Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development.

Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/-) mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/-) mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/-) mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.

Effects of cryoprotectants and cryopreservation on germinal vesicle-stage cumulus-oocyte complexes of rhesus monkeys.

OBJECTIVE: To determine the effect on rhesus germinal vesicle-stage oocytes enclosed within cumulus cells (COCs) of cryopreservation either by slow, equilibrium cooling or by rapid, non-equilibrium cooling. DESIGN: Experimental study. SETTING: University primate research center. SUBJECT(S): Twelve female rhesus monkeys. INTERVENTION(S): Monkeys were stimulated with recombinant FSH, and COCs were aspirated from follicles by an ultrasound-guided procedure. MAIN OUTCOME MEASURE(S): Rhesus COCs were examined by confocal microscopy to evaluate integrity of microtubules and intactness of transzonal processes between cumulus cells and the oocytes. RESULT(S): Exposure to 1.5 mol/L propylene glycol + 0.3 mol/L sucrose caused disruption of microtubules in all but 1 of 24 COCs and of transzonal processes in more than half of the COCs. Cryopreservation of 11 COCs by slow freezing disrupted microtubules and transzonal processes in all of them. Exposure alone to 2.7 mol/L ethylene glycol + 2.2 mol/L dimethylsulfoxide + 0.5 mol/L sucrose caused disruption of microtubules and transzonal processes in 7 of 19 COCs. Cryopreservation of COCs by rapid, non-equilibrium cooling caused disruption of microtubules and transzonal processes in 14 of 20 complexes. CONCLUSION(S): Maturation of rhesus COCs at the germinal vesicle stage may be seriously impaired because intracytoplasmic microtubules and transzonal processes are likely to be irreversibly damaged by cryopreservation.

Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients.

BACKGROUND: The protamine 1-to-protamine 2 ratio (P1/P2) is altered in the sperm cells of some infertile patients. Also, evidence for increased protamine 2 precursors (pre-P2) in a few patients has been reported. But so far, there have been no studies measuring simultaneously these two variables in a large number of patients. METHODS: We measured the P1/P2 ratio and the presence of pre-P2 using, for the first time, an antibody specific to the precursor pre-P2, together with other sperm parameters in 224 infertile patients. Additionally, the DNA integrity was assessed by terminal transferase dUTP nick-end labelling (TUNEL) in a subset of the samples. RESULTS: Pre-P2 levels show a significant positive correlation with the P1/P2 ratio, with the presence of other proteins and, at low pre-P2 levels, with TUNEL-positive sperm. An inverse correlation with sperm count, normal morphology and motility was detected. CONCLUSIONS: The levels of pre-P2 may provide clues into the pathogenic mechanisms of infertility. The increased proportion of pre-P2 in some patients with increased P1/P2 ratio suggests an involvement of pre-P2 processing. The positive correlation between TUNEL-positive sperm and pre-P2 at low pre-P2/P2 ratios also suggests a link between deficient protamine processing and decreased DNA integrity.

Transaldolase is essential for maintenance of the mitochondrial transmembrane potential and fertility of spermatozoa.

Fertility of spermatozoa depends on maintenance of the mitochondrial transmembrane potential (Deltapsi(m)), which is generated by the electron-transport chain and regulated by an oxidation-reduction equilibrium of reactive oxygen intermediates, pyridine nucleotides, and glutathione (GSH). Here, we report that male mice lacking transaldolase (TAL)(-/-) are sterile because of defective forward motility. TAL(-/-) spermatozoa show loss of Deltapsi(m) and mitochondrial membrane integrity because of diminished NADPH, NADH, and GSH. Mitochondria constitute major Ca(2+) stores; thus, diminished mitochondrial mass accounts for reduced Ca(2+) fluxing, defective forward motility, and infertility. Reduced forward progression of TAL-deficient spermatozoa is associated with diminished mitochondrial reactive oxygen intermediate production and Ca(2+) levels, intracellular acidosis, and compensatory down-regulation of carbonic anhydrase IV and overexpression of CD38 and gamma-glutamyl transferase. Microarray analyses of gene expression in the testis, caput, and cauda epididymidis of TAL(+/+), TAL(+/-), and TAL(-/-) littermates confirmed a dominant impact of TAL deficiency on late stages of sperm-cell development, affecting the electron-transport chain and GSH metabolism. Stimulation of de novo GSH synthesis by oral N-acetyl-cysteine normalized the low fertility rate of TAL(+/-) males without affecting the sterility of TAL(-/-) males. Whereas TAL(-/-) sperm failed to fertilize TAL(+/+) oocytes in vitro, sterility of TAL(-/-) sperm was circumvented by intracytoplasmic sperm injection, indicating that TAL deficiency influenced the structure and function of mitochondria without compromising the nucleus and DNA integrity. Collectively, these data reveal an essential role of TAL in sperm-cell mitochondrial function and, thus, male fertility.

Nucleoprotein transitions during spermiogenesis in mice with transition nuclear protein Tnp1 and Tnp2 mutations.

Chromatin remodeling during spermiogenesis is characterized by a series of nuclear protein replacements. Histones are replaced by transition nuclear proteins, which are in turn replaced by protamines. The transition nuclear proteins, TP1 and TP2, and the protamines, PRM1 and PRM2, are the major nuclear proteins involved in this process. Biochemical studies of mice with null mutations in one of the Tnp genes showed that the absence of one TP led to an apparent elevation in the amount of the remaining TP in the testis. To investigate the mechanism of changes of protein levels and effects of one Tnp mutation on other nuclear proteins, we used immunohistochemistry techniques to determine the distribution of these nuclear proteins. In contrast to previous biochemical analyses, which indicated that nuclear protein replacement was sequential with little overlap between the protein types, we found considerable overlap in the nucleoprotein types during spermiogenesis. The TPs, which appear in the nucleus before histone displacement is complete, were shared among genetically inequivalent spermatids. The absence of one TP did not affect the time of appearance of the other TP or of the protamines, but it did affect the displacement of the other TP, leading to its abnormal retention in the nucleus. The elevated levels of the remaining TP in Tnp-mutant mice appeared to be a consequence of the prolonged retention, rather than increased synthesis. Thus the absence of one of the TPs did not significantly affect transcription or translation of the other basic proteins, but it did affect posttranslational events.

Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice.

Previous studies have demonstrated the importance of transition nuclear proteins, TP1 and TP2, in spermatogenesis and male fertility. However, importance of the overall level of transition proteins and their level of redundancy in the production of normal sperm is not clear. Epididymal sperm from the nine possible Tnp1 and Tnp2 null genotypes demonstrated a general decrease in normal morphology, motility, chromatin condensation, and degree of protamine 2 processing with decreasing levels of transition proteins in mutant sperm. Nuclei of some mutant epididymal sperm stained poorly with hematoxylin and DNA fluorochromes, suggesting that the DNA of these sperm underwent degradation during epididymal transport. When epididymal sperm were injected directly into oocytes, fertilization and embryonic development were reduced only in the two most severely affected genotypes. These phenotypes indicated some functional redundancy of transition proteins; however, redundancy of transition protein function was not complete, as, for example, sperm from double heterozygous males had fewer abnormalities than sperm from males homozygous for a single Tnp null mutation. Our study suggests that each TP fulfills some unique function during spermiogenesis even though sperm phenotypes strongly indicate defects are largely attributable to an overall gene dosage effect. Similarities between sperm defects found in Tnp mutants and infertile patients make the Tnp mutants a valuable tool with which to study outcomes following fertilization using sperm with compromised DNA integrity.

Roles of transition nuclear proteins in spermiogenesis.

The transition nuclear proteins (TPs) constitute 90% of the chromatin basic proteins during the steps of spermiogenesis between histone removal and the deposition of the protamines. We first summarize the properties of the two major transition nuclear proteins, TP1 and TP2, and present concepts, based on their time of appearance in vivo and in vitro properties, regarding their roles. Distinct roles for the two TPs in histone displacement, sperm nuclear shaping, chromatin condensation, and maintenance of DNA integrity have been proposed. More definitive information on their roles in spermiogenesis has recently been obtained using mice with null mutations in the Tnp1 or Tnp2 genes for TP1 and TP2, respectively. In these mice, histone displacement and sperm nuclear shaping appear to progress quite normally. Spermatid nuclear condensation occurs, albeit in an abnormal fashion, and the mature sperm of the Tnp -null mutants are not as condensed as wild-type sperm. There is also evidence that sperm from these mutant mice contain an elevated level of DNA strand breaks. The mutant sperm showed several unexpected phenotypes, including a high incidence of configurational defects, such as heads bent back on midpieces, midpieces in hairpin configurations, coils, and clumps, other midpiece defects, reduced levels of proteolytic processing of protamine 2 during maturation, and reduced motility. The two TPs appear partly to compensate for each other as both Tnp1 - and Tnp2 -null mice were able to produce offspring, and appear to have largely overlapping functions as the two mutants had similar phenotypes.

Transition nuclear proteins are required for normal chromatin condensation and functional sperm development.

The histone-to-protamine transition is important in the formation of spermatozoa. In mammals this involves two steps: replacement of histones by transition nuclear proteins (TPs) and replacement of TPs by protamines. To determine the functions of the TPs and their importance for sperm development, we generated mice lacking both TPs, since mice lacking only TP1 or TP2 were fertile. Our results indicated that TP1 and TP2 had partially complemented each other. In mice lacking both TPs, nuclear shaping, transcriptional repression, histone displacement, and protamine deposition proceeded relatively normally, but chromatin condensation was irregular in all spermatids, many late spermatids showed DNA breaks, and protamine 2 was not posttranslationally processed. Nevertheless, genomic integrity was maintained in mature spermatids, since efficient fertilization and production of offspring were achieved by intracytoplasmic sperm injection. However, many mature spermatids were retained in the testis, epididymal spermatozoa were drastically reduced in number and were highly abnormal, and the mice were sterile. Most epididymal spermatozoa were incapable of fertilization even using intracytoplasmic sperm injection. Thus, in mammals TPs are required for normal chromatin condensation, for reducing the number of DNA breaks, and for preventing the formation of secondary defects in spermatozoa, eventual loss of genomic integrity, and sterility.