The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the mu-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida.

The acrosome is a unique organelle that plays an important role at the site of sperm-zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ-and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia.

Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that spermegg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9 ؊/؊ eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9 ؊/؊ eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9 ؊/؊ eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.

Hybrid breakdown is a type of reproductive failure that appears after the F 2 generation of crosses between different species or subspecies. It is caused by incompatibility between interacting genes. Genetic analysis of hybrid breakdown, particularly in higher animals, has been hampered by its complex nature (i.e., it involves more than two genes, and the phenotype is recessive). We studied hybrid breakdown using a new consomic strain, C57BL/6J-X MSM , in which the X chromosome of C57BL/6J (derived mostly from Mus musculus domesticus) is substituted by the X chromosome of the MSM/Ms strain (M. m. molossinus). Males of this consomic strain are sterile, whereas F 1 hybrids between C57BL/6J and MSM/Ms are completely fertile. The C57BL/6J-X MSM males showed reduced testis weight with variable defects in spermatogenesis and abnormal sperm head morphology. We conducted quantitative trait locus (QTL) analysis for these traits to map the X-linked genetic factors responsible for the sterility. This analysis successfully detected at least three distinct loci for the sperm head morphology and one for the testis weight. This study revealed that incompatibility of interactions of X-linked gene(s) with autosomal and/or Y-linked gene(s) causes the hybrid breakdown between the genetically distant C57BL/6J and MSM/Ms strains. H YBRIDS between individuals of two genetically di-Hybrid breakdown is another type of reproductive isolation, defined as inviability or sterility observed only verged populations show different extents of rein the F 2 or later generations of interspecific or intersubproductive failure; this failure is known as reproductive specific crosses, while F 1 hybrids are viable and fully isolation. This reduction of fecundity of the hybrids fertile. Hybrid breakdown may be due to disruption prevents gene flow across the two different populations, of interaction of genes at different loci as the genes accelerating genetic differentiation and eventually consegregate after the F 1 generation. Assuming that two tributing to speciation. Thus, the study of reproductive diverging populations have different alleles at each of isolation is essential for understanding the process of two loci that genetically interact, it is inferred that the speciation.proper interaction may occur only in the allele combinaIn mice, genetic studies on reproductive isolation tions that occur in each of the two diverging populahave focused mostly on male sterility in F 1 hybrids betions. Hybrid breakdown is thus hypothesized to arise tween subspecies or closely related species. This hybrid when genetic segregation causes alleles of each intersterility is likely caused by interallelic incompatibility at acting locus to become homozygous in an improper way. a given locus or at several different loci. In such cases, Therefore, the hybrid breakdown appears as a recessive one allele or a set of alleles is fixed or predominates trait (Muller 1940; Orr 1993). Little is known about in each genetically differentiating population. Hybrid the gene...

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility 1 . Here we show that Cnot7 -/-males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor 2 , is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7 -/-mice. Transplantation of spermatogonial stem cells from male Cnot7 -/-mice to seminiferous tubules of Kit mutant mice (Kit W/W-v ) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7 -/-condition. The testicular phenotypes of Cnot7 -/-mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb) 3 . We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

We cloned a testis-specific cDNA from mice that encodes a histone H1-like, haploid germ cell-specific nuclear protein designated HANP1/H1T2. The HANP1/H1T2 protein was specifically localized to the nuclei of murine spermatids during differentiation steps 5 to 13 but not to the nuclei of mature sperm. HANP1/H1T2 contains an arginine-serine-rich domain and an ATP/GTP binding site, and it binds to DNA, ATP, and protamine. To investigate the physiological role of HANP1/H1T2, we generated Hanp1/H1T2-disrupted mutant mice. Homozygous Hanp1/H1T2 mutant males were infertile, but females were fertile. Although a substantial number of sperm were recovered from the epididymides, their shape and function were abnormal. During sperm morphogenesis, the formation of nuclei was disturbed and protamine-1 and -2 were only weakly detectable in the nuclei. The chromatin packaging was aberrant, as demonstrated by electron microscopy and biochemical analysis. The mutant sperm exhibited deficient motility and were not competent to fertilize eggs under in vitro fertilization conditions; however, they were capable of fertilizing eggs via intracytoplasmic sperm injection that resulted in the birth of healthy progeny. Thus, we found that HANP1/H1T2 is essential for nuclear formation in functional spermatozoa and is specifically involved in the replacement of histones with protamines during spermiogenesis. At the time of submission of the manuscript, we found an independent publication by The complex process of spermatogenesis includes three major events: proliferation and differentiation of the spermatogonia, meiotic prophase in the spermatocytes, and drastic morphological changes during differentiation from the haploid round spermatids to the mature sperm (24). These events begin after birth, and approximately 35 days are required for the development of mature sperm in the mouse. The differentiation of the haploid germ cells (spermiogenesis) begins at 17 days of age in the mouse. Spermiogenesis involves diverse and complex processes, such as packaging and remodeling of the haploid germ cell nucleus, rearrangement of mitochondria, development of the flagellum, and formation of the acrosome. During this phase, the composition of the chromatin is altered dramatically (29). The changes in the nuclear proteins occur in association with the displacement of general nucleohistones by transition proteins (TNP) and other proteins, including a number of testis-specific histones and nonhistone chromosomal proteins (3,12,13,17,31) that are subsequently replaced with protamines to form nucleoprotamines (2, 20). The transition from histones to protamines in the chromatin of the haploid germ cells is accompanied by epigenetic changes (19) and the specific formation of nuclei in the sperm (25); these changes are associated with chromosome condensation and the shaping of the nucleus. Recently, mice with null mutations in TNP1 or TNP2 were found to be subfertile (32, 33), and mice with null mutations in both TNP1 and TNP2 were infertile (34). The nuclei of...

The proper folding of newly synthesized membrane proteins in the endoplasmic reticulum (ER) is required for the formation of functional mature proteins. Calnexin is a ubiquitous ER chaperone that plays a major role in quality control by retaining incompletely folded or misfolded proteins. In contrast to other known chaperones such as heat-shock proteins, BiP and calreticulin, calnexin is an integral membrane protein. Calmegin is a testis-specific ER protein that is homologous to calnexin. Here we show that calmegin binds to nascent polypeptides during spermatogenesis, and have analysed its physiological function by targeted disruption of its gene. Homozygous-null male mice are nearly sterile even though spermatogenesis is morphologically normal and mating is normal. In vitro, sperm from homozygous-null males do not adhere to the egg extracellular matrix (zona pellucida), and this defect may explain the observed infertility. These results suggest that calmegin functions as a chaperone for one or more sperm surface proteins that mediate the interactions between sperm and egg. The defective zona pellucida-adhesion phenotype of sperm from calmegin-deficient mice is reminiscent of certain cases of unexplained infertility in human males.

Using homologous recombination, we have previously produced male mice carrying a disruptive mutation (Acr ؊/؊ ) in the acrosin gene. Although Acr ؊/؊ mouse sperm lacking the acrosin protease activity still penetrated the zona pellucida and fertilized the egg, the mutant sperm exhibited a delay in penetration of the zona pellucida solely at the early stages after insemination. To further elucidate the role of acrosin in fertilization, we have examined the involvement of acrosin in the acrosome reaction of sperm using the Acr ؊/؊ mutant mice. When the ability of sperm to adhere (attach) and bind to the zona pellucida of cumulus-free eggs was assessed in vitro, no significant difference was observed among Acr ؉/؉ , Acr ؉/؊ , and Acr ؊/؊ mouse sperm. Immunocytochemical analysis demonstrated that the release of several acrosomal proteins from the acrosome of Acr ؊/؊ mouse sperm was significantly delayed during the calcium ionophore-and solubilized zona pellucidainduced acrosome reaction, despite normal membrane vesiculation. These data indicate that the delayed sperm penetration of the zona pellucida in the Acr ؊/؊ mouse results from the altered rate of protein dispersal from the acrosome and provide the first evidence that the major role of acrosin is to accelerate the dispersal of acrosomal components during acrosome reaction.The acrosome reaction of sperm, a fusion (vesiculation) event between the overlying plasma and outer acrosomal membranes, occurs following the binding of sperm to the zona pellucida (ZP), 1 an extracellular glycoprotein matrix surrounding the egg. This exocytotic reaction is required for fertilization, because only acrosome-reacted sperm are capable of penetrating ZP and of fusing with the egg plasma membrane (for review see Ref. 1). The acrosomal components, including hydrolytic enzymes, are released by the acrosome reaction and then interact initially with ZP to facilitate the sperm penetration of the glycoprotein matrix.Acrosin, an endoprotease with a trypsin-like substrate specificity, is localized in the acrosomal matrix as an enzymatically inactive zymogen, proacrosin, that is then converted into the active form as a consequence of the acrosome reaction (2-4). The physiological role of acrosin in fertilization has long been believed to be the limited proteolysis of the ZP, thus enabling the sperm to penetrate the ZP. Using homologous recombination, we have successfully produced male mice carrying a disruptive mutation in the acrosin gene (Acr) and found that the mouse sperm lacking the acrosin protease activity (Acr Ϫ/Ϫ ) still penetrate ZP and normally fertilize the egg (5). These data provide evidence that acrosin is not essential for sperm penetration of the ZP. However, as compared with Acr ϩ/ϩ and Acr ϩ/Ϫ mice, Acr Ϫ/Ϫ mouse sperm showed a delay in sperm penetration of the ZP solely at the early stages after insemination (5). A recent report using separate lines of Acr Ϫ/Ϫ mice (6) has confirmed that sperm lacking acrosin exhibit the delayed fertilization. Thus, these results imply ...