In Vitro Maturation Of Oocytes

PrefaceOur Knowledge of Reproductive medicine has been expanded rapidly since the birth of Louise Brown, the first baby to be conceived by In vitro Fertilization in 1978. Hardly a year goes by without the development of a new or a modification of an existing method of assisted reproduction.

The recent surge of interest in ovarian tissue banking and in vitro maturation of oocytes reflects the importance and the need of fertility conservation for many women facing premature ovarian failure, particularly those with cancer:

The aim of this work is to give a better under standing by reviewing and updating our knowledge in this emerging technology.

Cairo - Jan: 2002

Asem Anwar

The Oocyte

The origin & development :
The primordial germ cells are known to originate from the endoderm of the yolk sac (near the caudal end of the embryo). At this site, they can be identified as early as the end of the third week of gestation by alkaline phosphates staining. Migration of germ cells toward the genital ridge, occurs by ameboid movements, with the aid of pseudopodia.²  The route of migration along the dorsal mesentery of the hindgut is interrupted only by the required lateral crossing of the coelomid angle at the level of the genital ridge. Whereas some chemotaxis is dearly operational, the precise cellular mechanisms underlying the guidance of germ cells to the genital ridge remain uncertain. Importantly, germ cells appear unable to persist outside the genital ridge, which may thus be viewed as the only region competent to sustain gonadal development. By the same token, germ cells play an indispensable role in the induction of gonadal development. In fact, no functional gonad, is to be expected in the absence of germ cells.

On arrival at the genital ridge by the fifth week of gestation, the premeiotic germ cells are referred to as oogonia[³] During the subsequent 2 weeks of intrauterine life (weeks. 5 to 7 of gestation), the "indifferent stage," the primordial gonadal structure constitutes no more than; a bulge on the medial aspect of the urogenital ridge. This protuberance is created by proliferation of surface (coelomic) germinal epithelium, by growth of the underlying mesenchyme, and by oogonial multiplication. The oogonia total 10,000 by around 6 to 7 weeks of intrauterine life. Because meiosis and oogonial atresia are not operational, the actual number of germ cells is dictated by mitotic division at this time.

It is during this indifferent phase that the gonadal cortex and medulla are first delineated. However, short of cytogenetic evidence, the precise sexual identity of the gonadal ridge cannot be ascertained at this point. Nevertheless, the absence of testicular development beyond 7 weeks of gestation is generally considered presumptive evidence of I ovarian formation. Additional clues to the sexual identity of the gonad can be derived from the detection of oogonial meiosis at about 8 weeks of gestation because no comparable process will be observed in the testis until puberty. The sexual identity of the gonadal ridge is clear by 16 weeks of gestation, when the first primordial follicles can be - 9 Visualized.

By about 8 weeks of intrauterine life, persistent mitosis increases the total number of -oogonia to .600,000. From this point on, the oogonial endowment is subject to three simultaneous ongoing processes: mitosis, meiosis, and oogonial atresia. Stated differently, the onset of oogonial meiosis and oogonial atresia is now superimposed on oogonial mitosis. As a result of the combined impact of these processes, that is, mitosis counterbalanced by meiosis and oogonial atresia, the number of germ cells peaks at 6 to 7 × 106 by 20 weeks of gestation. At this time, two thirds of the total germ cells are intrameiotic primary oocytes; the remaining third can still be viewed as oogonial. The midgestational peak (and the post peak decline) are accounted for, if only in part, by the progressively decreasing .j rate of oogonial mitosis, a process destined to end entirely by about 7 months of intrauterine life. Equally relevant is the increasing rate of oogonial atresia, which peaks at about month 5 of gestation. During this period, regulation of the ovarian developmental process could be hypothesized to be complex, including the involvement-of peptide e growth factors.

Prom midgestation onward, relentless and irreversible attrition progressively diminishes the germ cell endowment of the gonad ultimately, some 50 years later, this is finally exhausted. For the most pan, this is accomplished through follicular atresia, rather than oogonial atresia, and begins around month 6 of gestation and continues throughout life.

In contrast, oogonial atresia (see later) is destined to end I at 7 months of intrauterine life as follicular atresia sets in.

As expected, follicular atresia does not and. cannot start c~ until follicles have formed. However, once follicular atresia .

Is motion, there is little question that it has a profound effect on germ cell endowment, given that only 1 -to 2 x lot 2erm cells are present at birth.'⁰ Remarkably, this dramatic depletion of the germ cell mass occurs during a period as short as 20 weeks. No similar rate of depletion will be seen again. Consequently, newborn females enter life still far from realizing reproductive potential, having lost as much as 80 percent of their germ cell endowment.

This decreases further to approximately 300,000 by the onset of puberty. Of these follicles, only 400 to 500 (i.e., less than 1 percent of the total) will ovulate in the course of a reproductive life span.
Between weeks 8 and 13 of fetal life, some of the oogonia depart from the mitotic cycle to enter the prophase of the first meiotic division. It is this change that marks the conversion of these cells to primary oocytes well before actual follicle formation. Meiosis (beginning at about 8 weeks of gestation) provides temporary protection from oogonial atresia, thereby allowing the germ cells to invest themselves with granulosa cells and to form primordial follicles. Accordingly, oogonia that persist beyond the seventh month of gestation and have not entered meiosis will be subject to oogonial atresia. Consequently, no oogonia are usually present at birth.

Once formed, the primary oocyte persists in prophase of the first meiotic division until the time of ovulation, when meiosis is resumed and the first polar body is formed and extruded. Although the exact cellular mechanisms responsorial for this meiotic arrest remain uncertain, it is generally presumed that a genulosa cell-derived putative meiosis inhibitor is in play. This hypothesis is based on the observation that denuded (granulosa-free) oocytes are capable of spontaneously completing meiotie maturation in vitro.

The primary oocyte is converted into a secondary oocyte by completion of the first meiotic metaphase and formation of the first polar body, before actual ovulation but after the Iuteinizin2 hormone (LH) surge. At ovulation, the secondary oocyte and the surrounding granulosa cells (cumulus oophorus) are extruded and enter the fallopian tube. If sperm penetration occurs, the secondary oocyte undergoes a second meiotic division, after which the second polar body is eliminated.


Table 3. Number or germ cells from the embryonic stage to puberty . In the reproductive years of a woman, only 400 - 500 oocytes reach ovulation

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References

1. Baker TG. A quantitative and cytological study of germ cells in human ovaries. Proc R Soc Biol 158:417, 1963.
2. Witschi E. Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal folds. Contrib Embryol 32:67, 1948.
3. Baker TO, Franchi LL. The fine structure of oogonia and oocytes - in human ovaries. 3 Cell Sci 2:2 13, 1967.
4. Ohno 5, Klinger H, Atkin N. Human oogenesis. Cytogenetics 1:42, 1962. hnm.

Oocyte Maturation : 
Oocyte maturation is defined as the reinitiating and completion of the first meiotic division, subsequent progression to metaphase II, and the nuclear and cytoplasmic processes, which become essential for fertilization and early embryo development. Oocytes are arrested in prophase I of meiosis during the fetal period. Completion of the first meiotic division takes place when oocytes have undergone extensive growth in cellular interaction with the granulosa and theca cells. The oocyte undergoes asymmetric cytokinesis and extrudes the first polar body containing a haploid chromosome complement. The first meiotic division is completed, the second meiotic division is initiated, but oocytes remain arrested in metaphase II until contact is made with a spermatozoon. The initiation of maturation in fully-grown oocytes, which are present in astral follicles, is based on the mid-cyclic onset of the luteinizing hormone (LH) surge or the external administration of human chorionic gonadotropin (HCG). Mechanisms of oocyte maturation in vivo and in vitro are still under investigation. In vitro animal models provided insight into the importance of substances affecting oocyte maturation and its inhibition, such as cAMP, calcium, cell-cycle proteins, growth factors, GnRH, gonadotropins, purines, and steroids.

Structural Changes during Oocyte Maturation

Germinal Vesicle Breakdown

The nucleus of an oocyte is the germinal vesicle (GV) (Fig. I). The most striking event of the reinitiation of meiosis is the disappearance or breakdown of the GV (GVB). The acquisition of competence to undergo GVB is a multistep or atretic degeneration of follicles, but when they are removed from their antral follicles or after removal of the entire oocyte-cumulus cell complex, spontaneous gonadotropin-independent maturation may occur in culture media as well. Within a few hours of culture in vitro fully-grown oocytes undergo complete GVB [3, 4]. GVB begins with undulations of the nuclear envelope (Fig. 2) which continues for approximately 1-2 b. These undulations may be correlated to the onset of chromosome condensation. Breaks in the nuclear envelope can be detected within 2 h, and after approximately 3 h the nuclear envelope has completely disappeared in rabbit, rat, and mouse oocytes; it is likely to be involved later in formation of the pronuclear membrane. In human beings this process initiating chromosome condensation takes 20 - 24 h. 


Meiotic Maturation and Chromosome Formation

Chromosome condensation and spindle formation are the following steps in the scheduled program of the maturing oocyte to complete meiosis. Directly subsequent to GVB - when the nuclear envelope and its inner lining, the fibrillar network of Iaminae, start to dissolve - chromosomes have moved from the center of the nucleus towards the undulating membranes, where condensation takes place. Chiasmata move to the ends of the chromosomes and chromatin becomes heterochromatic. After completion of condensation the chromosomal bivalents appear V-shaped and telocentric. They are often attached to fragments of the nuclear envelope. Being highly condensed, chromosomes become arranged in the center of the oocyte, waiting to line up on the metaphase spindle. During GVB and chromosome condensation, kinetochores and the microtubule system appear to organize the spindle formation. The spindle does not display centrioles as is typical for mitotic cells; rather, it derives from so-called pericentriolar material which forms the spindle poles during prometaphase. The spindle apparatus increases in size and moves to the periphery of the oocyte. The barrel-shaped spindle is surrounded by mitochondria, vacuoles, and granules. Metaphase I (Fig. 3) lasts for a few hours and leads to anaphase I, when chromosomal bivalents move towards the opposite ends of the spindle and the whole spindle rotates 900. During telophase I the extrusion of the first polar body is prepared. Homologous chromosomes become separated, and one half is extruded with cytoplasmic material such as mitochondria, ribosomes, and cortical granules into the perivitelline space (Fig. 4). This takes place in late telophase. The oocyte has reached metaphase II (Fig. 5). Progressive maturation beyond metaphase II marks the beginning of fertilization or indicates parthenogenetic activation of the oocyte.

Regulation of Oocyte Maturation

Gonodotropins and Intercellular Communication

In human and animal oocytes the preovulatory LH surge or the administration of human chorionic gonadotropin (hCG) induces GVB in vivo or when follicles are placed in culture. But there are no receptors for LH on the oocytes. Therefore, it is hypothesized that LB probably induces GVB indirectly by the action of granulosa cells [⁵]. LB apparently induces a block of intrafollicular communication and reduces the transfer of maturation-arresting substances to the oocyte. According to this hypothesis, LB-induced GVB is initiated the same way as spontaneous GVB when oocytes have lost their contact to cumulus and granulosa cells. It is known that oocytes enclosed in an antral follicle, when removed from their environment, do not resume meiosis. But adding gonadotropin or removing the oocyte from the follicle results in resumption of metosis.

Another hypothesis is that LH triggers production of a GVB-inducing signal in the granulosa cells being transmitted into the oocyte trough so-called gap junctions [⁶]. Gap junctions are regions of physical continuity between cellular membranes. They resemble a network connecting granulosa cells, cumulus cells, and the oocyte. The cells are metabolically and ionically coupled through the gap junctions. They consist of proteins called connections, which act as channels permitting the rapid passage of small molecules. It is assumed that LH-induced GVB, transferred through the gap-junction system, may be mediated by calcium. LH induces an increase of free inositol l,4,5-triphosphate (1P3) [⁷] and calcium in the granulosa cell. Both substances are transferred via gap junctions to the oocyte. The role of calcium will be discussed later. Follicle-stimulating hormone (FSH) is assumed to promote signals for GVB induction to a much greater extent in cumulus-enclosed oocytes than in cumulus-denuded oocytes. This would mean that maintenance of gap-junction communication between cumulus cells and the oocyte is essential for FSH stimulation of maturation [81.


The Role of Cyclic Adenosine Monaphosphate (cAMP)

Cyclic AMP, which is present in the oocyte, appears to be a candidate involved in maintenance of meiotic arrest (Fig. 6), since the mode of cAMP action is known. Cyclic AMP activates a cAMP-dependent protein kinase (PK-A). An inhibitory basal level of cAMP within the oocyte activates PK-A. The active form of the heterotetramer PK-A is the catalytic subunits after binding of cAMP to the inhibitory subunits of the PK-A complex. PK-A phosphorylates oocyte proteins which are necessary for GVB. Continuous phosphorylation of proteins maintains the nieiotic arrest. Resumption of meiosis, on the other hand, is triggered by a decrease in the inhibitory level of oocyte cAMP mediated through the action of cAMP phosphodiesterase (PDE) (Fig. 7). cAMP phosphodiesterase promotes the reassociation of the active catalytic subunits of PK-A with its regulatory subunits, which prevents PK-A from carrying on with phosphorylation. Oocyte proteins become dephosphorylated and meiotic maturation will be initiated. Some experiments indicate the involvement of different substances in regulating the oocyte cAMP levels. Spontaneous maturation of mouse oocytes does not occur in vitro in the presence of cAMP analogues such as dbcAMP or SbcAMP or phosphodiesterase inhibitors such as isobutyl methylxanthine (IBMX) and theophylline [9, 101. Forskolin maintains oocytes in meiotic arrest via direct stimulation of adenylate cyclase to increase the cAMP level [^{11, 12}]. Phosphodiesterase activity to increase or decrease cAMP levels in the oocyte is responsible for the onset or arrest of maturation and is modulated itself by calmodulin. The origin of active cAMP is still unclear. Cumulus cell-free oocytes produce cAMP on stimulation by forskolin [^13], but forskolin treatment produces only a delay of GVB. Thus, higher amounts of cAMP are needed to maintain meiotic arrest. It is still unclear if sufficient cAMP is created by the oocyte itself or originates in granulosa cells. As mentioned above, oocytes are coupled to granulosa and cumulus cells by gap junctions. Gap junctions allow diffusion of small molecules from one cell to another, so cAMP may possibly diffuse from granulosa cells to the oocyte. Another theory is that stimulated granulosa cells promote the oocyte to produce cAMP itself.

Furthermore, it is suggested that other types of protein kinases (PK) may be involved in the regulation of oocyte maturation. Stimulation of the P1K-C system with phorbol esters and diacylglycerol also results in a transient inhibition of GVB [⁴]. Purines. 

Although cAMP seems to play a major role in oocyte maturation andmeiotic arrest, other substances present in the follicle are also likely to participate in meiotic arrest. Hypoxanthine and adenosine were detected in mouse oocytes and follicu]ar fluid [^{14, 15}]. Hypoxanthine inhibits GVB when applied to denuded mouse oocytes. Moreover, the inhibitory effect of purines is increased in cumulus cell-enclosed oocytes. This observation suggests that the intercellular gap-junction pathway to the oocyte provides regulation of uptake and metabolism of those putative granulosa cell-generated substances. Adenosine displays a transiently inhibitory effect on GVB similar to that of forskolin in culture, but when augmented by hypoxanthine the inhibitory effect persists. Direct injection of adenosine into the oocyte does not show any effect. The theory is that hypoxanthine inhibits cAMP phosphodiesterase and therefore prevents hydrolysis of oocyte cAMP. Adenosine promotes the cAMP formation, acting at the oocyte surface by stimulating the adenylate cyclase [¹⁶]. Purines may also be involved, to regulate the suppressive effects of guanyl compounds on oocyte maturation. Guanosine monophosphate (GMP), a product of the conversion of inosine rnonophosphate (IMP), plays an essential role in the maintenance of xneiotic arrest. GMP is itself converted ta guanosine triphosphate (GTP), which intcrocta subsequently with G-proteins present on the oolemma and membranes of the cumulus cells, GTP is able to suppress GVB as well, as binding to G-proteins mediates an increase of cAMP levels within the oocyte.

Maturation-Promoting Factor

Maturation-promoting factor (MPP) is a cytopiasmic factor and was detected in - both mitotic and meiotic cells. Another name for this substance is M-phase promoting factor, owing to its ability to promote a G2- to an M-phase transition [¹⁷]. MPF apparently regulates chromosome condensation and nuclear envelope breakdown. MPF was initially demonstrated by injecotion of cytoplasm from progesterone-treated oocytes into untreated recipients, which resulted in maturation of the untreated oocytes [¹⁸]. It should be noted that progesterone plays a key role in the onset of oocyte maturation in amphibians. The formation of MPF seems to be independent of nuclear activity. It was demonstrated that treating enucleated Xenopus oocytes with progesterone and injecting their cytoplasm into nucleated non-progesterone-treated oocytes resulted in MPF activity in these cells, with subsequent maturation [¹⁸]. Protein synthesis is necessary for the initial production of MPF, but it is not required for further maturation-promoting activity. Cycloheximide fails to prevent the autocatalytic amplification of MPP as well as treatment with cytochalasin does [¹⁹]. These findings suggest that oocytes contain a store of inactive MPF. Protein synthesis is merely essential to activate it. Small amounts of active MPF are able to amplify MPF activity without any further protein synthesis. MPF is a protein kinase consisting of two components, cyclin B and cdc2 The small subunit which is called p34 is a protein homologue of the cdc2+ gene product of fission yeast necessary for the G2-M transition. Cdc2 homologues have been detected in cells of a variety of organisms, including mammals [²⁰]. The kinase activity of p34 remains high during M phase, resulting in the phosphorylation of di-verse proteins. Histone Hi is one target for p34 kinase [²¹]. Phosphorylation of histone Hl is suspected to be involved in the condensation process of chromatin that takes place during M phase [²²]. Another substrate for p34 are the lam-mae forming the fibrillar network of the inner surface of the nuclear envelope. Phosphorylation of the ]aminae is assumed to be responsible for the nuclear envelope breakdown. Moreover, p34 is likely to interact with the microtubular system and enhances maturation by organizing the spindle apparatus through phosphory]ation.

The other subunit of MPF is cyclin B, a protein representing the group of cyclins which accumulate during interphase and are destroyed by proteolysis after mitosis during each cell cycle [²³]. The role of cyclin B is to activate p34 kinase when they complex with p34. Cyclins, like p34, also seem to be universal cell-cycle regulators in eukaryotes. Cyclins are homologues to the gene product of cdcl3 in fission yeast. While p34 requires dephosphorylation for activation, cyclin B has to be phosphorylated to become active when preexisting in complexes with p34, whereas it forms directly active MPP when synthesized de novo. c-MOS (p39 mos), which is a product of the c-mos proto-oncogene, apparently triggers cyclin B activation. C-MOS is a kinase that is necessary for induction of maturation. Studies on Xenopus showed that injecting c-MOS mRNA into the oocyte induces maturation, while inhibiting c-MOS prevents maturation [²⁴]. c-MOS phosphorylates cyclin B in vitro. 

Briefly, c-MOS activates cyclin, which in turn activates p34. There is some question regarding how MPF activity is controlled in the oocyte. There are some good reasons for assuming that active p34 is directly controlled by PKA activity resulting from a decreased concentration of cAMP in the oocyte. Elevation of cAMP inhibits the formation of p34/cyclic B complex and p34 remains inactive. MPF exhibits its activity as long as cyclins are present. Degradation of cyclins at the end of the M phase in-activates MPF, allowing the cell cycle to continue. Arrest of the mature ?ocyte at metaphase II is probably due to a cytostatic factor (CSF) present in the cytoplasm which stabilizes MPF by blocking cyclin degradation (Fig. 8). It is assumed that CSF exhibits its function in the absence of calcium. Application of the calcium chelator EGTA retains the CSF-mediated arrest. There is evidence that CSF is actually c-MOS protein [²⁵]. It remains to be clarified why c-MOS has different functions, activating maturation and promoting metaphase arrest.


Growth Factors, GnRH, and Steroids

There is some evidence that growth factors are involved in oocyte maturation. Epidermal growth factor (EGF) and transforming growth factor (TGF) ? and seem to promote resumption of meiosis in follicle-enclosed oocytes, but the mechanisms of inducing GVB are still unclear to date. The activation of phosphatidyl inositol and calcium pathways is associated with the action of angiotensin II, which is likely to be regulated by TGF/1 GnRH induces maturation of oocytes, but much more slowly than LI-I. A possible mode of action for (AnRH is to activate PK-C. The effective role of steroids for oocyte maturation renimns unclear. Nonphysiological high concentrations of testosterone, progesttront, and pregnenolone seem to inhibit oocyte maturation either alone or in con junction with agents increasing the cAMP level. Estradiol, on the other hand, seems to improve induction of maturation.

Calcium

As it is known that calcium inhibitors such as verapamil transiently prevent GVB by elevating cAMP levels within the oocyte, a most striking role of calcium in the initiation of oocyte maturation must be considered. Calcium is essential in culture medium to sustain completion of meiosis in maturing oocytes. Activation of calcium within the oocyte starts via the phosphoinositide (1P43) pathway. Calcium may be released by the granulosa cells through gap junctions to the oocyte responding to the LH surge. GVB is mediated by calcium. 1P3 induces the mobilization of calcium stores within the oocyte. Free calcium activates cAMP phosphodiesterase, resulting in a decrease of cAMP concentration below the threshold needed to maintain meiotic arrest, and initiates GVB.

References:

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