Haploid-specific transcription of protamine-myc and protamine-T-antigen fusion genes in transgenic mice.

Abstract

The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.During spermatogenesis in mammals, the histones are ultimately replaced by protamines, which are small, basic proteins containing 40 to 70% arginine (for reviews, see references 6, 17, and 18). This change occurs during the late-haploid phases of male germ cell differentiation and produces a DNA-protamine complex that is compact and genetically inactive. The mammalian protamines are translated from RNA transcripts that are detectable only in the haploid cells of the testes.The nuclei of all mammalian sperm examined contain the P1 protamine, and a comparison of the complete sequence analysis of P1 protamines from several species has revealed identical lengths of 50 amino acids and strong sequence homologies (2, 10, 22-25, 28, 29, 35, 38). However, in mice and hamsters there are at least two distinct protamines, and in humans there are three protamine variants (2,3,24,25,27,30). The mouse P1 protamine, mPl, is similar to the P1 protamines of other mammalian species and is synthesized as the mature protein (22). The mouse P2 protamine, mP2, is distinguished from mPl by its amino acid composition: mPl has no histidine residues, whereas mp2 has 13 histidine residues (41). In addition, mP2 is synthesized as a larger protein of 106 amino acids that is processed into the mature protein of 63 amino acids found in spermatozoa (2, 41).The genes for the mouse protamines are present as singlecopy genes on chromosome 16 (19). They are transcribed only in the testis; the mRNAs are first detected in the round spermatids, the first haploid precursors of mature spermatozoa (20,21). These stable, very abundant mRNAs are then stored for up to 8 days before being translated in the elongating spermatids. A shortening of the poly(A) tail coincides with the presence of the mRNAs on polysomes (21). Mouse spermatids contain the two variant protamines in a ratio of approximately 1:1, but in the maturation process the ratio changes to 2:1 (mP2 to mPl) for the mature sperm (3). Whether these changes ...

An upstream region of the mouse ZP3 gene directs expression of firefly luciferase specifically to growing oocytes in transgenic mice.

“…During a period of 2-3 weeks, as oocytes arrested in dictyate of the first meiotic prophase increase 300-fold in volume, ZP3 mRNA increases from undetectable levels in nongrowing oocytes to "250,000 copies per fully grown oocyte (6,15). During this same period, ZP3 synthesis increases in growing oocytes from undetectable levels to as much as 2-3% of total protein synthesis (1,22 Results reported here on the ZP3/luciferase transgene provide additional support for oocyte-specific expression of the ZP3 gene and complement previous reports of germ cell-specific expression of transgenes in males (23)(24)(25). We have found that an upstream region of the mouse ZP3 gene directs expression of a reporter gene (that encoding firefly luciferase) specifically to growing oocytes of transgenic mice harboring the ZP3/luciferase construct.…”

Human protamines and male infertility

“…However, patients possessing a high percentage of mature sperm with single-stranded DNA are still capable of fertilization, as Ab patients had a median fertilization rate of 25.0%. Previous studies have supported this finding (23,36)° Studies carried out on animals which possess the P2 gene but produce no or low levels of protamine 2 (12,13,38,39) have suggested that reduction in either P 1 or P2 may be explained by either reduced protamine transcription and translation or unstable products as a result of failed posttranscriptional modification. Lack of P2 in the boar has been shown to be due to mutations within the P2 gene (31).…”

Analysis of the mouse protamine 1 promoter in transgenic mice.

“…Transgenic approaches have been used to demonstrate that as little as 465 bp of Prm-1 5' flanking sequence is sufficient to confer spermatid-specific expression on a marked Prm-1 gene (15,16). Likewise, when 859 bp of Prm-2 promoter sequence was fused to a c-myc gene, transcripts from this construct were detected only in round spermatids (17). Recently, in vitro transcription studies have been carried out on both mouse protamine genes (18,19).…”