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.

Ethanol embryotoxicity: direct effects on mammalian embryos in vitro.

Exposure to ethanol retards growth and differentiation in cultured rat embryos during organogenesis. The development of untreated embryos is indistinguishable from growth in utero. These data suggest that the hypoplastic features of children born to chronically alcoholic mothers are due, at least in part, to a direct action of ethanol, which causes reduced embryonic cellular proliferation early in gestation.

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.

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.

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.

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.

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.

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.

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.

Dipterex teratogenicity in the rat, hamster, and mouse when given by gavage.

Dipterex was teratogenic after administration by gavage (t.i.d.) at a dose level of 480 mg/kg-day to the CP rat on days 6 through 15 of gestation, but not when administered only on days 8 or 10 of gestation. A positive teratogenic response also occurred in the hamster after administration on days 7 through 11 of gestation at 400 mg/kg-day; the apparent no-effect level for the criteria studied was 200 mg/kg-day. Embryotoxicity, but not teratogenicity, occurred after administration of 400 mg/kg-day on day 8 of gestation. In both species, the teratogenicity seen was not merely due to reduced maternal food consumption during the period of exposure. The mouse was less susceptible to Dipterex than were the rat and hamster, but a significant increase in the incidence of cleft palates resulted from exposure on days 10 through 14, or on days 12 through 14 of gestation.

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.

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.

Isotretinoin teratogenicity in mouse whole embryo culture.

Recent clinical observations strongly suggest that isotretinoin [13-cis-retinoic acid (cis RA)] is a human teratogen causing primarily heart and craniofacial malformations including ear and palatal defects. The purpose of the present study was to determine if cis RA could induce similar craniofacial malformations in mouse embryo culture. Day 8 CD-1 mouse embryos were cultured for 48 hours in rat serum in the presence or absence of various concentrations of cis RA dissolved in DMSO. DMSO by itself had no effect on embryonic development; however, cis RA at 2 X 10(-5) M (6 micrograms/ml) was clearly toxic. At 2 X 10(-6) M cis RA, growth retardation was minimal, and approximately one-third of the embryos exhibited very specific defects including a dramatic reduction in the size of the first and second visceral arches, which eventually give rise to the maxilla, mandible, and ear. Similar observations were also made with 4-oxo-13-cis RA, which is a major metabolite of cis RA in the mouse and human. These malformations would be expected to result in defects similar to those observed in the human, and preliminary observations suggest these defects are due to cis RA-induced inhibition of cranial neural crest cell migration. Using day-10 mouse embryos cultured for 48 hours in Waymouth's medium containing 50% fetal calf serum, we observed that cis RA at 2 X 10(-5) M produced a high percentage of embryos with limb defects and median cleft lip. Our results demonstrate that labeled cis RA enters the tissues of the embryo both in vivo and in vitro. Cis RA inhibited proliferation of the frontonasal mesenchyme cells in primary culture with 31% inhibition occurring at 2 X 10(-5) M cis RA.

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.

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.

Retinoic acid inhibits migration of cranial neural crest cells in the cultured mouse embryo.

Clinical observations have demonstrated that isotretinoin (13-cis-retinoic acid; cis-RA) is a human teratogen causing primarily heart and craniofacial malformations. Isotretinoin exposure to the early postimplantation mouse embryo in culture results in specific defects in craniofacial development that may be due to an interference in the early migration of cranial neural crest (CNC) cells [Goulding and Pratt, 1986]. The present study was designed to test this hypothesis by examining the migration of these cells in whole embryo culture. Day 8 CD-1 mouse embryos were cultured for 6-48 hr in the presence or absence of cis-RA at 2 X 10(-6) to 2 X 10(-5) M. Embryos either were fixed for light microscopy using Nichols' method for localization of CNC cells or were processed for scanning and transmission electron microscopy. At the light microscopic level, CNC cells in the mid-brain region of control embryos had migrated to the region of the first and second visceral arches after 6 hr in culture. Cis-RA interfered with this migration; CNC cells in treated embryos either did not leave the neuroepithelium (NE) or were aggregated near the NE. Autoradiographic studies indicated that cis-RA did not affect the overall viability or DNA synthesis of the CNC cells. However, at the TEM level, there was a dramatic increase in the number of cellular blebs in the CNC cells. Our results demonstrate a direct effect of 13-cis-RA on the CNC cells and suggest that this effect is due to alterations in the cell surface.

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.

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.

Glucocorticoid teratogenesis in mouse whole embryo culture.

Glucocorticoids, such as triamcinolone acetonide (TAC-A) and triamcinolone hexacetonide (TAC-HA), are potent inducers of cleft palate in vivo in various mouse strains when administered on day 11 of gestation, whereas they are poor or ineffective inducers of cleft lip when given on day 7. The purpose of the present study was to determine whether glucocorticoids are capable of interfering with early embryonic development in culture. CD-1 mouse embryos were cultured for 48 hours starting either on day 8 (plug day 0) with the embryo inside the yolk sac, or on day 10 with the embryo exteriorized from its functional yolk sac. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered DNA and protein levels. With the day 8 cultures, TAC-A produced a dose-dependent inhibition of growth along with malformations consisting of cardiac irregularities, abnormal rotation, and irregular neural tube closure. With the day 10 cultures, these malformations were not observed, presumably due to the advanced stage of development when the embryos were exposed to TAC-A; however, TAC-A did produce growth inhibition along with cleft lip. When TAC-HA was administered in vivo to pregnant donor females on day 7, in combination with TAC-A added on day 10 to the culture medium, there was a dramatic increase in the frequency of cleft lip along with other alterations in craniofacial appearance. Our results demonstrate that glucocorticoids are capable of directly affecting embryonic growth and development during the early stages of organogenesis.

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.