Gonadal steroid fluctuations and reproduction results of European grayling (Thymallus thymallus L.) from broodstock farmed in a recirculation aquaculture system.

The initial experiment of this study was conducted to determine whether sex steroid concentrations varied before, during and after the reproductive season of European grayling (Thymallus thymallus L.) farmed broodstock confined in a recirculation aquaculture system (RAS). The results indicated that the plasma sex steroid (testosterone - T, progesterone - P4 and estradiol - E2) concentrations varied (P < 0.05) during these reproductive periods. There were greater concentrations of T, P4 and E2 before and during the grayling reproductive season indicating there are important functions of these steroid hormones associated with gonadal maturation, gamete production and reproductive behavior during the spawning period. In the second experiment of this study, the effectiveness of European grayling controlled reproduction treatment regimens was analyzed and there were 34.69 % and 68.18 % having ovulations in the 2 and 3-year-old broodfish, respectively. Of the embryos developing to the eyed-egg stage, there were 3.70 % and 6.87 % derived from eggs of 2- and 3-year-old grayling females, respectively. Proportions of embryos developing from the eyed-egg stage to hatching were 38.1 % and 52.1 % from eggs of 2- and 3-year-old grayling females, respectively, when there was culturing in a RAS. The results indicate that with grayling broodfish there is greater efficacy in induction of reproduction when there is imposing of the controlled reproduction treatment regimen on 3- rather than 2-year-old broodfish.

The Transcription Factor FOXN1 Regulates Skin Adipogenesis and Affects Susceptibility to Diet-Induced Obesity.

FOXN1, a transcription factor expressed in the epidermis, regulates keratinocyte differentiation and participates in skin wound healing. In this study, we explored the impact of FOXN1 insufficiency on diet-stimulated weight gain and dermal white adipose tissue regulation in the intact and wounded skin of FOXN1eGFP/+ (heterozygotes, FOXN1-insufficient) mice in the context of age and diet. The results showed that on a high-fat diet, FOXN1eGFP/+ mice gained significantly less body weight than their FOXN1+/+ counterparts (FOXN1-sufficient mice). The intact and wounded skin of FOXN1eGFP/+ mice displayed abrogated expression of the master regulators of adipogenesis, PPARγ, FABP4, and leptin, which decreased with age in FOXN1+/+ mice. FOXN1 insufficiency also resulted in a decreased percentage of adipocyte-committed precursor cells (CD24+) in the skin. The proadipogenic pathway genes Bmp2, Igf2, and Mest showed a gradual decrease in expression that accompanied the gradual inactivation of FOXN1 in the skin of FOXN1+/+, FOXN1eGFP/+, and FOXN1eGFP/eGFP (lack of FOXN1) mice. Bone morphogenetic protein 2 and insulin-like growth factor 2 signals colocalized with FOXN1-eGFP in the epidermis and in hair follicles. These data demonstrated that FOXN1 initiates the cascade of adipogenic signaling that regulates skin homeostasis and wound healing and affects susceptibility to diet-induced obesity.

De novo characterization of placental transcriptome in the Eurasian beaver (Castor fiber L.).

Our pioneering data provide the first comprehensive view of placental transcriptome of the beaver during single and multiple gestation. RNA-Seq and a de novo approach allowed global pattern identification of C. fiber placental transcriptome. Non-redundant beaver transcriptome comprised 211,802,336 nt of placental transcripts, grouped into 128,459 contigs and clustered into 83,951 unigenes. An Ensembl database search revealed 14,487, 14,994, 15,004, 15,267 and 15,892 non-redundant homologs for Ictidomys tridecemlineatus, Rattus norvegicus, Mus musculus, Homo sapiens and Castor canadensis, respectively. Due to expression levels, the identified transcripts were divided into two sets: non-redundant and highly expressed (FPKM > 2 in at least three examined samples), analysed simultaneously. Among 17,009 highly expressed transcripts, 12,147 had BLASTx hits. GO annotations (175,882) were found for 4301 transcripts that were assigned to biological process (16,386), cellular component (9149) and molecular function (8338) categories; 666 unigenes were also classified into 122 KEGG pathways. Comprehensive analyses were performed for 411 and 3078 highly expressed transcripts annotated with a list of processes linked to 'placenta' (31 GO terms) or 'embryo' (324 GO terms), respectively. Among transcripts with entire CDS annotation, 281 (placenta) and 34 (embryo) alternative splicing events were identified. A total of 8499 putative SNVs (~ 6.2 SNV/transcript and 1.7 SNV/1 kb) were predicted with 0.1 minimum frequency and maximum variant quality (p value 10e-9). Our results provide a broad-based characterization of the global expression pattern of the beaver placental transcriptome. Enhancement of transcriptomic resources for C. fiber should improve understanding of crucial pathways relevant to proper placenta development and successful reproduction.

Identification of pregnancy-associated glycoprotein family (PAG) in the brown bear (Ursus arctos L.).

Pregnancy-associated glycoproteins (PAGs) are abundant embryo-originated products expressed in the pre-placental trophoblast and, later, in the post-implantational chorionic epithelium of some mammalian species. This paper describes the identification and cellular immunolocalization of the chorionic PAG family in the discoidal-type placenta of the brown bear (Ursus arctos L. - Ua), in which the PAGs were named 'UaPAG-Ls'. The study used: 1) Western blot for total placental glycoproteins; and 2) cross-species heterologous double fluorescent immunohistochemistry (IHC) for cellular immune-localization of the PAGs. This is the first study reporting the identification and immunolocalization of the UaPAG-L family in placental cells during early pregnancy in the brown bear. Our Western analysis revealed a dominant mature 72 kDa UaPAG-L isoform was expressed in all Ua placentas during early pregnancy. Various other UaPAG-L isoforms (16-66 kDa) were also identified. Using IHC, the UaPAG-L proteins were localized to trophectodermal cells (TRD), where signal intensity resembled intense TRD proliferation within developing placenta. The data increases our general knowledge of PAG proteins localized in discoidal-type placenta during early pregnancy in the brown bear.

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.

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.

Transcript variations, phylogenetic tree and chromosomal localization of porcine aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) genes.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and/or different activation pathways.

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.

Gene structure of the pregnancy-associated glycoprotein-like (PAG-L) in the Eurasian beaver (Castor fiber L.).

The pregnancy-associated glycoprotein-like family (PAG-L) is a large group of chorionic products, expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium, and are involved in proper placenta development and embryo-maternal interaction in eutherians. This study describes identification of the PAG-L family in the genome of the Eurasian beaver (Castor fiber L.), named CfPAG-L. We identified 7657 bp of the CfPAG-L gDNA sequence (Acc. No. KX377932), encompassing nine exons (1-9) and eight introns (A-H). The length of the CfPAG-L exons (59-200 bp) was equivalently similar to the only known counterparts of bPAG1, bPAG2, and pPAG2. The length of the CfPAG-L introns ranged 288-1937 bp and was completely different from previously known PAG introns. The exonic CfPAG-L regions revealed 50.3-72.9% homology with equivalent segments of bPAG1 and pPAG2 structure. The intronic CfPAG-L regions alignments revealed a lack of homology. Within the entire CfPAG-L gene, 31 potential single nucleotide variants (SNV: 7 transversions and 24 transitions) were predicted. The identified exonic polymorphic loci did not affect the amino acid sequence of the CfPAG-L polypeptide precursor. This is the first report describing the CfPAG-L gene sequence, structural organization, and SNVs in the Eurasian beaver, one of the largest rodents.

Novel SNPs and InDels discovered in two promoter regions of porcine pregnancy-associated glycoprotein 2-like subfamily (pPAG2-Ls) in crossbreed pigs.

This is a pioneer study of single nucleotide polymorphisms (SNPs) within the entire promoter region (1204 bp) of the dominant pPAG2-L subfamily in the pig. The pPAG2-L subfamily was sequenced/examined using genomic deoxyribonucleic acid (gDNA) templates of crossbreed pigs (Landrace x Large White), and compared to two bacterial artificial chromosome (BAC) clones containing gDNA of the Duroc breed (as the positive controls). Our analysis of the pPAG2-L promoter identified 31 SNPs and one InDel mutation in crossbreed pigs. Among 42 SNPs identified in two BAC clones, 24 SNPs had not been previously detected in crossbreed pigs. The sequence alignment of pPAG2-L promoter, performed with Lasagne-Search 2.0, Cluster Bluster and MatInspector software, revealed a total of 28 transcription factor binding sites (TFBS) and 10 TFBS (AP-1, CCAAT, CHOP:C, FOXP1, LSF, MRF-2, Myc, NF1, NF-Y, TGIF) within SNPs in the core sequences. It was noted that TFBS (NF1) was found to be unique to the pPAG2 promoter sequence containing SNPs: g.-1100G>A(R), g.-1101T>C(Y), represented by GA and TC genotypes (p x = 0.12). Our broad-based novel database thus provides an SNP PAG2-L pattern for modern genotyping of female and male progenitors. This is required for further studies of various potential correlations between guiding SNP genotypes of the pPAG2-L subfamily in the sows of many breeds, in which the most economically important reproductive traits are properly documented on each farm.

Identification of the pregnancy-associated glycoprotein family (PAGs) and some aspects of placenta development in the European moose (Alces alces L.).

This study describes the identification and a broad-based characterization of the pregnancy-associated glycoprotein (PAG) genes expressed in the synepitheliochorial placenta of the Alces alces (Aa; N = 51). We used: (1) both size measurements (cm) of various Aa embryos/fetuses (crown-rump length) and placentomes (PLCs); (2) PCR, Southern and sequencing; (3) Western-blot for total placental glycoproteins; (4) deglycosylation of total cotyledonary proteins; and (5) double heterologous IHC for cellular immune-localization of the PAGs as pregnancy advanced (50-200 days post coitum). The crown-rump length and PLC size measurements permitted a novel pattern estimation of various pregnancy stages in wild Aa. The PLC number varied (5-21) and was the greatest at the mid and late stages of gestation in females bearing singletons or twins. The genomic existence of the identified PAG-like family was named AaPAG-L. Amplicon profiles of the AaPAG-L varied in the number and length (118-2000 bp). Southern with porcine cDNA probes confirmed specificity and revealed dominant AaPAG-L amplicons in males and females. Nucleotide sequences of the AaPAG-L amplicons shared 86.27% homology with the bovine PAG1 (bPAG1) gene. Amino acid AaPAG sequences revealed in silico 88.23% to 100% homology with the bPAG1 precursor. Western-blots revealed a dominant mature 55 kDa AaPAG fraction, and the major ∼48 kDa glycosylated form that was deglycosylated to ∼44 kDa. The AaPAG-Ls was immuno-localized to mono- and bi-nucleated trophectodermal cells (TRD-chorionic epithelium), where signal intensity resembled intense TRD proliferation within developing PLCs as pregnancy advanced. This is the first study identifying the AaPAG-L family in the largest representative among the Cervidae.

Plasma-Glucocorticoids and ACTH Levels During Different Periods of Activity in the European Beaver (Castor fiber L.).

Glucocorticoids (GCs) and adrenocorticotropic hormone (ACTH) are major components of the classic endocrine stress response. Free-living vertebrates are characterized by circannual changes in the baseline and/or stress-induced secretion of GCs and ACTH. In mammalian species, GC and ACTH levels vary seasonally but there is no consensus to the season in which animals have elevated GC and ACTH levels. The aim of our study was to determine, for the first time, the type and amount of glucocorticoids produced in free-living beaver (Castor fiber L.)--the largest rodent in Eurasia, and to find out whether stress-induced plasma GC and ACTH levels show seasonal variations. Blood samples were obtained from animals under general anesthesia in April (pregnancy in females), July (offspring rearing) and November (preparing for the winter). The adrenals of beavers produce both cortisol and corticosterone, and plasma cortisol levels were higher than corticosterone. In the current experiment, plasma cortisol concentrations in beavers were affected by the season. The highest stress-associated cortisol levels were noted in males in July during offspring rearing. Corticosterone and ACTH concentrations in beavers remained generally constant, regardless of the season and sex. In conclusion, seasonal changes were observed only in relation to stress-induced plasma cortisol levels in the beaver.

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

Co-expression of dopamine ß-hydroxylase (DßH) 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-DßH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DßH immunoreactive nerve fibers (DßH-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 DßH, while some DßH-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 DßH 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 DßH 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.

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.

Pregnancy-associated glycoprotein (PAG) family localized in chorionic cells within the epitheliochorial/diffuse placenta of the alpaca (Lama pacos).

Pregnancy-associated glycoproteins (PAGs) are abundant embryo-originated products expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium of some ungulate species. This paper describes the cellular immunolocalization of the chorionic PAG family in the epitheliochorial placenta type of the alpaca (Lama pacos-Lp), in which the PAGs were named 'LpPAGs'. Placental Lp sections (5 µm) of different females near mid-pregnancy (150 days post coitum; dpc), advanced pregnancy (244-263 dpc) and late pregnancy (347 dpc) were used for cross-species (heterologous-ht) double fluorescent immunohistochemistry (htdF-IHC). The htdF-IHC was performed with primary rabbit polyvalent anti-porcine PAG polyclonals. The LpPAG immuno-complexes were visualized with secondary goat anti-rabbit immunoglobulins-conjugated with Alexa 488 fluorophore (green), among all nuclei of placental cells stained with propidium iodide (red). This is the first study reporting the immunolocalization of the LpPAG family identified by htdF-IHC at the feto/maternal interface during different pregnancy stages of the alpaca. The most dominant and strongest immune-positive LpPAG signals were found in the well-developed chorionic cell layer. Our htdF-IHC indicated relatively high epitope resemblance to that of the PAGs in camelids and pigs. These data increase our general knowledge of chorionic PAG localization during pregnancy-stage dependent development of the epitheliochorial diffuse placenta type in the alpaca.

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.

Immunolocalisation of oestrogen receptors alpha (ERalpha) and beta (ERbeta) 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.

Isolation of pregnancy-associated glycoproteins from placenta of the American bison (Bison bison) at first half of pregnancy.

This paper describes the successful purification and characterisation of pregnancy-associated glycoproteins (PAG) extracted from placenta (3-4 months) of American bisons (Amb). Chorionic AmbPAG proteins were purified from foetal cotyledonary tissues (CT) and liquid cotyledonary-carrying proteins (LCP) leaking from damaged cells. Our protocols successfully indicated the usefulness of AmbPAG protein identification, especially from LCP fraction. The AmbPAGs were extracted, precipitated and eluted during DEAE cellulose chromatography. The richest protein fractions were further chromatographed on VVA (Vicia villosa agglutinin affinity column), then characterised by mono- and bi-dimensional electrophoresis, Western blot and N-terminal amino acid (aa) sequence. After being transferred to PVDF membranes, three selected VVA-purified AmbPAG isoforms differing in molecular masses and isoelectric points (Ip 4-4.6) were selected for sequencing. One identified N-terminal 25aa sequence of AmbPAG72kDa CT form was identified as completely new (RGSNLTSLPLQNVIDLFYVGNITIG). Two other AmbPAG proteins purified from different sources (74kDa CT and 76kDa LCP forms; RGSNLTIHPLRNIRDIFYVGNITIG) were identical or corresponded to N-terminus of various bovine PAGs (boPAG). The two AmbPAGs (74kDa CT and 76kDa LCP) revealed identical micro-sequence to boPAG7; and were similar mainly to bovine PAG4, -6, -15 and -17 precursors that were identified by full-length sequencing derived from cDNA cloning. The novel sequence of the AmbPAG (72kDa CT) was related to some boPAG and various other ruminant PAG precursors (caprine and ovine). All three identified AmbPAG sequences were also relatively similar to mature forms of purified native boPAG(56-75kDa) proteins. This is the first report indicating aa sequences of native AmbPAG proteins purified from placenta (CT and LCP) of bison species. The N-terminal sequences of the AmbPAGs have been deposited in the EMBL-EBI database (UniProtKB; Accession Nos.: P84916, P84917 and P84918).

Cellular localisation of the pregnancy-associated glycoprotein family (PAGs) in the synepitheliochorial placenta of the European bison.

This paper describes the cellular immuno-localisation of the PAG family in synepitheliochorial (cotyledonary) placenta of the European bison (Eb). Uteri were harvested from pregnant wild Eb (n=4; 45-150 days post coitum-dpc); and additionally from cattle (30, 45 dpc) and pigs (42 dpc)--both domestic species were used as positive controls for cellular PAG immunodetection. Placentas were sectioned, fixed, dehydrated and subjected to double fluorescent immunohistochemistry (dF-IHC) with the use of Alexa 488 fluorochrom (A488) and propidium iodide (PI). Native positive EbPAG signals were detected by heterologous (ht; cross-species) dF-IHC with primary rabbit anti-PAG polyclonals against native or recombinant porcine PAG antigens (anti-pPAG); then visualised with secondary anti-rabbit goat immunoglobulins--conjugated to A488. Our htdF-IHC indicated an unequivocal localisation to the mono- and bi-nuclear trophectoderm (chorionic epithelium) cells expressing the PAGs (A488-green) among all placental cells, in which PI (red) stained nuclei. This is the first paper reporting the EbPAG family expression examined by htdF-IHC at the feto-maternal interface in wild Pecoran species. The cross-reactivity of anti-pPAG polyclonals with the EbPAGs suggests that shared epitopes are present in these molecules. It seems that the EbPAG family, which is robustly expressed in mono- and bi-nucleated trophectoderm cells, is associated with events taking place during placenta development. Our study also provided a proficient ht-system to identify various PAGs that could be useful as prenatal protein markers for pregnancy diagnoses, which is essential for effective reproductive management of endangered mammals.

Biodiversity of multiple Pregnancy-Associated Glycoprotein (PAG) family: gene cloning and chorionic protein purification in domestic and wild eutherians (Placentalia)--a review.

This review presents a broad overview of chorionic glycoproteins encoded by the Pregnancy-Associated Glycoprotein (PAG) gene family and also serves to illustrate how the recent discovery of the PAG family has contributed to our general knowledge of genome evolution, placental transcription and placental protein expression. The complex and large PAG family is restricted to the Artiodactyla order, although single PAG-like genes have also been identified in species outside the Artiodactyla. The PAGs are members of the aspartic proteinase (AP) superfamily. Unexpectedly, however, some members of the PAG family possess amino acid substitutions within and around the active site that likely render them unable to act as proteinases. This paper summarises the available information regarding biodiversity of PAG gene expression based on cDNA cloning, mRNA localisation studies and the structural organisation of the PAG genes with a particular emphasis on PAG promoters. It also compares available data regarding PAG protein purifications, sequencing and their N-glycodiversity. Finally, it discusses the scientific relevance, possible functional roles of the PAGs and describes possible profitable applications related to the detection of PAG proteins in the blood of pregnant domestic and wild species.