Prion infected rhesus monkeys to study differential transcription of Alu DNA elements and editing of Alu transcripts in neuronal cells and blood cells.

BACKGROUND: Rhesus monkeys were used as a non-human primate model to study small non-coding RNA after infection with human sporadic and variant Creutzfeldt-Jakob prions. METHODS: Tissue-specific Alu DNA element transcription and editing of transcripts were assessed in neuronal - and blood cells (Buffy Coat). RESULTS: Tissue/cell-specific transcription and editing patterns were obtained. Active Alu DNA elements belonged to several Alu DNA families, they could be located on several chromosomes, and their genomic sites were identified. Deamination by adenosine deaminase acting on RNA and apolipoprotein B editing complex was found. CONCLUSIONS: Different Alu transcription and editing programmes exist and may depend on the infection status.

Progress toward understanding follicle development in vitro: appearances are not deceiving.

The interactive factors that influence the developmental progress of a follicle and determine whether it will progress to ovulation or toward atresia, are highly complex. In vitro models are being developed that are intended to provide a simplified environment to facilitate understanding of the dynamics of the processes involved. The purpose of this overview is to evaluate progress to date and to focus attention on issues that need more careful consideration to improve the usefulness of the models. Basically, two approaches exist. One, attached follicle culture, employs either enzyme-digested or mechanically harvested follicles depending on the method but allows attachment of the follicles to the culture surface. This produces a rounded or flattened structure (depending on culture conditions) that is no longer an intact follicle. During this culture, the cells reorganize themselves, some remaining in contact with the oocyte and others attaching to the culture surface and proliferating. The other approach, intact 3-dimensional follicle culture, employs mechanically dissected preantral follicles that are cultured as free-floating intact structures. Intact follicle culture emulates the in vivo developmental pattern of the follicle more closely than a non-intact structure can, and thereby provides a favorable model to investigate the interaction between hormonal and paracrine factors in the development of the follicle in isolation from systemic effects. For example, intact follicle culture has begun to be used to investigate the local effects of several different steroids. In addition, the local effects of inhibin, activin, and follistatin and their interactions with locally produced growth factors and steroids as well as synergy with gonadotrophins are beginning to be investigated. In our laboratory, the focus is on the roles of gonadotrophins at different stages of follicle development, particularly the effect of FSH isoforms in modulating follicle development in vitro. Finally, an important issue that urgently needs to be addressed, for future studies of in vitro follicle development, is the rationalization and standardization of follicle culture conditions.

Luteinizing hormone has a stage-limited effect on preantral follicle development in vitro.

Although it is known that LH receptors are present from the time of thecal differentiation, the role of LH during early follicle development is not yet clear. The effect of LH on preantral follicle development has therefore been investigated in vitro using a culture system that supports the development of intact follicles. We have previously shown that although preantral follicles 150 micrometer in diameter (2-3 granulosa cell layers) do not require LH to proceed through antral development, smaller follicles (1-2 granulosa cell layers, 85-110 micrometer in diameter) do not develop beyond the large preantral stage in the presence of only FSH and 5% mouse serum. Follicles of this size were therefore used to determine the effects of LH and serum on their development in vitro. The results showed that although FSH must be continuously present, a low concentration of LH together with a slight increase in serum concentration was necessary, specifically during the primary stage of follicle development (from 85 micrometer in diameter until the follicles had reached 150 micrometer in diameter) to induce the capacity for subsequent LH-independent rapid growth and antral development. The in vitro development of maturable oocytes with normal spindle and chromatin morphology was also supported. These results indicate that LH probably induces changes in the early differentiating thecal cells, which are critical for the completion of subsequent follicular and oocyte development.

Isoforms of human recombinant follicle-stimulating hormone: comparison of effects on murine follicle development in vitro.

The effects of three isoforms derived from recombinant human FSH on ovarian follicle development in vitro were characterized for the first time. The three subfractions comprised discrete pI ranges of 3. 6-4.6 (acid), 4.5-5.0 (mid), and 5.0-5.6 (least acidic). Follicular growth, estradiol secretion, and antral formation were assessed for each fraction of isoforms in a range of concentrations over a 5-day culture period. Least acidic FSH produced, at and above 1.5 ng/ml, a high percentage of follicles growing above the size threshold necessary for antral formation, whereas mid and acid FSH induced similar growth only at higher concentrations (7.5 ng/ml and 50 ng/ml, respectively). Least acidic FSH specifically induced the most rapid growth of follicles during preantral development. Acid FSH at all concentrations stimulated estradiol-17ss secretion later during culture and antral formation in a lower proportion of follicles than did least acidic and mid FSH. It can be concluded 1) that the least acidic isoform induced fastest preantral growth, producing the largest antral follicles at the lowest dose of all three fractions and 2) that the less and mid acidic isoforms had more impact on stimulation of estradiol production and antral formation than the acid isoform.

Abnormal in vitro development of ovarian follicles explanted from mice exposed to tetrachlorvinphos.

A system of mouse ovarian follicle culture in which follicles can be grown from a preantral stage of development through antral formation has been developed and modified recently by Nayudu and colleagues. Follicles have been shown to grow in this culture system at a relatively constant rate and show responsiveness to LH at the end of the culture by ovulation of mature oocytes. Reported here are the distinctly different in vitro growth patterns of follicles explanted from 22- to 24-day-old mice during a period when the colony was being treated for skin parasites with tetrachlorvinphos (TCVP) (Rabond). There is to date no information on the effects of this compound on the mammalian female reproductive system. For follicles from the TCVP treated group, the duration of growth as intact follicles was markedly reduced in comparison to mice of the same strain and source not treated with TCVP. In the treated group, premature termination of follicular growth was also associated with the spontaneous expulsion of oocytes with immature nuclei and without cumulus cells. For those follicles from treated mice that did remain in culture until the day luteinizing hormone was given, the ovulatory response was poor and the maturation response of the oocytes was low in comparison with the follicles from untreated mice. The effect of the treatment on the follicles was further characterized by obvious differences in the patterns of growth. Follicles in the untreated group grew in a linear pattern at around 25 microns/day; a single phase, fast pattern for the whole culture period.(ABSTRACT TRUNCATED AT 250 WORDS)