Bozena Szafranska

The placental expression of the porcine pregnancy-associated glycoprotein (pPAG) gene family examined in situ and in vitro

The objective of the study was to define the expression of the porcine pregnancy-associated glycoprotein (pPAG) gene family, including pPAG1, pPAG2, pPAG3, pPAG4 and pPAG6 that belong to the aspartic proteinase family. Porcine pPAG2, PAG4 and pPAG6 are members of a subfamily (pPAG2-like), which all have highly conserved sequences to pepsins, within two catalytic domains, suggesting enzymatic activity of these molecules. In contrast, pPAG1 and pPAG3 have catalytic sites with critical amino acid substitutions that likely render these molecules enzymatically inactive. The expression of pPAG mRNA was examined by using in situ hybridisation (ISH) in placental tissues or cultured cells and by ribonuclease protection assay (RPA). The pPAG protein family, secreted in vitro during long-term cultures, was examined using Western blotting. The trophoblastic pPAG mRNA expression starts around implantation and is continued in chorionic epithelium (trophectoderm) throughout pregnancy. ISH performed on porcine placental sections with pPAG antisense cDNA probes revealed an expression of pPAG transcripts, locally restricted only to trophectoderm. The pPAG2-like mRNA expression occurred in different trophectoderm cells. Some trophoblast cells were bigger than others and were involved in local rearrangements of maternal epithelium layer, especially in developing placental folds. A high similarity of dominating pPAG2-like transcript expression was confirmed by RPA analysis. Cultures of trophoblast cells revealed their differentiation to multinucleated forms that were not observed in situ. This confirms a strong inhibitory effect of the maternal microenvironment of uterine lumen on mononuclear trophoblast within porcine placental units that was not present during the development of multinucleated trophoblast cells in vitro. Long-term cultures of chorionic explants revealed a very efficient system of pPAG protein production in vitro. Western blotting of secretory pPAG proteins indicated similar immunologic epitope(s) of these molecules and pregnancy-stage dependent profile of chorionic secretion. Thus, some of the subpopulation(s) of porcine trophoblast cells expressing pPAG2-like transcripts and their secretory products can play an important role(s) in the mechanism(s) of the confrontation between trophoblast/trophectoderm cells and maternal endometrial epithelium during implantation, placenta formation and successful pregnancy maintenance in the pig.

IMMUNOLOCALISATION OF OESTROGEN RECEPTORS ALPHA (ERα) AND BETA (ERβ) IN PORCINE EMBRYOS AND FETUSES AT DIFFERENT STAGES OF GESTATION

The sites of oestrogen action can be shown by the localisation of their receptors in the target tissues. The aim of the present study was to show the localisation of oestrogen receptors in porcine embryos and fetuses obtained on days 18, 22, 32, 40, 50, 60, 71 and 90 post coitum (p.c.). The visualisation of proteins was conducted in embryos and various fetal organs such as gonads, uterus, lung, kidney, intestine and adrenal gland. Both ERs were observed in the blastocysts on day 18 p.c. In the male, ERbeta was detected in the testis and epididymis, whereas ERalpha was present in the efferent ductules. In the female, ERbeta was detected in the ovarian stromal cells investing the oocyte nests, while ERalpha protein was detected in the surface epithelium. In the uterus, ERs were present in the stromal cells, while ERbeta was present in the luminal epithelium. In the non-reproductive fetal porcine tissues ERbeta was localised in the lungs, kidneys, adrenal glands and in the umbilical cords. Both ERs were observed in the intestine. It is possible that ERbeta may play important roles in the development of the adrenal gland, testis, kidney and lungs, while both ERs are involved in the development of the ovary, uterus, epididymis and intestine of the porcine fetus.

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.

Persistent Müllerian duct syndrome (PMDS) in the Polish free-ranged bull populations of the European bison (Bison bonasus L.)

This study describes the diversity of vestigial male uteri of the European bison (Eb) examined for: (1) morphology, (2) glycoprotein localization, (3) total protein and glycoprotein profiles, (4) steroid concentrations, and (5) PMDS based on the mutation of AMH and AMHR2 genes. Uteri of adult bulls (5-12 years old) were compared to a uterus of a juvenile female (6 months old). Male uterine proteins were analyzed in parallel to secretory endometrial proteins of pseudo-pregnant pig (PsEND) and BSA used as profile-controls. Hematoxylin/eosin-staining revealed the diversity of male uterine morphology, including lumen size/shape, endometrial (END) gland density, luminal knob-like epithelial structures and multiple intrauterine cells proliferating within the lumen. PAS-staining revealed the presence of glycoproteins restricted to luminal epithelial cells and END glands. Heterologous total protein PAGE-profiles (20-66kDa) revealed two dominant fractions (66 and 45kDa), similar to PAS-profiles (67 and 47kDa) in male and female uterine tissues. In male uterine tissues, androstendione and progesterone, but not testosterone, estrone or estradiol concentrations were lower than in the female. Sequencing of AMH- and AMHR2-like amplicons allowed identification of these gene mutations in Eb. Our results provide novel data regarding PMDS, demonstrating the diversity of uterine morphology, glycoprotein mass/profile, steroid concentration and AMH/AMHR2 mutations in Eb bulls.

Co-expression pattern of dopamine beta-hydroxylase (DβH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.).

Co-expression of dopamine β-hydroxylase (DβH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45-120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DβH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DβH immunoreactive nerve fibers (DβH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DβH, while some DβH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DβH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DβH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.

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.