Expression and cellular pattern of relaxin mRNA in porcine corpora lutea during pregnancy

Kohsaka, Tetsuya; Singh, Udai P.; Yogo, Keiichiro; Sasada, Hiroshi; Taya, Kazuyoshi; Hashizume, Kazuyoshi

doi:10.1007/s00441-007-0492-5

Cited by 6 publications

(6 citation statements)

References 25 publications

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“…Furthermore, timing and site of detections suggest putative roles of relaxin during oocyte maturation and embryo development. The primer pairs were effective for the detection of relaxin and its receptors expression in ovarian follicular and corpora lutea cells used as positive controls [12,18,25]. However, we were unable to detect the presence of relaxin messenger RNA in male and female gametes or developing embryos.…”

Section: Discussionmentioning

confidence: 97%

Examination of relaxin and its receptors expression in pig gametes and embryos

Feugang

Rodríguez-Muñoz

Willard

et al. 2011

Reprod Biol Endocrinol

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BackgroundRelaxin is a small peptide also known as pregnancy hormone in many mammals. It is synthesized by both male and female tissues, and its secretions are found in various body fluids such as plasma serum, ovarian follicular fluid, utero-oviduct secretions, and seminal plasma of many mammals, including pigs. However, the presence and effects of relaxin in porcine gametes and embryos are still not well-known. The purpose of this study was to assess the presence of relaxin and its receptors RXFP1 and RXFP2 in pig gametes and embryos.MethodsImmature cumulus-oocyte complexes (COCs) were aspirated from sows' ovaries collected at the abattoir. After in vitro-maturation, COCs were in vitro-fertilized and cultured. For studies, immature and mature COCs were separately collected, and oocytes were freed from their surrounding cumulus cells. Denuded oocytes, cumulus cells, mature boar spermatozoa, zygotes, and embryos (cleaved and blastocysts) were harvested for temporal and spatial gene expression studies. Sections of ovary, granulosa and neonatal porcine uterine cells were also collected to use as controls.ResultsUsing both semi-quantitative and quantitative PCRs, relaxin transcripts were not detected in all tested samples, while RXFP1 and RXFP2 mRNA were present. Both receptor gene products were found at higher levels in oocytes compared to cumulus cells, irrespective of the maturation time. Cleaved-embryos contained higher levels of RXFP2 mRNA, whereas, blastocysts were characterized by a higher RXFP1 mRNA content. Using western-immunoblotting or in situ immunofluorescence, relaxin and its receptor proteins were detected in all samples. Their fluorescence intensities were consistently more important in mature oocytes than immature ones. The RXFP1 and RXFP2 signal intensities were mostly located in the plasma membrane region, while the relaxin ones appeared homogeneously distributed within the oocytes and embryonic cells. Furthermore, spermatozoa displayed stronger RXFP2 signal than RXFP1 after western-immunoblotting.ConclusionAll together, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond.

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Section: Discussionmentioning

confidence: 97%

Examination of relaxin and its receptors expression in pig gametes and embryos

Feugang

Rodríguez-Muñoz

Willard

et al. 2011

Reprod Biol Endocrinol

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“…For histological examination basically according to the previous reports [16,17], the samples were pre-embedded in 2% (w/v) agar (Nippon gene, Tokyo, Japan) and fixed with 10% (v/v) formaldehyde (Wako Co., Tokyo, Japan) in phosphate buffered saline (PBS) (Wako Co., Tokyo, Japan) up to 48 hours at room temperature. After dehydration with alcohol, they were embedded in paraffin at 60°C.…”

Section: Methodsmentioning

confidence: 99%

Incorporation of Phosphatase Inhibitor in Culture Prompts Growth Initiation of Isolated Non-Growing Oocytes

et al. 2013

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In vitro folliculogenesis of primordial and early preantral follicles is necessary for increment of reproductive efficiency in domestic animals, humans and endangered species. Recent study in phosphatase and tensin homolog (Pten) -knockout mice has revealed that this phosphatase acts as an inhibitory factor in follicle activation of primordial pool with the resultant inhibition of oocyte growth. To test in vitro effect of a phosphatase inhibitor on growth initiation of isolated non-growing oocytes in neonatal ovaries, we applied a specific inhibitor (bpV (HOpic)) for PTEN in culturing system. Non-growing oocytes isolated from the ovaries of newborn BDF1 (C57BL/6 × DBA/2) pups were divided to four culture groups. Five days after culture, the oocytes in 14 μmol/l bpV only, 14 μmol/l bpV plus 100 ng/ml Kit Ligand (KL), and 100 ng/ml KL groups showed significantly (P<0.05) growth (19.3±0.55, 25.8±0.53 and 21.6±0.29 μm, respectively) compared with that of the control (no additive) (16.9±0.53 μm). In addition, western blotting in those groups showed enhanced expression of phosphorylated Akt. In conclusion, we clearly demonstrate that isolated non-growing oocytes develop in phosphatase inhibitor, especially to PTEN, incorporated culturing system, and show first as we know that oocytes with zona Pellucidae can be obtained in vitro from isolated non-growing oocytes.

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“…The partial sequence (324 bp) of boar RLF/INSL3 cDNA (GenBank® accession number X68369) was PCR-amplified using forward (5′-CAGGAGGCGCCAGAGAAGCTGT-3′) and reverse (5′-GGGACAGAGGGTCAGCAAGTCTTG-3′) primers with Taq DNA polymerase (New England BioLabs). cRNA probes for in situ hybridization were prepared according to the method of Kohsaka et al [21]. Briefly, the RLF/INSL3 construct in pGEM-T Easy (Promega) was digested with NocI and SpeI, and then the product was used as a template for SP6 and T7 RNA polymerase (Promega) to generate antisense and sense cRNA probes, which were labelled with DIG (digoxigenin)-11-UTP (Roche Diagnostics).…”

Section: Dot-blot and Western Blot Analysesmentioning

confidence: 99%

“…The specificity of the DIG-labelled cRNA probes was verified by Northern blot analysis, in which the anti-sense probe hybridized to a 0.9-kb RLF/INSL3 mRNA, whereas the sense probe yielded no signal. Testicular cryosections (6 μm) were cut, and in situ hybridization was performed as described previously [21]. …”

Section: Dot-blot and Western Blot Analysesmentioning

confidence: 99%

Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars

Minagawa

Fukuda²,

Ishige

et al. 2011

Biochemical Journal

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RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity.

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Expression and cellular pattern of relaxin mRNA in porcine corpora lutea during pregnancy

Cited by 6 publications

References 25 publications

Examination of relaxin and its receptors expression in pig gametes and embryos

Examination of relaxin and its receptors expression in pig gametes and embryos

Incorporation of Phosphatase Inhibitor in Culture Prompts Growth Initiation of Isolated Non-Growing Oocytes

Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars

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