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Selenoprotein P (SEPP1), an extracellular glycoprotein of unknown function, is a unique member of the selenoprotein family that, depending on species, contains 10-17 selenocysteines in its primary structure; in contrast, all other family members contain a single selenocysteine residue. The SEPP1-null (Sepp1(-/-)) male but not the female mice are infertile, but the cellular basis of this male phenotype has not been defined. In this study, we demonstrate that mature spermatozoa of Sepp1(-/-) males display a specific set of flagellar structural defects that develop temporally during spermiogenesis and after testicular maturation in the epididymis. The flagellar defects include a development of a truncated mitochondrial sheath, an extrusion of a specific set of axonemal microtubules and outer dense fibers from the principal piece, and ultimately a hairpin-like bend formation at the midpiece-principal piece junction. The sperm defects found in Sepp1(-/-) males appear to be the same as those observed in wild-type (Sepp1(+/+)) males fed a low selenium diet. Supplementation of dietary selenium levels for Sepp1(-/-) males neither reverses the development of sperm defects nor restores fertility. These data demonstrate that SEPP1 is required for development of functional spermatozoa and indicate that it is an essential component of the selenium delivery pathway for developing germ cells.
The Cres (cystatin-related epididymal spermatogenic) gene encodes the defining member of a new subgroup within the family 2 cystatins of cysteine protease inhibitors. Cres expression is highly tissue- and cell-specific, with messenger RNA (mRNA) present in the testicular round/elongating spermatids, proximal caput epididymal epithelium, gonadotroph cells in the anterior pituitary gland, and corpus luteum of the ovary. To begin to elucidate the molecular mechanisms controlling the tissue- and cell-specific expression of the Cres gene, transgenic mice were generated containing 1.6 kilobases (kb) of the mouse Cres promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. A CAT enzyme-linked immunosorbent assay detected CAT protein in the testis, epididymis, isolated cauda epididymal spermatozoa, and anterior pituitary gland from mice heterozygous and homozygous for the transgene. However, reverse transcription (RT)-PCR did not detect CAT mRNA in any regions of the epididymis, suggesting that the CAT protein detected in the epididymis was from spermatozoa. RT-PCR also did not detect CAT mRNA in the ovary. RT-PCR analysis of the testes from mice of different postnatal ages showed CAT mRNA first detected at day 22, which correlated with the first appearance of Cres mRNA and with the presence of round spermatids. These studies demonstrate that 1.6 kb of Cres promoter contains the DNA elements necessary for germ cell and pituitary gland-specific expression but lacks critical sequences necessary for expression in the epididymis and ovary.
Mammalian spermatogenesis is a highly ordered process that occurs in mitotic, meiotic, and postmeiotic phases. The unique mechanisms responsible for this tightly regulated developmental process suggest the presence of an intrinsic genetic program composed of spermatogenic cell-specific genes. In this study, we analyzed the mouse round spermatid UniGene library currently containing 2124 gene-oriented transcript clusters, predicting that 467 of them are testis-specific genes, and systematically identified 28 novel genes with evident testis-specific expression by in silico and in vitro approaches. We analyzed these genes by Northern blot hybridization and cDNA cloning, demonstrating the presence of additional transcript sequences in five genes and multiple transcript isoforms in six genes. Genomic analysis revealed lack of human orthologues for 10 genes, implying a relationship between these genes and male reproduction unique to mouse. We found that all of the novel genes are expressed in developmentally regulated and stage-specific patterns, suggesting that they are primary regulators of male germ cell development. Using computational bioinformatics tools, we found that 20 gene products are potentially involved in various processes during spermatogenesis or fertilization. Taken together, we predict that over 20% of the genes from the round spermatid library are testis-specific, have discovered the 28 authentic, novel genes with probable spermatogenic cell-specific expression by the integrative approach, and provide new and thorough information about the novel genes by various in vitro and in silico analyses. Thus, the study establishes on a comprehensive scale a new basis for studies to uncover molecular mechanisms underlying the reproductive process.
We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.
In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.
Different aspects of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) have been used as discovery tools to obtain global and time-correlated information on the local proteomic composition of the sexually mature mouse epididymis from both qualitative and semiquantitative points of view. Tissue sections and laser captured microdissected cells and secretory products were analyzed by MALDI-MS and from the recovered protein profiles, over 400 different proteins were monitored. Over 50 of these, some of which have been identified, displayed regionalized behavior from caput to cauda within the epididymis. Combining the information obtained from high-resolution imaging mass spectrometry and laser captured microdissection experiments, numerous proteins were localized within the epididymis at the cellular level. Furthermore, from the signal intensities observed in the different protein profiles organized in space, semiquantitative information for each protein was obtained.
BACKGROUND - The aim of the present study was to define the effect of apoptosis on sperm quality and function.
METHODS - The apoptotic features in sperm were assessed in 60 subfertile subjects, using Annexin-V staining for phosphatidylserine (PS) externalization and Tdt-mediated dUTP nick end labelling (TUNEL) assay for DNA fragmentation.
RESULTS - On average, about 45% of the sperm were found to be apoptotic based on the results from Annexin-V staining, including both early (Annexin-V-positive, PI-negative) and late apoptosis (Annexin-V-positive, PI-positive). TUNEL-positive cells (median value 15%) significantly correlated to late apoptosis but not early apoptosis, indicating that DNA fragmentation only occurs at the later stage of sperm apoptosis. TUNEL-positive and late apoptotic cells (Annexin-V-positive, PI-positive) were found to be inversely correlated to sperm motility and vitality, and positively to abnormal sperm morphology. On the other hand, it is surprising to note that the apoptotic alterations in sperm positively correlated to sperm concentration or total sperm counts.
CONCLUSIONS - Overall results from this study support the abortive apoptosis theory; apoptosis in mature sperm is initiated during spermatogenesis, after which some cells earmarked for elimination via apoptosis may escape the removal mechanism and contribute to poor sperm quality.
Caenorhabditis elegans oocytes, like those of most animals, arrest during meiotic prophase. Sperm promote the resumption of meiosis (maturation) and contraction of smooth muscle-like gonadal sheath cells, which are required for ovulation. We show that the major sperm cytoskeletal protein (MSP) is a bipartite signal for oocyte maturation and sheath contraction. MSP also functions in sperm locomotion, playing a role analogous to actin. Thus, during evolution, MSP has acquired extracellular signaling and intracellular cytoskeletal functions for reproduction. Proteins with MSP-like domains are found in plants, fungi, and other animals, suggesting that related signaling functions may exist in other phyla.
The fragile X syndrome is one of more than a dozen genetic diseases attributed to the amplification of a trinucleotide repeat. Despite the number of these disease loci, relatively little is known about the mechanism(s) that cause a stable allele to become unstable. Population and family studies of the fragile X CGG repeat have identified a number of factors that may play a role in repeat instability including the number of AGG interruptions, purity of the 3' and 5' end of the repeat and cis-acting factors as related to haplotype background. However, studies that assess whether these factors have an impact on the rate and magnitude of change of the repeat are lacking, mainly due to the lack of an appropriate model system. Therefore, in order to dissect the factors involved in the initial mutations of the CGG repeat, small pool (SP)-PCR was performed on DNA derived from sperm and blood from seven unaffected males whose repeat sizes range from 20 to 33. Using the SP-PCR-derived data, regression analyses suggested that components of the repeat structure such as the number of interruptions and purity of the 3' end of the repeat are important determinants of germline repeat instability. In contrast, elements other than repeat structure, such as haplotype background, seemed to have an impact on somatic repeat instability. The factors identified for either cell type, however, explained only a small portion of the variance, suggesting that other factors may be involved in this process.
Mammalian CYP51 encodes lanosterol 14alpha-demethylase (P45014DM) that is involved in the postsqualene part of cholesterol biosynthesis. This enzyme removes the 14alpha-methyl group from lanosterol and 24,25-dihydrolanosterol producing intermediates in cholesterol biosynthesis, the oocyte meiosis-activating sterols FF-MAS and MAS-412. Human and rat CYP51 messenger RNAs (mRNAs) are expressed in all tissues, with highest levels in the testis due to the presence of an additional shorter CYP51 transcript in this tissue. In situ hybridization shows the highest CYP51 mRNA levels in seminiferous tubules, with only background levels in Leydig cells. The rat testis-specific CYP51 mRNA arises from the use of an upstream polyadenylation site and is restricted to germ cells, being most abundant in elongating spermatids in stages VII-XIV, whereas somatic CYP51 transcripts are present in all cells. In contrast, the mRNA levels of squalene synthase are maximal in round spermatids, and no germ cell-specific transcript is observed. The rat male germ cell-specific CYP51 transcript is translated in vitro to two proteins of approximately 55 and 53.5 kDa. CYP51 activity is higher in protein extracts of testes and germ cells of sexually mature rats than in prepubertal animals, in which postmeiotic germ cells are not yet present. This shows increased capacity for the production of MAS sterols by male germ cells that have already completed meiosis, suggesting that they serve a role different from meiosis activation.