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viewS, cOmmeNtarieS & OpiNiONS Spermatogenesis 2:4, 238-244; October/November/December 2012; © 2012 Landes Bioscience In vitro spermatogenesis A long journey to get tails Hye-Won Song and Miles F. Wilkinson* Department of Reproductive Medicine; School of Medicine; University of California, San Diego; La Jolla, CA USA The generation of functional sperm with germ cells to drive spermatogenesis. in vitro has been a goal for almost a Other somatic cells also participate in century. Until recently, researchers have spermatogenesis, including the peritu- only succeeded in reproducing the early bular myoid cells, which help form the steps of spermatogenesis. This is not seminiferous tubule structure, and the tes- surprising given that spermatogenesis is tosterone-producing Leydig cells, which a complicated process that requires the are located in the interstitial area between coordinated efforts of germ cells and tubules.1 several somatic cells within the tubu- Given that spermatogenesis normally lar structure of the testis. Finally—last occurs in seminiferous tubules and that year—Sato et al. reported the success- many somatic cell types participate in ful in vitro production of functional spermatogenesis, spermatogenesis has sperm, thereby potentially opening been difficult to reproduce in vitro. up a new era of reproductive biology. Nevertheless, this has not prevented Here, we summarize the history of researchers from trying! Indeed, as we research directed toward reproducing summarize in this commentary, there steps of spermatogenesis in vitro, detail have been scores of studies attempt- the seminal findings of Sato et al., and ing to achieve this goal. Driving these suggest ways that their approach can attempts have been the large rewards. be applied toward clinical applications For example, if a robust and efficient in and addressing fundamental questions vitro spermatogenesis procedure could be about the underlying mechanism of created, this would have potential clini- spermatogenesis. cal applications for treating some cases of Keywords: in vitro spermatogenesis, infertility and it could also greatly speed organ culture, spermatogonial stem cells, Introduction up the efforts to decipher the cellular and infertility, germ cells, stem cells, trans- molecular mechanisms underlying sper- plantation To become a functional sperm, an imma- matogenesis. Given these potential pay- Abbreviations: siRNA, small interfer- ture male germ cell undergoes a coordi- offs, there was much fanfare when Sato ing RNA; ISCI, intracytoplasmic sperm nated set of events that are collectively et al. recently reported the first success injection; ROSI, round spermatid injec- called spermatogenesis. This process in generating sperm from immature germ tion; SSCs, spermatogonial stem cells; occurs in tubules within the testis that cells in vitro.2 Using an organ culture iPS cell, induced pluripotent stem cell; harbor what is known as the seminiferous method that relies on defined medium IVF, in vitro fertilization; KSR, knock- epithelium. The heart of the seminiferous containing a putative spermatogenesis- out serum replacement; PGCs, primor- epithelium is a somatic cell—the Sertoli promoting factor that also maintains dial germ cells cell—that is in intimate contact with all embryonic stem (ES) cells, Sato et al. Submitted: 08/11/12 of the germ cells in the seminiferous epi- were able to generate elongated sperma- thelium, regardless of whether they are tids that were capable of fertilizing eggs Accepted: 09/03/12 undergoing mitosis, meiosis, or matura- and generating progeny. Here, we discuss http://dx.doi.org/10.4161/spmg.22069 tion. Sertoli cells provide essential factors the findings of Sato et al. and suggest for spermatogenesis and evidence suggests basic science and clinical applications for *Correspondence to: Miles F. Wilkinson; Email: [email protected] that they communicate back and forth their technique. 238 Spermatogenesis volume 2 issue 4 viewS, cOmmeNtarieS & OpiNiONS viewS, cOmmeNtarieS & OpiNiONS A Long Journey to Achieve testicular biopsies were able to give rise mature male germ cells from stem cells. In Vitro Spermatogenesis to male germ cells that progressed to the In one approach, investigators found that telophase-II stage, thereby casting some culturing mouse spermatogonial stem Organ culture. Since the first report— doubt on the reproducibility of their cul- cells (SSCs) with glial cell line-derived in 1920—providing evidence for limited turing method. neurotrophic factor (GDNF) allowed the differentiation of male germ cells when Culture of dissociated cells. Starting SSCs to grow for a long period of time cultured in vitro,3 there have been many in the 1980s, there was a burst in publica- and give rise to functional sperm when attempts to come up with ways to drive tions attempting to reproduce spermato- transplanted into germ cell-depleted further differentiation of germ cells. For genesis using dissociated testicular cells testes of wild-type mice.16,17 This was a example, in 1937, it was reported that a rather than organ culture. For example, major breakthrough, as the transplanted clot composed of fowl plasma and embryo it was reported that co-culture of imma- mice were able to father progeny by natu- extract permitted some of the mitoti- ture rat germ cells with primary Sertoli ral conception. Another group reported cally active spermatogonia within cul- cells for 11 d promoted their maturation that SSCs can be differentiated into early tured newborn mouse testes to progress into late pachytene spermatocytes.10 In the meiotic germ cells by simply culturing to the pachytene stage.4 In the 1960s, the 1990s, there were two reports providing the SSCs in retinoic acid (RA).18 It will Steinberger group identified several mea- evidence that immature germ cells could be interesting to determine whether RA sures that promoted long-term survival be pushed all the way to the haploid stage. in combination with appropriate culture of cultured testes, including the addition These studies,11,12 which revolved around conditions can drive later steps of male of pyruvate to the culture medium as an using immortalized Sertoli cell or germ germ cell differentiation. Other studies energy source, reducing the incubation cell lines, respectively, held great promise, have reported the successful generation temperature to 31°C to better simulate but, to our knowledge, no follow-up stud- of haploid germ cells from human and the intratesticular temperature in vivo, ies using these techniques have been pub- mouse embryonic stem (ES) cells.19,20 One and using a gas-liquid interphase method lished. Later—in 2002— it was reported of these studies demonstrated that haploid to increase oxygen exchange.5 Using tri- that immortalized mouse type-A sper- cells generated from the mouse ES cells tiated-thymidine to tag immature prolif- matogonial cells in the presence of stem were capable of triggering blastocyst for- erating male germ cells (spermatogonia cell factor (SCF) are capable of undergo- mation when injected into the cytoplasm and preleptotene spermatocytes) present ing differentiation into haploid spermatids of oocytes.19 However, whether these in rat testes explants, they demonstrated in vitro.13 However, no fully differentiated blastocysts can generate progeny when that some of these cells differentiated into germ cells (e.g., tailed spermatozoa) were injected into hormonally receptive female pachytene spermatocytes after culture in observed, it was not tested whether the mice has not yet been reported. vitro for 2 to 3 weeks.6 They also achieved round spermatids that are generated are In summary, the concerted efforts of similar results using human testicular functional competent for fertilization, many investigators over several decades biopsies.7 While encouraging, no germ and no follow-up studies have been subse- has led to the development of culture cells beyond the pachytene stage of meio- quently published. In the human system, a conditions for promoting specific steps sis were observed by either Steinberger and study reported that dissociated cells from in spermatogenesis in vitro. However, the colleagues or most other research groups testicular biopsy samples from humans ultimate goal of generating functional that attempted to improve culture con- with premeiotic maturation arrest at the sperm in vitro was not accomplished until, ditions during this early phase of the “in primary spermagocytes stage could be as described below, the Ogawa group vitro spermatogenesis” field. An exception coaxed into becoming haploid germ cells revisited the old organ culture method was one group that reported conditions after culture for 48 h.14 Remarkably, the using newly developed stem cell culture that allowed tritiated thymidine-labeled elongating spermatids generated in these techniques and a media supplement that human preleptotene spermatocytes to in vitro cultures were functional compe- they discovered is essential for spermatid differentiate into spermatocytes that pro- tent, as oocytes injected with them were maturation. gressed to the end of meiosis: the telo- shown to form healthy babies when trans- Complete spermatogenesis in vitro. phase-II stage. In their protocol, testicular planted into hormonally receptive human As a precursor to their groundbreaking biopsies were cultured for 14 d in media females.14 While promising, whether this work, the Ogawa group identified condi- containing deproteinized coconut milk, approach could be safely used in the clinic tions for culturing mouse testes fragments follicle-stimulating hormone (FSH) and is not clear since the time frame these from neonatal mice that allowed some of luteinizing hormone (LH).8 While poten- investigators found was sufficient for dif- the male germ cells in them to undergo tially promising, a concern with regard ferentiation of human meiotic germ cells a significant degree of maturation.21 To to physiological relevance is that these into elongating spermatids in vitro was assess maturation, they made the tes- investigators found that the speed of con- abnormally short (24–48 h); this multi- tes cultures from transgenic mice that version of the pre-leptotene germ cells to step process normally takes 20–22 d in express green fluorescent protein (GFP) telophase-II germ cells in vitro was more vivo.15 from either a mid-meiosis promoter or a rapid than normally occurs in vivo.9 A More recently, approaches have been late-meiosis promoter. This allowed them further concern is that only one out of 16 identified that drive the differentiation of to detect even small numbers of cells that www.landesbioscience.com Spermatogenesis 239 progressed to specific stages of meio- tubules of germ cell-depleted testes (made explants from neonatal testes2 and in only sis. Using this and other methods, they germ cell deficient by either genetic or 3 out of 17 cultured explants from SSC- detected some cells in the cultured testes chemical means), followed by culture of transplanted testes.22 Only the periph- chunks that completed meiosis and pro- fragments from these testes in the KSR- eral region of testes fragments exhibited gressed to the round spermatid stage. containing medium that they had shown advanced spermatogenesis in their system, While a significant advance, Ogawa promotes spermatogenesis in their ear- while the center lost normal morphology and coworkers were not able to detect any lier study.2 Remarkably, they found that, and had large numbers of degenerating germ cells in the testes chunks that pro- under these conditions, the transplanted cells after 40 d of culture.2 Furthermore, gressed past the round spermatid stage.21 SSCs migrated into the basement mem- they found a gradual decrease in germ cell They considered the possibility that the brane after only ~3 d of culture, began numbers over the 40-d culture period.2 fetal bovine serum in the culture medium proliferating a few days later, and then, Thus, creative approaches to improve was not supportive for this final stage of after almost two months, some of the germ cell viability are required, such as male germ cell maturation and thus they cells differentiated into elongated sperma- the addition of an “in vitro vascular” sys- screened for other tissue culture supple- tids. Some of the haploid spermatids were tem. Given that human application will ments that might have such activity. This functionally competent, as assessed by typically require using testes from ado- screen revealed that a supplement normally ROSI. These investigators also performed lescent or adult individuals, a practical used to maintain ES cells—knock out a proof-of-principal study in which they concern is whether explants from adult serum replacement (KSR) media—was asked whether their in vitro culture system testes can maintain spermatogenesis using able to promote the maturation of male was capable of correcting spermatogenic their culture conditions. In their mouse germ cells in cultured testes fragments failure.22 As a model system, they used experiments, Ogawa and colleagues used to the elongated spermatid stage.2 At steel mutant mice, which contain SSCs testes from neonatal mice2,21 or postnatal least some of these elongated spermatids but lack later stage male germ cells because (3-week-old) mice in the case of SSC- were functionally competent, as judged of a mutation in the gene encoding stem transplanted testes.22 Another issue for using intracytoplasmic sperm injection cell factor, a Sertoli-cell product essential human application is whether the sperma- (ICSI), a method that involves injection for the proliferation and differentiation tids generated from cryopreserved testes of elongated spermatids or spermatozoa of spermatogonial cells. To determine are functionally competent. In their mouse into mature oocytes, followed by in vitro whether they could drive the differen- studies, Ogawa and colleagues did not test culture of the fertilized oocytes to form tiation of SSCs from steel mutant testes whether explants from cryopreserved tes- two-cell embryos, and finally transfer of using their organ culture system, they tes can gave rise to functional spermatids, the embryos into the oviducts of pseudo- transplanted them into germ cell-deficient as judged, for example, by ISCI or ROSI.2 pregnant females. Importantly, Ogawa testes. They found that the transplanted In summary, the in vitro spermatogenesis and colleagues found that the females SSCs could be successfully differentiated approach described herein shows much implanted with these in vitro-generated into haploid germ cells in their in vitro promise. With improvements in efficiency spermatozoa-derived embryos delivered system. This result complemented an ear- and germ cell viability, along with altered live and fertile offspring. Even the less lier study from Brinster and colleagues, conditions to permit usage of mature tes- differentiated spermatids generated in which demonstrated restoration of sper- tes as input material, their technique is their organ culture system were capable matogenesis when SSCs from steel mutant likely to be useful for a variety of basic sci- of generating progeny, as they showed testes are transplanted into germ cell- ence and clinical applications, as described using a round spermatid injection (ROSI) deficient testes in vivo.23 Together, this below. technique. These investigators also tested earlier study23 and the more recent study whether their technique could be applied from Ogawa and colleagues,22 provide two Future Applications to a practical application: cryoprervation. independent paths—one in vitro and one In particular, they examined whether their in vivo—for assisted reproduction in large An efficient system for studying sper- system would support the proliferation host animals and humans. matogenesis. One of the technical chal- and differentiation of germ cells present While the identification of culture con- lenges holding back our understanding in cryopreserved neonatal testes tissues. ditions that permit immature male germ of spermatogenesis has been the lack of Indeed, they found that when thawed cells to mature into functional sperm is a an in vitro system that recapitulates sper- and cultured in KSR-containing medium, major breakthrough, there are still several matogenesis. Because of this deficit, many some immature germ cells in frozen testes issues that require attention. Not surpris- researchers have been forced to study samples proliferated and formed elongated ingly, efficiency is a major concern. Far mechanisms of spermatogenesis using in spermatids. fewer elongated spermatids and spermato- vivo systems. For example, to identify the In follow-up work, Ogawa and col- zoa are produced per input immature male role of specific genes in spermatogenesis, leagues tested whether they could use their germ cell using the currently available in it has been necessary to generate geneti- technique to differentiate mature germ vitro system2 than in a normal testis in cally modified mice harboring targeted cells from SSCs.22 They tested this by vivo. Ogawa and colleagues observed flag- mutations in specific genes. While this is transplanting SSCs into the seminiferous ellated sperm in only 5 out of 11 cultured an essential approach to understand the 240 Spermatogenesis volume 2 issue 4 lentiviruses can deliver transgenes into germ cells when injected into seminifer- ous tubules and that transgene expression in germ cells persisted for up to 3 mo.26 Given the contrasting results of these two studies, the question of whether or not lentiviruses efficiently infect germ cells in testes organ culture is an issue that needs to be revisited. For somatic cell types out- side of the seminiferous tubules—peritu- bular myoid cells and Leydig cells—it is likely that they will be highly susceptible to lentiviral infection since these cells are accessible to the culture media in cultured testes fragments. Thus, lentiviral vectors can probably be effectively used to test the role of genes in these two somatic cell types. In the case of germ cells, an alterna- tive approach is to introduce the vector or small interfering (si) RNA of interest into SSCs, followed by transplantation of these Figure 1.Studying genes involved in spermatogenesis using the in vitro spermatogenesis tech- SSCs into testes and then in vitro culture nique developed by Ogawa and colleagues. of explants generated from these testes. While this “in vivo-in vitro” approach has role of genes in vivo, it is extremely time using multiphoton fluorescence micros- not yet been tried, in principal it should be consuming and expensive. Furthermore, it copy, would allow germ cell develop- feasible as in vivo transplantation has been is often difficult to use such mouse mod- ment to be visualized in real time (Fig 1). used to identify genes involved in SSC els to identify molecular mechanisms. Another challenge is to work out how to self-renewal or differentiation.18,27 Another approach to study spermatogen- efficiently deliver vectors into the cells in How might in vitro approaches be esis has been to use immortalized cell organ culture in order to manipulate the specifically applied to studying sper- lines, but the number of cell lines available expression of genes of interest and thereby matogenesis? By coupling in vitro cul- for such studies is limited—particularly address their functional role. Two deliv- ture approaches with live tissue imaging, in the case of germ cells—and how well ery approaches are electroporation and the vector/siRNA-expressing cells can be such cell lines recapitulate events in nor- viral infection. Electroporation has been monitored, allowing for earlier detection mal cells is uncertain at best. Use of pri- successfully used to introduce genes into of defects than might be observed in vivo mary cells solves some of these problems, the testis in vivo,24 but it causes extensive (Fig. 1). To specifically study the cells but they are not trivial to prepare or main- tissue damage, limiting its use for many in which a given gene has been manipu- tain. Furthermore, the culture conditions applications. Also, electroporation typi- lated in explant cultures, one could use required for fully recapitulating spermato- cally only delivers genes transiently, so it two different reporters. As an example genesis with primary cells have not been is not generally suitable for determining application, a vector that co-expresses a identified, nor is it clear whether testicular functions occurring over long periods siRNA against the gene of interest and red cells—even if freshly isolated—placed in of time. Lentiviruses have the intrinsic fluorescent protein (RFP) could be intro- isolation on a plastic surface act in a physi- advantage of being able to integrate into duced into testes explants from a trans- ologically normal manner. the genome and thus infection of cells genic mouse containing a transgene that The ability to recapitulate spermato- with lentiviruses can provide long-term expresses GFP from a promoter active in genesis in organ culture2 solves many of gene expression. Indeed, lentiviruses have the germ cell stage of interest. Following these problems, but it also brings up new been used to successfully express genes for delivery of the vector, the testis fragments challenges. One challenge is how to detect long periods of time in cells in the semi- would be cultured as described2 and moni- germ cell maturation in an in vitro organ niferous tubule in vivo.25,26 However, a tored for GFP and RFP expression. If, for culture system. One approach to assay potential drawback of lentiviruses is that example, knockdown of the gene of inter- germ cell development is to use a battery of they may not be able to efficiently infect est by the siRNA causes a defect in pro- reporters (expressing different versions of most of the cells in organ culture. Indeed, gression to the stage monitored by GFP, GFP) under the control of promoters that one study found that lentiviruses infect there will be less RFP+/GFP+ double-pos- drive gene expression in different stages only Sertoil cells, not germ cells, when itive cells compared with testes fragments of germ cells. This advancement, cou- injected into seminiferous tubules.25 In delivered negative-control siRNAs. If cell pled with live organ imaging technology contrast, a recent report showed that type- or stage-specific expression of the www.landesbioscience.com Spermatogenesis 241 siRNA is required, this can be achieved by system could then be used to examine the spermatogenesis in cryopreserved juvenile using the appropriate promoter to drive its functional role of genes suspected to be testis biopsies since human spermatogen- expression.28 involved in human spermatogenesis by the esis typically does not initiate until after Not only is it likely to be much easier approaches described above for the mouse 10 y of age. If indeed human spermato- to examine the role of genes using in vitro system (Fig. 1). genesis can be safely reproduced in vitro, spermatogenesis procedures than generat- Infertility treatment. In vitro sper- then in principal it should be possible to ing and characterizing knockout mice, but matogenesis procedures have several use either the round or elongated sperma- in vitro procedures may provide opportu- potential applications in the clinic, includ- tids generated from cryopreserved juvenile nities that are not readily feasible with the ing: (1) restoration of fertility in juvenile testis biopsies to successfully fertilize eggs latter. For example, analysis of the redun- patients requiring chemotherapy, (2) gen- by either ISCI or ROSI, respectively (Fig. dant role of members of clustered gene eration of functional sperm from induced 2A). families cannot be easily accomplished pluripotent stem (iPS) cells, and (3) gene The discovery of methods to gener- using a standard knockout approach. This therapy approaches to generate fertile ate ES-like cells from fully differentiated is because the recombination frequencies sperm from infertile men. cells—iPS cells33,34—has opened up the of closely linked genes are too low to per- In the first application, the goal is to possibility of another means to generate mit feasible generation of double knock- provide juvenile cancer patients who functional spermatids in vitro (Fig. 2B). out progeny mice from single knockout have undergone high-dose chemotherapy The beauty of using iPS cells is that no tes- mice. In contrast, by introducing multiple with a source of functional spermatids ticular tissues from the donor are required. siRNAs in testes explants, one can rela- when they are ready to father children. In the way we envisage this approach, iPS tively easily examine the independent and The number of long-term survivors of cells generated from the individual would redundant roles of gene clusters. Another childhood cancer has increased greatly first be coaxed down the early male germ advantage is that one could feasibly per- in recent years, highlighting the need for cell lineage using in vitro methods that form genome-wide screening for factors identifying methods to recover the fertil- have already been developed for human involved in spermatogenesis using siRNA ity of these patients. One means to achieve ES cells.20,33 Following purification of the libraries (Fig. 1). this is to isolate SSCs from these patients male germ cells that have partially differ- Human spermatogenesis in vitro. prior to chemotherapy and then use them entiated in vitro from the iPS cells, they Studying mouse spermatogenesis will no for “auto-transplantation” in vivo (Fig. would be pushed further toward final doubt provide many insights into human 2A). This seems feasible given the recent maturity by transplanting them into tes- spermatogenesis, but ultimately, for the success of long-term culture of human ticular explants from another individual. purposes of human health, it will be cru- SSCs from cryopreserved testis biopsy.31 This latter step—which would involve in cial to develop systems to decipher pre- To judge the spermatogenic potential of vitro culture of testicular explants by the cisely how this process occurs in humans. such SSC cells, they can be transplant- methods described herein by Ogawa and This need is underscored by the extremely edinto testicular explant biopsies cultured colleagues2—is necessary because cur- high rate of evolution of the male repro- in vitro using the culture method devel- rent in vitro methods for differentiating ductive tract and the genes that function oped by the Ogawa group (Fig. 2A). A male germ cells from human pluripotent in male reproduction.29,30 This high rate of challenge with the “SSC approach” is to cells are extremely inefficient and generate evolution casts a doubt on the mouse being reduce its inherent risks. For example, few cells that progress beyond the haploid an appropriate model for many aspects of SSCs have the potential to become plu- stage.20,33 Following in vitro culture of the human spermatogenesis.36 There are both ripotent stem cells that generate terato- explants, the mature germ cells would be ethical and practical concerns with study- mas after transplantation.32 It is possible isolated and used to fertilize human eggs ing spermatogenesis in humans in vivo that this risk can be judged by injecting by ICSI or ROSI (Fig. 2B). While a poten- and thus it is essential to instead develop the SSCs into testes explants and assaying tially powerful method for certain clinical in vitro approaches to study this process. for tumors, but whether the latter sup- applications, it should be stressed that this In principal, the procedure developed by port tumor growth remains to be seen. An iPS/testes explant approach is fanciful at Ogawa and colleagues2 could be applied alternative approach is to skip the SSC iso- present. Even though conditions for dif- to human testis biopsies to achieve this lation step and instead directly use testes ferentiating human ES and iPS cells down goal. However, it is currently unknown biopsies from patients for in vitro culture. the haploid male germ-cell lineage have whether the culturing conditions that per- This requires that testes explant culture been established,20,33 the functionality of mit complete spermatogenesis to occur in conditions are discovered to support com- the differentiated spermatids produced mice explants can be applied to human plete human spermatogenesis, which may from these cultures has never been tested. explants. It might be more challenging to not be trivial to achieve given that human Another potential challenge is that the accomplish this goal with human explants, spermatogenesis requires more time than recipient heterologous testes explants for as human spermatogenesis takes much mouse spermatogenesis, at least in vivo.9 the donor iPS-derived male germ cells longer in vivo (~64 d) than does mouse Moreover, it may be necessary—in the would need to be treated to kill the pro- spermatogenesis (~35 d).9 Yet, if it can be case of very young individuals—to pro- genitor germ cells in them so that only accomplished, such a human testes culture vide external endocrine signals to trigger the donor germ cells provided would 242 Spermatogenesis volume 2 issue 4 Figure 2. potential clinical applications of the in vitro testis culture system. Blue arrows indicate germ cell transplantation in vivo or in vitro. SScs, spermatogonial stem cells; ipS cells, induced pluripotent stem cells; pGcs, primordial germ cells; ivF, in vitro fertilization. progress to maturity. While methods to the gene that randomly integrates into the reserve.35 Since the cytoplasm includes the do this have been established (e.g., using genome, but this insertion event could mitochondria and its associated genome, busulfan), this step adds another layer lead to a deleterious mutation in another this is an example where a portion of the of complexity to this approach. Despite gene and would also irreversibly alter human genome—the mitochondrial por- these concerns, this iPS/testes explant the genome organization of the human tion—was purposefully altered in future approach is worth attempting to establish, germ line. So, we consider an endogenous generations. Clearly, there is likely to be a as it has the potential to fill niches that gene targeting approach the only viable long debate about whether it is appropriate are currently empty in assisted reproduc- “gene therapy” approach for male germ for the betterment of humankind to cor- tion practice. For example, this approach cells. An advantage of this approach is its rect mutations in human male germ cells would allow cancer patients given high- affects can be tested in vitro before using that are used for fertilization. Since we are dose chemotherapy the ability to father it in vivo (Fig. 2C). Of course, such an getting closer to the point where this will children even if they did not bank frozen approach will raise ethical concerns given be technically feasible, perhaps it is time to testicular biopsies prior to chemotherapy that it involves passing on a manipulated start debating this issue in earnest. treatment (Fig. 2B). gene to future generations. But as long as Finally, we propose that the in vitro the only manipulation is a “positive one” Closing Remarks spermatogenesis procedure could be used that converts a mutant gene to a wild-type for personalized fertility therapy. For form, one can make a case for its util- After almost a century of effort, we are example, if an infertile male had a known ity outweighing such ethical concerns. now in a new phase of male germ cell mutation in germ cells that was respon- In contrast, currently available assisted studies in which all steps of spermatogen- sible for his infertility, this mutation could reproductive technologies are likely to esis can be recapitulated in vitro. This has be corrected in male germ cell progeni- be having the opposite effect. By allow- opened up a plethora of new opportuni- tors in vitro, followed by their expansion ing individual with infertility to have ties to better understand the underlying and differentiation in testes explants so children, these technologies are allowing mechanisms of spermatogenesis and to that spermatids could be harvested for in mutant genes that negatively affect fertil- treat male infertility. vitro fertilization procedures (Fig. 2C). ity to be passed on to future generations. It Optimally, a targeting vector would be is worth noting that there is already prec- Disclosure of Potential Conflicts of Interest used to correct the mutation in the endog- edent for foreign gene transfer through The authors have nothing to declare. enous gene. This would involve select- the human germ line. In particular, to ing for rare homologous recombinants in improve the efficiency of in vitro fertil- Financial Support which the wild-type sequences from the ization of oocytes from aged women with This work was supported by NIH grants targeting vector have replaced the mutant poor ovarian reserve, these ooctyes were R01-HD053808 and R01-HD45595. sequences. A technically simpler approach injected with the cytoplasm of oocytes would be to provide a wild-type copy of from younger women with good ovarian www.landesbioscience.com Spermatogenesis 243 References 16. Kubota H, Avarbock MR, Brinster RL. Growth 27. Oatley JM, Avarbock MR, Telaranta AI, Fearon factors essential for self-renewal and expansion of DT, Brinster RL. Identifying genes important for 1. Griswold MD. The central role of Sertoli cells mouse spermatogonial stem cells. Proc Natl Acad Sci spermatogonial stem cell self-renewal and sur- in spermatogenesis. 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Maintenance of adult human testicu- tion. Nature 2009; 462:222-5; PMID:19865085; http://dx.doi.org/10.1001/jama.2009.1689. lar tissue in culture. Anat Rec 1967; 157:327-8. http://dx.doi.org/10.1038/nature08562. 32. Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto 8. Ghatnekar R, Lima-de-faria A, Rubin S, Menander 21. Gohbara A, Katagiri K, Sato T, Kubota Y, Kagechika M, Ogonuki N, Miki H, et al. Generation of plu- K. Development of juman male meiosis in vitro. H, Araki Y, et al. In vitro murine spermatogenesis in ripotent stem cells from neonatal mouse testis. Cell Hereditas 1974; 78:265-72; PMID:4463180; http:// an organ culture system. Biol Reprod 2010; 83:261-7; 2004; 119:1001-12; PMID:15620358; http://dx.doi. dx.doi.org/10.1111/j.1601-5223.1974.tb01448.x. PMID:20393168; http://dx.doi.org/10.1095/biolre- org/10.1016/j.cell.2004.11.011. 9. Clermont Y. The cycle of the seminiferous epi- prod.110.083899. 33. Panula S, Medrano JV, Kee K, Bergström R, Nguyen thelium in man. Am J Anat 1963; 112:35-51; 22. Sato T, Katagiri K, Yokonishi T, Kubota Y, Inoue HN, Byers B, et al. Human germ cell differentia- PMID:14021715; http://dx.doi.org/10.1002/ K, Ogonuki N, et al. In vitro production of fertile tion from fetal- and adult-derived induced pluripo- aja.1001120103. sperm from murine spermatogonial stem cell lines. tent stem cells. Hum Mol Genet 2011; 20:752-62; 10. Tres LL, Kierszenbaum AL. Viability of rat spermato- Nat Commun 2011; 2:472; PMID:21915114; http:// PMID:21131292; http://dx.doi.org/10.1093/hmg/ genic cells in vitro is facilitated by their coculture dx.doi.org/10.1038/ncomms1478. ddq520. with Sertoli cells in serum-free hormone-supplement- 23. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. 34. Park TS, Galic Z, Conway AE, Lindgren A, van ed medium. Proc Natl Acad Sci USA 1983; 80:3377- Transplantation of male germ line stem cells restores Handel BJ, Magnusson M, et al. Derivation of 81; PMID:6407012; http://dx.doi.org/10.1073/ fertility in infertile mice. Nat Med 2000; 6:29-34; primordial germ cells from human embryonic pnas.80.11.3377. PMID:10613820; http://dx.doi.org/10.1038/71496. and induced pluripotent stem cells is significantly 11. Rassoulzadegan M, Paquis-Flucklinger V, Bertino 24. Yomogida K, Yagura Y, Nishimune Y. Electroporated improved by coculture with human fetal gonadal B, Sage J, Jasin M, Miyagawa K, et al. Transmeiotic transgene-rescued spermatogenesis in infertile cells. Stem Cells 2009; 27:783-95; PMID:19350678; differentiation of male germ cells in culture. Cell mutant mice with a sertoli cell defect. Biol Reprod http://dx.doi.org/10.1002/stem.13. 1993; 75:997-1006; PMID:7504588; http://dx.doi. 2002; 67:712-7; PMID:12193376; http://dx.doi. 35. Barritt JA, Brenner CA, Malter HE, Cohen J. org/10.1016/0092-8674(93)90543-Y. org/10.1095/biolreprod.101.001743. Mitochondria in human offspring derived from 12. Hofmann MC, Hess RA, Goldberg E, Millán 25. Ikawa M, Tergaonkar V, Ogura A, Ogonuki N, ooplasmic transplantation. Hum Reprod 2001; JL. Immortalized germ cells undergo meiosis in Inoue K, Verma IM. Restoration of spermatogenesis 16:513-6; PMID:11228222; http://dx.doi. vitro. Proc Natl Acad Sci USA 1994; 91:5533- by lentiviral gene transfer: offspring from infertile org/10.1093/humrep/16.3.513. 7; PMID:8202522; http://dx.doi.org/10.1073/ mice. Proc Natl Acad Sci USA 2002; 99:7524- 36. Swanson WJ, Vacquier VD. The rapid evolution of pnas.91.12.5533. 9; PMID:12032316; http://dx.doi.org/10.1073/ reproductive proteins. Nat Rev Genet 2002; 3:137- 13. Feng LX, Chen Y, Dettin L, Pera RA, Herr JC, pnas.072207299. 44; PMID:11836507; http://dx.doi.org/10.1038/ Goldberg E, et al. Generation and in vitro dif- 26. Kim TS, Choi HS, Ryu BY, Gang GT, Kim SU, nrg733. ferentiation of a spermatogonial cell line. Science Koo DB, et al. Real-time in vivo bioluminescence 2002; 297:392-5; PMID:12077424; http://dx.doi. imaging of lentiviral vector-mediated gene transfer org/10.1126/science.1073162. in mouse testis. Theriogenology 2010; 73:129-38; 14. Tesarik J, Bahceci M, Ozcan C, Greco E, Mendoza PMID:19837451; http://dx.doi.org/10.1016/j.therio- C. Restoration of fertility by in-vitro spermato- genology.2009.07.028. genesis. Lancet 1999; 353:555-6; PMID:10028987; http://dx.doi.org/10.1016/S0140-6736(98)04784-9. 15. Heller CH, Clermont Y. Kinetics of the Germinal Epithelium in Man. Recent Prog Horm Res 1964; 20:545-75; PMID:14285045. 244 Spermatogenesis volume 2 issue 4

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viewS, cOmmeNtarieS & OpiNiONS Spermatogenesis 2:4, 238-244; October/November/December 2012; © 2012 Landes Bioscience In vitro spermatogenesis A long journey to get tails Hye-Won Song and Miles F. Wilkinson* Department of Reproductive Medicine; School of Medicine; University of California, San Diego; La Jolla, CA USA The generation of functional sperm with germ cells to drive spermatogenesis. in vitro has been a goal for almost a Other somatic cells also participate in century. Until recently, researchers have spermatogenesis, including the peritu- only succeeded in reproducing the early bular myoid cells, which help form the steps of spermatogenesis. This is not seminiferous tubule structure, and the tes- surprising given that spermatogenesis is tosterone-producing Leydig cells, which a complicated process that requires the are located in the interstitial area between coordinated efforts of germ cells and tubules.1 several somatic cells within the tubu- Given that spermatogenesis normally lar structure of the testis. Finally—last occurs in seminiferous tubules and that year—Sato et al. reported the success- many somatic cell types participate in ful in vitro production of functional spermatogenesis, spermatogenesis has sperm, thereby potentially opening been difficult to reproduce in vitro. up a new era of reproductive biology. Nevertheless, this has not prevented Here, we summarize the history of researchers from trying! Indeed, as we research directed toward reproducing summarize in this commentary, there steps of spermatogenesis in vitro, detail have been scores of studies attempt- the seminal findings of Sato et al., and ing to achieve this goal. Driving these suggest ways that their approach can attempts have been the large rewards. be applied toward clinical applications For example, if a robust and efficient in and addressing fundamental questions vitro spermatogenesis procedure could be about the underlying mechanism of created, this would have potential clini- spermatogenesis. cal applications for treating some cases of Keywords: in vitro spermatogenesis, infertility and it could also greatly speed organ culture, spermatogonial stem cells, Introduction up the efforts to decipher the cellular and infertility, germ cells, stem cells, trans- molecular mechanisms underlying sper- plantation To become a functional sperm, an imma- matogenesis. Given these potential pay- Abbreviations: siRNA, small interfer- ture male germ cell undergoes a coordi- offs, there was much fanfare when Sato ing RNA; ISCI, intracytoplasmic sperm nated set of events that are collectively et al. recently reported the first success injection; ROSI, round spermatid injec- called spermatogenesis. This process in generating sperm from immature germ tion; SSCs, spermatogonial stem cells; occurs in tubules within the testis that cells in vitro.2 Using an organ culture iPS cell, induced pluripotent stem cell; harbor what is known as the seminiferous method that relies on defined medium IVF, in vitro fertilization; KSR, knock- epithelium. The heart of the seminiferous containing a putative spermatogenesis- out serum replacement; PGCs, primor- epithelium is a somatic cell—the Sertoli promoting factor that also maintains dial germ cells cell—that is in intimate contact with all embryonic stem (ES) cells, Sato et al. Submitted: 08/11/12 of the germ cells in the seminiferous epi- were able to generate elongated sperma- thelium, regardless of whether they are tids that were capable of fertilizing eggs Accepted: 09/03/12 undergoing mitosis, meiosis, or matura- and generating progeny. Here, we discuss http://dx.doi.org/10.4161/spmg.22069 tion. Sertoli cells provide essential factors the findings of Sato et al. and suggest for spermatogenesis and evidence suggests basic science and clinical applications for *Correspondence to: Miles F. Wilkinson; Email: [email protected] that they communicate back and forth their technique. 238 Spermatogenesis volume 2 issue 4 viewS, cOmmeNtarieS & OpiNiONS viewS, cOmmeNtarieS & OpiNiONS A Long Journey to Achieve testicular biopsies were able to give rise mature male germ cells from stem cells. In Vitro Spermatogenesis to male germ cells that progressed to the In one approach, investigators found that telophase-II stage, thereby casting some culturing mouse spermatogonial stem Organ culture. Since the first report— doubt on the reproducibility of their cul- cells (SSCs) with glial cell line-derived in 1920—providing evidence for limited turing method. neurotrophic factor (GDNF) allowed the differentiation of male germ cells when Culture of dissociated cells. Starting SSCs to grow for a long period of time cultured in vitro,3 there have been many in the 1980s, there was a burst in publica- and give rise to functional sperm when attempts to come up with ways to drive tions attempting to reproduce spermato- transplanted into germ cell-depleted further differentiation of germ cells. For genesis using dissociated testicular cells testes of wild-type mice.16,17 This was a example, in 1937, it was reported that a rather than organ culture. For example, major breakthrough, as the transplanted clot composed of fowl plasma and embryo it was reported that co-culture of imma- mice were able to father progeny by natu- extract permitted some of the mitoti- ture rat germ cells with primary Sertoli ral conception. Another group reported cally active spermatogonia within cul- cells for 11 d promoted their maturation that SSCs can be differentiated into early tured newborn mouse testes to progress into late pachytene spermatocytes.10 In the meiotic germ cells by simply culturing to the pachytene stage.4 In the 1960s, the 1990s, there were two reports providing the SSCs in retinoic acid (RA).18 It will Steinberger group identified several mea- evidence that immature germ cells could be interesting to determine whether RA sures that promoted long-term survival be pushed all the way to the haploid stage. in combination with appropriate culture of cultured testes, including the addition These studies,11,12 which revolved around conditions can drive later steps of male of pyruvate to the culture medium as an using immortalized Sertoli cell or germ germ cell differentiation. Other studies energy source, reducing the incubation cell lines, respectively, held great promise, have reported the successful generation temperature to 31°C to better simulate but, to our knowledge, no follow-up stud- of haploid germ cells from human and the intratesticular temperature in vivo, ies using these techniques have been pub- mouse embryonic stem (ES) cells.19,20 One and using a gas-liquid interphase method lished. Later—in 2002— it was reported of these studies demonstrated that haploid to increase oxygen exchange.5 Using tri- that immortalized mouse type-A sper- cells generated from the mouse ES cells tiated-thymidine to tag immature prolif- matogonial cells in the presence of stem were capable of triggering blastocyst for- erating male germ cells (spermatogonia cell factor (SCF) are capable of undergo- mation when injected into the cytoplasm and preleptotene spermatocytes) present ing differentiation into haploid spermatids of oocytes.19 However, whether these in rat testes explants, they demonstrated in vitro.13 However, no fully differentiated blastocysts can generate progeny when that some of these cells differentiated into germ cells (e.g., tailed spermatozoa) were injected into hormonally receptive female pachytene spermatocytes after culture in observed, it was not tested whether the mice has not yet been reported. vitro for 2 to 3 weeks.6 They also achieved round spermatids that are generated are In summary, the concerted efforts of similar results using human testicular functional competent for fertilization, many investigators over several decades biopsies.7 While encouraging, no germ and no follow-up studies have been subse- has led to the development of culture cells beyond the pachytene stage of meio- quently published. In the human system, a conditions for promoting specific steps sis were observed by either Steinberger and study reported that dissociated cells from in spermatogenesis in vitro. However, the colleagues or most other research groups testicular biopsy samples from humans ultimate goal of generating functional that attempted to improve culture con- with premeiotic maturation arrest at the sperm in vitro was not accomplished until, ditions during this early phase of the “in primary spermagocytes stage could be as described below, the Ogawa group vitro spermatogenesis” field. An exception coaxed into becoming haploid germ cells revisited the old organ culture method was one group that reported conditions after culture for 48 h.14 Remarkably, the using newly developed stem cell culture that allowed tritiated thymidine-labeled elongating spermatids generated in these techniques and a media supplement that human preleptotene spermatocytes to in vitro cultures were functional compe- they discovered is essential for spermatid differentiate into spermatocytes that pro- tent, as oocytes injected with them were maturation. gressed to the end of meiosis: the telo- shown to form healthy babies when trans- Complete spermatogenesis in vitro. phase-II stage. In their protocol, testicular planted into hormonally receptive human As a precursor to their groundbreaking biopsies were cultured for 14 d in media females.14 While promising, whether this work, the Ogawa group identified condi- containing deproteinized coconut milk, approach could be safely used in the clinic tions for culturing mouse testes fragments follicle-stimulating hormone (FSH) and is not clear since the time frame these from neonatal mice that allowed some of luteinizing hormone (LH).8 While poten- investigators found was sufficient for dif- the male germ cells in them to undergo tially promising, a concern with regard ferentiation of human meiotic germ cells a significant degree of maturation.21 To to physiological relevance is that these into elongating spermatids in vitro was assess maturation, they made the tes- investigators found that the speed of con- abnormally short (24–48 h); this multi- tes cultures from transgenic mice that version of the pre-leptotene germ cells to step process normally takes 20–22 d in express green fluorescent protein (GFP) telophase-II germ cells in vitro was more vivo.15 from either a mid-meiosis promoter or a rapid than normally occurs in vivo.9 A More recently, approaches have been late-meiosis promoter. This allowed them further concern is that only one out of 16 identified that drive the differentiation of to detect even small numbers of cells that www.landesbioscience.com Spermatogenesis 239 progressed to specific stages of meio- tubules of germ cell-depleted testes (made explants from neonatal testes2 and in only sis. Using this and other methods, they germ cell deficient by either genetic or 3 out of 17 cultured explants from SSC- detected some cells in the cultured testes chemical means), followed by culture of transplanted testes.22 Only the periph- chunks that completed meiosis and pro- fragments from these testes in the KSR- eral region of testes fragments exhibited gressed to the round spermatid stage. containing medium that they had shown advanced spermatogenesis in their system, While a significant advance, Ogawa promotes spermatogenesis in their ear- while the center lost normal morphology and coworkers were not able to detect any lier study.2 Remarkably, they found that, and had large numbers of degenerating germ cells in the testes chunks that pro- under these conditions, the transplanted cells after 40 d of culture.2 Furthermore, gressed past the round spermatid stage.21 SSCs migrated into the basement mem- they found a gradual decrease in germ cell They considered the possibility that the brane after only ~3 d of culture, began numbers over the 40-d culture period.2 fetal bovine serum in the culture medium proliferating a few days later, and then, Thus, creative approaches to improve was not supportive for this final stage of after almost two months, some of the germ cell viability are required, such as male germ cell maturation and thus they cells differentiated into elongated sperma- the addition of an “in vitro vascular” sys- screened for other tissue culture supple- tids. Some of the haploid spermatids were tem. Given that human application will ments that might have such activity. This functionally competent, as assessed by typically require using testes from ado- screen revealed that a supplement normally ROSI. These investigators also performed lescent or adult individuals, a practical used to maintain ES cells—knock out a proof-of-principal study in which they concern is whether explants from adult serum replacement (KSR) media—was asked whether their in vitro culture system testes can maintain spermatogenesis using able to promote the maturation of male was capable of correcting spermatogenic their culture conditions. In their mouse germ cells in cultured testes fragments failure.22 As a model system, they used experiments, Ogawa and colleagues used to the elongated spermatid stage.2 At steel mutant mice, which contain SSCs testes from neonatal mice2,21 or postnatal least some of these elongated spermatids but lack later stage male germ cells because (3-week-old) mice in the case of SSC- were functionally competent, as judged of a mutation in the gene encoding stem transplanted testes.22 Another issue for using intracytoplasmic sperm injection cell factor, a Sertoli-cell product essential human application is whether the sperma- (ICSI), a method that involves injection for the proliferation and differentiation tids generated from cryopreserved testes of elongated spermatids or spermatozoa of spermatogonial cells. To determine are functionally competent. In their mouse into mature oocytes, followed by in vitro whether they could drive the differen- studies, Ogawa and colleagues did not test culture of the fertilized oocytes to form tiation of SSCs from steel mutant testes whether explants from cryopreserved tes- two-cell embryos, and finally transfer of using their organ culture system, they tes can gave rise to functional spermatids, the embryos into the oviducts of pseudo- transplanted them into germ cell-deficient as judged, for example, by ISCI or ROSI.2 pregnant females. Importantly, Ogawa testes. They found that the transplanted In summary, the in vitro spermatogenesis and colleagues found that the females SSCs could be successfully differentiated approach described herein shows much implanted with these in vitro-generated into haploid germ cells in their in vitro promise. With improvements in efficiency spermatozoa-derived embryos delivered system. This result complemented an ear- and germ cell viability, along with altered live and fertile offspring. Even the less lier study from Brinster and colleagues, conditions to permit usage of mature tes- differentiated spermatids generated in which demonstrated restoration of sper- tes as input material, their technique is their organ culture system were capable matogenesis when SSCs from steel mutant likely to be useful for a variety of basic sci- of generating progeny, as they showed testes are transplanted into germ cell- ence and clinical applications, as described using a round spermatid injection (ROSI) deficient testes in vivo.23 Together, this below. technique. These investigators also tested earlier study23 and the more recent study whether their technique could be applied from Ogawa and colleagues,22 provide two Future Applications to a practical application: cryoprervation. independent paths—one in vitro and one In particular, they examined whether their in vivo—for assisted reproduction in large An efficient system for studying sper- system would support the proliferation host animals and humans. matogenesis. One of the technical chal- and differentiation of germ cells present While the identification of culture con- lenges holding back our understanding in cryopreserved neonatal testes tissues. ditions that permit immature male germ of spermatogenesis has been the lack of Indeed, they found that when thawed cells to mature into functional sperm is a an in vitro system that recapitulates sper- and cultured in KSR-containing medium, major breakthrough, there are still several matogenesis. Because of this deficit, many some immature germ cells in frozen testes issues that require attention. Not surpris- researchers have been forced to study samples proliferated and formed elongated ingly, efficiency is a major concern. Far mechanisms of spermatogenesis using in spermatids. fewer elongated spermatids and spermato- vivo systems. For example, to identify the In follow-up work, Ogawa and col- zoa are produced per input immature male role of specific genes in spermatogenesis, leagues tested whether they could use their germ cell using the currently available in it has been necessary to generate geneti- technique to differentiate mature germ vitro system2 than in a normal testis in cally modified mice harboring targeted cells from SSCs.22 They tested this by vivo. Ogawa and colleagues observed flag- mutations in specific genes. While this is transplanting SSCs into the seminiferous ellated sperm in only 5 out of 11 cultured an essential approach to understand the 240 Spermatogenesis volume 2 issue 4 lentiviruses can deliver transgenes into germ cells when injected into seminifer- ous tubules and that transgene expression in germ cells persisted for up to 3 mo.26 Given the contrasting results of these two studies, the question of whether or not lentiviruses efficiently infect germ cells in testes organ culture is an issue that needs to be revisited. For somatic cell types out- side of the seminiferous tubules—peritu- bular myoid cells and Leydig cells—it is likely that they will be highly susceptible to lentiviral infection since these cells are accessible to the culture media in cultured testes fragments. Thus, lentiviral vectors can probably be effectively used to test the role of genes in these two somatic cell types. In the case of germ cells, an alterna- tive approach is to introduce the vector or small interfering (si) RNA of interest into SSCs, followed by transplantation of these Figure 1.Studying genes involved in spermatogenesis using the in vitro spermatogenesis tech- SSCs into testes and then in vitro culture nique developed by Ogawa and colleagues. of explants generated from these testes. While this “in vivo-in vitro” approach has role of genes in vivo, it is extremely time using multiphoton fluorescence micros- not yet been tried, in principal it should be consuming and expensive. Furthermore, it copy, would allow germ cell develop- feasible as in vivo transplantation has been is often difficult to use such mouse mod- ment to be visualized in real time (Fig 1). used to identify genes involved in SSC els to identify molecular mechanisms. Another challenge is to work out how to self-renewal or differentiation.18,27 Another approach to study spermatogen- efficiently deliver vectors into the cells in How might in vitro approaches be esis has been to use immortalized cell organ culture in order to manipulate the specifically applied to studying sper- lines, but the number of cell lines available expression of genes of interest and thereby matogenesis? By coupling in vitro cul- for such studies is limited—particularly address their functional role. Two deliv- ture approaches with live tissue imaging, in the case of germ cells—and how well ery approaches are electroporation and the vector/siRNA-expressing cells can be such cell lines recapitulate events in nor- viral infection. Electroporation has been monitored, allowing for earlier detection mal cells is uncertain at best. Use of pri- successfully used to introduce genes into of defects than might be observed in vivo mary cells solves some of these problems, the testis in vivo,24 but it causes extensive (Fig. 1). To specifically study the cells but they are not trivial to prepare or main- tissue damage, limiting its use for many in which a given gene has been manipu- tain. Furthermore, the culture conditions applications. Also, electroporation typi- lated in explant cultures, one could use required for fully recapitulating spermato- cally only delivers genes transiently, so it two different reporters. As an example genesis with primary cells have not been is not generally suitable for determining application, a vector that co-expresses a identified, nor is it clear whether testicular functions occurring over long periods siRNA against the gene of interest and red cells—even if freshly isolated—placed in of time. Lentiviruses have the intrinsic fluorescent protein (RFP) could be intro- isolation on a plastic surface act in a physi- advantage of being able to integrate into duced into testes explants from a trans- ologically normal manner. the genome and thus infection of cells genic mouse containing a transgene that The ability to recapitulate spermato- with lentiviruses can provide long-term expresses GFP from a promoter active in genesis in organ culture2 solves many of gene expression. Indeed, lentiviruses have the germ cell stage of interest. Following these problems, but it also brings up new been used to successfully express genes for delivery of the vector, the testis fragments challenges. One challenge is how to detect long periods of time in cells in the semi- would be cultured as described2 and moni- germ cell maturation in an in vitro organ niferous tubule in vivo.25,26 However, a tored for GFP and RFP expression. If, for culture system. One approach to assay potential drawback of lentiviruses is that example, knockdown of the gene of inter- germ cell development is to use a battery of they may not be able to efficiently infect est by the siRNA causes a defect in pro- reporters (expressing different versions of most of the cells in organ culture. Indeed, gression to the stage monitored by GFP, GFP) under the control of promoters that one study found that lentiviruses infect there will be less RFP+/GFP+ double-pos- drive gene expression in different stages only Sertoil cells, not germ cells, when itive cells compared with testes fragments of germ cells. This advancement, cou- injected into seminiferous tubules.25 In delivered negative-control siRNAs. If cell pled with live organ imaging technology contrast, a recent report showed that type- or stage-specific expression of the www.landesbioscience.com Spermatogenesis 241 siRNA is required, this can be achieved by system could then be used to examine the spermatogenesis in cryopreserved juvenile using the appropriate promoter to drive its functional role of genes suspected to be testis biopsies since human spermatogen- expression.28 involved in human spermatogenesis by the esis typically does not initiate until after Not only is it likely to be much easier approaches described above for the mouse 10 y of age. If indeed human spermato- to examine the role of genes using in vitro system (Fig. 1). genesis can be safely reproduced in vitro, spermatogenesis procedures than generat- Infertility treatment. In vitro sper- then in principal it should be possible to ing and characterizing knockout mice, but matogenesis procedures have several use either the round or elongated sperma- in vitro procedures may provide opportu- potential applications in the clinic, includ- tids generated from cryopreserved juvenile nities that are not readily feasible with the ing: (1) restoration of fertility in juvenile testis biopsies to successfully fertilize eggs latter. For example, analysis of the redun- patients requiring chemotherapy, (2) gen- by either ISCI or ROSI, respectively (Fig. dant role of members of clustered gene eration of functional sperm from induced 2A). families cannot be easily accomplished pluripotent stem (iPS) cells, and (3) gene The discovery of methods to gener- using a standard knockout approach. This therapy approaches to generate fertile ate ES-like cells from fully differentiated is because the recombination frequencies sperm from infertile men. cells—iPS cells33,34—has opened up the of closely linked genes are too low to per- In the first application, the goal is to possibility of another means to generate mit feasible generation of double knock- provide juvenile cancer patients who functional spermatids in vitro (Fig. 2B). out progeny mice from single knockout have undergone high-dose chemotherapy The beauty of using iPS cells is that no tes- mice. In contrast, by introducing multiple with a source of functional spermatids ticular tissues from the donor are required. siRNAs in testes explants, one can rela- when they are ready to father children. In the way we envisage this approach, iPS tively easily examine the independent and The number of long-term survivors of cells generated from the individual would redundant roles of gene clusters. Another childhood cancer has increased greatly first be coaxed down the early male germ advantage is that one could feasibly per- in recent years, highlighting the need for cell lineage using in vitro methods that form genome-wide screening for factors identifying methods to recover the fertil- have already been developed for human involved in spermatogenesis using siRNA ity of these patients. One means to achieve ES cells.20,33 Following purification of the libraries (Fig. 1). this is to isolate SSCs from these patients male germ cells that have partially differ- Human spermatogenesis in vitro. prior to chemotherapy and then use them entiated in vitro from the iPS cells, they Studying mouse spermatogenesis will no for “auto-transplantation” in vivo (Fig. would be pushed further toward final doubt provide many insights into human 2A). This seems feasible given the recent maturity by transplanting them into tes- spermatogenesis, but ultimately, for the success of long-term culture of human ticular explants from another individual. purposes of human health, it will be cru- SSCs from cryopreserved testis biopsy.31 This latter step—which would involve in cial to develop systems to decipher pre- To judge the spermatogenic potential of vitro culture of testicular explants by the cisely how this process occurs in humans. such SSC cells, they can be transplant- methods described herein by Ogawa and This need is underscored by the extremely edinto testicular explant biopsies cultured colleagues2—is necessary because cur- high rate of evolution of the male repro- in vitro using the culture method devel- rent in vitro methods for differentiating ductive tract and the genes that function oped by the Ogawa group (Fig. 2A). A male germ cells from human pluripotent in male reproduction.29,30 This high rate of challenge with the “SSC approach” is to cells are extremely inefficient and generate evolution casts a doubt on the mouse being reduce its inherent risks. For example, few cells that progress beyond the haploid an appropriate model for many aspects of SSCs have the potential to become plu- stage.20,33 Following in vitro culture of the human spermatogenesis.36 There are both ripotent stem cells that generate terato- explants, the mature germ cells would be ethical and practical concerns with study- mas after transplantation.32 It is possible isolated and used to fertilize human eggs ing spermatogenesis in humans in vivo that this risk can be judged by injecting by ICSI or ROSI (Fig. 2B). While a poten- and thus it is essential to instead develop the SSCs into testes explants and assaying tially powerful method for certain clinical in vitro approaches to study this process. for tumors, but whether the latter sup- applications, it should be stressed that this In principal, the procedure developed by port tumor growth remains to be seen. An iPS/testes explant approach is fanciful at Ogawa and colleagues2 could be applied alternative approach is to skip the SSC iso- present. Even though conditions for dif- to human testis biopsies to achieve this lation step and instead directly use testes ferentiating human ES and iPS cells down goal. However, it is currently unknown biopsies from patients for in vitro culture. the haploid male germ-cell lineage have whether the culturing conditions that per- This requires that testes explant culture been established,20,33 the functionality of mit complete spermatogenesis to occur in conditions are discovered to support com- the differentiated spermatids produced mice explants can be applied to human plete human spermatogenesis, which may from these cultures has never been tested. explants. It might be more challenging to not be trivial to achieve given that human Another potential challenge is that the accomplish this goal with human explants, spermatogenesis requires more time than recipient heterologous testes explants for as human spermatogenesis takes much mouse spermatogenesis, at least in vivo.9 the donor iPS-derived male germ cells longer in vivo (~64 d) than does mouse Moreover, it may be necessary—in the would need to be treated to kill the pro- spermatogenesis (~35 d).9 Yet, if it can be case of very young individuals—to pro- genitor germ cells in them so that only accomplished, such a human testes culture vide external endocrine signals to trigger the donor germ cells provided would 242 Spermatogenesis volume 2 issue 4 Figure 2. potential clinical applications of the in vitro testis culture system. Blue arrows indicate germ cell transplantation in vivo or in vitro. SScs, spermatogonial stem cells; ipS cells, induced pluripotent stem cells; pGcs, primordial germ cells; ivF, in vitro fertilization. progress to maturity. While methods to the gene that randomly integrates into the reserve.35 Since the cytoplasm includes the do this have been established (e.g., using genome, but this insertion event could mitochondria and its associated genome, busulfan), this step adds another layer lead to a deleterious mutation in another this is an example where a portion of the of complexity to this approach. Despite gene and would also irreversibly alter human genome—the mitochondrial por- these concerns, this iPS/testes explant the genome organization of the human tion—was purposefully altered in future approach is worth attempting to establish, germ line. So, we consider an endogenous generations. Clearly, there is likely to be a as it has the potential to fill niches that gene targeting approach the only viable long debate about whether it is appropriate are currently empty in assisted reproduc- “gene therapy” approach for male germ for the betterment of humankind to cor- tion practice. For example, this approach cells. An advantage of this approach is its rect mutations in human male germ cells would allow cancer patients given high- affects can be tested in vitro before using that are used for fertilization. Since we are dose chemotherapy the ability to father it in vivo (Fig. 2C). Of course, such an getting closer to the point where this will children even if they did not bank frozen approach will raise ethical concerns given be technically feasible, perhaps it is time to testicular biopsies prior to chemotherapy that it involves passing on a manipulated start debating this issue in earnest. treatment (Fig. 2B). gene to future generations. But as long as Finally, we propose that the in vitro the only manipulation is a “positive one” Closing Remarks spermatogenesis procedure could be used that converts a mutant gene to a wild-type for personalized fertility therapy. For form, one can make a case for its util- After almost a century of effort, we are example, if an infertile male had a known ity outweighing such ethical concerns. now in a new phase of male germ cell mutation in germ cells that was respon- In contrast, currently available assisted studies in which all steps of spermatogen- sible for his infertility, this mutation could reproductive technologies are likely to esis can be recapitulated in vitro. This has be corrected in male germ cell progeni- be having the opposite effect. By allow- opened up a plethora of new opportuni- tors in vitro, followed by their expansion ing individual with infertility to have ties to better understand the underlying and differentiation in testes explants so children, these technologies are allowing mechanisms of spermatogenesis and to that spermatids could be harvested for in mutant genes that negatively affect fertil- treat male infertility. vitro fertilization procedures (Fig. 2C). ity to be passed on to future generations. It Optimally, a targeting vector would be is worth noting that there is already prec- Disclosure of Potential Conflicts of Interest used to correct the mutation in the endog- edent for foreign gene transfer through The authors have nothing to declare. enous gene. This would involve select- the human germ line. In particular, to ing for rare homologous recombinants in improve the efficiency of in vitro fertil- Financial Support which the wild-type sequences from the ization of oocytes from aged women with This work was supported by NIH grants targeting vector have replaced the mutant poor ovarian reserve, these ooctyes were R01-HD053808 and R01-HD45595. sequences. A technically simpler approach injected with the cytoplasm of oocytes would be to provide a wild-type copy of from younger women with good ovarian www.landesbioscience.com Spermatogenesis 243 References 16. Kubota H, Avarbock MR, Brinster RL. Growth 27. Oatley JM, Avarbock MR, Telaranta AI, Fearon factors essential for self-renewal and expansion of DT, Brinster RL. Identifying genes important for 1. Griswold MD. The central role of Sertoli cells mouse spermatogonial stem cells. Proc Natl Acad Sci spermatogonial stem cell self-renewal and sur- in spermatogenesis. 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