You have recently accepted a position as Human Resource Director for Jupiter Industries, a multi-national organization operating across 5 business segments, specifically, industrial valves and controls, plastics, fire suppression equipment, electronic components, and specialized medical products. Jupiter Industries has established worldwide headquarters in Bremen, Germany, and has groups located in the Americas, Europe, Asia and Pacific Rim countries. The MENA group is the most recently formed division within the Jupiter organization, and is composed of various concentric businesses that fit into one of the five organizational business segments, all of which have been acquired over the past two years in both the Middle East region (Saudi Arabia, UAE, Oman, Bahrain, and Jordan) as well as North Africa (Egypt, Morocco, Tunisia, and Algeria). You will be working at the MENA headquarters located in Manama, Bahrain and will be responsible for all divisions located in the MENA (Middle East, North Africa) region. You will report directly to the VP of Operations in Manama, with dotted-line reporting responsibility to Chief Human Resources Officer (CHRO) in Bremen. Specifically, you are being tasked with establishing Human Resource policy and practice in regard to strategic collaboration with divisional heads, utilizing HRIS systems in addressing areas such as recruitment and retention, training and development, performance management programs, compensation management, employee benefit programs and employee relations, including motivational programs, incentive programs, and disciplinary procedures. You will be working on policies and procedures that can be utilized throughout the region, to bring some semblance of order and uniformity in dealing with strategic HR employee programs. After a thoughtful review of current HR programs for the region, you will prepare a report for the VP of Operations and CHRO and distribute to all division heads, formulating your HR strategic goals across all HR areas of responsibility, highlighting recommendations for improving the overall impact of these programs on talent management initiatives including attraction and retention of top talent and the methodologies used to do so. This report will be inclusive of all Human Resource functions and provide both strategic and operational goals of the desired outcomes and, the means or “how” to reach these deliverables.

What type of international assignee compensation plan would you suggest for Bremen expat employees appointed to 3-year assignments in Egypt and Morocco? Explain. What type of international assignee compensation plan would you suggest for Bremen expat employees appointed to 3-year assignments in Saudi Arabia and Jordan?

You have recently accepted a position as Human Resource Director for Jupiter Industries, a multi-national organization operating across 5 business segments, specifically, industrial valves and controls, plastics, fire suppression equipment, electronic components, and specialized medical products. Jupiter Industries has established worldwide headquarters in Bremen, Germany, and has groups located in the Americas, Europe, Asia and Pacific Rim countries. The MENA group is the most recently formed division within the Jupiter organization, and is composed of various concentric businesses that fit into one of the five organizational business segments, all of which have been acquired over the past two years in both the Middle East region (Saudi Arabia, UAE, Oman, Bahrain, and Jordan) as well as North Africa (Egypt, Morocco, Tunisia, and Algeria). You will be working at the MENA headquarters located in Manama, Bahrain and will be responsible for all divisions located in the MENA (Middle East, North Africa) region. You will report directly to the VP of Operations in Manama, with dotted-line reporting responsibility to Chief Human Resources Officer (CHRO) in Bremen. Specifically, you are being tasked with establishing Human Resource policy and practice in regard to strategic collaboration with divisional heads, utilizing HRIS systems in addressing areas such as recruitment and retention, training and development, performance management programs, compensation management, employee benefit programs and employee relations, including motivational programs, incentive programs, and disciplinary procedures. You will be working on policies and procedures that can be utilized throughout the region, to bring some semblance of order and uniformity in dealing with strategic HR employee programs. After a thoughtful review of current HR programs for the region, you will prepare a report for the VP of Operations and CHRO and distribute to all division heads, formulating your HR strategic goals across all HR areas of responsibility, highlighting recommendations for improving the overall impact of these programs on talent management initiatives including attraction and retention of top talent and the methodologies used to do so. This report will be inclusive of all Human Resource functions and provide both strategic and operational goals of the desired outcomes and, the means or “how” to reach these deliverables.

Considering employee retention, how would you make a convincing argument to senior management at Jupiter of the escalating costs of employee turnover. Give details. Explain how to conduct exit interviews and why this gathered information can be utilized to identify issues and reduce turnover. Considering the core activities associated with HR, which activities can be leveraged to promote employee retention strategies? (Example: Compensation & Benefits, Career Development, etc.) Describe how employee engagement goes beyond simple job satisfaction

PLEASE ANSWER ALL NOT UNDERSTANDING

1.An unsuspecting bird is coasting along in an easterly direction at 3.00 mph when a strong wind from the south imparts a constant acceleration of 0.200 m/s2. If the wind\'s acceleration lasts for 3.20 s, find the magnitude r and direction θ (measured counterclockwise from the easterly direction) of the bird\'s displacement over this time interval. (HINT: assume the bird is originally travelling in the x direction and there are 1609 m in 1 mile.)

Now, assume the same bird is moving along again at 3.00 mph in an easterly direction but this time the acceleration given by the wind is at a 45.0 degree angle to the original direction of motion. If the magnitude of the acceleration is 0.500 m/s2, find the displacement vector , and the angle of the displacement, θ1. Enter the components of the vector and angle below. (Assume the time interval is still 3.20 s.)

2 .Volumetric expansion coefficients of simple materials are often well catalogued. However, the thermal expansion coefficient β of a human body is less well known. This could affect the human body\'s specific gravity and, therefore, measurements of its body/fat ratio. Suppose that a human body of weight w0 (on dry land) is placed on a scale while completely immersed in formaldehyde of temperature T1. Once the temperature increases by ΔT, the scale reading drops by Δw. Derive an expression for β in terms of ΔT, w0, and Δw by assuming that the ratio of the formaldehyde ρf and the initial density of the body ρb is R = ρf /ρb. Assume also that ρf does not change when heated.

If the body weighs 224.3 lb on dry land and his weight reading lowers by 0.237 lb when the formaldehyde is heated from 66.40oF to 88.60oF, calculate the coefficient of volume expansion of the body.* Assume R = 0.720.

* The coefficient β will likely vary widely from one human body to the next. The numerical value computed here should not be considered factual.

3. If 39.5 mol of an ideal gas occupies 89.5 L at 77.00 °C, what is the pressure of the gas?

4. How many moles of gas are in 17.5 L N2 at 25 °C and 125 kPa?

How many moles of gas are in 41.9 L O2 at 25 °C and 125 kPa?

5. A 2250-kg space station orbits Earth at an altitude of 495000 m. Find the magnitude of the force with which the space station attracts Earth. The mass and mean radius of Earth are 5.98 × 1024 kg and 6370000 m, respectively.

Please respond to the discussion post 1 and 2 in your own words: (Must respond to both)

Discussion post 1

How do you think most people see the majority of human interactions, as opportunities to compete or to cooperate?

I believe that the majority of human interactions are seen as opportunities to compete. The reason I say this is the competitive nature that we have in us as humans. This nature is exemplified daily in our school and our work. Let’s think about today’s workforce. It is extremely competitive and dog eats dog. The old days of job security with the same company for forty years is long gone. Being that Michigan is an at-will state, employers can let employees go for no reason at any time that they want. This makes employees want to be competitive, because they feel like their job is on the line. Some people will do anything to make sure that they are being looked at with a keen eye by management. They will do anything in their power to make sure they are getting credit for things they do, and even things that they don’t.

Sports also bring out this extremely competitive nature in humans. Sports are one of the most competitive activities in this world. Two teams giving their all to win. That feeling of winning is what drives the competitive nature of the players.

Can Covey's "Win-Win or No Deal" approach be "strategic" in the ultimate effects it may accrue?

I think that this approach can be strategic in the effects it accrues. The reason I say this is because in this approach everybody is happy in some way. This is a great thing for personal relationships, as stated in the examples that Covey talks about in the text. I think that this approach builds trust between people as well, whether that be in a personal or professional relationship. Unlike some of the other approaches that Covey discusses, this is the least-selfish of the approaches.

Discussion post 2

How do you think most people see the majority of human interactions, as opportunities to compete or to cooperate?

I think that most people see the majority of human interactions as opportunities to compete. I believe that at one point, it was to cooperate but as the society we live in have become more competitive, so have the individuals who live in it. This isn't to say that our human interactions are not genuine in wanting to understand others and get along amongst each other by any means. I also find that competing and cooperating can go hand in hand with one or the other playing a more heavy role in the interaction depending on the circumstances. This often happens at work during promotion time. When a position is available and all of the women are competing amongst each other, they want to cooperate on a human level as they are friends but many of their interactions are competitive, trying to "out shine" each other while discussing their successes that they feel the other may not have. As promotions are constantly reoccurring where I work, I find in my specific situation that the interaction is more for the opportunity to compete. These circumstances vary depending on your environment so I certainly believe that in other circumstances such as volunteer work the interaction would be more to cooperate as everyone is working for one common goal rather than competing.

Can Covey's "Win-Win or No Deal" approach be "strategic" in the ultimate effects it may accrue?

I believe so. I find it to be strategic in the effects it may accrue because no person or wants to give others a "win" and find nothing coming their way in return. By having a "win-win or no deal" approach, everyone involved is satisfied in some way which could be influential for positive relationships, both personal and professional. I think by having a win-win approach, it also allows for trust in a building relationship. By making sure both parties are satisfied and win, a stronger bond is built. I think this approach can be highly strategic in business because having business partners or organizations where you and they share mutual trust and carry a "win-win or no deal" mentality allows everyone involved to know they will be satisfied with the outcome. This makes you as a person (or business) more approachable and trustworthy when others know you have this kind of mentality.

1. If you were creating a library using the restriction endonucleases SbfI and EcoRI, for which restriction site would you design a fork-tailed adapter? Can you imagine any characteristic of a genome that would cause you to design a fork-tailed adapter for the other restriction site? If you were to use this set of enzymes/adapters to create a library in corn, how many reads would you need for adequate coverage of each individual?

2. How many reads would you need using SbfI/EcoRI in zebrafish to provide adequate cover for each individual? How many would you need for a stickleback fish? Does this make sense, given the size of the respective genomes? How could you explain this observation? Does looking at the results for different enzyme combinations for these two species help to explain the observation?

3. How many individuals could you analyze on a single MiSeq run, if you were using the combination of NlaIII and MluCI on mice?

4. If you were interested in doing a study of population structure in corn, which enzyme combination would you choose?  How many individuals could you analyze with a single MiSeq run? How about with a single lane of an Illumina HiSeq 2000?

5. Suppose you were using paired-end sequencing with 250 cycles. How much of the genome of a stickleback fish would you expect to sequence at >7X coverage if you used EcoRI/MspI?

6. You amplify an EcoRI/MspI fragment using MspI 2.1.0 and EcoRI 1.1.0 adapters and the the PCR primers NGS_i5_S505 and NGS_i7_N719. What would the final amplicon look like? (Write the sequence.)  

7. What happens to MspI/MspI fragments in the ddRadSeq protocol we are following? How about EcoRI/EcoRI fragments?

8. We are using three different MspI and EcoRI adapters in our protocol. There are a couple of advantages to using more than one adapter for each end; what are they?

9. Taking into consideration the different indices available, and the use of three different adapters for each end of genomic fragments, how many different individuals could be analyzed with a single sequencing run?

10. Suppose you were analyzing mice just as depicted in Table 1. of Petersen et al., using the enzyme pair EcoRI-MspI  You want to increase the number of fragments you are analyzing by 50%; how would you adjust your protocol?

11. For SNP discovery, there are concerns that ddRadSeq may limit analysis to regions of the genome with low point mutation rates. Please explain why this would be so.

12. Explain the similarities between ddRadSeq and two-step PCR. Why isn’t a PCR step (instead of ligation) used on the genomic fragments in ddRadSeq?

13. If you were using single step PCR, how many different primers would you need to take full advantage of the indices available from Illumina (S502, N701, etc.) for one locus? How many would be needed if you were using two-step PCR for one locus? How would the answers change for 10 loci?

14. What are the advantages of two-step PCR over one-step PCR? Are there any conditions under which one-step PCR is more efficient?

15. A typical number of cycles for the second PCR in two-step PCR is 10. How many copies of each input molecule would be generated in ten cycles?

16. What would happen if you used two-step PCR, and the primers used in the first step were both compatible with i7 primers? Would amplification happen in the second step of the two-step PCR process? Could the amplicon be sequenced?

The table below represents the production function for Hawg Wild, a small catering company specializing in barbecued pork. The numbers in the cells represent the number of customers that can be served with various combinations of labor and capital.

a.Is this production function a short-run or long-run production function? How can you tell?

b.Suppose that Hawg Wild employs 5 units of capital and 2 workers. How many diners will be served?

c.Suppose that Hawg Wild employs 5 units of capital and 2 workers, but that the owner, Billy Porcine, is considering adding his nephew to the payroll. What will the marginal product of Billy’s nephew be?

d.Notice that when Hawg Wild uses 1 unit of capital, the marginal product of the fifth unit of labor is 16. But when Hawg Wild uses 5 units of capital, the marginal product of the fifth unit of labor is 43. Does this production function violate the law of diminishing marginal product of labor? Why or why not?

e.Suppose that Hawg Wild employs 5 units of capital and 2 workers, but that the owner, Billy Porcine, is considering adding another meat smoker to the kitchen (which will raise the amount of capital input to 6 units). What will the marginal product of the smoker be?

f. Hawg Wild employs 5 units of capital and 2 workers. Billy is considering the choice between hiring another worker or buying another smoker. If smokers cost $8 and workers $12, then at the margin, what is the most cost-effective choice for Billy to make?

1. Assuming the underlying demand for cases of O Be Joyful beverages is a linear function of O Be Joyful per-case price and state income (000s), use the data to obtain least squares estimates for each state's beverage demand function (three separate regressions). Report each estimated demand function in your memo. Please feel free to attach summary results to your memo. Your memo should stand alone, however, as communication. Do not ask the reader to go to attachments to justify any argument.
   1. Estimate own-price and income elasticities for each state and interpret the results. (Please see three tables below)

Wisconsin

Kindly write a one page summary of the content below including important details. Thank you.

Abstract Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion [1], but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody [2] and gene cloning, we identify a mouse sperm fusion related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a genedisrupted mouse line. Izumo −/− mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zonafree hamster eggs. Identification of Izumo To identify factors involved in sperm–egg fusion, we used a monoclonal antibody, OBF13, against mouse sperm that specifically inhibits the fusion process [2]. The antigen was identified by separation of the crude extracts from mouse sperm by two-dimensional gel electrophoresis and subsequent immunoblotting with the monoclonal antibody. We named the antigen ‘Izumo’ after a Japanese shrine dedicated to marriage. The identified spot was analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS), and ten peptides that were 100% identical to a part of the sequence listed in the RIKEN full-length database were found. The registered DNA sequence was confirmed by sequencing after polymerase chain reaction with reverse transcription (RT–PCR) with total RNA prepared from the testis. A human homologue was found as an unverified gene in the NCBI database. The gene encodes a novel immunoglobulin superfamily (IgSF), type I membrane protein with an extracellular immunoglobulin-like domain that contains one putative glycosylation site (Fig. 1a). Mouse Izumo was shown to be a testis (sperm)-specific 56.4-kDa antigen by western blotting with a polyclonal antibody raised against recombinant mouse Izumo (Fig. 1b). Izumo was also detectable as a 37.2-kDa protein by western blotting of human sperm with anti-human Izumo antibody (Fig. 1c). Izumo was not detectable on the surface of fresh sperm. Coinciding with the fact that mammalian sperm are incapable of fertilizing eggs when ejaculated and that fertilization occurs only after an exocytotic process called the acrosome reaction, both mouse and human Izumo became detectable on sperm surface only after the acrosome reaction (Fig. 1d, e). This would probably be because Izumo is not localized on plasma membrane of fresh spermatozoa but is hidden under plasma membrane and accessible after the acrosome reaction, as occurs with CD46 on mouse sperm [3]. Figure 1 Identification and characterization of Izumo. a, Izumo is a typical type I membrane glycoprotein with one immunoglobulin-like domain and a putative N-glycoside link motif (Asn 204). b, Izumo was detected exclusively in testis and sperm by western blotting. The tissues examined are, from left to right: brain, heart, thymus, spleen, lung, liver, muscle, kidney, ovary, testis and sperm. The arrowhead indicates mouse Izumo protein. c, Western blotting analysis of human Izumo protein from human sperm. The arrow indicates human Izumo protein. d, Immunostaining of Izumo in sperm from an acrosin-promoter-driven transgenic mouse line that has enhanced green fluorescent protein in the acrosome. Izumo was not detected in fresh sperm with intact acrosomes expressing EGFP (indicated by green arrows), but was revealed on acrosome-reacted (non-green fluorescent) sperm (stained red, shown by white arrowheads), when stained with the polyclonal antibody against mouse Izumo. e, Human sperm were also stained with polyclonal anti-human Izumo antibody (red). Acrosome-reacted human sperm (stained green with anti-CD46 antibody) were reactive to the antibody against human Izumo but the same antibody did not react to acrosome-intact (CD46-negative) sperm. Scale bar, 10 mm. 40 ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005 Establishment of Izumo-deficient mice To address the physiological role of Izumo in vivo we generated Izumo-deficient mice by homologous recombination. An Izumo targeting construct was designed to replace exons 2–10 with a neomycin-resistant gene (neor ). Both the targeting event in D3 embryonic stem cells and the germline transmission of targeted genes were confirmed by Southern blot analysis. In the homozygous mutant mice, the full-length messenger RNA and the Izumo protein were not detected. Because the disruption of a gene can cause a concomitant increase or decrease in some related genes, we examined CD46, sp56, CD55, CD147, and ADAM2, which were reported to be involved in sperm–egg interactions. We could not find a significant change in these protein levels in sperm after the deletion of Izumo gene. The fecundity of Izumo-deficient males Izumo −/−mutant mice were healthy and showed no overt developmental abnormalities. Izumo -/- females demonstrated normal fecundity. Izumo +/− males also showed normal fertilizing ability. However, Izumo −/− males were sterile despite normal mating behaviour and ejaculation, with normal vaginal plug formations. After observation of 28 plugs, nine pairs of Izumo −/− male and wild-type females were kept for another 4 months but no pregnancies were observed. In at least four different cases of gene knockouts that resulted in male sterility attributed to impaired zonabinding ability, the sperm also failed to migrate into the oviduct. However, disruption of Izumo did not cause any defect in sperm migration into the oviduct (data not shown, and there was no reduction of sperm motility in Izumo −/− sperm motility was measured 120 min after incubation by computer-aided sperm analysis (CASA; mean; s.e.m.=81.7±7.7% in Izumo +/− sperm and 77±8.9% in Izumo −/− sperm)). The sterile nature of Izumo −/− sperm was shown in the in vitro fertilization system (Fig. 2a). The impaired fertilization step undoubtedly followed zona penetration because sperm penetrated the zona pellucida and accumulated in the perivitelline space of the eggs (Fig. 2b). Fusion ability in Izumo-deficient sperm Syngamy can be considered to occur to two stages: binding of the sperm plasma membrane to that of the egg, and actual membrane fusion. Izumo −/− sperm were capable of binding to the plasma membranes of eggs whose zona pellucida had been mechanically removed [4] (Fig. 2c). In this system, the Izumo +/− sperm incubated for 2 and 6 h fused to eggs in approximate ratios of 4.5 and 6 sperm per egg, respectively, but no Izumo −/− sperm fused with eggs (Fig. 2c). Sperm can not fuse with eggs unless the former have undergone the acrosome reaction. To verify the acrosomal status of Izumo −/− sperm, we stained the sperm accumulated in perivitelline spaces with the MN9 monoclonal antibody, which immunoreacts only to the equatorial segment of acrosomereacted sperm [5]. The staining indicated that the Izumo −/− sperm had undergone the acrosome reaction (Fig. 2b) but failed to fuse with eggs. Development of eggs after intracytoplasmic sperm injection (ICSI) with Izumo-deficient sperm Because no offspring were fathered by Izumo −/− male mice, it was unclear whether the defect was limited to fusion or extended to later developmental stages. To address this question, we used ICSI to insert Izumo −/− sperm directly into the cytoplasm of wild-type eggs and bypass the fusion step. Eggs injected with Izumo −/− sperm were successfully activated and the fertilized eggs were transplanted into the oviducts of pseudopregnant females. The eggs implanted normally and the resulting embryos developed appropriately to term with rates similar to those of heterozygous mice. Human Izumo is also involved in sperm-egg fusion Sperm–egg fusion is known to be less species-specific than sperm–zona interaction. For example, human sperm can not penetrate the hamster zona pellucida but they can fuse with zona-free hamster eggs, and this system (zona-free hamster-egg sperm penetration test) has been used for the assessment of human sperm fertility. We first examined the contribution of mouse Izumo in a zona-free hamster-egg sperm penetration assay. As indicated in Fig. 3a, the mouse Izumo was essential not only in the homologous fusion system but also for heterologous fusion with hamster eggs. Similarly, when the anti-human Izumo polyclonal antibody was added to the incubation mixture, no fusion was observed, whereas the sperm treated with control IgG fused with eggs at an average of 5.9±0.7 sperm per egg. The total numbers of eggs observed were 23 and 29, respectively (n=3). These results indicated that human Izumo is involved in the fertilization process in human sperm (Fig. 3b). Rescued fertility of Izumo-deficient male by transgene The phenotypes of gene knockout mice are not always related Figure 2 Male infertility caused by Izumo disruption. a, In vitro fertilization of sperm from Izumo +/−and Izumo −/−mice. Unlike Izumo +/−, the eggs inseminated with Izumo −/− sperm had many sperm on their zona pellucida, owing to the failure of sperm–egg fusion that probably leads to the absence of zona-reaction to lessen the sperm-binding ability of the zona pellucida. b,Upper panel, accumulation of many sperm in the perivitelline space of the eggs recovered from the females mated with Izumo −/− males. Lower panel, sperm in perivitelline space labelled with acrosome reacted, spermspecific monoclonal antibody MN9. c, Fused sperm stained by Hoechst 33342 preloaded into the egg. The arrowheads show the fused sperm. to the disrupted genes but are sometimes caused by disruption of a neighbouring gene. To examine whether the phenotype was directly derived from the lack of Izumo on sperm, we performed a rescue experiment by crossing Izumo −/− mice with transgenic mouse lines generated to express Izumo by using the testis-specific calmegin promoter [6]. The sterile phenotype was rescued with the transgenically expressed Izumo on mouse sperm (Fig. 4). Discussion In the search for sperm surface proteins that function in sperm–egg plasma-membrane binding and fusion, various candidates such as DE, CD46, equatorin, Sperad and SAMP32 have been reported. ADAM family proteins are given the most attention for their possession of a putative fusion peptide (ADAM1) and disintegrin domain (ADAM2 and ADAM3). None of the mice possessing disrupted ADAM1a, ADAM2 and ADAM3 show a significant defect in the ability to fuse with eggs [7-9], but do show an impairment of sperm–zona binding ability. Similarly, CD46 disruption does not diminish fusion [3]. In contrast, CD9 on the egg surface is essential for the fusing ability of eggs [1] and some indications for the involvement of the binding of integrins to CD9 are postulated in reference to sperm–egg fusion. However, the disruptions of the most probable candidate integrin α6β1 cause no major influence on the fusing ability of eggs. Thus, for several years, postulated fertilization mechanisms were repeatedly changed as a result of gene disruption experiments. This suggests that the essential nature of the candidate gene must be judged after observing the phenotype of the gene-disrupted mice. In this context, Izumo is the first sperm membrane protein shown to be essential for fusion. It is not yet known whether sperm Izumo interacts with egg CD9, as occurs with placental IgSF protein PSG17; neither do we know why the localization of Izumo after acrosome reaction is not limited to the 41 Osaka University 100 Papers : 10 Selected Papers ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005 Figure 4 Transgene to express mouse Izumo under the control of calmegin promoter. a, The locations of primers A to E were indicated in this figure. b, lane 1; Izumo +/− mouse with intrinsic Izumo, lane 2 and 3; Izumo −/− mouse with transgenically expressed Izumo and Izumo His-tag, respectively. c, Litter size obtained by mating male mice with C57BL/6 wild-type mice. The group numbers are equal to those shown in b. The numbers in parentheses indicate the numbers of matings. References 1. Miyado, K. et al., Requirement of CD9 on the egg plasma membrane for fertilization. Science, 287, 321-4 (2000). 2. Okabe, M. et al., Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. J Reprod Immunol, 11, 91-100 (1987). 3. Inoue, N. et al., Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm. Mol Cell Biol, 23, 2614-22 (2003). 4. Yamagata, K. et al., Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane. Dev Biol, 250, 348-57 (2002). 5. Manandhar, G. & Toshimori, K., Exposure of sperm head equatorin after acrosome reaction and its fate after fertilization in mice. Biol Reprod, 65, 1425-36 (2001). 6. Ikawa, M. et al., Calmegin is required for fertilin alpha/beta heterodimerization and sperm fertility. Dev Biol, 240, 254-61 (2001). 7. Cho, C. et al., Fertilization defects in sperm from mice lacking fertilin beta. Science, 281, 1857-9 (1998). 8. Nishimura, H., Cho, C., Branciforte, D. R., Myles, D. G. & Primakoff, P., Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta. Dev Biol, 233, 204-13 (2001). 9. 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, 279, 34957-62 (2004). equatorial segment where fusion initially takes place. All we can say now is that continued study of this protein’s function will undoubtedly lead to a fuller understanding of the cell–cell fusion process in fertilization and perhaps in other somatic systems such as muscle cells or trophoblasts. The finding not only provides insight into the enigmatic fusion mechanism but also promises benefits in the clinical treatment of infertility and the potential development of new contraceptive strategies

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Abstract Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion [1], but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody [2] and gene cloning, we identify a mouse sperm fusion related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a genedisrupted mouse line. Izumo −/− mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zonafree hamster eggs. Identification of Izumo To identify factors involved in sperm–egg fusion, we used a monoclonal antibody, OBF13, against mouse sperm that specifically inhibits the fusion process [2]. The antigen was identified by separation of the crude extracts from mouse sperm by two-dimensional gel electrophoresis and subsequent immunoblotting with the monoclonal antibody. We named the antigen ‘Izumo’ after a Japanese shrine dedicated to marriage. The identified spot was analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS), and ten peptides that were 100% identical to a part of the sequence listed in the RIKEN full-length database were found. The registered DNA sequence was confirmed by sequencing after polymerase chain reaction with reverse transcription (RT–PCR) with total RNA prepared from the testis. A human homologue was found as an unverified gene in the NCBI database. The gene encodes a novel immunoglobulin superfamily (IgSF), type I membrane protein with an extracellular immunoglobulin-like domain that contains one putative glycosylation site (Fig. 1a). Mouse Izumo was shown to be a testis (sperm)-specific 56.4-kDa antigen by western blotting with a polyclonal antibody raised against recombinant mouse Izumo (Fig. 1b). Izumo was also detectable as a 37.2-kDa protein by western blotting of human sperm with anti-human Izumo antibody (Fig. 1c). Izumo was not detectable on the surface of fresh sperm. Coinciding with the fact that mammalian sperm are incapable of fertilizing eggs when ejaculated and that fertilization occurs only after an exocytotic process called the acrosome reaction, both mouse and human Izumo became detectable on sperm surface only after the acrosome reaction (Fig. 1d, e). This would probably be because Izumo is not localized on plasma membrane of fresh spermatozoa but is hidden under plasma membrane and accessible after the acrosome reaction, as occurs with CD46 on mouse sperm [3]. Figure 1 Identification and characterization of Izumo. a, Izumo is a typical type I membrane glycoprotein with one immunoglobulin-like domain and a putative N-glycoside link motif (Asn 204). b, Izumo was detected exclusively in testis and sperm by western blotting. The tissues examined are, from left to right: brain, heart, thymus, spleen, lung, liver, muscle, kidney, ovary, testis and sperm. The arrowhead indicates mouse Izumo protein. c, Western blotting analysis of human Izumo protein from human sperm. The arrow indicates human Izumo protein. d, Immunostaining of Izumo in sperm from an acrosin-promoter-driven transgenic mouse line that has enhanced green fluorescent protein in the acrosome. Izumo was not detected in fresh sperm with intact acrosomes expressing EGFP (indicated by green arrows), but was revealed on acrosome-reacted (non-green fluorescent) sperm (stained red, shown by white arrowheads), when stained with the polyclonal antibody against mouse Izumo. e, Human sperm were also stained with polyclonal anti-human Izumo antibody (red). Acrosome-reacted human sperm (stained green with anti-CD46 antibody) were reactive to the antibody against human Izumo but the same antibody did not react to acrosome-intact (CD46-negative) sperm. Scale bar, 10 mm. 40 ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005 Establishment of Izumo-deficient mice To address the physiological role of Izumo in vivo we generated Izumo-deficient mice by homologous recombination. An Izumo targeting construct was designed to replace exons 2–10 with a neomycin-resistant gene (neor ). Both the targeting event in D3 embryonic stem cells and the germline transmission of targeted genes were confirmed by Southern blot analysis. In the homozygous mutant mice, the full-length messenger RNA and the Izumo protein were not detected. Because the disruption of a gene can cause a concomitant increase or decrease in some related genes, we examined CD46, sp56, CD55, CD147, and ADAM2, which were reported to be involved in sperm–egg interactions. We could not find a significant change in these protein levels in sperm after the deletion of Izumo gene. The fecundity of Izumo-deficient males Izumo −/−mutant mice were healthy and showed no overt developmental abnormalities. Izumo -/- females demonstrated normal fecundity. Izumo +/− males also showed normal fertilizing ability. However, Izumo −/− males were sterile despite normal mating behaviour and ejaculation, with normal vaginal plug formations. After observation of 28 plugs, nine pairs of Izumo −/− male and wild-type females were kept for another 4 months but no pregnancies were observed. In at least four different cases of gene knockouts that resulted in male sterility attributed to impaired zonabinding ability, the sperm also failed to migrate into the oviduct. However, disruption of Izumo did not cause any defect in sperm migration into the oviduct (data not shown, and there was no reduction of sperm motility in Izumo −/− sperm motility was measured 120 min after incubation by computer-aided sperm analysis (CASA; mean; s.e.m.=81.7±7.7% in Izumo +/− sperm and 77±8.9% in Izumo −/− sperm)). The sterile nature of Izumo −/− sperm was shown in the in vitro fertilization system (Fig. 2a). The impaired fertilization step undoubtedly followed zona penetration because sperm penetrated the zona pellucida and accumulated in the perivitelline space of the eggs (Fig. 2b). Fusion ability in Izumo-deficient sperm Syngamy can be considered to occur to two stages: binding of the sperm plasma membrane to that of the egg, and actual membrane fusion. Izumo −/− sperm were capable of binding to the plasma membranes of eggs whose zona pellucida had been mechanically removed [4] (Fig. 2c). In this system, the Izumo +/− sperm incubated for 2 and 6 h fused to eggs in approximate ratios of 4.5 and 6 sperm per egg, respectively, but no Izumo −/− sperm fused with eggs (Fig. 2c). Sperm can not fuse with eggs unless the former have undergone the acrosome reaction. To verify the acrosomal status of Izumo −/− sperm, we stained the sperm accumulated in perivitelline spaces with the MN9 monoclonal antibody, which immunoreacts only to the equatorial segment of acrosomereacted sperm [5]. The staining indicated that the Izumo −/− sperm had undergone the acrosome reaction (Fig. 2b) but failed to fuse with eggs. Development of eggs after intracytoplasmic sperm injection (ICSI) with Izumo-deficient sperm Because no offspring were fathered by Izumo −/− male mice, it was unclear whether the defect was limited to fusion or extended to later developmental stages. To address this question, we used ICSI to insert Izumo −/− sperm directly into the cytoplasm of wild-type eggs and bypass the fusion step. Eggs injected with Izumo −/− sperm were successfully activated and the fertilized eggs were transplanted into the oviducts of pseudopregnant females. The eggs implanted normally and the resulting embryos developed appropriately to term with rates similar to those of heterozygous mice. Human Izumo is also involved in sperm-egg fusion Sperm–egg fusion is known to be less species-specific than sperm–zona interaction. For example, human sperm can not penetrate the hamster zona pellucida but they can fuse with zona-free hamster eggs, and this system (zona-free hamster-egg sperm penetration test) has been used for the assessment of human sperm fertility. We first examined the contribution of mouse Izumo in a zona-free hamster-egg sperm penetration assay. As indicated in Fig. 3a, the mouse Izumo was essential not only in the homologous fusion system but also for heterologous fusion with hamster eggs. Similarly, when the anti-human Izumo polyclonal antibody was added to the incubation mixture, no fusion was observed, whereas the sperm treated with control IgG fused with eggs at an average of 5.9±0.7 sperm per egg. The total numbers of eggs observed were 23 and 29, respectively (n=3). These results indicated that human Izumo is involved in the fertilization process in human sperm (Fig. 3b). Rescued fertility of Izumo-deficient male by transgene The phenotypes of gene knockout mice are not always related Figure 2 Male infertility caused by Izumo disruption. a, In vitro fertilization of sperm from Izumo +/−and Izumo −/−mice. Unlike Izumo +/−, the eggs inseminated with Izumo −/− sperm had many sperm on their zona pellucida, owing to the failure of sperm–egg fusion that probably leads to the absence of zona-reaction to lessen the sperm-binding ability of the zona pellucida. b,Upper panel, accumulation of many sperm in the perivitelline space of the eggs recovered from the females mated with Izumo −/− males. Lower panel, sperm in perivitelline space labelled with acrosome reacted, spermspecific monoclonal antibody MN9. c, Fused sperm stained by Hoechst 33342 preloaded into the egg. The arrowheads show the fused sperm. to the disrupted genes but are sometimes caused by disruption of a neighbouring gene. To examine whether the phenotype was directly derived from the lack of Izumo on sperm, we performed a rescue experiment by crossing Izumo −/− mice with transgenic mouse lines generated to express Izumo by using the testis-specific calmegin promoter [6]. The sterile phenotype was rescued with the transgenically expressed Izumo on mouse sperm (Fig. 4). Discussion In the search for sperm surface proteins that function in sperm–egg plasma-membrane binding and fusion, various candidates such as DE, CD46, equatorin, Sperad and SAMP32 have been reported. ADAM family proteins are given the most attention for their possession of a putative fusion peptide (ADAM1) and disintegrin domain (ADAM2 and ADAM3). None of the mice possessing disrupted ADAM1a, ADAM2 and ADAM3 show a significant defect in the ability to fuse with eggs [7-9], but do show an impairment of sperm–zona binding ability. Similarly, CD46 disruption does not diminish fusion [3]. In contrast, CD9 on the egg surface is essential for the fusing ability of eggs [1] and some indications for the involvement of the binding of integrins to CD9 are postulated in reference to sperm–egg fusion. However, the disruptions of the most probable candidate integrin α6β1 cause no major influence on the fusing ability of eggs. Thus, for several years, postulated fertilization mechanisms were repeatedly changed as a result of gene disruption experiments. This suggests that the essential nature of the candidate gene must be judged after observing the phenotype of the gene-disrupted mice. In this context, Izumo is the first sperm membrane protein shown to be essential for fusion. It is not yet known whether sperm Izumo interacts with egg CD9, as occurs with placental IgSF protein PSG17; neither do we know why the localization of Izumo after acrosome reaction is not limited to the 41 Osaka University 100 Papers : 10 Selected Papers ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005 Figure 4 Transgene to express mouse Izumo under the control of calmegin promoter. a, The locations of primers A to E were indicated in this figure. b, lane 1; Izumo +/− mouse with intrinsic Izumo, lane 2 and 3; Izumo −/− mouse with transgenically expressed Izumo and Izumo His-tag, respectively. c, Litter size obtained by mating male mice with C57BL/6 wild-type mice. The group numbers are equal to those shown in b. The numbers in parentheses indicate the numbers of matings. References 1. Miyado, K. et al., Requirement of CD9 on the egg plasma membrane for fertilization. Science, 287, 321-4 (2000). 2. Okabe, M. et al., Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. J Reprod Immunol, 11, 91-100 (1987). 3. Inoue, N. et al., Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm. Mol Cell Biol, 23, 2614-22 (2003). 4. Yamagata, K. et al., Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane. Dev Biol, 250, 348-57 (2002). 5. Manandhar, G. & Toshimori, K., Exposure of sperm head equatorin after acrosome reaction and its fate after fertilization in mice. Biol Reprod, 65, 1425-36 (2001). 6. Ikawa, M. et al., Calmegin is required for fertilin alpha/beta heterodimerization and sperm fertility. Dev Biol, 240, 254-61 (2001). 7. Cho, C. et al., Fertilization defects in sperm from mice lacking fertilin beta. Science, 281, 1857-9 (1998). 8. Nishimura, H., Cho, C., Branciforte, D. R., Myles, D. G. & Primakoff, P., Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta. Dev Biol, 233, 204-13 (2001). 9. 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, 279, 34957-62 (2004). equatorial segment where fusion initially takes place. All we can say now is that continued study of this protein’s function will undoubtedly lead to a fuller understanding of the cell–cell fusion process in fertilization and perhaps in other somatic systems such as muscle cells or trophoblasts. The finding not only provides insight into the enigmatic fusion mechanism but also promises benefits in the clinical treatment of infertility and the potential development of new contraceptive strategies