J. Endocrinol. Invest. 23: 584-591, 2000
Lessons from knockout and transgenic mice for infertility in men J.P. Venables and H.J. Cooke Medical Research Council, Human Genetics Unit, Western General Hospital, Edinburgh, UK
ABSTRACT. This review concentrates on the clear cases where knocking out a gene in mice has caused male infertility and thus comes near to proving that the gene plays a role in the development of sperm. Knockout mice have been created with primary defects at every stage of spermatogenesis thus creating a framework for decoding the genetic hierarchy that causes male germ cell differentiation. As well as defining es-
sential genes in vivo experiments have defined promoter and untranslated sequences responsible for the expression of proteins at all the spermatogenic stages. In conclusion knockout mice remain the ultimate test of spermatogenic hypotheses as well as providing detailed information about this complex process. (J. Endocrinol. Invest. 23: 584-591, 2000) ©2000,
Controlled mutagenesis in mice, through the embryonal stem cell route or by addition of genes through pronuclear injection, is the only way of proving that a gene has an essential function. If the gene can then be reintroduced in order to restore wild type phenotype, modifications can be made to the gene and protein to gain some understanding of its mechanism of action. Here we concentrate on the simple cases where proof has been obtained that a gene is important for male fertility and not on the more complicated cases of ectopic or over-expression of genes in testes, many of which have been well covered before (1). We have further confined the area we cover to genes whose disruption causes a phenotype primarily or only in the process of gametogenesis. The “knock in” mouse can be proof of involvement in fertility if the gene in question is reintroduced into one of many naturally occurring mutants (2), such as the ‘hypogonadal’ mouse lacking gonadotropin-releasing hormone (GnRH). When the GnRH gene was reintroduced into these mutant mice full fertility was restored (3). A similar proof was gained for the identity of the testis determining factor Sry from the Y chromosome, a transgene of which could confer maleness on XX embryos (4). More recently de-
Editrice Kurtis
tailed questions have been asked about the domains of Sry, and the glutamine rich putative transactivating domain of Sry was found to be essential for Sry function, by the knockout mouse experiment (5), although this domain is not conserved in humans. Several genes involved in spermatogenesis have been knocked out in mice without causing infertility. These included Xist (6), acrosin (7) and the testis- specific histone H1t, which must be redundant with the other H1 genes as the knockout mice displayed totally normal spermatogenesis (8). These unexpected findings can be in part explained by redundancy but it should be remembered that phenotypes need to be relatively crude to be detected in the laboratory. In the wild, absence of proteins such as acrosin can be expected to reduce the fitness (in genetic terms) of the animal substantially. By contrast, widely expressed genes not originally thought to be specifically involved in spermatogenesis give rise to infertile males when knocked out. The breast cancer gene BRCA1 knockout was for the most part inviable and the few survivors were small, but against this background there was male sterility (9). In such cases it is not clear whether the gene is directly involved in spermatogenesis or this defect is secondary to the health of the mouse. Fertility problems in knockout mice result from lesions in genes of a wide range of functions and expression profiles, so they have been categorized here according to the stage of fertilization at which things start to go wrong (Fig. 1).
Key-words: Spermatogenesis, meiosis, spermiogenesis, infertility, knockout, transgenic, promoter, UTR. Correspondence: Dr. J.P. Venables, Medical Research Council, Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. E-mail:
[email protected]
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THE ENDOCRINOLOGICAL CONTEXT
to the forming embryonic genital ridge from day E11.5 and reduced survival therein, so that by day E13.5 there were no germ cells left (12). Presumably due to hormonal feedback between the developing gonad and the hypothalamus TIAR deficient mice were small and usually inviable. By contrast the homeobox gene Lhx9 is expressed throughout the embryos of mice but only in the somatic cells of the genital ridge, and the only defect of the Lhx9 knockout mouse was a failure of these cells to form a gonad around day E12E13.5 in both sexes (13). The remainder of this review therefore only concerns cases such as Lhx9, where knocking out a gene causes male sterility but has little or no effect on viability, as these are more likely to be models of human infertility as presented in the clinics.
Germ cell development is a complex process which is dependent on hormonal interplay between the hypothalamus and developing gonad, e.g. GnRH (3), and mice with impaired gene expression in the hypothalamus are frequently hypogonadal, with additional downstream effects on germ-cell differentiation. The Ahch transcription factor which causes the “adrenal hypoplasia congenita” condition is expressed in both the developing urogenital ridge and the hypothalamus. Correspondingly, sterility in the ahch knockout results from retarded gonad development as well as degeneration of surviving germ cells from meiosis onwards (10). Knocking out the hypothalamic transcription factor Nhlh2 also caused underdevelopment of genitalia and sterility in males (11). Interestingly, homozygous females were only infertile if raised alone. Environmental hormone levels were considered high enough to render them fertile if they were brought up in cages with male mice. Again this emphasizes the need for careful analysis of the phenotype of the animals.
SPERMATOGONIAL PROLIFERATION AND SURVIVAL p27(kip) is a Sertoli expressed gene whose knockout mice have a defect in spermatogonial proliferation, so presumably p27(kip) is important for molecular transactions between Sertoli cells and spermatocytes (14). Dazl is an RNA-binding protein expressed in spermatogonia that is required for male and female germ-cell progression into meiosis (15). In Drosophila the Dazl homologue Boule is required for translation of the cdc25 message (16).
PRIMORDIAL GERM CELL DEVELOPMENT/MIGRATION TIAR is an RNA-binding protein expressed in primordial germ cells (PGCs). Deleting TIAR from the mouse genome led to reduced migration of PGCs
TIAR
Lhx9
Primordial germ cell
Dazl p27 (kip)
Spermatogonia
SCP3 ATM TLS Vasa Dmc1 Pms2 Msh5
A-myb Mlhl
Synapsis Pachytene De synapsis
Maturation and fertilization
Elongation
Round Spermatid
Spermatozoon
C-ros Fertilin β
Hsp70-2 Cyclin A1
Nectin-2
Pplcγ Hr6b CREM
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Fig 1. The stages of spermatogenesis are shown along with the gene knockouts in mice that impact them.
Knockout mice and infertility
Table 1 - Studies of fusions of testes specific promoters to reporter transgenes in transgenic mice showing the cell type in which expression occurs. Promoter of Reporter/coding region Expression pattern Reference Rat ABP Rat ABP Sertoli cells (47) ACEt lacZ Spermatids (48) Rabbit ACEt CAT Spermatids (49) β-4-galactosyl transferase LacZ Pachytene-round spermatids (50) β4Gal-t lacZ Late pachytene-round spermatids (51) Calmegin CAT Testes (52) Rat calmodulin lacZ Spermatocytes (53) Rat H1t LacZ, luciferase and ratH1t Spermatocytes and round spermatids (54-56) Human HSLtes CAT Round spermatids (57) HSP70-2 lacZ Meiosis (58) Rat hst70 CAT, lacZ Spermatocytes (59, 60) Inhibin α subunit SV40 Leydig cells (61) Intracisternal-A particle lacZ Gonocytes (62) c-kit lacZ Spermatids (63) LAMB1 lacZ Prospermatogonia (64) Ldh3 lacZ Leptotene-pachytene (65) Human LdhC Ldh1 Leptotene-pachytene (66) MIS SV40-T antigen Sertoli cells (67) MIS promoter mutation MIS Abrogates expression (68) OCT-4 GFP Female and male germcells pre-type A spermatogonia (69) Pdha-2 CAT Testes (70) PGK2 CAT, luciferase Meiosis and after (71) Proacrosin CAT, SV40 Pachytene-round spermatids (72) Rat proenkephalon CAT Male germ cells (73) Prm-1 Prm-1, human growth hormone, toxin Spermatids (74, 75) Prm2 Boar proacrosin, c-myc, SV40 Spermatids (76, 77) SP-10 GFP Round spermatids (78) Sycp1 LacZ, luciferase Pachtene (79) Tcp-10p(t) lacZ Male germ cells (80) Xist luciferase pachytene (81) Zfy lacZ Germ cells (82)
EARLY PROPHASE
in primordial germ cells up to meiosis, and mice lacking this gene have reduced germ-cell numbers and a complete block to meiosis during zygotene (21). This protein is predicted to have an RNA helicase function but its targets are not known. Also there is zygotene block in an insertional mutant of the germ cell specific Morc gene (22). Again the function of this gene remains to be determined as the sequence gives few clues. Knocking out synaptonemal complex protein SCP3 causes apoptosis before pachytene of male germ cells, while females are not totally infertile (23). Synapsis failure is also observed in several mutants for homologues of bacterial genes known to be involved in that process. These include RecA homologue Dmc1 (24, 25), which has a similar phenotype
Three ATM-deficient mouse models have been developed in order to gain insights into human disease ataxia Telangiectasia, and their block to spermatogenesis starts as early as leptotene and results in a near total failure of synapsis (17). Most patients are infertile and the knockout mice are completely infertile. ATM and ATR are related proteins involved in DNA repair and have been extensively reviewed elsewhere (18, 19). TLS is an RNA-binding protein implicated in homologous recombination, and the first defect in the TLS knockout is failure to pair and sometimes mispairing of chromosomes (20). The mouse homologue of Drosophila DEAD box containing translational regulator Vasa is expressed
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in yeast, and mutL homologue Pms2 (26). The MutS homologue 5 (Msh5) knockout’s chromosomes also fail to pair properly (27).
normal acrosome reaction but then failed to fuse with egg plasma membranes (41). TESTICULAR GENE EXPRESSION STUDIES
APOPTOSIS
As well as showing what happens when a testis gene is absent, transgenic mice can also answer questions about which sequences are responsible for the manner in which genes in the testes are expressed. In testes many mRNAs including Protamine 1 are transcribed during meiosis and translated later, presumably because of the action of RNAbinding proteins (42). A Protamine 1 transgene lacking its 3’ UTR led to infertility just because it was no longer translationally repressed without that element (43). Another transgene containing a CAT reporter with a protamine 3’ UTR element was also translationally repressed (44). Reporter genes have frequently been fused to testes-specific promoters in order to define promoter elements and transcription factors responsible for testes-specific expression (Table 1). Knowledge of promoter elements gained in this way is now being used to direct ectopic expression of genes at strategic points in the spermatogenic process for sorting and/or studying these stages. For example a transgenic mouse expressing green fluorescent protein in the acrosome of sperm has enhanced video images of the fertilization process (45).
ATM, Vasa and Mlh1 knockout mice all displayed an increase in apoptosis of meiotic cells, and two genes known to be directly involved in apoptosis are required for male fertility. Apoptosis regulators Bcl-w (28) and Bax (29) both cause a gradual attrition of germ cells from meiosis onwards and sterility when knocked out. PACHYTENE A-myb transcription factor knockout mice also displayed increased apoptosis and their germ cells were arrested at the very beginning of pachytene (30). Another homologue of mutS called Mlh1 led to a block during pachytene (31, 32) and human MLH1 can complement Mlh1 deficiency in fibroblasts from Mlh1 knockout mice (33). Hsp70-2 is a pachytene spermatocyte-specific chaperone for Cdc2 necessary for its association with CyclinB1 and hence for the first division in male meiosis and male but not female knockouts for Hsp70-2 are indeed blocked from progression to the first meiotic division (34). Cyclin A1 knockout mice also failed to desynapse properly in meiosis (34, 35).
CONCLUSIONS Altering gene expression in the mouse is the ultimate experimental test of gene function, and has resulted in models of infertility at every developmental stage (Fig. 1). Mutations in a large number of genes impact this complex process. These and future models will be an invaluable foundation for deciphering the biology of gametogenesis in man. New technologies such as microarrays can be used to monitor differences in gene expression between wild type and knockout animals at the precise developmental stage when disruption occurs, thus providing clues to the exact mechanisms and pathways at work. One caveat is that the mouse is not a man! Two Y chromosome examples of this are that DAZ genes are absent from the mouse Y and, whereas the DBY gene in humans is deleted in about 5% of idiopathic azoospermic males, this gene does not seem to play a role in the infertile Sxrb mouse model (46). How useful will these insights be in dealing with male infertility? If large numbers of genes each contribute a small number of cases then health economics may mean that even a diagnostic test is in-
SPERMIOGENESIS FAILURE MHR6b and CREM knockout mice have spermiogenic failure and are discussed elsewhere in this issue (36, 37). Protein phosphatase 1c γ knockout mice are severely impaired at the round spermatid stage (38). Nectin-2 is a widely expressed plasma membrane protein that may be involved in Sertoli/germ cell interactions. In germ cells Nectin–2 is expressed in elongating spermatids and the Nectin-2 knockout has aberrant sperm morphology (39). FERTILIZATION The C-ros tyrosine kinase is a protooncogene expressed in the epididiymis. When C-ros was knocked out sperm were formed that presumably did not mature properly at the very final stages, as they could fertilize eggs in vitro despite the mice being sterile (40). Sperm with a defect in fertilization itself came from the Fertilin β knockout mouse which underwent a
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11. Good D.J., Porter F.D., Mahon K.A., Parlow A.F., Westphal H., Kirsch I.R. Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene. Nat. Genet. 1997, 15: 397-401. 12. Beck A.R.P., Miller I.J., Anderson P., Streuli M. RNA-binding protein TIAR is essential for primordial germ cell development. Proc. Natl. Acad. Sci. USA 1998, 95: 2331-2336. 13. Birk O.S., Casiano D.E., Wassif C.A., Cogliati T., Zhao L., Zhao Y., Grinberg A., Huang S., Kreidberg J.A., Parker K.L., Porter F.D., Westphal H. The LIM homeobox gene Lhx9 is essential for mouse gonad formation. Nature 2000, 403: 909-913. 14. Beumer T.L., Kiyokawa H., Roepers Gajadien H.L., van den Bos L.A.C., Lock T.W., Gademan I.S., Rutgers D.H., Koff A., de Rooij D.G. Regulatory role of P27(Kip1) in the mouse and human testis. Endocrinology 1999, 140: 1834-1840. 15. Ruggiu M., Speed R., Taggart M., Mckay S.J., Kilanowski F., Saunders P., Dorin J., Cooke H.J. The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 1997, 389: 73-77. 16. Maines J.Z., Wasserman S.A. Post-transcriptional regulation of the meiotic Cdc25 protein twine by the Dazl orthologue boule. Nat. Cell Biol. 1999, 1: 171-174. 17. Barlow C., Liyanage M., Moens P.B., Tarsounas M., Nagashima K., Brown K., Rottinghaus S., Jackson S.P., Tagle D., Ried T., Wynshaw-Boris A. Atm deficiency results in severe meiotic disruption as early as leptonema of prophase I. Development 1998, 125: 4007-4017. 18. Brown E.J., Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 2000, 14: 397-402. 19. Dasika G.K., Lin S.C., Zhao S., Sung P., Tomkinson A., Lee E.Y. DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene 1999, 18: 7883-7899. 20. Kuroda M., Sok J., Webb L., Baechtold H., Urano F., Yin Y., Chung P., de Rooij D.G., Akhmedov A., Ashley T., Ron D. Male sterility and enhanced radiation sensitivity in TLS(-/-) mice. EMBO J. 2000, 19: 453-462. 21. Tanaka S.S., Toyooka Y., Akasu R., Katoh-Fukui Y., Nakahara Y., Suzuki R., Yokoyama M., Noce T. The mouse homolog of drosophila vasa is required for the development of male germ cells. Genes Dev. 2000, 14: 841-853.
viable. Individual genes would have to contribute to approaching the level of Y chromosome deletions before the clinical impact would be significant. It seems most likely that the major role of the knockout mouse will be in understanding the basic biology and that significant diagnostic/therapeutic approaches are some way off.
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