Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice

Abstract

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitary-gonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5'-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3') encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymus-pituitary-gonadal axis.

Fig. 1

DNA constructs encoding the thymulin analog methionine-FTS (metFTS) and a recombinant adenoviral (RAd) vector for metFTS (RAd-metFTS). A DNA sequence coding for native serum thymus factor (FTS) was designed for optimal expression in rat cells. By adding an ATG starting codon upstream and 2 stop codons downstream of this sequence, we converted it into an open reading frame (ORF) for the analog metFTS (A). This metFTS ORF was used to generate a construct to be cloned in the shuttle vector pDC515. The construct included the phage T7 promoter primer binding site, which was used for sequencing purposes (B). The shuttle pDC515-metFTS was generated by inserting the T7-metFTS sequence into the BamHI-SalI sites of the multiple cloning site of the shuttle pDC515. This construct was used to generate RAd-metFTS (C). For further details, see MATERIALS AND METHODS and Ref. 25. PmCMV, mouse cytomegalovirus promoter; frt, recognition element for the yeast FLP recombinase; ITR, inverted terminal repeats; ΔE1 and ΔE3, deletions in the Ad5 genome; SV40, simian virus 40 polyadenylation signal; Ψ, packaging signal.

Fig. 2

Effect of long-term thymulin quenching on gonadotropin serum levels in mice. Experimental animals (shaded bars) received weekly intraperitoneal injections of rabbit anti-FTS serum beginning at birth. Control mice (solid bars) received normal rabbit serum. LH, luteinizing hormone; FSH, follicle-stimulating hormone. All mice were killed at 33 days postinjection. Numbers over bars represent the number of mice assessed for the corresponding data point. Inset: in vivo immunoneutralization of serum thymulin in mice. Effects of a single intraperitoneal injection of undiluted rabbit anti-FTS serum (8 μl/g body wt) on thymulin serum level in 15-day-old mice were determined. Error bars represent SE. *P < 0.05; **P < 0.01, significance of differences between control and experimental mice.

Fig. 3

Short-term immunoneutralization of serum thymulin in mice. Experimental animals (shaded bars) were intraperitoneally injected at postnatal days 1 and 2 with rabbit anti-FTS serum, which was followed by 2 intraperitoneal injections of synthetic thymulin (FTS-Zn; 60 pg/g body wt) on postnatal days 8 and 9. Control mice (solid bars) were submitted to the same treatment except that they received a rabbit anti-keyhole limpet hemocyanin (KLH) serum instead of anti-FTS. Asterisks represent significance of differences between control an experimental mice at each time point.

Fig. 4

Effect of short-term thymulin quenching on gonadotropin serum levels in mice. Short-term thymulin quenching was performed as described in Fig. 3. Control mice received anti-KLH rabbit serum, whereas experimental mice received rabbit anti-FTS serum. All mice were killed at 45 days of age. Other details are as described in Fig. 2. **P < 0.01, significance of differences between control and experimental mice.

Fig. 5

Impact of short-term serum thymulin quenching on gonadotropic cell morphology in C57BL/6 mice. Mice were submitted to short-term thymulin immunoneutralization as described in Fig. 3 and MATERIALS AND METHODS. Mice were killed at 45 days of age. Pituitary sections were submitted to immunohistochemistry for LH and FSH (see MATERIALS AND METHODS). A trend toward hypertrophy and a decrease in numbers in gonadotrophs can be noticed in the representative experimental (EXP) sections shown compared with the control (CTR) counterparts. Scale bar, 25 μm.

Fig. 6

Effect of neonatal thymulin gene therapy on serum levels of thymulin in heterozygous (nu/+) and homozygous (nu/nu) nude mice. Vectors were intramuscularly injected on the day of birth or the day after (arrow). At postnatal day 13, 3 mice per group were bled and serum thymulin was assayed. On postnatal days 51 and 52, the remainder of the animals were bled and thymulin was assayed. Since the volume of serum that can be obtained from newborn mice is exceedingly small, thymulin could not be reliably determined on postnatal days 1 and 2. Serum thymulin values (fg/ml) are expressed as means ± SE; n values for data points at days 51 and 52 are shown above symbols. **P < 0.01, significance of differences between RAd-GFP/TK-injected animals and RAd-metFTS-injected counterparts.

Fig. 7

Effect of neonatal thymulin gene therapy on serum LH and FSH in nude mice. The indicated vectors were intramuscularly injected at birth, and the animals were bled 51-52 days afterward for serum LH and FSH determination. Other details are as described in Fig. 2. *P < 0.05; **P < 0.01, significance of differences between RAd-GFP/TK-injected animals and RAdmetFTS-injected counterparts.