scholarly journals Induction of sperm maturation in vitro in epididymal cell cultures of the tammar wallaby (Macropus eugenii): disruption of motility initiation and sperm morphogenesis by inhibition of actin polymerization

Reproduction ◽  
2002 ◽  
pp. 107-117 ◽  
Author(s):  
M Lin ◽  
R Hess ◽  
RJ Aitken
Keyword(s):  
Culture System ◽  
Actin Polymerization ◽  
Tammar Wallaby ◽  
Sperm Maturation ◽  
Actin Organization ◽  
Cell Monolayers ◽  
Macropus Eugenii ◽  
Maturation In Vitro ◽  
Marsupial Species

A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of spermatozoa from a marsupial species, the tammar wallaby (Macropus eugenii). This system was able to maintain wallaby epididymal epithelial cells in vitro for more than 2 months. The system also enabled immature wallaby spermatozoa to differentiate from a T-shaped to a streamlined form, accompanied by the development of progressive motility after co-culture with epididymal cell monolayers that had been cultured for 7 days. The addition of inhibitors of actin polymerization (latrunculin A or B) to the co-culture system showed that wallaby sperm maturation was impaired by the interruption of actin organization within the immature spermatozoa. These results indicate that actin filaments play a significant role in sperm transformation during post-testicular maturation in marsupials. These observations also indicate that the marsupial co-culture system has the potential to greatly increase understanding of sperm-epididymal cell interactions and the mechanism of sperm maturation in these species.

In vitro culture of brushtail possum (Trichosurus vulpecula) epididymal epithelium and induction of epididymal sperm maturation in co-culture

Reproduction ◽  
2000 ◽  
pp. 1-14 ◽  
Author(s):  
M Lin ◽  
X Zhang ◽  
R Murdoch ◽  
RJ Aitken
Keyword(s):  
Sperm Maturation ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Epididymal Sperm ◽  
Cell Monolayers ◽  
Progressive Motility ◽  
Cell Culture Systems ◽  
Marsupial Species ◽  
Morphological Maturation

A medium modified from eutherian systems was used to culture epididymal epithelial cells of the brushtail possum (Trichosurus vulpecula) for more than 2 months. Epididymal tubule fragments from the caput, corpus and cauda epididymides were used to generate cell monolayers. All three epididymal cell culture systems supported maturational changes in marsupial spermatozoa and enabled immature possum spermatozoa to differentiate from a T-shape to a streamlined shape, accompanied by the development of progressive motility after co-culture with 7-day-old cultured epididymal cell monolayers. This epididymal cell and sperm co-culture system for marsupial species may facilitate the identification of specific epithelial factors that affect sperm maturation, particularly in a species in which morphological maturation is readily visible.

Hormones of oestrus and ovulation and their manipulation in marsupials

1996 ◽  
Vol 8 (4) ◽  
pp. 661 ◽  
Author(s):  
LA Hinds ◽  
TP Fletcher ◽  
JC Rodger
Keyword(s):  
In Vitro Fertilization ◽  
Tammar Wallaby ◽  
Monodelphis Domestica ◽  
Ovarian Activity ◽  
Vitro Fertilization ◽  
Macropus Eugenii ◽  
Australian Species ◽  
Maturation In Vitro ◽  
American Laboratory

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.

Biochemical studies of intrauterine components of the tammar wallaby Macropus eugenii during pregnancy

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 325-338
Author(s):  
Elizabeth J. Thornber ◽  
Marilyn B. Renfree ◽  
Gregory I. Wallace
Keyword(s):  
Protein Concentration ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Tammar Wallaby ◽  
Macropus Eugenii ◽  
Tenfold Increase ◽  
Specific Proteins ◽  
Wet Weight

The in vitro uptake and incorporation of [3H]ui idine by blastocysts of the tammar wallaby showed a 16- and 30-fold increase from day 0 to day 10 after removal of pouch young, respectively. Two of the six non-expanded blastocysts recovered on day 5 showed a tenfold increase in incorporation. During the first ten days after removal of pouch young the diameter of the blastocyst increased threefold. Endometrial exudate from gravid uteri had a higher protein concentration than exudate from nongravid uteri (39·5 ± 0·9 and 32·0 ± 2·0 mg/ml (mean ± s.e.m.), respectively). Endometrial exudates from uteri where the blastocyst was actively growing were found to contain six uterine-specific proteins. These were separated by gradient polyacrylamide gel electrophoresis. Two of the proteins were pre-albumins and the others were larger molecules (M.W. 153000–670000). Two proteins were only present at particular stages of pregnancy: the other four were present at all stages from diapause to birth, in exudate from gravid and nongravid uteri. The specific binding of progesterone and androstenedione to proteins in endometrial exudates or uterine flushings from pregnant wallabies was less than one per cent of the value obtained from day-5 pregnant rabbits. The ability of mouse blastocysts to take up and incorporate [3H]uridine into acidinsoluble material increased threefold in the presence of day-10 endometrial exudates from wallabies. However, this was less than ten percent of the values obtained in the presence of bovine serum albumin. The concentration of calcium in endometrial exudates increased from 23·6 to 45·2 μg/ml during pregnancy; in endometrium it remained at 88·7 μg/g (wet weight) throughout pregnancy, and in plasma it was 53·3 μg/ml. The concentration of zinc in endometrial exudates was 4·5 μg/ml; in endometrium it decreased from 21·8 to 13·3 μg/g (wet weight) during pregnancy and in plasma it was 0·6 μg/ml.

scholarly journals Negative Regulation of Yeast Eps15-like Arp2/3 Complex Activator, Pan1p, by the Hip1R-related Protein, Sla2p, during Endocytosis

2007 ◽  
Vol 18 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Jiro Toshima ◽  
Junko Y. Toshima ◽  
Mara C. Duncan ◽  
M. Jamie T.V. Cope ◽  
Yidi Sun ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Affinity Purification ◽  
Coiled Coil ◽  
Actin Assembly ◽  
Related Protein ◽  
Loss Of Function ◽  
Actin Organization ◽  
Coiled Coil Region ◽  
Kinase Phosphorylation

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites ∼25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification–tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2ΔCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.

Pellet-freezing spermatozoa of two marsupials: the tammar wallaby, Macropus eugenii, and the brushtail possum, Trichosurus vulpecula

1996 ◽  
Vol 8 (4) ◽  
pp. 681 ◽  
Author(s):  
FC Molinia ◽  
JC Rodger
Keyword(s):  
Room Temperature ◽  
Tammar Wallaby ◽  
Brushtail Possum ◽  
Vitro Fertilization ◽  
Macropus Eugenii ◽  
Progressive Motility ◽  
Viable Population ◽  
Phosphate Buffered Saline ◽  
Breeding Techniques

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.

Low MHC class II variability in a marsupial

1994 ◽  
Vol 6 (6) ◽  
pp. 721 ◽  
Author(s):  
LM McKenzie ◽  
DW Cooper
Keyword(s):  
Genetic Variation ◽  
Mhc Class Ii ◽  
Fragment Length Polymorphism ◽  
Tammar Wallaby ◽  
Class Ii ◽  
Mixed Lymphocyte Culture ◽  
Lymphocyte Culture ◽  
Macropus Eugenii ◽  
Histocompatibility Complex ◽  
Marsupial Species

The major histocompatibility complex (MHC) loci have been shown to be highly polymorphic in most eutherian ('placental') species studied. Several hypotheses have been advanced for the maintenance of this exceptional level of genetic variation, one of which suggests that it is necessary for successful eutherian reproduction. Marsupials (metatherians) and eutherians are the only two groups of viviparous mammals, but their modes of reproduction are quite distinct. Although marsupials have placentae, they are generally shorter lived and less invasive than in eutherians. Other investigations have shown that genetic variation at marsupial MHC class I loci is probably high. Weak or non-existent mixed lymphocyte culture responses previously reported in several marsupial species have suggested a lack of class II variation. Data have therefore been collected on the level of restriction fragment length polymorphism at MHC class II beta-chain encoding loci of a marsupial, Macropus eugenii (the tammar wallaby). This level is shown to be low, between the level of MHC variation found in cheetahs and a population of lions with a restricted genetic base. Attention is drawn to the need to collect more data on the level of class II variability in both eutherians and marsupials, and to the potential of marsupials for understanding the relation, if any, between mode of reproduction and MHC variability.

Effect of Testosterone on Sperm Maturation in vitro

Nature ◽  
1973 ◽  
Vol 245 (5424) ◽  
pp. 328-329 ◽  
Author(s):  
M. C. ORGEBIN-CRIST ◽  
P. L. TICHENOR
Keyword(s):  
Sperm Maturation ◽  
Maturation In Vitro

scholarly journals In-vitro secretion of progesterone by the corpus luteum of the tammar wallaby, Macropus eugenii

Reproduction ◽  
1983 ◽  
Vol 67 (1) ◽  
pp. 57-63 ◽  
Author(s):  
L. A. Hinds ◽  
S. M. Evans ◽  
C. H. Tyndale-Biscoe
Keyword(s):  
Corpus Luteum ◽  
Tammar Wallaby ◽  
Macropus Eugenii
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