Marta Majewska

Pregnancy-Associated Glycoprotein (PAG) family: transcripts and gene amplicons in camelids

In this study, the placental localization of PAG-like transcripts and genomic existence of PAG-like amplicons in new-world (Lp, Lama pacos, alpaca) and old-world camelids (Cb, Camelus bactrianus, bactrian; Cd, Camelus dromedarius; dromedary) are reported for the first time. Sections of Lp (150-347 days post coitum), Cd (43-90 cm crown-rump length) and Cb (term) placentas were used for heterologous (ht; cross-species) autoradiographic in situ hybridization (aISH) with single-stranded diagnostic (antisense) or control (sense) [alpha-(35)S]dATP-labeled 323 nt porcine PAG8 (pPAG8) cDNA probes produced by asymmetric PCRs. The aISH with antisense (35)S-pPAG8 probe identified camelid PAG-like (LpPAG, CbPAG and CdPAG) mRNA expression restricted to chorionic epithelium cells within placentas of camelids. In addition, genomic DNA (gDNA), isolated from placental sections were used as templates for camelid PAG-like gene amplicon production by PCR. Specificity of the obtained multiple camelid gDNA PAG-like amplicons was confirmed by double ht-Southern hybridizations with [alpha-(32)P]dATP-labeled 611 bp pPAG5 and pPAG10 double-stranded cDNA probes. The double ht-Southern hybridizations of camelid gDNA amplicons (with pPAG5 and -10 probes) allowed the identification of length-polymorphism of LpPAG, CbPAG and CdPAG genes, coding catalytically active and potentially inactive forms. Such an application of porcine PAG probes may be advantageous for future identification of still undiscovered PAG-like families in other eutherian species.

Chromosomal assignment of porcine pregnancy-associated glycoprotein gene family

This study presents the chromosomal assignment of a multiple pregnancy-associated glycoprotein (PAG) gene family in the domestic pig (pPAG). The pPAG locus was identified by physical mapping (fluorescent in situ hybridisation-FISH; with various probes), and additionally confirmed by Southern hybridisation of pPAG amplicons using laser microdissected Sus scrofa chromosome 1 (SSC1), as genomic templates. Various pPAG probes were produced with the use of diverse identified templates: pPAG1-6, -8, -10 cDNAs (GenBank: L34360-1, AF315377, AF272734, AY188554, AF272735, AY373029 and AY775784, respectively), or genomic DNA (gDNA) probes of pPAG2 gene and its promoter (GenBank: U39198-9, U39762-3, U41421-4). All probes, including long gDNA probes (approximately 9.2kbp GpPAG2 gene; approximately 2.8kbp GpPAG2 promoter), a shorter cDNA probe (PlpPAG4, 1385bp) and amplified pPAG2-like probes (ApPAG2L) specific for cDNA inserts of pPAG2-like gene subfamily (pPAG2, -4, -6, -8 and -10; 1283-1385bp) were produced by random priming using biotin-labelled deoxynucleotides (16-dUTP). Numerous FISH mappings with various pPAG probes revealed the chromosomal assignment of the pPAG gene family to the long arm of porcine chromosome 1 (SSC1q16-q24 region). This cytogenetic assignment was confirmed by Southern hybridisation (with (32)P-labelled pPAG10 probe) of multiple distinct pPAG amplicons (603-3943bp) produced with the use of 25 laser microdissected SSC1, as gDNA templates. This is the first study identifying the chromosomal locus of the pPAG gene family in the pig.

Expression of pregnancy-associated glycoprotein family in the epitheliochorial placenta of two Camelidae species (C. dromedarius and C. bactrianus).

This study describes placental morphology and immunolocalization of the placental pregnancy associated glycoprotein-like family (PAGs) identified in two selected taxa of Old-World camels of the Camelidae family: Camelus dromedarius (Cd) and Camelus bactrianus (Cb). Placental tissues of Cd from days 140-293 post-coitum (dpc), term (404 dpc); and of Cb from term (440 dpc) were examined. Histological staining (hematoxylin/eosin and propidium iodine) revealed the development of the placental structure, while chorionic folding increased the feto-placental surface during the progress of pregnancy. The camelid placenta during early pregnancy is similar to the diffuse epitheliochorial type, and during later stages of pregnancy resembles the synepitheliochorial (cotyledonary) type. Placental expression of the PAGs was detected (Alexa 488 - green) within camelid trophectoderm cells (TRD - chorionic epithelium as outer layer of embryonic cells) among all placental cells with nuclei stained by propidium iodide (red). The PAGs, identified in both Camelidae taxa, were named CbPAGs and CdPAGs. Placental CbPAG and CdPAG expression is restricted to the TRD cells, which are differentially developed throughout gestation. Cross-reactivity of polyvalent anti-pPAG polyclonals with the CbPAGs and CdPAGs revealed high structural similarities of the PAG-like epitopes in pigs and camels. This is the first study identifying PAG expression in chorionic cells of the camel placenta.

Identification of Novel Placentally Expressed Aspartic Proteinase in Humans

This study presents pioneering data concerning the human pregnancy-associated glycoprotein-Like family, identified in the genome, of the term placental transcriptome and proteome. RNA-seq allowed the identification of 1364 bp hPAG-L/pep cDNA with at least 56.5% homology with other aspartic proteinases (APs). In silico analyses revealed 388 amino acids (aa) of full-length hPAG-L polypeptide precursor, with 15 aa-signal peptide, 47 aa-blocking peptide and 326 aa-mature protein, and two Asp residues (D), specific for a catalytic cleft of the APs (VVFDTGSSNLWV91-102 and AIVDTGTSLLTG274-285). Capillary sequencing identified 9330 bp of the hPAG-L gene (Gen Bank Acc. No. KX533473), composed of nine exons and eight introns. Heterologous Western blotting revealed the presence of one dominant 60 kDa isoform of the hPAG-L amongst cellular placental proteins. Detection with anti-pPAG-P and anti-Rec pPAG2 polyclonals allowed identification of the hPAG-L proteins located within regions of chorionic villi, especially within the syncytiotrophoblast of term singleton placentas. Our novel data extend the present knowledge about the human genome, as well as placental transcriptome and proteome during term pregnancy. Presumably, this may contribute to establishing a new diagnostic tool for examination of some disturbances during human pregnancy, as well as growing interest from both scientific and clinical perspectives.

The Influence of Resiniferatoxin (RTX) and Tetrodotoxin (TTX) on the Distribution, Relative Frequency, and Chemical Coding of Noradrenergic and Cholinergic Nerve Fibers Supplying the Porcine Urinary Bladder Wall

The present study investigated the influence of intravesically instilled resiniferatoxin (RTX) or tetrodotoxin (TTX) on the distribution, number, and chemical coding of noradrenergic and cholinergic nerve fibers (NF) supplying the urinary bladder in female pigs. Samples from the bladder wall were processed for double-labelling immunofluorescence with antibodies against cholinergic and noradrenergic markers and some other neurotransmitter substances. Both RTX and TTX caused a significant decrease in the number of cholinergic NF in the urinary bladder wall (in the muscle coat, submucosa, and beneath the urothelium). RTX instillation resulted in a decrease in the number of noradrenergic NF in the submucosa and urothelium, while TTX treatment caused a significant increase in the number of these axons in all the layers. The most remarkable changes in the chemical coding of the NF comprised a distinct decrease in the number of the cholinergic NF immunoreactive to CGRP (calcitonin gene-related peptide), nNOS (neuronal nitric oxide synthase), SOM (somatostatin) or VIP (vasoactive intestinal polypeptide), and an increase in the number of noradrenergic NF immunopositive to GAL (galanin) or nNOS, both after RTX or TTX instillation. The present study is the first to suggest that both RTX and TTX can modify the number of noradrenergic and cholinergic NF supplying the porcine urinary bladder.

Novel effects of identified SNPs within the porcine Pregnancy-Associated Glycoprotein gene family (pPAGs) on the major reproductive traits in Hirschmann hybrid-line sows

This is the first study describing identification of SNPs within the multiple and polymorphic Pregnancy-Associated Glycoprotein gene family (PAGs) in the genome of the domestic pig (pPAGs). We identified pPAG-like (pPAG-L) genotypes in primiparous and multiparous farmed hybrid-line JSR Hirschmann (Hrn) sows (N=159), in which various novel associations with their phenotypes for the major reproductive traits have been discovered. Genomic DNA templates were isolated from the blood and different pPAG-L primers were used to amplify various regions by PCR. Electrophoretically-separated amplicons were selected, purified and sequenced. All identified SNPs were verified for possible pPAG2-L genotype associations with the major reproductive traits. In total, 196 SNPs were identified within the entire structure of the pPAG2-Ls, encompassing 9 exons and 8 (A-H) introns, resembling all aspartic proteinases. It was discovered that among all SNPs, one diplotype localized in exon 6 (657C>T/749G>C; pPAG2 ORF cDNA numbering; L34361) caused amino acid substitutions (Asp220→Asn and Ser250→Thr) in the polypeptide precursors and was associated with an increase in the number of live-born piglets (P≤0.05) in Hrn sows. In turn, co-localized SNP (504g>a; KF537535 numbering) in the intron F of the pPAG2-Ls, but only in the homozygotic genotype (gg), was associated with an increased number of live-born (P≤0.01) and weaned (P≤0.05) piglets in the Hrn sows. These results qualify the pPAG2-Ls as candidate genes of the main QTLs. The novel pPAG SNP profiles provide the basis for a diagnostic genotyping test required for early pre-selection of female/male piglets, presumably mainly useful in various breeding herds.

Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.)

The Eurasian beaver is one of the largest rodents that, despite its high impact on the environment, is a non-model species that lacks a reference genome. Characterising genes critical for pregnancy outcome can serve as a basis for identifying mechanisms underlying effective reproduction, which is required for the success of endangered species conservation programs. In the present study, high-throughput RNA sequencing (RNA-seq) was used to analyse global changes in the Castor fiber subplacenta transcriptome during multiple pregnancy. De novo reconstruction of the C. fiber subplacenta transcriptome was used to identify genes that were differentially expressed in placentas (n=5) from two females (in advanced twin and triple pregnancy). Analyses of the expression values revealed 124 contigs with significantly different expression; of these, 55 genes were identified using MegaBLAST. Within this group of differentially expressed genes (DEGs), 18 were upregulated and 37 were downregulated in twins. Most DEGs were associated with the following gene ontology terms: cellular process, single organism process, response to stimulus, metabolic process and biological regulation. Some genes were also assigned to the developmental process, the reproductive process or reproduction. Among this group, four genes (namely keratin 19 (Krt19) and wingless-type MMTV integration site family - member 2 (Wnt2), which were downregulated in twins, and Nik-related kinase (Nrk) and gap junction protein β2 (Gjb2), which were upregulated in twins) were assigned to placental development and nine (Krt19, Wnt2 and integrin α7 (Itga7), downregulated in twins, and Nrk, gap junction protein β6 (Gjb6), GATA binding protein 6 (Gata6), apolipoprotein A-I (ApoA1), apolipoprotein B (ApoB) and haemoglobin subunit α1 (HbA1), upregulated in twins) were assigned to embryo development. The results of the present study indicate that the number of fetuses affects the expression profile in the C. fiber subplacental transcriptome. Enhancement of transcriptomic resources for C. fiber will improve understanding of the pathways relevant to proper placental development and successful reproduction.

The Influence of Tetrodotoxin (TTX) on the Distribution and Chemical Coding of Caudal Mesenteric Ganglion (CaMG) Neurons Supplying the Porcine Urinary Bladder

The treatment of micturition disorders creates a serious problem for urologists. Recently, new therapeutic agents, such as neurotoxins, are being considered for the therapy of urological patients. The present study investigated the chemical coding of caudal mesenteric ganglion (CaMG) neurons supplying the porcine urinary bladder after intravesical instillation of tetrodotoxin (TTX). The CaMG neurons were visualized with retrograde tracer Fast blue (FB) and their chemical profile was disclosed with double-labeling immunohistochemistry using antibodies against tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), calbindin (CB), galanin (GAL) and neuronal nitric oxide synthase (nNOS). It was found that in both the control (n = 6) and TTX-treated pigs (n = 6), the vast majority (92.6% ± 3.4% and 88.8% ± 2%, respectively) of FB-positive (FB+) nerve cells were TH+. TTX instillation caused a decrease in the number of FB+/TH+ neurons immunopositive to NPY (88.9% ± 5.3% in the control animals vs. 10.6% ± 5.3% in TTX-treated pigs) or VIP (1.7% ± 0.6% vs. 0%), and an increase in the number of FB+/TH+ neurons immunoreactive to SOM (8.8% ± 1.6% vs. 39% ± 12.8%), CB (1.8% ± 0.7% vs. 12.6% ± 2.7%), GAL (1.7% ± 0.8% vs. 10.9% ± 2.6%) or nNOS (0% vs. 1.1% ± 0.3%). The present study is the first to suggest that TTX modifies the chemical coding of CaMG neurons supplying the porcine urinary bladder.

Preliminary RNA-Seq Analysis of Long Non-Coding RNAs Expressed in Human Term Placenta

Development of particular structures and proper functioning of the placenta are under the influence of sophisticated pathways, controlled by the expression of substantial genes that are additionally regulated by long non-coding RNAs (lncRNAs). To date, the expression profile of lncRNA in human term placenta has not been fully established. This study was conducted to characterize the lncRNA expression profile in human term placenta and to verify whether there are differences in the transcriptomic profile between the sex of the fetus and pregnancy multiplicity. RNA-Seq data were used to profile, quantify, and classify lncRNAs in human term placenta. The applied methodology enabled detection of the expression of 4463 isoforms from 2899 annotated lncRNA loci, plus 990 putative lncRNA transcripts from 607 intergenic regions. Those placentally expressed lncRNAs displayed features such as shorter transcript length, longer exon length, fewer exons, and lower expression levels compared to messenger RNAs (mRNAs). Among all placental transcripts, 175,268 were classified as mRNAs and 15,819 as lncRNAs, and 56,727 variants were discovered within unannotated regions. Five differentially expressed lncRNAs (HAND2-AS1, XIST, RP1-97J1.2, AC010084.1, TTTY15) were identified by a sex-bias comparison. Splicing events were detected within 37 genes and 4 lncRNA loci. Functional analysis of cis-related potential targets for lncRNAs identified 2021 enriched genes. It is presumed that the obtained data will expand the current knowledge of lncRNAs in placenta and human non-coding catalogs, making them more contemporary and specific.

Identification of Placental Aspartic Proteinase in the Eurasian Beaver (Castor fiber L.)

Aspartic proteinases (AP) form a multigenic group widely distributed in various organisms and includes pepsins (pep), cathepsins D and E, pregnancy associated glycoproteins (PAGs) as well as plant, fungal, and retroviral proteinases. This study describes the transcript identification and expression localization of the AP within the discoid placenta of the Castor fiber. We identified 1257 bp of the AP cDNA sequence, encoding 391 amino acids (aa) of the polypeptide precursor composed of 16 aa signal peptide, 46 aa pro-piece, and 329 aa of the mature protein. Within the AP precursor, one site of potential N-glycosylation (NPS119–121) and two Asp residues (D) specific for the catalytic cleft of AP were identified (VLFDTGSSNLWV91–102 and GIVDTGTSLLTV277–288). The highest homology of the identified placental AP nucleotide and aa sequence was to mouse pepsinogen C (75.8% and 70.1%, respectively). Identified AP also shared high homology with other superfamily members: PAGs, cathepsins, and napsins. The AP identified in this study was named as pepsinogen/PAG-Like (pep/PAG-L). Diversified pep/PAG-L protein profiles with a dominant 58 kDa isoform were identified. Immune reactive signals of the pep/PAG-L were localized within the trophectodermal cells of the beaver placenta. This is the first report describing the placental AP (pep/PAG-L) in the C. fiber.