Table of Contents  
INVITED RESEARCH HIGHLIGHT
Year : 2015  |  Volume : 17  |  Issue : 4  |  Page : 646-652

Mechanisms of fertilization elucidated by gene-manipulated animals


Center for Genetic Analysis for Biological Responses, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565 0871, Japan

Date of Web Publication07-Apr-2015

Correspondence Address:
Masaru Okabe
Center for Genetic Analysis for Biological Responses, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565 0871
Japan
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1008-682X.153299

Rights and Permissions
  Abstract 

Capacitation and the acrosome reaction are key phenomena in mammalian fertilization. These phenomena were found more than 60 years ago. However, fundamental questions regarding the nature of capacitation and the timing of the acrosome reaction remain unsolved. Factors were postulated over time, but as their roles were not verified by gene-disruption experiments, widely accepted notions concerning the mechanism of fertilization are facing modifications. Today, although in vitro fertilization systems remain our central research tool, the importance of in vivo observations must be revisited. Here, primarily focusing on our own research, I summarize how in vivo observations using gene-manipulated animals have elucidated new concepts in the mechanisms of fertilization.


How to cite this article:
Okabe M. Mechanisms of fertilization elucidated by gene-manipulated animals. Asian J Androl 2015;17:646-52

How to cite this URL:
Okabe M. Mechanisms of fertilization elucidated by gene-manipulated animals. Asian J Androl [serial online] 2015 [cited 2019 Sep 18];17:646-52. Available from: http://www.ajandrology.com/text.asp?2015/17/4/646/153299 - DOI: 10.4103/1008-682X.153299

FNx01This article was presented at the 12th International Symposium on Spermatology, August 10-14, 2014, Newcastle, Australia.


Studies of the mechanisms of fertilization date back to Aristotle (384-322 BCE), who thought that the woman provided fertile ground for the male seed to grow. By the 17 th century, however, it was recognized that females produce eggs. Leeuwenhoek's microscope provided the next insight, making it possible to visualize the spermatozoa in semen. Using this microscopic observation, Hartsoeker (one of the first spermatologists) claimed that he could observe a small person residing in the head of spermatozoa. Then in 1876, Hertwig found that the nuclei of the sperm and egg fuse during fertilization in sea urchin. [1] In the 1950s, mammalian spermatozoa were found to undergo a physiological change called capacitation [2],[3] and a subsequent morphological change known as the acrosome reaction. [4] Thus, when we look back the history, the comprehension of the mechanisms of fertilization sometimes went in the wrong direction, but gradually nearing the true figure by modifying or abandoning old notions. In this process, the evolution of experimental tools such as light microscopy, antibodies, electron microscopy, etc., played important roles. Today, powerful investigative aids such as transgenic animals and/or gene-disrupted KO animals have become available. We can create an animal deficient in a given gene of interest or one with a "designer gene." For example, the latter includes spermatozoa with a green fluorescent protein (GFP) in their acrosome to report acrosomal integrity. These gene-manipulated animals give us deeper insight into the mechanisms of fertilization. In the present article, I describe the new findings, most of which have depended on the use of gene-manipulated animals.


  The In vitro Fertilization System Top


After the discovery of capacitation [2],[3] and the acrosome reaction, [4] it took more than 15 years until Yanagimachi and Chang reported in vitro fertilization (IVF) in hamsters, [5] and for mice, it required another 15 years until an efficient fertilization system became available. [6] A few years later, human IVF was successfully achieved, and the first test tube baby was born, which led Robert Edwards receiving a Nobel Prize in 2010. IVF was supplemented by another discovery that fertilization could be achieved by injecting sperm directly into the egg cytoplasm by a pipette (Intra-Cytoplasmic Sperm Injection). [7],[8] These findings boosted assisted fertilization for infertile couples, and today, a significant number of IVF babies are born worldwide.

Although IVF showed great clinical success, it had weaknesses as a probe to study the mechanisms of fertilization. One reason may be that a suitable medium for mouse fertilization did not emerge until 20 years after the discovery of capacitation. Even fertile spermatozoa failed to fertilize eggs unless they were incubated in a proper medium. Moreover, there is no consensus as to which currently-used media is the best during IVF. For example, once we learned that frozen C57BL/6 sperm were prone to lose their fertilizing ability in IVF, Takeo et al. developed a medium for these spermatozoa allowing them to penetrate eggs by the addition of methyl-beta-cyclodextrin. [9] This indicates that IVF results are significantly affected by the constitution of the medium. It also implies that the addition of various factors in the IVF medium may affect the results of IVF.


  The Emergence of a New Technique - Knockout Mice Top


After the discovery and establishment of pluripotent embryonic stem cells (ES cells) from the inner cell mass of a blastocyst, [10] Capecchi [11] and Smithies [12] independently demonstrated that a gene of interest could be disrupted by homologous recombination using ES cells. Their finding became a powerful tool in analyzing the role of genes in living mice.

Before describing the results of gene-disruption experiments, I would like to mention the drawbacks of this technique.

Existence of cumulatively functioning genes

If no phenotype is seen after gene disruption, one may conclude that the gene of interest is not essential to the phenomenon one is studying. However, when some genes are paired with others and cumulatively form an essential gene set, a single gene disruption may not result in an apparent phenotype. G1 cyclins in yeast are an example of this. These proteins (CLNI, CLN2 and CldV3) are encoded by three individual genes and are expressed in the G1 phase of the cell cycles, but cells mutant for any two of the three genes are phenotypically wild type and G1 arrest could be observed only in the triple mutant yeasts. [13]

Effects on neighboring genes

When myogenic regulatory factor 4 (Mrf4), a basic helix-loop-helix Mrf family member, was disrupted, Braun and Arnold declared that the mice die at birth, [14] Zhang et al. indicated that the mice survive, [15] and Patapoutian et al. reported that the mice occasionally die. [16] Afterward, it was found that insertion of a neo gene was detrimental to the neighboring Myf5 gene and that Mrf4 disruption was not the cause of the neonatal death. [17] A similar case was reported in the disruption of the prion gene, which is responsible for bovine spongiform encephalopathy. Some groups reported the disruption caused an ataxia phenotype, whereas others claimed they found no phenotype. The difference was that when some of the targeting vectors were used, it caused an exon skip and connected the prion gene to the neighboring doppel gene to express an aberrant fusion protein ectopically. [18]

Involvement of microRNAs

MicroRNAs (miRNAs) often reside in the intron area of certain genes, and it is known that the disruption of miRNA (s) sometimes causes a severe phenotype in the mouse. [19] Therefore, when we design the targeting vector, we must be careful not to eliminate miRNA (s) unintentionally from the modified area. [20]

Subtle effects

When we observe the phenotype of KO mice, the experimental time frame is limited. Although the gene disruption may not show a significant phenotype, the mice might have a subtle disadvantage. To discover a 5% fitness reduction, the corresponding sample size should be over 2000 and if it were 1%, it might require 600,000. [21] In other words, it is difficult to clarify the subtle effect (s) of gene disruption in normal experimentation. However, these subtle differences could ultimately affect the life of a species from an evolutionary point of view.

In this article, I neglected to describe most genes showing subtle differences and classified them as "nonessential" for the sake of simplicity in describing the fundamental mechanisms of fertilization.


  Verification of Various Factors in KO Mouse Lines Top


After IVF had become available in mice, various fertilization-related factors were identified using the IVF systems. These factors were subjected to gene KO experiments, and their respective roles were verified in vivo. The first gene examined in the KO mouse system in the field of fertilization research was acrosin, a sperm acrosomal enzyme. Acrosin was widely thought to play an important role in sperm penetration of the zona pellucida. Thus, acrosin-null spermatozoa were believed to become fertilization incapable. However, to everyone's surprise, acrosin KO mice were fertile, although a slight delay was observed in zona penetration. [22]

Another example was "fertilin," which attracted the attention of many researchers. [23] Fertilin is a heterodimer consisting of two subunits: Adam1b and Adam2. Initially, fertilin was disrupted by eliminating Adam2, and the fertilin-disrupted male mice showed an infertile phenotype. [24] Fertilin was thought to be a fusion protein, but strangely, the phenotype was loss of zona binding ability of the spermatozoa. As also shown in this example, gene function in vivo does not necessarily correspond to expectations. Later, when fertilin was disrupted by eliminating Adam1b instead of Adam2, the fertilin-null males showed normal fertility. [25] As mentioned above, when a KO mouse showed two different phenotypes, the wild-type phenotype was normally the true phenotype and any others were caused by disruption of an unrelated factor (s). In this particular case, it was learned that Adam2 was essentially required in testis (not in spermatozoa) to make fertile spermatozoa by forming a heterodimer with Adam1a. [26] Other factors, demonstrated not to be essential using KO mice, are summarized in [Table 1].
Table 1: Most gene KO mice showed no, subtle or unexpected phenotypes


Click here to view



  Essential Factors Found by KO Mouse Lines Top


Although various genes predicted to be important for IVF experiments were shown to be dispensable in vivo, others were serendipitously found as essential factors for fertilization. The first case was the calmegin KO. Calmegin is a testis-specific molecular chaperone, which is expressed mainly in pachytene stage spermatocytes and disappears from spermatozoa upon spermiation. We expected a phenotype in spermatogenesis, but no abnormality was found in calmegin KO mice. However, we discovered that the males were infertile despite having normal spermatozoa in terms of number and motility. [27] Further investigation revealed that the spermatozoa lost their zona-binding ability. We made two more testis-specific molecular chaperone KO mouse lines, calsperin KO and Pdilt KO. Lacking these genes, the spermatozoa again became incapable of binding to zona. [28],[29] If these genes were only expressed during spermatogenesis, how then was sperm-zona binding affected? As of now, we are aware of at least 13 genes involved in the formation of sperm zona-binding ability, and in all 13 cases, the spermatozoa lack Adam3 (or have aberrant Adam3). Since the Adam3-disrupted male mice are infertile [30] without affecting other gene products, Adam3 could be an ultimately essential factor in all of the gene-disrupted mouse lines as shown in [Table 2]. Interestingly, these gene KO mouse lines shared common phenotypes, with (i) no migration into the oviduct and (ii) aberrant zona-binding ability in vitro.
Table 2: KO mice with impaired zona binding ability


Click here to view



  An Inconvenient Truth Top


Although the data in [Table 2] indicated Adam3 on spermatozoa as a key protein in the fertilization process, Adam3 is surprisingly a pseudogene in humans. Therefore, to place Adam3 in the center of the general fertilization scheme may not be appropriate. Do humans have a completely different mechanism of fertilization from mice? Considering the fact that most of the genes in [Table 2] are conserved in human, we could assume the general schema is similar in humans and mice. Our current hypothesis is that we are still missing the ultimate factors contributing to sperm-zona binding. In this context, Ly6k is very interesting as spermatozoa from the Ly6k KO mice lost zona-binding ability while Adam3 remains present on spermatozoa. [31] However, Ly6k could not be the ultimate key molecule, as it disappears from mature spermatozoa even in wild-type mice. I think we are coming closer to the ultimate factors, but the process of spermatozoa-egg encounters requires further investigation.


  Is 'Sperm-Zona Binding' Dispensable? Top


In mice, the uterus and oviduct meet in a structure called the uterotubal junction (UTJ), which significantly reduces the number of spermatozoa reaching the eggs. In order to elucidate the mechanisms of UTJ penetration by spermatozoa, we produced chimeric mice that ejaculate both wild-type spermatozoa and GFP-tagged, calmegin-disrupted spermatozoa, and we mated them with wild-type females. We found that only wild-type spermatozoa migrated into the oviduct, while the equally motile calmegin-disrupted spermatozoa remained in the uterus. [32] This indicated that some unknown recognition mechanisms function in the UTJ region. Although spermatozoa from the gene-disrupted mouse lines in [Table 2] fail to migrate into the oviduct, we do not know the reason why the zona-binding ability is always associated with UTJ penetrating ability. What would happen if spermatozoa were directly injected into the oviduct, bypassing the UTJ? We tried this experiment using Pdilt, [29] Tex101[33] and Ly6k[31] KO mouse spermatozoa. To our surprise, the spermatozoa of these three KO mouse lines fertilized the eggs. In other words, spermatozoa could fertilize eggs in the oviduct without the so-called "zona-binding ability." A similar case was reported in Adam1a −/− mice; the sperm from Adam1a −/− mice could fertilize eggs in vitro when they were covered with cumulus layers. [26]


  Should the 'Zona-Induced Acrosome Reaction' Be Renounced? Top


Many reports indicated that the acrosome reaction was induced upon contact with the zona pellucida, and many researchers considered that spermatozoa undergoing the acrosome reaction before zona contact had no fertilizing ability. [34] In this context, zona-binding proteins were assumed to initiate the signaling cascade leading to the acrosome reaction. [35] We made a transgenic mouse line that expressed GFP in the acrosome. This allowed us to observe the moment of the acrosome reaction. Spermatozoa on the zona pellucida were observed, but zona-binding spermatozoa did not acrosome react under a live imaging system. [36],[37] In addition, a recent study by Jin et al. indicated that most of the fertilizing spermatozoa were acrosome-reacted before reaching the zona pellucida. [38] The experiments using gene-manipulated animals renounce the "zona-induced acrosome reaction" theory, at least in the mouse.

What about acrosomal exocytosis? If the acrosomal enzymes were released before spermatozoa approach the zona pellucida, it would be difficult for released enzymes to facilitate zona penetration. This question was also investigated using gene-manipulated animals. We previously generated Izumo1[39] and Cd9 KO mouse lines. [40],[41],[42] Spermatozoa from the Izumo1 KO line and eggs from the Cd9 KO line were not able to fuse with wild-type gametes of the opposite sex. Therefore, we could observe many spermatozoa from Izumo1 KO males in wild-type eggs or wild-type spermatozoa inside the perivitelline space of Cd9 KO eggs. We recovered both of these acrosome-reacted and zona-penetrated spermatozoa from the perivitelline space by cracking the zona with a piezo-driven micropipette. The spermatozoa swam out from the perivitelline space and were added to freshly recovered cumulus covered eggs. We found that these spermatozoa could penetrate egg investments (cumulus layers and zona pellucida) a second time and, in the case of wild-type spermatozoa recovered from Cd9 KO eggs, fuse with the eggs. [43] Thus, the timing of the acrosome reaction before zona binding seemed to be considerably flexible. This re-penetration experiment indicated that if enzymes are released from the sperm during the acrosome reaction, all enzymes are dispensable for the sperm penetration of the zona pellucida. If enzymes were involved in zona penetration, they might not be the kind released from the acrosome; rather, they remained on the spermatozoa even after the acrosome reaction. In the mouse, it was reported that the acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis. [44],[45] If acrosomal enzymes (s) were involved, they should have remained on the sperm surface even after zona penetration, sperm recovery, and during the repeated penetration of the fresh egg investments.

In any case, the timing of the acrosome reaction is flexible, as indicated long ago in the rabbits. [46] These findings also indicated that the significant "sperm-zona binding" must occur between acrosome-reacted spermatozoa and the zona pellucida, while most of the classical "sperm-zona binding" assays were observing binding between acrosome-intact spermatozoa and the zona pellucida [47] ([Figure 1]).
Figure 1: The mechanisms of fertilization, elucidated by gene-manipulated animals. ( a ) Spermatozoa that present Adam3 (or some unknown factor(s)) can migrate into the oviduct and reach the vicinity of the eggs. Acrosome reaction is induced before spermatozoa reach the zona pellucida and the fusion-related sperm protein Izumo1 on the outer acrosomal membrane migrates out to sperm surface (indicated by red color). ( b ) Spermatozoa bind to zona pellucida when mixed with cumulus-free oocytes.74 However, this binding (mostly observed between the acrosome-intact spermatozoa and zona pellucida) was dispensable. The spermatozoa that lost the so-called "zona-binding" ability remained able to fertilize eggs in vivo once the oviduct migration step was bypassed.29,31,33 Moreover, the timing of the acrosome reaction is flexible, as acrosome-reacted spermatozoa recovered from the perivitelline space could penetrate the zona pellucida a second time and fertilize eggs.43 The mechanism of sperm penetration of zona pellucida is largely unknown. ( c ) Only acrosome-reacted spermatozoa can fuse with eggs. Spermatozoa without Izumo1 never fused with eggs.39 Cd9 on the egg played an important role in fertilization,40-42 but Cd9-disrupted females were not completely infertile. In addition, no direct interaction between Cd9 and Izumo1 was observed. This led us to predict a real counterpart for Izumo1. Using the newly established AVEXIS assay, JUNO was recently found to be a counterpart for Izumo1 on the egg.51 Modified from review.75

Click here to view



  Factors Essential for Sperm-Egg Fusion Top


The first fusion-related factor, Cd9, was discovered serendipitously. A tetraspanin protein coding Cd9 was initially disrupted by researchers in other fields to examine its role in immunology. However, the Cd9-disrupted females were infertile, due to the eggs requiring Cd9 for sound fusion ability with spermatozoa. [40],[41],[42] On the sperm side, we had a monoclonal antibody OBF13, which inhibited sperm-egg fusion. [48] This was one of the fertilization inhibitory antibodies as shown in [Table 1]. While most of the factors in [Table 1] are shown to be nonessential as a result of KO experiments, the role of the OBF13 antigen remained unexamined by KO experiments for a long time. This was due to OBF13 being an IgM class antibody; therefore, there were technical difficulties in identifying the antigen. Once western blot sensitivity improved, we could finally identify the antigen and succeeded in cloning the gene. From its sequence, it was found to be a member of the immunoglobulin superfamily with a single Ig-like domain. We named this gene Izumo1 based on a Japanese shrine dedicated to marriage. As mentioned in an earlier section, the Izumo1-disrupted spermatozoa could acrosome react and penetrate both cumulus and zona pellucida layers, but were unable to fuse with eggs as we expected. [39]

The fusing ability of Cd9-disrupted eggs was severely impaired, but it was not entirely lost, differing from the complete infertility seen in Izumo1 disruption. In addition, the binding of the putative functional fragment of Izumo1 in the N-terminus region (Izumo1: 57-113) to the egg surface was not altered by disruption of Cd9. [49] Thus, Izumo1 binding to a protein other than Cd9 was expected on the egg surface. However, as the number of eggs that we can use for the experiment is quite limited, the purification of Izumo1 binding protein from eggs seemed difficult by conventional means. However, a method called the AVEXIS assay (avidity-based extracellular interaction screen) was invented. [50] Using this method, a soluble, biochemically active, highly avid recombinant mouse Izumo1 ectodomain was prepared and the reactivity against HEK293 cells transfected with a normalized mouse oocyte cDNA expression library was analyzed and Bianchi et al. successfully identified the Izumo1 binding protein on the egg and named it JUNO after the goddess of marriage. [51] The Juno-disrupted female mice were completely infertile. Now that interacting components Izumo1 and JUNO have been found, rapid progress in the elucidation of the sperm-egg fusion mechanism is expected to follow ([Figure 2]).
Figure 2: Factors involved in sperm-egg fusion. Izumo1, migrated outward from the outer acrosomal membrane to the sperm surface, tending to localize in the equatorial segment of spermatozoa. Various segments of Izumo1 were examined for their binding ability to eggs and residue 57-113 was indicated to contain an active binding site.49 Using the AVEXIS assay, JUNO was identified as an Izumo1 binding protein and its role in fusion was verified by gene-disruption experiments. JUNO is a 244-residue protein but is cleaved at 222 to form a GPI (glycosylphosphatidylinositol)-anchored protein. GPI-anchored proteins are initially formed on the cytosolic side and flipped over to the outer membrane side in the final maturation stage. The next helpful piece of information will be the elucidation of the active site of JUNO. Since Izumo1 (57-113) bound to Cd9-disrupted eggs normally, the elucidation of Cd9's role(s) will offer further clarification.

Click here to view



  Live Imaging of Fertilization Top


Observation of fertilization using gene-manipulated animals has given us a new insight. To investigate the role of Izumo1 in fusion, we made a transgenic mouse line containing the Izumo1-mCherry fusion protein and visualized the dynamic movement of Izumo1 during the fertilization process. [52]

Although OBF13 was a monoclonal antibody, various staining patterns were obtained in spermatozoa before and after the acrosome reaction. [53] Our long-standing question was how Izumo1 changed its localization from under the plasma membrane to the sperm surface during the acrosome reaction. Two possibilities were postulated: (i) migration via two steep curves in the equatorial sheath and (ii) Re-adsorption of the antigen after acrosomal vesiculation. However, both hypotheses had their own shortcomings. [54] Moreover, the exact localization of Izumo1 in live spermatozoa was unclear because it resided under the plasma membrane. First, the red fluorescent protein-tagged Izumo1-bearing spermatozoa were observed under a confocal microscope where it was revealed that Izumo1 was in the acrosomal cap area of both the inner and outer acrosomal membrane. The migration of Izumo1 upon acrosome reaction was then imaged in live cells. Apparently, Izumo1 migrated on the sperm surface, not by adsorption of vesicles formed by the acrosome reaction. It was further confirmed that Izumo1 did not migrate via the acrosomal sheath. This introduced the new hypothesis that Izumo1 migrated out from the outer acrosomal membrane to the plasma membrane at the beginning of the acrosome reaction when the two membranes fused making tiny holes ([Figure 1]a). Izumo1 migrated out to the plasma membrane and spread all over the head, but tended to associate in the equatorial segment. [52]

The dynamic movement of Izumo1 at fusion was also observed using the same transgenic mouse line. Izumo1 mainly localized to the equatorial segment dispersed in the first step of sperm-egg fusion. However, Izumo1 on the inner acrosomal membrane did not disperse but was incorporated into the cytoplasm of the egg, together with the inner acrosomal membrane structure. These Izumo1 movements were recorded in real time. [52] In conjunction with electron microscopic observations reported by many researchers, we realized that the sperm-egg fusion is apparently divided into two different phases as explained in [Figure 3].
Figure 3: Fertilization requires two independent fusions. Intact spermatozoa have a plasma membrane (blue) and an acrosomal membrane (orange). After the acrosome reaction, these two membranes fuse and form a new sperm membrane (pink). The first fusion takes place between the pink membrane and egg plasma membrane (black). After the first fusion, egg and sperm membrane form a new consecutive membrane (green). If fusion is accomplished in this step, Izumo1 on the acrosomal cap of the inner acrosomal membrane (indicated by red) should spread on the newly-formed egg surface (green). However, the second fusion (invagination) follows the first fusion that separates the acrosomal cap and acrosomal sheath areas (light blue) from the fused membrane (green). Thus, Izumo1 on the inner acrosomal membrane is invaginated into the cytoplasm of the eggs. From live imaging, Izumo1 seems to be required for the first fusion. The nature of the second fusion remains totally unknown.

Click here to view



  Conclusion Top


Observation of fertilization using gene-manipulated animals has brought us a new schematic diagram in mammalian fertilization ([Figure 1]). Note that the classical theories of the zona-induced acrosome reaction are not included in the figure. In order to understand the molecular mechanisms of fertilization, we apparently need more information. Reflecting on the progress in fertilization research, the role of gene-manipulated animals seems all the more important. Fortunately, the Crisper/Cas9 system has opened a new (wide) door for gene-disruption experiments. [55] The method is both quick and easy and applicable to mammals, fish, insects, and even to plants. In one sense, gene disruption is easier than antibody production. Use of gene-manipulated animals will soon become as routine as gel-electrophoresis.


  Acknowledgments Top


I am grateful to Dr. Martin M Matzuk and Ms. Samantha Young for helpful discussion and critical reading of the manuscript. This work was supported in part by grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan.


  Competing Financial Interests Top


The authors would declare no competing interests, or other interests that might be perceived to influence the results and/or discussion reported in this article.[75]

 
  References Top

1.
Clift D, Schuh M. Restarting life: fertilization and the transition from meiosis to mitosis. Nat Rev Mol Cell Biol 2013; 14: 549-62.  Back to cited text no. 1
    
2.
Austin CR. Observations on the penetration of the sperm in the mammalian egg. Aust J Sci Res B 1951; 4: 581-96.  Back to cited text no. 2
    
3.
Chang MC. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 1951; 168: 697-8.  Back to cited text no. 3
    
4.
Dan J. Studies on the acrosome reaction. I. Reaction to egg water and other stimuli. Biol Bull 1952; 103: 54-66.  Back to cited text no. 4
    
5.
Yanagimachi R, Chang MC. Fertilization of hamster eggs in vitro. Nature 1963; 200: 281-2.  Back to cited text no. 5
    
6.
Toyoda Y, Yokoyama M, Hoshi T. Studies on the fertilization of mouse eggs in vitro. Jpn J Anim Reprod 1971; 16: 147-57.  Back to cited text no. 6
    
7.
Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod 1976; 15: 467-70.  Back to cited text no. 7
    
8.
Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17-8.  Back to cited text no. 8
    
9.
Takeo T, Hoshii T, Kondo Y, Toyodome H, Arima H, et al. Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biol Reprod 2008; 78: 546-51.  Back to cited text no. 9
    
10.
Robertson E, Bradley A, Kuehn M, Evans M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 1986; 323: 445-8.  Back to cited text no. 10
    
11.
Capecchi MR. Generating mice with targeted mutations. Nat Med 2001; 7: 1086-90.  Back to cited text no. 11
    
12.
Smithies O. Forty years with homologous recombination. Nat Med 2001; 7: 1083-6.  Back to cited text no. 12
    
13.
Reed SI. G1-specific cyclins: in search of an S-phase-promoting factor. Trends Genet 1991; 7: 95-9.  Back to cited text no. 13
    
14.
Braun T, Arnold HH. Inactivation of Myf-6 and Myf-5 genes in mice leads to alterations in skeletal muscle development. EMBO J 1995; 14: 1176-86.  Back to cited text no. 14
    
15.
Zhang W, Behringer RR, Olson EN. Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies. Genes Dev 1995; 9: 1388-99.  Back to cited text no. 15
    
16.
Patapoutian A, Yoon JK, Miner JH, Wang S, Stark K, et al. Disruption of the mouse MRF4 gene identifies multiple waves of myogenesis in the myotome. Development 1995; 121: 3347-58.  Back to cited text no. 16
    
17.
Olson EN, Arnold HH, Rigby PW, Wold BJ. Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 1996; 85: 1-4.  Back to cited text no. 17
    
18.
Rossi D, Cozzio A, Flechsig E, Klein MA, Rülicke T, et al. Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain. EMBO J 2001; 20: 694-702.  Back to cited text no. 18
    
19.
Hasuwa H, Ueda J, Ikawa M, Okabe M. miR-200b and miR-429 function in mouse ovulation and are essential for female fertility. Science 2013; 341: 71-3.  Back to cited text no. 19
    
20.
Osokine I, Hsu R, Loeb GB, McManus MT. Unintentional miRNA ablation is a risk factor in gene knockout studies: a short report. PLoS Genet 2008; 4: e34.  Back to cited text no. 20
    
21.
Brookfield J. Can genes be truly redundant? Curr Biol 1992; 2: 553-4.  Back to cited text no. 21
    
22.
Baba T, Azuma S, Kashiwabara S, Toyoda Y. Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization. J Biol Chem 1994; 269: 31845-9.  Back to cited text no. 22
    
23.
Blobel CP, Wolfsberg TG, Turck CW, Myles DG, Primakoff P, et al. A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion. Nature 1992; 356: 248-52.  Back to cited text no. 23
    
24.
Cho C, Bunch DO, Faure JE, Goulding EH, Eddy EM, et al. Fertilization defects in sperm from mice lacking fertilin beta. Science 1998; 281: 1857-9.  Back to cited text no. 24
    
25.
Kim E, Yamashita M, Nakanishi T, Park KE, Kimura M, et al. Mouse sperm lacking ADAM1b/ADAM2 fertilin can fuse with the egg plasma membrane and effect fertilization. J Biol Chem 2006; 281: 5634-9.  Back to cited text no. 25
    
26.
Nishimura H, Kim E, Nakanishi T, Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface. J Biol Chem 2004; 279: 34957-62.  Back to cited text no. 26
    
27.
Ikawa M, Wada I, Kominami K, Watanabe D, Toshimori K, et al. The putative chaperone calmegin is required for sperm fertility. Nature 1997; 387: 607-11.  Back to cited text no. 27
    
28.
Ikawa M, Tokuhiro K, Yamaguchi R, Benham AM, Tamura T, et al. Calsperin is a testis-specific chaperone required for sperm fertility. J Biol Chem 2011; 286: 5639-46.  Back to cited text no. 28
    
29.
Tokuhiro K, Ikawa M, Benham AM, Okabe M. Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility [corrected]. Proc Natl Acad Sci U S A 2012; 109: 3850-5.  Back to cited text no. 29
    
30.
Shamsadin R, Adham IM, Nayernia K, Heinlein UA, Oberwinkler H, et al. Male mice deficient for germ-cell cyritestin are infertile. Biol Reprod 1999; 61: 1445-51.  Back to cited text no. 30
    
31.
Fujihara Y, Okabe M, Ikawa M. GPI-anchored protein complex, LY6K/TEX101, is required for sperm migration into the oviduct and male fertility in mice. Biol Reprod 2014; 90: 60.  Back to cited text no. 31
    
32.
Nakanishi T, Isotani A, Yamaguchi R, Ikawa M, Baba T, et al. Selective passage through the uterotubal junction of sperm from a mixed population produced by chimeras of calmegin-knockout and wild-type male mice. Biol Reprod 2004; 71: 959-65.  Back to cited text no. 32
    
33.
Fujihara Y, Tokuhiro K, Muro Y, Kondoh G, Araki Y, et al. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa. Proc Natl Acad Sci U S A 2013; 110: 8111-6.  Back to cited text no. 33
    
34.
Bleil JD, Beall CF, Wassarman PM. Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2. Dev Biol 1981; 86: 189-97.  Back to cited text no. 34
    
35.
Gong X, Dubois DH, Miller DJ, Shur BD. Activation of a G protein complex by aggregation of beta-1,4-galactosyltransferase on the surface of sperm. Science 1995; 269: 1718-21.  Back to cited text no. 35
    
36.
Nakanishi T, Ikawa M, Yamada S, Parvinen M, Baba T, et al. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett 1999; 449: 277-83.  Back to cited text no. 36
    
37.
Baibakov B, Gauthier L, Talbot P, Rankin TL, Dean J. Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. Development 2007; 134: 933-43.  Back to cited text no. 37
    
38.
Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, et al. Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proc Natl Acad Sci U S A 2011; 108: 4892-6.  Back to cited text no. 38
    
39.
Inoue N, Ikawa M, Isotani A, Okabe M. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 2005; 434: 234-8.  Back to cited text no. 39
    
40.
Kaji K, Oda S, Shikano T, Ohnuki T, Uematsu Y, et al. The gamete fusion process is defective in eggs of Cd9-deficient mice. Nat Genet 2000; 24: 279-82.  Back to cited text no. 40
    
41.
Le Naour F, Rubinstein E, Jasmin C, Prenant M, Boucheix C. Severely reduced female fertility in CD9-deficient mice. Science 2000; 287: 319-21.  Back to cited text no. 41
    
42.
Miyado K, Yamada G, Yamada S, Hasuwa H, Nakamura Y, et al. Requirement of CD9 on the egg plasma membrane for fertilization. Science 2000; 287: 321-4.  Back to cited text no. 42
    
43.
Inoue N, Satouh Y, Ikawa M, Okabe M, Yanagimachi R. Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs. Proc Natl Acad Sci U S A 2011; 108: 20008-11.  Back to cited text no. 43
    
44.
Hardy DM, Oda MN, Friend DS, Huang TT Jr. A mechanism for differential release of acrosomal enzymes during the acrosome reaction. Biochem J 1991; 275 (Pt 3): 759-66.  Back to cited text no. 44
    
45.
Kim KS, Foster JA, Gerton GL. Differential release of guinea pig sperm acrosomal components during exocytosis. Biol Reprod 2001; 64: 148-56.  Back to cited text no. 45
    
46.
Valdivia M, Sillerico T, De Ioannes A, Barros C. Proteolytic activity of rabbit perivitelline spermatozoa. Zygote 1999; 7: 143-9.  Back to cited text no. 46
    
47.
Gahlay G, Gauthier L, Baibakov B, Epifano O, Dean J. Gamete recognition in mice dependes on the cleavage status of an egg's zona pellucida protein. Scinece 2010; 329: 216-9.  Back to cited text no. 47
    
48.
Okabe M, Yagasaki M, Oda H, Matzno S, Kohama Y, et al. Effect of a monoclonal anti-mouse sperm antibody (OBF13) on the interaction of mouse sperm with zona-free mouse and hamster eggs. J Reprod Immunol 1988; 13: 211-9.  Back to cited text no. 48
    
49.
Inoue N, Hamada D, Kamikubo H, Hirata K, Kataoka M, et al. Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion. Development 2013; 140: 3221-9.  Back to cited text no. 49
    
50.
Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, et al. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature 2011; 480: 534-7.  Back to cited text no. 50
    
51.
Bianchi E, Doe B, Goulding D, Wright GJ. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 2014; 508: 483-7.  Back to cited text no. 51
    
52.
Satouh Y, Inoue N, Ikawa M, Okabe M. Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1. J Cell Sci 2012; 125 (Pt 21): 4985-90.  Back to cited text no. 52
    
53.
Okabe M, Adachi T, Takada K, Oda H, Yagasaki M, et al. Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. J Reprod Immunol 1987; 11: 91-100.  Back to cited text no. 53
    
54.
Toshimori K. Dynamics of the mammalian sperm membrane modification leading to fertilization: a cytological study. J Electron Microsc (Tokyo) 2011; 60 Suppl 1: S31-42.  Back to cited text no. 54
    
55.
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013; 153: 910-8.  Back to cited text no. 55
    
56.
Lu Q, Shur BD. Sperm from beta 1, 4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly. Development 1997; 124: 4121-31.  Back to cited text no. 56
    
57.
Asano M, Furukawa K, Kido M, Matsumoto S, Umesaki Y, et al. Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells. EMBO J 1997; 16: 1850-7.  Back to cited text no. 57
    
58.
Baba D, Kashiwabara S, Honda A, Yamagata K, Wu Q, et al. Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg. J Biol Chem 2002; 277: 30310-4.  Back to cited text no. 58
    
59.
Inoue N, Ikawa M, Nakanishi T, Matsumoto M, Nomura M, et al. Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm. Mol Cell Biol 2003; 23: 2614-22.  Back to cited text no. 59
    
60.
Ensslin MA, Shur BD. Identification of mouse sperm SED1, a bimotif EGF repeat and discoidin-domain protein involved in sperm-egg binding. Cell 2003; 114: 405-17.  Back to cited text no. 60
    
61.
Hanayama R, Tanaka M, Miyasaka K, Aozasa K, Koike M, et al. Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science 2004; 304: 1147-50.  Back to cited text no. 61
    
62.
Lin YN, Roy A, Yan W, Burns KH, Matzuk MM. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis. Mol Cell Biol 2007; 27: 6794-805.  Back to cited text no. 62
    
63.
Da Ros VG, Maldera JA, Willis WD, Cohen DJ, Goulding EH, et al. Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1). Dev Biol 2008; 320: 12-8.  Back to cited text no. 63
    
64.
Sutton KA, Jungnickel MK, Florman HM. A polycystin-1 controls postcopulatory reproductive selection in mice. Proc Natl Acad Sci U S A. 2008; 105: 8661-6.  Back to cited text no. 64
    
65.
Tardif S, Wilson MD, Wagner R, Hunt P, Gertsenstein M, et al. Zonadhesin is essential for species specificity of sperm adhesion to the egg zona pellucida. J Biol Chem 2010; 285: 24863-70.  Back to cited text no. 65
    
66.
Muro Y, Buffone MG, Okabe M, Gerton GL. Function of the acrosomal matrix: zona pellucida 3 receptor (ZP3R/sp56) is not essential for mouse fertilization. Biol Reprod 2012; 86: 1-6.  Back to cited text no. 66
    
67.
Hagaman JR, Moyer JS, Bachman ES, Sibony M, Magyar PL, et al. Angiotensin-converting enzyme and male fertility. Proc Natl Acad Sci U S A 1998; 95: 2552-7.  Back to cited text no. 67
    
68.
Yamaguchi R, Yamagata K, Ikawa M, Moss SB, Okabe M. Aberrant distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace)- and calmegin (Clgn)-deficient mice. Biol Reprod 2006; 75: 760-6.  Back to cited text no. 68
    
69.
Shen C, Kuang Y, Liu J, Feng J, Chen X, et al. Prss37 is required for male fertility in the mouse. Biol Reprod 2013; 88: 123.  Back to cited text no. 69
    
70.
Yamaguchi R, Muro Y, Isotani A, Tokuhiro K, Takumi K, et al. Disruption of ADAM3 impairs the migration of sperm into oviduct in mouse. Biol Reprod 2009; 81: 142-6.  Back to cited text no. 70
    
71.
Marcello MR, Jia W, Leary JA, Moore KL, Evans JP. Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm. J Biol Chem 2011; 286: 13060-70.  Back to cited text no. 71
    
72.
Yamaguchi R, Fujihara Y, Ikawa M, Okabe M. Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa. Mol Biol Cell 2012; 23: 2671-9.  Back to cited text no. 72
    
73.
Krutskikh A, Poliandri A, Cabrera-Sharp V, Dacheux JL, Poutanen M, et al. Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility. FASEB J 2012; 26: 4198-209.  Back to cited text no. 73
    
74.
Hartmann JF, Gwatkin RB, Hutchison CF. Early contact interactions between mammalian gametes in vitro: evidence that the vitellus influences adherence between sperm and zona pellucida. Proc Natl Acad Sci U S A 1972; 69: 2767-9.  Back to cited text no. 74
    
75.
Okabe M. The cell biology of mammalian fertilization. Development 2013; 140: 4471-9.  Back to cited text no. 75
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]


This article has been cited by
1 An update of the regulatory factors of sperm migration from the uterus into the oviduct by genetically manipulated mice
Wenfeng Xiong,Zhugang Wang,Chunling Shen
Molecular Reproduction and Development. 2019;
[Pubmed] | [DOI]
2 The female reproductive tract contains multiple innate sialic acid-binding immunoglobulin-like lectins (Siglecs) that facilitate sperm survival
Eillen Tecle,Hector Sequoyah Reynoso,Ruixuan Wang,Pascal Gagneux
Journal of Biological Chemistry. 2019; 294(31): 11910
[Pubmed] | [DOI]
3 The fate of spermatozoa in the female reproductive tract: A comparative review
J.P. Rickard,K.R. Pool,X. Druart,S.P. de Graaf
Theriogenology. 2019; 137: 104
[Pubmed] | [DOI]
4 Increase of germ cell nuclear factor expression in globozoospermic Gopc -/- knockout mice
M. Bizkarguenaga,L. Gomez-Santos,J. F. Madrid,F. J. Sáez,E. Alonso
Andrology. 2019;
[Pubmed] | [DOI]
5 Influence of the genetic background on the reproductive phenotype of mice lacking Cysteine-Rich Secretory Protein 1 (CRISP1)†
Mariana Weigel Muñoz,María A Battistone,Guillermo Carvajal,Julieta A Maldera,Ludmila Curci,Pablo Torres,Daniel Lombardo,Omar P Pignataro,Vanina G Da Ros,Patricia S Cuasnicú
Biology of Reproduction. 2018;
[Pubmed] | [DOI]
6 Deficiency of fibroblast growth factor 2 (FGF-2) leads to abnormal spermatogenesis and altered sperm physiology
Lucía Saucedo,Regina Rumpel,Cristian Sobarzo,Dietmar Schreiner,Gudrun Brandes,Livia Lustig,Mónica Hebe Vazquez-Levin,Claudia Grothe,Clara Marín-Briggiler
Journal of Cellular Physiology. 2018;
[Pubmed] | [DOI]
7 Beware of memes in the interpretation of your results - lessons from gene-disrupted mice in fertilization research
Masaru Okabe
FEBS Letters. 2018;
[Pubmed] | [DOI]
8 Factors controlling sperm migration through the oviduct revealed by gene-modified mouse models
Yoshitaka Fujihara,Haruhiko Miyata,Masahito Ikawa
Experimental Animals. 2018; 67(2): 91
[Pubmed] | [DOI]
9 Human sperm acrosomal status, acrosomal responsiveness, and acrosin are predictive of the outcomes of in vitro fertilization: A prospective cohort study
Fang Xu,Hailun Zhu,Wenbing Zhu,Liqing Fan
Reproductive Biology. 2018;
[Pubmed] | [DOI]
10 CatSper? regulates the structural continuity of sperm Ca2+ signaling domains and is required for normal fertility
Jean-Ju Chung,Kiyoshi Miki,Doory Kim,Sang-Hee Shim,Huanan F Shi,Jae Yeon Hwang,Xinjiang Cai,Yusuf Iseri,Xiaowei Zhuang,David E Clapham
eLife. 2017; 6
[Pubmed] | [DOI]
11 Exosomes versus microexosomes: Shared components but distinct functions
Kenji Miyado,Woojin Kang,Kenji Yamatoya,Maito Hanai,Akihiro Nakamura,Toshiyuki Mori,Mami Miyado,Natsuko Kawano
Journal of Plant Research. 2017;
[Pubmed] | [DOI]
12 Microfluidic devices for the study of sperm migration
S. S. Suarez,M. Wu
Molecular Human Reproduction. 2016;
[Pubmed] | [DOI]
13 GPI-AP release in cellular, developmental, and reproductive biology
Yoshitaka Fujihara,Masahito Ikawa
Journal of Lipid Research. 2016; 57(4): 538
[Pubmed] | [DOI]
14 Behavior of Mouse Spermatozoa in the Female Reproductive Tract from Soon after Mating to the Beginning of Fertilization1
Yuko Muro,Hidetoshi Hasuwa,Ayako Isotani,Haruhiko Miyata,Kazuo Yamagata,Masahito Ikawa,Ryuzo Yanagimachi,Masaru Okabe
Biology of Reproduction. 2016; 94(4)
[Pubmed] | [DOI]
15 D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein
T Leahy,JP Rickard,RJ Aitken,SP de Graaf
Reproduction. 2016; 151(5): 491
[Pubmed] | [DOI]
16 Heat Shock Protein member A2 forms a stable complex with angiotensin converting enzyme and protein disulfide isomerase A6 in human spermatozoa
Elizabeth G. Bromfield,Eileen A. McLaughlin,Robert John Aitken,Brett Nixon
Molecular Human Reproduction. 2016; 22(2): 93
[Pubmed] | [DOI]
17 Mechanical tuning of mammalian sperm behaviour by hyperactivation, rheology and substrate adhesion: a numerical exploration
Kenta Ishimoto,Eamonn A. Gaffney
Journal of The Royal Society Interface. 2016; 13(124): 20160633
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
The In vitro<...
The Emergence of...
Verification of ...
Essential Factor...
An Inconvenient ...
Factors Essentia...
Live Imaging of ...
Conclusion
Acknowledgments
Competing Financ...
Is 'Sperm-Zona B...
Should the 'Zona...
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed4774    
    Printed43    
    Emailed0    
    PDF Downloaded551    
    Comments [Add]    
    Cited by others 17    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]