Neonatal thymulin gene therapy prevents ovarian dysgenesis and attenuates reproductive derangements in nude female mice.

Congenitally athymic (nude) female mice show severe ovarian dysgenesis after puberty, which seems to be consequential to a number of neuroendocrine derangements described in these mutants. Thus, considerable evidence suggests that thymulin, a thymic peptide, may be involved in thymus-pituitary communication. In order to clarify the relevance of thymulin for the maturation of the female reproductive system, we assessed at hypothalamic, pituitary, ovarian, and uterine level the preventive action of neonatal thymulin gene therapy (NTGT) on the changes that typically occur after puberty in congenitally athymic female mice. We injected (im) an adenoviral vector harboring a synthetic DNA sequence encoding a biologically active analog of thymulin, methionine-serum thymic factor, in newborn nude mice (which are thymulin deficient) and killed the animals at 70-71 d of age. NTGT in the athymic mice restored the serum thymulin levels. Morphometric analysis revealed that athymic nudes have reduced numbers of brain GnRH neurons and pituitary gonadotropic cells as compared with heterozygous controls. NTGT prevented these changes and also rescued the premature ovarian failure phenotype typically observed in athymic nude mice (marked reduction in the number of antral follicles and corpora lutea, increase in atretic follicles). Serum estrogen, but not progesterone, levels were low in athymic nudes, a reduction that was partially prevented by NTGT. Little to no morphological changes were observed in the endometrium of female nudes. The delay in the age of vaginal opening that occurs in athymic nudes was significantly prevented by NTGT. Our results suggest that thymulin plays a relevant physiologic role in the thymus-hypothalamo-pituitary-gonadal axis.

Peripheral and mesencephalic transfer of a synthetic gene for the thymic peptide thymulin.

Thymulin is a thymic peptide with antiinflammatory activity in the brain. We constructed a recombinant adenoviral vector, RAd-FTS, expressing a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin and used it for peripheral and central gene transfer in rats. Thymulin concentration in serum and brain tissue was determined by bioassay. Reporter gene expression in the substantia nigra (SN) was quantitated by enzymohistochemistry or fluorescence microscopy using an appropriate image analysis software. A single intramuscular injection (10(8) plaque forming units (pfu)/animal) of RAd-FTS in thymectomized rats (nondetectable serum thymulin) induced supraphysiologic serum thymulin levels for at least 110 days (123+/-22 fg/ml versus 598+/-144 fg/ml in intact and vector-injected rats, respectively). Stereotaxic intranigral injection of RAd-FTS induced steady expression levels of met-FTS for at least 90 days, whereas expression of adenovirally transferred reporter genes coding for green fluorescent protein fused to HSV thymidine kinase (GFP-TK)(fus) or E.coli beta-galactosidase (beta-gal), declined drastically within a month (% transgene expression in the SN on post-injection day 30 relative to day 2 was: 18, <1 and 125%, for beta-gal, (GFP-TK)(fus) and met-FTS, respectively). We conclude that RAd-FTS constitutes a suitable biotechnological tool for the assessment of peripheral and central thymulin gene therapy in animal models of nigral dopaminergic neurodegeneration induced by pro-inflammatory agents.