contribution of the oocyte to embryo quality- sirard 2006- theriogenology

Contribution of the oocyte to embryo quality

Marc-Andre Sirard *, Francois Richard, Patrick Blondin,Claude Robert

Centre de Recherche en Biologie de la Reproduction, Department of Animal Sciences,

Laval University, Pav. Comtois, Sainte-Foy, Que., Canada G1K 7P4

Abstract

The ability of a bovine embryo to develop to the blastocyst stage, to implant and to generate a healthy

offspring is not a simple process. To clarify the importance of the contribution of the oocyte to the

embryo quality, it is important to define more precisely the different types of competence expressed by

oocytes. The ability to resume meiosis, to cleave upon fertilization to develop into a blastocyst, to induce

pregnancy and to generate an healthy offspring are all separate events and succeeding in the first events

does not ensure the success of subsequent ones. Furthermore, these events are associated with the three

types of maturation processes observed in the oocyte: meiotic, cytoplasmic and molecular. These

abilities vary also upon the type of follicle the oocytes is removed from. Larger or slow-growing follicles

have been shown to foster better eggs than small or actively growing follicles. Hormonal stimulation can

also affect oocyte competence with the nature of the effect (positive or negative) depending on timing

and dose. This complex situation requires better definition of the contribution of each factor affecting the

oocyte competence and the resulting embryo quality.

# 2005 Elsevier Inc. All rights reserved.

Keywords: Oocyte competence; Follicle; RNA; Differentiation

1. Introduction

The literature concerning bovine embryo quality has become very abundant. The

influence of the oocyte quality on the developmental potential of the embryo has been

recognized in the cow more clearly that in any other species. Because multiple factors are

involved and a vast amount of data have been published, it may seem difficult to have a

www.journals.elsevierhealth.com/periodicals/the

Theriogenology 65 (2006) 126136

* Corresponding author. Tel.: +1 418 656 2131x7359; fax: +1 418 656 3766.

E-mail address: [email protected] (M.-A. Sirard).

0093-691X/$ see front matter # 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.theriogenology.2005.09.020

clear idea of the concept of competence and the factors acting upon it. Therefore, it is

timely to clarify the concepts of oocyte competence, oocyte maturation and follicular

differentiation with regards to resulting embryo quality.

2. Oocyte competence

The following is a comprehensive enumeration and a short description of the five levels

of oocyte competence. They represent key steps that characterize developmental

competence:

(1) Ability to resume meiosis.

(2) Ability to cleave following fertilization.

(3) Ability to develop to the blastocyst stage.

(4) Ability to induce a pregnancy and bring it to term.

(5) Ability to develop to term in good health.

2.1. Ability to resume meiosis

Even if the ability to resume meiosis is probably the easiest to measure, it underlies a

spatio-temporal synchrony of cell cycle molecules. When mammalian oocytes, including

those from the cow, are removed from their follicles, they have the ability to spontaneously

resume meiosis [1]. No stimulating agents are required. Meiotic resumption can be

visualised under the microscope by the first polar body extrusion or with specific dyes to

stain the metaphase. This resumption of meiosis is believed to be a consequence of the

absence of a follicular inhibitor still unidentified in domestic animals [2,3]. In the cow, the

oocyte acquires the ability to form a metaphase plate when reaching its full size in the

growing follicle just before antrum formation [4]. In some species, the capacity to reach

metaphase I is acquired before the capacity to reach metaphase II [5]. In cows, the two

seems to be acquired at the same time, but the low abundance of cell cycle activator can

result in an metaphase I arrest [6], indicating that the two capacities require distinct

molecules.

2.2. Ability to cleave following fertilization

The capacity to cleave is almost automatic and is an intrinsic potential within the fully

grown oocytes of large mammals because it can occur in absence of fertilization by a

simple activation stimulus (electrical current, ethanol) [7]. When cleavage does not occur,

it is not clear if it is the consequence of a dysfunctional sperm that failed to activate the

oocyte or the oocyte itself did not have the ability to undergo the first cell division.

The assessment of hundreds of presumptive zygotes that did not cleave 36 h post-

insemination indicates that most of them were not fertilized and only a small proportion

had an incomplete decondensed sperm or asynchronized pronuclei (Sirard, personal

observation). This observation indicates that a negligible subpopulation of full-size oocytes

obtained from slaughterhouse ovaries are incompetent to trigger the downstream processes

M.-A. Sirard et al. / Theriogenology 65 (2006) 126136 127

following fertilization and cleave. This situation is different than in the mouse where the

ability to activate/cleave following fertilization is acquired later than the capacity to

resume meiosis [8].

2.3. Ability to develop to the blastocyst stage

This is the most controversial and yet key marker of oocyte competence commonly used

by most laboratories. It is obvious that a fertilized oocyte must reach the blastocyst stage

within 69 days in the proper culture conditions to have a significant chance of inducing a

pregnancy and producing an offspring. The ability to sustain the first week of embryonic

development is clearly influenced by the follicular status from which the oocyte is obtained

(see below) indicating that this developmental potential is inherent within certain oocytes.

Since most early embryos that do not reach the blastocyst stage are blocked at or close to

the maternal to zygotic transition (MZT)-stage, which occurs at the eight-cell stage in

cattle [9], one could speculate that incompetent oocytes fail to appropriately activate the

embryonic genome.

The early developmental program embedded in the oocyte through the accumulation of

proteins and RNA is likely to be responsible for the proper execution of the embryonic

genome activation. The use of transcription inhibitors such as a-amanitin during the firstfew days following fertilization results in normal cleavage until the four- to eight-cell stage

[9]. Thus, transcriptional activation of the new embryonic genome is required for

development beyond MZT [9]. Such activation may well depend on the activation or

translation of some maternal transcription factors already stored in competent oocytes that

make this activation possible [10].

Since blastocysts can be classified based on their morphology, it is clear that their

quality varies depending on criteria such as number of cells, trophectoderm to inner-cell

mass ratio, blastocoele expansion, overall appearance, etc. (IETS Manual). Their ability to

survive cryopreservation and induce a pregnancy is also affected by their apparent

morphology as well as by their origin (in vitro or in vivo). From these visual assessments,

one can approximately predict pregnancy rates supporting the perception that good

blastocysts can result in pregnancy. Therefore the competence to achieve the blastocyst

stage is more an assessment of the normalcy of the oocyte than anything else. Even in

cloning experiments, most often the blastocyst rate obtained is lower or closer to the IVF

controls but never above, indicating the intrinsic limitation, of a proportion of immature

oocytes obtained from slaughterhouse ovaries [11]. Incidentally, none of the attempts to

improve culture conditions for bovine embryos has produced consistent blastocyst

production rates above 3040%. Only modifications of superovulatory treatments to

control follicular growth have had a significant effect on increasing these rates [12]. This

suggests that developmental competence is mainly acquired during oocyte growth within

the follicle by an unknown mechanism [13].

2.4. Ability to induce a pregnancy

As mentioned above, all blastocysts are not equal and do not always result in a

pregnancy once transferred in suitable recipients. Part of this failure can be attributed to the

M.-A. Sirard et al. / Theriogenology 65 (2006) 126136128

recipient. However, blastocysts that originate from oocytes matured in vitro result in

lower rates of gestation compared to their in vivo counterparts [14]. If we exclude the

influence of genetics (some oocyte donors are better than others) or sperm selection

(some bulls also achieve higher blastocyst rate and quality than others [15]), the quality

of blastocysts resulting from in vivo or in vitro matured oocytes collected from small

growing follicles and submitted to identical fertilization and embryo culture procedures

should be comparable. Therefore what could explain the higher rate of gestation failure

[14] associated with the in vitro procedure? It is reasonable to think that the ability to go

to term is influenced by events occurring before the blastocyst stage and could be

explained by either faulty culture conditions and/or by the incomplete oocyte

programming before aspiration from its follicle. Up to now, most of the negative

consequences of culture have been associated with the culture media post-fertilization,

but what about in vivo conditions in the follicle before the oocyte is removed as well as

culture conditions during maturation?

Could we be using oocytes that have not completed their imprinting by collecting

them from small growing follicles? There are only a few studies with in vivo matured

oocytes in cows [1618] and they were not designed to explain if the deleterious effect

observed following in vitro maturation comes from incomplete oocyte programming or

inadequate culture conditions. Recent studies indicate that induction of follicular

differentiation by manipulation of the ovarian stimulation protocol, namely FSH

starvation or coasting, can result in the recovery of germinal-vesicle stage (immature)

oocytes where most are capable of developing to the blastocyst stage following

completion of in vitro procedures. Moreover, the embryonic developmental rates

obtained from this procedure equal or even surpass the blastocyst rates obtained with

in vivo matured oocytes submitted to in vitro fertilization and culture although the two

were not compared in the same experiment [12]. Overall, the comparison between the

in vivo matured and the in vitro matured oocyte recovered at different times before the

LH surge support a progressive influence of the follicular differentiation on oocyte

competence.

2.5. Ability to develop to term in good health

One of the most surprising discoveries from the use of in vitro production of

embryos in cattle is the effect of the procedure on the health of the offspring. The most

studied of these effects, the Large Calf Syndrome (LCS), does not systematically occur

after vitro culture and varies in frequency according to the different laboratories [19].

Its presence is a warning that culture conditions at the early embryonic stage may have

direct impacts much later in life. The principal cause could be related to the oocyte

maturation period, possibly due to an incomplete acquisition of developmental

competence at onset of maturation as mentioned above, or as a consequence of

suboptimal culture conditions. Therefore the follicular environment could have an

influence not only on oocyte quality and female fertility but on the offsprings health as

well. It is known that the uterine environment can affect fetal development and impact

the offsprings health but the ovarian influence is becoming an additional source of

epigenetic influence that must be explored [20].


3. Oocyte maturation

The three levels of oocyte maturation are described here to improve clarity and to allow

dissection of the association of follicular to intra-oocytes events. These levels are:

(1) Meiotic maturation

(2) Cytoplasmic maturation

(3) Molecular maturation

3.1. Meiotic maturation

As described above for meiotic competence, meiotic maturation is the cascade of

nuclear events that is induced either by the LH surge or by the removal of the oocyte

from its follicular environment. These events are programmed to occur in the oocyte

upon the removal of a still unidentified inhibitory substance. Once allowed to proceed,

maturation promoting factor (MPF), a protein complex composed of cyclin B1 and

P32cdc2, is synthesized and/or activated depending on the species and thereafter, the cell

cycle machinery becomes activated and the oocyte undergoes the first metaphase and

extrusion of the first polar body before arresting after the formation of the second

metaphase under the influence of the cytostatic factor (CSF). The timing of meiotic

maturation is quite precise and defined [21] allowing it to be used as a reference for other

intra-oocyte events.

3.2. Cytoplasmic maturation

Cytoplasmic maturation is not as clearly defined as the meiotic process since it

encompasses events both visible and invisible to the microscope. The early description of

cytoplasmic maturation is based on ultrastructural observations during the few days before

the LH surge when the oocytes awaits the ovulation signal. The first evidence of

cytoplasmic competence occurs when the oocyte stop its preparation phase (RNA and

protein synthesis) by modifying the transcription and translation machinery (nucleolus

condensation and ribosome depletion) [4,22,23]. A second series of changes occur close to

the LH surge which result in a re-distribution of organelles such as the mitochondria and

the cortical granules along with the changes occurring with the cell progression to

metaphase [24]. The second aspect of maturation that is often included as part of the

cytoplasmic maturation process is the accumulation of specific molecules, largely

unidentified, which prepare the oocyte for post-fertilization events. This component has

been referred to before as oocyte capacitation [18] but for the purpose of this explanation it

will be referred to as molecular maturation.

3.3. Molecular maturation

Molecular maturation is the least defined of the three types of oocyte maturation. As

indicated above, most of fully grown oocytes undergo normal meiotic and cytoplasmic

maturation although only a subset of them will develop to the blastocyst stage. The


difference between a developmentally capable oocyte and an incompetent one can be

related to the differentiation state of the follicle of origin and these differences are not

always visible in the oocyte at the ultrastructural level. Currently, the most popular

hypothesis is that specific mRNA and possibly some proteins are produced and added to the

oocytes stockpile in the last few days before ovulation. These unique capacitators

would give the ovary a last word on the outcome of ovulation by altering the developmental

ability of the gamete produced. Logically, there are several good evolutionary reasons for

such a mechanism that are beyond the scope of this chapter. What comes out of all these

observations is that special instructions, coming from a specific follicular environment and

accumulated in the oocyte, are essential to promote the proper molecular cascades for

embryonic genome activation and the development to the blastocyst stage. Since no clear

discrepancies associated with the first two types of oocyte maturation have been identified

to be responsible for developmental competence, it is believed that molecular maturation

represents the closest association with the intrinsic capacity of an oocyte to reach the

blastocyst stage and probably beyond.

4. Follicular influence on oocyte competence

To dissect the influence of the follicular environment on the oocytes acquisition of

developmental competence, it is important to distinguish a number of relevant follicular

phases as follows:

(1) The preantral phase

(2) The growing phase

a. FSH dependent

b. FSH independent

(3) The early atretic phase

(4) The late atretic phase

(5) The dominant phase

(6) The plateau phase

(7) The preovulatory phase

(8) The post LH phase

4.1. The preantral phase

As described before [4], oocytes from preantral follicles cannot complete meiosis

because of incomplete meiotic, cytoplasmic and molecular maturation.

4.2. The growing phase

4.2.1. FSH dependent

This category includes the cohort of small antral follicles that can respond to FSH either

in a normal estrous cycle leading to a single dominant follicle or in an ovarian stimulation

scheme which leads to ovulation in a multi-dominant paradigm. These follicles contain


oocytes with two to four layers of cumulus cells and very little, if any, signs of atresia.

Because these follicles are actively growing, it is very difficult to aspirate them from living

animals using ultrasound or laparoscopy and when removed in the growing phase (under

FSH stimulation) they display very limited competence [25,26]. The oocytes from these

follicles are partially competent only if maintained in the follicles post-mortem at body

temperature [27].

4.2.2. FSH independent

Once follicles reach a diameter of 8.5 mm in non-stimulated animals, they acquire LH

receptor in the granulosa layers and become less dependent of the FSH support [28].

Normally, in cows, only the follicle corresponding to the dominant follicle reaches this

status during each follicular wave. It is possible to induce the production of several of these

follicles with exogenous FSH support and, surprisingly, when oocytes are collected in the

active growing phase, they also display low developmental competence even if their size

often exceeds 79 mm [13].

4.3. The early atretic phase

The subordinate follicles and the dominant follicle in its demise contain oocytes of

relatively high developmental potential as measure by the blastocyst rate [29]. This

situation is potentially explained by similarities between maturing dominant follicle and

follicles in their early phases of atresia which might send similar maturation promoting

signals to the oocyte [27]. The increased competence level displayed by oocytes collected

from early atretic follicles and the reduced competence level of oocytes collected from late

atretic follicles can be induced artificially by prolonging folliculogenesis with external

FSH support followed by FSH withdrawal for 2448 h to obtain early atretic follicles or for

more than 72 h to collect oocytes from late atretic follicles [27].

4.4. The late atretic phase

The follicles entering the late atretic phase contain oocytes with a disrupted cumulus

layer which can be easily classified and result in a poor development rate to the blastocyst

stage [25]. A surprising observation is that atretic follicles above 5 mm (large subordinates

that were striving for dominance) often contain oocytes with partially expanded outer

layers of cumulus (as if the oocyte was trying to mature) and consequently display the

cumulus morphology change normally occurring after the LH surge.

4.5. The dominant phase

The dominant follicle is sustaining a fast growing rate for a few days and then reaches a

slower growth rate correlated with a higher estradiol output indicative of further follicular

differentiation [28]. Once these changes occur, the developmental potential of the oocyte

rapidly increases [18,29]. If this dominant follicle then faces a high progesterone level from

a persistent corpus luteum, it will not ovulate and the next follicular wave will emerge. The

potential of the oocyte within the non-ovulating dominant follicle is probably maintained a


few days but very few studies have analysed these oocytes in non-stimulated conditions.

The fact that prostaglandin injection can lead to ovulation of these follicles and subsequent

pregnancy following insemination provides additional evidence that these follicles

contains good eggs [30].

4.6. The plateau phase

This phase is naturally occurring between the establishment of dominance and the

resultant reduction of growth and the preovulatory period where progesterone

concentrations decrease sharply and LH pulsatility increases [31]. This phase can be

induced artificially by withdrawing FSH for 48 h after three consecutive days of

stimulation [12] and result in oocytes with a high competence to reach the blastocyst

stage.

4.7. The preovulatory phase

This phase is coincident with low progesterone concentrations and can be obtained

without stimulation on days 1920 of the oestrous cycle or with prostaglandin injections

any day after wave emergence or with FSH stimulation and prostaglandin. The oocytes

obtained in these conditions have a competence level close to that of oocytes that mature in

vivo [18].

4.8. The post LH phase

Oocytes collected at different times after the LH surge posses a high degree of

competence to reach the blastocyst stage although the variability across animals reflects the

delicate equilibrium between the normal ovulation and the stimulated multiple ovulation

obtained with FSH. Because pregnancy rates are quite high (>85%) in non-stimulated andinseminated heifers, it is assumed that most, if not all, of the oocytes produced in the

normal non-stimulated conditions are fully competent. By contrast, it is well known that a

significant proportion of eggs obtained after ovarian stimulation are not retrieved at the

blastocyst stage on day 7 post-insemination, supporting a variable level of competence

within an oocyte pool from different follicles. Such a result would suggest that, upon

stimulation, the ovary produces some late-growing follicles that ovulate oocytes of low

competence. Since follicles acquire the ability to ovulate with the appearance of LH

receptors in granulosa cells at relatively small diameter [28], follicles still growing under

the influence of FSH may be rushed to ovulation without the appropriate differentiation

stage so important to oocyte quality.

5. The effect of ovarian stimulation on oocyte competence

As indicated in previous sections, further evidence of follicular influence on oocyte

potential can be obtained by hormonal stimulation. Developmental competence is on

hold during growth and this is seen in follicles from all size. To activate that competence,


a process of follicular differentiation must be induced either through dominance or early

atresia. Therefore to maximize the assisted reproductive success, it is important to better

understand the different physiological steps that trigger the passage from follicular growth

to differentiation and ideally to reproduce this normal pattern using the right combination

of gonadotropins. For example, it could be physiologically logical to increase the LH

support and decrease the FSH support as stimulation progresses to better reflect the in vivo

processes and promote the final slow follicular growth accompanied with high

differentiation that results in better eggs.

By using molecular approaches to define the changes occurring in these follicles, it will

become possible to understand the differentiation processes that lead to the oocytes

increased developmental capacity. Preliminary analysis using subtractive molecular

approaches has revealed a number of good candidates both from the follicle side [32] and

the oocyte itself [33]. These early analyses are pointing to the early luteinization events as

indicators of the type of differentiation associated with increased competence.

The signalling from the follicle to the oocyte is certainly more complex than only one

given factor at one given threshold. The analysis of the competence of oocyte from

different follicles also indicates that it is not an all or nothing event. When oocytes originate

from differentiated follicles, they have a higher ability to produce a morphologically

healthy blastocyst which is normally associated with higher pregnancy rates. Although we

have not found in the literature a report that show directly the comparison of blastocyst

from IVP following ovarian stimulation versus in vivo, the fact that the ovarian stimulation

results in more blastocyst of good quality is supportive of an higher quality. The molecular

dissection of all aspects of both follicular and oocyte differentiation is the only way to

understand the complexity of these processes and to bring significant improvements to the

assisted reproduction procedures in such animals as the cow.

To illustrate the window of the acquisition of competence within the follicle, a figure

may be helpful (Fig. 1). During each follicular waves a number of follicles enter the rapid

growth phase and as they exit from it, the oocyte inside them acquires part or all the


Fig. 1. Schematic illustration of the growth of follicles in waves during the estrous cycle in cows. The grey-shaded

areas represent windows in which the competence of the oocyte is likely to increase significantly. The darker

follicles are the more differentiated ones and the stippled follicles are atretic.

information required to proceed to the blastocyst stage once fertilized. In regular cycle,

only the second or third wave will result in ovulation of an oocyte with the maximum

potential. The distinction between the growing follicles and the receding ones or the ones

reaching the plateau phase is probably resulting in difference of competence according to

the size of the follicle at inflexion.

It must be added that competence is temporary and maybe lost with more advanced

stages of atresia. It is not known if the competence factors disappears or other components

comes into play to prevent the development of an oocyte from a follicle in a more advance

stages of degeneration.

6. Conclusion

It is clear that the follicle can profoundly influence the quality of the oocyte obtained at

ovulation and, as a result, the quality of embryo obtained. There is also growing evidence

that the ovary resists ovarian stimulation by decreasing the quality of the oocytes it

produces. This brings back in perspective the importance of moderating the hormonal

stimulation protocols based on physiological considerations to optimize the yield of high

quality, transferable embryos in animals as well as in humans.

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Contribution of the oocyte to embryo qualityIntroductionOocyte competenceAbility to resume meiosisAbility to cleave following fertilizationAbility to develop to the blastocyst stageAbility to induce a pregnancyAbility to develop to term in good health

Oocyte maturationMeiotic maturationCytoplasmic maturationMolecular maturation

Follicular influence on oocyte competenceThe preantral phaseThe growing phaseFSH dependentFSH independent

The early atretic phaseThe late atretic phaseThe dominant phaseThe plateau phaseThe preovulatory phaseThe post LH phase

The effect of ovarian stimulation on oocyte competenceConclusionReferences

contribution of the oocyte to embryo quality- sirard 2006- theriogenology

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