G2/M transition of pig oocytes: How do oocytes initiate maturation?

Abstract

In the ovary, mammalian oocytes resume meiosis and mature to the second metaphase when they are stimulated with gonadotrophins. Similarly, oocytes can mature in vitro when they are liberated from ovarian follicles and cultured under appropriate conditions. Early in the process of maturation, oocytes undergo dramatic but well‐ordered changes at the G2/M transition in the cell cycle including: (i) chromosome condensation; (ii) nucleolus disassembly; (iii) germinal vesicle breakdown (GVBD); and (iv) spindle formation in the first metaphase (MI‐spindle). These events have been thought to be induced by MPF (maturation‐promoting factor or M‐phase promoting factor), now known as Cdc2 kinase or Cdk1 kinase, which consists of a catalytic subunit, Cdc2, and a cyclin B regulatory subunit. In fact, nuclear lamins are phosphorylated by Cdc2 kinase, and nuclear membrane breakdown occurs concomitantly with the activation of Cdc2 kinase in the M‐phase of both somatic cells and oocytes. Based on the classical and recent studies of the pig oocyte, however, the chromosomes start to condense and the nucleolus disassembles before full activation of Cdc2 kinase, and the MI‐spindle is formed after activation of both Cdc2 kinase and MAP kinase; another kinase known to become activated during oocyte maturation. These findings suggest that chromosome condensation and nucleolus disassembly in oocytes are induced by either some kinase(s) other than Cdc2 kinase and MAP kinase or some phosphatase(s). The accumulation of new results regarding the molecular nature of oocyte maturation is important for improving the reproductive technologies in domestic animals as well as in humans. (Reprod Med Biol 2003; 2: 91–99)

INTRODUCTION

Over a decade has passed since the molecular nature of maturation‐promoting factor or M‐phase promoting factor (MPF) was discovered, and MPF (Cdc2 kinase or Cdk1 kinase) is now generally accepted as a key molecule involved in the regulation of M‐phase transition in somatic cells as well as oocytes. The mechanisms of MPF activation and inactivation have been described in relation to molecular changes in Cdc2, cyclin B, and other associated molecules. Oocyte maturation in mammals has also been explained in terms of these molecules.

Oocyte maturation is an important process that prepares the egg for the fertilization by spermatozoa. The process can be seen as dynamic changes in the oocyte nucleus or germinal vesicle (GV). These changes include chromosome condensation, nucleolus disassembly, nuclear membrane breakdown (germinal vesicle breakdown: GVBD), and metaphase I (MI) spindle formation. In this review, we dissect out these early events of oocyte maturation, and discuss the possible involvement of Cdc2 kinase based on our recent findings in pig oocytes.

MATURATION OF MAMMALIAN OOCYTES

In mammalian fetal ovaries, oogonia enter the meiotic cell cycle to become primary oocytes and duplicate their chromosomes (S‐phase). They progress through the first meiotic prophase to the diplotene stage, a point at which they stop the meiotic progression and wait until puberty. During this diplotene arrest at the G2‐phase of the first meiotic cell cycle, some oocytes start to grow. They increase in volume, the chromosomes decondense and an active transcription is detected, which results in the accumulation of stockpiles of materials including RNAs and proteins that are required for the later events; maturation, fertilization and early embryonic development. This G2‐arrest is maintained until immediately before ovulation when the oocytes resume meiosis and enter the M‐phase.

When animals reach puberty, a limited number of fully grown oocytes in the ovary periodically resume meiosis in response to gonadotrophic stimuli, while the majority of oocytes are maintained in the prolonged diplotene stage of the first meiotic prophase. After meiotic resumption, oocytes undergo a dramatic series of changes: condensation of chromosomes, disassembly of nucleoli, GVBD, and assembly of the MI spindle. The first meiotic division is completed when the first polar body is extruded in anaphase I (AI) and telophase I (TI). The oocytes then enter the second meiotic cell cycle without an intervening interphase, and spontaneously arrest at the metaphase of the second meiosis (MII) awaiting the fertilization or parthenogenetic activation. These sequential processes are called ‘oocyte maturation’. Oocyte maturation can be induced in vitro by culturing fully grown follicular oocytes from various mammalian species under appropriate conditions. ¹ , ²

Cdc2 KINASE IN THE OOCYTE

Like the M‐phase of somatic cells, it is now commonly accepted that meiotic resumption of oocytes is controlled by a protein kinase generally known as MPF. The MPF activity was first discovered by Masui and Markert in 1971 as the activity in the cytoplasm of metaphase‐arrested frog (Rana pipience) oocytes. ³ In their experiment, transferred cytoplasm from the metaphase‐arrested frog oocytes induced the recipient oocytes to resume meiosis. The MPF activity rises rapidly to a peak at MI and then falls around the time of chromosome segregation at AI/TI. The MPF is now known to be a serine/threonine protein kinase consisting of a 34‐kDa catalytic subunit Cdc2 (p34^cdc2) and a cyclin B regulatory subunit; it is also referred to as Cdc2 kinase or Cdk1 kinase. Cyclins were originally identified in sea urchin eggs ⁴ and clam eggs ⁵ as proteins displaying a striking periodicity in synthesis and degradation during the early embryonic cell cycle. B‐type cyclins are components of Cdc2 kinase; ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ they act through the association with the highly conserved catalytic subunit, Cdc2, the product of the cell division cycle gene cdc2 firstly described in the fission yeast Schizosaccaromyces pombe. ¹¹

Fully grown Xenopus oocytes contain both Cdc2 and cyclin B, and increased synthesis of cyclin B occurs in the cytoplasm during oocyte maturation. ¹² Cyclin B molecules associate with Cdc2 molecules, and the complex accumulates as an inactive pre‐MPF whose Cdc2 is phosphorylated at specific tyrosine and threonine residues (T14 and Y15; Fig. 1a). Progesterone, the maturation‐inducing hormone in amphibians, stimulates Cdc25 phosphatase to dephosphorylate T14/Y15, and thus Cdc2 kinase becomes activated. The increased activity is maintained during MI and MII with a temporal decrease at the AI/TI transition, which then declines sharply after fertilization or artificial activation of the oocytes. The disappearance of kinase activity is thought to be associated with degradation of the cyclin B subunit via the ubiquitin pathway. ¹³

Figure 1.

Models for the activation process of Cdc2 kinase at G2/M transitions in oocytes. (a) Xenopus, (b) goldfish oocytes, as proposed by Yamashita. ²² In Xenopus, immature oocytes contain inactive pre‐maturation‐promoting factor (preMPF), cyclin B‐bound Cdc2, the T14 and Y15 sites of which are phosphorylated. At the G2/M transition, Cdc2 kinase is activated by dephosphorylation of the inhibitory sites by Cdc25 phosphatase. In contrast, immature oocytes of goldfish contain only monomeric Cdc2 but not cyclin B. At the G2/M transition, cyclin B is accumulated and forms a complex with Cdc2. The cyclin B‐bound Cdc2 is then phosphorylated and activated by Cdk‐activating kinase (CAK).

Cell cycle studies have also been conducted in mammalian oocytes. ¹⁴ , ¹⁵ , ¹⁶ During the oocyte maturation, the activity of Cdc2 kinase increases around GVBD, peaks at MI and MII, and then declines sharply after fertilization. It has been reported that pre‐MPF exists in mouse oocytes. ¹⁷ , ¹⁸ , ¹⁹ In contrast, pre‐MPF is suggested to be absent in immature bovine and pig oocytes. ²⁰ , ²¹ Yamashita has thoroughly investigated the activation mechanism of Cdc2 kinase during G2/M transition in fish and amphibian oocytes. ²² He found that immature goldfish oocytes contain only monomeric Cdc2, and that cyclin B is not detectable (Fig. 1b). After stimulation by a maturation‐inducing hormone, cyclin B is synthesized and binds to the pre‐existing Cdc2. The binding of Cdc2 and cyclin B enables CAK (Cdk‐activating kinase) to phosphorylate cyclin B‐bound Cdc2 on T161 and activates Cdc2 kinase. Neither T14/Y15 phosphorylation nor dephosphorylation is involved in the kinase activation in this species. ²³ Similar mechanisms have also been suggested for other fishes and amphibians, except for Xenopus. ²⁴ According to our findings in the pig, the mechanisms of Cdc2 kinase activation during oocyte maturation are likely to be similar to those in the goldfish. ²⁵

DOES Cdc2 KINASE INDUCE EARLY EVENTS OF OOCYTE MATURATION?

As in mitosis of somatic cells, it is generally accepted that active MPF/Cdc2 kinase induces chromosome condensation, GVBD, and spindle formation during the meiotic resumption (G2/M transition) in oocytes. But is the hypothesis tenable for mammalian oocytes?

Cell cycle progression from the G2 to M‐phase occurs in a relatively short time in somatic cells (within 30 min) and in Xenopus oocytes (4 h). In contrast, mammalian oocytes take much longer to progress from the G2 stage (the germinal vesicle stage: GV stage) to the M‐phase of the first meiosis (MI); for example, this transition takes 24–27 h in the pig. Therefore, we can examine separately the early sequential events including chromosome condensation, disassembly of the nucleolus, GVBD, and formation of the MI‐spindle. Mammalian oocytes are too small (70–120 µm in diameter) to study biochemical changes in the cell cycle molecules. However, the characteristic lack of egg yolk in mammalian oocyte cytoplasm makes it easier to research morphological changes of the nucleus and chromosome by examining them as whole‐mount specimens.

In 1976, Motlik and Fulka described the precise and sequential changes in the nucleus (GV) of pig oocytes during maturation. ²⁶ Just after collection from the ovary, pig oocytes have an intact, clearly visible nuclear membrane and nucleolus surrounded by ring‐ or horseshoe‐shaped chromatin (Fig. 2a). Before GVBD, chromosomes start to condense and the nucleolus disappears completely, at least when evaluated under the light microscope. This study pointed out that the events during the G2/M transition of pig oocytes, that is, chromosome condensation, disassembly of nucleolus and GVBD, do not occur simultaneously, but rather separately and sequentially.

Figure 2.

Photomicrographs of pig oocytes cultured in media either containing or not containing cycloheximide (a protein synthesis inhibitor) for 27 h. (a) Horseshoe‐like heterochromatin was stained with aceto‐orcein around the nucleolus in the germinal vesicle (GV) of pig oocytes before culture. (b) Oocytes cultured in cycloheximide‐free medium resumed meiosis and reached the first metaphase (MI) after 27 h of culture. (c) Oocytes cultured in cycloheximide‐supplemented medium for 27 h have condensed chromosomes in an intact GV.

We have also used in vitro‐cultured pig oocytes for cell cycle studies. Pig oocytes are almost freely available from commercially slaughtered pigs. Under our culture conditions, all pig oocytes dissected from healthy antral follicles, which are 4–5 mm in diameter, are at the GV stage, and over 90% of the oocytes progress to MII synchronously in response to hMG (human menopausal gonadotropin) stimulation. ²⁷ , ²⁸ Soon after the culture has started, oocyte chromosomes start to condense; this is consistent with the findings of Motlik and Fulka. ²⁶ The oocyte nucleolus becomes invisible, and the oocytes then undergo GVBD at 21–24 h and reach MI by 27 h (2, 3). Thereafter, the oocytes pass through AI and TI at 30–33 h, and maturation is completed in MII after 36 h of culture.

Figure 3.

An autoradiogram showing the changes in Cdc2 kinase and mitogen‐activated protein (MAP) kinase activities during maturation of pig oocytes. Histone H1 and myelin basic protein are used for the substrates of Cdc2 kinase and MAP kinase, respectively. The lower figures represent the hours of maturation culture. GVBD, germinal vesicle breakdown, MI, metaphase I.

We have also investigated changes in Cdc2 kinase and MAP kinase (ERK1/ERK2) activities. The MAP kinase is another kinase in which activation during oocyte maturation has been reported in the mouse, ²⁹ , ³⁰ , ³¹ pig, ³² goat, ³³ bovine, ³⁴ rat, ³⁵ and human oocytes. ³⁶ Pig oocytes at the GV stage have undetectable cyclin B1 molecules and express a low activity of Cdc2 kinase (Fig. 3). Following the stimulation with gonadotropins, oocytes start to accumulate cyclin B1 at 21 h, and Cdc2 kinase activity gradually increases around GVBD at 24 h and reaches a maximum level at MI. The GV oocytes have MAP kinase in the inactive form, and this kinase becomes phosphorylated and activated after GVBD. These results suggest that GVBD occurs in close association with the activation of Cdc2 kinase. However, the chromosome condensation and disassembly of the nucleolus occurs much earlier than the activation of Cdc2 kinase. In contrast, the MI spindle is formed later, when both Cdc2 kinase and MAP kinase are fully active. In the following sections of the paper, we discuss the possible molecular mechanisms that have recently proposed to underlie: (i) chromosome condensation; (ii) nucleolus disassembly; (iii) GVBD; and (iv) spindle formation in oocytes.

CHROMOSOME CONDENSATION

Chromosome condensation is the first step of oocyte maturation, and consequently is an important process necessary for correct chromosome distribution into the secondary oocyte and the first polar body. It has been known that mouse oocytes can undergo GVBD without synthesis of new proteins, ³⁷ however, oocytes of all domestic animals require new protein synthesis for GVBD. ³⁸ , ³⁹ , ⁴⁰ The difference in the necessity for protein synthesis may reflect the accumulation of cyclin B molecules during the growth phase of the oocytes. Nevertheless, pig oocytes cultured in the medium containing a protein synthesis inhibitor form highly condensed chromosomes in the nucleoplasm, although they are still arrested in the GV stage (Fig. 2c). ⁴¹ In the oocytes, neither Cdc2 kinase nor MAP kinase is activated. This implies that neither synthesis of new proteins nor activation of Cdc2 kinase is required for chromosome condensation. This conclusion raises the question: what molecule or molecules induce chromosome condensation?

Protein complexes called condensins have recently been identified to play a major role in chromosome condensation in Xenopus egg extracts. ⁴² Condensins appear to promote positive supercoiling of DNA to compact chromosomes into a condensed structure. However, it has been suggested that condensins are phosphorylated and activated by Cdc2 kinase. ⁴³ Recent studies have also revealed an enzyme system, conserved among diverse eukaryotes, that regulates chromosome dynamics and controls histone phosphorylation. The basic unit of chromatin structure is the nucleosome, which consists of ∼200 b.p. of DNA wrapped around a histone core that contain two molecules each of histones H2A, H2B, H3, and H4. The N‐terminal tail of histone H3 is phosphorylated at serine 10 (Ser 10) during mitosis and meiosis in a wide range of eukaryotes; this phosphorylation is thought to be essential for proper chromosome condensation and segregation. ⁴⁴ , ⁴⁵ It has been shown that a protein Ipl1/aurora‐B kinase and its related phosphatase, Glc7/PP1, are responsible for the balance of histone H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans. ⁴⁶ Chromosome assembly system using Xenopus egg cytoplasmic extracts has revealed that mitotic histone H3 Ser 10 kinase activity is associated with mitotic chromosomes, and that the kinase is not affected by inhibitors of cyclin‐dependent kinases (Cdks). ⁴⁷ It is thought that such a histone H3 kinase/PP1 system may be responsible for inducing chromosome condensation in oocytes, although its upstream cascade remains to be elucidated.

NUCLEOLUS DISASSEMBLY

Small growing oocytes from ovarian follicles contain fibrillogranular nucleoli, which actively transcribe ribosomal RNAs. As the oocyte approaches full size, nucleoli change their structure and are visible as a compact round‐shaped fibrillar mass with no detectable transcriptional activity. ⁴⁸ , ⁴⁹ Consequently, fully grown oocytes contain a prominent nucleolus inside the GV that is not stained with aceto‐orcein in whole mount specimens but is stained dark blue with hematoxylin in histological sections.

Following chromosome condensation, the nucleolus disappears; this occurs just before GVBD. During the MI and MII stages and the MI/MII transition, the nucleolus never appears, but it reassembles again in the female pronucleus after the oocyte is released from MII arrest by sperm penetration or artificial stimuli. Fibrillar nucleolar material is presumably released into the GV nucleoplasm at the time of the nucleolar disassembly, then into the cytoplasm at GVBD. It has been speculated that this process is controlled by reversible phosphorylation and dephosphorylation of nucleolar proteins, although the process has not been fully understood.

Recently, Fulka et al. have reported that the microsurgical removal of nucleoli from fully grown pig oocytes permits chromosome condensation and GVBD, and that the enucleolated oocytes mature to MII with a normal MII spindle. ⁵⁰ Moreover, the enucleolated oocytes are able to activate and inactivate Cdc2 kinase and MAP kinase in nearly the same time‐course as the intact oocytes (Ogushi et al., unpubl. data, 2003). Therefore, the nucleolus in fully grown oocytes may be dispensable, at least for oocyte maturation. Further fate of enucleolated oocytes remains to be determined; that is, reappearance of nucleoli in pronuclei after fertilization and the eventual embryonic development.

GERMINAL VESICLE BREAKDOWN

Germinal vesicle breakdown is one of the most prominent events in the early stages of oocyte maturation. Thus, GVBD has been commonly used as the first sign of meiotic resumption in the mouse and other mammalian oocytes. The Cdc2 kinase is activated around GVBD in all mammalian species examined so far; a causal connection between the two has been found to exist.

The nuclear envelope has a double‐membrane structure of lipid bilayer, and inner and outer nuclear membranes. The inner nuclear membrane is supported by the nuclear lamina, a meshwork of intermediate‐filament lamins, located adjacent to the inside face of the nuclear envelope. ⁵¹ Three nuclear lamin, A, B, and C, are present in the vertebrate cells, and have been identified as evident targets for Cdc2 kinase.

Germinal vesicle breakdown requires the disassembly of the nuclear lamina. Active Cdc2 kinase phosphorylates all three types of lamin molecules on specific serine residues, causing the lamin filaments to depolymerize into individual lamin dimers. ⁵² This disintegration of the lamina meshwork leads to the fragmentation of the nuclear envelope into small vesicles. The endoplasmic reticulum, whose membrane is continuous with the outer nuclear membrane, similarly fragments into small vesicles during oocyte maturation in the pig. ⁵³ It is thought that the breakdown of these membranes is also induced by active Cdc2 kinase.

Germinal vesicle breakdown, without Cdc2 kinase activation, has been reported under some special conditions. For example, when the pig oocytes are treated by okadaic acid (an inhibitor of protein phosphatases 1 and 2A) and butyrolactone I (a potent and specific inhibitor of Cdks) at the same time, chromosomes condense and the nuclear membrane breaks down. ⁵⁴ In these oocytes, Cdc2 kinase remains inactive, but MAP kinase is active. As active MAP kinase has a weak activity to phosphorylate nuclear lamins, MAP kinase is likely to induce GVBD in this case. ⁵⁵

METAPHSE I SPINDLE FORMATION

During oocyte maturation, the regular metaphase spindle is formed twice, at MI and MII. The MI spindle is assembled after the completion of chromosome condensation and GVBD; formation starts concomitantly with activation of Cdc2 kinase and is completed after both Cdc2 kinase and MAP kinase have been activated. Increased activity of Cdc2 kinase is thought to phosphorylate microtubule‐associated proteins directly or indirectly, thereby inducing in the somatic cells a rapid increase in the rate of turnover of microtubules, which are composed of α‐ and β‐tubulin heterodimers. In frog oocytes (Rana japonica), it has recently been revealed that GVBD is induced by Cdc2 kinase without MAP kinase activity, and that reorganization of microtubules at GVBD is induced by MAP kinase without Cdc2 kinase activation. ⁵⁶

The formation of mitotic spindle in the somatic cells is well explained in basic textbooks of cell biology. Briefly, the centrosome, a pair of centrioles surrounded by electron‐dense pericentriolar materials, is replicated during interphase. As mitosis proceeds, the replicated centrosomes separate and move away from each other to serve as the two spindle poles (Fig. 4). A large number of microtubules radiating from the opposite spindle poles search and capture the two kinetochores of sister chromatids, which are located on opposite sides of each condensed chromosome. ⁵⁷ Consequently, the polar and kinetochore microtubules form the ‘rugby ball‐shaped’ spindle. A combination of microtubule motor proteins at the kinetochore and microtubule dynamics is thought to align the chromosomes at the spindle equator. It has been reported that cyclin B binds to microtubules, ⁵⁸ and active MAP kinase (ERK1/ERK2) binds strongly to the spindle poles. ⁵⁹

Figure 4.

Shape and formation of mitotic and meiotic spindles. Immunofluorescent localization of α‐tubulin (a, c), γ‐tubulin (b), and nuclear mitotic apparatus protein (NuMA) (d) in pig aortic endothelial cells at the M‐phase (a, b) and in pig oocytes at MI (c, d). α‐Tubulin, γ‐tubulin, and NuMA appear in green (by the use of fluorescein isothiocyanate (FITC)), and DNA appears in red (by propidium iodide). (e) Postulated organization models of the mitotic and meiotic spindles. In somatic cells, two mictrotubule‐organizing centers, the centrosomes, exist prior to the mitotic spindle formation and are used as the two poles of the spindle. In pig oocytes, however, there are no microtubule‐organizing centers prior to the formation of meiotic spindles. In the oocytes, microtubules are nucleated randomly, probably from cytoplasmic microtubule nucleating factors containing γ‐tubulin. The cytoplasmic microtubules are then thought to be bundled at the minus end by a NuMA‐containing motor complex.

The pattern of MI spindle formation in mammalian oocytes is markedly different from that in the mitosis of somatic cells. The difference is caused by the lack of a centrosome in oocytes, similar to that of plant cells. ⁶⁰ In the pig, just after GVBD, bivalent chromosomes make a single clump, the stage first described by Motlik and Fulka as ‘late diakinesis’. ²⁶ Spindle microtubules are nucleated toward random directions around the clump of chromosomes because there are no centrosomes in pig oocytes. ²⁸ Although it is unknown how microtubules are nucleated in the absence of centrosomes, it has been speculated that the randomly arrayed microtubules are initially nucleated from the microtubule‐nucleating factor containing γ‐tubulin ²⁸ found in the centrosomes. ⁶¹ , ⁶² At this stage, the nuclear mitotic apparatus protein (NuMA), which is associated with the dynein/dynactin motor complex, ⁶³ is found in association with the randomly arrayed microtubules. As meiosis proceeds, NuMA moves away from the condensed chromosomes and bundles the microtubules at the minus ends to integrate both spindle poles and form a ‘barrel‐shaped’ MI spindle on which bivalents align at the equator (Fig. 4c,d). ²⁸ Kinetochores of sister chromatids of each homologous chromosome are adjacent to each other and oriented in the same direction, while the kinetochores of homologous chromosomes are directed toward opposite spindle poles. ⁶⁴ In AI/TI, the homologous chromosomes separate while sister chromatids remain associated; one of each homologous pair consisting of two sister chromatids is then distributed into the secondary oocyte or the first polar body. It has been reported that active MAP kinases (ERK1/ERK2) are present at the periphery of the condensed chromosomes in the MI spindle, ⁶⁵ and cyclin B1 is located at the spindle poles in MII pig oocytes. ⁶⁶ As NuMA has four predicted Cdc2 kinase phosphorylation sites in the C‐terminal domain, ⁶⁷ the formation of oocyte spindle poles may be regulated by Cdc2 kinase through NuMA's function.

PERSPECTIVES

Herein, we have described the molecular aspects and possible involvement of Cdc2 kinase in early events of oocyte maturation: (i) chromosome condensation; (ii) nucleolus disassembly; (iii) GVBD; and (iv) MI‐spindle formation. Of these events, only GVBD is thought to be induced by the direct action of active Cdc2 kinase, and MI‐spindle formation is presumed to be induced by the coupled action of active Cdc2 kinase and MAP kinase. In the Xenopus, the first signal of oocyte maturation in response to progesterone leads to the Mos/MEK/MAP kinase cascade. ⁶⁸ , ⁶⁹ Through one or more mechanisms, activation of this cascade leads to the activation of Cdc2 kinase, which in turn seems to act positively on the MAP kinase cascade. Although the Mos/MAP kinase cascade plays an important role in initiating Xenopus meiotic maturation, MAP kinase (ERK1/ERK2) in mammalian oocytes is not activated prior to Cdc2 kinase activation during the G2/M transition. What induces the chromosome condensation and nucleolus disassembly, and what is the first signal in mammalian oocytes? Another question is how the above‐mentioned four events are coupled during early stages of oocyte maturation. Does the commencement or completion of the first process trigger the next process as in a chain of falling dominoes, or do the four processes occur in succession independently? Accumulation of additional findings regarding the molecular mechanisms underlying each meiotic event in mammalian oocyte maturation is important for improving the reproductive technologies in domestic animals as well as in humans, because some oocytes are unable to undergo GVBD and others are incompetent to proceed through to the MI/MII transition.