Tribbles (TRIB) proteins, a family of evolutionary conserved psuedokinase proteins, modulate various signalling pathways within the cell. The regulatory roles of TRIB make them an important part of a number of biological processes ranging from cell proliferation to metabolism, immunity, inflammation and carcinogenesis. Innate immune system plays a pivotal role during the regulation of reproductive processes that allows successful creation of an offspring. Its involvement initiates from fertilization of the oocyte by spermatozoon and lasts throughout early embryonic development, pregnancy and labour. Therefore, there is a close cooperation between the reproductive system and the innate immune system. Evidence from our lab has demonstrated that improper activation of the innate immune system can reduce embryo implantation, thus leading to infertility. Therefore, control mechanisms regulating the innate immune system function can be critical for successful reproductive events.
Tribbles (TRIB), first identified in Drosophila, have gained their names from a fluffy creature in Star Trek Sci-Fi television series. Mutation in the tribbles genes gives the fruit fly a look reminiscent to the fictitious creatures in this series. A rapidly growing body of literature highlights the involvement of TRIB proteins in different physiological and pathological processes ranging from cancer to immune system regulation [1,2]. In recent years, we have gathered evidence that TRIB proteins may have an effect on reproductive processes, too [Basatvat Shaghayegh, Carter Deborah Angela Louise, Kiss-Toth Endre and Fazeli Alireza, unpublished work]. However, understanding of the involvement of TRIB in different reproductive processes is currently scarce and an under investigated area of reproductive biology. Here, in this short review, we will at first discuss potential different functions mediated by different TRIB proteins, present a summary of different reproductive processes and cite the relevant literature investigating TRIB function in these processes or hypothesize the potential involvement of TRIB in reproductive functions. Since TRIB seems to have a major function as regulators of innate immune system, we will particularly highlight the reproductive processes that involve innate immune responses and can be potentially mediated or influenced by TRIB.
Structure and function of tribbles proteins family
Members of TRIB family are encoding evolutionarily conserved psuedokinase proteins. These proteins are involved in modulation of cell functions by regulating intracellular signalling pathways . All TRIB proteins have a central serine/threonine kinase-like domain but they seem to lack robust enzymatic activity. Therefore they may be unable to effectively phosphorylate their target proteins . Hence, it is believed that TRIB may be working with a wide range of targets as scaffold proteins and controlling various signalling pathways . Scaffold/adaptor proteins smooth the flow of signals by helping with translocation and facilitate proximity of different components of a signalling cascade. They facilitate signal transduction, as for the optimal output of signalling network, there should be a balance between the concentrations of scaffold proteins and their targets . The psuedokinase domain of TRIB protein plays an essential role in the protein-protein interactions with their targets.
TRIB proteins are believed to regulate various events within the cell such as proliferation, metabolism, carcinogenesis, inflammation, cell differentiation, apoptosis and managing cellular stress [5,6]. All three members of the TRIB family, Trib1, Trib2 and Trib3 have been reported as regulators of the innate immune system by interacting with toll-like receptor (TLR)-mediated nuclear factor kappa-B (NF-KB) signalling pathways [7–9]. NF-KB is a group of key transcription factors that control the expression of various cytokines, chemokines, growth factors and adhesion molecules linked to innate immunity and different inflammatory processes .
Trib3 is a negative regulator of TLR2 response to Helicobacter pylori lipopolysaccharide (LPS) in human epithelial cells via the inhibition of NF-KB signal transduction leading to reduced IL-8 expression . Trib3 in this regard is considered as a modulator of inflammatory processes. Indeed, Trib3 was identified as a regulator of the NF-KB signalling pathway earlier by another group. They have discovered that SINK/Trib3 is negatively modulating NF-KB function by inhibiting p65/RelA phosphorylation. As a result transcriptional activity of NF-KB but not its nuclear translocation would be disrupted .
Another member of the TRIB family, Trib2 has also been shown to negatively regulate TLR5 ligand-stimulated NF-KB signalling pathway . Wei et al. have demonstrated that Trib2 can act as an anti-inflammatory factor and can affect both effector arms of TLR5 signalling; mitogen-activated protein kinase (MAPK) pathway and NF-KB pathways in opposite ways . The knockdown of Trib2 reduced the phosphorylation of C-jun N-terminal kinase (JNK) and P38 mitogen-activated kinase (P38) but did not affect the phosphorylation of extracellular signal-regulated kinase (ERK). On the other hand, the siTrib2 treatment of the same cell type increased the NF-KB luciferase activity in response to flagellin.
In conclusion, TRIB family proteins seem to have diverse roles in different cell properties ranging from cytoskeletal scaffolding functions to mediating innate immune-response. The rest of this review paper will focus on describing briefly different reproductive processes leading to production of an offspring and will highlight the literature reporting involvement of TRIB family in these processes.
Brief overview of different reproductive processes and events
In mammals, reproductive processes leading to the creation of an offspring involve production of gametes (spermatozoa and oocyte), transfer of the spermatozoa into the female reproductive tract (FRT), sperm and egg transport to the site of fertilization in FRT, conception, embryo development, implantation, pregnancy and the birth of the offspring.
Spermatozoa are produced during a process termed spermatogenesis. During coitus/mating spermatozoa are deposited in FRT and transported to the site of fertilization . The female gamete (oocyte) is generated within the ovaries through a process named oogenesis. Ovaries have another crucial task which is the production of sex hormones essential for the proper function of female reproductive organs. Ovulation is the last stage of oogenesis and is associated with the release of the oocyte into fallopian tubes [13,14].
Maternal interactions with gametes  are described as the signals swapped either between the mother and spermatozoa following mating or between the mother and oocyte immediately after ovulation . Interestingly spermatozoa and oocyte alter the proteomic profile of oviductal cell in dissimilar and definite way [17,18]. In other words, the oviductal cells are able to distinguish between spermatozoa and oocyte that helps to provide the appropriate microenvironment for sperm transportation, sperm storage and capacitation, fertilization and early embryo development [19–22].
Fertilization is defined as the fusion of oocyte and a spermatozoon which in mammals is taking place in fallopian tube/oviduct. During fertilization, spermatozoa bind to the zona pellucida and go through acrosome reaction, which facilitates penetration of the zona pellucida. Finally the sperm and egg content combine and form the zygote . Upon syngamy, the zygote starts cleavage to ultimately form the blastocyst. The blastocyst moves towards the uterine cavity and hatches from zona pellucida to gain the implantation fitness . Implantation of the embryo is an essential part of a successful pregnancy. It depends on three factors; receptive endometrium, healthy embryo at blastocyst stage and the effective communication between them.
Endometrial receptivity is a time limited process, the endometrium is only ready for the embryo to implant during a specific period of time, named window of implantation (WOI). In human WOI is during the mid-secretory phase of menstrual cycle between days 19 and 23. During this time, the endometrial cells undergo fundamental molecular and morphological changes. The molecular adaptations in endometrial cells that make them receptive for embryo implantation are a result of alterations in the expression of different cytokines, chemokines, growth factors and adhesion molecules with the aim of acquiring the adhesion ligands and losing the inhibitory factors [25,26]. The floating blastocyst commences the communication with the receptive endometrium through growth factors and local hormones in order to implant and establish pregnancy followed by apposition, adhesion and invasion .
Maintenance of the pregnancy and the growth and development of the conceptus is hugely dependent on the maternal recognition of the pregnancy. This process requires progesterone and placental hormones. The embryo needs to hint its presence to the mother in order to increase the existence of corpus luteum and as a result, the production of progesterone. Secretion of progesterone regulates the uterine endometrial functions, supports the early embryonic development and placentation .
Importance/regulation of innate immune system in males and females
Innate immune system in FRT protects the maternal tract against invading pathogens on one hand and support the growth and survival of the fetus on the other hand . Generally, the innate immune system with the help of its well-known receptors, TLRs, have the ability to rapidly recognize non-self-entities like bacterial and viral particles and further activate the adaptive immunity. Following stimulation by their specific ligands, TLRs induce intracellular signalling pathways leading to the activation of various transcription factors and expression of cytokines, chemokines and anti-microbial factors . It has been reported that presence of an infection in maternal tract is significantly related to pregnancy disorders and infertility . Hence, in normal pregnancy, the maternal innate immune system has a significant role in establishment of the mother–embryo communication as immune cells are abundantly present at the implantation site . The active role of the innate immune system at the site of embryo implantation and its contribution in setting up the interaction between the maternal endometrium and the implanting blastocyst is portrayed in Figure 1. Indeed, the crucial part that the innate immune system is playing in FRT starts earlier and upon deposition of semen into the maternal tract. Insemination stimulates series of immunological and inflammatory reactions that determine the outcome of successful pregnancy. Since spermatozoa are non-self-entities, it is logical to expect spermatozoa to elicit an immune reaction in FRT. However the innate immune process is truly in favour of the pregnancy. For instance, the cytokine expression in response to semen in FRT works as a mediator in facilitating the ovulation process .
The Role of immune system at the implantation site
It is well documented that sex hormones (oestradiol and progesterone) can affect both the function and expression of the TLRs in FRT and the expression of nearly all TLRs are at their peak during the secretory phase of female menstrual cycle which is associated with lower levels of oestradiol presence in FRT compared with the proliferative phase . Oestradiol can reduce the inflammatory response of pathogens by inhibiting NF-KB function and limit the transcription of proinflammatory cytokines such as macrophage inhibitory factor (MIF) and IL-6 and chemokines like IL-8. Oestradiol blocks the nuclear translocation of NF-KB or suppresses the degradation of its cytoplasmic inhibitors [34,35].
The expression and function of TLRs have been investigated in epithelial cells of female  and male  reproductive tract. Results from both in vivo  and in vitro experiments [39,40] showed that stimulation of endometrial TLRs with their specific agonists, have decreased the binding of embryo to the maternal endometrial cells. In the above mentioned studies, the activation of TLRs with their ligand at the time of implantation, significantly affects the endometrial receptivity which hugely decreases the rate of successful embryo implantation. Treatment of murine uterine horn with TLR2/6 ligand (FSL-1, a synthetic diasylated lipoprotein) has altered the tissue structure which affects the receptivity of the endometrial cells to embryo . Stimulation of TLR3 in endometrial cell-line, RL95-2 with its ligand poly I:C (synthetic double-stranded RNA) has reduced actin polymerization and CD98 expression. These modifications in response to TLR3 activation hugely disturb the endometrial receptivity and reduce the number of attached embryos to the endometrial cells . CD98 expression has been reported as an important molecule, up-regulated at the implantation site in endometrial cells and is mandatory for blastocyst binding to the endometrium .
Similar to their female counterparts, presence of bacterial, viral or yeast infection in male reproductive organs is also associated with inflammatory environment that is contributed to impaired sperm production and sperm maturation that consequently leads to infertility . It has also been reported that testosterone, the sex hormone produced in male reproductive tract, has an inhibitory effect on TLR expression namely TLR4 in testis. Testosterone seems to have immunosuppressive control role by affecting the balance between anti- and pro-inflammatory cytokine expressions in non-immune cells such as sertoli cells .
Potential involvement of tribbles in infertility
Although substantial research has been performed on TRIB function in different biological process [2,3,5], to the best of our knowledge, only one study reported on the involvement of TRIB proteins in mammalian reproduction. Brisard et al.  have investigated the expression pattern of all three members of TRIB protein family during oocyte maturation. They have confirmed the expression of these proteins in cumulus cells (CC) surrounding mouse and bovine oocyte with Trib2 showing the highest and Trib3 the lowest level of expression in CC of both species. Their experiments showed a remarkable reduction in Trib2 expression during oocyte maturation whereas Trib1 and Trib3 expression was significantly increased during this process. Differences in the expression patterns of different TRIB proteins during oocyte maturation could be explained by the different roles TRIB may have in different biological processes. Trib2 is involved in cell proliferation and that is explained by its over expression in different cancers and its down-regulation in oocyte maturation process .
Considering the fundamental function of innate immunity in both the female and male reproductive system and the previous studies on the regulatory role of TLRs signalling pathway by TRIB proteins, we speculate that TRIB may have significant modulatory roles in both the male and female reproductive system. Their regulation could cover a range of events within FRT such as implantation and establishment of pregnancy. Hence, it is plausible to suggest that TRIB may be involved in processes such as sperm transport and embryo implantation, as both these processes have potential to trigger the innate immune system in FRT. In male reproductive system they could affect the production and maturation of spermatozoa.
The diagram in Figure 2 shows the possible regulatory interactions TRIB protein may have in TLRs signalling pathway as a well-known family of innate immune system.
The potential involvement of Tribbles proteins in TLR-induced NF-KB and MAPK pathways
TRIB proteins expression and function are cell-type specific. They are involved in various cell functions and act as regulators of different signalling pathways, MAPK and NF-KB signal transduction for instance [3,45,46]. TRIB expression is modulated by different stimuli [47,48] as an example, TRIB-2 expression can be stimulated in response to TLR ligands . Consequently any alteration in the expression of TRIB can affect their function and the signalling pathway which they control. As explained earlier both male and female reproductive tracts are exposed to entities that can stimulate the innate immune system and lead to the transcriptional activation of downstream genes. Unnecessary activation of the immune system impairs the balanced milieu within the reproductive tract and affects the fertility outcome. This demands a control system to guarantee the successful reproduction which is where the TRIB protein can greatly participate.
Authors acknowledge the support of the European Cost Action, Epiconcept (FA1201) towards this investigation.
This investigation was partially funded by an Epiconcept European Cost Action (FA1201) short term scientific mission award to Shaghayegh Basatvat.
Tribbles Pseudokinases on the Crossroads of Metabolism, Cancer, Immunity and Development: Held at The Aquincum Hotel, Budapest, 22–24 April 2015