bluuebunny/crossref_metadata_2025 huggingface dataset:
Egg maturation and fertilization in marsupials
Authors: Breed WG
Published: October 2005

This brief review summarizes our knowledge of the morphological events that are associated with oocyte maturation and fertilization in marsupials in which it has been suggested that there are marked differences from eutherians in both the developmental timetable of oocyte maturation and in some of the processes associated with sperm-egg interaction. Most of the data have been obtained from studies on four species: Monodelphis domestica, Sminthopsis crassicaudata, Sminthopsis macroura, and Trichosurus vulpecula. Differences between the species have been described for: (1) the arrangement of 'yolk' in the oocyte cytoplasm; (2) the time of formation of cortical granules: (3) the mode of sperm penetration through the zone pellucida: (4) the sperm membrane involved in sperm-egg fusion: (5) the fate of inner acrosomal and sperm plasma membranes: and (6) the rapidity of sperm chromatin decondensation in the ooplasm. Such differences suggest considerable variation in these processes between different marsupial species although some of the variation described may be due to technical differences in the obtaining of the data. Thus, whether there are fundamental differences between the two major extant infraclasses of mammals, marsupials and eutherians, in some of the processes associated with fertilization is conjectural at the present time. The interspecific variation in the results obtained cautions one in extrapolating from observations on one or two 'model' species to the infraclass as a whole: a conclusion that might not, on reflection, be too surprising bearing in mind the long and separate evolutionary history of the major extant marsupial lineages.
Possum biocontrol: prospects for fertility regulation
Authors: Cowan PE
Published: October 2005

Research has begun recently into biocontrol of brushtail possums as the only long-term, cost-effective solution to the possum problem in New Zealand, where possums cause significant damage to native forests, threaten populations of native plants and animals, and infect cattle and deer with bovine tuberculosis. Fertility regulation as a means of biocontrol has the support of major animal welfare and conservation groups in New Zealand. Systems are being investigated, mostly in reproduction and development, with the ultimate aim of developing immunologically-based fertility regulation (immunocontraception), but much basic information essential to such an approach for possums is lacking. The key components for the success of this approach--suitable vectors expressing possum-specific reproductive antigens sufficiently to block reproduction--are reviewed. The social and political issues of local, national and international risk and acceptability arising from such an approach are also discussed.
Hormones of oestrus and ovulation and their manipulation in marsupials
Authors: Hinds LA , Fletcher TP , Rodger JC
Published: October 2005

Oestrus and ovulation occur spontaneously in the majority of marsupials, with behavioural oestrus usually occurring 1-2 days before ovulation. The hormone changes that occur at this time have been described in the most detail for the monovular tammar wallaby Macropus eugenii. The respective roles of the Graafian follicle, corpus luteum and the pituitary in the events leading up to oestrus and ovulation in this species are also reviewed. Recently, various protocols have been developed for superovulation of marsupials, including Australian species, such as the brush-tailed possum, fat-tailed dunnart, brush-tailed bettong and tammar wallaby, and the American laboratory opossum, Monodelphis domestica. These protocols provide an opportunity for studying the regulation of ovarian activity and for the collection of larger quantities of material for the study of gamete maturation, in vitro fertilization and embryonic development.
Pellet-freezing spermatozoa of two marsupials: the tammar wallaby, Macropus eugenii, and the brushtail possum, Trichosurus vulpecula
Authors: Molinia FC , Rodger JC
Published: October 2005

A protocol was developed for pellet-freezing spermatozoa of the tammar wallaby and the brushtail possum. Seren was collected by electro-ejaculation and wallaby spermatozoa were washed by 'swim-up' into phosphate-buffered saline (PBS), whereas possum spermatozoa were not washed. Wallaby spermatozoa were screened for toxicity in diluents containing a range of cryoprotectants (0-10%): dimethyl sulfoxide (DMSO), ethylene glycol and propanediol. Possum spermatozoa were tolerant of diluents containing 17.5% glycerol. Wallaby and possum spermatozoa were diluted 1:1 with the most promising cryoprotective diluents (final concentrations in PBS: possum, 17.5% glycerol; wallaby, 7.5% glycerol + 10% DMSO) and, after 5 min equilibration at room temperature, were pellet-frozen. Pellets were thawed (35 degrees C) and wallaby spermatozoa were washed by centrifugation (200 g for 5 min) and resuspended in PBS to minimize cryoprotectant toxicity. A high proportion of possum spermatozoa was recovered after freezing (67.5%), having good progressive motility (3.6 on a 0-5 scale). The progressive motility of frozen-thawed wallaby spermatozoa was also high (3.0), but only 10% of motile spermatozoa were recovered. The pellet-freezing method in conjunction with the post-thaw washing procedure (wallaby) may produce a viable population of cryopreserved marsupial spermatozoa suitable for use in assisted-breeding techniques such as in vitro fertilization and artificial insemination.
Nutrient uptake and culture of Sminthopsis macroura (stripe-faced dunnart) embryos
Authors: Gardner DK , Selwood L , Lane M
Published: October 2005

Glucose and pyruvate uptake by individual embryos were measured in a marsupial species (stripe-faced dunnart) and a eutherian species (mouse). At each stage of development, nutrient uptake by the dunnart embryo was around an order of magnitude greater than that of the mouse embryo. The pattern of glucose uptake by the dunnart embryo was not like that for any eutherian embryo, all of which have a low glucose uptake before the blastocyst stage. Rather, in the dunnart embryo there was a significant increase in glucose uptake after the third cleavage division, increasing from 13.6 pmol embryo h-1 at the 4-cell stage to 34.9 pmol embryo h-1 by the 8-cell stage. This increase in glucose uptake before blastocyst formation may be attributed to an increased energy demand associated with the movement of cells within the dunnart embryo. Using a new culture system, it was possible to culture 66% of dunnart embryos at the 2-4-cell stage and 80% of those at the 8-16-cell stage to the unilaminar blastocyst stage. Embryos cultured from the 2-cell to the 4-cell stage were retarded by around 12 h when they reached the blastocyst stage. Developmental retardation was also reflected in the pattern of nutrient uptake, which lagged behind that of embryos developed in vivo. The present study has shown that it is possible to culture the early marsupial embryo to the blastocyst stage in a serum-free culture system, while concomitantly quantifying embryonic nutrient requirements. Such an approach is essential for species where there is a paucity of material for study.
The blastocyst epithelium is not a protoderm in dasyurid marsupials: a review of the evidence
Authors: Selwood L
Published: October 2005

Evidence from studies of cleavage and blastocysts in dasyurids in reviewed to show that the unilaminar blastocyst is not a protoderm but consists of two cell types. Cleavage and blastocyst formation in marsupials has been most comprehensively studied in dasyurids, in which the secondary oocyte and zygote are polarized with respect to the position of the nucleus, cytoskeletal elements and cytoplasmic vesicles. Polarity is reinforced by fertilization. Early cleavage divisions are associated with the polarized elimination of a yolk mass and many vesicular structures into the perivitelline space. Because secretion of the vesicular structures, of which several types are found, facilitate blastomere-zona then blastomere-blastomere associations during cleavage, a unilaminar blastocyst forms without an intervening morula stage. Polarization of the cleavage cavity is related to the appearance of two cell lineages, pluriblast and trophoblast, at the 16-cell stage. In species in which the yolk mass persists, the tier of eight cells lying nearest the yolk mass forms the pluriblast (future embryonic and extra-embryonic lineages), and the other tier forms the trophoblast (future extra-embryonic ectoderm of the yolk sac). Thus, the unilaminar blastocyst epithelium is not a protoderm. Blastocyst expansion is associated with increased cell numbers mainly in the trophoblast. Pluriblast cells begin to increase just before the appearance of the hypoblast. The two cell populations can be distinguished by ultrastructural and histological features at the end of cleavage. During blastocyst expansion they differ in appearance and behaviour in vitro and in cell-doubling time. Two populations of cells have also been found in cleavage and unilaminar blastocyst stages in other marsupials, such as opossums, brushtail possums and bandicoots.
Cloning, sequencing and expression of stem cell factor (c-kit ligand) cDNA of brushtail possum (Trichosurus vulpecula)
Authors: Greenwood PJ , Seamer C , Tisdall DJ
Published: October 2005

By means of reverse transcription polymerase chain reaction (RT-PCR), three stem cell factor (SCF) cDNAs (822-738 bp in size) were amplified from brushtail possum ovarian poly (A)+ RNA. The largest and smallest of these cDNAs were cloned and sequenced. Characterization of these cDNAs has revealed that possum SCF has approximately 75% and 66% homology to SCF of eutherian mammals at the nucleotide level and the predicted amino acid level respectively. Nucleotide sequencing shows that the 738-bp cDNA represents an mRNA splice variant, equivalent to that found in eutherian mammals, in which an exon (84 bp) encoding a potential proteolytic cleavage site is removed. Comparison of the predicted possum SCF amino acid sequence with the predicted SCF amino acid sequences from eutherian mammals reveals conservation of all cysteine residues and 3 of 4 potential N-linked glycosylation sites. In addition, the hydropathicity profile of the possum SCF protein is similar to that of eutherian SCF suggesting that protein conformation is conserved. Northern analysis was used to characterize possum SCF gene expression in adult ovary and testis. A major transcript of 9 kb was observed in both ovarian and testicular tissue. The conservation of the SCF gene and its predicted protein, suggests that SCF in the possum has similar biological activities to SCF in eutherian mammals.
Duration of embryonic diapause in the brush-tailed bettong, Bettongia penicillata (Potoroidae): effect of age of quiescent corpus luteum
Authors: Smith MJ
Published: October 2005

It has been shown that changes to the frequency of sucking by the pouch young do not affect the time of reactivation of the quiescent corpus luteum and diapause embryo in Bettongia penicillata; these observations led to the suggestion that the corpus luteum may have an inherent maximum duration of quiescence. The aim of the present study was to investigate the effect of the age of the corpus luteum on the timing of its reactivation. Ovulation fails to occur post partum in female B. penicillata isolated from males, and the introduction of a male B. penicillata induces oestrus in females suckling a young in the pouch. Oestrus was induced from Day 23 of lactation to Day 76, in different females, and the corpus luteum became quiescent. All parturitions occurred between Day 95 and Day 103 of lactation (average, Day 98.8), at the time expected if the corpus luteum had been formed post partum. Duration of quiescence of the corpus luteum ranged from 60 days to 10 days, compared with 83.9 +/- 0.43 days in females that mated post partum. The results show that the age of the corpus luteum does not affect the timing of its redevelopment and release of the embryo from diapause near the end of the period of the young being suckled within the pouch.
Glycosaminoglycans of the uterine and vaginal cul-de-sac tissue in the brushtail possum (Trichosurus vulpecula)
Authors: Legge M , Hill BL , Shackell GH , McLeod BJ
Published: October 2005

Live-captured feral brushtail possums (Trichosurus vulpecula) received slow-release progestagen implants for 16 days after removal of pouch young. At the time of implant removal one group was injected intramuscularly with pregnant mare serum gonadotrophin (PMSG, 2 I.U. kg-1 in 0.2 mL saline) and another group received an equivalent volume of sterile saline. Animals from each group were killed on Days 0, 3, 4, 5 or 6 after implant removal (PMSG, n = 4 per day; saline controls, n = 3 per day). The uterus and vaginal cul-de-sac were excised and tissue was analysed for glycosaminoglycans (GAGs) and keratan sulfate content. GAG concentration increased progressively over the six-day period following implant removal. Mean uterine GAG concentrations were significantly higher than those in vaginal cul-de-sac from Day 3 to Day 6 in PMSG-treated and saline-treated animals following progestagen implant removal (P < 0.05). Keratan sulfate concentrations showed a similar response--concentrations were higher in uterine tissue than in vaginal cul-de-sac tissue and PMSG-treated animals showed a greater response than did saline controls in the uterine tissue, but the same response in cul-de-sac tissue (P < 0.05 for all groups). The progressive increase in GAG concentrations of reproductive tract tissue following removal of the progesterone implant is indicative of hormonal stimulation and suggests that GAGs may have a supportive role in gamete and embryo nutrition and growth in the brushtail possum.
Urinary excretion of prostacyclin in a rat model of uteroplacental vasculature occlusion: implications for fetal growth retardation
Authors: Hophy M , Harel S , Yavin E
Published: October 2005

An experimental model was devised in the pregnant rat to study by a combined high pressure liquid chromatography and radioimmunoassay technique the accumulation of prostanoids (PNs) in the urine after transient-complete or permanent-partial interruption of the maternal-fetal blood flow. After 8 min of complete restriction of the blood flow in the pregnant rat at 18 days of gestation, the urinary concentration of 6-keto-prostaglandin F1 alpha (6k-PGF1 alpha, the stable prostacyclin metabolite) increased from 4.97 +/- 1.27 ng mg-1 creatinine to 8.09 +/- 2.47 ng mg-1 creatinine and 13.02 +/- 4.5 ng mg-1 creatinine after the second and third post-operative day respectively. The urinary concentration of the 2,3-dinor derivative of prostacyclin reached 12.35 +/- 5.44 ng mg-1 creatinine after the second post-operative day and was reduced to 4.71 +/- 1.94 ng mg-1 creatinine after the third post-operative day. The concentration of thromboxane B2 (TxB2, the stable thromboxane A2 metabolite) increased approximately 7-fold and 13-fold over that of the control after the second and third post-operative day respectively. The urinary concentration of the 2,3-dinor derivative of TxB2 (d-TxB2) increased from about 1.42 +/- 0.3 ng mg-1 creatinine to 4.49 +/- 0.9 ng mg-1 creatinine and 7.76 +/- 2.63 ng mg-1 creatinine under the same experimental conditions. Increases in the urinary concentrations of 6k-PGF1 alpha and d-TxB2 to 94 +/- 27.76 ng mg-1 creatinine and 12.05 +/- 2.26 ng mg-1 creatinine, respectively, were observed on the second post-operative day, after the restriction time was increased to 30 min. Permanent-partial occlusion of the maternal fetal circulation resulted in excretion of PNs in the urine to similar levels produced after transient-complete restriction. High concentrations of prostacyclin (range, 0.8 ng min-1 mg-1 wet weight) were produced in vitro by uterine preparations from restricted animals after the second post-operative day. Placenta preparations from restricted animals generally exhibited a lower ability to synthesize PNS (up to 0.006 ng min-1 mg-1 wet weight) compared with uterine tissue but produced more thromboxane than their sham counterparts. The data suggest that the uterus constitutes the main source for urinary PN excretion following short episodes of maternal-fetal blood flow interruption.
Thyroid hormone as a regulator of basal and human chorionic gonadotrophin-stimulated steroidogenesis by cultured porcine theca and granulosa cells isolated at different stages of the follicular phase
Authors: Gregoraszczuk EL , Skalka M
Published: October 2005

To characterize thyroid hormone action in the ovary, the direct effect of triiodothyronine (T3) was investigated in vitro using a culture system of porcine theca cells (Tcs) and granulosa cells (Gcs) in mono- and co-culture (GT), the latter resembling follicles in vivo. The cells were cultured in the absence or presence of human chorionic gonadotrophin (hCG) with or without T3 (10(-7), 10(-9) or 10(-11) M). Follicular cells were obtained from follicles of different size (small, medium and large), and steroid secretion into the culture medium was detected by radioimmunoassay. T3 alone did not influence steroid secretion by Tcs and Gcs isolated from follicles that were small and medium in size. In preovulatory follicles, an increase in basal androgen secretion and a simultaneous decrease in oestradiol secretion were observed with Tcs and Gcs in both mono- and co-culture. T3 together with hCG decreased hCG-stimulated androgen secretion in Tcs isolated from medium-sized follicles and had a simultaneous stimulatory effect on hCG-stimulated oestradiol secretion by Gcs. In cultures of follicular cells obtained from large follicles, T3 decreased hCG-stimulated secretion of both androgen and oestrogen by Tcs and simultaneously stimulated oestradiol secretion in GT co-cultures. Thus, the interaction of T3 with gonadotrophin hormone modulated follicular steroidogenesis, depending on follicle size and cell type used in culture. The observed T3-induced increase in basal androgen secretion by Tcs could account for the atresia of follicles, since it is accompanied by a decrease in oestradiol secretion in GT co-culture. In its co-activity with hCG, an adequate level of T3 prevents excessive androgen production by Tcs, probably influencing aromatization processes in the follicle. An increase in hCG-stimulated oestradiol secretion in GT co-cultures is then observed. Further investigations are required to clarify whether this is linked with an effect on the aromatization processes occurring in the follicle.
Digoxin transfer across the isolated placenta is influenced by maternal and fetal albumin concentrations
Authors: Schmolling J , Jung S , Reinsberg J , Schlebusch H
Published: October 2005

The aim of this study was to evaluate the influence of different maternal and fetal albumin concentrations on the transplacental transfer and the placental tissue accumulation of digoxin. Digoxin passage across the isolated lobules of 15 human placentae was calculated from repeated fetal and maternal perfusate samples, and placental tissue digoxin concentrations were measured at the end of the experiments. Metildigoxin (Lanitop) was added to the maternal medium at a concentration of 5.70 +/- 0.73 ng mL-1, and maternal and fetal perfusate albumin (BSA) concentrations were kept equal either at a high concentration of 21 g L-1 (Group I; n = 5) or at a low concentration of 3 g L-1 (Group III; n = 5), or differed with a materno-fetal gradient of 21:3 g L-1 (Group II; n = 5). In the experiments with low maternal albumin concentrations (Group III), digoxin concentrations in the maternal circuit decreased to 3.56 ng mL-1, whereas digoxin concentrations in the fetal circuit reached 2.59 ng mL-1 over a 3-h period. With maternal BSA concentrations of 21 g L-1 (Group I and Group II), the decrease in digoxin concentration in the maternal circuit was lower (P < 0.05), and digoxin tissue concentrations at the end of the experiments were smaller (0.45 +/- 0.07 and 0.42 +/- 0.03 v. 0.82 +/- 0.32 ng mg-1 protein, Group I and Group II v. Group III respectively; P < 0.05). Comparing only those lobules with similar high concentrations of maternal protein, fetal BSA concentrations of 21 g L-1 resulted in a greater increase in digoxin concentrations in the fetal circuit (end-feto to initial-maternal digoxin concentrations of 0.44 +/- 0.08 v. 0.37 +/- 0.04 ng mg-1 protein (Group I v. Group II respectively), although this was not significant. The data suggest that maternal and fetal serum albumin concentrations may have an influence on transplacental digoxin transfer, and this should be considered when treating fetuses with cardiac disease transplacentally with glycosides.
Osmolality of equine blastocyst fluid from day 11 to day 25 of pregnancy
Authors: Waelchli RO , Betteridge KJ
Published: October 2005

Horse conceptuses collected between Day 11 and Day 18 of pregnancy float in isotonic media. To investigate this phenomenon, blastocyst fluids from 30 conceptuses from 13 mares were analysed for osmolality and for concentrations of Na+, Cl-, K+, glucose, urea and creatinine. In conceptuses from Group A, samples from Day 11 to Day 16 yielded the following results (mean +/- s.e.m.): osmolality, 121.4 +/- 1.5 mOsm kg-1; Na+, 11.0 +/- 2.2 mM; Cl-, 29.3 +/- 2.5 mM; K+, 26.2 +/- 2.6 mM; glucose, 0.6 +/- 0.1 mM; urea, 6.0 +/- 0.6 mM; creatinine, 9.6 +/- 1.1 microM. Between Day 16 and Day 25, the osmolalities and Na+ concentrations increased gradually with age but the former never exceeded 255 mOsm kg-1. Fluids from Group B were obtained from eight conceptuses exposed to saline solutions of different osmolalities for various periods of time. An increased perivitelline space of 1-3 mm became evident at the lower pole of the floating conceptus after 45 min of exposure to solutions with osmolalities of > or = 300 mOsm kg-1, suggesting that Na+ and Cl- diffuse freely through the capsule but not through the trophoblast. The significance of the hypo-osmolality of equine blastocyst fluid is discussed but remains unclear.
Effect of fetal thyroidectomy on brown adipose tissue and thermoregulation in newborn lambs
Authors: Schermer SJ , Bird JA , Lomax MA , Shepherd DA , Symonds ME
Published: October 2005

The effect of fetal thyroidectomy on thermoregulation in newborn lambs was investigated. Seven of 14 lambs born normally at term were thyroidectomized at Day 127 of gestation. Colonic temperature and oxygen consumption were measured during non-rapid eye movement sleep 6-45 h after birth. All lambs were then killed and perirenal brown adipose tissue was sampled for measurement of thermogenic activity (guanosine diphosphate binding), uncoupling protein and lipid contents. Thyroidectomized lambs tended to have a mean colonic temperature 2.35 degrees C lower (P = 0.067) than controls and two became hypothermic (i.e. colonic temperature < 35 degrees C). Thyroidectomized lambs exhibited lower rates of oxygen consumption (P = 0.05) and an increased incidence of shivering thermogenesis. The perirenal adipose tissue of these lambs had a lower thermogenic activity (P < 0.01), less uncoupling protein (P < 0.01) and higher lipid content (P = 0.072) compared with intact controls. It is concluded that fetal thyroidectomy results in a decreased ability of newborn lambs to utilize nonshivering thermogenesis in brown adipose tissue as well as increasing the incidence of hypothermia. These changes are associated with decreased synthesis of uncoupling protein and functional development of brown adipose tissue in the late gestation fetus.
Developmental capacity, energy metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep
Authors: O'Brien JK , Dwarte D , Ryan JP , Maxwell WM , Evans G
Published: October 2005

Development to the blastocyst stage was assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The proportion of cleaved oocytes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal sheep than for those from adult sheep (7.4% and 24.6% respectively). There were no differences in the metabolism of glucose, glutamine or pyruvate between oocytes matured in vivo and in vitro, or of glucose or pyruvate between oocytes from prepubertal and adult sheep. Glutamine metabolism by mature oocytes from prepubertal sheep was significantly lower than that by oocytes from adult sheep. Ultrastructural studies revealed no differences in the morphology of cytoplasmic organelles of oocytes matured in vitro from prepubertal and adult sheep, but differences in the volume fraction and size of mitochondria and cortical granules were observed. These data suggest that mature oocytes from prepubertal sheep do not possess the developmental potential of their adult-derived counterparts, and this phenomenon may be associated with metabolic and ultrastructural anomalies.
Effect of maternal glucose infusion on brown adipose tissue and liver development in the neonatal lamb
Authors: Clarke L , Andrews DC , Lomax MA , Symonds ME
Published: October 2005

The effect of maternal glucose infusion over the final 5-7 days of gestation in under-fed ewes on perirenal brown adipose tissue (BAT) and liver development in lambs over the first month of neonatal life was examined. During glucose infusion, higher maternal plasma concentrations of glucose and thyroid hormones, and lower plasma concentrations of non-esterified fatty acids and 3-hydroxybutyrate were observed, compared with saline-infused controls. These differences were not observed 1-1.5 h before parturition when plasma concentrations of glucose, lactate, cortisol and thyroid hormones all increased in control ewes. Lamb birthweight and liver and BAT weights were similar between groups, but lambs born to glucose-infused ewes had a higher hepatic glycogen content and greater iodothyronine 5'deiodinase activities in liver and BAT. The norepinephrine, epinephrine and dopamine contents were also greater in BAT sampled from lambs born to glucose infused ewes. Three lambs born to glucose-infused ewes failed to survive beyond the second week of life and exhibited abnormally low plasma triiodothyronine concentrations. It is concluded that maternal glucose infusion stimulates development of the fetal sympathetic nervous system during late gestation but this adaptation does not appear to improve postnatal survival.
Changes in distribution of labile zinc in mouse spermatozoa during maturation in the epididymis assessed by the fluorophore Zinquin
Authors: Zalewski PD , Jian X , Soon LL , Breed WG , Seamark RF , Lincoln SF , Ward AD , Sun FZ
Published: October 2005

The Zn(II)-specific fluorophore Zinquin was used to determine the regional distribution of free or loosely-bound Zn(II) in mouse spermatozoa. Spermatozoa from the testes exhibited bright fluorescence over the entire head; those from the caput epididymides generally fluoresced more brightly in the post-acrosomal region; and spermatozoa from the caudae epididymides fluoresced less brightly, with foci of fluorescence over the sperm head which were lost after extraction with Triton X-100 and hence appeared to be membrane-associated. Treatment of cauda sperm with sodium dodecyl sulfate resulted in a bright uniform Zinquin fluorescence in the heads, similar to that observed in caput sperm, indicating that the two types of sperm have similar amounts of head Zn(II) but that the availability of Zn(II) for binding Zinquin is different. By contrast, the intensity of tail fluorescence was similar in spermatozoa from different regions of the male reproductive tract and was largely unaffected by Triton X-100 extraction, consistent with an intracellular location. Similar differences were observed between caput sperm and cauda sperm in the rat. It is concluded that visualization and measurement of free or loosely-bound Zn(II) in subcellular compartments of spermatozoa should facilitate investigation of the role of this metal in the development and function of spermatozoa and abnormalities that might accompany infertility and Zn(II) deficiency.
Luteinizing hormone, follicle-stimulating hormone, testosterone and dihydrotestosterone during testicular descent in the pig fetus
Authors: Visser JH , Heyns CF
Published: October 2005

To investigate the relationship between gonadotrophins, androgens and testicular descent in the pig fetus, serum concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and serum concentrations and testicular content of testosterone (T) and 5 alpha-dihydrotestosterone (DHT) were determined by radioimmunoassay in 95 fetuses (48 males, 47 females) between Day 60 and Day 90 post coitum (p.c.). The testes were descended in 0% of fetuses at Day 60, 8% of fetuses at Day 70, 40% of fetuses at Day 80 and 87% of fetuses at Day 90 p.c. There was a significant increase in mean serum concentrations of LH in both male and female fetuses from Day 60 to Day 90, with a significant difference in mean serum concentrations of LH between male fetuses with a body mass < 350 g (4% of testes descended) and those with a body mass > 450 g (87% of testes descended), but not between male and female fetuses < 350 g or > 450 g in body mass. There was no significant difference in mean serum concentrations of FSH between male and female fetuses. Mean serum concentrations of T were significantly higher in male fetuses at Day 60 than in those at Days 70-90, with no significant difference in serum concentrations of DHT from Day 60 to Day 90 p.c. Testicular content of T and DHT showed a non-linear increase from Day 60 to Day 90 p.c. Although the serum concentration of T is elevated before, and decreased during, the period of descent, the testicular content of T and DHT increases during the period of descent, indicating that serum concentrations of LH and FSH may have an indirect effect on descent by stimulating testicular androgen synthesis.
High concentrations of immunoreactive inhibin in the plasma of mares and fetal gonads during the second half of pregnancy
Authors: Nambo Y , Nagata S , Oikawa M , Yoshihara T , Tsunoda N , Kohsaka T , Taniyama H , Watanabe G , Taya K
Published: October 2005

Plasma concentrations of immunoreactive (ir)-inhibin were measured in seven pregnant mares from around Day 140 of gestation to Day 2 after parturition using a heterologous bovine-based radioimmunoassay (RIA). Concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), oestradiol-17 beta, progesterone and relaxin were also measured in the same samples. A marked increase in plasma concentrations of ir-inhibin, FSH and LH occurred between Day 220 and Day 300 of gestation but the concentrations of all three hormones returned to baseline by about Day 320 (three weeks before parturition). In contrast, circulating concentrations of the three placental hormones, oestradiol-17 beta, progesterone and relaxin, increased during the final weeks of pregnancy and then decreased markedly to basal values within two days of parturition. There was a positive correlation between circulating concentrations of ir-inhibin and FSH (r = 0.75, P < 0.01) rather than the expected negative correlation. ir-inhibin was not detected in homogenates obtained at Day 190 of pregnancy and form term placenta, but high concentrations of ir-inhibin were present in homogenates of fetal and newborn gonads. Despite the high concentrations of ir-inhibin in these homogenates, they failed to exert any suppressive bioactivity on FSH secretion by rat pituitary cells cultured in vitro. Furthermore, immunohistochemical staining revealed the presence of inhibin in the interstitial cells of equine fetal gonads at Day 190 of gestation. These findings demonstrate for the first time that high concentrations of ir-inhibin, LH and FSH are secreted into the peripheral circulation of the mare during the second half of pregnancy. However, ir-inhibin present in the plasma of pregnant mares appears to be biologically inactive. This hormone is not presumed to be of placental origin but it is proposed that either the enlarged fetal gonads or the maternal ovaries, or both of these organs, may be a source of inhibin in response to the coincident increase in circulating concentrations of LH and FSH.
Effect of culture conditions on artificial activation of porcine oocytes matured in vitro
Authors: Yamauchi N , Sasada H , Sugawara S , Nagai T
Published: October 2005

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.
Evidence against a significant role for mast cells in blastocyst implantation in the rat and mouse
Authors: Salamonsen LA , Jeziorska M , Newlands GF , Dey SK , Woolley DE
Published: October 2005

Rats were treated with the highly potent stabilizer of mast cells, FPL 55618, before and during the first seven days of pregnancy to establish whether stabilization of mast cells resulted in impaired blastocyst implantation. There was no significant reduction in either the number of ovulations or the number of implantation sites in treated rats compared with controls; 11 of 15 treated rats were pregnant compared with 5 of 6 control rats. The distribution of mast cells was examined in uterine tissues, implantation sites and interimplantation sites in both rats and mice using highly sensitive immunohistochemical techniques. Virtually all of the mast cells in rat uterine tissue stained for rat mast cell protease-I (RMCP-I; connective tissue type), whereas few stained for RMCP-II (mucosal type). Most of the mast cells were present in the myometrium with very sparse distribution in the endometrium and there were no differences in numbers of mast cells between implantation and inter-implantation sites on Day 7 of pregnancy. In tissue sections of mouse uteri sampled from Day 1 to Day 8 of pregnancy there were virtually no mast cells in the endometrium or deciduum adjacent to implantation sites. Mouse uterine mast cells also stained predominantly for the connective tissue-type mast cell protease MMCP-4, the murine equivalent of RMCP-I. Thus, mast cells and their products appear to play little, if any, role in blastocyst implantation in murid rodents. Since mast cells are a prominent feature of human endometrium, this study emphasizes the important consideration of species differences when choosing animal models for implantation studies.
Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm
Authors: Glogowski J , Babiak I , Goryczko K , Dobosz S
Published: October 2005

Milt of brown, rainbow and brook trout was cryopreserved. Activity of aspartate aminotransferase (AspAT) and acid phosphatase was assayed both in supernatants and in spermatozoa obtained from thawed sperm samples; additionally, post-thaw motility was evaluated. Enzyme activities differed according to fish species and were strongly affected by the type of cryoprotectant used. The activity in supernatants was usually higher than that in spermatozoa because of protein leakage from injured cells. AspAT activity in cryopreserved spermatozoa correlated positively with fertilization success in all three species. There was a negative correlation between activity of extracellular (supernatant) AspAT and fertilization rates in variants with dimethyl sulfoxide and dimethylacetamide-based extenders. The motility of thawed sperm, determined microscopically, provided some information on the cryopreservation efficiency of trout milt.
Defects in the allocation of cells to the inner cell mass and trophectoderm of parthenogenetic mouse blastocysts
Authors: Mognetti B , Sakkas D
Published: October 2005

Diploid parthenogenetic mouse embryos (which possess two maternally-derived genomes) can develop only as far as the 25-somite stage when transferred in utero and exhibit a substantial reduction in trophoblast tissue. The loss of cultured parthenogenetic embryos during postimplantation indicates that a defect in cell lineage may be evident as early as the blastocyst stage. The possibility that a defect may already be reflected at the preimplantation stage was investigated by examining the allocation of cells to the trophectoderm (trophoblast progenitor cells) and the inner cell mass of haploid and diploid parthenogenetic mouse blastocysts. Utilizing a differential labelling technique for counting cells, diploid parthenogenetic blastocysts were found to have fewer inner cell mass cells and trophectoderm cells than their haploid counterparts and normal blastocysts. In addition, both haploid and diploid parthenogenetic blastocysts had a lower inner cell mass: trophectoderm ratio than normal blastocysts. Thus, the relatively poor development of the trophectoderm lineage at the postimplantation stage is not reflected by a reduction in its allotment of cells at its first appearance. Nevertheless, the findings indicate that parthenogenetic development is already compromised at the blastocyst stage, and provide evidence that the expression of imprinted genes has significance for the development of the embryo at the preimplantation stage.
Using faecal pellet counts along transects to estimate quokka (Setonix brachyurus) population density
Authors: Hayward Matt W. , de Tores Paul J. , Dillon Michael J. , Fox Barry J. , Banks Peter B.
Published: October 2005

A study was conducted to determine the validity of using transect counts of faecal pellet groups to estimate population densities of a threatened, macropodid marsupial – the quokka (Setonix brachyurus (Quoy & Gaimard, 1830)). Mark–recapture estimates of population density were regressed against counts of faecal pellet groups at six sites with and three sites without fox control within the northern jarrah forest of Western Australia. Significant linear relationships were found between population density and pellet counts for all sites (r2 = 0.56, P < 0.02) and when all unbaited sites were excluded (r2 = 0.98, P < 0.01). We suggest that this method could be used for broad-scale monitoring of this threatened species.
Terrestrial avifauna of the Gippsland Plain and Strzelecki Ranges, Victoria, Australia: insights from Atlas data
Authors: Radford James Q. , Bennett Andrew F.
Published: October 2005

The rate and spatial scale at which natural environments are being modified by human land-uses mean that a regional or national perspective is necessary to understand the status of the native biota. Here, we outline a landscape-based approach for using data from the ‘New Atlas of Australian Birds’ to examine the distribution and status of avifauna at a regional scale. We use data from two bioregions in south-east Australia – the Gippsland Plain and the Strzelecki Ranges (collectively termed the greater Gippsland Plains) – to demonstrate this approach. Records were compiled for 57 landscape units, each 10′ latitude by 10′ longitude (~270 km2) across the study region. A total of 165 terrestrial bird species was recorded from 1870 ‘area searches’, with a further 24 species added from incidental observations and other surveys. Of these, 108 species were considered ‘typical’ of the greater Gippsland Plain in that they currently or historically occur regularly in the study region. An index of species ‘occurrence’, combining reporting rate and breadth of distribution, was used to identify rare, common, widespread and restricted species. Ordination of the dataset highlighted assemblages of birds that had similar spatial distributions. A complementarity analysis identified a subset of 14 landscape units that together contained records from at least three different landscape units for each of the 108 ‘typical’ species. When compared with the 40 most common ‘typical’ species, the 40 least common species were more likely to be forest specialists, nest on the ground and, owing to the prevalence of raptors in the least common group, take prey on the wing. The future status of the terrestrial avifauna of the greater Gippsland Plains will depend on the extent to which effective restoration actions can be undertaken to ensure adequate representation of habitats for all species, especially for the large number of species of conservation concern.
Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism
Authors: Castro Marián , Nikolaev Viacheslav O. , Palm Dieter , Lohse Martin J. , Vilardaga Jean-Pierre
Published: October 2005

Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within ≈1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (τ ≈ 140 ms) and then with slower kinetics (τ ≈ 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHR
CFP/YFP
). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).
Genetic variation of folate-mediated one-carbon transfer pathway predicts susceptibility to choline deficiency in humans
Authors: Kohlmeier Martin , da Costa Kerry-Ann , Fischer Leslie M. , Zeisel Steven H.
Published: October 2005

Choline is a required nutrient, and some humans deplete quickly when fed a low-choline diet, whereas others do not. Endogenous choline synthesis can spare some of the dietary requirement and requires one-carbon groups derived from folate metabolism. We examined whether major genetic variants of folate metabolism modify susceptibility of humans to choline deficiency. Fifty-four adult men and women were fed diets containing adequate choline and folate, followed by a diet containing almost no choline, with or without added folate, until they were clinically judged to be choline-deficient, or for up to 42 days. Criteria for clinical choline deficiency were a more than five times increase in serum creatine kinase activity or a >28% increase of liver fat after consuming the low-choline diet that resolved when choline was returned to the diet. Choline deficiency was observed in more than half of the participants, usually within less than a month. Individuals who were carriers of the very common 5,10-methylenetetrahydrofolate dehydrogenase-1958A gene allele were more likely than noncarriers to develop signs of choline deficiency (odds ratio, 7.0; 95% confidence interval, 2.0-25;
P
< 0.01) on the low-choline diet unless they were also treated with a folic acid supplement. The effects of the C677T and A1298C polymorphisms of the 5,10-methylene tetrahydrofolate reductase gene and the A80C polymorphism of the reduced folate carrier 1 gene were not statistically significant. The most remarkable finding was the strong association in premenopausal women of the 5,10-methylenetetrahydrofolate dehydrogenase-1958A gene allele polymorphism with 15 times increased susceptibility to developing organ dysfunction on a low-choline diet.
Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice
Authors: Wang Xiangchun , Inoue Shinya , Gu Jianguo , Miyoshi Eiji , Noda Katsuhisa , Li Wenzhe , Mizuno-Horikawa Yoko , Nakano Miyako , Asahi Michio , Takahashi Motoko , Uozumi Naofumi , Ihara Shinji , Lee Seung Ho , Ikeda Yoshitaka , Yamaguchi Yukihiro , Aze Yoshiya , Tomiyama Yoshiaki , Fujii Junichi , Suzuki Keiichiro , Kondo Akihiro , Shapiro Steven D. , Lopez-Otin Carlos , Kuwaki Tomoyuki , Okabe Masaru , Honke Koichi , Taniguchi Naoyuki
Published: October 2005

The core fucosylation (α1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated α1,6-fucosyltransferase (
Fut8
)-null mice and found that disruption of
Fut8
induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that
Fut8
-/-
mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from
Fut8
-/-
mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-β1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact,
Fut8
-/-
mice have a marked dysregulation of TGF-β1 receptor activation and signaling, as assessed by TGF-β1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-β1 receptor defects found in
Fut8
-/-
cells can be rescued by reintroducing
Fut8
into
Fut8
-/-
cells. Furthermore, exogenous TGF-β1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in
Fut8
-/-
lung. We propose that the lack of core fucosylation of TGF-β1 receptors is crucial for a developmental and progressive/destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.