Recent potential interest has arisen in the use of enucleated GV, MI and MII oocytes as recipientscytoplasts for GV nucleus, embryonic and somatic nuclei in addition to germ cells in the last couple of decades for different purposes
(Mohammed et al., 2019; Melo et al., 2022). The stages of enucleated oocyte include germinal vesicle, metaphase I and metaphase II stage oocytes. In addition, the types of introduced cells or nuclei include embryonic cells, somatic cells, pluripotent stem cells and germ cells. Furthermore, the cell cycle stages of donor nuclei include G0/G1, S-phase and G2/M stages (
Mohammed 2008,
2014a,
b).
Several techniques were adapted to increase the efficiency of creation artificial oocytes and their further embryo developmental competence and the resulting produced offspring (
Al Jubran et al., 2023; Al Zeidi et al., 2022a,
b). The adapted techniques include the recipient cytoplast
(Mohammed et al., 2006a,b, Mohammed et al., 2008, 2010), the technique of enucleation
(Mohammed et al., 2019), activation method
(Montgomery et al., 2022), nucleolus transfer (
Benc et al., 2019)
. The reconstructed “artificial oocytes” seems to be an interesting model for studying meiotic progression of the introduced nuclei or cells, reproductive disorder treatments or for embryonic/somatic cloning. Therefore, the recipient GV, MI and MII cytoplasts and donor GV, embryonic and somatic nuclei at G0/G1, S-phase and G2/M stages affect the maturation or activation efficiency in addition to the developmental competence of the resulting embryos. Furthermore, the factors that increase the quality of recipient cytoplasts were presumably increase the quality of reconstructed oocytes and their developmental competence as
in vivo cytoplast transfer and culture conditions
(Mohammed et al., 2005). Therefore, this review is designed to collect and discuss the progress in creation of “artificial oocytes” in addition to factors affecting the developmental competence of reconstructed oocytes.
The current study was carried out according to the procedure approved by Deanship of Scientific Research, King Faisal University, Saudi Arabi from August to February 2024. Ethical approval for this study was not required and the data were obtained from science direct databases.
Artificial oocytes are required for infertility treatment of people around the world and the number increasing every year
(Oqani et al., 2022). In recent years, assisted reproductive technologies have been widely developed (
Pavlović et al., 2024;
Desy and Marre 2024).
Differences among mammalian oocytes
Mammalian oocytes are similar in some ways, but there are also some key differences between them (
Albertini 2015). These differences are important to consider when using these models to investigate reconstruction of oocytes. The differences will be concentrated on mouse, bovine in addition to human oocytes. The diameter of mouse oocytes is about 70 micrometers, bovine oocytes is ~120 micrometers while human oocytes is ~100 micrometers. In addition, mouse oocytes have a spherical shape, while bovine and human oocytes have a more oval shape
(Mohammed et al., 2022). Bovine oocytes also have a unique feature of containing lipid droplets making the germinal vesicle (GV) invisible in the cytoplasm versus mouse and human oocytes, which contain visible GV nucleus. The timing of maturation for bovine and human oocytes lasts 24h whereas that of mouse oocytes lasts 17h.
Protein synthetic patterns in immature and mature mammalian oocytes were recorded on cytoplasmic lattices (
Schultz and Wassarman 1977;
Goff et al., 2001; Jentoft et al., 2023) to store proteins for the early embryo. Protein synthesis is required for germinal vesicle breakdown in bovine oocytes
(Li et al., 2021), human oocytes (
Cha and Chian 1998), but not in mice
(Li et al., 2022). In addition, the cumulus cells is tightly attached to bovine and human oocytes compared to those loosely attached to mouse human oocytes
(Mohammed et al., 2019).
Cumulus-enclosed germinal vesicle (GV) oocyte aspiration or collection
Cumulus-enclosed GV oocyte could be aspirated or collected from slaughterhouse materials or live females
(Mohammed et al., 2005; Kagawa et al., 2022). There are three methods for collecting oocytes from slaughterhouse materials include aspiration, dissection and slicing (
Gordon 2003). There are two main methods of cumulus germinal vesicle (GV) oocyte collection include transvaginal ultrasound-guided oocyte retrieval and laparoscopic oocyte retrieval. The oocyte could be collected form pre-pubertal
(Currin et al., 2021), pubertal and pregnant animals (
Pongsuthirak and Vutyavanich, 2018).
Exposure to compounds in relation to reproductive dysfunctions
Endocrine-disrupting chemicals can interfere with the normal functioning of the endocrine system, which is responsible for synthesizing and regulating hormone production
(Santos et al., 2014; Chen et al., 2023). These compounds can be found in a wide range of products, including pesticides, herbicides, plastics, personal care products and food additives. Exposure of animals and human as well can occur through variety of routes, including ingestion, inhalation and skin contact. The symptoms of reproductive dysfunctions might include interfere with the production and release of estrogen hormone, estrous or menstrual cycle irregularities, endometriosis, polycystic ovary syndrome (Fig 1). In addition, the compounds have also been linked to an increased risk of miscarriage, stillbirth and premature birth. The effects of toxic compounds on reproductive health are depending on the type of compound, the dose and the timing of exposure
(Santos et al., 2014).
Artificial oocytes for dysfunctions’ repair
Abnormalities of mammalian oocytes could be nucleus or cytoplasmic in natures due to different factors as chromosome misalignment
(Cui et al., 2005; Tatíèková et al., 2023) or ultrastructure dysfunctions including mitochondrial dysfunction, spindle disorganization
(Jiang et al., 2021; Xing et al., 2021) and mitochondrial and glucose metabolic dysfunctions in granulosa cells
(Zhang et al., 2022) and nucleolus dysfunction
(Fulka et al., 2004). Various trials were applied to repair dysfunction oocytes
(Jiang et al., 2021; Xing et al., 2021; Zhang et al., 2022). The trials include the recipient cytoplasts (GV, MI and MII stages)
(Mohammed et al., 2019, Al Jubran et al., 2023), the donor nuclei and their cell cycle stage (G1, S-phase and G2/M stages) in addition to the modification of
in vitro culture media and conditions
(Mohammed et al., 2005; Sharma et al., 2024).
The question arose, is it possible for the reconstructed oocytes to repair dysfunction oocytes through different combinations of recipient cytoplasts and donor nuclei in addition to the culture conditions. The low efficiency of success in cloning of different mammalian species with MII cytoplasts as recipient of embryonic/somatic nuclei gave reason for 1) new micromanipulation techniques 2) modified culture systems of oocytes or 3) new combination between the recipient cytoplasts and donor nuclei
(Mohammed et al., 2019; Al Jubran et al., 2023).
Thus, for better understanding the background of difficulties in co-operation between foreign nucleus and cytoplasm in reconstructed oocytes, the development of new micromanipulation techniques and/or new culture systems of oocytes are required which might also help to overcome the existing problems and to increase the developmental competence of resulting embryos
(Mohammed et al., 2008, 2010,
2019).
Obtaining recipient enucleated cytoplasts
The efficiency of obtaining recipient enucleated cytoplasts is dependent on the oocyte stage and the technique used
(Mohammed et al., 2008, 2010,
2019; Al
Zeidi et al., 2022a,b; Al Jubran et al., 2023). There are two main methods for obtaining recipient enucleated cytoplasts either the manual enucleation or the chemical enucleation. The oocytes are enucleated at germinal vesicle (GV) (Fig 2), metaphase I (MI) and metaphase II (MII) stage (Fig 3).
To the best of our knowledge, the success in assisted reproduction has been achieved using MII cytoplasts (
Wilmut et al., 1997;
Son et al., 2021). Therefore, the idea is to introduce the donor nuclei at earlier stage of development “GV or MI oocytes”. Hence, techniques were adapted for enucleation of those oocytes. The MI cytoplasts gave success with germ cell donor nuclei
(Ogura et al., 1997; Tao et al., 2022). On the other hand, removal the whole GV from oocyte upon enucleated resulted in lack of cytoplasm from nuclear materials, nuclear sap and nucleolus. Upon reconstruction with somatic nuclei, the development has been blocked at one cell-stage and the nucleus without nucleoli. Therefore, our adapted techniques “selective enucleation of GV oocytes” is targeted to leave in the GV cytoplast the nuclear sap. In addition, we were able to enucleated GV oocytes cumulus-enclosed, which promote developmental competence upon reconstruction with embryonic/somatic cells. The developmental competence exceeded one cell-stage embryos to hatching/hatched blastocyst
(Mohammed et al., 2008, 2010). In my point of view, such technique needs further studies to apply in other species with different donor nuclei.
Type of donor cells and the cycle stage for reconstruction oocytes
The types of donor cells and their cell cycle-stage for reconstruction oocytes are important factors that affect the success of either maturation or further embryonic development (
Al Jubran et al., 2023). The most common types of donor cells used for oocyte reconstruction of oocytes are 1) embryonic cells such as blastomeres or inner cell mass cells, 2) somatic cells such as skin or blood cells, granulosa cells
(Nguyen et al., 2021) in addition to polar body for generation of functional human oocytes
(Ma et al., 2017).
Regarding to the cell cycle stage of donor nuclei, the synchronization between the donor cell and recipient cytoplast is necessitated for success in maturation and embryonic development
(Wani et al., 2018). The donor cells should be in the G0/G1 phase of the cell cycle when they are introduced into the enucleated MII oocyte
(Mohammed et al., 2019). This is the resting phase of the cell cycle and it is the time when the DNA is most stable and reduced the risk of chromosomal abnormalities. In addition, the G0/G1 phase is the longest phase of the cell cycle, which gives the donor cell more time to reprogram to the embryonic state. With the emergence of GV and MI cytoplast, S-phase and G2/M phase were investigated for maturation and further embryonic development. The s-phase donor nuclei showed the matured oocytes with chromosomal abnormalities in addition to blocked embryos at one-cell stage. Overall, the the type of donor cells and the cell cycle stage are important factors that affect the success of oocyte reconstruction. G0/G1 phase donor cells are the preferred choice, but S -phase and G2/M phase donor cells may be used in some cases.
Developmental potential of artificial oocyte after activation or fertilization
Oocyte activation of matured oocytes is a fascinating and crucial process for further embryonic development. Out of sperm-mediated oocyte activation, the methods of oocyte activation used in assisted reproductive technologies include electrical or chemical stimulation
(Mohammed et al., 2019; AlJubran et al., 2023). Human oocyte activation over round spermatid injection is resulting healthy babies
(Tao et al., 2022).
The development of artificial oocytes to embryos and offspring is a rapidly evolving field and there is significant potential for this technology to have a major impact on reproductive medicine. Development of artificial oocyte after activation or fertilization is a complex process include a rise in intracellular calcium levels, completion of meiosis II and completion of meiosis II, decondensation of the chromosomes and formation of the female and/or male pronuclei and proceed to embryo cleavages in definite time
(Mohammed et al., 2005, 2008,
2010). To the best of our knowledge, cytoplasts of MI reconstructed with germ cells and MII cytoplasts reconstructed embryonic/somatic cells were developed to full term offspring whereas GV cytoplasts reconstructed embryonic/somatic cells were developed only to hatching/hatched blastocysts
(Mohammed et al., 2019).
