Cancer treatment can diminish fertility in women and men. The need for fertility preservation is growing as increasing numbers of people survive cancer. Cryostorage of reproductive material to preserve potential for conception for cancer survivors has moved from being experimental to being a part of clinical management of women and men who are diagnosed with cancer in their reproductive years. There is little existing evidence about how fertility preservation services can be enhanced to meet the complex needs of patients who are diagnosed with cancer in their reproductive years. The aim of this research was to inform clinical practice development by drawing on the collective experience and knowledge of staff at well-established clinics that offer fertility preservation before cancer treatment.A qualitative research model was adopted using semi-structured interviews with professionals involved in the care of people who freeze reproductive material before cancer treatment. In the state of Victoria, Australia, two large assisted reproductive technology (ART) centres have been providing fertility preservation services for more than two decades. An invitation to participate in a semi-structured interview about clinical care in the context of fertility preservation was emailed to past and current staff members. To capture diverse perspectives, informants were sought from all relevant professions: fertility specialists, andrologists, nurses, embryologists/scientists, counsellors, and administrative staff. Transcripts were analysed thematically.communication between oncology and ART specialists; managing urgency; managing patients’ expectations; establishing and implementing protocols, systems, and data bases; and maintaining contact with patients.Thirteen key informants were interviewed from August 2013 to February 2014. The identified themes relating to enhancing clinical care in a fertility preservation service were The collective knowledge of this study’s informants, who represent multidisciplinary teams with more than two decades’ experience in fertility preservation, yields important insights into strategies that fertility preservation services can employ to promote the integration of oncology and fertility care, the psychosocial care of patients, data recording and monitoring, and reporting of outcomes.The online version of this article (doi:10.1186/s12913-017-2292-2) contains supplementary material, which is available to authorized users. Many people of reproductive age who are diagnosed with cancer hope to have children after completing treatment , 2. CancMost techniques for retaining potential for parenthood after cancer involve cryopreservation of reproductive material. The disease, the urgency of treatment, and patients’ characteristics  can limit the options . Sperm bThe likelihood of pregnancy resulting from cryopreserved oocytes has improved as a result of the increasing use of the vitrification freezing technique, with pregnancy rates now similar to those achieved with fresh oocytes . WhetherThe need for fertility preservation is growing as increasing numbers of people survive cancer . FurtherWhile there is ample evidence about advances in the technical aspects of fertility preservation, less is known about the psychosocial aspects of fertility preservation in the context of cancer. Emerging evidence indicates that there are psychological benefits for women in being counselled about fertility preservation and the possibility of storing reproductive material , that coIncreasingly, ART clinics offer fertility preservation as part of their service. Clinical guidelines relating to fertility preservation before cancer treatment recommend a multidisciplinary approach to information provision, counselling, and clinical care; that the potential threat to fertility posed by cancer treatment is discussed as early as possible in the treatment process; that women and men are offered fertility preservation and informed about available options; and that advice to patients takes into account the cancer diagnosis and prognosis, treatment plan, expected outcome of subsequent fertility treatment, and viability of thawed material , 31–33. In the state of Victoria, Australia, two large ART centres have been providing fertility preservation services for more than two decades. Between them they store reproductive material for several thousand individuals. Melbourne IVF and Monash IVF together undertake more than 90% of all fertility preservation procedures in Victoria and have significant clinical and scientific expertise in this area.Fertility preservation in the context of cancer treatment requires a multidisciplinary team and clinical practice evolves over time. In order to capture the diverse knowledge and experiences of the team, informants were sought from all relevant professions involved in establishing fertility preservation services at Melbourne IVF and Monash IVF or currently providing this service: fertility specialists, andrologists, nurses, embryologists/scientists, counsellors, and administrative staff. An invitation to participate in a semi-structured interview about the development of clinical care and their role in fertility preservation was emailed to past and current staff members who had been or currently were involved in fertility preservation. Those who expressed interest were sent a detailed explanatory statement and a consent form. An appointment was made with those who consented to be interviewed.A discussion guide was developed, informed by the investigators’ clinical and research experience, the published literature in the field of fertility preservation care , 27, 35,Face-to-face interviews were conducted from August 2013 to February 2014 by KH and JF in a location chosen by the informants and audio-recorded with their consent.Recordings were transcribed by a skilled transcriber and identifying details removed or amended. Transcripts were entered into NVivo 10 to assist with data management. The established techniques of thematic analysis  were appThirteen key informants were interviewed, of whom seven were associated with Melbourne IVF and six with Monash IVF. They represented embryologists/scientists (4), fertility specialists (3), andrologists (2), counsellors (2), and administrative staff (2). No nurses volunteered to be interviewed. To maintain confidentiality, informants are identified hereafter by their profession. Interviews lasted between 40 and 75 min.communication between oncology and ART specialists; managing urgency; managing patients’ expectations; establishing and implementing protocols, systems, and data bases; and maintaining contact with patients. Apart from the comments about male fertility preservation offered by the two andrologists, respondents’ comments almost exclusively related to fertility preservation for women diagnosed with cancer.The identified themes, evident across disciplines, were You know, sometimes, in the early days, a lot of these doctors weren’t comfortable about talking about fertility issues anyway, so they would shy away from bringing them up. But that’s changed, so I think that all people are pretty used to doing this now, and some of them have even made comments that—you know, actually diverting their mind onto the fertility preservation often seems to help them a bit from the point of view of facing the illness itself. (Andrologist 1)Informants, the fertility specialists and andrologists in particular, spoke of the fundamental need for good communication between oncologists and fertility specialists. Historically, according to informants, oncologists did not regard fertility preservation as a priority, which meant that they did not discuss fertility preservation options with their patients nor refer them to a fertility specialist. Patients who requested fertility preservation in the early days were mostly self-referred. There was consensus that referral patterns had changed over time as oncologists’ attitudes to fertility preservation moved from apprehensive to facilitative.We gave a lot of talks at different oncology hospitals, to try and get them on board. … It was also that thing for me to say, “Yes, we can do this immediately. We’ll attend the case. There’s no need for a second anaesthetic. Call me the day before, and I’ll attend”. (Scientist 1)This change was attributed to fertility specialists’ and scientists’ dedicated efforts to make oncologists aware of the existence of the fertility preservation service and their willingness to accommodate the need to avoid delaying cancer treatment.It’s the dialogue, and seeing patients promptly, writing good letters, ringing the oncologist, asking them what they think, keeping them informed, because it requires intense collaboration if you’re going to start chemo and you’re doing egg freezing. Active engagement with oncologists was also seen as vital in building collaborative relationships.[for fertility preservation] but, to us, the important thing is the conversation. Not all patients will choose an option, but it’s important that, even if they choose not to go ahead with an option, that they have really good counselling. If the oncologists can do that—or the oncology team and the oncology nurse specialist—that’s great. If they want to come to us, that’s great. It’s now, I think, almost universally accepted that all patients should have the discussion, not necessarily being referred Through these efforts, it was thought that most oncologists in Victoria now routinely discuss the potential loss of fertility and possible options to preserve fertility with patients of reproductive age who are facing cancer treatment and refer them, where appropriate, to a clinical fertility preservation service.You couldn’t make an appointment for 3 weeks’ time; it had to be dealt with that day or that week. So it was always very quick. (Counsellor 2)I think the key concern and the key issue is usually the time limit that we have that we have to work within. Often, by the time that it’s been brought up—which is not usually the first thing that you talk about, which is understandable—the patient is already well into the planning of her chemotherapy, and so we are left with only a small amount of time. It was trying to get some sense of what they understood about what was possible with these options and what it was all about, and I think also quite strongly saying, “Yeah, you can say ‘no’ to treatment if you want to. You don’t have to do it”. And for some that was a relief. (Counsellor 2)To avoid delaying initiation of cancer treatment, clinics need be able to see patients at very short notice and to give them information about the available options that helps them decide whether or not to proceed with fertility preservation. Respondents, counsellors in particular, emphasised the need to be non-directive and to frame declining fertility preservation as a legitimate option.One of my fears and concerns was who was actually driving the decision to store the material. (Counsellor 2)Informants also discussed the difficulties that can arise when young women are accompanied by their mothers. The need to ensure that the young woman is not coerced has to be balanced against the benefits of a supportive and involved parent.All professions were acutely aware that careful management of patients’ expectations is an integral aspect of offering fertility preservation procedures. They perceived the need to temper hopes as an important part of their role, because of the uncertain outcomes of both cancer treatment and fertility preservation.If their doctor has referred them now, then that represents hope to them, and that is very powerful. They always turn up for their appointments, and they don’t make foolhardy decisions. You think, well, how can you make a decision in 2 days or 1 day? But they do, and they can, and we would never let anyone make a foolhardy decision. But it is amazing the buy-in you get from these patients, because, for them, it’s a way of looking forward. And they never whinge and complain and say, “Oh, I’ve got so much to think about; oh my god, my cancer!” They say, “Great. I want to really focus on this”. It’s almost universal, and I presume they behave the same way with their oncologist, but I don’t know. Informants emphasised that, for women who freeze eggs or ovarian tissue, the procedure does not only represent hope for future children but also embodies hope of survival.I mean, as counsellors in the area, it was very important to us to see the people who were involved, and not just the technology, so I think we wanted to explain the technology, too, in easy-to-understand terms. And that’s what we’d done with IVF for years. … But this was different, wasn’t it, because these people weren’t infertile; they’d never been near any reproductive—they’d just been plucked out of nowhere so fast that it was crisis work. (Counsellor 1)And we’ve always been so paranoid about making sure we don’t over-sell something and give people false hope, that we’ve possibly been overly pessimistic, I think. Informants underscored the importance of explaining the process of fertility preservation in a way that enabled patients, women in particular, to be fully aware of what it involves and they expressed concern that some women appear too optimistic that the procedure will guarantee future children. Counselling women and providing factual information was thus seen as a crucial part of good clinical practice.They don’t see a counsellor; no, they don’t. Why would they? They’ve got enough counsellors from the cancer support. (Andrologist 2)Men we didn’t see; the men we didn’t counsel for sperm freezing. That was, I think, for a number of reasons, mainly because there was no technology involved. (Counsellor 2)However, respondents did not see the need to extend counselling to men who freeze sperm before being treated for cancer.When I started, there was a database existing there, and, to me, it was just very much a telephone book: lots of patients’ names on there with a little bit of background about them, but all in a kind of ‘Notes’ field, and … you can’t do anything with text fields. You need fields that you can pull data from. (Administrator 1)So, for research, … you need good-quality data, so that’s how I’ve sort of picked up that role. And I’m also involved with the research, and that includes the cryostorage, so I guess, over the time, we’ve developed more efficient systems. (Administrator 2)The importance of establishing protocols, systems, and data bases that allow research as well as monitoring and evaluation of clinical practice, outcomes of the procedures, and patient outcomes was emphasised by all informants. They reported from their experience that data collection in the experimental phase of fertility preservation proved inadequate once procedures were incorporated into a clinical service.It’s about understanding what the fertility outcomes are post-treatment. What were the treatment types that they had? How old were they? They’re the kind of fields that we felt were important to be able to draw from and analyse. … And that’s also important information for us in terms of knowing what the prognosis is for some diagnoses, and some chemotherapy strengths and whatever; for us to be able to counsel around fertility preserving options if they’re given a particular diagnosis. (Administrator 1)It was viewed as essential to have comprehensive data collection that allows evidence to accumulate about the types of cancer and cancer treatments for which fertility preservation improves the chance of future conception, and about factors that influence successful use of stored reproductive material. These data would allow clinics to provide evidence-based, individualised information to patients about the options available to them.[To avoid writing to patients who have died], I would like to have that cross-match with the Births, Deaths and Marriages Registry. … For the ones that do have their eggs frozen, with the Births, Death and Marriages cross-match, it will be important information for us how many of those did spontaneously conceive. (Administrator 1)The need to link clinic fertility preservation data with records in the state-based Births, Deaths and Marriages (BDM) Registry has become evident, as this provides crucial information about deaths and births after spontaneous pregnancies among people who do not return to use their stored reproductive material.We’re at the point now of setting up a National Fertility Preservation Registry. The value in having a national registry, the value in getting those numbers, is so you can develop protocols and guidelines a lot quicker, is what the aim is of the national database. (Administrator 1)Informants discussed the value of a national registry for all fertility preservation procedures and were supportive of an initiative to establish such a registry.This is also a way for us to keep up with our patients. There’s a little thing at the back saying, “If your information has changed, please contact us if you—”. So this is just, you know, if we can keep in contact with the patients, they can let us know if they’ve changed their address or—. (Administrator 1)[of a regular newsletter] was to do an update of where technology was, but it was also to, I suppose, cull out people a little bit. If you’ve got return mail, you knew they’d changed their address. But also you’d get those who—you’d then find out they’d died by that method, so I think that was important. (Counsellor 1)One of the purposes [send newsletters] was that there was this fear that, at the time of the crisis, they actually hadn’t heard everything properly, and it was important at a later date to follow it up with more information and proper contact details and all of that stuff. (Counsellor 2)But part of the reason to do it Informants pointed out that storage of reproductive material can continue for decades and that it is essential to have systems for maintaining contact with patients and avoiding loss to follow-up. One commonly suggested method for ensuring up-to-date contact details was regular distribution of a newsletter to patients, which was seen as fulfilling several purposes.I think what is important is to follow up with patients, and I think we were pretty slow about that. We used to say, “Oh yeah, come back”, but now we really say “Come back for us to talk to you, and see what your ovarian function is like.” Because we really need to manage their fertility in the next 5 to 10 years after their treatment, and so we’re getting more proactive about that, so we’ll probably see more usage of eggs and tissue. It was also seen as important to have an active system for clinical follow-up to assess fertility and discuss potential need to use the stored material.This investigation makes an original contribution to knowledge in the opportunity it provides to learn from experts in fertility preservation. In the last 20 years, procedures for cryostorage of reproductive material to preserve potential for conception for cancer survivors have moved from being experimental to being a part of the clinical management of cancer. The insights shared by the key informants who participated in this study can be used to inform clinical practice and enhance the care and management of patients seeking fertility preservation before treatment for cancer. Learning from them is essential for what has come to be known as ‘knowledge management’ which aids the creation, transfer, and application of knowledge in an organisation .To enable patients to consider fertility preservation, it is necessary for their treating oncologists to inform them about the potential impact of treatment on fertility, discuss the available options for storing reproductive material for future use, and refer them for consultation with a fertility specialist. It is known that young women and men who are diagnosed with cancer want to be informed about the consequences of gonadotoxic treatment for future fertility and about fertility preservation options but that this is not routinely offered , 28, 38.Being informed about the option of fertility preservation and referred to a facility where this can be done is essential to allow people to make informed decisions about whether to proceed. Men interviewed about their experience of sperm banking before cancer treatment emphasized the vital role their oncologist had played in their decision to do so . HoweverAfter a diagnosis of cancer, confronting a decision to freeze reproductive material can be overwhelming, coinciding as it does with such an existential threat. Although counselling about the possible impact of cancer treatment on fertility would seem to be invaluable for women, men, and adolescents, it is not always provided , 50. InfThe informants’ views that optimum psychosocial care helps women to make informed decisions about whether to proceed with fertility preservation is supported by a study of more than 1000 women who had been diagnosed with cancer in their reproductive years. It found that women who had received pre-cancer treatment infertility counselling by a fertility specialist reported less long-term decision regret about having or not having preserved fertility than those who did not receive counselling .Informants also viewed counselling as essential for managing potentially over-optimistic expectations. They acknowledged the need to maintain the psychological benefits of hope inherent in cryopreservation, but stressed the importance of simultaneously conveying a realistic appreciation of the chance of conception from cryopreserved material. Similar cautions have been identified in relation to pregnancy and miscarriage . NeverthBecause storage time for reproductive material frozen before cancer treatment can span decades, informants made it clear that accurate records are both a necessity and more than usually difficult to gather and maintain. A regular newsletter and invitations to follow-up consultations were suggested as strategies to reduce the risk of loss to follow-up and improve the ability to monitor survival rates and fertility outcomes. Birth and death registries were identified as important sources of information, contributing evidence about the safety and benefits (or lack thereof) of fertility preservation for various cancer diagnoses. However, despite strategies to maintain contact with people who store reproductive material, there are inherent challenges in long-term storage including maintaining contact and loss to follow-up. Studies of men who had stored sperm before cancer treatment report low rates of return for semen analysis to monitor fertility despite invitations to do so  and low Informants also asserted that national and international registries of large numbers of patients with diverse cancers and treatments offer the best opportunity to generate evidence about fertility after cancer treatment and the potential benefits of fertility preservation. Informants in this study are among those establishing such a registry. The Australasian Oncofertility Registry is the world’s first web-based database collecting data from oncology and fertility centres in Australia and New Zealand [www.futurefertility.com.au]. The cumulative data in this registry will allow clinicians to provide personalised advice to patients about the potential benefits of fertility preservation options which, in turn, will help patients to make informed decisions. Cancer-specific decision aids for fertility preservation may also be helpful .Strengths of this study are that respondents included professionals from most disciplines involved in fertility preservation; the respondents’ extensive collective experience of fertility preservation; and that the semi-structured interviews allowed respondents to describe their own thoughts and experiences as members of a team caring for people who contemplate fertility preservation in the context of cancer. This study also has limitations: Respondents were drawn from two clinical services in one state in Australia and their views and experiences may be different from those of clinicians in other parts of Australia and in other countries; there were relatively few respondents; and no nurses participated.The collective knowledge of this study’s informants, who represent multidisciplinary teams with more than two decades of experience in fertility preservation, allows important insights into strategies that can be employed by ART clinics that offer fertility preservation in order to promote the integration of oncology and fertility care, the psychosocial care of patients who have to make difficult decisions at a very distressing time and under considerable time constraint, data recording and monitoring, and reporting of outcomes. Future research should include explorations of people’s experiences of fertility preservation to inform the development and testing of a measure of quality of fertility preservation care. Follow-up studies of the reproductive outcomes of people who store reproductive material before cancer treatment are also needed.


In recent years, it has become apparent that the Wnt signaling pathway, known for its essential functions in embryonic development and tissue homeostasis, exerts immunomodulatory functions during inflammation and infection. Most functional studies indicate that Wnt5a exerts pro-inflammatory functions on its cellular targets, which include various types of immune and non-immune cells. Wnt5a expression has also been linked to the pathogenesis of chronic inflammatory diseases. Activation of beta-catenin-dependent Wnt signaling, e.g., by Wnt3a, has however been shown to limit inflammation by interfering with the nuclear factor kappa-light chain-enhancer of activated B-cells (NF-kappaB) pathway. This review focuses on the regulation of Wnt5a, Wnt3a, and the recently identified Wnt6 and their functional role in bacterial infections with a primary focus on pulmonary tuberculosis, a leading infectious cause of morbidity and mortality worldwide. Mycobacteria and to the Mycobacterium tuberculosis complex, which comprises of several closely related pathogenic sub-species (Mycobacterium tuberculosis (M. tuberculosis) is a non-motile, slow growing, gram-positive bacterium with a lipid-rich cell wall. The pathogen is adapted to an intracellular lifestyle, allowing it to survive and replicate within its main host cell, the macrophage  as the leading-cause of death from infectious disease worldwide  of the immune system [recently extensively reviewed in Ref. ]. Among ividuals  that recividuals . TLR-individuals . Antigenrculosis , 23. It rculosis , which srculosis . Synergirculosis  and enabrculosis , 27. Altrculosis , 29.The Wingless/Integrase 1 (Wnt) signaling pathway, which is evolutionary highly conserved in multicellular eukaryotic animals (metazoa), regulates basic cellular processes such as proliferation apoptosis, differentiation, polarization, and motility [reviewed in Ref. –32]. ExeWith regard to the immune system, there is evidence that Wnt signaling is necessary for normal hematopoiesis . Moreove2+ pathway, and Wnt/Planar cell polarity pathway [reviewed in Ref.  are present in lung biopsies of TB-patients, that mycobacteria induce Wnt5a in macrophages in a TLR-NF-kappaB dependent manner, and that Wnt5a exerts a distinct immunomodulatory function on immune cells . This obmegmatis . Notablymegmatis . In addiebrafish , demonstM. tuberculosis-induced expression of Wnt proteins in macrophages.Beside the abovementioned TLRs and dectin-1, a whole variety of PRRs contribute to host cell activation by pathogenic mycobacteria and were shown to be involved in mounting an anti-mycobacterial response. This includes the macrophage inducible C-type lectin (MINCLE), macrophage scavenger receptor A (SRA), macrophage receptor with collagenous structure (MARCO), and the intracellularly located nucleotide-binding oligomerization domain-containing protein 2 (NOD2), NLR family, pyrin domain-containing 3 (NLRP3), as well as the dsDNA sensor cyclic GMP-AMP synthase . It is c2+ pathway , and Calcineurin. Independent laboratories have shown that there is an intense crosstalk between the Wnt5a-induced signaling cascade and the NF-kappaB as well as certain mitogen-activated protein kinase pathways (purified protein derivative of M. tuberculosis (PPD) reactive healthy donors is altered by blocking Wnt5a or its putative receptor Fzd5   . Moreove1 (Fzd1) . In a vanditions –86. Due nditions , we hypoal cells . Along tal cells .M. tuberculosis) are often associated with severe tissue damage. This leads to the release of various intracellular factors such as adenosin triphosphate (ATP), heat shock proteins, mitochondrial components and several alarmins, a group of proteins which include high-mobility group box 1 protein, IL-1α, IL-33, and the Ca2+-binding S100 proteins [reviewed in Ref.  exhibited a decrease in levels of Wnt3a, Fzd1, phosphorylated GSK3β at Ser9, total β-catenin, and nuclear beta-catenin, demonstrating that major constituents of the Wnt3a signaling machinery in the lung were downregulated. In contrast, Wnt5a, Fzd5, total CaMKII, and phosphorylated CaMKII were upregulated, indicating that Wnt5a dependent, pro-inflammatory Camic rats . These fmic rats . These dmic rats , 86, andmic rats . On a cemic rats , 80, butmic rats . Furthermic rats . During mic rats  during wmic rats .M. tuberculosis infection  c-Myc is described as a well-known Wnt/beta-catenin-dependent target gene get gene , 102. Reget gene , showingeceptors . Moreoveeceptors . Only Wneceptors , 105, wheceptors , downstrSo far, Wnt6 was described in the context of early embryogenesis , 107, duSalmonella infection induces Wnt6 expression in intestinal epithelial cells  representing another gram-negative bacterium which exploits Wnt/beta-catenin signaling during infection. These bacteria disturb epithelial tissue homeostasis in fallopian tubes. The authors demonstrate that acute Ctr infection activates the paracrine Wnt signaling pathway, leading to profound disruption of epithelial cell structure and function, which facilitates the dissemination of damage beyond that of infected cells  infection . In additaplasia . With retaplasia . Based oM. tuberculosis infection is regulated by members of the Wnt signaling cascade. And still new factors and related functions are being identified. A simplified view on the systemic expression and functional role of each of the 19 individual Wnts in M. tuberculosis infection is currently not possible, since to our understanding local determinants define either the induction or repression of a given Wnt signaling factor and its related functions. The detailed characterization of the underlying signaling pathways and the identification of molecular interactions will lead to better understanding of the disease mechanisms and may even lead to the identification of new target structures in order to develop a shorter and more efficient therapy of pulmonary TB.Pulmonary TB is one of the most complex infectious diseases known, characterized by severe chronic inflammation being important for bacterial growth restriction but also being responsible for severe immunopathology. In the last 10 years, it has become clear that the immune function during All the authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Now, the transceptor concept is further enlarged to includemicronutrient sensing via the iron and zinc high-affinity transporters ofSaccharomyces cerevisiae. Interestingly, what seems tounderline the transport and/or sensing function of receptors, transporters andtransceptors is ligand-induced conformational alterations recognized bydownstream intracellular effectors.Cells need to communicate with their environment in order to obtain nutrients,grow, divide and respond to signals related to adaptation in changingphysiological conditions or stress. A very basic question in biology is howcells, especially of those organisms living in rapidly changing habitats, sensetheir environment. Apparently, this question is of particular importance to allfree-living microorganisms. The critical role of receptors, transporters andchannels, transmembrane proteins located in the plasma membrane of all types ofcells, in signaling environmental changes is well established. A relativenewcomer in environment sensing are the so called  Well-studied cases ofenvironmental sensing via typical receptors concern pH 4bona fidae receptors, transportersand channels mediate the uptake of solutes, metabolites, drugs or ions, whichthemselves can act as molecular signals, once intracellularly accumulated. Aprominent case of transport-mediated signaling is described in filamentousascomycetes, particularly in Aspergillus nidulans, where dormantconidiospores transferred to a fresh medium sense their new environment viatransient transcriptional activation of most of their nutrient-related transportersand subsequently adapt their gene expression to the nutrients available Three well-known types of molecular machines are employed for the sensing of theenvironment. Receptors, transporters and channels. All are polytopic transmembraneproteins, monomeric or oligomeric, located in the plasma membrane of all types ofcells. Their importance is reflected by the fact that 10-15% of all genomes encodefor such membrane proteins and by the numerous genetic diseases related to theirmalfunction. Receptors are proteins that interact with an external chemical ligand,which could be any simple molecule, including nutrients, ions, hormones or drugs andxenobiotics. Ligand binding induces a receptor conformational change, which acts asthe primary molecular signal leading to two alternative downstream outcomes. In mostcases, ligand-binding leads to the transduction of the receptor conformationalchange, th Small Meeting of Yeast Transport andEnergetics (SMYTE) held in Cordoba (Spain), Johan Thevelein presented evidence for anew type of sensing mechanism, operating however, through known transporters. Inparticular, he presented evidence that the general amino acid permease Gap1 ofSaccharomyces cerevisiae is necessary for cAMP-independentactivation of the PKA (Protein Kinase A) pathway under conditions of re-addition ofamino acids to cells previously starved for amino acids transceptor (transport andreceptor), and became the founder member ofall transporters classified as transceptors today (see later). The basic originalidea on the role of Gap1 in PKA activation was that signaling to the PKA pathwaydoes not depend on the transport activity of Gap1. In other words, the role of Gap1in activating PKA was independent from its role in amino acid transport. Theevidence that Gap1 acts as a sensor was basically genetic. Strains carrying null orspecific mutations in GAP1 showed no PKA activation or distincteffects on transport and signaling, although these mutants could still transporthigh concentrations of amino acids that are Gap1 substrates via other specificpermeases.In 1999, in the course of the 14Microbial Cell, Thevelein and his colleagues further expand thefamily of transceptors by providing evidence that the iron (Ftr1) and zinc (Zrt1)high-affinity transporters can also signal PKA activation In the years to follow, Thevelein’s group has provided additional evidence supportingthe concept of transceptors 1011121314In summary, highlights of the evidence supporting the idea of transceptors include a)maintenance of signaling in specific transceptor transport-deficient versions, bestexemplified by mutations in residues tentatively involved in proton-coupling in theSul1 transceptor, b) loss of signaling in null mutants of transceptors despiteaccumulation of the relevant substrates by other transporters with similarspecificity, c) identification of competitive and non-competitive inhibitors ofGap1-mediated transport, either with or without agonist action for signaling,including non-transported agonists, d) identification of analogues with distincteffects on Gap1 transport, transporter ubiquitination, endocytosis and signaling(transporter ubiquitination and endocytic turnover in response to substrate excessis a common phenomenon discussed below), and e) involvement of highly induciblescavenging transporters, as expected for proteins acting in signaling.Overall, these results further support the hypothesis that different substrates, orsubstrate analogues, or ligands, bind to partially overlapping binding sites in thesame general substrate-binding pocket of transceptors, triggering divergentconformations, resulting in distinct downstream processes, including ubiquitination,endocytic turnover or signaling A. nidulans or in the Can1arginine transporter in S. cerevisiae) that are active, or evenhyperactive, but still not endocytosed, and some inactive mutants that areendocytosed 18181919In my opinion, the idea of transceptors has gained unexpected conceptual support fromstudies addressing the phenomenon of activity-dependent transporter endocytosis. Infungi and mammalian cells many transporters are down-regulated in response to excesssubstrates by a mechanism involving ubiquitination, followed by immediate endocyticinternalization and subsequent sorting in early endosomes and degradation in thevacuole/lysosome. Importantly, substrate-elicited transporter endocytosis, unlikeendocytosis in response to other signals, such as stress or changes in N or C sourcesupply, is activity-dependent and in most cases requires several transport cycles tobe completed. This conclusion is mostly based on the observation that inactivetransporters are, in most cases, not internalized in the presence of substrates. Inline with the idea of the necessity of full transport cycles to be completed inorder to allow endocytosis to occur, is the observation that substrate analoguesthat act as non-transported ligands do not elicit internalization. However, thereare some exceptions to this rule. For example there are specific transporter mutants.As proposed in signaling through transceptors, transporter activity is thusassociated with a distinct mechanism, endocytosis, via specific conformationalchanges accompanying transport catalysis. In the case of transporters,conformational changes are ‘read’ by Rsp5/HulA/Nedd4 ubiquitin ligase adaptors(α-arrestins), which promote their internalization and turnover. In the case oftransceptors, unknown effectors transduce conformational changes into the PKAsignaling pathway. It thus seems that the cell has the capacity to sense subtleconformational nanochanges in transporters/transceptors, while they function at itsplasma membrane. What is really fascinating about the discovery and the generalconcept of transceptors is that transceptors seem to provide a functionalintermediate link in the apparent evolution of proper receptors from transportersNeurospora crassa andUstilago maydis), plants , protozoa (Leishmania mexicana) and in humancells  and especially with low-affinity substrates. A different butcomplementary approach to direct uptake measurements approach is the use of growthtests in cases where the uptake of a substrate leads to a growth phenotype. Forexample, a strain of S. cerevisiae or A. nidulansexpressing a transporter specific for adenine will grow well in growth mediacontaining this purine as a sole nitrogen source, while it will also be sensitive toadenine toxic analogues, such 8-azaadenine. In contrast, inactivation of the sametransporter will lead to absence of growth on adenine media and resistance to8-azaadenine. Importantly, if similar growth tests are performed with a transporterover-expressed via a strong promoter, they can even allow the detection oftransporters or transport mutants possessing very low activities. In respect to theidentification of substrate analogues that only act as ligands, but still inducetransporter-mediated signaling, rigorous evidence should be obtained that asubstrate analogue is not accumulated, even at very low concentrations, in the cell,by another transporter, or even by the same transporter acting in signaling.In bacteria, yeast, filamentous fungi and other microorganisms, transport activitycan be measured directly using radiolabeled substrates in living cells, a rigorousapproach as the transporter activity is measured in its proper lipid environment.The only limiting factor in this approach might be the simultaneous expression ofseveral transporters with similar or overlapping specificities and/or transportkinetics with the transporter studied. This apparent drawback is however overcomewhen a specific transporter can be studied in a genetically ‘clean’ backgroundlacking, due to null mutations, transporters with similar specificities, as is thecase in the model fungi cis-acting elements arenecessary for signaling in different transceptors? Are the extended cytoplasmicterminal regions of some transceptors  sufficient or critical forsignaling, as it is in proper receptors (e.g. the Ssy1) or more complex synergisticintermolecular interactions determine signaling? And of course, which are thedownstream effectors mediating transceptor signaling? Designing the proper geneticand unbiased screens in model fungi might lead to some answers to thesequestions.Several fascinating questions remain to be answered in respect to the mechanism oftransceptor function. What distinguishes a transporter that acts solely in transportfrom a transporter that also acts as a receptor? Is this difference intrinsicallybased on structural determinants, or is it also a matter of transporter/transceptorlocalization in specific PM microdomains, and thus differentially accessible todownstream cellular effectors? Which


To gain a better understanding of the role of Pcgf1 in transcriptional control of differentiation, we analysed mRNA profiles from Pcgf1 deficient cells using RNA-seq. Interestingly, we found that Pcgf1 positively regulated expression of essential transcription factors involved in ectoderm and mesoderm differentiation, revealing an unexpected function of Pcgf1 in gene activation during ES cell lineage specification. Chromatin immunoprecipitation experiments demonstrated that Pcgf1 deletion caused a decrease in Ring1B and its associated H2AK119ub1 mark binding to target genes. Altogether, our results suggested an unexpected function of Pcgf1 in gene activation during ES cell maintenance.The Polycomb repressive complex 1 (PRC1) is essential for fate decisions of embryonic stem (ES) cells. Emerging evidence suggests that six major variants of PRC1 complex, defined by the mutually exclusive presence of Pcgf subunit, regulate distinct biological processes, yet very little is known about the mechanism by which each version of PRC1 instructs and maintains cell fate. Here, we disrupted the Pcgf1, also known as Nspc1 and one of six Pcgf paralogs, in mouse ES cells by the CRISPR/Cas9 technology. We showed that although these mutant cells were viable and retained normal self-renewal, they displayed severe defects in differentiation  Polycomb group (PcG) proteins are known as epigenetic chromatin modifiers that regulate gene expression in multiple cell types and tissue contexts and are critical for cell fate decisions and development1571011Pcgf1 is also known as Nspc1 which is mainly expressed in nervous system1820Here, we use CRISPR/Cas9 strategy to establish Pcgf1 null ES cells. Although the deletion of Pcgf1 does not impair ES cell proliferation and EB formation, mutant cells display profound defects in differentiation. Importantly, RNA-seq analysis reveals that Pcgf1 plays an unexpected role in transcriptional activation, in contrast to the repressive role of canonical PRC1. Mechanistically, this process is initiated by Pcgf1-mediated noncanonical PRC1.1 complex assembly.−/− zebra fishPcgf1 gene and cloned them into the PX330 vector which encodes the Cas9 nuclease  amplification. Sanger sequencing of the truncated transcript showed the deletion of 259 bp in Pcgf1 mRNA and also introduction of a frame shift to the truncated coding sequence  in hanging drops at the first three days and subsequently maintained them in rotating conditions in the absence of LIF  were markedly downregulated in EBs derived from Pcgf1 knockout ES cells . Together, these criteria revealed a set of 1929 Pcgf1 target genes. 1491 (77%) genes were downregulated in the absence of Pcgf1 while only 438 genes (23%) were upregulated  and PRC2 subunit Suz12, as well as their associated H2AK119ub1 and H3K27me3, were enriched at these 6 targets. The enrichment of BcoR, Ring1B and Suz12 were greatly reduced in Pcgf1−/− ES cells. In contrast, deletion of Pcgf1 did not alter the binding of Rybp  and PRC2 core subunit Suz12 was performed on these targets. Additionally, ChIP-qPCR was done on the Pcgf1 of Rybp . Notablyin vitro  and the absence of transcriptional activation of lineage marker genes during differentiation in the same cells. Accordingly, our results indicate that Pcgf1 and Kdm2b share common biological functions. Moreover, Kdm2b knockdown in ES cells also demonstrates a critical function of Kdm2b in recruiting PRC1 to CpG Islands of developmental regulators19Although the Pcgf1-deleted ES cells do not show detectable proliferation defects and form EBs with an efficiency similar to that of wild-type cells, mutant cells exhibit severe defects in differentiation in vitro that ES cells deficient in Pcgf1 display severe defects in ectoderm and mesoderm differentiation. Additionally, Pcgf1 has been involved in the proliferation and differentiation of tumor cellsin vivo contributions of Pcgf1 during early development, homeostasis of the tissue and tumorigenesis.We observed 2.ES cells were co-cultured with mitomycin-inactivated murine embryonic fibroblasts (MEFs) on gelatinized tissue culture plates in DMEM (Gibico) supplemented with 15% fetal calf serum (Gibico), non-essential amino acids (Gibico), leukemia inhibitory factor (LIF), penicillin/streptomycin (Sunshine Biotechnology), L-glutamine (Sunshine Biotechnology) and 0.1 mM β-mercaptoethanol (sigma) as described−/− ES cells were generated by Cas9 technology as describedhttp://crispr.mit.edu/). SgRNAs were cloned into the pX330-U6-Chimeric-BB-CBh-hSpCas9  vector. The sgRNA expression constructs were verified by sequencing. SgRNA-Cas9 vectors were co-transfected with a plasmid encoding puromycin (Puro) resistance into ES cells. After 24 hours, ES cells were treated with puromycin for 48 hours and then seeded on MEF feeder to form single colony. The Pcgf1−/− ES cell colonies were identified via DNA-PCR, RT-PCR and Western blot.Pcgf1−/− ES cells with polybrene . Puromycin was used to screen positive Pcgf1−/− rescued ES cell line and the FLAG-tagged Pcgf1 expression levels were examined by Western Blot. The primers used for PCR are shown in The Pcgf1 full-length cDNA (NM_197992) was modified by adding N-terminal Flag-tag (DYKDDDDK) sequence into the foward PCR-primer, followed by cloning into pBluescript KS (-). The complete coding sequence was verified by sequencing. The correct inserts were cloned into lentiviral vectorES cell cultures were fixed with 4% PFA (Solarbio) and stained for alkaline phosphatase activity using an Alkaline Phosphatase Stain Kit (Yeasen) according to the manufacturer’s instructions.ES cells were trypsinized, washed three times with PBS, fixed in ice-cold 75% ethanol  for 30 minutes, and stored at −20 °C for at least 4 hours. Subsequently, cells were washed twice with PBS, harvested and incubated for 30 min at 37 °C with RNase A , and stained with the propidium iodine (20 μg /ml) protected from light for 60 min at 37 °C followed by analysis on a FACS LSRFortessa (BD Biosciences) as described2). Three days later, EBs were harvested and cultured on a rotating shaker . Fresh medium was replaced every 2 days to avoid medium exhaustion. Total RNA was collected from day 3, 7 and 12  and analysed by RT-qPCR.ES cells were trypsinized and resuspended in medium without LIF® Green Master Mix (Invitrogen) on a StepOneTM Software v2.3 (Applied Biosystems). The relative expression of genes was analysed based on the 2−∆∆Ct method using the Actin gene as a control. The primers used for RT-qPCR are shown in Total RNA collected from ES cells and EBs was purified using Trizol (Invitrogen). RNA was reverse transcribed into cDNA with oligodT or random primers using the HiScriptTM 1st Strand cDNA Synthesis Kit (Vazyme Biotech). Quantitative real-time PCR (RT-qPCR) was performed using PowerUp™ SYBR2 and 0.5 mM β-mercaptoethanol, 10 mg/ml PMSF, Protease Inhibitor Mix (Sigma)). Nuclear Extracts were prepared from nuclei using lysis buffer . Nuclear extracts from the Pcgf1 Flag-tagged ES cells were incubated with M2 agarose beads , in binding buffer  and 1/3 volume nuclei lysis buffer) overnight at 4 °C. The beads were washed 3 times with washing buffer  and subsequently the bound proteins were dissolved in gel loading buffer. Total proteins were separated by SDS-PAGE. The proteins were transferred on polyvinylidene fluoride (PVDF) membrane and the membrane was blocked with 5% (w/v) non-fat milk for one hour at room temperature and then incubated overnight at 4 °C with antibodies against Pcgf1 , Flag , BcoR , Rybp , Ring1B , Nanog , Oct4 , Sox2 , Pcgf5 , β-Actin , Suz12 , H2AK119ub1 , H3K27me3 , H3 . Then, the membrane was incubated with a horseradish peroxidase-conjugated goat anti-mouse lgG-HRP , goat anti-rabbit lgG-HRP  for 1 h. Chemiluminescence was detected using the ECL blot detection system. Histone Extraction was performed as describedES cells were harvested and lysed in hypotonic lysis buffer  according to the manufacturer’s protocol. The preparation of whole RNA-seq libraries and deep sequencing were performed by the Annoroad Gene Technology Corporation . RNA integrity number (RIN) and the concentration were measured using a 2100 RNA Nano 6000 Assay Kit . The mRNA was enriched with Oligo (dT) mRNA magnetic beads. RNA-seq libraries were prepared using 6 bp random primers and libraries were sequenced on the IlluminaHiSeq X-Ten with 150 bp paired-end reads. RNA-seq reads were mapped to the mouse genome (mm10) using TopHat v2.0.12. Reads per Kilobase Millon Mapped Reads (RPKM) were used to quantitatively estimate gene expression values7 ES cells were cross-linked with formaldehyde (37%) to a final concentration of 1% for 10 min at room temperature−ΔCt, where ΔCt = Ct (ChIP) − Ct (Input). The primers used for ChIP-qPCR are shown in ChIP was performed essentially as describedHow to cite this article: Yan, Y. et al. Loss of Polycomb Group Protein Pcgf1 Severely Compromises Proper Differentiation of Embryonic Stem Cells. Sci. Rep.7, 46276; doi: 10.1038/srep46276 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing.CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions.Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate < 1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes Despite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.The online version of this article (doi:10.1186/s12864-017-3673-y) contains supplementary material, which is available to authorized users. Mammalian infertility is of concern to both human couples seeking to establish a family and also in the dairy industry to meet production demand. In couples seeking assisted reproductive technology, male infertility accounts for 40% of all couples’ infertility diagnosis , and it PLCZ1 and CRISP2, both of which have roles in fertilization. The authors concluded that full-length transcripts within transcriptionally inactive sperm could plausibly be translated after spermatogenesis to have roles in the early development of the embryo.The genetic contribution of sperm in relation to fertility has been of recent interest across mammalian species. It is now well understood that, at the time of fertilization, the spermatozoa delivers more than just paternal DNA, but rather an entire package including RNAs, transcription factors, and cell signaling molecules . Indeed,Several studies have sought to characterize the differences in sperm RNA between males of differing fertility –15. In hSire fertility has been evaluated in terms of physical quality parameters, including motility and morphology as well as the RNA profile associated with a given fertility index. While the sperm transcriptome has been characterized across sire fertility indices, previous studies have not determined whether the embryonic transcriptome is influenced by the “RNA package” delivered by sires of differing fertility status at the time of fertilization. We hypothesized that bulls of differing fertility will have different DNA methylation signatures and will affect not only the development of the early preimplantation embryo, but also the transcriptome of the embryo. Here, we first aimed to assess whether embryo morphology and development, in terms of fertilization and blastocyst rate, differed between embryos derived from high and low fertility sires. The second aim was to characterize the embryonic transcriptome of embryos derived from high and low fertility sires to determine whether the sire’s fertility has a genetic effect on the embryo and to potentially identify differentially expressed genes. The third aim was to characterize DNA methylation signatures of bulls differing in their fertility status. Utilizing an in vitro fertilization (IVF) system, embryos were generated from either a high or low fertility sire allowing for the analysis of the paternal influence on the embryonic transcriptome. Understanding how the preimplantation embryonic transcriptome may be impacted by paternal factors could facilitate the identification of paternal RNAs, microRNAs and transcription factors that drive embryonic development. These factors attributed to the paternal genome may be implicated in “differential fetal programming”, and could serve as biomarkers of bull fertility.https://aipl.arsusda.gov/reference/arr-scr1.htm). Semen from 12 bulls was donated by Genex Cooperative, Inc., where six bulls were deemed as high fertility bulls and six were deemed as low fertility bulls. The bulls selected represent the extreme sires for the SCR measure within the company’s marketed Holstein sire pool. The measure of SCR and corresponding percent accuracies were as follows: high fertility sires were 5 (97%), 4.1 (93%), 3.9 (95%), 3.8 (95%), 3.7 (97%), 3.4 (99%) whereas the low fertility sires were -2.3 (81%), -2.7 (72%), -3.8 (94%), -3.9 (86%), -5.3 (78%), -7.5 (90%).Sires were chosen based on extreme SCR which is defined as a percent increase or decrease in conception rate for a given sire relative to the herd’s average. The SCR is an evaluation performed on bulls with greater than 300 mating records within the last 4 years across a minimum of 10 herds  supplemented with 0.22 mM sodium pyruvate, 25 μg/mL gentamicin sulfate and 6 mg/ml essentially fatty acid-free bovine serum albumin . It is important to note that prior to fertilization, COCs from each maturation culture plate were divided between two fertilization culture plates in order to randomize the oocyte population prior to fertilization. These fertilization plates were then fertilized with frozen-thawed semen by either a high fertility or low SCR bull, where a total of 150–350 oocytes per bull were fertilized per IVF replicate. Semen was prepared using a Percoll discontinuous gradient as described by Parrish et al. , and adjwww.r-project.org/) using mixed models taking into account the IVF replicate and bull effect. The significance of the bull effect was tested using a likelihood ratio test comparing the full model against a model without the treatment effect, analyzing the response variables fertilization and blastocyst rates.Statistical analysis of development data was performed in the program R (n = 46–63) using the RNaqueous Micro-Kit  and then underwent one round of linear amplification using the MessageAmp II aRNA amplification kit (Life Technologies). Samples were quantified and quality checked using a Qubit® 2.0 Fluorometer (Life Technologies) and Agilent 2100 Bioanalyzer , respectively.Total RNA was extracted from each pool of blastocysts  following the recommended protocol for the kit. Libraries of cDNA were then quantified and quality checked using a Qubit® 2.0 Fluorometer (Life Technologies) and Agilent 2100 Bioanalyzer (Agilent), respectively. Libraries were then sequenced using an Illumina HiSeq 2000 at the University of Wisconsin-Madison Biotechnology Center.Data analysis was performed by the University of Wisconsin-Madison Biotechnology Center. Sequencing reads were trimmed to remove sequencing adaptors and low-quality bases and were then aligned to the bovine reference genome UMD 3.1 utilizing the default parameters of the alignment software STAR v2.4.0j . Quantifn = 14–54 blastocysts) using an RNaqueous Micro-Kit- (Life Technologies) and cDNA was generated using an iScript cDNA synthesis kit . Equal amounts of cDNA from each pool of blastocysts were used to generate a pool of cDNA representative of embryos derived from high fertility or low fertility sires. Primers for qRT-PCR reactions were designed to span exon-exon junctions to minimize amplification of genomic DNA, where the sequences are listed in Additional file GAPDH), was used as in our previous work [-ΔΔCt method by Livak and Schmittgen [To confirm the differential expression results obtained by RNA-Seq, gene expression was assessed by qRT-PCR in three additional pairs of high/low SCR derived embryo pools. Total RNA was extracted from each embryo pool  was designed by and purchased from Exiqon, Inc. . Embryos were generated by IVF as aforementioned. At the time point in which the presumptive zygotes were placed into culture media, either 1 μM gapmer, 5 μM gapmer, or water  was supplemented to the medium. On day 8 of development, fertilization rate and blastocyst rate were assessed for each of the gapmer supplemented experimental groups as well as the control. Blastocysts were pooled and collected for each experimental group. To assess gene expression following supplementation, total RNA was extracted, cDNA was generated, and qRT-PCR was carried out utilizing the same methodology as described above for gene expression validation. Statistical analysis was performed using the program OriginLab  in which a paired t-test was used to compare the ΔCt values between blastocyst samples for each gene.As a proof-of-concept, the effect of a differentially expressed gene on embryonic development was assessed using an antisense oligonucleotide, gapmer, supplemented to the culture medium of presumptive zygotes. Gapmer supplementation to media is an effective means to reduce gene expression as cells effectively take up the antisense oligonucleotide and specifically target an mRNA for degradation –29. The n = 2 bulls per pool, with the exception of 1 high pool of n = 1 bull).DNA was extracted using a phenol:choloroform extraction method . Extract-ΔΔCt method; which compares enrichment values of a positive (TGFB3) to a negative (MON2) primer pair, between experimental and input DNA samples.To capture differences in methylated regions, a methyl binding domain capture assay combined with next generation sequencing method was employed. A MethylCap kit  was used to purify methylated DNA based on high-affinity binding of methyl domain binding proteins. In brief, DNA was dissolved in GenDNA TE to a concentration of 0.1 μg/μl. DNA was then cut into 300–500 base pair fragments using a Bioruptor® sonicator (Diagenode) and was then run on an agarose gel to confirm the presence and size of the fragmented DNA. Fragmented DNA was captured per kit recommendation using magnetic beads to wash unbound DNA followed by elution. Eluted DNA was purified using a MiniElute PCR Purification kit . To assess the enrichment of methylated DNA, qRT-PCR was used where duplicates of each sample were tested using the iCycler iQ PCR system . The relative fold enrichment levels were calculated following the 2To prepare the sequencing libraries, fragmented DNA was end repaired using a NEBNext® End Repair Module  followed by addition of a 3’A to the repaired end of DNA using DNA Polymerase I, Large (Klenow) Fragment (NEB). Paired Solexa adaptors were ligated to the repaired ends of DNA by T4 ligase . The DNA was loaded onto an agarose gel, and DNA fragments containing adaptors were selected that were between 200 and 500 bp in size. PCR of the selected DNA fragments was performed using Phusion® Hot Start High-Fidelity DNA Polymerase (NEB) to enrich the purified DNA. The library DNA was quality checked and then quantified using a Qubit Fluorometer (Life Technologies). Cluster generation and sequencing were then performed using a Solexa 1G Genome Analyzer  following the manufacturer’s recommendations.http://genome.ucsc.edu). For the alignment process, Bowtie  was employed [FASTQ sequence files were examined for quality assurance. After a satisfactory quality confirmation, files were aligned to the bosTau6 (Bos_taurus_UMD_3.1) reference genome obtained from the UCSC browser  . The sofDNA was extracted as described above from an additional semen straw for each high and low SCR bull. DNA was pooled for high and low fertility bulls, respectively, and each pool was bisulfite converted utilizing an EZ DNA Methylation-Lightning™ kit . As per kit recommendation, a total of 500 ng of DNA per pool were used as input for bisulfite conversion. The bisulfite converted DNA was amplified by PCR for 35 cycles using primers listed in Additional file P > 0.05) between all SCR sires as 70.28% of the oocytes fertilized with a high SCR bull cleaved and 72.74% of the oocytes cleaved following fertilization with a low SCR bull. Similarly, the blastocyst rate or the percentage of cleaved embryos that developed to the blastocyst stage was not significantly different between high and low SCR bulls as the rates were 29.41% and 27.01%, respectively. Notably, the blastocysts derived from high and low SCR bulls were of similar morphology. Blastocysts of stage 7, grades 1 and 2 as morphologically described by Bo and Mapletoft [To assess whether morphology and development are different between embryos generated from high and low fertility sires, IVF was carried out in two biological replicates for each high and low bull pair for a total of six pairs. Herein, a bull pair will refer to one IVF replicate in which oocytes were randomly split and fertilized with either a high or low SCR sire. In terms of preimplantation embryonic development, embryos that were fertilized with either a high or low SCR sire did not differ in fertilization rate or blastocyst rate Table . The cleapletoft  were colGiven that morphology and development rates were similar between embryos produced from high and low fertility bulls, it was intriguing to test whether or not the transcriptomic profiles of these embryos were different. The embryonic transcriptome was profiled at the blastocyst stage through RNA-Seq. A summary of the read alignments is illustrated in Table CYCS, TFB2M, MEPCE, EEA1, and SLC16A7 was assessed by qRT-PCR  which flank DNA monomers specific to a target mRNA of interest [P < 0.001), which was about 70% of the control embryos (Table P < 0.05) in comparison to control non-supplemented zygotes . Overall, a higher degree of methylation was observed for spermatozoa of high fertility sires as evident by the greater number of methylated binding domains.MBD-Sequencing (MBD-Seq) was performed for three pools derived from high fertility spermatozoa and three pools derived from low fertility spermatozoa, where To determine whether the captured methylated regions differed between high and low fertility sires, analysis of DMRs was performed. The DMRs width ranged from 250 to 3423 bp, with a mean of 521.97 ± 408.52 bp. The DMRs were distributed across 23 chromosomes in which chromosome 5 had the largest number of DMRs (8/76 or 10.5% of the DMRs) and 7 DMRs were unmapped to a specific chromosome . Comparatively, the CHR19 DMR had a significantly higher level of methylated CpG sites for sperm DNA of lower fertility sires with 34.0% methylated CpG sites, whereas higher fertility sires exhibited 20.8% methylated CpG sites within this DMR (Table P < 0.0001). Overall, the MBD-Seq results were validated for both DMRs, thus confirming the observation that sperm DNA of high and low fertility sires differ in their epigenetic signature.To validate the MBD-Seq results, methylation was assessed by bisulfite conversion of DNA followed by Sanger sequencing of two DMRs identified by MBD-Seq analysis. DMRs identified on chromosome 12 (CHR12) and chromosome 19 (CHR19) were selected as they represent DMRs that were highly methylated in high fertility bulls and highly methylated in low fertility bulls, respectively. Table Reproductive performance of sires varies greatly in mammals. However, the influence and roles of the paternal genetic component on embryonic development are not well understood. We hypothesized that sires of differing fertility have different epigenetic signatures that affect not only embryonic development, but also the embryonic transcriptome. This study revealed that sire field fertility status did not affect preimplantation embryonic development in terms of both fertilization and blastocyst rate. In contrast, embryos derived from either a high or low fertility sire that were of similar morphology by day 8 of development displayed significant transcriptomic profiles.Several semen quality parameters such as morphology, motility, and binding ability have been evaluated with limited success for in vitro prediction of sire fertility . FertiliMEPCE) gene catalyzes the addition of a methyl phosphate cap to 7sk snRNA, a gene that participates in transcription regulation at the transition from initiation to elongation [TFB2M) gene is a mitochondrial transcription factor [CYCS), which codes a heme protein that participates in electron transfer within the mitochondrial electron transport chain in addition to promoting apoptosis through activation of Caspase 9 [TFB2M and CYCS, and also NDUFA1 [SFXN4 [Despite similar embryonic development and morphology, RNA-Seq revealed significantly different transcriptomic profiles of embryos derived from differing fertility sires. Many differentially expressed genes were more highly expressed in embryos derived from high fertility sires and functionally have roles in metabolic processes and catalytic activities. For example, the methyl phosphate capping enzyme , was previously detected in mouse preimplantation development where it acts to shuttle lactate and also plays a role in regulating redox in the early mouse embryo [SLC16A7 become upregulated during oxidative stress [GADD45A) gene also indicates stress as genes within the GADD45 family are stress sensors with roles in DNA repair, cell cycle regulation, and apoptosis as well as DNA demethylation [Several genes more highly expressed in embryos from low fertility sires may explain poorer development beyond the blastocyst stage. The expression of solute carrier family 16 (monocarboxylate transporter), member 7 . Further characterization of sperm-derived factors, such as microRNAs, should, therefore, be explored as potential contributors to embryonic reprogramming and may be developed as biomarkers of reproductive performance.As a proof-of-concept that the differentially expressed genes identified are important to embryonic development, expression knockdown of of mtDNA . As the of mtDNA . Gapmer iR-2284x , which tWhile the transcriptomic landscape depicts embryonic differences influenced by the sire, it is still unclear why or how sire field fertility is correlated with the embryonic transcriptome. Multiple components may attribute to the differences observed within the embryonic transcriptome, including the impact of paternal allelic variation as well as the delivery of paternal factors at the time of fertilization. Several studies have indicated that the “RNA package” delivered at the time of fertilization is strikingly different between bulls of differing fertility , 13–15. The investigation into the epigenetic signature of the sperm between the high and low fertility sires, revealed 76 regions to be differentially methylated between sires of differing fertility status. Similarly, a study by Verma et al.  reportedKCK4 gene, a member a two-pore domain potassium (K2P) channel family [KCK4 localizes to the equatorial region of acrosome reacted sperm, and that inhibitor of the K2P channels reduces not only fertilization but also development of bovine and mouse embryos in vitro. Interestingly, a DMR was located in the CTCF gene, which has been associated with spermatogenesis and male fertility [CTCF gene plays a critical role in genome-wide gene regulation and has roles in epigenetic reprogramming, gametogenesis and embryo development and is also associated with fertility, as reviewed by Franco et al. [CTCF gene had smaller testis and spermatogenesis was impacted as several males were infertile. Moreover, spermatozoa of the CTCF-conditional knock-out mice also demonstrated aberrant histone retention and disrupted chromatin compaction. While the functions of the DMRs identified within the present study remain to be elucidated, several of the DMRs identified are within genes associated with roles in spermatogenesis, fertility, and embryonic development.Several DMRs identified between high and low fertility sires are located within genes with functional roles in spermatogenesis and fertilization that may underpin the differences in field fertility. For instance, a study by Ferrer et al.  found thl family , where pl family . In bulll family  reportedertility , 71. Theo et al. . Indeed,It is well understood that during the process of spermatogenesis the chromatin structure undergoes drastic remodeling by replacing histones with protamines to achieve a high condensation of DNA. Also, sites, where histones are retained, are within key developmental gene regions . Errors Male fertility had received less attention in comparison to female fertility, yet it has been demonstrated that the male gamete contributes not only DNA but also RNA and signaling factors to the oocyte at fertilization. The present study identified transcriptomic differences within embryos derived from bulls of differing fertility status at the preimplantation stage of development. While transcriptomic differences within the embryos were observed at the preimplantation stage, the association between male fertility and embryonic development following embryo transfer should be investigated in the future. In addition, it is vital to explore whether DNA condensation and integrity as well as alterations in epigenetic signatures within the spermatozoa contribute to sire fertility and its effect on embryo development.Additional file 1: Table S1.Primer sequences utilized for qRT-PCR gene expression analysis to validate the RNA-Seq results. (DOC 33 kb)Additional file 2: Table S2.Primer sequences for PCR of bisulfite-converted DNA. (DOC 29 kb)Additional file 3: Table S3.Genomic distribution of aligned reads in embryos derived from high and low fertility bulls. Represented as a percentage of the total mapped reads. (DOC 27 kb)Additional file 4: Table S4.Differentially expressed genes between embryos derived from high and low fertility sires (FDR <5%). (DOCX 27 kb)Additional file 5: Figure S1.Distribution of the DMRs across chromosomes. The histogram represents the number of DMRs located on each chromosome. unk: represents regions with an unknown location as they do not map to a chromosome. (PDF 34 kb)Additional file 6: Table S5.Differentially Methylated Regions between high and low SCR sires (FDR <10%). (XLS 40 kb)Additional file 7: Figure S2.Principal component analysis for high and low fertility pools. (PDF 21 kb)