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Maternal communication with gametes and embryos
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Theriogenology 70 (2008) 1182–1187 www.theriojournal.com
Maternal communication with gametes and embryos A. Fazeli * Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, United Kingdom
Abstract Mechanisms for gametes and embryos to interact with their maternal environment are crucial in achieving reproductive success, both in livestock and the human. Long-range (hormones) and short-range signalling molecules play important roles in mediating cell–cell maternal interactions/communications with gametes and embryos. Slight malfunctions or disturbances of the environment that host this interaction can retard embryonic development. This may lead to creation of a memory for the embryo leading to offspring prone to degenerative diseases in adulthood. Despite an overwhelming amount of research and the literature, not all signalling molecules involved and their relationship with each other are known. Progress in the application of high-throughput genomic and proteomic analytical tools, such as microarrays and quantitative proteomic technologies has had a positive impact on our understanding of various aspects of maternal communication with gametes and embryos. Recent advances point to the presence of a local mechanism in the female reproductive tract capable of recognising the arrival of gametes and embryos and modulating the tract’s environment accordingly for the next stage. Further investigations are underway to characterise the details of this system. It is important to consider spatial or temporal components of maternal communication with gametes and embryos that may confer consequences for developmental potential. Finally, it seems that the application of a systems biology approach for creation of an interactome map of maternal communication with gametes and embryos is essential and provides an excellent opportunity for an inter-disciplinary collaboration with engineers and mathematical modellers. # 2008 Elsevier Inc. All rights reserved. Keywords: Gametes; Embryo; Maternal communication; Systems biology
1. Introduction Reproductive fitness and offspring health are major players in determining the economic profitability of the livestock industry. Both parameters are highly complex and are deeply influenced by a wide array of physiological and environmental variables. Maternal interaction with gametes and embryos underpins pregnancy and influences the future health of offspring.
* Tel.: +44 114 2268195. E-mail address: [email protected]. URL: http://alirezafazeli.staff.shef.ac.uk/ 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.06.010
This communication with gametes and embryos is a key factor in early embryonic development, implantation, maintenance of pregnancy and the future health of offspring. Therefore, understanding how the maternal tract communicates with gametes and embryos at a molecular level is of major scientific, economic and health importance. Understanding this interaction is advantageous and has a number of different applications. For example, in many species including humans, females have the ability to store spermatozoa after mating or artificial insemination. The duration of sperm storage varies between females of different taxa (for a comprehensive review in reptiles, birds and mammals see [1]). The
A. Fazeli / Theriogenology 70 (2008) 1182–1187
honey bee queen (Apis mellifera) stores spermatozoa for several years [2] and domestic chickens (Gallus domesticus) store spermatozoa for more than a month [3]. In mammals, Noctule bats (Nyctalus noctula) have been reported to store spermatozoa for the longest duration (198 days, [4]). The task of sperm storage in mammals is mainly accomplished by maternal interaction/communication with spermatozoa through the oviduct. The isthmic region of the oviduct and the uterotubal junction are considered as the main sites of sperm storage in different mammalian species [5]. Elucidating the mechanisms involved in sperm storage within the oviduct might help to identify oviductal components that could be harnessed for semen preservation. A number of candidate molecules and strategies based on maternal interaction with gametes have already been proposed for improving semen preservation in the artificial insemination industry (for a review see [6]). This strategy improves upon the empirical approaches to sperm diluent development that have traditionally been employed. In cattle, up to 40% of total embryonic losses occur between days 8 and 17 of pregnancy, indicating that early embryonic mortality is the main source of reproductive wastage [7,8]. This high embryonic mortality rate has enormous economic implications, increasing the number of days open and retarding genetic progress. The high rate of pregnancy failure is assumed to be a consequence of insufficient communication between the conceptus and the maternal environment. The ability of embryonic interferon tau (IFNt) to inhibit uterine secretion of prostaglandin F2a, is critical to the establishment of pregnancy in cattle. The critical period for this signal appears to be between days 15 and 17, when the endometrium in the nonpregnant situation initiates a biochemical reaction cascade resulting in luteolysis [8]. Although embryonic secretions and uterine receptivity for IFNt are undoubtedly key factors of pregnancy recognition and maintenance in cattle, other factors may be involved that either modulate the IFNt system or act independently of IFNt. Further characterisation of components involved in maternal–embryo communication will lead to the identification of these factors. This will allow strategies to be devised that will reduce early embryonic and economic losses. The majority of adult chronic diseases such as coronary heart disease, hypertension, stroke, type 2 diabetes, obesity, osteoporosis and cognitive impairment have been shown in numerous epidemiological studies from diverse world populations to have in utero origins, associated with poor maternal nutrition and
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physiology (‘Foetal Origins’ hypothesis, [9]). Using different animal models, the initiation of altered developmental potential associated with postnatal disease can be traced back to the periconceptional period and the cross-talk between the maternal reproductive tract and the preimplantation embryo. Thus, several studies have shown that cleavage stage embryos are sensitive to environmental conditions which can permanently alter the developmental programme leading to abnormal postnatal growth, metabolism, physiology and behaviour (reviewed in [10]). Identifying the environmental factors that can influence embryonic maternal communication and can adversely affect the developmental potential of offspring through to adulthood would have major public health implications, and provide a basis for the prevention of common diseases. Research performed in livestock on long-term effects of alterations in maternal communication with gametes and embryos is limited. However, one can assume, like humans, alterations in the maternal communication conditions should have a profound influence on the health of the offspring and on its vulnerability to infectious disease. Understanding the optimum conditions for maternal communication with gametes and embryos may allow for production of healthy livestock, resistant to degenerative and infectious disease. The above examples highlight some important aspects of maternal communication with gametes and embryos. It also emphasises the need for further research to characterise these processes and define conditions/factors that can influence its optimum condition/performance. 2. What is maternal communication with gametes and embryos? It is impossible with the current state of knowledge to explain all different aspects of maternal communication with gametes and embryos. A holistic view of all actions and interactions taking place during this crosstalk between the gametes, embryos and the female reproductive tract does not exist. Maternal interaction with gametes is initiated from the moment that mating/ artificial insemination and/or ovulation takes place. Once fertilization occurs, the embryo takes the interaction over. In specific situations, such as during embryo transfer, maternal communication with the embryo is not preceded by the usual exposure of the female reproductive tract to one or both of the gametes. Gamete maturation and transport, fertilization, early embryonic development, implantation, and mainte-
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A. Fazeli / Theriogenology 70 (2008) 1182–1187
nance of pregnancy are the main events taking place during maternal communication with gametes and embryo. All these processes are critically dependent on intact and efficient communication between gametes, embryo and the maternal tract. So far, some of the signals involved in these interactions have been identified. For example, bovine IFNt exhibits antiluteolytic activity by preventing the transcription of the oxytocin receptor gene and oxytocin-induced luteolytic pulses of PGF2a, thus, acting as a pregnancy recognition signal (for review see [11]). Another long-range signalling molecule is progesterone, which is able to reduce ciliary beating in the oviduct [12]. Short-range signalling molecules in cell–cell maternal interactions/ communications with gametes and embryos are less defined. Recently Georgiou et al. [13] demonstrated that the presence of either of the gametes in the oviduct results in gamete specific alterations of oviductal secretory proteome. This is in agreement with previous reports demonstrating a change in gene expression in the female reproductive tract in different species as a result of the arrival of the gametes in the tract [14–16]. Growth hormones, growth factors, such as the insulinlike growth factor system, the hyaluronic acid system (reviewed in [17]), and several lectins have also been shown to support gamete interaction and early embryonic development. General endocrine signalling pathways and local factors produced by the gametes, the embryo, and the maternal epithelium, form a complex interactome network (molecular interactions needed to achieve functions) that leads to the establishment and maintenance of pregnancy. Disturbances of this network can result in adverse outcomes such as sub-fertility, infertility, loss of pregnancy, and even offspring with poor health status in adulthood [10]. 3. The scope of this paper In this paper, I will focus on some ongoing research projects within my laboratory which aim to provide a systematic view of maternal interaction with gametes. In addition, I will discuss a current European strategy for defining a comprehensive interactome map of maternal communication with gametes and embryos. 4. Gamete alteration of the oviductal transcriptome Until recently, the accepted view in the field has been that the oviductal environment and the composition of oviductal fluid are solely under the influence of the hormonal changes in the oviduct [18,19]. However, in
recent years, several investigations from our laboratory and others have challenged this view by providing evidence of transcriptional changes in the oviduct in response to gametes irrespective of oviductal hormonal status [14–16]. We tested the hypothesis that the oviduct has a recognition system for spermatozoa that can detect the arrival of spermatozoa in the oviduct after insemination, resulting in alterations of the oviductal transcriptome (collection of all gene transcripts, or mRNA) [15]. We initially performed a global screening of the oviductal transcriptome using affymetrix oligonucleotide arrays in mice, at the time of estrus (mating) and 6 h after mating. The results indicated transcriptional alterations in the oviduct after mating. However these alterations could have been attributed to the presence of spermatozoa in the oviduct after mating and also to changes in the hormonal environment as female mice underwent the transition from estrus to diestrus. To distinguish these possibilities, female mice were then mated with T145H mutant mice, which because of spermatogenic arrest, produce seminal plasma but no spermatozoa. Focusing on two molecules that in the first experiment were upregulated after mating, it was found that adrenomedullin and prostaglandin endoperoxidase synthase 2 transcripts were upregulated in the oviducts of mice only after mating with fertile males. Those mated with T145H infertile males showed significantly less upregulation. These results indicate that it is the arrival of spermatozoa in the oviduct that activates one or more signal transduction pathways and leads to changes in the oviductal transcriptome profiles. Oocytes also seem to induce oviductal gene expression. Bauersachs et al. [14] reported changes in oviduct epithelial cell gene transcription activity by comparing gene expression profiles of ipsilateral and contralateral oviductal epithelia of individual cows after ovulation. These changes are likely to have resulted from the direct contact of oviductal epithelial cells with ovulated cumulus–oocyte complexes. In agreement with this observation Lee et al. [16] had previously reported that oocytes and embryos induce a different transcriptomic profile in mice oviducts. Finally, in a recent investigation [20], it was observed that motile bull spermatozoa increased the secretion of prostaglandin in oviductal epithelial cells as well as cellular expression of mRNA for cyclooxygenase, prostaglandin E and F synthases in a dose- and time-dependent manner. A maximum three- to fivefold increased secretion of prostaglandin was observed with a dose of 105 spermatozoa/ml after a 12 h co-incubation. Neither killed spermatozoa nor truncated sperma-
A. Fazeli / Theriogenology 70 (2008) 1182–1187
tozoa heads stimulated oviductal biosynthesis and secretion of prostaglandins at any dose or time point observed. The results that live spermatozoa in the oviduct upregulate the local prostaglandin system, and thereby, enhance oviductal contractions. Thus, spermatozoa may bear a role in accelerating their own transport into the fertilization site. 5. Gamete alteration of the oviductal secretory proteome Although data based on transcriptomic analysis provide strong evidence suggesting the modulation of the oviductal environment by gametes, transcriptomes lack information regarding the exact changes to the oviductal proteomic profile, for example, the secretory profile. In mammals, not all the changes in the transcriptome are translated into proteomic alterations due to post-translational modifications. Ellington et al. [21] and Thomas et al. [22] provide the initial evidence that at least spermatozoa can influence the (secretory) proteomic profile of oviductal epithelial cells in vitro. These investigations have reported de novo protein synthesis in oviductal epithelial cell monolayers in response to spermatozoa in vitro. However, they failed to obtain the identity of the de novo synthesized proteins. In a recent investigation [13], we hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography–tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of the oviductal secretory proteins altered by gametes were regulators of protein folding and stability, such as various chaperones, protein isomerases, and proteolytic enzymes. Most of the identified proteins were uniquely regulated by either sperm or oocyte presence in the oviduct and are known to have influence on gamete maturation, viability, and function. There is also evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. The question arises as to how the oviduct is able to recognise the presence of gametes and alter its environment in response to them. This question becomes more complex, taking into consideration that
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sperm itself is a non-self entity for the female reproductive tract and as such should initiate an immune response to repel spermatozoa. However, the maternal response presented to gametes can be regarded as a favourable response to maintain their viability and facilitate their function. Presence of a gamete recognition system in the female reproductive tract, able to distinguish between spermatozoa and oocytes, would be intriguing. If such a system did exist, then it would most likely be conserved in different species that reproduce utilizing internal fertilization. Such a system for recognition of gametes in the female reproductive tract can be compared with Toll-like receptors for recognition of non-self entities by the innate immune system [23]. We may further speculate that a special form of Toll-like receptor molecule may exist in the female reproductive tract for recognition of spermatozoa that has yet to be discovered. At the time of ovulation and mating, nearly all mammals are in estrus, and therefore their oviducts/ female reproductive tracts are under dramatic hormonal influence. Nearly all proteomic investigations carried out to investigate maternal communication with gametes to date have been performed in vitro. It is not known if hormones can influence the maternal communication with gametes. This is an important point that requires further investigation. In addition no conclusive information exists regarding the number of spermatozoa that traverse the oviduct following natural mating. It is estimated that from the billions of spermatozoa that are deposited into the female reproductive tract after natural mating, only tens to hundreds of sperm reach the upper regions of the female reproductive tract [24]. In nearly all proteomic studies conducted so far in vitro, oviducts or epithelial cells were exposed to far more than the physiological number of spermatozoa at the site of fertilization after natural mating (106–107 sperm/ml [13,21]). Finally, there is always a need for caution when attempting to extrapolate in vitro observations to in vivo systems [25]. Providing answers to the above questions requires performing in vivo designed experiments. Such experiments are currently running in our laboratory in collaboration with colleagues from the University of Murcia, Spain. The preliminary results [26] using an in vivo model at the time of mating/estrus under the influence of reproductive hormones confirm our previous conclusions [15]. Of note, under hormonal influence, proteins other than those reported in in vitro systems were altered by gametes, suggesting that hormones are vital to the appropriate communication system.
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6. Towards creation of an interactome map of maternal communication with gametes and embryos Our ultimate aim of characterisation of maternal communication with gametes and embryos is to create a so-called ‘‘Interactome’’ map incorporating both short-range (cell-to-cell interactions) as well as long-range (endocrine) signalling, during different stages of the reproductive cycle and pregnancy. This map should cover normal and altered conditions, such as maternal diet, age and physiology, and should be multi-species so that outcomes will have impact across the spectrum from livestock breeding to human health. Although several aspects of the gamete/embryo– maternal interaction have been studied as mentioned, so far no systematic analysis of this interactome has been performed. Such studies are vital for creation of a comprehensive interactome map of maternal interaction with gametes and embryos. Obviously such an ambitious task cannot be fulfilled by an individual laboratory or discipline. Therefore, a number of laboratories from European member states currently involved in investigating different aspects of maternal communication with gametes and embryos have joined forces to establish a research network. The main goal of this network is to define the basic requirements for production of an interactome map of maternal communication with gametes and embryos. Some of the current objectives of the group are (i) to establish the optimum in vivo model(s) for creation of an interactome map/database of maternal interaction/ communication with gametes and embryos, (ii) to establish the optimum in vitro model(s) for creation of an interactome map/database of maternal interaction/ communication with gametes and embryos, and (iii) to establish strategies for progress from in vivo towards in vitro models for creation of the interactome map. Furthermore, this network hopes to establish guidelines and standards for the use of in vivo and in vitro models to investigate the effect of maternal interaction/communication with gametes and embryos allowing investigators in different laboratories to share data and information. This is an ongoing project and further information regarding its progress can be obtained by visiting http://alirezafazeli.staff.shef. ac.uk/COST.html. We hope the work of this network further advances knowledge in the field and establishes the conditions for creation of an interactome map of maternal interaction with gametes and embryos.
7. Concluding remarks Progress in the development of high-throughput genomic and proteomic analytical tools, such as microarrays or quantitative proteomic technologies, has had a positive impact on our understanding of various aspects of maternal communication with gametes and embryos. We have realised that maternal communication with gametes and embryos is a complex and continuous interaction. Each stage of this interaction must prepare the maternal environment for the next stage. We have learned that slight malfunctions or disturbances of the environment that host this interaction can retard embryonic development. It is possible that this creates a ‘memory’ within an embryo leading to offspring who are prone to degenerative diseases in adulthood. Research in the field indicates the need for applying systems biology approaches to understand different dimensions of maternal communication with gametes and embryos. A systems biology approach would allow mathematical modelling of maternal communication with gametes and embryos, and construction of an in silico model for the temporal sequence of events involved. Nearly all investigations so far have studied this interaction in isolation, without consideration of spatial or temporal components that may confer consequences for developmental potential. It would be crucial to interact with engineers and mathematical modellers and utilize their experiences in application of a systems biology approach to our model. Creation of an interactome map of maternal communication with gametes and embryos is an excellent opportunity for an inter-disciplinary collaboration with engineers and mathematical modellers. Acknowledgments I would like to thank Miss Emma Pewsey for critical proof reading and correction of this manuscript. I am in debt to the members of my lab for their help in preparation of this manuscript. References [1] Birkhead TR, Moller AP. Sexual selection and the temporal separation of the reproductive events: sperm storage data from reptiles, birds and mammals. Biol J Linn Soc 1993;50:295–311. [2] Koeniger G. Reproduction and mating behavior. In: Rinderer TE, editor. Bee genetics and breeding. New York: Academic Press; 1986. p. 255–80. [3] Nalbandov A, Card LE. Effect of stale sperm on fertility and hatchability of chicken eggs. Poult Sci 1943;5:451–2.
A. Fazeli / Theriogenology 70 (2008) 1182–1187 [4] Racey PA. The viability of spermatozoa after prolonged sperm storage by male and female European bats. Period Biol 1973; 75:201–5. [5] Hunter RH. The Fallopian tubes in domestic mammals: how vital is their physiological activity? Reprod Nutr Dev 2005;45: 281–90. [6] Holt WV, Elliott RM, Fazeli A, Sostaric E, Georgiou AS, Satake N, et al. Harnessing the biology of the oviduct for the benefit of artificial insemination. Soc Reprod Fertil Suppl 2006;62: 247–59. [7] Humblot P. Use of pregnancy specific proteins and progesterone assays to monitor pregnancy and determine the timing, frequencies and sources of embryonic mortality in ruminants. Theriogenology 2001;56:1417–33. [8] Thatcher WW, Guzeloglu A, Mattos R, Binelli M, Hansen TR, Pru JK. Uterine–conceptus interactions and reproductive failure in cattle. Theriogenology 2001;56:1435–50. [9] Barker DJ.In: Mothers, babies and health in later life. Edinburgh: Churchill Livingstone; 1998. [10] Fleming TP, Kwong WY, Porter R, Ursell E, Fesenko I, Wilkins A, et al. The embryo and its future. Biol Reprod 2004;71:1046–54. [11] Spencer TE, Johnson GA, Bazer FW, Burghardt RC, Palmarini M. Pregnancy recognition and conceptus implantation in domestic ruminants: roles of progesterone, interferons and endogenous retroviruses. Reprod Fertil Dev 2007;19:65–78. [12] Wessel T, Schuchter U, Walt H. Ciliary motility in bovine oviducts for sensing rapid non-genomic reactions upon exposure to progesterone. Horm Metab Res 2004;36:136–41. [13] Georgiou AS, Sostaric E, Wong CH, Snijders AP, Wright PC, Moore HD, et al. Gametes alter the oviductal secretory proteome. Mol Cell Proteomics 2005;4:1785–96. [14] Bauersachs S, Blum H, Mallok S, Wenigerkind H, Rief S, Prelle K, et al. Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach. Biol Reprod 2003;68: 1170–7. [15] Fazeli A, Affara NA, Hubank M, Holt WV. Sperm-induced modification of the oviductal gene expression profile after natural insemination in mice. Biol Reprod 2004;71:60–5.
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[16] Lee KF, Yao YQ, Kwok KL, Xu JS, Yeung WS. Early developing embryos affect the gene expression patterns in the mouse oviduct. Biochem Biophys Res Commun 2002;292:564–70. [17] Wolf E, Arnold GJ, Bauersachs S, Beier HM, Blum H, Einspanier R, et al. Embryo–maternal communication in bovine: strategies for deciphering a complex cross-talk. Reprod Domest Anim 2003;38:276–89. [18] Buhi WC. Characterization and biological roles of oviductspecific, oestrogen-dependent glycoprotein. Reproduction 2002;123:355–62. [19] Leese HJ. The formation and function of oviduct fluid. J Reprod Fertil 1988;82:843–56. [20] Kodithuwakku SP, Miyamoto A, Wijayagunawardane MP. Spermatozoa stimulate prostaglandin synthesis and secretion in bovine oviductal epithelial cells. Reproduction 2007;133: 1087–94. [21] Ellington JE, Ignotz GG, Ball BA, Meyers-Wallen VN, Currie WB. De novo protein synthesis by bovine uterine tube (oviduct) epithelial cells changes during co-culture with bull spermatozoa. Biol Reprod 1993;48:851–6. [22] Thomas PG, Ignotz GG, Ball BA, Brinsko SP, Currie WB. Effect of coculture with stallion spermatozoa on de novo protein synthesis and secretion by equine oviduct epithelial cells. Am J Vet Res 1995;56:1657–62. [23] Aflatoonian R, Fazeli A. Toll-like receptors in female reproductive tract and their menstrual cycle dependent expression. J Reprod Immunol 2008;77:7–13. [24] Kunavongkrit A, Sang-Gasanee K, Phumratanaprapin C, Tantasuparuk W, Einarsson S. A study on the number of recovered spermatozoa in the uterine horns and oviducts of gilts, after fractionated or non-fractionated insemination. J Vet Med Sci 2003;65:63–7. [25] Hunter RH, Rodriguez-Martinez H. Analysing mammalian fertilisation: reservations and potential pitfalls with an in vitro approach. Zygote 2002;10:11–5. [26] Georgiou AS, Sostaric E, Wong CH, Snijders APL, Wright PC, Vazquez JM, et al. Gametes alter the oviductal secretory proteome in vivo. In: Endocrine society for Australia and society for reproductive biology annual scientific meeting; 2005 [Abstract number 293].
Theriogenology 70 (2008) 1182–1187 www.theriojournal.com
Maternal communication with gametes and embryos A. Fazeli * Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, United Kingdom
Abstract Mechanisms for gametes and embryos to interact with their maternal environment are crucial in achieving reproductive success, both in livestock and the human. Long-range (hormones) and short-range signalling molecules play important roles in mediating cell–cell maternal interactions/communications with gametes and embryos. Slight malfunctions or disturbances of the environment that host this interaction can retard embryonic development. This may lead to creation of a memory for the embryo leading to offspring prone to degenerative diseases in adulthood. Despite an overwhelming amount of research and the literature, not all signalling molecules involved and their relationship with each other are known. Progress in the application of high-throughput genomic and proteomic analytical tools, such as microarrays and quantitative proteomic technologies has had a positive impact on our understanding of various aspects of maternal communication with gametes and embryos. Recent advances point to the presence of a local mechanism in the female reproductive tract capable of recognising the arrival of gametes and embryos and modulating the tract’s environment accordingly for the next stage. Further investigations are underway to characterise the details of this system. It is important to consider spatial or temporal components of maternal communication with gametes and embryos that may confer consequences for developmental potential. Finally, it seems that the application of a systems biology approach for creation of an interactome map of maternal communication with gametes and embryos is essential and provides an excellent opportunity for an inter-disciplinary collaboration with engineers and mathematical modellers. # 2008 Elsevier Inc. All rights reserved. Keywords: Gametes; Embryo; Maternal communication; Systems biology
1. Introduction Reproductive fitness and offspring health are major players in determining the economic profitability of the livestock industry. Both parameters are highly complex and are deeply influenced by a wide array of physiological and environmental variables. Maternal interaction with gametes and embryos underpins pregnancy and influences the future health of offspring.
* Tel.: +44 114 2268195. E-mail address: [email protected]. URL: http://alirezafazeli.staff.shef.ac.uk/ 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.06.010
This communication with gametes and embryos is a key factor in early embryonic development, implantation, maintenance of pregnancy and the future health of offspring. Therefore, understanding how the maternal tract communicates with gametes and embryos at a molecular level is of major scientific, economic and health importance. Understanding this interaction is advantageous and has a number of different applications. For example, in many species including humans, females have the ability to store spermatozoa after mating or artificial insemination. The duration of sperm storage varies between females of different taxa (for a comprehensive review in reptiles, birds and mammals see [1]). The
A. Fazeli / Theriogenology 70 (2008) 1182–1187
honey bee queen (Apis mellifera) stores spermatozoa for several years [2] and domestic chickens (Gallus domesticus) store spermatozoa for more than a month [3]. In mammals, Noctule bats (Nyctalus noctula) have been reported to store spermatozoa for the longest duration (198 days, [4]). The task of sperm storage in mammals is mainly accomplished by maternal interaction/communication with spermatozoa through the oviduct. The isthmic region of the oviduct and the uterotubal junction are considered as the main sites of sperm storage in different mammalian species [5]. Elucidating the mechanisms involved in sperm storage within the oviduct might help to identify oviductal components that could be harnessed for semen preservation. A number of candidate molecules and strategies based on maternal interaction with gametes have already been proposed for improving semen preservation in the artificial insemination industry (for a review see [6]). This strategy improves upon the empirical approaches to sperm diluent development that have traditionally been employed. In cattle, up to 40% of total embryonic losses occur between days 8 and 17 of pregnancy, indicating that early embryonic mortality is the main source of reproductive wastage [7,8]. This high embryonic mortality rate has enormous economic implications, increasing the number of days open and retarding genetic progress. The high rate of pregnancy failure is assumed to be a consequence of insufficient communication between the conceptus and the maternal environment. The ability of embryonic interferon tau (IFNt) to inhibit uterine secretion of prostaglandin F2a, is critical to the establishment of pregnancy in cattle. The critical period for this signal appears to be between days 15 and 17, when the endometrium in the nonpregnant situation initiates a biochemical reaction cascade resulting in luteolysis [8]. Although embryonic secretions and uterine receptivity for IFNt are undoubtedly key factors of pregnancy recognition and maintenance in cattle, other factors may be involved that either modulate the IFNt system or act independently of IFNt. Further characterisation of components involved in maternal–embryo communication will lead to the identification of these factors. This will allow strategies to be devised that will reduce early embryonic and economic losses. The majority of adult chronic diseases such as coronary heart disease, hypertension, stroke, type 2 diabetes, obesity, osteoporosis and cognitive impairment have been shown in numerous epidemiological studies from diverse world populations to have in utero origins, associated with poor maternal nutrition and
1183
physiology (‘Foetal Origins’ hypothesis, [9]). Using different animal models, the initiation of altered developmental potential associated with postnatal disease can be traced back to the periconceptional period and the cross-talk between the maternal reproductive tract and the preimplantation embryo. Thus, several studies have shown that cleavage stage embryos are sensitive to environmental conditions which can permanently alter the developmental programme leading to abnormal postnatal growth, metabolism, physiology and behaviour (reviewed in [10]). Identifying the environmental factors that can influence embryonic maternal communication and can adversely affect the developmental potential of offspring through to adulthood would have major public health implications, and provide a basis for the prevention of common diseases. Research performed in livestock on long-term effects of alterations in maternal communication with gametes and embryos is limited. However, one can assume, like humans, alterations in the maternal communication conditions should have a profound influence on the health of the offspring and on its vulnerability to infectious disease. Understanding the optimum conditions for maternal communication with gametes and embryos may allow for production of healthy livestock, resistant to degenerative and infectious disease. The above examples highlight some important aspects of maternal communication with gametes and embryos. It also emphasises the need for further research to characterise these processes and define conditions/factors that can influence its optimum condition/performance. 2. What is maternal communication with gametes and embryos? It is impossible with the current state of knowledge to explain all different aspects of maternal communication with gametes and embryos. A holistic view of all actions and interactions taking place during this crosstalk between the gametes, embryos and the female reproductive tract does not exist. Maternal interaction with gametes is initiated from the moment that mating/ artificial insemination and/or ovulation takes place. Once fertilization occurs, the embryo takes the interaction over. In specific situations, such as during embryo transfer, maternal communication with the embryo is not preceded by the usual exposure of the female reproductive tract to one or both of the gametes. Gamete maturation and transport, fertilization, early embryonic development, implantation, and mainte-
1184
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nance of pregnancy are the main events taking place during maternal communication with gametes and embryo. All these processes are critically dependent on intact and efficient communication between gametes, embryo and the maternal tract. So far, some of the signals involved in these interactions have been identified. For example, bovine IFNt exhibits antiluteolytic activity by preventing the transcription of the oxytocin receptor gene and oxytocin-induced luteolytic pulses of PGF2a, thus, acting as a pregnancy recognition signal (for review see [11]). Another long-range signalling molecule is progesterone, which is able to reduce ciliary beating in the oviduct [12]. Short-range signalling molecules in cell–cell maternal interactions/ communications with gametes and embryos are less defined. Recently Georgiou et al. [13] demonstrated that the presence of either of the gametes in the oviduct results in gamete specific alterations of oviductal secretory proteome. This is in agreement with previous reports demonstrating a change in gene expression in the female reproductive tract in different species as a result of the arrival of the gametes in the tract [14–16]. Growth hormones, growth factors, such as the insulinlike growth factor system, the hyaluronic acid system (reviewed in [17]), and several lectins have also been shown to support gamete interaction and early embryonic development. General endocrine signalling pathways and local factors produced by the gametes, the embryo, and the maternal epithelium, form a complex interactome network (molecular interactions needed to achieve functions) that leads to the establishment and maintenance of pregnancy. Disturbances of this network can result in adverse outcomes such as sub-fertility, infertility, loss of pregnancy, and even offspring with poor health status in adulthood [10]. 3. The scope of this paper In this paper, I will focus on some ongoing research projects within my laboratory which aim to provide a systematic view of maternal interaction with gametes. In addition, I will discuss a current European strategy for defining a comprehensive interactome map of maternal communication with gametes and embryos. 4. Gamete alteration of the oviductal transcriptome Until recently, the accepted view in the field has been that the oviductal environment and the composition of oviductal fluid are solely under the influence of the hormonal changes in the oviduct [18,19]. However, in
recent years, several investigations from our laboratory and others have challenged this view by providing evidence of transcriptional changes in the oviduct in response to gametes irrespective of oviductal hormonal status [14–16]. We tested the hypothesis that the oviduct has a recognition system for spermatozoa that can detect the arrival of spermatozoa in the oviduct after insemination, resulting in alterations of the oviductal transcriptome (collection of all gene transcripts, or mRNA) [15]. We initially performed a global screening of the oviductal transcriptome using affymetrix oligonucleotide arrays in mice, at the time of estrus (mating) and 6 h after mating. The results indicated transcriptional alterations in the oviduct after mating. However these alterations could have been attributed to the presence of spermatozoa in the oviduct after mating and also to changes in the hormonal environment as female mice underwent the transition from estrus to diestrus. To distinguish these possibilities, female mice were then mated with T145H mutant mice, which because of spermatogenic arrest, produce seminal plasma but no spermatozoa. Focusing on two molecules that in the first experiment were upregulated after mating, it was found that adrenomedullin and prostaglandin endoperoxidase synthase 2 transcripts were upregulated in the oviducts of mice only after mating with fertile males. Those mated with T145H infertile males showed significantly less upregulation. These results indicate that it is the arrival of spermatozoa in the oviduct that activates one or more signal transduction pathways and leads to changes in the oviductal transcriptome profiles. Oocytes also seem to induce oviductal gene expression. Bauersachs et al. [14] reported changes in oviduct epithelial cell gene transcription activity by comparing gene expression profiles of ipsilateral and contralateral oviductal epithelia of individual cows after ovulation. These changes are likely to have resulted from the direct contact of oviductal epithelial cells with ovulated cumulus–oocyte complexes. In agreement with this observation Lee et al. [16] had previously reported that oocytes and embryos induce a different transcriptomic profile in mice oviducts. Finally, in a recent investigation [20], it was observed that motile bull spermatozoa increased the secretion of prostaglandin in oviductal epithelial cells as well as cellular expression of mRNA for cyclooxygenase, prostaglandin E and F synthases in a dose- and time-dependent manner. A maximum three- to fivefold increased secretion of prostaglandin was observed with a dose of 105 spermatozoa/ml after a 12 h co-incubation. Neither killed spermatozoa nor truncated sperma-
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tozoa heads stimulated oviductal biosynthesis and secretion of prostaglandins at any dose or time point observed. The results that live spermatozoa in the oviduct upregulate the local prostaglandin system, and thereby, enhance oviductal contractions. Thus, spermatozoa may bear a role in accelerating their own transport into the fertilization site. 5. Gamete alteration of the oviductal secretory proteome Although data based on transcriptomic analysis provide strong evidence suggesting the modulation of the oviductal environment by gametes, transcriptomes lack information regarding the exact changes to the oviductal proteomic profile, for example, the secretory profile. In mammals, not all the changes in the transcriptome are translated into proteomic alterations due to post-translational modifications. Ellington et al. [21] and Thomas et al. [22] provide the initial evidence that at least spermatozoa can influence the (secretory) proteomic profile of oviductal epithelial cells in vitro. These investigations have reported de novo protein synthesis in oviductal epithelial cell monolayers in response to spermatozoa in vitro. However, they failed to obtain the identity of the de novo synthesized proteins. In a recent investigation [13], we hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography–tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of the oviductal secretory proteins altered by gametes were regulators of protein folding and stability, such as various chaperones, protein isomerases, and proteolytic enzymes. Most of the identified proteins were uniquely regulated by either sperm or oocyte presence in the oviduct and are known to have influence on gamete maturation, viability, and function. There is also evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. The question arises as to how the oviduct is able to recognise the presence of gametes and alter its environment in response to them. This question becomes more complex, taking into consideration that
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sperm itself is a non-self entity for the female reproductive tract and as such should initiate an immune response to repel spermatozoa. However, the maternal response presented to gametes can be regarded as a favourable response to maintain their viability and facilitate their function. Presence of a gamete recognition system in the female reproductive tract, able to distinguish between spermatozoa and oocytes, would be intriguing. If such a system did exist, then it would most likely be conserved in different species that reproduce utilizing internal fertilization. Such a system for recognition of gametes in the female reproductive tract can be compared with Toll-like receptors for recognition of non-self entities by the innate immune system [23]. We may further speculate that a special form of Toll-like receptor molecule may exist in the female reproductive tract for recognition of spermatozoa that has yet to be discovered. At the time of ovulation and mating, nearly all mammals are in estrus, and therefore their oviducts/ female reproductive tracts are under dramatic hormonal influence. Nearly all proteomic investigations carried out to investigate maternal communication with gametes to date have been performed in vitro. It is not known if hormones can influence the maternal communication with gametes. This is an important point that requires further investigation. In addition no conclusive information exists regarding the number of spermatozoa that traverse the oviduct following natural mating. It is estimated that from the billions of spermatozoa that are deposited into the female reproductive tract after natural mating, only tens to hundreds of sperm reach the upper regions of the female reproductive tract [24]. In nearly all proteomic studies conducted so far in vitro, oviducts or epithelial cells were exposed to far more than the physiological number of spermatozoa at the site of fertilization after natural mating (106–107 sperm/ml [13,21]). Finally, there is always a need for caution when attempting to extrapolate in vitro observations to in vivo systems [25]. Providing answers to the above questions requires performing in vivo designed experiments. Such experiments are currently running in our laboratory in collaboration with colleagues from the University of Murcia, Spain. The preliminary results [26] using an in vivo model at the time of mating/estrus under the influence of reproductive hormones confirm our previous conclusions [15]. Of note, under hormonal influence, proteins other than those reported in in vitro systems were altered by gametes, suggesting that hormones are vital to the appropriate communication system.
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6. Towards creation of an interactome map of maternal communication with gametes and embryos Our ultimate aim of characterisation of maternal communication with gametes and embryos is to create a so-called ‘‘Interactome’’ map incorporating both short-range (cell-to-cell interactions) as well as long-range (endocrine) signalling, during different stages of the reproductive cycle and pregnancy. This map should cover normal and altered conditions, such as maternal diet, age and physiology, and should be multi-species so that outcomes will have impact across the spectrum from livestock breeding to human health. Although several aspects of the gamete/embryo– maternal interaction have been studied as mentioned, so far no systematic analysis of this interactome has been performed. Such studies are vital for creation of a comprehensive interactome map of maternal interaction with gametes and embryos. Obviously such an ambitious task cannot be fulfilled by an individual laboratory or discipline. Therefore, a number of laboratories from European member states currently involved in investigating different aspects of maternal communication with gametes and embryos have joined forces to establish a research network. The main goal of this network is to define the basic requirements for production of an interactome map of maternal communication with gametes and embryos. Some of the current objectives of the group are (i) to establish the optimum in vivo model(s) for creation of an interactome map/database of maternal interaction/ communication with gametes and embryos, (ii) to establish the optimum in vitro model(s) for creation of an interactome map/database of maternal interaction/ communication with gametes and embryos, and (iii) to establish strategies for progress from in vivo towards in vitro models for creation of the interactome map. Furthermore, this network hopes to establish guidelines and standards for the use of in vivo and in vitro models to investigate the effect of maternal interaction/communication with gametes and embryos allowing investigators in different laboratories to share data and information. This is an ongoing project and further information regarding its progress can be obtained by visiting http://alirezafazeli.staff.shef. ac.uk/COST.html. We hope the work of this network further advances knowledge in the field and establishes the conditions for creation of an interactome map of maternal interaction with gametes and embryos.
7. Concluding remarks Progress in the development of high-throughput genomic and proteomic analytical tools, such as microarrays or quantitative proteomic technologies, has had a positive impact on our understanding of various aspects of maternal communication with gametes and embryos. We have realised that maternal communication with gametes and embryos is a complex and continuous interaction. Each stage of this interaction must prepare the maternal environment for the next stage. We have learned that slight malfunctions or disturbances of the environment that host this interaction can retard embryonic development. It is possible that this creates a ‘memory’ within an embryo leading to offspring who are prone to degenerative diseases in adulthood. Research in the field indicates the need for applying systems biology approaches to understand different dimensions of maternal communication with gametes and embryos. A systems biology approach would allow mathematical modelling of maternal communication with gametes and embryos, and construction of an in silico model for the temporal sequence of events involved. Nearly all investigations so far have studied this interaction in isolation, without consideration of spatial or temporal components that may confer consequences for developmental potential. It would be crucial to interact with engineers and mathematical modellers and utilize their experiences in application of a systems biology approach to our model. Creation of an interactome map of maternal communication with gametes and embryos is an excellent opportunity for an inter-disciplinary collaboration with engineers and mathematical modellers. Acknowledgments I would like to thank Miss Emma Pewsey for critical proof reading and correction of this manuscript. I am in debt to the members of my lab for their help in preparation of this manuscript. References [1] Birkhead TR, Moller AP. Sexual selection and the temporal separation of the reproductive events: sperm storage data from reptiles, birds and mammals. Biol J Linn Soc 1993;50:295–311. [2] Koeniger G. Reproduction and mating behavior. In: Rinderer TE, editor. Bee genetics and breeding. New York: Academic Press; 1986. p. 255–80. [3] Nalbandov A, Card LE. Effect of stale sperm on fertility and hatchability of chicken eggs. Poult Sci 1943;5:451–2.
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