Interleukin-6 modulates in vitro development of the cattle oocytes and embryo: A review

Introduction

The use of in vitro embryo development to produce calves with the correct genetic composition is being quickly adopted by the dairy industry. This can be achieved by using assisted reproductive technologies such as embryo transfer (ET), ovum pickup (OPU), and in vitro production (IVP). Over the past years, there has been a significant increase in the use of in vitro-produced (IVP) bovine embryos worldwide; according to the most recent estimate, over 1.6 million IVP embryos were transferred in 2022 (Viana, 2022) [53] . Since 1981, it has been possible to produce calves from in vitro produced (IVP) bovine embryos (Brackett et al., 1982) [11] , resulting in a breakthrough in cattle reproductive biotechnology. However, there are two main drawbacks to in vitro embryo technologies for production. Firstly, the generation of embryos is currently not very efficient. Numerous factors, including the culture media's composition, the environment (such as oxygen content), seasonal fluctuations, and the degree of technical expertise, can cause the success rate to fluctuate significantly. Only between 20 to 45 percent of oocytes employed in in vitro embryo development are known to effectively grow into transferable blastocyst-stage embryos (Lorengan et al., 2003 and Lorengan et al., 2008) [25,26] . Secondly, after embryo transfer, a significant number of transferable IVP embryos do not survive. Wooldridge et al (2019) [54] found that only 27 percent of IVP embryo transfers produced live calves in dairy industry. Some researchers hypothesize that adding biologically active compounds made by the reproductive system in the early stages of pregnancy to media formulations could improve post-transfer IVP embryo competency and in vitro embryo production efficiency. Hansen and colleagues introduced the term "embryokine to refer to substances that control embryo growth that are produced by There are various growth factors, signalling molecules and cytokines identified as an embryokine. Growth factors such as IGF1, EGF, and FGF greatly impact the in vitro embryo production when it is used in without combination (Xie et al., 2017) [57] . In cattle, insulin-like growth factor 1 (IGF1) supplementation increases the amount of blastocyst cells, IVP blastocyst development rates, and post-transfer pregnancy rates throughout the summer (Block et al., 2003, Jousan et al., 2007 and Xie et al., 2017) [10,23,57] . A pleiotropic cytokine, interleukin-6 (IL-6) is essential for inflammation, immunological responses, and cellular communication. Cytokines that have been identified and explored as an embryokines basically belong to two cytokine families, first the beta common cytokine family which includes CSF2 or GM-CSF, IL3 and IL5. Members of the second cytokine family belong to the interleukin 6 (IL6) family which are differ from the β-common cytokines in both structure and function. IL6, IL11, IL27, IL31, leukemia inhibitory factor (LIF), oncostatin-M (OSM), and ciliary neurotropic factor (CNTF) are all members of the IL6 family (Eulenfeld et al., 2012 and Rose-John, 2018) [18,37] . However, in the IL6 family, IL6 and LIF are the molecules that attract the most interest as embryokines. This review provides insights into in vitro embryo development and livestock productivity by supplementing interleukin 6.

Evidence of IL-6 involvement in folliculogenesis, oocyte maturation, and embryo development in vivo

In reproductive biology, IL-6 has been connected to follicular development, oocyte maturation, and early embryogenesis. For decades, preimplantation embryos of various species, including humans, mice, rabbits, and cattle, have been cultured in vitro to the blastocyst stage (Tervit et al., 1972) [51] . However, in vitro culture systems lack the complexity of the oviductal environment, which can hinder ICM development. It is therefore not surprising that defects of the ICM can result in lethality (Feldman et al., 1995 and Takeda et al., 1997; Arman et al., 1998) [19,50,7] . Interleukin 6 (IL-6) is a pleiotropic cytokine; It has multiple cellular and tissue effects and leads to pro-inflammatory response. IL-6 may also play a crucial role in follicular development (Yang et al., 2020) [58] . Some studies have shown that a higher level of IL-6 correlated with worse quality of embryos, and patients were less likely to get pregnant (Semeniuk et al., 2018) [41] . In contrast, high levels of IL-6 in the follicular fluid were good for oocyte maturation and were associated with increased rates of clinical pregnancy and embryo implantation (Wu et al., 2017) [55] . Indeed, during the periimplantation period, immunological balances must be regulated, including regulation of pro-inflammatory/antiinflammatory chemokines and IL17/regulatory T cells (Ali et al., 2021) [5] . Some studies have shown that high levels of IL-6 in follicular fluid have a positive effect on oocyte maturation (Kawasaki et al., 2003 and DeMatos et al., 2008) [15, 16] . High levels of IL-6 in embryo culture are associated with an increased rate of clinical pregnancies and implantation of embryos (Wu et al., 2017 and Qin et al., 2016 and Wu et al., 2007) [55,35,56] Higher levels of IL-6 correlate with poor embryo quality, and patients are less likely to become pregnant (Altun et al., 2011 and Semeniuk et al., 2018) [6,41] . It has been concluded that IL-6 inhibits oocyte development (Banerjee et al., 2012) [9] . In addition, some studies have suggested that IL-6 does not affect the clinical rate of pregnancy, as well as the quality of oocytes (Alhilali et al., 2019 and Asimakopoulos et al., 2008) [4,8] . On the basis of studies, that IL-6 might be improve oocyte developmental competence and embryo quality in in vitro conditions by mimicking in vivo conditions.

Expression of IL-6 receptors (IL-6R/gp130) and Signaling in bovine ovarian cells and embryos

Cytokines may be significant autocrine and paracrine regulators of ovarian activities, as evidenced by their production in the ovary, accumulation in ovarian follicular fluid, and impact on some ovarian processes (Qiao et [34,36,47,43] . Among all Interleukins (ILs) are cytokines that seem to play a key role in regulating reproductive function and a variety of physiological and pathological processes in the ovary (Smolikova et al., 2012) [47] . Leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), IL-11, and cardiotrophin-like cytokine factor 1 (CLCF1) are among the members of the IL-6 family (Senaldi et al., 1999) [42] . They have similar biological roles and share the receptor IL-6 signal transducer (IL-6ST, also called gp130) (Taga and Kishimoto, 1997) [49] . LIF has been linked to the early development of follicles (Skinner, 2005) [46] and oocyte quality (De Matos et al., 2008) [15,16] and implantation (Hu et al., 2007) [22] . Since oocytes can be direct targets of cytokine action, mice with a conditional deletion of the gp130 receptor in their oocytes show reduced zygotic cleavage to the two-cell stage (Molyneaux et al., 2003) [31] . In granullosa cells, by attaches itself to the IL-6 receptor, which is made up of IL-6Rα and another protein on the cell membrane. The precise ligands that control JAK/STAT3 activity in bovine embryos are unknown. The JAK/STAT3 pathway is crucial for the ICM lineage in mice and calves (Takeda et al., 1997; Meng et al., 2015) [50,29] . Members of the IL6 cytokine family appear to fulfill this function in a number of ways. Certain members of the family act as pluripotency factors for mouse embryonic stem cells (Shmitt et al., 1991; Conover et al., 1993; Nichols et al., 1994) [39,14,32] . Additionally, in pig blastocysts, IL6 supplementation promotes STAT3 phosphorylation and raises the quantity of ICM cells (Shen et al., 2012) [44] . Because embryonic genome activation had already taken place by this stage of development (the 8-16 cell stage), allowing embryos to respond to external signals, and because these embryos were getting ready for compaction and embryonic lineage specification, the majority of the studies started on day 5 of embryonic development (Graf et al., 2014) [20] . This is an ICM-specific activity. In trophectoderm (TE) cells, no similar IL6-induced STAT3 activation is seen. Additionally, ICM development in bovine embryos is inhibited by pharmacological suppression of JAK2, which cannot be reversed by IL6 supplementation. (Wooldridge et al., 2019 and Meng et al., 2015) [54,29] .

Interleukin 6 supplementation effect on in vitro maturation of bovine oocytes

Bovine embryos created in vitro (IVP) have lower posttransfer survival and developmental potential. The absence of growth, differentiation, and cytokine factors that are present in the follicle but not in oocyte maturation culture systems could be one explanation for this. The cumulusoocyte complex (COC) gene expression, fertilisation success, embryo development potential, and maturation success are all impacted when certain members of the interleukin-6 (IL6) cytokine family are supplemented during in vitro bovine oocyte maturation. According to Vendrell-Flotats et al. 2020 [52] supplementation of IL6 or IL11 during IVM enhanced the production of many microRNAs that promote the maturation of bovine oocytes. According to a different study, IL6 might play a role in regulating when meiosis completion occurs in cattle under heat stress (Rowinski et al., 2021) [38] . McKinley et al. (2023) [28] reported that Oocyte maturation and COC gene expression were all negatively impacted by IL6 administration results could be explained by the fact that IVM produces enough endogenous IL6 to eliminate the requirement for further supplementation. [28] discovered that although supplementing with LIF and IL-11 boosted blastocyst growth rates and the expression of many transcripts linked to oocyte competency, it had no appreciable impact on the embryo's development or the success of fertilization. According to a different study on IL-6 supplementation during in vitro embryo development, it increased the number of inner cell mass cells and appeared to have a tendency to increase the number of trophectoderm cells in blastocysts, but it had no effect on blastocyst formation rates.

Interleukin 6 supplementation effect on in vitro fertilisation of bovine oocytes

Interleukin 6 supplementation effect on in vitro culture of bovine embryo inner cell mass and trophectoderm

Supplementation with various growth factors and cytokines during in vitro embryo production can have positive effects on pregnancy retention, calf birth weight and dystocia (Loureiro et al., 2009; Denicol et al., 2014) [27,17] . It was intriguing to see that on day nine, IL6 treatment increased the amount of undifferentiated cells. This might be a result of IL6's capacity to increase ICM cell counts before epiblast/ primitive endoderm specification, as demonstrated most recently in research done on blastocysts analyzed on day 7 after fertilization (Wooldridge et al., 2019) [54] . However, after they have committed to one of the two cell fates, they lose this flexibility. The TE was slightly impacted by IL6 treatment as well. Given that the majority of studies did not identify effects of IL6 on TE cell counts, this finding is noteworthy even though just a statistical tendency for increased TE cell numbers was found (Wooldridge et al., 2019) [54] . However, when comparing the EPI and PE figures at days 9 and 10 to those at day 8, no improvements were found. Therefore, it's possible that undifferentiated cell differentiation only partially restored the missing PE and/or EPI cells. Alternatively, apoptosis of undifferentiated cells might have taken place. When undifferentiated cells continue to exist in the ICM, this is a typical occurrence (Plusa B, et al., 2008) [33] When 100 ng/ml of recombinant bovine IL6 was added to in vitroproduced bovine embryos on days 1, 3, or 5, the number of inner cell mass (ICM) cells and the ICM: trophectoderm (TE) ratio increased (p<0.05), but not the number of TE cells. After taking 1 or 10 ng/ml of IL6 starting on day 1 or day 5, there was no increase in the number of ICM or TE cells. The number of ICM cells was raised (p<0.05) by sequential supplementation with 100 ng/ml IL6 on days 1 and 5 (for a total of 200 ng/ml IL6) more than using IL6 supplements for a single time in one research but not in another. Furthermore, compared to supplementing with 100 ng/ml IL6, giving 200 ng/ml IL6 starting on day 1 or day 5 did not result in any additional increase in ICM cell counts. In group culture, cleavage and blastocyst formation were unaffected by IL6 therapy. However, when bovine embryos were cultivated separately, IL6 treatment enhanced cleavage and the development of day 8 blastocysts (Wooldridge et al., 2019) [54] . Once [3,30] .

Effect of the IL 6 on the expression of gene

Additionally, STAT3 activation in mouse blastocysts is decreased by antibody-based immunoneutralization of embryo-derived IL6 (Do et al., 2013) [61] Additionally, there are some signs that IL6 may promote the pluripotency of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESC) (Do et al., 2013; Tang et al., 2020) [61,59] . When human fibroblasts are reprogrammed, IL6 mRNA increases 50-fold (Brady et al., 2013) [2] . The same study found that IL6 supplementation activated a number of STAT3-induced targets, including c-Myc, one of the four elements necessary to cause human and mouse somatic cells to become pluripotent (Liu et al., 2008). When the other three components (Oct4, Klf4, and Sox2) were overexpressed, cells were able to undergo pluripotency by substituting IL6 for c-Myc expression. RNA sequencing results did not show the SOCS3 rise (Wooldridge et al., 2019) [54] . RNA sequencing investigation involved embryos exposed to IL6 for 72 hours and that SOCS3 expression is known to increase initially but then decline following prolonged IL6 treatment, this is not surprising (Starr et al., 1997) [48] . Additionally, the RNA sequencing dataset demonstrated that IL6 supplementation had negligible effects on the development of embryo lineages, early pluripotency, and differentiation potential. Little information on the processes behind IL6's effects on the ICM was revealed by the RNA profiling study. To interpret changes in gene expression linked to IL6 treatment, time-response studies and studies that specifically look at the ICM are probably required. The most widely expressed ligand-specific receptor was IL6R. Furthermore, IL6R expression improved in response to IL6 supplementation. While changes in PE lineage markers were observed (Wooldridge et al., 2019) [54] . IL6 supplementation had no effect on the expression of lineage markers linked to ICM and TE development. The yolk sac will be formed by the PE, and compared to embryos created in vivo, the yolk sac of IVP bovine embryos is not as well vascularized (Alberto et al., 2013, Mess et al., 2017) [3,30] . IL 6 treatment control the expression of the transcription regulator gene like STAT1, STAT3 and OTX2 and it is expected to detect these responses to IL6 treatment in bovine embryos (Brocke-Heidrich et al., 2004 and Zang et al., 2005) [12,60] . 7. Limitations and future challenges using IL6 as supplement in bovine embryo production IL6 acts as an embryokine in bovine embryos, promoting STAT3 phosphorylation in the ICM. Its effects on ICM growth are blocked by JAK/STAT3 inhibition, but its reliance on this pathway remains unclear, as STAT3 is essential for ICM survival (Wooldridge et al., 2019) [54] . IL6 transcripts are highly expressed in the oviduct on days 3 and 5 and in the endometrium on day 5 during early pregnancy (Starr et al., 1997; Shen et al., 2012; Wooldridge & Alan, 2019) [48,44,54] . While IL6 enhances ICM development in vitro, its necessity for overall embryo development is uncertain. IL6-treated embryos show no adverse effects on fetal or placental development during the first 10 weeks of gestation. Instead, it may support embryo and fetal development, though studies with limited pregnancies prevent definitive conclusions (Wooldridge et al., 2019) [54] . Further research is needed to assess its impact on neonatal health and post-transfer pregnancy survival. Individual embryos showed varied responsiveness to IL6 supplementation, indicating that not all embryos may benefit equally from this treatment, which could limit its effectiveness in practical applications IL6 supplementation did not significantly affect cleavage rates or blastocyst formation in group cultures, suggesting that its benefits may be context-dependent and not universally applicable Future studies are necessary to explore the optimal conditions for IL6 supplementation, including timing and concentration, to maximize its benefits for embryo development in various culture environments (Wooldridge et al., 2019) [54] . Recently in a study examined cytokines IL6, IL11, and LIF in embryos post-fertilization. Results showed IL6 and LIF increased ICM cell quantity, indicating better embryo quality. However, no treatments affected IVP embryo survival post-freezing and thawing (Wooldridge et al., 2019) [54] .

Conclusion

Interleukin-6 (IL-6) plays a complex role in embryo development, exerting both beneficial and detrimental effects on oocyte maturation and embryo quality. It, along with related cytokines like LIF and IL-11, regulates ovarian function, folliculogenesis, and embryogenesis via pathways such as JAK-STAT3. In bovine models, IL-6 supplementation can enhance developmental competence and inner cell mass (ICM) formation; however, excessive levels may impair maturation due to adequate endogenous IL-6. While LIF and IL-11 improve oocyte competence and blastocyst development, IL-6's influence on embryo quality and post-transfer outcomes remains promising yet inconclusive. IL-6 also affects ICM and trophectoderm cell numbers, pluripotency markers, and early gestation, but its role in ensuring overall embryo viability appears contextdependent. Despite its potential to improve in vitro embryo production and fertility outcomes, the precise mechanisms and optimal conditions for IL-6 use remain unclear. Thus, balanced cytokine supplementation and further research are crucial to harness IL-6's full reproductive potential.

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