{"paper_id":"d8c56000-a686-4617-8377-c9196cf7bbd6","body_text":"Oocyte maturation and embryo development\nare controlled by steroid hormones as well as intra-\novarian factors such as cytokines and growth\nfactors ( 1 - 4 ). In vivo, oocyte maturation takes\nplace in the presence of follicular fluid which\nis composed of plasma exudates and secretions\nof follicular cells. With each follicular developmental\nstage, the steroid contents of follicular\nfluids change and the ratio of progesterone (P4)\nto estradiol (E2) is related to the maturation\nstage of the oocytes ( 5 - 7 ).\nDuring folliculogenesis the oocyte gains its\ndevelopmental competence in a gradual and sequential\nmanner, after which it becomes a fully\nmature oocyte with the capability to become\nfertilized and develop into a high quality embryo\n( 8 ).\nThe process of mammalian oocyte meiosis\ntakes place in several steps. Initiation of the\nfirst meiotic division leads to primary oocytes\nthat occur in the fetal development period or\naround the time of birth. Oocytes progress\nthrough zygotene, pachytene and early diplotene\nstages but arrest at the dictyate stage of\nprophase I. At puberty the first meiotic division\nis completed by a surge of luteinizing hormone\nduring the menstrual cycle; the second meiotic\narrest of the oocytes occurs at ovulation. Resumption of the second meiotic division occurs\nafter penetration of the sperm ( 9 ).\nP4 is a cholesterol-derived, phylogenetically old\nsteroid hormone ( 10 ). It is synthesized during the\nsteroid hormone metabolizing pathways from androgens,\nestrogens, and glucocorticoids within several\ncell types such as the corpus luteum, placenta\nand adrenal gland. In addition it is produced from a\nplant steroid precursor, diosgenin ( 11 ,  12 ).\nWithin the ovary, cholesterol is converted by an\nenzyme to pregnenolone, another precursor steroid,\nafter which it can follow one of two pathways (Δ4 or\nΔ5). In the Δ4 path way pregnenolone is converted\nto P4. P4 not only serves as aprecursor for other steroids,\nbut enters the female’s blood and acts as a hormone\non target tissue ( 13 ).\nThe level of plasma P4 varies with sex and reproductive\nage. P4 is mainly bound to albumin,\nhowever it has an affinity to bind to corticosteroidbinding\nglobulin. In the normal menstrual cycle,\nits levels rise during the follicular phase and reach\na maximum level after ovulation. Its half-life in serum\nis about 5 minutes ( 10 - 12 ,  14 ).\nP4 is an intra follicular steroid that plays critical\nroles in ovulation, implantation and maintenance\nof pregnancy ( 15 ,  16 ). P4 is the dominant\ncontent of follicular fluid steroids in mammalian\npreovulatory follicles, which are temporary\nand elevated at 18 hours after the luteinizing\nhormone (LH) surge ( 17 ).\nP4 was initially studied as a contraceptive agent\nby inhibition of the luteinizing hormone surge and\novulation ( 18 ). However, it has a critical function\nin pregnancy maintenance and in the regulation of\ndifferent biological functions in the ovarian tissue\nand feto-maternal unit such as resumption of\nmeiosis, fertilization, embryonic development and\nimplantation ( 19 - 21 ).\nClinically, it can be used in the female reproductive\nsystem as luteal support during  in vitro  fertilization\n(IVF) ( 22 ), hormone replacement therapy\nfor older women ( 23 ), and as treatment for endometriosis\nand polycystic ovarian syndrome in\nyounger women ( 24 ). In addition, P4 has immunological\nfunctions for the maintenance of a fetomaternal\nallograft ( 19 ).\nSome investigations showed that P4 administration\nfor luteal support improved uterine receptivity\nat the ultra structure levels ( 25 - 27 ) and enhanced\nthe implantation rate in mice ( 28 ).\nIn our experiments we used ovariectomized animal\nmodels and injected exogenous hormones to\nevaluate the effects of P4 on endometrial morphology\nand gene expression. Our observations showed\nthat exogenous P4 administration affected expression\nof endometrial integrin molecules ( 29 ,  30 ).\nThe biological actions of P4 are mediated by\nthree genomic isoforms of P4 receptors (PR), PRA,\nPR-B and PR-C, in addition tothree non-genomic\nisoforms, alpha, beta, and gamma ( 31 ,  32 ). Although\nPR-A and PR-B arise from a single gene, PR-A is a\nmore important repressor than PR-B ( 33 ). The PR-C\nisoform is the shortest isoform. PR-C does not have\ntranscriptional activity, however it has a role in decidual\ncells during late pregnancy ( 31 ,  34 ).\nBoth PR-A and PR-B are expressed in preovulatory\nfolliclegranulosa cells ( 35 - 37 ). The membrane\nPR or non-genomic PGR are particularly\nnotable as promoters of oocyte meiosis. They are\nexpressed in neural, kidney, and intestinal tissues\nin addition to the reproductive tract ( 32 ).\nMice that lack PR-A and PR-B isoforms are infertile\nas a result of ovulation failure ( 38 ). It has\nbeen suggested that induction of PR isoforms in\ncumulus cells and their binding to P4 appear to affect\nfollicular growth, oocyte maturation, and embryo\ndevelopment ( 39 ,  40 ).\nAlthough PR was identified in granulosa cells,\nthere was no evidence of this receptor on the oocyte\n( 41 ,  42 ) with the exception of one report\nwhich observed the PR receptor in Xenopus laevis\noocytes ( 43 ). Canine oocytes have been shown to\nexpress estrogen receptors during the estrous cycle,\nhowever, there is a lack of PR expression in\nall phases ( 41 ).\nPR membrane component 1 (PGRMC1) is another\npotential mediator of P4 action ( 44 ). Possibly\nPGRMC1 mediatesanti-apoptoticactions of P4\n( 45 ). Western blot analysis has demonstrated the presence of PGRMC1in bovine germinal vesicle\n(GV) and metaphase II (MII) oocytes ( 46 ) as well\nas rat ( 44 ), and human oocytes ( 47 ). PGRMC1\nexpression is not only associated with male and\nfemale pronuclear formation, it is also highly expressed\nin blastocysts ( 46 ).\nDespite high level expression of PGRMC1, little\nis known about its role on oocyte function, however\nit may be directly involved in the regulation\nof meiotic maturation ( 48 ).\nDespite the lack of PGR ( PR) expression in the\noocyte, both P4 and estrogen receptor mRNA and\nproteins have been detected in mature cumulus oocyte\ncomplexes (COCs) and embryonic cells from\nseveral mammalian species ( 49 - 55 ).\nAparicio and colleagues ( 39 ) have shown decreased\nbovine embryo development as a result of\nblocking genomic PR and non-genomic PR alpha\nactivity. This result indicates that P4 intracellular\nsignaling is mediated by its interaction with nuclear\nand membrane PRs and is also important for\noocyte developmental competence.\nLimited work has been performed on embryo\nPR expression however there were varied, contradictory\nresults. Expression of PR during the preimplantation\nstages of pig and mouse embryos has\nbeen shown ( 48 ,  49 ,  51 - 54 ,  56 ,  57 ). P4 receptor\nmRNA was present during all stages of bovine\nembryo development ( 51 ). PR mRNA and protein\nwere expressed in pre-implantation pig embryos\nprior to the fifth cell division but not at later stages\nthrough blastocyst formation. P4 receptor mRNA\nwas undetectable until the blastocyst stage ( 54 ).\nWe have located no studies on PR expression\nduring early organogenesis. However, mRNA and\nprotein are expressed in increasing amounts in the\nfemale reproductive tract of the rat after organogenesis\n( 58 ). In the female reproductive system, PR\nis expressed in the uterus, mammary gland, ovarian\ntissue, fallopian tubes ( 57 ) and placenta ( 59 ).\nResumption of meiosis in oocytes is triggered by\nsteroid hormones, specifically P4, in certain species\n( 60 ). The resumption of meiosis and its progression\nto MII in several mammalian species such\nas cows, sheep and pigs is steroid dependent and\nthe inhibition of steroidogenesis in ovine follicles leads to impairment of resumption of meiosis and\nprogression to MII. According to research, levels of\nP4in follicular fluid and its ratio to the estrogen levels\nare strongly associated with oocyte quality and\nmaturity ( 61 ). However, controversy exists regarding\nthe effect of P4 on  in vitro  oocyte maturation\n(IVM). Our investigations have shown that addition\nof P4 (10, 38, 50, 100 μM) to the  in vitro  maturation\nmedia of mouse GV oocytes could not improve\nmaturation rates and developmental competence of\nGV in COCs and cumulus denuded oocytes (CDOs)\nat any of the tested concentrations when compared\nto the control groups. When we increased P4 from\n10 to 100 μM in the culture medium, the maturation\nrate decreased in a dose dependent manner and the\nGV arrested rates increased. Research has shown\nthat the effect of P4 in inhibition of meiotic resumption\nwas more effective in COC than CDO ( 62 ). It\nseems there were intensive interactions between oocytes\nand the surrounding cumulus cells. Oocytes\ncould affect cumulus cell functions.\nVanderhyden et al. ( 63 ) have shown that mouse oocytes\nmodulate steroid production by the surrounding\ncumulus cells. These observations have suggested\nthat oocytes secret a factor (s) which control cumulus\ncell production of E2 and P4. In contrast, Jamnongjit\net al. ( 64 ) observed that testosterone or P4 and epidermal\ngrowth factor induced meiotic resumption in\nmouse oocytes during their  in vitro  maturation; the\neffect of these steroids could have been inhibited by\nspecific receptor antagonists. Fukui et al. ( 65 ) demonstrated\nthat P4 supplementation of IVM culture\nsystems decreased the rate of bovine oocyte maturation\nand that addition of P4 to fertilization culture medium\ndid not improve the number of cleavage stage\nembryos. Carter et al. ( 66 ) have shown that addition\nof P4 to culture medium did not affect the proportion\nof  in vitro  matured/ in vitro  fertilized zygotes that developed\nto the blastocyst stage  in vitro . There was no\neffect on conceptus elongation following transfer to\nsynchronized recipient heifers.\nElsewhere, the role of P4 on bovine oocyte developmental\ncompetence has been investigated\nby inhibiting P4 production of cumulus cells. Research\nhas shown that supplementation of oocyte\nmaturation medium with trilostane, an inhibitor of\n3 β-hydroxy steroid dehydrogenase, caused a significant\ndecrease in the blastocyst formation rate,\nwhich was completely reversed by the addition of\nP4 or a P4 agonist. This observation might support\na positive rolefor P4 in oocyte quality ( 39 ).\nSupplementation of canine oocyte culture media\nwith steroid hormones stimulates their nuclear\nmaturation ( 67 ,  68 ). However, this effect of steroid\nhormones has not been shown in anestrus bitches\n( 69 ). In rhesus monkeys, the improvement of in\nvitro oocyte development was demonstrated in the\npresence of P4 and E2 ( 68 ).\nOverall, these inconsistent results may be due\nto different experimental strategies that have been\nused. It seems that the length of time between LH\nor human chorionic gonadotropin (hCG) stimulation\nand GV breakdown (GVBD) might explain\ndifferences among mammalian species. Possibly,\nmaintaining the healthiness of the oocyte and its\nability to mature in species with a long dormant\nperiod between LH surge and GVBD requires steroid\nsupport.\nIt has been shown that the P4 antagonist mifepristone\n(RU486) which occupies PR could not reverse\nthe inhibitory effect of P4 on oocyte maturation\n( 62 ). Therefore, it has been concluded that the\ninhibition of mouse oocyte maturation by P4 is not\nreceptor dependent. It appears that P4 could inhibit\ncAMP phosphodiesterase (PDE) activity through\nbinding to the purine-binding site of this enzyme,\nwhich in turn inhibits meiosis by increasing oocyte\ncAMP levels ( 70 ,  71 ).\nThe role of steroids has been shown to be involved\nin the acquirement of meiotic competence\nand the ability to undergo normal fertilization and\ndevelopment to the blastocyst stage ( 33 ). In humans\nand rhesus monkeys, high ratios of P4 to E2\nin follicular fluid were associated with better embryo\ndevelopment ( 72 ).\nIn this regard we attempted to investigate the effect\nof P4 in concentrations similar to that of preovulatory\nfollicular fluid (10 and 38 μM) on developmental\ncompetence of mouse GV oocytes and\nsubsequent fertilization potential. Our experiments\nshowed that P4 could not increase the fertilization\nrate and development of the embryo to the blastocyst\nstage ( 62 ). The result of this experiment was\ninconsistent with other studies ( 73 - 75 ). Silva and\nKnight ( 76 ) have shown that the addition of P4 to\nbovine oocyte  in vitro  maturation medium reduced\nthe rate of blastocyst formation.\nMattioli et al. ( 77 ) reported that presence ofP4\nin porcine oocyte maturation medium increased\nsubsequent sperm head decondensation and\nmale pronuclei formation. Zhang and Armstrong\n( 78 ) reported that the addition of P4 to porcine\noocyte maturation medium could increase both\nfertilization and cleavage rates, whereas E2\ncould not. P4 had the opposite effect in ovineoocytes\n( 74 ).\nThe embryo develops in tubal and uterine microenvironments\nthat are mainly controlled by\nP4. P4 may act directly as a survival factor or\nindirectly promote the production and secretion\nof cytokines which contribute to embryonic survival\nand development ( 79 ). It has been shown\nthat P4 elevation occurs when the embryo does\nnot reach the uterus, thus this finding proposes\nthat the effect of P4 on embryo development is\nmediated via P4-induced changes in the endometrial\ntranscriptome ( 80 ).\nGranulocyte macrophage colony stimulating\nfactor is a cytokine secreted by the embryo and\nendometrium under control of P4 ( 81 ) which\npromotes embryo development ( 82 ). Lessey et\nal. ( 83 ) have shown an increase in growth factor\nproduction in the stromal cells in response to P4\nadministration.\nLow P4 levels have been linked to early pregnancy\nfailure ( 84 ) and poor embryo development\n( 85 ), while in cattle administration of P4 enhances\nconceptus development ( 66 ,  86 ).\nSome studies have shown that endogenous P4\nis a main factor in the preparation of the endometrium\nfor embryo implantation ( 87 ,  88 ). Due\nto the effects of P4 on improving pregnancy rates\nof IVF patients in the ART clinic, thus exogenous\nP4has been used as luteal support to enhance implantation\nrates ( 89 ). Although P4 is essential for\ncontinuation of pregnancy in all mammals, expressions\nof P4 receptors cease prior to implantation.\nIt seems the loss of P4 receptors is important for\nmaternal recognition and embryo development in\nearly pregnancy ( 90 ).\nSeveral studies have described the effect of exogenous\nP4 supplementation on embryo development\nwith varying results, according to the time and duration of P4 treatment ( 91 - 95 ).\nInitiation of P4 supplementation at the time of\nonset of the postovulatory rise (between days 4\nand 5) resulted in consistent increases in pregnancy\nrate, however when P4 was administered later\nthere was no improvement in pregnancy rate ( 92 ).\nThe results of another study have suggested that\nthe time of/or strength of the postovulatory P4 rise\nis critical for embryo development rather than the\nfinal concentration of P4in the luteal phase ( 91 ).\nUse of supraphysiological levels of P4 during early\npregnancy in the mouse has resulted in a similar\nconclusion ( 96 ).\nIn our previous study we compared embryo\nquality and implantation rate in pregnant mice\nin superovulated, P4 treated and superovulated-\nP4 treated groups. Our observation showed a\nhigh survival rate of blastocysts (97.68%) and\nimplantation rate (92.06%, p< 0.001) in pregnant\nmice from the P4-treated group compared to the\nP4 superovulated group, which meant that injections\nof 1 mg/mouse of progesterone in un-stimulated\nmice significantly improved implantation\nrates compared to the control and super ovulated\ngroups ( 28 ).\nTo answer the question of whether P4 directly\naffects embryo development or there is an indirect\neffect via changes in the endometrium, some researchers\nhave added P4 to embryo culture medium\n in vitro  and examined development to the blastocyst\nstage, with contradictory results ( 2 ,  97 - 100 ). These\ncontradictory observations might be attributed to\ndifferent culture systems which have been used.\nIn vitro  and in vivo experiments by Clemente et\nal. ( 57 ) showed that the effects of P4 on conceptus\nelongation could be due to a direct effect of P4 on\nthe embryo. They demonstrated that a P4 receptorwas\nexpressed in all stages of embryo development.\nThese researchers showed the direct effect\nof P4 on embryo development. Supplementation\nof simple or co-culture embryo culture systems\nwith P4 did not affect on the embryo development\nand blastocyst cell number. However,  in vitro -derived\nembryo transfer to a recipient treated with P4\nresulted in a four-fold increase in conceptus length\non day 14. These data confirmed the hypothesis that conceptus elongation in cattle was related to P4-\ninduced changes in the uterine environment ( 57 ).\nThis finding agreed with a study by Geisert et al.\n( 101 ) who showed that administration of P4 early\nin the estrous cycle advanced uterine receptivity for\nthe transfer of older asynchronous embryos. Supplementation\nof embryo culture medium with lipidsoluble\nP4 resulted in an increase in the numbers of\nembryos that developed to the blastocyst stage ( 28 ,\n 99 ,  102 - 106 ). However different observations were\nreported by other studies ( 76 ,  99 ,  107 ).\nFerguson et al. ( 97 ) have demonstrated that addition\nof physiological concentrations of P4 to\nembryo culture medium at three days post-insemination\nbenefitted embryo development in several\nways. Thus, they have concluded that P4 has a\ndirect positive effect on the developing  in vitro \nculture of bovine embryos. P4 supplementation increased\nthe number of  in vitro  culture embryos that\ndeveloped to the grade 1 blastocyst stageas well as\nthe number of hatched blastocysts.\nIt was shown that co-culture of an embryo with\nendometrial tissue cultured in the presence of P4\nand E2 benefitted embryo development ( 108 ).\nAlso, our investigations led to similar results in\nwhich  in vitro  culture of mouse 2-cell embryos\nin the presence of 20 ng/ml P4 resulted in a high\nproportion of embryos that reached the blastocyst\nstage. In this study,embryo quality was less affected\nby P4 ( 28 ).\n\nThese results have shown that P4 could be a\nfactor for embryonic survival and improve in\nvivo embryo development and implantation,\nboth directly and indirectly. on its concentration\nand the mammalian species. The effect of P4\non oocyte maturation and embryo development\nmay be dependent on its concentration and the\nmammalian species.","source_license":"CC-BY-4.0","license_restricted":false}