levels are
diminished. SIN-1 (300 µМ), a nitric oxide donor, significantly
enhanced PGE generation by control OCC, but was unable to modify the PGE
levels in type I diabetic OCC. L-NMMA, a nitric oxide inhibitor that
diminished PGE values in type II diabetic OCC, did not modify PGE generation
in either control and type I diabetic OCC. Superoxide dismutase (SOD, 1000 U
mL–1), and SOD (1000 U
mL–1) plus SIN-1 (300 µМ), enhanced PGE
generation by both control and diabetic OCC. The present results suggest that
even when nitric oxide (NO) is overproduced in diabetic ovaries, the
NO–PGE pathway is impaired in type I diabetic OCC. As SOD additions are
able to increase PGE generation by diabetic OCC, high concentrations of free
oxygen radicals might be quenching the NO, impairing its physiological
functions.
Relationship between motility and oxygen consumption of sperm from the cauda epididymides of the rat
Authors: Murdoch R. N. , Jones R. C. , Armstrong V. L. , Clulow J.
Published: November 2003

The oxygen consumption of rat sperm was low (2.7 µL
O2 108
sperm–1h–1) in caudal
epidi-dymal semen (CES) when stimulation of motility was avoided. The addition
of 1 µL of Krebs Ringer phosphate buffer (KRP) to 40 µL of CES
(CES : KRP = 40 : 1) did not activate motility, but stimulated oxygen
consumption 2-fold. Inclusion of 1–5 mМ glucose, acetate, pyruvate
or lactate in the KRP further stimulated respiration rate (up to 4.3-fold)
without activating motility, but respiration was reduced when 2- deoxyglucose
replaced energy substrates. Inclusion of dibutyryl cAMP (1 mM) activated sperm
motility in all samples and stimulated oxygen consumption 2.9-fold. Dilution
of CES at the ratio of CES : KRP = 40 : 1000 also activated sperm
motility and stimulated respiration rate 2.9-fold. The combined effect of
dibutyryl cAMP and glucose in stimulating respiration was greater than their
individual effects. However, the response to cAMP or substrates was not
altered by incubation in KRP containing either 0 or 0.5 mM
Ca2+. It was concluded that the motility and
metabolism of rat epididymal sperm are suppressed
in vivo. Respiration can be stimulated by a small
(1.025-fold) dilution and further stimulated by the inclusion of energy
substrate, without activating motility. However, a larger dilution or
inclusion of cAMP activated motility and simultaneously stimulated metabolism,
with exogenous substrate being required to stimulate respiration to the
maximum rate. This suggests that prior to activation, the rate of oxygen
consumption and sperm motility are not coupled.
Pregnancy in a marsupial, the Tasmanian pademelon (Thylogale billardierii)
Authors: Rose R. W. , Horak J. A. A. , Shetewi A. D. , Jones S. M.
Published: November 2003

The Tasmanian pademelon, Thylogale billardierii, is a
medium-sized wallaby that adapts well to captivity and, unlike the
well-studied tammar wallaby, is capable of breeding all year round. It may,
there-fore, be a useful model species for research into the reproductive
biology of macropod marsupials. This paper presents necessary background data
on histological changes in the reproductive organs and the rate of embryonic
growth during gestation in T. billardierii. After Day 4
RPY (removal of young from the pouch) the gravid and non-gravid uteri differ
significantly in some histological parameters. The corpus luteum becomes
active by Day 6 RPY and is fully developed by Day 14 RPY; it begins to
degenerate from Day 19 RPY. Plasma progesterone concentrations through
gestation follow a pattern similar to that in the tammar wallaby. There is an
early, smaller, peak at Day 5 RPY, with plasma concentrations of progesterone
then falling until the larger pre-partum peak occurs several days before
birth.
Maternal caffeine consumption during pregnancy does not affect preimplantation development but delays early postimplantation growth in rat embryos
Authors: Jacombs Anita , Pollard Irina , Ryan John , Loupis Anne
Published: November 2003

Models for studying prenatal drug-induced
intrauterine growth retardation (IUGR) have, without exception, measured
growth-related factors in the postimplantation embryo, fetus or neonate.
Therefore, it is not known whether effects of drug exposure on growth and
metabolism begin early in the preimplantation embryo, or whether IUGR is
exclusively a postimplantation phenomenon. The present study investigates
whether caffeine, a drug known to induce a dose-dependent fetal IUGR, affects
embryo development before and/or after implantation or is exclusively a
fetal phenomenon. Preimplantation embryo assessment (with treatment from Days
2 to 4 of pregnancy) included glucose utilization, cell number evaluation and
stage of development (morula to hatched blastocyst); whereas, postimplantation
embryo assessment (treatment from Days 2 to 10, 10.5 or 11 of pregnancy)
included somite number evaluation and extent of neural tube closure, as seen
using scanning electron microscopy. Comparing control preimplantation embryos
with those exposed to 30 and 60 mg kg –1 caffeine did not reveal any
effects of caffeine exposure, as assessed on Day 5 of gestation. However,
postimplantation embryo development assessed on Day 12 of gestation revealed
that caffeine exposure of 15 and 30 mg kg –1 significantly reduced, at
both dosage levels, somite number and the extent of neural tube closure. In
addition, comparisons of control and experimental groups revealed that in the
high-dose caffeine group the forebrain cavity was significantly enlarged and
bounded by a reduced, irregularly aligned neuroepithelium. The findings
suggest that IUGR is a phenomenon first identifiable during late
postimplantation embryogenesis and continues in fetal life.