Transition nuclear proteins (TPs), the major proteins found in chromatin of condensing spermatids, are believed to be important for histone displacement and chromatin condensation during mammalian spermatogenesis. We generated mice lacking the major TP, TP1, by targeted deletion of the Tnp1 gene in mouse embryonic stem cells. Surprisingly, testis weights and sperm production were normal in the mutant mice, and only subtle abnormalities were observed in sperm morphology. Electron microscopy revealed large rod-like structures in the chromatin of mutant step 13 spermatids, in contrast to the fine chromatin fibrils observed in wild type. Steps 12–13 spermatid nuclei from the testis of Tnp1-null mice contained, in place of TP1, elevated levels of TP2 and some protamine 2 (P2) precursor. Most of the precursor was processed to mature P2, but high levels of incompletely processed forms remained in epididymal spermatozoa. Sperm motility was reduced severely, and ≈60% of Tnp1-null males were infertile. We concluded that TP1 is not essential for histone displacement or chromatin condensation. The absence of TP1 may partially be compensated for by TP2 and P2 precursor, but this dysregulation of nucleoprotein replacement results in an abnormal pattern of chromatin condensation and in reduced fertility.

https://www.pnas.org/content/pnas/97/9/4683.full.pdf

Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice

The expanding family of CREB/CREM transcription factors that are involved with spermatogenesis.

Male germ-cell differentiation requires spermatogenic stage- and cell-specific gene expression that is achieved by unique chromatin remodeling, transcriptional control and the expression of testis-specific genes or isoforms. Recent findings have shown that the testis has specialized transcription complexes that coordinate the differentiation program of spermatogenesis. There are male germ cell-specific differences in the components of the general transcription machinery. These include upregulated expression of the TATA-binding protein (TBP) family and its associated cofactors. Importantly, a member of the TBP family, TBP-like factor (TLF), has a distribution pattern that is dependent on the spermatogenic cycle and is essential for spermatogenesis. Interestingly TBP-associated factor (TAF7), a factor of the transcription factor (TF)IID complex, is exchanged at a critical stage in germ cell development for the testis-specific paralogue TAF7L. A compelling amount of data has established that cAMP-response-element modulator (CREM), a transcription factor responsive to the cAMP signal transduction pathway, drives expression of key testis-specific genes. In this review we summarize recent advances in the transcription machinery that is testis-specific, gene-selective and necessary for the process of spermatogenesis.

https://rep.bioscientifica.com/downloadpdf/journals/rep/128/1/1280005.pdf

Testis-specific transcription mechanisms promoting male germ-cell differentiation.

During spermiogenesis, DNA-binding histones are replaced by protamines. DNA-protamine-interactions result in chromatin condensation causing cessation of transcription in elongating spermatids. Thus, in haploid spermatids, processes of transcription and translation need to be temporally uncoupled in order to ensure protein synthesis in transcriptionally silent germ cells. Post-transcriptional events, such as processing, transport, and storage of mRNAs, therefore, play important roles in determining when transcripts become functionally available for translation. This review focuses on mechanisms of translational regulation in haploid spermatids and male infertility caused by defects of translational control processes.

Haploid spermatids exhibit translationally repressed mRNAs.

Histone-to-protamine exchange in haploid spermatids is preceded by hyperacetylation of core histones resulting in decreased DNA-histone interaction. During normal spermatogenesis, immunohistochemistry with a polyclonal antihyperacetylated histone H4 antibody displayed a strong signal in nuclei of elongating spermatids and, in addition, spermatogonia. Quantitative analysis revealed 98.2 +/- 1.1% of immunopositive spermatids. The percentage of positive spermatids was significantly reduced in infertile men exhibiting at least qualitatively normal spermatogenesis (scores 10-8, 93.1 +/- 6.6%) and impaired spermatogenesis (scores 7-1, 74.9 +/- 23.4%). In seminiferous tubules showing spermatogenic arrest at the level of round spermatids, only 59.5 +/- 16.5% of spermatids were immunopositive for hyperacetylated histone H4. These data demonstrate that the decrease of histone acetylation in spermatids associated with impaired spermatogenesis corresponds with the well known reduction of protamine expression in these cells and confirms the essential role of histone hyperacetylation for correct histone-to-protamine exchange. In seminiferous tubules exhibiting round spermatid maturation arrest, there was an additional signal in nuclei of spermatocytes, suggesting that premature hyperacetylation of histone H4 may result in precocious histone-to-protamine exchange followed by infertility. This is in accordance with data from transgenic mice, where it has been demonstrated that premature expression of protamine-1 results in precocious chromatin condensation followed by sterility.

Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis

During spermiogenesis, histones are replaced by transition proteins, which in turn are replaced by protamines. The TNP1 gene-encoding TP1 (transition protein 1) protein contains a cAMP-responsive element (CRE) that serves as binding site for the CRE modulator (CREM). To gain further insight into the complex regulation of nucleoprotein exchanges in haploid spermatids and its potential role for spermatogenic impairment, we studied the gene expression of testis-specific histone H1t, CREM, and TNP1 in testicular biopsies from men with normal spermatogenesis (n = 20) and with round spermatid maturation arrest (n = 16). During normal spermatogenesis, H1t messenger RNA (mRNA) was present in 86.2%+/-8.7% of pachytene spermatocytes (stages III-V), whereas H1t protein was synthesized in 83.5%+/-13.0% of pachytene spermatocytes (stages III-V) and persisted in 95.2%+/-3.1% of spermatids (steps 1-5). CREM mRNA was detectable in 74.2%+/-9.4% of pachytene spermatocytes (stages IV-V) and in 78.7%+/-10.0% of spermatids (steps 1-3). CREM protein was synthesized in 81.2%+/-14.2% of spermatids (steps 1-3). TNP1 mRNA was present in 80.0%+/-13.5% of spermatids (steps 2-4), whereas TP1 protein was synthesized in 89.7%+/-5.3% of spermatids (steps 3-4). In men with round spermatid maturation arrest, spermatids only develop to step 3 of differentiation. These spermatids were devoid of both CREM and TP1 but did contain H1t. These results indicate that TP1 is likely to be an important parameter in the histone-to-protamine exchange and in the initiation of spermatid elongation. CREM is involved in the regulation of TNP1 gene expression and consequently plays a vital role in the correct differentiation step from round spermatids to mature spermatozoa.

Round spermatids show normal testis-specific H1t but reduced cAMP-responsive element modulator and transition protein 1 expression in men with round-spermatid maturation arrest.

Volker Schaller

Papers

Round spermatids show normal testis-specific H1t but reduced cAMP-responsive element modulator and transition protein 1 expression in men with round-spermatid maturation arrest.