Protamines are the major DNA-binding proteins in the nucleus of sperm in most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. Many mammals have one protamine, but a few species, including humans and mice, have two. Here we use gene targeting to determine if the second protamine provides redundancy to an essential process, or if both protamines are necessary. We disrupted the coding sequence of one allele of either Prm1 or Prm2 in embryonic stem (ES) cells derived from 129-strain mice, and injected them into blastocysts from C57BL/6-strain mice. Male chimeras produced 129-genotype sperm with disrupted Prm1 or Prm2 alleles, but failed to sire offspring carrying the 129 genome. We also found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function. Our studies show that both protamines are essential and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.

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Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.

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MicroRNAs play important roles in regulating development at both transcriptional and posttranscriptional levels. Here, we report 29 microRNAs from mouse testis that are differentially expressed as the prepubertal testis differentiates to the adult testis. Using computational analyses to identify potential microRNA target mRNAs, we identify several possible male germ cell target mRNAs. One highly conserved sequence in the 3'-untranslated region (UTR) of transition protein 2 (Tnp2) mRNA, a testis-specific and posttranscriptionally regulated mRNA in postmeiotic germ cells, is complementary to Mirn122a. Mirn122a is enriched in late-stage male germ cells and is predominantly on polysomes. Mirn122a, but not another noncomplementary microRNA, inhibits the activity of a luciferase reporter construct containing the 3'-UTR of Tnp2. Site-directed mutations of Mirn122a indicate that base pairing of the 5'-region of Mirn122a to its complementary site in the 3'-UTR of Tnp2 mRNA is essential for the downregulation of luciferase activity. Real-time reverse transcription-polymerase chain reaction and ribonuclease protection assays reveal that the Mirn122a-directed decrease of the Tnp2 reporter gene activity results from mRNA cleavage. We propose that specific microRNAs, such as Mirn122a, could be involved in the posttranscriptional regulation of mRNAs such as Tnp2 in the mammalian testis.

T he testis contains a diverse population of somatic and germ cell types. As spermatogenesis proceeds, diploid spermatogonia differentiate into meiotic spermatocytes, which divide twice without additional DNA replication, producing haploid round spermatids (1, 2). These spermatids transform into highly polarized and uniquely shaped spermatozoa. As the germ cells differentiate, the changing amounts and populations of mRNAs in the germ cells and somatic cells have been well documented by microarray analyses (3-7) and by the cloning and sequencing of cDNA libraries prepared from highly purified populations of individual cell types (8,9).Although these microarray and cloning studies provide valuable insight into the temporal appearance͞disappearance of individual mRNAs, they do not address the question of when the proteins encoded by the mRNAs are synthesized. In the germ cells of the testis, a temporal disconnect between mRNA transcription and protein synthesis is especially common, in part because RNA synthesis terminates during midspermiogenesis long before the spermatid completes its differentiation into the spermatozoon (1). Thus, posttranscriptional mechanisms play major roles in the temporal regulation of protein synthesis in developing male gametes.

Here we report the isolation and characterization of mouse testicular cDNAs encoding the mammalian homologue of the Xenopus germ cell-specific nucleic acid-binding protein FRGY2 (mRNP3+4), hereafter designated MSY2. MSY2 is a member of the Y box multigene family of proteins; it contains the cold shock domain that is highly conserved among all Y box proteins and four basic/aromatic islands that are closely related to the other known germline Y box proteins from Xenopus, FRGY2, and goldfish, GFYP2. Msy2 undergoes alternative splicing to yield alternate N-terminal regions upstream of the cold shock domain. Although MSY2 is a member of a large family of nucleic acid-binding proteins, Southern blotting detects only a limited number of genomic DNA fragments, suggesting that Msy2 is a single copy gene. By Northern blotting and immunoblotting, MSY2 appears to be a germ cell-specific protein in the testis. Analysis of Msy2 mRNA expression in prepubertal and adult mouse testes, and in isolated populations of germ cells, reveals maximal expression in postmeiotic round spermatids, a cell type with abundant amounts of stored messenger ribonucleoproteins. In the ovary, MSY2 is present exclusively in diplotene-stage and mature oocytes. MSY2 is maternally inherited in the one-cell-stage embryo but is not detected in the late two-cell-stage embryo. This loss of MSY2 is coincident with the bulk degradation of maternal mRNAs in the two-cell embryo.

MSY2, a germ-cell-specific member of the Y-box family of DNA-͞ RNA-binding proteins, is proposed to function as a coactivator of transcription in the nucleus and to stabilize and store maternal and paternal mRNAs in the cytoplasm. In mice lacking Msy2, a normal Mendelian ratio is observed after matings between heterozygotes with equal numbers of phenotypically normal but sterile male and female homozygotes (Msy2 ؊/؊ ). Spermatogenesis is disrupted in postmeiotic null germ cells with many misshapen and multinucleated spermatids, and no spermatozoa are detected in the epididymis. Apoptosis is increased in the testes of homozygotes, and real-time RT-PCR assays reveal large reductions in the mRNA levels of postmeiotic male germ cell mRNAs and smaller reductions of meiotic germ cell transcripts. In females, there is no apparent decrease in either the number of follicles or their morphology in ovaries obtained from 2-and 8-day-old Msy2 ؊/؊ mice. In contrast, follicle number and progression are reduced in 21-day-old Msy2 ؊/؊ ovaries. In adult Msy2 ؊/؊ females, oocyte loss increases, anovulation is observed, and multiple oocyte and follicle defects are seen. Thus, Msy2 represents one of a small number of germ-cell-specific genes whose deletion leads to the disruption of both spermatogenesis and oogenesis.he highly conserved family of Y-box proteins, expressed in organisms ranging from bacteria to humans, contains a cold-shock domain essential for nucleic-acid binding and variable N and C termini that confer binding specificity (1). As DNA-binding proteins, Y-box proteins serve as transcription coactivators, recognizing DNA motifs such as CTGATTGGC͞ TC͞TAA (2). As RNA-binding proteins, Y-box proteins help stabilize mRNAs and, depending on their concentration, can inhibit or stimulate mRNA translation (3, 4).Among DNA-͞RNA-binding proteins, the mouse Y-box protein MSY2 is one of the most abundant, constituting 0.7% ( Fig. 1) and 2% of total protein in male germ cells and fully grown oocytes, respectively (5, 6). MSY2 is the mouse ortholog of the Xenopus laevis FRGY2 (2) and human Contrin proteins (7), Y-box proteins proposed to be solely expressed in germ cells (8). In the testis, Msy2 is expressed in meiotic and postmeiotic germ cells, where it is believed to function in long-term mRNA storage and stabilization because cessation of transcription in postmeiotic germ cells necessitates posttranscriptional regulation for many mRNAs encoding late-stage germ cell and spermatozoan proteins (8). In addition, MSY2 marks specific mRNAs in the nucleus for storage in the cytoplasm, providing a linkage between transcription and mRNA storage for a subset of male germ cell mRNAs (9).In the female, MSY2 protein accumulates during oocyte growth, but after fertilization, it is totally degraded by the late two-cell stage (6). As in postmeiotic male germ cells, MSY2 is located in the cytoplasm in oocytes, but, in contrast to male germ cells, where MSY2 is soluble, Ϸ70% of MSY2 is retained after permeabilization procedures that release Ͼ70...