title: Prolactin affects bovine oocytes through direct and cumulus-mediated pathways.
abstract: The available evidence points to participation of PRL in regulation of mammalian oocyte maturation. The aim of the present study was to characterize pathways of PRL action on bovine oocytes. We analyzed (1) the presence of the PRL receptor and its mRNA isoforms in oocytes and cumulus cells; (2) the effect of PRL on meiosis resumption and the role of cumulus cells, the NO/NO synthase system, protein kinase C, and tyrosine kinases in this effect; and (3) PRL effects in the presence of gonadotropins on the developmental capacity of cumulus-free and cumulus-enclosed oocytes. The transcript and protein expression of the PRL receptor in the cells were detected by reverse transcription polymerase chain reaction and immunocytochemistry, respectively. The nuclear status of oocytes was assessed after culture of cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) with or without PRL (5-500 ng/mL) for 7, 14, or 24 hours. Besides, DOs were incubated for 7 hours in the absence or the presence of PRL (50 ng/mL) and/or L-NAME (an inhibitor of NO synthase), genistein (an inhibitor of tyrosine kinases), or calpostin C (a protein kinase C inhibitor). After IVM in 2 different systems containing PRL (50 ng/mL) and/or gonadotropic hormones, a part of oocytes underwent IVF and IVC and the embryo development was tracked until the blastocyst stage. Messenger RNA of long and short isoforms of the PRL receptor was revealed in both oocytes and cumulus cells. Immunocytochemistry confirmed the presence of the PRL receptor in oocytes and the cumulus investment. In the absence of gonadotropins (system 1), PRL retarded meiosis resumption in DOs but not in cumulus-enclosed oocytes, with this effect being short term, dose dependent, suppressed by L-NAME and genistein, and unaffected by calpostin. In systems containing gonadotropins, PRL did not affect nuclear maturation and the cleavage rate of cumulus-free and cumulus-enclosed oocytes. However, in the case of COCs, it raised the blastocyst yield both in system 2 (from 20.5%-40.9%, P < 0.01) and in system 3 (from 21.7%-33.9%, P < 0.05). The findings show for the first time the functioning of the direct pathway of PRL signaling into bovine oocytes, as confirmed by the expression of receptors of PRL and its direct meiosis-retarding effect involving activation of tyrosine kinases and NO synthase. Furthermore, this is the first demonstration that the beneficial effect of PRL on the oocyte developmental capacity is achieved via cumulus cells containing PRL receptors.
authors.affiliation: Center of Animal Biotechnology and Molecular Diagnostics, Russian Research Institute of Animal Breeding, Dubrovitsy-Podolsk, Russia; Department of Large Animal Sciences, Veterinary Reproduction and Obstetrics, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address: irledv@mail.ru.
authors.forename: Irina Y
authors.initials: IY
authors.lastname: Lebedeva
authors.affiliation: Center of Animal Biotechnology and Molecular Diagnostics, Russian Research Institute of Animal Breeding, Dubrovitsy-Podolsk, Russia.
authors.forename: Galina N
authors.initials: GN
authors.lastname: Singina
authors.affiliation: Center of Animal Biotechnology and Molecular Diagnostics, Russian Research Institute of Animal Breeding, Dubrovitsy-Podolsk, Russia.
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Volkova
authors.affiliation: Department of Large Animal Sciences, Veterinary Reproduction and Obstetrics, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Vejlsted
authors.affiliation: Center of Animal Biotechnology and Molecular Diagnostics, Russian Research Institute of Animal Breeding, Dubrovitsy-Podolsk, Russia.
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Zinovieva
authors.affiliation: Department of Large Animal Sciences, Veterinary Reproduction and Obstetrics, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D054885:Cumulus Cells / Q000737:chemistry / Q000502:physiology*; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008540:Meiosis / Q000187:drug effects; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry / Q000187:drug effects*; D011388:Prolactin / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011981:Receptors, Prolactin / Q000032:analysis / Q000235:genetics; D015398:Signal Transduction
chemical_list: D012333:RNA, Messenger; D011981:Receptors, Prolactin; D009569:Nitric Oxide; D011388:Prolactin; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C
keywords: Cumulus cells; Meiosis resumption; Oocyte developmental capacity; PRL receptor; Signaling pathways