Decreased home cage movement and oromotor impairments in adult Fmr1-KO mice.

Fragile X syndrome (FXS) is a common inherited disorder that significantly impacts family and patient day-to-day living across the entire life span. The childhood and adolescent behavioral consequences of FXS are well appreciated. However, there are significantly fewer studies (except those examining psychiatric comorbidities) assessing behavioral phenotypes seen in adults with FXS. Mice engineered with a genetic lesion of fragile X mental retardation 1 (Fmr1) recapitulate important molecular and neuroanatomical characteristics of FXS, and provide a means to evaluate adult behavioral phenotypes associated with FXS. We give the first description of baseline behaviors including feeding, drinking, movement and their circadian rhythms; all observed over 16 consecutive days following extensive environmental habituation in adult Fmr1-KO mutant mice. We find no genotypic changes in mouse food ingestion, feeding patterns, metabolism or circadian patterns of movement, feeding and drinking. After habituation, Fmr1-KO mice show significantly less daily movement during their active phase (the dark cycle). However, Fmr1-KO mice have more bouts of activity during the light cycle compared with wild types. In addition, Fmr1-KO mice show significantly less daily water ingestion during the circadian dark cycle, and this reduction in water intake is accompanied by a decrease in the amount of water ingested per lick. The observed water ingestion and circadian phenotypes noted in Fmr1-KO mice recapitulate known clinical aspects previously described in FXS. The finding of decreased movement in Fmr1-KO mice is novel, and suggests a dissociation between baseline and novelty-evoked activity for Fmr1-KO mice.

Estrogen receptor-alpha is required by the supporting somatic cells for spermatogenesis.

The gene for estrogen receptor-alpha (ERalpha) was disrupted in embryonic stem cells by homologous recombination and these cells were used to generate mice with a targeted mutation in the ERalpha gene (alphaERKO mice). It was found that males homozygous for the mutation are infertile, indicating that estrogen signaling through this nuclear hormone receptor is required for male reproductive function. Although spermatogenesis appears normal in juvenile and young adult alphaERKO mice, the sperm produced are unable to fertilize eggs in vitro. To determine whether ERalpha is required by somatic or germ cells in the male reproductive tract, we transplanted germ cells from homozygous mutant (ERalpha(-/-)) males to the testes of wild-type (ERalpha(+/+)) males depleted of germ cells by busulfan treatment. The recipients ('surrogate fathers') sired offspring heterozygous for the mutation (ERalpha(+/-)) and carrying the coat-color marker of the infertile donor males. This indicated that ERalpha(-/-) germ cells are able to produce sperm competent to fertilize when they are supported by ERalpha(+/+) somatic cells. When ERalpha(+/-) offspring produced by germ cell transplantation were mated to produce ERalpha(-/-) males, these mice were found to have the same phenotype as originally reported for alphaERKO males. These studies showed that male germ cells do not require ERalpha for regulation of their own genes for development and function, and strongly imply that somatic cells of the male reproductive tract require ERalpha to support the production of sperm that are capable of fertilization.

Haploinsufficiency of protamine-1 or -2 causes infertility in mice.

Protamines are the major DNA-binding proteins in the nucleus of sperm in most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. Many mammals have one protamine, but a few species, including humans and mice, have two. Here we use gene targeting to determine if the second protamine provides redundancy to an essential process, or if both protamines are necessary. We disrupted the coding sequence of one allele of either Prm1 or Prm2 in embryonic stem (ES) cells derived from 129-strain mice, and injected them into blastocysts from C57BL/6-strain mice. Male chimeras produced 129-genotype sperm with disrupted Prm1 or Prm2 alleles, but failed to sire offspring carrying the 129 genome. We also found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function. Our studies show that both protamines are essential and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.

Spermatogenic cells do not require estrogen receptor-alpha for development or function.

Estrogen receptors alpha (ERalpha) and beta (ERbeta) are ligand-dependent transcription factors and members of the nuclear hormone receptor superfamily encoded by separate genes. Male mice homozygous for a mutation in the gene encoding ERalpha are infertile. To determine whether germ cells or somatic cells require ERalpha, germ cells were transplanted from donor males homozygous for the mutation (ERalpha-/-) to testes of wild-type (ERalpha+/+) recipient mice depleted of germ cells. The recipients served as "surrogate fathers" for the infertile ERalpha-/- males. When mated to wild-type females, the recipients sired offspring heterozygous for the mutation (ERalpha+/-) and carrying the coat-color marker of the ERalpha-/- donor mice. These studies show that male germ cells do not require ERalpha for development or to function in fertilization, and imply that male ERalpha-/- mice are infertile due to disruption of estrogen action within somatic cells of the male reproductive system.

Fertilization defects in sperm from mice lacking fertilin beta.

Fertilin, a member of the ADAM family, is found on the plasma membrane of mammalian sperm. Sperm from mice lacking fertilin beta were shown to be deficient in sperm-egg membrane adhesion, sperm-egg fusion, migration from the uterus into the oviduct, and binding to the egg zona pellucida. Egg activation was unaffected. The results are consistent with a direct role of fertilin in sperm-egg plasma membrane interaction. Fertilin could also have a direct role in sperm-zona binding or oviduct migration; alternatively, the effects on these functions could result from the absence of fertilin activity during spermatogenesis.

Mouse spermatogenic cell-specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1-S protein is localized mainly in the sperm tail.

Unique type 1 hexokinase (HK1) mRNAs are present in mouse spermatogenic cells (mHk1-s). They encode a spermatogenic cell-specific sequence region (SSR) but not the porin-binding domain (PBD) necessary for HK1 binding to porin on the outer mitochondrial membrane. This study determined the origin of the multiple Hk1-s transcripts in mouse spermatogenic cells and verified that they are translated in mouse spermatogenic cells. It also showed that a single mHk1 gene encodes the mHk1 transcripts of somatic cells and the mHk1-sa and mHk1-sb transcripts of spermatogenic cells, that alternative exons are used during mHk1 gene expression in mouse spermatogenic cells, and that mHK1-S is translated in mouse spermatogenic cells and is localized mainly with the fibrous sheath in the tail region, not with the mitochondria in the midpiece of mouse sperm.

HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes.

Spermatogenic cells synthesize a unique 70-kDa heat shock protein (HSP70-2) during prophase of meiosis I, and targeted disruption of the Hsp70-2 gene has shown that this protein is required for spermatogenic cell differentiation in adult mice. HSP70-2 is associated with synaptonemal complexes formed between paired homologous chromosomes during meiotic prophase. The present study focuses on the nearly synchronous first wave of spermatogenesis in 12- to 28-day old juvenile mice to determine more precisely when HSP70-2 is required and what meiotic processes are affected by its absence. Spermatogenesis in homozygous mutant mice (Hsp70-2[-/-]) proceeded normally until day 15 when increasing numbers of pachytene spermatocytes became apoptotic and differentiation of cells beyond the pachytene stage began to falter. Synaptonemal complexes assembled in Hsp70-2(-/-) mice and spermatocytes developed through the final pachytene substage. However, synaptonemal complexes failed to desynapse and normal diplotene spermatocytes were not observed. Metaphase spermatocytes were not seen in tissue sections from testes of Hsp70-2(-/-) mice, and expression of mRNAs and antigens characteristic of late pachytene spermatocytes (e.g., cyclin A1) and development of spermatids did not occur. Thus, HSP70-2 is required for synaptonemal complex desynapsis, and its absence severely impairs the transition of spermatogenic cells through the late meiotic stages and results in apoptosis beginning with the first wave of germ cell development in juvenile mice.

Allosteric activation and tuning of ligand efficacy in cyclic-nucleotide-gated channels.

Despite recent advances in the identification of ligand-binding and voltage-sensing regions of ion channels, the domains that couple such regions to channel opening have not been identified. Moreover, it is uncertain whether ligand binding or depolarization are obligatory steps that must precede channel opening (according to linear reaction schemes) or whether they act to stabilize the channel in an open state that can exist independently of ligand binding or depolarization (according to cyclic allosteric models). By comparing ligand-independent and ligand-dependent channel openings, we now show that retinal and olfactory cyclic-nucleotide-gated channels are activated by a cyclic allosteric mechanism. We further show that an amino-terminal domain, distinct from the pore and ligand-binding motifs, participates in the allosteric gating transition, accounting for differences in the free energy of gating of the two channels. The allosteric transition provides an important mechanism for tuning the physiological response of ligand-binding proteins, such as cyclic-nucleotide-gated channels, to different biological signals.

Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility.

The reproductive system of male mice homozygous for a mutation in the estrogen receptor (ER) gene (ER knock-out; ERKO) appears normal at the anatomical level. However, these males are infertile, indicating an essential role for ER-mediated processes in the regulation of male reproduction. Adult ERKO male mice have significantly fewer epididymal sperm than heterozygous or wild-type males. Although spermatogenesis is occurring in some seminiferous tubules of 3- to 5-month-old ERKO males, other tubules either have a dilated lumen and a disorganized seminiferous epithelium with few spermatogenic cells or lack a lumen and contain mainly Sertoli cells. There are no obvious differences in seminiferous tubules at 10 days of age between wild-type and ERKO mice, but the lumen in ERKO males is dilated in all seminiferous tubules by 20 days. However, spermatogenesis progresses and similar numbers of sperm are present in the cauda epididymis of ERKO and wild-type males until 10 weeks of age. Disruption of spermatogenesis and degeneration of the seminiferous tubules become apparent after 10 weeks in the caudal pole of the testis and progresses in a wave to the cranial pole by 6 months. However, the seminal vesicles, coagulating glands, prostate, and epididymis do not appear to be altered morphologically in ERKO mice. Serum testosterone levels are somewhat elevated, but LH and FSH levels are not significantly different from those in wild-type males. Sperm from 8- to 16-week-old mice have reduced motility and are ineffective at fertilizing eggs in vitro. In addition, ERKO males housed overnight with hormone-primed wild-type females produce significantly fewer copulatory plugs than do heterozygous or wild-type males. These results suggest that estrogen action is required for fertility in male mice and that the mutation of the ER in ERKO males leads to reduced mating frequency, low sperm numbers, and defective sperm function.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis.

Germ cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation of Hsp70-2, we used mRNA primer- extension, reverse transcriptase PCR (RT-PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5'-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression of Hsp70-2 during spermatogenesis. Results with multiple lines of transgenic mice containing promoter-reporter transgenes with varying lengths of Hsp7-2 sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required for Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene beta- galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution of Hsp7O-2 and Hsp7O-2/lacZ mRNAs was determined by Northern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp7O-2 and Hsp7O-2/lacZ was specific to the meiotic phase of spermatogenesis.

Boar proacrosin expressed in spermatids of transgenic mice does not reach the acrosome and disrupts spermatogenesis.

Transgenic mice that express boar proacrosin were produced to examine mechanisms for targeting hydrolytic enzymes to the acrosome. A 2.3 kb transgene was constructed by ligating the cDNA for boar preproacrosin with the mouse protamine 2 promoter region. Six founder mice that incorporated the transgene were identified by polymerase chain reaction and Southern blot analysis. Northern blots indicated that the two male founders (Ac.2 and Ac.5) and male progeny from three female founders (Ac.3, Ac.4, Ac.6) expressed the transgene mRNA in testis, but not in somatic tissues. In these transgenic animals boar proacrosin was detected by immunohistochemistry in condensing spermatids, but was not localized in the acrosome. This acrosomal targeting defect of the transgene product may result from its delayed expression during the later steps of haploid differentiation. Furthermore, both male founders and all Ac.4 and Ac.6 males were infertile, as determined by multiple matings for at least 2 months. Ac.3 males were either infertile or rarely transmitted the transgene to their offspring. The infertile males mated, produced copulatory plugs, and had seminal vesicle weights and testosterone levels within the normal range. However, they produced significantly fewer spermatozoa and had lower testis weights than controls. Although the mitotic and meiotic phases of spermatogenesis appeared normal by histological criteria, condensing spermatids were missing from most tubules, and multinucleated cells were present in the lumen of seminiferous tubules and in the epididymis. We hypothesize that boar proacrosin which fails to reach the acrosome is activated in these transgenic mice, and that its proteolytic activity disrupts spermatogenesis during spermatid formation.

Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating.

In voltage-gated ion channels and in the homologous cyclic nucleotide-gated (CNG) channels, the loop between the S5 and S6 transmembrane segments (P region) is thought to form the lining of the pore. To investigate the structure and the role in gating of the P region of the bovine retinal CNG channel, we determined the accessibility of 11 cysteine-substituted P region residues to small, charged sulfhydryl reagents applied to the inside and outside of membrane patches in the open and closed states of the channel. The results suggest that the P region forms a loop that extends toward the central axis of the channel, analogous to the L3 loop of bacterial porin channels. Furthermore, the P region, in addition to forming the ion selectivity filter, functions as the channel gate, the structure of which changes when the channel opens.

Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration.

Cyclooxygenases 1 and 2 (COX-1 and COX-2) are key enzymes in prostaglandin biosynthesis and the target enzymes for the widely used nonsteroidal anti-inflammatory drugs. To study the physiological roles of the individual isoforms, we have disrupted the mouse Ptgs1 gene encoding COX-1. Homozygous Ptgs1 mutant mice survive well, have no gastric pathology, and show less indomethacin-induced gastric ulceration than wild-type mice, even though their gastric prostaglandin E2 levels are about 1% of wild type. The homozygous mutant mice have reduced platelet aggregation and a decreased inflammatory response to arachidonic acid, but not to tetradecanoyl phorbol acetate. Ptgs1 homozygous mutant females mated to homozygous mutant males produce few live offspring. COX-1-deficient mice provide a useful model to distinguish the physiological roles of COX-1 and COX-2.

Molecular mechanism of cyclic-nucleotide-gated channel activation.

Studies on the activation of ligand- and voltage-gated ion channels have identified regions involved in both ligand binding and voltage sensing, but relatively little is known about how such domains are coupled to channel opening. Here we investigate the structural basis for the activation of cyclic-nucleotide-gated channels, which are directly opened by cytoplasmic cyclic nucleotides but are structurally homologous to voltage-gated channels. By constructing chimaeras between cyclic-nucleotide-gated channels cloned from bovine retinal photoreceptors and catfish olfactory neurons, we identify two distinct domains that are important for ligand binding and channel gating. A putative alpha-helix in the carboxy-terminal binding domain determines the selectivity of the channel for activation by cGMP relative to cAMP. A domain in the amino-terminal region determines the ease with which channels open and thus influences agonist efficacy. We propose that channel opening is coupled to an allosteric conformational change in the binding site which enhances agonist binding. Thus, cyclic nucleotides activate the channel by binding tightly to the open state and stabilizing it.

Aberrant reproductive phenotypes evident in transgenic mice expressing the wild-type mouse estrogen receptor.

The estrogen receptor (ER) acts as a transcription factor to regulate multiple cellular functions involved in normal physiology, differentiation, and reproduction. To date, there is no known animal model for studying aberrant ER expression. Therefore, we created transgenic mice expressing the wild-type mouse ER under the control of the mouse metallothionein-I (MT) promoter to determine whether overexpression of the ER would disrupt normal reproductive processes. Five male and one female founder mice were produced, and all were fertile. The progeny from these mice were screened for MT-mER expression by the ribonuclease protection assay. Mice in all six lines were found to express the transgene in a variety of tissues, although generally at low levels. The highest level of expression was observed in the female reproductive tract of line E. Females in all six lines demonstrated aberrant reproductive phenotypes involving processes at parturition and, with some of the lines, a tendency toward reduced fertility. Gestational length was prolonged up to 4 days beyond the normal gestation of 19 days, providing evidence of delayed parturition. In addition, prolonged labor (up to 3 days in length to deliver all pups) and labors requiring cesarean sections for maternal survival demonstrated the occurrence of dystocia in the MT-mER females. As maternal age increased, the incidence of stillborn litters, delayed parturition, and dystocia approached 100% in the transgenic dams. Difficulties at parturition were not observed in nontransgenic control females. These phenotypes suggest that the mechanisms regulating parturition may be perturbed by improper expression of the ER. The MT-mER transgenic mice may provide a novel approach for studying the estrogen-regulated signals involved in parturition and fertility as well as a unique animal model for the human reproductive phenotypes of delayed parturition and dystocia.

Role of H5 domain in determining pore diameter and ion permeation through cyclic nucleotide-gated channels.

Ion permeation through membrane channels is thought to be governed by a narrow region of the channel pore termed the selectivity filter, which has been proposed to discriminate among ions by both specific binding and molecular sieving, as determined by pore diameter. Recent evidence suggests that a conserved domain (known as H5, P or SS1-SS2) in voltage-gated potassium, sodium and calcium channels contributes to the lining of the pore. Here we investigate whether the H5 domain determines pore diameter and examine the role of pore diameter in controlling ion permeation. These studies rely on differences in single channel conductance, ion selectivity and apparent pore diameter between cyclic nucleotide-gated channels cloned from bovine retina and catfish olfactory neurons. Using chimaeric retinal-olfactory channels, we find that the H5 domain determines these differences in permeation properties, providing structural evidence that the cyclic nucleotide-gated channels are indeed members of the voltage-gated channel family. Moreover, these results show directly that the H5 domain helps form the selectivity filter and that molecular sieving is important in controlling ion permeation.

Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.

We have cloned a functional cDNA encoding the cyclic nucleotide-gated channel selectively expressed in catfish olfactory sensory neurons. The cyclic nucleotide-gated channels share sequence and structural features with the family of voltage-gated ion channels. This homology is most evident in transmembrane region S4, the putative voltage sensor domain, and the H5 domain, thought to form the channel pore. We have characterized the single-channel properties of the cloned catfish channel and compared these properties with the channel in native catfish olfactory sensory neurons. The channel is activated equally well by cAMP and cGMP, shows only a slight voltage dependence of gating, and exhibits a pH- and voltage-dependent subconductance state similar to that observed for the voltage-gated L-type calcium channel.

Teratogenicity of benzoic acid derivatives of retinoic acid in cultured mouse embryos.

Isotretinoin (13-cis-RA) is a human teratogen and mouse embryos exposed to 13-cis-RA in vivo exhibit many of the same defects as humans. Early postimplantation mouse embryos exposed to 13-cis-RA in culture exhibit developmental alterations of the visceral arches, similar to those seen after in vivo exposure. Certain benzoic acid derivatives of retinoic acid have been shown to possess activity equal to or greater than retinoic acid in several in vitro systems. This study examines the teratogenic effects of some of these retinoids on mouse embryos in vitro. Day 8 CD-1 mouse embryos were cultured for 48 hours in the presence of these benzoic acid derivatives. With the exception of Ro-15-0778, all compounds produced visceral arch malformations similar to those seen in embryos exposed to 13-cis-RA, but at dramatically different effective concentrations. Extremely low concentrations of the retinoic acid-related compounds tested appear to have detrimental effects on embryonic development and these compounds may be poor candidates for therapeutic use.

Alterations in craniofacial growth induced by isotretinoin (13-cis-retinoic acid) in mouse whole embryo and primary mesenchymal cell culture.

Recent evidence has demonstrated that 13-cis-retinoic acid (13-cis-RA, or isotretinoin) is responsible for various craniofacial malformations in the rodent and human embryo. Our studies have been directed toward understanding this effect using mouse whole embryo and primary cell cultures. In whole embryo culture, 13-cis-RA caused significant overall embryonic growth retardation, especially in the primary and secondary palatal processes. In embryos explanted on day 10 of gestation and exposed for 24 or 48 hr, the mesenchyme beneath the epithelium of the nasal and maxillary processes contained pyknotic nuclei as well as a dramatically reduced number of nuclei incorporating 3H-thymidine. The secondary palatal processes and the roof of the oral-nasal cavity had fewer mesenchymal cells than control embryos. The incorporation of 3H-thymidine into TCA-insoluble macromolecules was 30% less in the retinoid-treated heads. In primary cell cultures from day-12 mouse secondary palatal mesenchyme, subsequent cell growth was decreased at concentrations of 13-cis-RA greater than 1 X 10(-5) M. After a 40-hr treatment period, labeling indices in retinoid-treated cells were significantly lower than control values (25% compared with 40%). Retinoic acid also caused a significant, concentration-dependent decrease in 3H-thymidine incorporation. The inhibitory effect of 13-cis-RA on proliferation of oral-nasal mesenchymal cells appears to be related to the production of craniofacial malformations.