Estrogen receptor 1 appears essential for fetal viability in a murine model of premature birth.

PROBLEM: Protective effects for adult neurological disorders have been attributed to sex hormones. Using a murine model of prematurity, we evaluated the role of estrogen signaling in the process of perinatal brain injury following exposure to intrauterine inflammation. METHOD OF STUDY: Intrauterine lipopolysaccharide (LPS) was used to invoke preterm labor and fetal neuroinflammation. Fetal brains were analyzed for changes in Esr1, Esr2 and Cyp19. Dams heterozygous for the Esr1 knockout allele were also given intrauterine LPS to compare delivery and offspring viability to wild type controls. RESULTS: The upregulation in inflammatory cytokines was accompanied by an increase in Esr1 and Esr2 transcripts, though protein levels declined. Cyp19 did not differ by mRNA or protein abundance. Offspring from Esr1 mutants were larger, had a longer gestation and significantly greater mortality. CONCLUSIONS: Estrogen signaling is altered in the fetal brains of preterm offspring exposed to neuroinflammatory injury. The reduction of Esr1 and Esr2 proteins with LPS suggests that these proteins are degraded. It is possible that transcriptional upregulation of Esr1 and Esr2 occurs to compensate for the loss of these proteins. Alternatively, the translation of Esr1 and Esr2 mRNAs may be disrupted with LPS while a feedback mechanism upregulates transcription. Intact Esr1 signaling is also associated with early preterm delivery following exposure to intrauterine LPS. A loss of one Esr1 allele delays this process, but appears to do so at the cost of fetal viability. These results suggest estrogen signaling plays opposing roles between maternal and fetal responses to preterm birth.

Fecundity is impaired in a mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by mutations in genes encoding type I collagen or proteins that process it. Women with OI have a small, but significant increase in risk of serious pregnancy complications including uterine rupture. Here, the OI mouse, Col1a2oim/oim , was used to examine the effects of collagen mutation on establishment and maintenance of pregnancy. Picrosirius birefringence was faint in Col1a2oim/oim uteri, indicating diminished collagen in the myometrium and endometrium. There was some evidence of increased uterine gland number (p = .055) and size (p = .12) in (p = .055) virgin uteri, though the they were not significantly different than controls. There were no differences in the number of corpora lutea, or the time from pairing to delivery of pups between Col1a2oim/oim and control dams, suggesting that ovulation and conception occur normally. However, when examined at Gestation Day 6.5 (postimplantation), gestation Day 10.5 (midpregnancy), and Postnatal Days 1-2, Col1a2oim/oim dams had significantly fewer viable pups than controls overall. In pairwise comparisons, the loss was only significant in the postnatal group, suggesting the gradual loss of pups over time. Overall, the Col1a2oim/oim mouse data suggest that OI impairs uterine function in pregnancy in a way that affects a small but significant number of fetuses.

Hypoxanthine phosphoribosyltransferase (HPRT)-deficiency is associated with impaired fertility in the female rat.

The purine hypoxanthine plays important role in regulating oocyte maturation and early embryonic development. The enzyme hypoxanthine phosphoribosyltransferase (HPRT) recycles hypoxanthine to generate substrates for nucleotide synthesis and key metabolites, and here we show that HPRT deficiency in the rat disrupts early embryonic development and causes infertility in females.

Diverse species-specific phenotypic consequences of loss of function sorting nexin 14 mutations.

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

DNA methylation and expression of imprinted genes are associated with the viability of different sexual cloned buffaloes.

The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS-PCR). The expression levels of these imprinted genes were also investigated by quantitative real-time PCR (QRT-PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes.

Genome-wide association studies and genomic prediction of breeding values for calving performance and body conformation traits in Holstein cattle.

BACKGROUND: Our aim was to identify genomic regions via genome-wide association studies (GWAS) to improve the predictability of genetic merit in Holsteins for 10 calving and 28 body conformation traits. Animals were genotyped using the Illumina Bovine 50 K BeadChip and imputed to the Illumina BovineHD BeadChip (HD). GWAS were performed on 601,717 real and imputed single nucleotide polymorphism (SNP) genotypes using a single-SNP mixed linear model on 4841 Holstein bulls with breeding value predictions and followed by gene identification and in silico functional analyses. The association results were further validated using five scenarios with different numbers of SNPs. RESULTS: Seven hundred and eighty-two SNPs were significantly associated with calving performance at a genome-wise false discovery rate (FDR) of 5%. Most of these significant SNPs were on chromosomes 18 (71.9%), 17 (7.4%), 5 (6.8%) and 7 (2.4%) and mapped to 675 genes, among which 142 included at least one significant SNP and 532 were nearby one (100 kbp). For body conformation traits, 607 SNPs were significant at a genome-wise FDR of 5% and most of them were located on chromosomes 5 (30%), 18 (27%), 20 (13%), 6 (6%), 7 (5%), 14 (5%) and 13 (3%). SNP enrichment functional analyses for calving traits at a FDR of 1% suggested potential biological processes including musculoskeletal movement, meiotic cell cycle, oocyte maturation and skeletal muscle contraction. Furthermore, pathway analyses suggested potential pathways associated with calving performance traits including tight junction, oxytocin signaling, and MAPK signaling (P < 0.10). The prediction ability of the 1206 significant SNPs was between 78 and 83% of the prediction ability of the BovineSNP50 SNPs for calving performance traits and between 35 and 79% for body conformation traits. CONCLUSIONS: Various SNPs that are significantly associated with calving performance are located within or nearby genes with potential roles in tight junction, oxytocin signaling, and MAPK signaling. Combining the significant SNPs or SNPs within or nearby gene(s) from the HD panel with the BovineSNP50 panel yielded a marginal increase in the accuracy of prediction of genomic estimated breeding values for all traits compared to the use of the BovineSNP50 panel alone.

Molecular signature and functional analysis of uterine ILCs in mouse pregnancy.

In addition to natural killer cells, other innate lymphoid cells have recently been identified in the mouse and human uterus, but their roles in successful pregnancy remain poorly defined. In this study, we examined the dynamic changes of uterine innate lymphoid cells throughout pregnancy in mice. We found that the total number of uterine innate lymphoid cells markedly increased at early-gestation. Among the three groups of uterine innate lymphoid cells, the number of the group 2 uterine innate lymphoid cells increased the most during pregnancy. We also determined that the depletion of uterine innate lymphoid cells in Rag1-/- mice resulted in impaired uterine spiral artery remodeling. These results suggest that uterine innate lymphoid cells may play an important role in mouse reproduction.

Maternal heterozygous NLRP7 variant results in recurrent reproductive failure and imprinting disturbances in the offspring.

It has been shown previously that homozygous and compound-heterozygous variants affecting protein function in the human NLRP genes impact reproduction and/or fetal imprinting patterns. These variants represent so-called 'maternal effect mutations', that is, although female variant carriers are healthy, they are at risk of reproductive failure, and their offspring may develop aberrant methylation and imprinting disorders. In contrast, the relevance to reproductive failure of maternal heterozygous NLRP7 variants remains unclear. The present report describes the identification of a heterozygous NLRP7 variant in a healthy 28-year-old woman with a history of recurrent reproductive failure, and the molecular findings in two of the deceased offspring. Next-generation sequencing (NGS) for NLRP variants was performed. In the tissues of two offspring (one fetus; one deceased premature neonate) methylation of imprinted loci was tested using methylation-specific assays. Both pregnancies had been characterized by the presence of elevated human chorionic gonadotropin (hCG) levels and ovarian cysts. In the mother, a heterozygous nonsense 2-bp deletion in exon 5 of the NLRP7 gene was identified (NM_001127255.1:c.2010_2011del, p.(Phe671Glnfs*18)). In the two investigated offspring, heterogeneous aberrant methylation patterns were detected at imprinted loci. The present data support the hypothesis that heterozygous NLRP7 variants contribute to reproductive wastage, and that these variants represent autosomal dominant maternal effect variants which lead to aberrant imprinting marks in the offspring. Specific screening and close prenatal monitoring of NLRP7 variant carriers is proposed. Egg donation might facilitate successful pregnancy in heterozygous NLRP7 variant carriers.

MCT8 Deficiency in Male Mice Mitigates the Phenotypic Abnormalities Associated With the Absence of a Functional Type 3 Deiodinase.

Mice deficient in the type 3 deiodinase (D3KO mice) manifest impaired clearance of thyroid hormone (TH), leading to elevated levels of TH action during development. This alteration causes reduced neonatal viability, growth retardation, and central hypothyroidism. Here we examined how these phenotypes are affected by a deficiency in the monocarboxylate transporter 8 (MCT8), which is a major contributor to the transport of the active thyroid hormone, T3, into the cell. MCT8 deficiency eliminated the neonatal lethality of type 3 deiodinase (D3)-deficient mice and significantly ameliorated their growth retardation. Double-mutant newborn mice exhibited similar peripheral thyrotoxicosis and increased brain expression of T3-dependent genes as mice with D3 deficiency only. Later in neonatal life and adulthood, double-mutant mice manifested central and peripheral TH status similar to mice with single MCT8 deficiency, with low serum T4, elevated serum TSH and T3, and decreased T3-dependent gene expression in the hypothalamus. In double-mutant adult mice, both thyroid gland size and the hypothyroidism-induced rise in TSH were greater than those in mice with single D3 deficiency but less than those in mice with MCT8 deficiency alone. Our results demonstrate that the marked phenotypic abnormalities observed in the D3-deficient mouse, including perinatal mortality, growth retardation, and central hypothyroidism in adult animals, require expression of MCT8, confirming the interdependent relationship between the TH transport into cells and the deiodination processes.

Lentiviral Vector-Mediated Complementation Restored Fetal Viability but Not Placental Hyperplasia in Plac1-Deficient Mice.

The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation, and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of overexpressed Plac1 on placental formation and function remained unaddressed. We complemented the Plac1 knockout placental dysfunction by lentiviral vector-mediated, placenta-specific Plac1 transgene expression. Whereas fetal development and the morphology of maternal blood sinuses in the labyrinth zone improved, placental hyperplasia remained, with an expanded the junctional zone that migrated and encroached into the labyrinth zone. Further experiments revealed that wild-type placenta with transgenically expressed Plac1 resulted in placental hyperplasia without the encroaching of the junctional zone. Our findings suggest that Plac1 is involved in trophoblast cell proliferation, differentiation, and migration. Its proper expression is required for normal placentation and fetal development.

Advances in ultrasound imaging for congenital malformations during early gestation.

BACKGROUND: With refinement in ultrasound technology, detection of fetal structural abnormalities has improved and there have been detailed reports of the natural history and expected outcomes for many anomalies. The ability to either reassure a high-risk woman with normal intrauterine images or offer comprehensive counseling and offer options in cases of strongly suspected lethal or major malformations has shifted prenatal diagnoses to the earliest possible gestational age. METHODS: When indicated, scans in early gestation are valuable in accurate gestational dating. Stricter sonographic criteria for early nonviability guard against unnecessary intervention. Most birth defects are without known risk factors, and detection of certain malformations is possible in the late first trimester. RESULTS: The best time for a standard complete fetal and placental scan is 18 to 20 weeks. In addition, certain soft anatomic markers provide clues to chromosomal aneuploidy risk. Maternal obesity and multifetal pregnancies are now more common and further limit early gestation visibility. CONCLUSION: Other advanced imaging techniques during early gestation in select cases of suspected malformations include fetal echocardiography and magnetic resonance imaging.

Loss of Extended Synaptotagmins ESyt2 and ESyt3 does not affect mouse development or viability, but in vitro cell migration and survival under stress are affected.

The Extended Synaptotagmins (Esyts) are a family of multi-C2 domain membrane proteins with orthologs in organisms from yeast to human. Three Esyt genes exist in mouse and human and these have most recently been implicated in the formation of junctions between endoplasmic reticulum and plasma membrane, as well as the Ca(2+) dependent replenishment of membrane phospholipids. The data are consistent with a function in extracellular signal transduction and cell adhesion, and indeed Esyt2 was previously implicated in both these functions in Xenopus. Despite this, little is known of the function of the Esyts in vivo. We have generated mouse lines carrying homozygous deletions in one or both of the genes encoding the highly homologous Esyt2 and Esyt3 proteins. Surprisingly, esyt2(-/-)/esyt3(-/-) mice develop normally and are both viable and fertile. In contrast, esyt2(-/-)/esyt3(-/-) mouse embryonic fibroblasts display a reduced ability to migrate in standard in vitro assays, and are less resistant to stringent culture conditions and to oxidative stress than equivalent wild type fibroblasts.

Genetic alterations affecting cholesterol metabolism and human fertility.

Single nucleotide polymorphisms (SNPs) represent genetic variations among individuals in a population. In medicine, these small variations in the DNA sequence may significantly impact an individual's response to certain drugs or influence the risk of developing certain diseases. In the field of reproductive medicine, a significant amount of research has been devoted to identifying polymorphisms which may impact steroidogenesis and fertility. This review discusses current understanding of the effects of genetic variations in cholesterol metabolic pathways on human fertility that bridge novel linkages between cholesterol metabolism and reproductive health. For example, the role of the low-density lipoprotein receptor (LDLR) in cellular metabolism and human reproduction has been well studied, whereas there is now an emerging body of research on the role of the high-density lipoprotein (HDL) receptor scavenger receptor class B type I (SR-BI) in human lipid metabolism and female reproduction. Identifying and understanding how polymorphisms in the SCARB1 gene or other genes related to lipid metabolism impact human physiology is essential and will play a major role in the development of personalized medicine for improved diagnosis and treatment of infertility.

Myocardin-like protein 2 regulates TGFß signaling in embryonic stem cells and the developing vasculature.

The molecular mechanisms that regulate and coordinate signaling between the extracellular matrix (ECM) and cells contributing to the developing vasculature are complex and poorly understood. Myocardin-like protein 2 (MKL2) is a transcriptional co-activator that in response to RhoA and cytoskeletal actin signals physically associates with serum response factor (SRF), activating a subset of SRF-regulated genes. We now report the discovery of a previously undescribed MKL2/TGFß signaling pathway in embryonic stem (ES) cells that is required for maturation and stabilization of the embryonic vasculature. Mkl2(-/-) null embryos exhibit profound derangements in the tunica media of select arteries and arterial beds, which leads to aneurysmal dilation, dissection and hemorrhage. Remarkably, TGFß expression, TGFß signaling and TGFß-regulated genes encoding ECM are downregulated in Mkl2(-/-) ES cells and the vasculature of Mkl2(-/-) embryos. The gene encoding TGFß2, the predominant TGFß isoform expressed in vascular smooth muscle cells and embryonic vasculature, is activated directly via binding of an MKL2/SRF protein complex to a conserved CArG box in the TGFß2 promoter. Moreover, Mkl2(-/-) ES cells exhibit derangements in cytoskeletal organization, cell adhesion and expression of ECM that are rescued by forced expression of TGFß2. Taken together, these data demonstrate that MKL2 regulates a conserved TGF-ß signaling pathway that is required for angiogenesis and ultimately embryonic survival.

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP(C)-deficiency.

The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrP(C)). Sho has been considered a candidate for the hypothetical π protein that supplies a PrP(C)-like function to maintain the viability of Prnp(0/0) mice lacking the PrP(C) protein. To understand these relationships more clearly we probed the cell biology of Sho and created knockout mice. Besides full-length and a "C1" C-terminal fragment, we describe a 6-kDa N-terminal Sho neuropeptide, "N1," which is present in membrane-enriched subcellular fractions of wild-type mice. Sprn null alleles were produced that delete all protein coding sequences yet spare the Mtg1 gene transcription unit that overlaps the Sprn 3' UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn(0/0) mice maintained in two genetic backgrounds weighed less than wild-type mice. Lack of Sho protein did not affect prion incubation time. Contrasting with lethality reported for knockdown of expression in Prnp(0/0) embryos using lentiviruses targeted against the Sprn 3' UTR, we established that double-knockout mice deficient in both Sho and PrP(C) are fertile and viable up to 690 d of age. Our data reduce the impetus for equating Sho with the notional π protein and are not readily reconciled with hypotheses wherein expression of PrP(C) and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrP(C), we infer that Sho's activity will prove germane to the maintenance of neuronal viability in postnatal life.

Akt1 and insulin-like growth factor 2 (Igf2) regulate placentation and fetal/postnatal development.

Phenotypic characterization of Akt1 and Igf2 null mice has revealed roles for each in the regulation of placentation, and fetal and postnatal growth. Insulin-like growth factor 2 (IGF2) is encoded by the Igf2 gene and influences cellular function, at least in part, through activation of an intracellular serine/threonine kinase called AKT1. Akt1 and Igf2 null mice were originally characterized on inbred and mixed genetic backgrounds, prohibiting direct comparisons of their phenotypes. The impact of loss of AKT1 or IGF2 on placental, fetal, and postnatal function were examined following transfer of Akt1 and Igf2 null mutations to an outbred CD1 genetic background. Disruption of IGF2 did not affect AKT expression or activation. Both Akt1-/- and Igf2-/- mice exhibited decreased placental weight, fetal weight and viability. Deregulation of placental growth was similar in Akt1 and Igf2 nulls; however, disruption of Igf2 had a more severe impact on prenatal survival and postnatal growth. Placental structure, including organization of junctional and labyrinth zones and development of the interstitial, invasive, trophoblast lineage, were similar in mutant and wild-type mice. Akt1 and Igf2 null mutations affected postnatal growth. The relative impact of each gene differed during pre-weaning versus post-weaning growth phases. AKT1 had a more significant role during pre-weaning growth, whereas IGF2 was a bigger contributor to post-weaning growth. Akt1 and Igf2 null mutations impact placental, fetal and postnatal growth. Placental phenotypes are similar; however, fetal and postnatal growth patterns are unique to each mutation.

A reduction of licensed origins reveals strain-specific replication dynamics in mice.

Replication origin licensing builds a fundamental basis for DNA replication in all eukaryotes. This occurs during the late M to early G1 phases in which chromatin is licensed by loading of the MCM2-7 complex, an essential component of the replicative helicase. In the following S phase, only a minor fraction of chromatin-bound MCM2-7 complexes are activated to unwind the DNA. Therefore, it is proposed that the vast majority of MCM2-7 complexes license dormant origins that can be used as backups. Consistent with this idea, it has been repeatedly demonstrated that a reduction (~60%) in chromatin-bound MCM2-7 complexes has little effect on the density of active origins. In this study, however, we describe the first exception to this observation. A reduction of licensed origins due to Mcm4 ( chaos3 ) homozygosity reduces active origin density in primary embryonic fibroblasts (MEFs) in a C57BL/6J (B6) background. We found that this is associated with an intrinsically lower level of active origins in this background compared to others. B6 Mcm4 ( chaos3/chaos3 ) cells proliferate slowly due to p53-dependent upregulation of p21. In fact, the development of B6 Mcm4 ( chaos3/chaos3 ) mice is impaired and a significant fraction of them die at birth. While inactivation of p53 restores proliferation in B6 Mcm4 ( chaos3/chaos3 ) MEFs, it paradoxically does not rescue animal lethality. These findings indicate that a reduction of licensed origins may cause a more profound effect on cell types with lower densities of active origins. Moreover, p53 is required for the development of mice that suffer from intrinsic replication stress.

Survival and size are differentially regulated by placental and fetal PKBalpha/AKT1 in mice.

The importance of placental circulation is exemplified by the correlation of placental size and blood flow with fetal weight and survival during normal and compromised human pregnancies in such conditions as preeclampsia and intrauterine growth restriction (IUGR). Using noninvasive magnetic resonance imaging, we evaluated the role of PKBalpha/AKT1, a major mediator of angiogenesis, on placental vascular function. PKBalpha/AKT1 deficiency reduced maternal blood volume fraction without affecting the integrity of the fetomaternal blood barrier. In addition to angiogenesis, PKBalpha/AKT1 regulates additional processes related to survival and growth. In accordance with reports in adult mice, we demonstrated a role for PKBalpha/AKT1 in regulating chondrocyte organization in fetal long bones. Using tetraploid complementation experiments with PKBalpha/AKT1-expressing placentas, we found that although placental PKBalpha/AKT1 restored fetal survival, fetal PKBalpha/AKT1 regulated fetal size, because tetraploid complementation did not prevent intrauterine growth retardation. Histological examination of rescued fetuses showed reduced liver blood vessel and renal glomeruli capillary density in PKBalpha/Akt1 null fetuses, both of which were restored by tetraploid complementation. However, bone development was still impaired in tetraploid-rescued PKBalpha/Akt1 null fetuses. Although PKBalpha/AKT1-expressing placentas restored chondrocyte cell number in the hypertrophic layer of humeri, fetal PKBalpha/AKT1 was found to be necessary for chondrocyte columnar organization. Remarkably, a dose-dependent phenotype was exhibited for PKBalpha/AKT1 when examining PKBalpha/Akt1 heterozygous fetuses as well as those complemented by tetraploid placentas. The differential role of PKBalpha/AKT1 on mouse fetal survival and growth may shed light on its roles in human IUGR.

Class 2 IGF-1 isoforms are dispensable for viability, growth and maintenance of IGF-1 serum levels.

Insulin-like growth factor 1 (IGF-1) is a pleiotropic factor involved in growth, cell survival and cellular differentiation. It exerts its functions through endocrine, paracrine or autocrine mechanisms. Circulating IGF-1 is essential for normal fetal and postnatal growth, although the published phenotypes of IGF-1 null animals have been only partially penetrant, presumably due to mixed genetic backgrounds. Molecular dissection of IGF-1 action is complicated by the existence of at least nine different IGF-1 isoforms, generated in both humans and rodents by usage of alternate promoters, differential splicing and different post-translational modifications. Several lines of evidence suggest that the Class 2 IGF-1 isoform is specifically destined for circulation, supporting an endocrine role of IGF-1 in normal growth processes. Using Cre/LoxP conditional gene targeting of exon 2 of the IGF-1 gene, we have generated a Class 2 IGF-1 knockout mouse line in a pure C57/Bl6 genetic background, where the specific removal of exon 2 ablated Class 2 IGF-1 isoform. Class 2 IGF-1 knockout mice exhibited normal development and postnatal growth patterns and had normal IGF-1 circulating levels, due to compensatory upregulation of Class 1 transcripts. In contrast, progeny of a total IGF-1 knockout line lacking exon 3 in the same genetic background were predictably smaller, displayed dramatically reduced IGF-1 receptor phosphorylation and all died perinatally, apparently due to respiratory failure. These results confirm that Class 2 signal peptide is not necessary for systemic circulation of IGF-1, revealing an internal compensation system for maintaining IGF-1 serum concentrations. We also uncover a vital requirement of IGF-1 for perinatal viability, previously obscured by modifiers in heterogeneous genetic backgrounds.