Busulfan administration replicated the characteristics of the epididymal initial segment observed in mice lacking testis-epididymis lumicrine signaling

Abstract

The physiological functions of the mammalian epididymis are typically regulated by the testes. In addition to sex steroids secreted by testicular Leydig cells, which act on the epididymis in an endocrine manner, there is a non-sex-steroidal signaling pathway known as the lumicrine pathway. This lumicrine signaling pathway involves ligand proteins secreted from germ cells within the testicular seminiferous tubules traversing the male reproductive tract, which induce epithelial differentiation in the epididymis. These findings prompted an inquiry into whether treatments influencing testis physiology can disrupt epididymal function by interfering with testis-epididymis communication. Busulfan, an alkylating agent commonly used to deplete testicular germ cells in reproductive biology, has not been sufficiently explored because of its effects on the epididymis. This study investigated the effects of busulfan administration on the proximal epididymis using histological and transcriptomic analyses. Notably, busulfan, as opposed to the vehicle dimethyl sulfoxide (DMSO), altered the morphology of the initial segment of the epididymis, leading to a reduction in the cell height of the luminal epithelium. RNA sequencing identified 185 significantly downregulated genes in the proximal epididymis of busulfan-administered mice compared to DMSO-administered mice. Comparative transcriptome analyses revealed similarities between the epididymal transcriptome of busulfan-administered mice and lumicrine-deficient mice, such as efferent-duct-ligated W/Wv and Nell2^-/- mice. However, this differed from that of bilaterally orchidectomized mice, in which both the endocrine and lumicrine signaling pathways were simultaneously ablated. Collectively, these results suggested that the harmful effects of busulfan on the proximal epididymis are secondary consequences of the ablation of testis-epididymis lumicrine signaling.

Keywords: Busulfan; Epididymis; Gene expression; Initial segment; Lumicrine.

Fig. 1.

Histology of the initial segment of the epididymis of busulfan-treated mice. A–G, Hematoxylin and eosin (HE)-stained sections of IS-caput of the epididymides from wild-type (WT) (A), dimethylsulfoxide (DMSO)-treated (B), and busulfan-treated (C) mice with efferent duct ligation (EDL) (D), W/Wv mice (E), Nell2^-/- mice (F), and bilaterally orchidectomized mice (G). L, luminal epithelium; I, interstitial tissue. Bars, 100 μm. (H) Average cell height of the initial segment (IS) of the luminal epithelium. All values are shown as the mean ± standard error of the mean (n = 3). The results of one-way analysis of variance were: F(6, 14) = [36.797], P = 8.67E-08. The P values of the two-tailed Student’s t-test are also shown. BOD, bilateral orchidectomy. N.S., not significant.

Fig. 2.

RNA sequencing analyses of the IS-caput of the epididymis of DMSO- or busulfan-treated mice. A–C, RNA sequencing of the IS-caput of the epididymis from untreated WT vs. DMSO-treated (A), untreated WT vs. busulfan-treated (B), and DMSO-treated vs. busulfan-treated (C) mice. Fragments per kilobase of exon per million mapped reads (FPKM) values are plotted. Statistically significantly downregulated (fold change < 0.1, and Student’s t-test P < 0.05) and upregulated (fold change > 10, and Student’s t-test P < 0.05) genes are represented in green and yellow, respectively. (D) The fold change in gene expression levels in DMSO-treated vs. untreated WT mice (n = 3) and busulfan-treated vs. untreated WT mice (n = 3). Green and magenta represent downregulation and upregulation, respectively.

Fig. 3.

Comparative representation of genes downregulated in IS-caput of epididymides by busulfan treatment and other experimental treatments. Fold change in gene expression levels in the IS-caput of the epididymis compared between busulfan-injected mice, mice with EDL, W/Wv mice, Nell2^-/- mice, and bilaterally orchidectomized mice. Green and magenta represent downregulation and upregulation, respectively. BOD, bilateral orchidectomy.

Fig. 4.

Protein expression levels in the IS-caput of the epididymis of busulfan-treated and other experimentally treated mice. Immunoblot analyses of lumicrine-signaling-associated proteins ADAM28, OVCH2, and RNASE10 and endocrine-signaling-associated proteins GPX5 and SPAG11B in IS-caput epididymal lysates from untreated WT mice, busulfan-treated mice, mice with EDL, W/Wv mice, and Nell2^-/- mice, and bilaterally orchidectomized (BOD) mouse. GAPDH immunodetection is also shown as an internal control.

Fig. 5.

A scheme representing the possible mechanism of action of busulfan on the IS of the epididymis. Lumicrine signaling is interfered with indirectly by busulfan administration, as a secondary consequence of testicular germ cell ablation. The endocrine action by Leydig cells appears to be unaffected by busulfan administration. The direct action of busulfan on the IS of the epididymis is also possible, although it is currently uncertain whether such an action causes IS defects.