Levonorgestrel enhances spermatogenesis suppression by testosterone with greater alteration in testicular gene expression in men

Abstract

Prior studies have demonstrated that combined treatment of testosterone with a progestin induces a more rapid and greater suppression of spermatogenesis than testosterone treatment alone. We hypothesized that the suppressive effects of the combination of testosterone undecanoate (TU) injections plus oral levonorgestrel (LNG) on spermatogenesis may be mediated through a greater perturbation of testicular gene expression than TU alone. To test this hypothesis, we performed open testicular biopsy on 12 different adult healthy subjects: 1) four healthy men as controls; 2) four men 2 wk after TU treatment; and 3) four men 2 wk after TU + LNG administration. RNA isolated from biopsies was used for DNA microarray using the Affymetrix Human Genome U133 Plus 2.0 oligonucleotide microarrays. Gene expression with >or=2-fold changes (P < 0.05) compared with control was analyzed using the National Institutes of Health Database for Annotation, Visualization, and Integrated Discovery 2008 resource. The TU treatment altered the gene expression in 109 transcripts, whereas TU + LNG altered the gene expression in 207 transcripts compared with control. Both TU and TU + LNG administration suppressed gene expression of insulin-like 3; cytochrome P450, family 17, subfamily A1 in Leydig cells; and inhibin alpha in Sertoli cells; they increased proapoptotic transcripts BCL2-like 14, insulin-like growth factor-binding protein 3; and they decreased X-linked inhibitor of apoptosis protein. In comparison with TU treatment alone, TU + LNG treatment upregulated insulin-like 6 and relaxin 1, and downregulated RNA-binding protein transcripts. We conclude that TU + LNG administration induces more changes in testicular gene expression than TU alone. This exploratory study provided a novel and valuable database to study the mechanisms of action of hormonal regulation of spermatogenesis in men and identified testicular-specific molecules that may serve as potential targets for male contraceptive development.

Fig. 1

Global gene expression distribution of each testicular RNA sample by principal component analysis (PCA), a vector space transformation method used to reduce multidimensional data sets to lower dimensions for visualization and analysis. The three principal components accounted for x-axis, 22.41%; y-axis, 14.29%; and z-axis, 11.4% of total variance, respectively. Principal component analysis showed that TU- and TU + LNG-treated samples were clustered distinguishably different from the control samples.

Fig. 2

Hierarchical clustering analysis showing the statistically significant (P < 0.05) transcripts with equal or more than 2-fold changes in gene expression in control and 2 wk after TU alone or TU + LNG treatment. Red color represents a high number of transcript expression. Green color represents a low number of transcript expression. Note the apparently greater alteration of testicular gene expression in TU + LNG than in TU treatment alone compared with controls.

Fig. 3

Venn diagram shows that TU treatment alone caused 51 gene changes that were different from 149 gene changes in TU+LNG treatment; whereas, there were 58 common gene changes shared by both TU and/or TU+LNG treatment. When compared with control, TU+LNG treatment caused more changes of testicular gene expression than TU alone.

Fig. 4

The expressions of Leydig cell-specific gene INSL3 (A) and Sertoli cell-specific gene INHA (B) were suppressed by both TU and TU + LNG treatment. Asterisks indicate significant difference (P < 0.05) from control (Con). Error bars indicate standard errors of normalized signal intensity.

Fig. 5

Representative light micrographs showing immunostaining of IGFBP3 on testicular sections from control (A), 2 wk after TU alone (B), TU + LNG treatment (C), and negative control (D). IGFBP3 was localized abundantly in Leydig cells (asterisks), moderately in Sertoli cells (red arrows), and spermatocytes and round spermatids (yellow arrows). After TU or TU + LNG treatment, IGFBP3 expression appeared to be increased in Sertoli cells. Original magnification ×250; bar = 20 μm.