Developing an integrated substance use and mental health service in the specialised setting of a youth detention centre.

This article describes the frequency of co-morbid substance use and mental health problems of young people within the youth justice system and demonstrates that mental health and drug and alcohol services can be integrated and work effectively. The establishment of an integrated Mental Health Alcohol Tobacco and Other Drugs Service (MHATODS) to juveniles in detention represents a shift away from the traditional paradigm of separate services frequently found throughout Australia. The development of referral procedures and adolescent-focused treatment programmes that are tailored to the specific needs of this disadvantaged population are discussed.

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection.

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years.

Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulfide status.

Mouse spermatozoa from the caudae epididymides could be freeze-dried without losing their ability to support normal development. Immature spermatozoa from the testes, in contrast, were damaged by freeze-drying. However, immature spermatozoa became resistant to freeze-drying after their treatment with diamide, which oxidizes free -SH groups.

Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa.

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA.

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa.

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains.

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation.

Role of facilitative glucose uptake in the glucose-inorganic phosphate-mediated retardation and inhibition of development in different strains of mouse embryos.

Mouse embryos from different strains develop differently in vitro depending on the composition of the culture medium, and in particular on the presence or absence of glucose and inorganic phosphate. Glucose is both stimulatory and inhibitory in certain conditions. Glucose uptake by cells can be passive, down concentration gradients, or active, through sodium driven pumps, or can occur through facilitative transport.

Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa.

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris.HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution.

Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries.

Fresh and frozen ovaries from 10 day old C57BL/6J-Gpi-1(a) mice were grafted orthotopically into ovariectomized B6CBF1 (homozygous Gpi-1(b)) recipients. The recipients were mated with B6CBF1 males. The birth and size of each litter was recorded.

The effect of follicle stimulating hormone and epidermal growth factor on the developmental capacity of in-vitro matured mouse oocytes.

This study investigates the effects of follicle stimulating hormone (FSH) or epidermal growth factor (EGF) on the development of mouse oocytes matured in vitro. The data show that addition of FSH or EGF does not significantly increase the proportion of oocytes maturing to metaphase II but does increase the ability of these oocytes to cleave to the 2-cell stage after fertilization. After transfer of 2-cell embryos to pseudopregnant recipients, 64-78% of the embryos implanted, which was significantly reduced compared to embryos from in-vivo matured controls (89%).

Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection.

Epididymal mouse spermatozoa were suspended in various physiological solutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffinose before cooling to -20 degrees, -50 degrees or -196 degrees C and storage for 1-28 days. After thawing, a few spermatozoa frozen with raffinose were partially motile (about 2%) but in all other treatments they were immotile and diagnosed as 'dead' by staining that differentiates between live and dead spermatozoa. Almost all oocytes injected with sperm heads (nuclei) from spermatozoa frozen with and without raffinose were fertilized normally (95-100%) and developed to the two-cell stage (89-100%).

Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries.

Ovaries from 10-day-old mice were exposed to 1.5 mol l-1 dimethylsulfoxide, 1,2-propanediol, ethanediol or glycerol for 5-60 min at room temperature before freezing. Follicles in fresh and frozen ovaries were counted and scored as normal or damaged in stained serial sections.

Capacitation-like changes occur in mouse spermatozoa cooled to low temperatures.

Previously we showed that >70% of mouse spermatozoa cooled slowly from 37 degrees C to 4 degrees C and warmed have undergone capacitation-like changes as examined by a chlortetracycline staining assay. These membrane changes are reflected in the ability of cooled spermatozoa to achieve fertilization rates in vitro similar to those of uncooled controls when added to oocytes immediately upon warming. The aim of this study was to determine the nature of these membrane changes.

Meiotic and mitotic Ca2+ oscillations affect cell composition in resulting blastocysts.

At fertilization periodic Ca2+ oscillations release oocytes from meiotic arrest. The present study examined whether these oscillations have a long-term role in pre- and postimplantation development, independent of their immediate effect. Sr(2+)-containing medium was used to induce oscillations during exit from meiosis and first embryonic mitosis and Sr(2+)-activated parthenotes were compared to ethanol-activated parthenotes and embryos generated by in vitro fertilization.

A comparison of sperm- and IP3-induced Ca2+ release in activated and aging mouse oocytes.

Ca2+ release mechanisms in oocytes are highly sensitive and a number of agents including sperm and inositol trisphosphate (IP3) generate Ca2+ transients. Recently it was shown that this sensitivity decreases after fertilization and subsequent entry into the first mitotic cell cycle (Jones et al., Development 121, 3259-3266, 1995).

Ca2+ release and the development of Ca2+ release mechanisms during oocyte maturation: a prelude to fertilization.

Oogenesis involves the production of an oocyte that can undergo fertilization and support early development. The stimulus that initiates embryogenesis is an increase in the concentration of intracellular Ca2+ in the cytoplasm of the oocyte at the time of fertilization. The development of the ability of the oocyte to release Ca2+ in response to the fertilizing spermatozoon is an essential step in the process of oogenesis.

Effect of cooling mouse spermatozoa to 4 degrees C on fertilization and embryonic development.

Attempts to freeze mouse spermatozoa in liquid nitrogen (-196 degrees C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4 degrees C was examined. Epididymal spermatozoa were collected into a variety of media at 37 degrees C, cooled slowly to 4 degrees C over 4 h and warmed in a water bath at 37 degrees C for 5 min.

Influence of genetic background and media components on the development of mouse embryos in vitro.

One-cell embryos from some inbred and random-bred mice, but not those derived from certain F1 hybrids, suffer from a block during in vitro development known as the two-cell block. This two-cell block can be overcome by removing glucose or inorganic phosphate from the culture system or by altering the ratio of other medium components such as sodium, potassium, or bicarbonate. This issue is made more complex by the fact that the rate of development is different for each strain of mouse and this rate of development is invariably slowed under in vitro culture conditions.

A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division.

We have used Ca2+-sensitive fluorescent dyes to monitor intracellular Ca2+ during mitosis in one-cell mouse embryos. We find that fertilized embryos generate Ca2+ transients at nuclear envelope breakdown (NEBD) and during mitosis. In addition, fertilized embryos arrested in metaphase using colcemid continue to generate Ca2+ transients.

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro.

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry.

Analysis of the chromosome complement of frozen-thawed mouse oocytes after parthenogenetic activation.

Frozen-thawed mouse oocytes were artificially activated with Sr2+ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen-thawed and freshly collected oocytes and the majority of oocytes (> 90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozen-thawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0 degree C or 37 degrees C for extended periods beyond those required for protection.

Cytogenetical analysis and developmental potential of vitrified mouse oocytes.

Mature mouse oocytes were cryopreserved by vitrification in 6 M dimethyl sulfoxide (VS). After warming they were either artificially activated with strontium (Sr2+), and the incidence of chromosome non-disjunction was assessed at first cleavage metaphase; or they were fertilized in vitro, and postimplantation survival was examined at Day 15 of gestation. Similar proportions of vitrified and freshly collected oocytes were activated with Sr2+ (75% and 82%, respectively).

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent.

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known.