Autophagy-related gene and protein expressions during blastocyst development

Abstract

Purpose

This study aims to examine the expression of autophagic genes and proteins during blastocyst development and differentiation.

Methods

This is a prospective cohort study. Between March 2018 and November 2019, 30 females aged 30.13 ± 4.83 years underwent an intracytoplasmic sperm injection (ICSI) cycle at Madina Fertility Center. ICSI was used to develop and incubate 82 leftover embryos to day 5. Then, the embryos were divided into two groups based on their developmental structure: group D (n = 49) included embryos that developed into blastocysts, whereas group A (n = 33) included arrested embryos. These embryos were used to investigate the autophagic gene and protein expressions. The current study was approved by the Clinical Trial Ethical Committee of the Faculty of Medicine, Alexandria University, following the ethical standards of scientific research (Registration no. 0303721).

Results

Embryos that developed into blastocysts on day 5 (group D) had significantly higher relative expression of the LC3 gene (1.11 ± 0.52) and beclin-1 gene (1.43 ± 0.34) and beclin-1 protein expression (3.8 ± 0.028) than those that did not develop into blastocysts on day 5 (group A) [0.72 ± 0.18 (P = 0.03), 0.35 ± 0.12 (P = 0.0001), and 3.14 ± 0.05, (P = 0.0001), respectively]. In contrast, mTOR and PIK3C3 protein expression was significantly higher in group A (arrested embryos) than those in group D (developed embryos) (P = 0.007 and P = 0.0001, respectively). Furthermore, the expression of the eIF4E gene was significantly lower in group D embryos (0.32 ± 0.07) than that in group A embryos (4.38 ± 1.16) (P = 0.0001).

Conclusions

This work identifies  autophagy as a well regulated process required to maintain cell allocation and differentiation during late preimplantation embryo developmental stages.

Introduction

Autophagy, derived from the Ancient Greek words “auto” and “phagy” meaning “self-eating,” is a necessary molecular mechanism for early embryonic development. It is a lysosome-mediated cytoplasmic degradation pathway [1]. Moreover, it has been identified as an adaptive response that provides nutrients and energy under various stresses and is a tool for preventing the aggregation of misfolded, long-lived proteins or damaged organelles [2]. Therefore, autophagy plays a vital role in physiological functions such as homeostasis and development [3].

Maternal factors such as mRNA and proteins accumulate in the oocyte cytoplasm during oogenesis. These maternal factors significantly contribute to embryonic development after fertilization [4]. In numerous species, maternal mRNA and proteins are rapidly degraded and replaced with embryo genome-derived mRNA and proteins after fertilization, a process known as the maternal-to-zygotic transition [5].

As the transition process occurs rapidly and quickly, an efficient system for degrading maternal proteins in bulk is required [6]. The ubiquitin–proteasome system and autophagy are two primary pathways for cytoplasmic protein degradation. Because proteasomes only target ubiquitinated proteins for degradation, the ubiquitin–proteasome pathway is regarded as a subjective degradation pathway [7]. In mammals, ubiquitin-mediated proteolysis has been associated with the resumption of oocyte meiosis and the maternal-to-zygotic transition [8].

Other proteins or cytoplasmic components that are not degraded by the ubiquitin–proteasome system are degraded via the second nonselective cytoplasmic degradation pathway of autophagy [7]. Compared to the ubiquitin–proteasome pathway, autophagy is a better pathway for the bulk degradation of maternal cytoplasmic contents during the maternal-to-zygotic transition and as a response to the rapid change in embryonic morphology and properties during different developmental stages. Moreover, the amino acids released by protein degradation during autophagy are recycled as substrates for nutrients and the synthesis of new proteins, which contribute to subsequent embryonic development [6].

Approximately 35 autophagy-related genes (ATG) have been identified [9]. Autophagy can be observed by the visualization of the microtubule-associated protein-1 light chain 3 (LC3), which is a homolog of yeast ATG8 and is related to the autophagosome [10]. Since LC3 is degraded in the lysosome, the amount of degraded LC3 protein, also known as autophagic flux assay, can be measured and used as a method of autophagy monitoring [11].

When autophagy is induced, an isolation membrane appears, elongates, and engulfs a portion of the cytoplasm, resulting in the generation of a double-membrane structure known as the autophagosome. After the fusion of the outer membrane of the autophagosome with a lysosome, the cytoplasmic contents sequestered by the autophagosome are degraded by lysosomal enzymes [12].

During autophagy, cytosolic LC3 (LC3-I) is converted to membrane-bound LC3 (LC3-II) via lipidation. While LC3-II is closely associated with autophagosomes, it is degraded after lysosome fusion. Therefore, LC3 protein turnover is an indicator of autophagic activity [11].

Autophagy is activated from the late two-cell stage to the eight-cell stage. Autophagy-deficient embryos were found to arrest at the four-cell to the eight-cell stage, indicating the importance of autophagy in embryogenesis [13]. On the other hand, experimentally delayed implantation was observed in a mouse model established by ovariectomy before blastocyst implantation, indicating that 17β-estradiol induced autophagy in blastocysts [14].

Moreover, progesterone can induce autophagy by decreasing the activity of the mammalian target of rapamycin (mTOR) in bovine mammary epithelial cells. Therefore, both E2 and progesterone are the primary regulators of autophagy [15].

When the mTOR pathway is activated, its downstream target proteins, eukaryotic initiation factor 4E-binding protein (4E-BP1) and p70 ribosomal S6 kinase 1 (S6K1), are phosphorylated. Under unphosphorylated conditions, 4E-BP1 tightly binds to eIF4E, forming the inactive eIF4E·4E-BP1 complex [16].

During anabolic conditions, mTORC1 induces the phosphorylation of 4E-BP1, resulting in the dissociation of eIF4E from the inactive complex and allowing eIF4E to form an active complex with eIF4G. The association between eIF4E and eIF4G is required for binding the 43S preinitiation complex to mRNA. S6K1 is another mTORC1 substrate that regulates mRNA translation [17].

Fertilization-induced autophagy was revealed to be molecularly unique, implying the involvement of other molecules [18]. The phosphoinositide-3 kinase (PI3K) complex, including beclin-1, ATG14 (L), Vps15, and VPS34, is essential for autophagosome formation and plays a vital role in regulating the autophagic cytoprotective function and, oppositely, the apoptotic cellular death process [19].

In most cell types, mTORC1 activity inhibits autophagy. However, in fertilized oocytes, the PI3K signaling pathway, rather than mTORC1, regulates fertilization-induced autophagy, indicating that unique mechanisms are involved [19].

The present study aims to investigate the expressions of autophagic genes and proteins during blastocysts development and differentiation.

Materials and methods

Patients and study design

This study is a prospective cohort study approved by the Clinical Trial Ethical Committee of the Faculty of Medicine, Alexandria University, following the ethical standards of scientific research (Registration no. 0303721). In total, 30 females aged 30.13 ± 4.83 years (Table 1) undergoing an intracytoplasmic sperm injection (ICSI) cycle in a private IVF center were enrolled. The inclusion criteria were as follows: good responder patients with normal male partners, unexplained infertility, and embryo transfer on day 5. The exclusion criteria were as follows: patients with azoospermia, cryopreserved semen samples, embryo transfer on day 3, cancelation of embryo transfer due to failed fertilization, or development of embryo degeneration after biopsy.

Table 1.

Patients and ICSI cycles demographic characteristics (n = 30)

Demographic characteristics and cycle details	30 females (82 leftover embryos tested)
Maternal age (years)	30.13 ± 4.83
Paternal age (years)	36.10 ± 5.50
Maternal BMI (kg/m2)	23.50 ± 5.10
AMH, ng/mL	2.4 ± 0.8
Final estradiol level	2500 ± 900
Days of stimulation	10.4 ± 2.2
Oocytes retrieved	17.33 ± 7.81
Number of mature oocytes	13.70 ± 6.65
Number of normal fertilized oocytes (2PN)	11.7 ± 6.30
Number of day 3 embryos	10.40 ± 5.16
Number of day 5 embryos	5.92 ± 3.38
Number of previous ICSI trials	1.40 ± 1.54
Sperm concentration, million/mL	30 ± 70
Sperm motility % (a, b, c)	60 ± 15.50
Normal sperm morphology %	5 ± 15

All patients signed consent forms between March 2018 and November 2019. The embryos used in this study were donated as leftover embryos by 30 patients who had ICSI after embryo transfer and freezing extra embryos. Developed embryos on day 5 were chosen when patients refused to freeze extra straws due to financial issues, whereas compact arrested embryos on day 5 were selected when patients decided to discard them and refused to give a chance for incubation on day 6 to examine delayed development. Developed and arrested leftover embryos were collected from each patient; both groups included sibling embryos, as shown in Fig. 1. All patients signed a consent form indicating their willingness to participate in scientific research with their leftover embryos. Leftover embryos were not transferred and were only used to study the effects of autophagy activity on blastocyst development and differentiation.

Fig. 1.

Study design

A sample size of 34 embryos per group (number of groups = 2; total sample size = 68) was adequate to detect a standardized effect size of 0.825 (minimum difference in the mean grade of the embryo) of the primary outcome [20, 21] as statistically significant with 90% power and at a significance level of 95% (accepted alpha error = 0.05). The sample size was calculated using GPower version 3.1.9.2 [22].

In this study, 82 leftover embryos were developed by ICSI and were incubated for 115 h (day 5) in culture in the Labotect C200 triple-gas incubator (Germany). On day 5, embryos were divided into two groups based on their developmental structure and blastocoele formation. Group D (developed embryos, n = 49) included embryos developed into blastocysts with blastocoele expansion graded from 2 to 5, trophectoderm, and inner cell mass graded as A or B according to the Gardner blastocyst grading system. On the other hand, group A (arrested embryos, n = 33) comprises embryos that failed to form a blastocoel and remained compact. These embryos were used to investigate the expressions of autophagic genes and proteins.

Ovarian stimulation

The protocols for controlled ovarian hyperstimulation (COH) were selected based on the serum antimullerian hormone (AMH) levels, antral follicle count, and response to previous stimulation. The pituitary downregulation was regulated using the gonadotropin-releasing hormone (GnRH) agonist or antagonist protocol. In this study, follicle-stimulating hormone (FSH) was used alone or combined with human menopausal gonadotropin (hMG) to stimulate follicular growth. According to the COH protocol, final maturation was triggered with human chorionic gonadotropin (hCG) or GnRH agonist in the antagonist protocol when at least three follicles reached a mean diameter of > 18 mm. After 36 h, all patients underwent ultrasound-guided transvaginal oocyte retrieval.

Seminal sample processing for ICSI

All semen samples were collected 1 h before oocyte retrieval time, liquified for up to 30 min, and processed using the sperm gradient method. Then, the supernatant was removed; 1 mL of fresh medium was layered carefully over the pellet and left for 15–30 min to allow the motile sperm to swim up. Finally, the supernatant was collected, and the final preparation was evaluated. All of the included semen samples had normal sperm parameters and were processed using the same method.

Oocyte retrieval, embryo incubation, and culture conditions

After 36 h of hCG injection, oocytes were collected and washed with a pre-equilibrated Global Total for Fertilization medium (LifeGlobal Group, Denmark). After incubating the oocytes for at least 20 min, they were placed in hyaluronidase solution (80 IU/mL) (SAGE, Denmark) to remove the cumulus cells. Subsequently, the oocytes were immediately placed in a clean drop of fertilization medium supplemented with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and human serum albumin (HSA). All the remaining coronal cells were removed mechanically using a fine-bore glass pipette, and then denuded oocytes were incubated at least 30 min before injection. Oocytes that had extruded a first polar body (metaphase II) were subjected to ICSI using conventional methods. ICSI was performed using an RI Integra Ti manipulator (Origio, Denmark) and an inverted microscope (Olympus Cooperation, Hamburg).

Injected oocytes were washed and cultured as groups (5 embryos/droplet) in a triple-gas incubator (Labotect C200, Germany) in Global Total medium (50 μL/droplet) (LifeGlobal) under paraffin oil, pre-equilibrated overnight at 37 °C, 5.0% CO₂, and 5.0% O₂ atmosphere. After 115 h of incubation, embryos were subsequently divided into two groups based on their developmental capacity to form blastocoele: group D, embryos developed into blastocyst stage, including those that developed to blastocysts with blastocoele expansion graded from 2 to 5, trophectoderm, and inner cell mass graded as A or B according to the Gardner blastocyst grading system; group A, arrested embryos, which remained compact. Embryos were biopsied, and biopsied cells and embryos were examined to determine the following:

1. Autophagy protein levels using leftover biopsied embryos: mTOR, beclin-1, and PI3K3 levels using the enzyme-linked immunosorbent assay (ELISA) technique

2. Autophagy gene expressions using biopsied cells: LC3, beclin-1, and eukaryotic translation initiation factor 4E (eIF4E) levels using the reverse transcription-polymerase chain reaction (RT-PCR) technique

Embryo biopsy protocols

Embryos were placed in a culture dish containing 8–10 microdroplets of HEPES-buffered Ca-Mg-free media (Global w/HEPES; CooperSurgical) overlaid with paraffin oil (LifeGlobal). In the case of group D embryos, a piece of 4–8 nucleated trophectodermal cells according to degree of blastocoele expansion away from the inner cell mass was aspirated. On the other hand, in group A, 2–3 nucleated blastomeres were aspirated after removing cell junctions and divalent cations from the culture medium by incubating it in Ca-Mg-free biopsy media for at least 1 min before aspiration. Aspiration was done using a biopsy pipette (internal diameter, 40 mm), and dissection was performed with 3–4 laser pulses using a laser system (RI Saturn 5 Laser System).

Determination of autophagy protein level (mTOR, beclin-1, and PI3K3) levels using the ELISA Technique

In the assay, the quantitative sandwich enzyme immunoassay technique was used. Leftover biopsied embryos were homogenized individually in 50 mM Tris–HCl buffer (pH 7.5) containing 0.15 M NaCl, 10 mM CaCl₂, and 0.02% Brij 35. The supernatants were prepared by centrifugation at 10,000 × g, and protein concentrations were measured according to the manufacturer’s instruction of Novus Biologicals Company (beclin-1 and mTOR) and Cloud-Clone Corp. (PI3K3). Separate microplates were precoated with antibodies specific to human mTOR, beclin-1, and PI3K3. After briefly spinning the vial of standard, 1 mL of biopsied embryo diluent was added to prepare a 40-pg/mL standard solution. Serial dilutions of standard recombinant mTOR, beclin-1, or PI3K3 were prepared separately, and sample diluent buffer served as the zero standards (0 pg/mL). In duplicate wells, 100 μL of each standard and sample were added to appropriate wells. Afterward, the plate was covered and incubated for 2 h at 37 °C. The solution was discarded, and 100 µL of biotin antibody (1 ×) was added to all wells. The microwell strips were covered and incubated for 1 h at 37 °C with gentle shaking. After discarding the solution, the wells were washed three times with a washing buffer. After the last wash, any remaining wash buffer was removed by decanting. The plate was inverted and blotted against clean paper towels. A volume of 100 µL of HRP-Avidin (1 ×) was added to all wells. The microwell strips were covered and incubated for 1 h at 37 °C. The solution was discarded, and the wells were washed with a washing buffer five times. A volume of 90 µL of substrate reagent was then added to each well and incubated for 30 min at 37 °C in the dark. Finally, 50 µL of stop solution was added to each well, and the color intensity was immediately measured at 450 nm [23].

Determination of autophagy gene expressions (LC3, beclin-1, and eIF4E) levels using reverse transcription-polymerase chain reaction (RT-PCR) technique

The biopsied cells were washed in 3 small drops (~ 15–20 µL) of phosphate-buffered saline (PBS) and then loaded into RNase-DNase polymerase chain reaction tubes in ~ 3 µL PBS. First, RNA was extracted using a denaturing solution that facilitates the lysis of fatty tissues and inhibits RNases [24]. Subsequently, RNA quantification was done in two steps. First, RNA was reverse-transcribed into cDNA as follows: template RNA (1 µL) was mixed with 1 µL of Oligo (dT)₁₈ Primer and completed to 12 µL with R6, then denatured for 5 min at 65 °C, and finally cooled on ice. Denatured RNA was gently mixed with reaction master mix (4 µL R3, 1 µL RNase inhibitor, 2 µL R4 mix, 1 µL R1, and 12 µL R6) and incubated for 60 min at 42 °C. The reaction was terminated by heating at 70 °C for 5 min. Then, quantitative real-time PCR was performed. The PCR master mix was thoroughly mixed and dispensed appropriately into PCR tubes, and then the set of each primer (Table 2), template cDNA, and water were added, as shown in Table 3 [25]. PCR tubes were capped and inverted several times to mix, and then centrifuged tubes were loaded into the real-time PCR instrument, and the run was started using the mentioned cycling conditions listed in Table 4. RNA extraction and the measurement of the relative gene expressions of EIF4E, beclin-1, and LC3II using real-time PCR were repeated two times for each group sample.

Table 2.

Primers and the corresponding annealing temperatures (AT)

Primer	Sequence	AT (°C)
EIF4E (F)	5′-TGGCGACTGTCGAACCG-3′	60
EIF4E (R)	5′-AGATTCCGTTTTCTCCTCTTCTGTAG-3′	60
Beclin-1 (F)	5′-GGCTGAGAGACTGGATCAGG-3′	60
Beclin-1 (R)	5′-CTGCGTCTGGGCATAACG-3′	60
LC3II (F)	5′-GAGAAGCAGCTTCCTGTTCTGG-3′	60
LC3II (R)	5′-GTGTCCGTTCACCAACAGGAAG-3′	60
GAPD (F)	5′-GGACTGACCTGCCGTCTAG-3′	60
GAPD (R)	5′-TAGCCCAGGATGCCCTTGAG-3′	60

Table 3.

PCR reaction mixture

SYBR SELECT MASTER MIX	12.5 µL
FORWARD PRIMER	0.3 µM
REVERSE PRIMER	0.3 µM
TEMPLATE CDNA	≤ 500 NG
WATER, NUCLEASE FREE	TO 25 µL
TOTAL VOLUME	25 µL

Table 4.

Cycling conditions

Step	Temperature, °C	Time	Number of cycles
Polymerase activation	95	10 min	1
Denaturation	95	5 s	30–45
Annealing and elongation	60	30 s

Real-time PCR data analysis

The general process for analyzing the data from gene expression assays involves the following: the comparative cycle threshold ΔΔC_T method was used for calculating the relative quantitation of the gene expression (RQ) as follows [26]:

$${\mathrm{\Delta }C}_T=C_{T}ofthestudiedgene-C_{T}ofthecontrolgene$$

$${\mathrm{\Delta }\mathrm{\Delta }C}_T={\mathrm{\Delta }C}_T-\mathrm{\Delta }calibrator$$

where Δ calibrator = mean C_T of the control gene − mean C_T of the control normalized gene.

$$\mathrm{Fold}\mathrm{change}\left(\mathrm{RQ}\right)={2^{-\mathrm{\Delta }\mathrm{\Delta }C}}_T$$

C_T values were calculated using the StepOne v2.2.1 software (Applied Biosystems, USA). The comparative cycle threshold (ΔC_T), ∆∆C_T, and the fold change = 2^−ΔΔC_T were calculated using Microsoft Excel.

Statistical analysis

Data were fed into the computer and analyzed using IBM SPSS software version 20.0 (Armonk, NY: IBM Corp). A paired-sample t-test was conducted to determine the significance difference between developed and arrested groups for protein levels and gene expression folds of autophagy and apoptotic regulating biomarkers. Quantitative data were described using mean and standard deviation, and the significance of the obtained results was determined at the 5% level.

Results

Between March 2018 and November 2019, we studied a total of 82 leftover embryos from 30 ICSI cycles. Thirty females aged 30.13 ± 4.83 years underwent the ICSI cycle, and only one cycle per patient was included in the study. The demographic characteristics of the involved patients are shown in Table 1.

Embryos cultured in a triple-gas incubator (n = 82) were evaluated on whether they developed to the blastocyst stage on day 5. After 115 h of incubation, 82 embryos were subsequently divided into two groups based on their developmental capacity to form blastocoele: group D (n = 49), developed embryos to the blastocyst stage, including those with blastocoele expansion graded from 2 to 5, trophectoderm, and inner cell mass graded as A or B according to the Gardner blastocyst grading system; group A (n = 33), arrested embryos that remained compact. The two groups were compared in terms of their autophagy activity.

When the autophagy protein levels in the two groups were compared, the beclin-1 protein level was significantly higher in group D embryos (3.8 ± 0.028 ng/mL) than in group A embryos (3.14 ± 0.05 ng/mL, P = 0.0001), as shown in Table 5.

Table 5.

The protein levels of autophagy and apoptotic regulating biomarkers

	Parameters	Developed N = 49	Arrested N = 33	P
Protein level (ng/mL)	mTOR	1.13 ± 0.053	1.23 ± 0.09*	0.007
	Beclin-1	3.8 ± 0.028	3.14 ± 0.05*	0.0001
	PIK3C3	18.6 ± 1.4	24.5 ± 1.5*	0.0001

P: P value for comparing between the two studied groups

^*Statistically significant at P ≤ 0.05

Conversely, mTOR and PIK3C3 proteins levels were significantly higher in group A (1.23 ± 0.09 and 24.5 ± 1.5, respectively) than those in group D (1.13 ± 0.053 and 18.6 ± 1.4, respectively; P = 0.007 and P = 0.0001) (Table 5).

Group D showed a significantly higher gene expression of LC3 (1.12 ± 0.51) than that in group A (0.72 ± 0.17, P = 0.03) and a statistically significant higher level of beclin-1 (1.43 ± 0.33) than that in group A (0.35 ± 0.12, P = 0.0001) (Table 6).

Table 6.

The gene expression folds of autophagy and apoptotic regulating biomarkers

	Parameters	Developed N = 49	Arrested N = 33	P
Relative gene expression fold	LC3	1.12 ± 0.51	0.72 ± 0.17*	0.03
	Beclin-1	1.43 ± 0.33	0.35 ± 0.12*	0.0001
	elf4e	0.32 ± 0.07	4.38 ± 1.16*	0.0001

P: P value for comparing between the two studied groups

^*Statistically significant at P ≤ 0.05

In addition, the relative gene expression of eIF4E in group D (0.32 ± 0.07 ng/mL) was significantly lower than its level in group A (4.38 ± 1.16 ng/mL, P = 0.0001) (Table 6).

Discussion

The molecular mechanisms and events that determine embryo viability between fertilization and embryo transfer are significant. As a result, developing a new method that relies on genetic integrity to reflect embryo viability is critical. Thus, a method that predicts embryo quality is becoming increasingly important for clinical applications to reduce the time to successful ART [27], avoid the high cost of repeated cycles, and avoid the psychological trauma of repeated failure.

Consequently, this study was designed to examine the expressions of autophagic genes and proteins during embryo development and differentiation within the two studied groups, as autophagy is a molecular mechanism involved in the preimplantation embryo development process [28]. The term “preimplantation embryo development” refers to the growth period from the zygotic stage to the blastocyst stage and the embryo’s attachment to the uterine wall [29]. These different stages of development involve many molecular mechanisms, which must be strictly regulated to maximize embryonic developmental potential.

Tsukamoto et al. demonstrated that autophagy machinery is activated after fertilization and is a fertilization-dependent pathway [3]. They revealed that the activation of autophagy immediately after fertilization is inversely related to the activation of the mTOR signaling pathway [30]. This initial activation of autophagy plays a significant role in reprogramming cell fate and further embryo development [31] by inducing zygotic genome activation (ZGA), where maternal proteins are degraded and zygotic proteins are synthesized [13].

In fertilized oocytes, autophagy is induced by the activation of the PIK3C3 signaling pathway during embryo development, resulting in increased levels of corresponding gene expressions and protein biosynthesis of beclin-1, PIK3C3, and LC3 and the inhibition of the mTOR signaling pathway, which leads to decreased levels of expression and protein biosynthesis of related genes, such as mTOR and eIF4E [30, 32].

Furthermore, activating autophagy at late developmental stages reduces significantly the PIK3C3 complex concentration, which is essential for autophagosome formation within developed embryos. Since beclin-1 is a significant component of the PIK3C3 complex [33], the high levels of beclin-1 protein and beclin-1 gene expressions, as shown in our results, were due to the degradation of the PIK3C3 complex, which is considered a good marker for better embryo development at late stages.

In contrast, the eIF4E relative gene expression was significantly decreased in the developed embryos, whereas it was significantly increased in arrested ones. This finding confirms our hypothesis that the mTOR signaling pathway is reduced or inhibited due to increased autophagy activity within developed viable embryos at late developmental stages. Furthermore, arrested embryos may show impaired autophagy activity, which is replaced with activation of the mTOR pathway as indicated by elevation of eIF4E expression and high mTOR protein level. This finding agrees with the fact that the transcription factor eIF4E plays a vital role in controlling cell proliferation and regulates the mTOR signaling pathway activation via phosphorylation and inhibition of S6K [34].

Since eIF4E-dependent transcription was modulated only during the early stages of the cell cycle, several experimental studies observed that the levels of eIF4E are rate-limiting for the G1/S transition [35]. This finding suggests that the roles of eIF4E and mTOR signaling pathway activation are essential in early developmental events, whereas the mechanism of autophagy activation is preferred for later developmental events.

Autophagy is highly activated within 4 h of fertilization [31], beginning with the engulfment of the targeted components, including macromolecules (proteins, glycogens, lipids, and nucleotides) and organelles (e.g., mitochondria, peroxisomes, and endoplasmic reticulum) in double membrane-bound autophagosomes; thus, cytosolic LC3 (LC3-I) is converted to a membrane-bound LC3 (LC3-II) via lipidation. LC3-II is stably associated with the autophagosome until lysosome fusion; once fusion occurs, LC3-II itself is degraded. Consequently, the turnover of LC3 protein is an indicator of autophagic activity [31, 36]. These findings are consistent with those of previous studies reporting that microinjection of LC3 fused with green fluorescence protein (GFP-LC3) induces normal embryonic development before implantation, which is proved by complete degradation of GFP-LC3 protein in embryos, indicating autophagy activation [6, 37]. The immunofluorescent LC3 counts per cell reflect the number of autophagosomes at each embryonic stage. This finding was supported by a recent study that investigated the mechanistic effects of free fatty acid treatment on autophagy and preimplantation mouse embryo development. It showed a gradual increase of LC3-II expression during the development progression from the one-cell stage to the blastocyst stage. At the same time, beclin-1 levels were significantly increased at the blastocyst stage [38].

In addition, it was reported that at late-stage blastocysts of embryonic development, both trophoblast and inner cell mass (ICM) express a high level of different autophagic markers [39].

In the current study, we found similar results for LC3 and beclin-1 expression as autophagy markers, which increase in blastocysts but decrease in arrested embryos at the compact stage, indicating that embryos with autophagy have strong developmental potential and high survival capacity.

According to the existing evidence, autophagy activation occurs at a variable rate. First, it is induced immediately after fertilization, then suppressed until the two-cell stage, and then increased gradually during preimplantation development to the late blastocyst stage. This variable rhythm of induction should be well regulated [40], as the imbalance in autophagic mechanisms can jeopardize embryo developmental potential [13], cell fate determination [41], and cell survival against stress or nutrient-deprived environments [42].

These findings are supported by the study of Lee et al., who applied autophagy modulators to investigate their effects on the developmental potential of preimplantation mouse embryos. They concluded that any disruption of either autophagy inducers or inhibitors would negatively impact the normal rhythm of autophagy activation during different developmental stages, resulting in apoptosis and disruption of blastocyst development [40].

Song et al. modulated the autophagy pathway in bovine embryos through the acute induction of autophagy with rapamycin, which significantly increased blastocyst development, trophectoderm cell number, and the viability of in vitro–produced bovine embryos. They also reported that short-term induction of autophagy alleviated bovine embryos from ER stress, which may have contributed to the improvement in blastocyst viability observed in the study [43].

Based on the previous findings, our data suggest that autophagy was impaired within arrested embryos (group A), as it demonstrated low expression of both LC3 and beclin-1 and could not develop further and form blastocoele.

Although no specific mechanism has been identified to explain how autophagy enhances embryo developmental potential [44], the current study investigated autophagy activity using different methods, namely, the analysis of autophagic gene expression via mRNA transcript analysis and the analysis of protein expression of autophagic markers.

However, one of our study’s limitations is the small sample size, which could be attributed to the high cost of genetic testing. A patient-specific assortment analysis must be performed to demonstrate that the differences discovered are not patient specific. Furthermore, our study only correlates embryo development with the various parameters involved. Therefore, future studies should follow a different approach to associate these parameters with more clinical information about the medical history of the involved patients and more ICSI outcome parameters, such as clinical pregnancy rate, live birth rate, and incidence of abortion.

The current findings may encourage further research and evaluation of autophagy function during preimplantation embryo development, as well as expand our understanding of the autophagy mechanism during this time to determine the best way to modulate autophagy activity to benefit embryonic development. We also recommend conducting studies to evaluate the possibility of releasing any autophagic products in the spent culture media of the preimplantation embryo during different stages to introduce autophagy modulators to the culture media to enhance embryo development competence. Further studies on gene integrity and corresponding protein analysis are required to fully understand the molecular mechanisms and cellular functions of developing embryos and find a method or additives for gene editing or enhancing a better molecular mechanism during the culture period of incubated embryos.

Finally, autophagic activity within embryos should be regulated and activated to maintain and improve cell differentiation during embryo late developmental stages. Moreover, embryos express a lower level of autophagic gene expression, are unable to form blastocoele at late embryonic developmental stages, and remain compact.

Conclusions

Among the studied autophagy genes and proteins, beclin-1 and LC3 expressions are essential and highly activated within embryos with high developmental potential that developed into blastocysts on day 5. Conversely, the elevation of mTOR protein level and high expression of eIF4E with arrested embryos indicate the activation of the mTOR signaling pathway, which may diminish cell differentiation to more advanced stages (blastocyst formation) and negatively impact the ICSI outcomes.

Data Availability

The primary data for this study are available from the authors on direct request.

Declarations

Conflict of interest

The author declares no competing interests.