Transgenic Mice Over-expressing Endothelin-1 in Testis Transactivated by a Cre/loxP System Showed Decreased Testicular Capillary Blood Flow

Abstract

It is generally believed that too high or low levels of endothelin-1 (ET-1), a strong vasoconstrictor, may be detrimental to animals. Therefore, in order to understand the in vivo function of ET-1, we used a conditional transgenic approach, Cre/loxP recombination system, to generate transgenic mice that over-express ET-1 in a tissue-specific manner. In such a strategy a single transgenic mouse line, ELSE, was initially generated where a general promoter, human elongation factor 1alpha (hEF1alpha) promoter, was used to drive the expression of a loxP-flanked sequence containing the lacZ reporter gene and a STOP cassette before the ET-1 cDNA, the recombinational competency of which was confirmed in an Escherichia coli test system. In ELSE mice, expression of the reporter lacZ was limited to spermatozoa and spermatogonia as well as Sertoli, Leydig and endothelial cells in the testis, thus confirming the suitability of these mice for the generation of testes-limited ET-1 expression. To generate transgenic progeny with ET-1 over-expression in the testis (successful recombination, ELSE/ELT), ELSE mice were mated with EIIa-cre mice expressing Cre recombinase in pre-implantation mouse embryos. These ELSE/ELT mice exhibiting testis-specific ET-1 over-expression had normal reproductive function and showed no obvious alterations in gross testicular morphology. Although over-expression of ET-1 leads to reduction of testicular blood flow, young adult ELSE/ELT mice showed no obvious signs of inflammation, fibrosis or abnormal proliferation of cells in the testes of young ELSE/ELT mice by histochemical analyses.

A Non-Specific Effect Associated with Conditional Transgene Expression Based on Cre-loxP Strategy in Mice

“…This has become the basic assumption since the Cre-loxP technology was first applied to conditionally express transgenes in mice in 1992 [1], [2]. In recent years, the use of loxP-containing transgenes in transgenic mice has increased but the possibility of unintended effects of this approach has not been taken into consideration [5], [7], [8], [9], [10], [11], [12], [13], [14], [37], [38], [39], [40], [41]. Our data demonstrate that, contrary to the widely held assumption that transgenes are integrated into the genome in a head-to-tail tandem array [5], [7], [8], [9], [10], [11], [12], [13], [14], [28], [29], [30], [37], [38], [39], [40], [41], inverted transgene integration is common when multiple copies of the transgene are integrated into the genome.…”

The “Hitchhiker’s Guide to the Galaxy” of Endothelial Dysfunction Markers in Human Fertility

“…Although these data demonstrated that ET-1 is involved in the regulation of human fertility, no evidence is currently available suggesting a physiological direct relationship between ET-1 and parameters indicative of sperm functions [190]. A genetically modified mouse model with ET-1 overexpression had reduced testicular blood flow, but neither increased inflammatory status, nor impaired cell proliferation [184]. Hypothetically, ET-1 dysfunction could impair sperm progression through the genital tract, negatively impacting the viability of gametes in the ejaculate.…”

“…In the male genital tract, endothelin-1 (ET-1) is detectable at testicular, epididymal and prostatic levels both in human and in rat males [184,185]. Within the testis, ET receptors were mainly found in Leydig cells, in which they promote testosterone secretion [186] In Sertoli cells, ET receptors are involved in micro-tubule contractility [187].…”