Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Abnormal spermatogenesis in RXR[ mutant mice Philippe Kastner, t Manuel Mark, ~ Mark Leid, 2 Anne Gansmuller, William Chin, 3 Jesus M. Grondona, Didier D6cimo, Wojciech Krezel, Andr6e Dierich, and Pierre Chambon 4 Institut de G6n4tique et de Biologie Mol6culaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)/Institut National de la Sant6 et de la Recherche M6dicale (INSERM)/Universit6 Louis Pasteur (ULP1, Collhge de France, BP 163-67404 ILLKIRCH-CEDEX, C.U. de Strasbourg, France We have generated mouse lines in which the RXRI3 gene was disrupted by homologous recombination. Approximately 50% of the RXR~ homozygous mutants died before or at birth, but those that survived appeared normal except that the males were sterile, owing to oligo-astheno-teratozoospermia. Failure of spermatid release occurred within the germinal epithelium, and the epididymis contained very few spermatozoa that, in addition, exhibited abnormal acrosomes and tails. There was a progressive accumulation of lipids within the mutant Sertoli cells, which were histochemically characterized as unsaturated triglycerides. In old mutant males, progressive degeneration of the germinal epithelium occurred, ending with the formation of acellular lipid-filled tubules. The selective expression of RXR[~ in Sertoli cells, together with the timing of appearence of the histological abnormalities, suggests that the primary defect resulting from the mutation resides in these cells. [Key Words: Sertoli cells; lipid metabolism defect; male sterility; seminiferous epithelium degeneration; retinoid X receptor] Received August 21, 1995; revised version accepted October 31, 1995. The three retinoid X receptors, RXRa, RXRB, and RXR~, receptor (VDR), and the peroxisome proliferator-acti- are members of the vertebrate nuclear receptor super- vated receptors (PPARs) (for review, see Mangelsdorf et family (Leid et al. 1992a, b; Mangelsdorf et al. 1992, 1994; al. 1992; Liu and Linney 1993; Doll4 et al. 1994; Nagata Chambon 1994; Giguhre 1994; Glass 1994; Kastner et al. et al. 1994; Desvergne and Wahli 1995). Finally, RXRs 1994b). In the mouse, RXRa and RXRB transcripts ap- may also be the heterodimeric partners of a number of pear to be widely expressed in the embryo and in adult orphan nuclear receptors, including members of the tissues, whereas the distribution of RXR-¢ transcripts is FXR/RLD-1/LXR/UR family (Apfel et al. 1994; Song et more restricted (Mangelsdorf et al. 1992; Liu and Linney al. 1994; Forman et al. 1995a; Seol et al. 1995; Teboul et 1993; Doll6 et al. 1994; Nagata et al. 1994). In vitro al. 1995; Willy et al. 1995; note that UR has also been DNA-binding studies and studies in transfected cells named OR-1 or RIP15), members of the NGFI-B/ cultured in vitro have provided evidence indicating that NURR-1 family (Forman et al. 1995b; Perlmann and RXRs could be involved in several signaling pathways. Jansson 1995), and the orphan receptor MB67 (Baes et al. First, as 9-cis retinoic acid (RA) (9C-RA)-dependent tran- 1994). scriptional regulators, RXR homodimers may transduce Whether RXRs engaged in heterodimeric associations some of the effects of the active retinoid derivatives of act as 9C-RA-dependent transcriptional regulators to vitamin A. Second, RXRs may also play a role in the synergistically control initiation of transcription or are retinoid signaling pathway, as heterodimeric partners for transcriptionally silent, simply allowing their partners the retinoic acid receptors (RARoL, RARf3, and RARe/) to bind efficiently to the response elements of their cog- that act as all-trans RA (T-RA)- or 9C-RA-dependent nate target genes, is largely unknown. Several studies transcriptional regulators. Third, RXRs may be involved performed in vitro and in transfected cultured cells have as heterodimeric partners in additional signaling path- suggested that the 9C-RA-dependent transcriptional ac- ways mediated by other nuclear receptors, which include tivity of the RXR partner is dependent on both the nature the thyroid hormone receptors (TRs), the vitamin D3 and the ligand occupancy of the heterodimeric partner, as well as on the nature of the bound response element (Durand et al. 1994; Kurokawa et al. 1994; Forman et al. tThese authors contributed equally. 1995b; Perlmann and Jansson 1995; Willy et al. 1995). Present addresses: 2College of Pharmacy,Oregon State University, Cor- Interestingly, Roy et al. (1995) have shown recently that, vallis, Oregon 97331 USA; SHarvardMedical School, Dept. of Medicine, Boston, Massachusetts 02115 USA. in P19 and F9 embryonal carcinoma cells, RAR- and 4Corresponding author. RXR-specific ligands have synergistic effects to activate 80 GENES& DEVELOPMENT 10:80-92 © 1996 by Cold Spring Harbor LaboratoryPress ISSN 0890-9369/96 $5.00 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Male sterility in RXR~ mutant mice endogenous RA target genes and to induce differentia- A BC (~ Probes I I , ,~ I--I 5' 3" tion, which indicates that, at least in some cases of RXR/ w-r Sp K XN?.B,.. KE B B,.S ep.Nh NhBB SpKX j .,, , , i ,(, RAR partnerships, both receptors can be implicated in RXR~ locus (+) ' transcriptional activation of target genes. In contrast to the wealth of information related to the targeting construct ~I , lkb 3' " 5' functions of RXRs in vitro, very little is known to date PGK-NEO(A +) concerning the actual physiological role of RXRs in vivo. K XN.': _=B=..I__~_EHB Sp ..'-"BB ,Sp.== BB, Sp,r" KX , Notably, it is unknown whether, as a receptor for 9C- mutant locus (-) S p RA, RXR transduces some of the multiple effects of ret- • 5 "1 inoids. In this respect, it is noteworthy that most of the (+)1 9kb I | Spel digest I J probe A abnormalities found in vitamin A-deficient fetuses or 7kb adult mice are reproduced in mice bearing mutations in (+) I~-~-~-I ] BamHI one or several RARs, which demonstrate that RARs are (-) ~1 digest 1kb probeB involved in the physiological transduction of the RA sig- nal (Lohnes et al. 1993, 1994; Lufkin et al. 1993; Men- delsohn et al. 1994). The possible function of RXRR has ''+' @ © +~i" +'~'~ been investigated by examining homozygous null mice .•&' generated by targeted disruption of the RXRa gene in -v 3kb embryonic stem (ES) cells (Kastner et al. 1994a; Sucov et probe A probe B Spe I 7kb BamHI al. 1994). These mutants exhibit fetal heart and eye de- 0-- lkb fects similar to those occurring in the fetal vitamin A-de- ficiency (VAD) syndrome, thus suggesting that RXR~ t 2 3 / 3kb mediates some of the effects of vitamin A. Moreover, P robe C BamHI [ these studies have revealed a strong synergy between mutations in RAR and RXR genes for the generation of 1234567 abnormalities that were either less severe in single RXR or RAR mutants or not present at all. It was also shown Figure 1. The RXR[3 mutation. (al Targeting the RXRff gene. A that some specific RXR/RAR pairs are much more effi- map of the wild-type RXRI3 gene is shown at the top. (.) Exons cient than others at generating some of the abnormali- (numbering as in Nagata et al. 1994). Probe A is a NotI-KpnI ties seen in the fetal VAD syndrome (Kastner et al. fragment; probe B is a 500-bp BamHI-HindIII fragment imme- 1994a; P. Kastner, M. Mark, and P. Chambon, unpubl.). diately upstream of the deleted region; probe C is the HindIII- EcoRI fragment (corresponding to the deleted region). (Sp) SpeI; Taken together, these data strongly suggest that RAR/ (K) KpnI; (N) NotI; (B) BamHI; (E) EcoRI; (Nh) NheI; (X)XhoI; RXR heterodimers are the major functional units respon- (H) HindIII. (b) Targeted ES cells. Southern blot analysis of SpeI- sible for transducing the retinoid signal during develop- restricted DNA from wild type (WT) and the HA67 and HA9 ES ment. cell clones, analyzed with the 5' probe A. (c) Southern blot anal- We now report the generation of RXR~ null mutant ysis of mutant mice. (Top) The analysis of a litter using BamHI- mice. Approximately 50% of these mutants die in utero restricted DNA and probe B. (Bottom) Corresponds to an iden- or very shortly after birth for unknown reasons. Surpris- tical blot hybridized with probe C. ingly, the remaining null mutants are externally indis- tinguishable from their wild-type or heterozygous litter- mates. However, RXR~-/- males are sterile, owing to abnormal germ cell maturation, leading to oligo-as- HA9) transmitted the mutation, thus generating two theno-teratozoospermia. These sperm abnormalities lines of mutant mice. For both lines, crosses between most probably reflect an indispensable function of RXR~ heterozygotes (RXRS +/-) yielded viable homozygote in the Sertoli cells, which in prepubertal mutant males mutants (RXR~-/-), which were externally indistin- start to accumulate lipid droplets. The possible involve- guishable from their wild-type and heterozygote litter- ment of PPARs in the generation of the testis abnormal- mates. Homozygote females were fertile, but males were ities seen in RXR~ null mutants is discussed. sterile (see below). As expected, a probe spanning the genomic region en- coding the RXR~ DNA-binding domain did not detect any hybridizing fragment in homozygotes {probe C in Results Fig. lc). Therefore, even though RXRI3 exons 1, 2, and 5-9 are still present in the genome of homozygotes, a RXR[3 mutant mice functional DNA-binding RXR~ protein cannot be pro- The RXR~ gene was disrupted by homologous recombi- duced in the mutants. RT-PCR analysis of total RNA nation in ES cells with a replacement-type vector in isolated from several tissues showed that transcripts cor- which the genomic sequences encoding the DNA-bind- responding to exon 2, as well as transcripts containing ing domain (3' region of exons 3 and 4) were replaced exons 5-9, were present at a low level in RXR~ - / - mice with a PGK-NEO(A +) cassette (Fig.