Elimination of Male Germ Cells in Transgenic Mice by the Diphtheria Toxin A Chain Gene Directed by the Histone H1t Promoter1

Abstract

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Characterization of the H1t Promoter: Role of Conserved Histone 1 AC and TG Elements and Dominance of the Cap-Proximal Silencer1

“…It shares all known regulatory elements of standard H1 genes, yet instead of widespread expression during the S phase, its mRNA is present in significant amounts only during the later part of the protracted G1 phase in primary spermatocytes. Trace expression of H1t mRNA reported in early spermatocytes or spermatogonia of mice [41] agrees with results from experiments in which H1t-directed transgene expression was inferred as early as the first type A spermatogonia [28]. However, significant levels of H1t do not accumulate until late in meiosis [7,[10][11][12][13][14][15][16].…”

“…We did not observe a distinct silencer activity in this region, and it may be that separate silencer elements are active in specific cell lines. We know from transgenic experiments that the Ϫ948 promoter region generates germ cell-specific expression with both lacZ and diphtheria toxin A chain reporters [27,28]. Therefore, silencers that are sufficient to repress H1t completely in non-germ line cells must lie downstream of Ϫ948.…”

“…Transgenic analysis demonstrated that the rat H1t gene will express correctly in mice [26,27], and that as little as 141 base pairs (bp) of the rat promoter is sufficient to direct correct expression of the H1t coding region [27]. More recently, it was shown that a 1-kilobase upstream sequence directs expression of the lethal diphtheria toxin A chain gene exclusively to the male germ line [28].…”

RFX2 Is a Potential Transcriptional Regulatory Factor for Histone H1t and Other Genes Expressed During the Meiotic Phase of Spermatogenesis1

“…The tightness of its transcriptional regulation was demonstrated by a transgenic study in which an H1t promoterdiphtheria toxin fusion gene led to complete elimination of germ cells in males, but had no other apparent effect [13]. This tight regulation is made more intriguing because the H1t promoter region is strikingly similar to those of the 5 standard H1 variants expressed ubiquitously in somatic cells [14][15][16][17].…”

Cell‐specific ablation in the testis: what have we learned?

“…2). In contrast, females are completely fertile (Bartell et al ., 2000). Although the testes in these animals are extremely small, seminal vesicle weights are normal, suggesting that loss of germ cells does not impact androgen production.…”