Lack of Spem1 causes aberrant cytoplasm removal, sperm deformation, and male infertility

We identified a previously uncharacterized gene, spermatid maturation 1 (Spem1), encoding a protein exclusively expressed in the cytoplasm of steps 14-16 elongated spermatids in the mouse testis. This protein contains no known functional domains and is highly conserved across mammalian species. Male mice deficient in Spem1 were completely infertile because of deformed sperm characterized by a bent head wrapped around by the neck and the middle piece of the tail. We show that lack of Spem1 causes failure of the cytoplasm to become loose and detach from the head and the neck region of the developing spermatozoa. Retained cytoplasmic components mechanically obstruct the straightening of the sperm head and the stretching of the growing tail, leading to the bending of the head in the neck, followed by the wrapping of the head by the neck or the middle piece of the sperm tail. Our study reveals that proper cytoplasm removal is a genetically regulated process requiring the participation of Spem1 and that lack of Spem1 causes sperm deformation and male infertility.     

Structural genes on the Y chromosome of Drosophila melanogaster.

Testis proteins of Drosophila melanogaster deficient for six different Y-chromosome regions were fractionated by means of a sodium dodecyl sulfate/polyacrylamide gel system designed to separate high molecular weight polypeptides (Mr, greater than 200,000). Analysis of the banding patterns indicates that the three regions containing fertility genes kl-2, kl-3, and kl-5 are responsible for three different high molecular weight polypeptides. Several observations indicate that these polypeptides are structural components of the sperm axoneme. They are present in seminal vesicles, which are highly enriched for mature sperm. They are first detected during development at a time when the first spermatids are elongating. Finally, deletion of either kl-5 or kl-3 leads to the absence of the outer dynein arm of the peripheral doublets of the axoneme. Although absence of the kl-2 region eliminates the third polypeptide, an associated structural defect in the axoneme has yet to be identified. The three polypeptides are in the Mr 300,000-350,000 range, and their mobilities are similar to those of dynein polypeptides from Chlamydomonas axonemes. Experiments using dosage variation and a temperature-sensitive sterile mutation in kl-5 suggest that the Y-chromosome regions contain the coding sequences for the polypeptides.     

Assisted reproductive technologies do not alter mutation frequency or spectrum

Assisted reproductive technologies (ARTs) have now contributed to the birth of >3 million babies worldwide, but concerns remain regarding the safety of these methods. We have used a transgenic mouse model to examine the effects of ARTs on the frequency and spectrum of point mutations in midgestation mouse fetuses produced by either natural reproduction or various methods of ART, including preimplantation culture, embryo transfer, in vitro fertilization, intracytoplasmic sperm injection, and round spermatid injection. Our results show that there is no significant difference in the frequency or spectrum of de novo point mutations found in naturally conceived fetuses and fetuses produced by in vitro fertilization, intracytoplasmic sperm injection, or round spermatid injection. These results, based on analyses of a transgenic mouse system, indicate that with respect to maintenance of genetic integrity, ARTs appear to be safe.     

Immortalized germ cells undergo meiosis in vitro.

Establishing mammalian germ-cell lines capable of differentiation in vitro would greatly facilitate the study of gametogenesis and the meiotic process that is so fundamental for reproduction and the maintenance of genetic diversity of the species. We have established two germ-cell lines [GC-2spd(ts) and GC-3spc(ts)] by cotransfecting primary mouse testicular germ cells with the simian virus 40 large tumor antigen gene and the gene coding for a temperature-sensitive mutant of p53. Both cell lines express the germ cell-specific lactate dehydrogenase C4 isozyme and cytochrome ct isoform. At the permissive temperature of 37 degrees C, the GC-2spd(ts) line generates cells with a haploid DNA content and morphologic and biochemical features of round spermatids, including the appearance of an acrosomic granule. The identification of a flagellar axoneme when these cells are cultured at 32 degrees C further indicates that these cells correspond to the early spermatid stages of spermiogenesis.     

Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22.

Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1/2 of 5 min at 30 degrees C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.     

带绦虫精子发生过程中精子变形的超微结构观察

目的：研究带绦虫在精子发生过程中的精子变形。方法：透射电镜。结果：32个玫瑰花样的次级精母细胞经第2次减数分裂后,产生64个精细原形体（64－spermatid－plasmodium）。精细胞的变形过程复杂,首先见精细胞胞质和核伸长,胞质增多由胞质桥“cytophore”相连;然后是核的进一步伸长,核染色质聚合成线束状,最后脱离胞质桥,形成成熟精子。成熟的精子呈细线状,长约16．2－18．6μm,宽0．35－0．45μm,可分为有核的头部及无核的尾部两部分。头部长约5－6μm,占体长的1／3,有一个较长的核缠绕着轴丝,无线粒体。尾部长约11．2－16．6μm。在尾部的前段及头部的后方,纵断面上见一些线粒体包绕着轴丝,全长约1．6－1．7μm。精子的尾部只含一条结构为“9＋1”的轴丝。精子横断面,质膜下见46条微管（周围微管）。结论：带绦虫精子子的变形是一个非常复杂的过程。     

日本血吸虫精子的超微结构

目的：了解日本血吸虫正常精子的超微结构。方法：半超薄切片定位雄虫睾丸和含有成熟精子的雌虫输卵管，常规透射电镜制样并观察。结果：日本血吸虫精子由头、尾两部分组成，头部呈长卵圆形，平均长6．2μm，宽1．4μm，无顶体构造，前端钝圆，后端尖细，质膜下有1圈纵行的微管，核1个，前端有少量线粒体，尾部鞭毛1根，在中、后段主体部分鞭毛轴丝外周为9组二联管，中央为一团弥散的电子致密物质，呈9×2＋《1》型；但在过渡区鞭毛轴丝中央无电子致密物质，呈9×2＋0型。结论：日本血吸虫精子超微结构与其他血吸虫相似，具有同源性，但明显区别于复殖目大多数吸虫的构造。     

Structure of Trypanosoma brucei flagellum accounts for its bihelical motion

Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting proteins. We show here, by cryoelectron tomography, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting proteins. During flagellar bending, the PFR crystallographic unit cell lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axoneme drives the expansion and compression of the PFR lattice. We propose that the PFR modifies the in-plane axoneme motion to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography.     

斯氏狸殖吸虫精子形成的透射电镜观察

用透射电镜观察了斯氏狸殖吸虫精细胞的变化和精子的主要形态结构特征。从而揭示了该虫精子形成的过程。证实了斯氏狸殖吸虫的精细胞和精子有顶体结构。成熟的精子可分为头部和尾部。头部由核质充满,核质可延伸至精子尾部中段的前端。头尾之间为连接段。尾部又由中段和末段组成。尾部的中段起始部的两侧各有一条轴丝。每条轴丝各由9对外周微管和2个中央微管组成,在每2条中央微管的外围,均由一个纤维鞘包绕,其间形成一个车轮样的9 2微管系统结构,其腹侧排列着线粒体。尾部的末段,主要为2条紧靠的轴丝。     

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

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 approximately 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.     

Thyroid hormone-dependent gene expression in differentiated embryonic stem cells and embryonal carcinoma cells: identification of novel thyroid hormone target genes by deoxyribonucleic acid microarray analysis

T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRalpha P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.     

Ultrastructural observation of spermatozoa and fertilization in Schistosoma japonicum

The ultrastructure of the sperm and the process of fertilization are described in Schistosoma japonicum. The sperm of S. japonicum has an elongated head and a single tail. The head measures 6.2 x 1.4 microm in average size. No acrosome is present. A mass of mitochondria locates in front of the nucleus. A layer of about 100-120 peripheral microtubules is parallel with the long axis of the head under plasma membrane. The nucleus is dense with some electron-lucent patches. The tail is a single flagellum with unique axoneme, which originates from a centriole. The structure of axoneme includes two types: 9 x 2 + in the main part of the flagellum, and 9 x 2 + 0 near the end of the flagellum. The sperm ultrastructure of S. japonicum is similar to that of other schistosomes, apart from the fact that two types of configuration coexisted in the same axoneme, and there is no striated root found in S. japonicum. The sperm differs distinctly from other Digenea. The aberrant ultrastructure of S. japonicum reflects that its evolution is far away from other genera in Digenea. Fertilization occurs at the posterior part of oviduct, in which region the oviduct wall lacks lamellae. Some cortical granules (CG) fuse with plasma membrane, and discharge their content on the surface of the fertilized ovum. The other CGs break down or degenerate in the cytoplasm. By the secondary mature division, the secondary oocyte finally divides to form a female pronucleus. During this period a male pronucleus also forms. The female and male pronucleus approach each other, come into contact in the central region and finally fuse to form a zygote. The function of CGs is discussed.     

Effect of torsinA on membrane proteins reveals a loss of function and a dominant-negative phenotype of the dystonia-associated DeltaE-torsinA mutant

Most cases of early-onset torsion dystonia (EOTD) are caused by a deletion of one glutamic acid in the carboxyl terminus of a protein named torsinA. The mutation causes the protein to aggregate in perinuclear inclusions as opposed to the endoplasmic reticulum localization of the wild-type protein. Although there is increasing evidence that dysfunction of the dopamine system is implicated in the development of EOTD, the biological function of torsinA and its relation to dopaminergic neurotransmission has remained unexplored. Here, we show that torsinA can regulate the cellular trafficking of the dopamine transporter, as well as other polytopic membrane-bound proteins, including G protein-coupled receptors, transporters, and ion channels. This effect was prevented by mutating the ATP-binding site in torsinA. The dystonia-associated torsinA deletion mutant (DeltaE-torsinA) did not have any effect on the cell surface distribution of polytopic membrane-associated proteins, suggesting that the mutation linked with EOTD results in a loss of function. However, a mutation in the ATP-binding site in DeltaE-torsinA reversed the aggregate phenotype associated with the mutant. Moreover, the deletion mutant acts as a dominant-negative of wild-type torsinA through a mechanism presumably involving association of wild-type and mutant torsinA. Taken together, our results provide evidence for a functional role for torsinA and a loss of function and a dominant-negative phenotype of the DeltaE-torsinA mutation. These properties may contribute to the autosomal dominant nature of the condition.     

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.     

Deletion of the Parkin coregulated gene causes male sterility in the quaking(viable) mouse mutant

Quaking(viable) (qk(v)) is a recessive neurological mouse mutation with severe dysmyelination of the CNS and spermiogenesis failure. The molecular lesion in the qk(v) mutant is a deletion of approximately 1 Mb on mouse chromosome 17 that alters the expression of the qk gene in oligodendrocytes. Complementation analysis between the qk(v) mutation and qk mutant alleles generated through chemical mutagenesis showed that the male sterility is a distinctive feature of the qk(v) allele. This observation suggested that the sperm differentiation defect in qk(v) is due to the deletion of a gene(s) distinct from qk. Here, we demonstrate that the deletion of Pacrg is the cause of male sterility in the qk(v) mutant. Pacrg is the mouse homologue of the human PARKIN-coregulated gene (PACRG), which encodes for a protein whose biochemical function remains unclear. We show that Pacrg is highly expressed in the testes in both mice and humans. In addition, the expression pattern of Pacrg during spermiogenesis suggests that it plays a role in sperm differentiation. In support of this hypothesis, we show that transgenic expression of Pacrg in testes restores spermiogenesis and fertility in qk(v) males. This finding provides the first in vivo evidence, to our knowledge, for the function of Pacrg in a model organism. Immunolocalization experiments on isolated spermatozoa show that the Pacrg protein is present in mature sperm. Remarkably, the mammalian Pacrg protein shares significant sequence similarities with gene products from flagellated protozoans, suggesting that Pacrg may be necessary for proper flagellar formation in many organisms.