Photoinduction of granulosa cell and oocyte co-culture to influence in vitro maturation and fertilisation

HJ378_GA

Infertility issues can be attended to by in vitro maturation (IVM) of an immature oocyte into a developmentally competent oocyte followed by in vitro fertilisation (IVF). The hypothesis presented here is a consolidated logical outcome of independent research publications concluding by suggesting photoirradiation of granulosa cumulus cells and oocytes as means to influence IVM and IVF in humans and animals with possible outcomes discussed.

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Introduction

Fertility issues are mostly associated with the quality of the oocyte (1) which in turn is determined by its maturation signals. One other factor influencing fertility is the fusion of sperm and egg which is determined by cell surface-associated proteins like CD81 and CD9 (2,3). Attending to these factors during in vitro maturation (IVM) or during in vitro fertilization (IVF) would increase the success rate of embryo formation. IVM has been suggested as an effective technique to solve infertility issues and also as a tool in wildlife or endangered species conservation (4). Fertility issues can be treated in these three ways (5): (i) Superovulation by hormones, which results in unnatural multiple egg formation, which are retrieved and used for IVF by either intracytoplasmic sperm injection (ICSI) or sperm-oocyte incubation overnight followed by embryo transfer. This technique is known for its health risks; (ii) Normal ovulation followed by egg retrieval which is used for IVF achieved by ICSI or incubation with sperm-egg overnight followed by embryo transfer; (iii) Instead of mature egg retrieval, sometimes the cumulus cells (transformed granulosa cells) with the oocyte, called COC or cumulus oocyte complex, is retrieved and cultured in vitro and the mature egg is used for IVF by ICSI or incubation with sperm-egg overnight followed by embryo transfer. The oocyte and a single layer of squamous granulosa cells, collectively called the primordial follicle, transits from primary (unilaminar) to secondary (multilaminar) to mature (tertiary) follicle, culminating with antrum formation and the release of the oocyte along with few granulosa cells called the corona radiata. The remnant of the granulosa cells get transformed into corpus luteum which is important for maintaining pregnancy through the release of progesterone (1,6). Antral follicles do retain cumulus cells with the oocyte (cumulus oocyte complex/COC) that can be cultured with the aid of gonadotrophins (7) and COC can be co-cultured with granulosa cells in the presence of follicle stimulating hormone (FSH) (8,9) or individually cultured in the presence of FSH (10). Other procedures include FSH priming in animals (monkeys) before the collection of granulosa cells to co-culture with oocytes (11) or unprimed animal cumulus oocytes that can also be used for IVM (12).

Hypothesis

Photoirradiation of granulosa cells and oocytes would help towards IVM and IVF.

(HJ378_STAR_FIG_1AIndependent references)

HJ378_IMG1

(Picture source: Sriram Kannan for buffalo and Onnu Reddy for monkey. The pictures were converted to gray scale by cartoonize.net)

Supporting arguments

Our approach includes hypothesizing photoinduction as a means to alter molecular factors which influence IVM/IVF.

i. Effect of light on cell adherence and proliferation of cultured cells:
The proliferation and differentiation of granulosa cells is important for follicular growth, ovulation and luteinization (13). Light has been shown to increase cell proliferation in epidermal cells and well adhered granulosa cells do produce better estradiol. Hence granulosa cell proliferation and adherence play a vital role in its secretions. RGC5 cells in the presence of serum have shown a survival rate of 82.7% under a 1000 lux light source (14). Hence while using a fluorescent LED light source of 3 v / 20 lux, which is far less intense than 1000 lux, the chances of cell death are negligible. Our system uses and suggests one such source of light of < 20 lux (3 v fluorescent LED light) per well in a 24/6 well cell culture dish for 30’ only at the 3rd and 18th hour of seeding and hence because of such a low exposure time and intensity the chances of detrimental effects could be less or negligible.

ii. Effect of light on ion channels and exosomes in granulosa cells:
Photoirradiation is known to increase HSP70 expression in cultured cells (15) and levels of TGF beta secretion in fibroblast cell culture (16). It also has been shown that HSP70 is secreted via exosomes (17) like TGF beta, which is packed either in the exosome (18) or on the surface of the exosomes (19). Both these factors, namely HSP70 (20) and TGF beta, can be independently hypothesized to be linked to steroidogenesis. HSP70 is known to increase the activity of potassium ion channels (21) and hence it can be assumed that light induction of HSP70 might enhance potassium channel activity and exosome trafficking, both of which are favorable for steroidogenesis. This is because K channel antagonists are known to decrease LH/FSH-based progesterone levels in porcine granulosa cells (22,23) and hence HSP70-based induction of K channels might act towards elevation of steroidogenesis. Similarly, TGF beta is another factor that is photoinductable and light has been shown to increase secretion of TGF beta in fibroblast cell culture (16). TGF beta is known to aid in estradiol synthesis via aromatization along with FSH (24) in granulosa cells.

Table 1. Experimental IVM with buffalo oocytes and granulosa cells (9)

HJ378_TBL_1

Experimental IVM with Rhesus monkey oocytes has shown the following outcomes (12):
Estradiol and progestrone added to CMRL1 medium improve IVM of rhesus monkey immature cumulus oocytes cultured in the presence or absence of gonadotrophins (FSH) that were collected from unstimulated prepubertal and adult rhesus monkeys.

In non-mammalian species like jellyfish, light has been shown to have positive effects on oocyte maturation (25,26). It is also well established that the oocyte and the granulosa cells do communicate bidirectionally (1,27) and through exosomes (28). The photoinduced changes in intracellular and extracellular levels of TGF beta and HSP70 by exosome shedding are discussed as follows with a practical model. Granulosa cells were attempted to be grown over paper clippings sputtered with gold exposed to a 3 v (max 20 lux) fluorescent LED light source for photoirradiation used at the 3rd hour (30’) and 17th hour (30’) of seeding the cells. Further, the gold-coated paper provides a 3D scaffold for granulosa cell development that might better influence estradiol production because of better cell adhesion. It also might benefit co-culture of oocytes, as inundations of the cellulose with granulosa cells attached might mimic a natural microenvironment, as shown in Figure 1.

HJ378_FIG_1

Figure 1 | Attempted model (cellulose paper sputtered with gold) seeded with granulosa cells. Top: Scanning Electron Microscopy image (5000x) of granulosa cell seeded on gold sputtered paper. Further additional coating on the gold-sputtered paper was also attempted with curcumin or isolated exosomes. (Acknowledgement for central SEM facility Dr. S. K. Tomar & Dr. Babar Ali, National Dairy Research Institute). Bottom: Model of granulosa cell mats placed in a cell culture dish grown on cellulose paper as scaffold.

Different mats can be treated differently in terms of light exposure and the scaffold (Figure 1) used to grow. It is known that TGF beta is packed on/in exosomes and HSP70 is collocated in the same exosomes (28) and hence as light can cause HSP70 shedding, it is quite probable to create a microenvironment in which the cells get a change in the intracellular and extracellular levels of TGF beta and HSP70 due to light-induced exosome shedding. It might be possible to produce mats with different phenotypes by light exposure—some producing more estradiol, some turned on towards progesterone and some for melatonin. Mimicking the dynamic microenvironment of oocyte maturation-granulosa cell mats is depicted in Figure 2.

HJ378_FIG_2

Figure 2 | Model of granulosa cell culture mats. Granulosa cells cultured on different coated scaffolds could be treated with different light intensities. Hypothesizing the relation between light intensities and its possible effect on pathways is shown in Figure 5 in detail.

Exposing different mats with cultured granulosa cells to different light intensities can result in different secretions, as shown in Figure 2, and these mats can be placed together in different ratios to co-culture with oocytes to mimic the dynamic microenvironment of maturing follicles to atresia. Hence, exposing few scaffolds with granulosa cells to higher intensity light (1000 lux) and lower intensity light (20 lux) might result in increased apoptosis of cells (14) that can be used to mimic follicular atresia, the time point wherein apoptosis of granulosa cells and NFKB activation through EGFR signaling in low intense light exposed cells occurs. These two types of cells could be introduced alongside in the same culture vessel to create a dynamic microenvironment, as shown in Figure 3.

HJ378_FIG_3

Figure 3 | Combination of different granulosa cell mats. The combination of different mats with progression of time could be standardized with trial and error but the advantage is that it can mimic the dynamic environment of oocyte maturation.

It has been reported that granulosa cells too are capable of synthesizing melatonin in late stages of folliculogenesis, apart from the pineal glands (30,31,32), and melatonin is considered favorable for IVM by increased progesterone synthesis (33). Such a process could be induced by activating NFKB, as has been shown in macrophages synthesizing melatonin (34). Instead of high intensity light, few scaffolds could be coated with curcumin which can be photoactivated to kill the cells (35) to create a mat of cells undergoing apoptosis to resemble late folliculogenesis. But it is acceptable that the ratio of placing differently treated mats will need to be standardized by trial and error to find out the best ratio for IVM. As it has been shown that thyroxine receptors (36) and EGF receptors play important roles in oocyte maturation (37,38) and as photogenetics is an upcoming field, with photocaging of receptors for control of pathways with attempts in thyroxine receptor photocaging (39) and optical genetic switches attempted in bacteria and cell lines (40), it could be possible to create granulosa cells with optical genetic switches to control pathways of interest as shown in Figure 4.

HJ378_FIG_4

Figure 4 | Combination of mats with photocaged system. Photocaged system (blue ovals) in granulosa cells could be used to influence specific pathways.

Positive and negative effects of light on molecular pathways are discussed as follows and in Figure 5. HSP70 interaction with Tid1 protein is essential for EGFR degradation (41) and EGFR signaling from granulosa cells and cumulus cells is important for oocyte maturation (37,38).

HJ378_FIG_5

Figure 5 | Possible effect of light irradiation on cumulus granulosa cells. Yellow lines represent possible pathways influenced by light. Tid1 protein is known to interact with smad7 through its MH2 domain and with HSP70 with its DNAJ domain. But if light can increase HSP70 and TGF beta exosome shedding, leading to decreased interaction with Tid1, possibilities of limiting EGFR degradation via HSP70-DNAJ domain-Tid1 would become limited, thus enhancing EGFR signaling. Similarly, shuttling TGF beta out of the cell might facilitate limiting TGF beta-smad7 interaction, thus limiting apoptosis. Gold circles: different granulosa cell mats with different light exposure, later placed together, or few mats coated with curcumin for photo activation of apoptosis to mimic atresia. 

It is known that HSP70 and TGF beta are located in exosomes and photoirradiation can cause exosome shedding, thus probably limiting the interaction products of HSP70 and TGF beta. TGF beta and smad7 signal apoptosis in granulosa cells. But HSP70, once outside the granulosa cell, could be beneficial, as it can activate K channels that can help in progesterone synthesis. Depleting the TGF beta by exosome shedding out of the cell could activate EGFR signaling extracellularly and prevent smad7 signaling intracellularly in granulosa cells. Further, by replacement of medium, transferring the granulosa cell mat to oocyte culture using trial and error and running at different timepoints, along with photoinduction, different microenvironments could be created.

Melatonin has been shown to be important in IVM. It is synthesized in the pineal glands and delivered to follicles via blood, but it is also known to be synthesized in granulosa cells (32) and in macrophages. In macrophages it has been shown that NFKB plays a positive role in synthesis of melatonin (34). Hence by photostimulating EGFR, NFKB could be upregulated and that could result in the possibility shown in Figure 6.

HJ378_FIG_6

Figure 6 | Possible effect of light on NFKB pathway in granulosa cell (41). Yellow lines indicate possible effects of light in granulosa cell culture systems.

Thus it can be logical to hypothesise that light can decrease EGFR degradation through tid1-HSP70, interfere with TGF beta-smad7 apoptosis and increase progesterone levels in cultured granulosa cells. TGF beta in extracellular conditions can activate EGFR, which can upregulate NFKB and melatonin synthesis. All these could be beneficial in oocyte maturation as it has been shown that EGFR signaling, melatonin and progesterone levels can improve in vitro maturation of oocytes (12,32,37,38). In terms of IVF, CD81 and CD9 expressed on the oocyte are required for the fusion of sperm (2,3) and light can possibly increase CD81 and CD9 expression in the oocytes as its HSP70 trafficking is through exosomes which is photoinductable. This might prove useful in in vitro fertilization by increased CD81 expression or in in vitro maturation, as exosomes secreted by co-cultured granulosa cells might influence the developmental competency of oocytes (28).

Conclusion

We hypothesized that light could influence granulosa/oocyte culture towards in vitro maturation of oocytes and in vitro fertilization by an increase in estradiol and progesterone from granulosa cells by HSP70 K channel activation. Further light-induced HSP70 and TGF beta extracellular shedding with exosomes could be useful to limit EGFR degradation by tid1-HSP70 intracellularly and TGF beta could be useful for activation of EGFR. Moreover, by using different light-treated granulosa cell mats, different microenvironments could be created to mimic the progression of follicular maturation to follicular atresia. Combination of such mats at different time points placed in co-culture with oocytes could give dynamic signals from granulosa cells that might facilitate oocyte maturation in vitro. By implementing photocaged receptors and optogenetic systems in granulosa cells, particular pathways could be regulated with light. Hence, instead of manually supplementing a multitude of factors to the oocyte culture, light and controlled supporting cells like granulosa and cumulus cells could be used for creating a complex and dynamic microenvironment for the in vitro maturation of oocytes. Working towards such processes would be useful in human infertility, domestic animal productivity and also in wildlife conservation with diminishing effects on cost and risk. H

Authors declare no conflicts of interest.

About the authors

Dr. Dheer Singh is the Principal Scientist in Animal Biochemistry with specialization in Molecular Endocrinology at National Dairy Research Institute (NDRI), India. His lab interests include analyses of functional regulation of trait-specific (fertility) genes with research works on Indian water buffalo follicular cells at the molecular level using in-vitro models with siRNA and nanotechnology interventions. Research work in these fields is undertaken by his PhD students, namely Anu Mehta, Onnu K Reddy and Vijay Simha. Binny P Kaur is a senior research fellow in gene expression project and Sriram Kannan handled nanotech project as a research associate under Dr. Singh. Dr. Suneel Onteru is a Senior Scientist in Animal Biochemistry Division, NDRI and co-PI in research projects with Dr. Singh and his interests are genome-wide association studies apart from fertility research in cattle and buffaloes.

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