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

Abstract

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.

Fig. 1.

Recombinant adenovectors expressing metFTS (thymulin) and GFP. A, A DNA sequence coding for the thymulin analog metFTS was cloned in a first generation adenovector backbone, thus generating RAd-FTS. B, A DNA sequence encoding a chimeric variant of enhanced GFP (GFP/TK) was cloned in an adenovector thus generating RAd-GFP, a control vector. PmCMV, Mouse cytomegalovirus promoter; T7, phage T7 promoter primer binding site; ITR, inverted terminal repeats; ΔE1 and ΔE3, deletions in the Ad5 genome; Sv40pA, simian virus 40 polyadenylation signal; ψ, packaging signal.

Fig. 2.

GnRH-producing neuron numbers in the brain of control and experimental hetero and homozygous nude females. GnRH perikarya were counted in one every four coronal sections of the whole brain, and the sum of all counted sections per mouse was multiplied by 4 (thus, values represent an estimate of total brain GnRH-producing neurons). Numbers above columns are in the format (N;CV) and represent the N value and the coefficient of variation per group, respectively. Asterisks refer to differences vs. corresponding control nu/nu; **, P < 0.01; *, P < 0.05.

Fig. 3.

Effect of thymulin gene therapy on ovarian morphology in nude mice. H&E-stained sections of ovaries from nu/+ (A) and nu/nu (C) mice treated with the control vector (RAd-GFP) and nu/+ (B) and nu/nu (D) mice submitted to NTGT. Scale bar, 200 μm.

Fig. 4.

Histomorphometric assessment of ovaries from control and experimental nude mice. The number of antral (secondary and tertiary) and atretic follicles (Fol.) as well as corpora lutea was assessed in the ovaries from nu/+ and nu/nu mice treated with the control vector (RAd-GFP) and nu/+ and nu/nu mice submitted to NTGT. Asterisks refer to differences vs. corresponding control nu/nu; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Other details are as in Fig. 2.

Fig. 5.

Serum E₂ and P₄ levels in control and experimental nude mice. Steroids were measured in the serum of RAd-GFP- and RAd-FTS-treated heterozygous and homozygous female nude mice. Asterisks refer to differences vs. corresponding control nu/+; **, P < 0.01. Serum E₂ levels in metFTS gene-treated nu/nu and control nu/+ mice did not differ significantly (Tukey test). Other details are as in Fig. 2.

Fig. 6.

Effect of thymulin gene therapy on the age of vaginal opening in nude mice. The age of vaginal opening was assessed in RAd-GFP- and RAd-FTS-treated heterozygous and homozygous nude mice. Asterisks refer to differences from corresponding control nu/nu; ***, P < 0.001; *, P < 0.05. Other details are as in Fig. 2.