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• W2023370492 abstract "Tyrosine sulfation is mediated by one of two Golgi isoenzymes, called tyrosylprotein sulfotransferases (TPST-1 and TPST-2). A relatively small number of proteins are known to undergo tyrosine sulfation, including certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, extracellular matrix proteins, and hormones. As one approach to explore the role of these enzymes in vivo and how they might interact in biological systems, we have generated TPST-1-deficient mice by targeted disruption of the Tpst1 gene.Tpst1 +/− mice appear normal and, when interbred, yield litters of normal size with a Mendelian genetic distribution and an equal sex distribution.Tpst1 −/− mice appear healthy but have ≈5% lower average body weight than Tpst1 +/+controls. In addition, we show that although fertility ofTpst1 −/− males and females per seis normal, Tpst1 −/− females have significantly smaller litters because of fetal death between 8.5 and 15.5 days postcoitum. These findings suggest that there are proteins involved in regulation of body weight and reproductive physiology, which require tyrosine sulfation for optimal function that are yet to be described. Our findings also strongly support the conclusion that TPST-1 and TPST-2 have distinct biological roles that may reflect differences in their macromolecular substrate specificity. Tyrosine sulfation is mediated by one of two Golgi isoenzymes, called tyrosylprotein sulfotransferases (TPST-1 and TPST-2). A relatively small number of proteins are known to undergo tyrosine sulfation, including certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, extracellular matrix proteins, and hormones. As one approach to explore the role of these enzymes in vivo and how they might interact in biological systems, we have generated TPST-1-deficient mice by targeted disruption of the Tpst1 gene.Tpst1 +/− mice appear normal and, when interbred, yield litters of normal size with a Mendelian genetic distribution and an equal sex distribution.Tpst1 −/− mice appear healthy but have ≈5% lower average body weight than Tpst1 +/+controls. In addition, we show that although fertility ofTpst1 −/− males and females per seis normal, Tpst1 −/− females have significantly smaller litters because of fetal death between 8.5 and 15.5 days postcoitum. These findings suggest that there are proteins involved in regulation of body weight and reproductive physiology, which require tyrosine sulfation for optimal function that are yet to be described. Our findings also strongly support the conclusion that TPST-1 and TPST-2 have distinct biological roles that may reflect differences in their macromolecular substrate specificity. tyrosylprotein sulfotransferase-1 adenosine 3′-phosphate,5′-phosphosulfate days postcoitum cholecystokinin phosphate-sulfate binding phosphate binding fluorescence in situ hybridization 4′,6-diamidino-2-phenylindole 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid 4-morpholinepropanesulfonic acid Tyrosine sulfation is mediated by one of two closely related and ubiquitously expressed Golgi isoenzymes, called tyrosylprotein sulfotransferases-1 and -2 (TPST-11 and TPST-2, EC2.8.2.20). TPSTs catalyze the transfer of sulfate from PAPS to the side chain hydroxyl of tyrosine residues within acidic motifs of proteins that transit the Golgi. Mouse and human cDNAs encoding TPST-1 were first isolated by Ouyang et al. (1Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (147) Google Scholar) using the amino acid sequence obtained from a purified rat liver TPST preparation. Subsequently, a second member of the gene family, TPST-2, was identified based on its high degree of homology to TPST-1 (2Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 3Beisswanger R. Corbeil D. Vannier C. Thiele C. Dohrmann U. Kellner R. Ashman K. Niehrs C. Huttner W.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11134-11139Crossref PubMed Scopus (121) Google Scholar). There is no evidence for the existence of additional mouse or humanTPST genes in the publicly accessible genomic or expressed sequence tag databases. Human and mouse TPST-1 are ≈96% identical, and human and mouse TPST-2 have a similar degree of identity. TPST-1 is ≈65% identical to TPST-2 in both mouse and man (4Moore K.L. Wiley Encyclopedia of Molecular Medicine. John Wiley & Sons, Inc., New York2001Google Scholar). Each enzyme is ≈370 amino acids in length with type II transmembrane topology and a lumenally oriented catalytic domain. Experimental evidence shows that TPST-1 has two N-linked sugars and indicates that the lumenal catalytic domain contain one or more disulfide bonds (1Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (147) Google Scholar, 2Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Based on Northern blot analyses of a variety of human and mouse tissues, TPST-1 and TPST-2 transcripts are ubiquitously expressed, although there are some differences in the abundance of TPST transcripts in certain tissues (1Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (147) Google Scholar, 2Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Expressed sequence tags corresponding to both enzymes are represented in many cDNA libraries derived from a variety of normal tissues, including libraries derived from mouse embryos as early as 8 days postcoitum (dpc). We have detected TPST-1 and TPST-2 transcripts in several clonal cell lines including HL-60 (promyelocytic leukemia), HeLa S3 (cervical adenocarcinoma), K-562 (chronic myelogenous leukemia), MOLT-4 (T cell acute lymphoblastic leukemia), SW480 (colon adenocarcinoma), A549 (lung carcinoma), and G361 (melanoma) cells, as well as human umbilical endothelial cells. 2Y.-B. Ouyang and K. L. Moore, unpublished observations. Taken together, these data strongly suggest that both enzymes are co-expressed in many and perhaps in all cell types. An examination of the amino acid residues surrounding known tyrosine sulfation sites in proteins reveals that the major characteristic feature is the presence of several acidic amino acid residues within ±5 residues of the sulfotyrosine. However, there are no invariant residues in the window surrounding known sulfotyrosines. Based on this information coupled with in vitro studies using synthetic peptide acceptors and crude enzyme preparations, consensus features for tyrosine sulfation have been described (PROSITE accession number PS00003, www.expasy.ch/prosite/) (5Huttner W.B. Baeuerle P.A. Mod. Cell Biol. 1988; 6: 97-140Google Scholar, 6Nicholas H.B., Jr. Chan S.S. Rosenquist G.L. Endocrine. 1999; 11: 285-292Crossref PubMed Google Scholar). However, the positive predictive value of these features is unknown. To date, only a couple dozen tyrosine-sulfated proteins have been described in man, and substantially fewer have been documented in the mouse, but there are certainly many others yet to be described (4Moore K.L. Wiley Encyclopedia of Molecular Medicine. John Wiley & Sons, Inc., New York2001Google Scholar, 5Huttner W.B. Baeuerle P.A. Mod. Cell Biol. 1988; 6: 97-140Google Scholar, 7Kehoe J.W. Bertozzi C.R. Chem. Biol. 2000; 7: R57-R61Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Among the known tyrosine-sulfated proteins are many that play important roles in inflammation, hemostasis, immunity, and other processes. These include certain adhesion molecules (P-selectin glycoprotein ligand-1, platelet glycoprotein Ibα), G-protein-coupled receptors (CCR5, CCR2B, CXCR4, and C5a receptor), coagulation factors (factors V, VIII, and IX), serpins (α2-antiplasmin and heparin cofactor II), extracellular matrix proteins (vitronectin, fibronectin, laminin, type III collagen, and microfibril-associated glycoprotein-1), hormones (gastrin and cholecystokinin), and others. Tyrosine sulfation has been shown to be required for the optimal function of some proteins. For example, tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for binding to P-selectin (8Wilkins P.P. Moore K.L. McEver R.P. Cummings R.D. J. Biol. Chem. 1995; 270: 22677-22680Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), tyrosine sulfation of coagulation factors V and VIII is required for optimal thrombin-mediated activation (9Pittman D.D. Tomkinson K.N. Michnick D. Selighsohn U. Kaufman R.J. Biochemistry. 1994; 33: 6952-6959Crossref PubMed Scopus (68) Google Scholar, 10Pittman D.D. Wang J.H. Kaufman R.J. Biochemistry. 1992; 31: 3315-3325Crossref PubMed Scopus (88) Google Scholar), and sulfation of cholecystokinin (CCK) is required for CCK-A receptor activation (11Steigerwalt R.W. Williams J.A. Endocrinology. 1981; 109: 1746-1753Crossref PubMed Scopus (74) Google Scholar). However, in many of the known tyrosine-sulfated proteins, a functional role for tyrosine sulfation has not been established. Little is known about the macromolecular substrate specificity of these two enzymes and whether their substrate repertoires are distinct or overlapping. However, the very limited data that have been published on this question suggest that the specificity of the two TPSTs toward small peptide acceptors differs (2Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This information, or lack thereof, makes it impossible to make cogent predictions regarding the phenotype(s) of TPST-deficient mice, although it would seem reasonable to expect that the phenotypic expression of TPST-1 or TPST-2 deficiency may be pleiotropic given the functional importance of tyrosine sulfation in the optimal function of several known and perhaps many unknown tyrosine-sulfated proteins. To gain a better understanding of TPST function in vivo we have developed TPST-1-deficient mice by targeted disruption of theTpst1 gene. Herein we describe the details of our work on the production and initial characterization of these mice. Mouse genomic clones were identified and plaque purified from a 129S6/SvEv spleen Lambda FIX IITM genomic library (Stratagene, La Jolla, CA) by high stringency hybridization with a full-length mouse TPST-1 cDNA probe. The genomic organization of the Tpst1 gene was partially characterized by a combination of Southern blotting, restriction mapping, and sequence analysis. Comparison of genomic and cDNA sequences showed that most of the TPST-1 open reading frame including the N-terminal 281 (out of 370) amino acids of the protein was encoded by a single 946-bp exon, which includes 101 bp of 5′-untranslated region and 845 bp of coding sequence. This was designated exon 3 by analogy to the genomic structure of the humanTPST2 gene (2Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and includes the initiating methionine, all six lumenal cysteine residues, and both N-linked glycosylation sites (Fig. 1 A). Most importantly this region contains both the 5′-phosphate-sulfate binding (PSB) motif and the 3′-phosphate binding (PB) motif described previously as being involved in binding the 5′- and 3′-sulfate groups of the substrate analog PAP in the four known sulfotransferase crystal structures (12Yoshinari K. Petrotchenko E.V. Pedersen L.C. Negishi M. J. Biochem. Mol. Toxicol. 2001; 15: 67-75Crossref PubMed Scopus (50) Google Scholar). A construct was engineered for targeted disruption of theTpst1 gene in which a 1.3-kb fragment spanning exon 3 was replaced by a 1.7-kb PGKneo cassette (Fig. 1 B). A 4.5-kbSspI-XbaI genomic fragment was used as the left arm for homologous recombination and was cloned into the pPGKneo vector upstream of the PGKneo cassette. The 1.1-kb right arm 3′ to exon 3 was generated by PCR and cloned downstream of the PGKneo cassette. Sequencing of both strands confirmed that the nucleotide sequence was identical to the genomic template used to generate it. The construct was linearized with NotI and electroporated into AB2.2 embryonic stem cells, an ES cell line derived from male 129S6/SvEv mice (Lexicon Genetics Inc., Houston, TX). After G418 selection, five positive clones were identified from 900 G418-resistant colonies by PCR screening. Surviving clones were further analyzed by Southern blotting to confirm the correct homologous recombination event, and random integration of extra copies of the targeting construct was excluded by hybridization with a neomycin probe (not shown). ES clones were injected into blastocysts, and the blastocysts were implanted into pseudopregnant mice to generate chimeras. Chimeric males were bred with wild-type 129S6/SvEv females (Taconic Farms, Germantown, NJ) to generate Tpst1 +/− mice on a 129S6/SvEv inbred genetic background, and Tpst1 +/− mice were interbred to generate mice with three Tpst1 genotypes (Tpst1 +/+, Tpst1 +/−,Tpst1 −/−). Genotyping was performed by PCR using the following primers: 5′-GGACTGTCTGACCAAGTGGAACCGGGC-3′ and 5′-GTTTGCTCCTGCCTCCCAAGTGCTGGC-3′ for the 348-bp fragment from the wild-type allele, and 5′-CCTCGTGCTTTACGGTATCGCCGC-3′ and 5′-GTTTGCTCCTGCCTCCCAAGTGCTGGC-3′ for the 600-bp fragment from the mutant allele. All experiments reported here were performed on mice in the 129S6/SvEv background. Mice were housed in ventilated racks under 12-h light/12-h dark cycles in a controlled environment with 50% relative humidity at 21 °C. All animals were maintained on sterilized pelleted feed (Harlan Teklad number 7913) containing 6% fat and 18% protein. Breeding cages were inspected daily to determine date of birth. In all breeding experiments mice were pairwise-mated, and pups were weaned at 21 days of age. All procedures were approved by the Animal Care and Use Committee of the Oklahoma Medical Research Foundation. Total RNA was isolated from tissues using Trizol Reagent (Invitrogen), separated on 1% denatured gels, and transferred to nitrocellulose membrane. For Northern blotting a 1205-bp fragment corresponding to nucleotides 212–1416 of the full-length cDNA was used as a probe. For Southern blotting, tail DNA was isolated (Qiagen genomic DNA kit), exhaustively digested with XbaI, and then separated on 1% agarose gels and transferred to nitrocellulose membranes. A 793-bp TPST-1 DNA fragment downstream of the right arm (Probe B) was used as a probe. Filters were prehybridized with Hybrisol solution (Oncor) for 60 min at 42 °C and hybridized with 32P-labeled probes overnight at 42 °C. The blots were washed twice with 2 × SSC, 0.1% SDS for 20 min at 22 °C and twice with 0.1 × SSC, 0.1% SDS for 20 min at 65 °C. The membrane was exposed to a phosphorimaging screen at room temperature. Mice were euthanized, and the livers were rapidly excised and immersed in cold homogenization buffer (10 mm Tris-HCl (pH 7.5), 1.5 mm MgCl2, 250 mmsucrose, 0.5 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride). Homogenates were centrifuged (10 min, 800 × g), and the postnuclear supernatants were centrifuged (30 min, 16,000 × g). The microsomal pellets were suspended in 1.5 ml/g of liver in 2% Triton X-100, 20 mm TAPS (pH 9.0), 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin/antipain and stirred for 1 h. Microsomal extracts were then clarified by centrifugation (30 min, 16,000 × g), and to the supernatants glycerol was added to 10% (w/v), MOPS (pH 7.5) to 50 mm, and phenylmethylsulfonyl fluoride to 0.5 mm. The samples were snap-frozen in liquid N2 and stored at −80 °C. TPST activity was determined by measuring the transfer of [35S]sulfate from [35S]PAPS to an immobilized peptide substrate (QATEYEYLDYDFLPE) modeled on the N-terminal 15 residues of the mature P-selectin glycoprotein ligand-1 polypeptide as previously described (1Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (147) Google Scholar). Chromosomal mapping was performed using fluorescence in situ hybridization (FISH) signal mapping and 4′,6-diamidino-2-phenylindole (DAPI) stain to assign chromosome number by SeeDNA Biotech Inc. (Windsor, Canada). Lymphocytes were isolated from mouse spleen and cultured at 37 °C in RPMI 1640 supplemented with 15% fetal calf serum and 3 μg/ml concanavalin A, 10 μg/ml lipopolysaccharide, and 50 μm2-mercaptoethanol. After 44 h the cells were treated with 0.18 mg/ml bromodeoxyuridine for an additional 14 h. Synchronized cells were washed and recultured at 37 °C for 4 h in α-minimal essential medium with 2.5 μg/ml thymidine, and chromosome slides were prepared. A 5-kb mouse genomic DNA fragment spanning exon 3 was used for FISH mapping. The probe was biotinylated with dATP using the Invitrogen BioNick labeling kit (15 °C for 1 h). The procedure for FISH detection was performed according to Heng et al. (13Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar) and Heng and Tsui (14Heng H.H.Q. Tsui L.-C. Chromosoma (Berl.). 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). Briefly, slides were baked at 55 °C for 1 h, RNase-treated, denatured (70% formamide in 2 × SSC, 2 min, 70 °C), then dehydrated with ethanol. Probes were denatured (75 °C, 5 min) in 50% formamide, 10% dextran sulfate, prehybridized for 15 min at 37 °C, and then hybridized overnight. FISH signals and DAPI banding patterns were captured using a CCD camera, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes. To disrupt the Tpst1 gene we constructed a targeting vector in which exon 3 of the Tpst1 gene was replaced with a PGKneo cassette (Fig. 1 B). Linearized targeting vector was used to electroporate ES cells, which were then subjected to positive selection. Homologous recombinants were detected using PCR and confirmed by Southern analysis ofEcoNI-digested genomic DNA using probe A andXbaI-digested DNA using probe B (Fig. 1 B). ES clones were injected into blastocysts, and the blastocysts were implanted into pseudopregnant mice using standard techniques. Male chimeras were mated with wild-type 129S6/SvEv females, and heterozygotes were interbred to generateTpst1 −/− mice. Transmission of the targeted mutation was confirmed by PCR and genomic Southern blot analysis of tail DNA isolated from gene-targeted animals as described under “Experimental Procedures.” Homologous recombination at theTpst1 locus resulted in the introduction of aXbaI site that results in a shorter XbaI genomic fragment (3.8 kb) than that from the wild-type allele (5 kb) that is detected by hybridization with Probe B (Fig. 1 C). Functional inactivation of the Tpst1 gene was confirmed by Northern blot analysis of total RNA isolated fromTpst1 +/+, Tpst1 +/−, andTpst1 −/− mouse organs (Fig. 1 D). This analysis showed an ≈50% reduction of full-lengthTpst1 mRNA (≈1.8 kb) in both spleen and testes ofTpst1 +/− mice compared with wild-type and failed to detect full-length transcripts inTpst1 −/− even in testes, which expresses abundant levels of Tpst1 mRNA (1Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (147) Google Scholar). A parallel analysis using a Tpst2 cDNA probe demonstrated that disruption of the Tpst1 gene had no detectable effect on expression of theTpst2 gene in these tissues (Fig.1 D). In Tpst1 +/− andTpst1 −/− mice, but not wild-type mice, a smaller mRNA species was detected in testes and was also apparent in the spleen when the blots were overexposed. To assess the structure of this transcript, Northern blots were reprobed with an exon 3-specific probe corresponding to nucleotides 212–740 of the full-length cDNA. As expected, this analysis detected the full-length message in Tpst1 +/+ andTpst1 +/− but not the smaller transcript inTpst1 +/− and Tpst1 −/−samples, thus confirming that this smaller transcript lacked exon 3 (not shown). Given that exon 3 encodes the first 281 of 370 amino acids of the mature protein, including the 5′-PSB motif and the 3′-PB motif that are critical for catalysis, it is certain that this translation of this transcript could not result in a functional protein. To determine the impact of TPST-1 deficiency on the total enzyme activity, assays of liver microsomal extracts were performed as described under “Experimental Procedures” using a P-selectin glycoprotein ligand-1 acceptor peptide that is an efficient acceptor for both TPST-1 and TPST-2 (2Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). We found that TPST activity in liver extracts from Tpst1 −/− mice was reduced to ≈12% of that observed in wild-type mice (Fig. 1 E). This suggests that TPST-1 is the predominant TPST in mouse liver. However, a firm conclusion cannot be made based on these data because we lack definitive knowledge regarding whether these enzymes differ with respect to their K m for PAPS and acceptor, and it is well established that PAPS is unstable in crude tissue extracts (15Rens-Domiano S. Roth J.A. J. Biol. Chem. 1989; 264: 899-905Abstract Full Text PDF PubMed Google Scholar). In the 129S6/SvEv background, Tpst1 +/− mice appeared normal. Male and female heterozygote mating pairs yielded litters of normal size (5.8 ± 2.1 (mean ± S.D., n = 57)) when compared with that reported for this strain by the supplier (Taconic Farms, Inc.). Among all offspring fromTpst1 +/− × Tpst1 +/−crosses, the ratio of the three Tpst1 genotypes was consistent with Mendelian inheritance, indicating thatTpst1 −/− mice develop normally in utero and that the sex ratio in each Tpst1 genotype was ≈50:50 (Table I).Table IGenotype and sex distribution among offspring from Tpst1 +/− × Tpst1 +/− crosses Tpst1 genotypeTotalRatio+/++/−−/−Male3477281390.52Female3356381270.48Total6713366266Ratio0.250.500.25Animals were mated, genotyped, and weaned as described under “Experimental Procedures.” Open table in a new tab Animals were mated, genotyped, and weaned as described under “Experimental Procedures.” To assess the growth of Tpst1 −/− mice, a large cohort of mice were weighed weekly from age 2 to 10 weeks (Fig.2). At 2 and 3 weeks of age, the mean weights of male and female Tpst1 −/− mice were not statistically different from wild-type animals. However, after weaning, the mean body weights of male and femaleTpst1 −/− mice lagged behind that of wild-type animals. Beginning at 5–6 weeks of age, male and femaleTpst1 −/− mice weighed 0.8–1.3 and 0.6–1.0 g less than the wild-type cohort, respectively. Although modest, these differences in weight constitute ≈5% of the body weight and were statistically significant beginning at 4–5 weeks (Fig. 2). The biological significance of this observation is substantially strengthened by the fact that these mice are on an inbred genetic background. The mean body weight of Tpst1 +/−mice was not statistically different from wild-type animals (not shown). Otherwise, Tpst1 −/− mice appeared healthy out to 12 months of age. Hematoxylin/eosin staining of tissues from 15-week-old mice showed no evidence of histological abnormalities in heart, liver, brain, spleen, kidney, lung, stomach, small or large bowel, testes, or ovary (not shown). Early in this study we noticed that litter sizes fromTpst1 −/− females appeared somewhat low. Moreover, in a 1-year period, we observed that the mean size of litters at birth from Tpst1 +/+ ×Tpst1 +/+, Tpst1 +/− ×Tpst1 +/−, and Tpst1 −/−× Tpst1 −/− crosses were 6.2 ± 2.0 (n = 55), 5.8 ± 2.1 (n = 57), and 3.8 ± 1.8 (n = 57), respectively. The lower mean litter size of Tpst1 −/− ×Tpst1 −/− crosses compared with the other groups was highly statistically significant (p < 10−7). To examine the reproductive performance ofTpst1−/− mice in more detail, virgin femaleTpst1 +/+ and Tpst1 −/−mice from 8 to 20 weeks of age were pair-mated with eitherTpst1 +/+ or Tpst1 −/−males. The results of these matings are shown in TableII. To determine whether and when copulation occurred, females were examined for vaginal plugs each morning. Vaginal plugs were detected in all 20Tpst1 +/+ females mated withTpst1 +/+ or Tpst1 −/−males and in 17 of the 20 Tpst1 −/− females mated with Tpst1 +/+ orTpst1 −/− males. The time between the set up of these matings and the detection of vaginal plugs was not different inTpst1 +/+ (2.6 ± 1.1 days,n = 20) and Tpst1 −/− females (2.6 ± 0.9 days, n = 17). These data indicate that the copulatory behavior of both Tpst1 −/−males and females is normal and that the estrous cycle length inTpst1 −/− females is normal.Table IIReproductive performance of Tpst1 −/− miceGroupMaleFemale nCopulationsLittersLitter size2-aValues are expressed as the mean ± S.D. of the indicated number of matings.Live birthsPerinatal deathsSurviving pups %1+ /++ /+101095.9 ± 1.8530532− /−+ /+101096.3 ± 1.7570573+ /+− /−10994.1 ± 1.5379 (24)284− /−− /−10863.2 ± 1.9196 (32)13Animals were mated as described under “Experimental Procedures.” One-way analysis of variance was used to determine the overall statistical differences in the litter sizes of each experimental group (p = 0.0032). Post-hoc significance of pairwise comparisons between groups were made using the least squares means test in the GLM procedure using Statistical Analysis Software (SAS version 10, SAS Institute, Inc.). Group 1 versus 2,p = 0.59. Group 1 versus 3,p = 0.035. Group 1 versus 4,p = 0.0052. Group 2 versus 3,p = 0.0099. Group 2 versus 4,p = 0.0015. Group 3 versus 4,p = 0.30.2-a Values are expressed as the mean ± S.D. of the indicated number of matings. Open table in a new tab Animals were mated as described under “Experimental Procedures.” One-way analysis of variance was used to determine the overall statistical differences in the litter sizes of each experimental group (p = 0.0032). Post-hoc significance of pairwise comparisons between groups were made using the least squares means test in the GLM procedure using Statistical Analysis Software (SAS version 10, SAS Institute, Inc.). Group 1 versus 2,p = 0.59. Group 1 versus 3,p = 0.035. Group 1 versus 4,p = 0.0052. Group 2 versus 3,p = 0.0099. Group 2 versus 4,p = 0.0015. Group 3 versus 4,p = 0.30. The size of the litters from Tpst1 +/+ ×Tpst1 +/+ crosses was indistinguishable from matings of Tpst1 −/− males andTpst1 +/+ females (group 1 versus 2,p = 0.59). This demonstrates that the fertility ofTpst1 −/− males is normal. However, when wild-type males were mated with Tpst1 −/−females, litter sizes were significantly reduced compared with the groups in which the females were wild-type irrespective of the genotype of the male (group 1 versus 3, p = 0.035; group 2 versus 3, p = 0.0099). WhenTpst1 −/− males were mated withTpst1 −/− females, litter sizes were similarly reduced (group 1 versus 4, p = 0.0052; group 2 versus 4, p = 0.0015) but were not significantly different from crosses of wild-type males andTpst1 −/− females (group 3 versus 4,p = 0.30). These results show that the reduced litter size observed in Tpst1 −/− females results exclusively from TPST-1 deficiency in the female and is independent of the genotype of the sire or the fetus. In addition, we observed that homozygosity in the female was associated with a striking incidence of perinatal mortality. In these experiments, 15 of 56 (28%) live births to Tpst1 −/− females were found dead within 48 h of birth, whereas no death occurred in litters from wild-type females (Table II). Furthermore, over a 1-year period of observation, we observed a perinatal death rate of 3.9% inTpst1 +/+ × Tpst1 +/+crosses (12 of 355 live births, 57 litters), 3.7% inTpst1 +/− × Tpst1 +/−crosses (12 of 327 live births, 57 litters), and 18.6% inTpst1 −/− × Tpst1 −/−crosses (40 of 218 live births, 57 litters). In all cases neonates found dead were morphologically normal. The synchrony of reproductive performance over time also appears to be disturbed in Tpst1 −/− females.Tpst1 +/+ × Tpst1 +/+(Group 1), Tpst1 −/− ×Tpst1 +/+ (Group 2), andTpst1 +/+ × Tpst1 −/−(Group 3) crosses were set up using 8–10-week-old virgin females, and the number and timing of litters and litter size were followed for 18 weeks (Fig. 3). Each mating pair was continuously housed together, and litters were weaned promptly at 21 days of age. Over 18 weeks the number of litters produced in each group was not statistically different. However, the number of pups per litter from Tpst1 −/− females (Group 3) was consistently lower over time compared with litters fromTpst1 +/+ females irrespective of the genotype of the male (groups 1 and 2). It was also noted that the timing of litters from Tpst1 +/+ females (groups 1 and 2) was quite synchronous over the 18-week observation period. In contrast, this was not the case with Tpst1 −/− females (group 3). To evaluate the cause of the low litter sizes from Tpst1 −/− females, timed matings between wild-type males and age-matched female wild-type orTpst1 −/− mice were set up. Females were examined for vaginal plugs each morning, plugged females were sacrificed at 15.5 dpc, and the uterine contents were examined. An embryo was considered viable if the size and morphology were consistent with 15.5 dpc. An embryo was considered to be resorbed if a placental remnant could be identified. The number of implantations was defined as the number of viable embryos plus the number of resorbed embryos. We observed that the frequency of fetal loss was three times" @default.
• W2023370492 created "2016-06-24" @default.
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• W2023370492 creator A5062873972 @default.
• W2023370492 creator A5073756699 @default.
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• W2023370492 date "2002-06-01" @default.
• W2023370492 modified "2023-10-16" @default.
• W2023370492 title "Reduced Body Weight and Increased Postimplantation Fetal Death in Tyrosylprotein Sulfotransferase-1-deficient Mice" @default.
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