Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

human embryos,

the majority caused by chromo-

somal errors arising during meiosis I. For instance,

chromosome non-disjunction in females that results

in tragic birth defects occurs during first meiosis,

probably because the stringency of metaphase-

anaphase checkpoint control is very low in the

maturing oocyte.

51,52

These findings have produced

clinically useful preconception genetic diagnostic

tests to predict chromosome errors, such as fluores-

cent in situ hybridization (FISH) of the first polar

body, a product of first meiosis,

as opposed to the

more typical preimplantation genetic diagnosis

(PGD) after embryo blastomere biopsy.

54,55

With the exception of rodents, all mammalian

oocytes reduce and lose their traditional, replicating

centrosomes, although the exact timing and nature

of this reduction is not well known. Extrapolation

from invertebrates

suggests that the maternal cen-

trosome is reduced before the completion of the



meiotic divisions.

In humans, all the mitotic divi-

sions of the oogonia are completed by mid-gestation

within female fetuses, i.e. 4–5 months prior to birth.

As reproduction cannot start for more than a

decade and the potential of eggs from women older

than 35 years declines swiftly, the nature of meiotic

spindle assembly and stability in oocytes aged over

decades is of both fundamental and clinical impor-

tance. With the advent of oocyte donation, women

beyond menopause can deliver healthy children if

the oocyte is obtained from a younger woman.

For reproductive biologists, questions persist

regarding the nature of meiotic spindle assembly

without the benefit of a maternal centrosome inside

a maturing oocyte.

As yet, it is unclear what distin-

guishes an oocyte that is reproductively viable in the

third decade of life from another oocyte in the

fifth decade that often produces aberrant meiotic

spindles and aneuploid embryos incapable of either

ABC

DEF

γ –Tub DNA

Mt γ –Tub DNA

Figure 26.6 Mitotic spindle formation in dispermic human zygotes. Dispermy in human zygotes results in a disorganized prophase

mitotic spindle (A) with four foci of ␥-tubulin ((B) location denoted by asterisks in (A)). This disorganized prophase spindle resolves

at metaphase resulting in a bipolar spindle (D) with well-organized chromosomes (F). Four foci of ␥-tubulin are still observed on the

spindle ((E) location denoted by asterisks in (D)). Mt, microtubules; ␥-Tub, ␥-tubulin. Arrows denote incorporated sperm axonemes

and asterisks denote ␥-tubulin foci. Bar ⫽ 10 ␮m. Reprinted with permission from Simerly et al.

HPE_Chapter26.qxp 7/13/2007 5:32 PM Page 346

THE SPERM CENTRIOLE: ITS EFFECT ON THE DEVELOPING EMBRYO

implantation or embryogenesis. Furthermore, female

meiosis is fundamentally distinct from that of male

meiosis for reasons not yet clear. Mammalian female

meiosis begins in utero, arrests until puberty, and is

then reinitiated once per month in a small pool of

immature oocytes until they are depleted and meno-

pause ensues. In contrast, mitosis in the male germ

line restarts with the onset of puberty, and continu-

ous waves of male meiosis as well as on-going prolif-

eration of male germ cells occur through the majority

of the life of the individual.

Clearly, understanding

these distinctions will be required to fully character-

ize gametogenesis and the special restrictions on

centrosome restorative properties in male and female

gametes so vital to ensure reproductive success.

THE SPECIAL PROBLEM OF

PARTHENOGENESIS: ROLES OF

MICROTUBULE MOTORS AND

SPINDLE STRUCTURAL PROTEINS

In the classical sense, the centrosomes direct mitotic

bipolar spindle assembly by acting as MTOCs at

their poles.

1,2

In rodents, the maternally derived cen-

trosomes inherited from oogenesis remain active for

directing the motility events crucial to the comple-

tion of the fertilization process during meiosis as

well as after insemination.

For other mammals,

the uniparental contribution of the centrosome is

strictly observed, as evidenced by multiple sperm

asters during polyspermic insemination as well as

by the lack of cytoplasmic asters after partheno-

genetic activation. Most species that follow a pater-

nal method of centrosome inheritance also support

parthenogenic activation,

and development of

cleavage stage embryos is observed.

61–64

Analyses of meiotic spindles in a variety of mam-

malian oocytes suggest that there is no maternal

centrosome at their poles, although functional

bipolar meiotic spindles assemble during meiosis.

20,65

Centrioles are absent in mammalian oocytes, but

can arise de novo in parthenogenetic embryos after

a few cleavage divisions.

13,61

Such observations pose a dilemma regarding the

restoration of the previously lost maternal centro-

some after parthenogenetic activation.

After pri-

mate and bovine parthenogenetic activation,

14,66

bipolar spindle with aligned chromosomes forms at

first mitotic metaphase (Figure 26.7A), but without

identifiable centrosomal proteins (i.e. ␥-tubulin or

pericentrin) at the poles.

These spindles accurately

segregate the chromosomes (Figure 26.7B) and can

complete preimplantation development to the

blastocyst stage.

Exactly how meiotic and parthenogenetic

mitotic spindles can assemble and direct cell divi-

sion without functional MTOCs is not well under-

stood.

Some evidence suggests that acentrosomal

systems assemble bipolar spindles by an ‘inside-out’

mechanism where random microtubules bound to

chromatin are sorted and assembled into a bipolar

structure using specific molecular motor proteins

related to the bim C and nod class of kinesin-related

proteins. After sorting these microtubules, the spin-

dle poles are then shaped by a host of other proteins,

including the minus-end directed motors dynein

and the Kin C kinesins, the plus-end directed

motors of the bim C family (i.e. Eg5) (Figure 26.7),

and the structural proteins dynactin and NuMA

(nuclear mitotic apparatus protein).

In mouse

oocytes, Eg5 (a Kin N plus end-directed kinesin)

and HSET (Kin C minus-end directed kinesin) are

present in meiotic spindles; perturbing their activity

following microinjection of function-blocking

antibodies demonstrates that they are required for

bipolar spindle assembly.

Similar crucial roles for

minus-end directed kinesin motors in organizing

female meiotic spindles have been described for

Drosophila mutants deficient in the minus-end

kinesin nonclaret disjunctional (ncd).

Thus, the

centrosome may be dispensable for meiotic and first

mitotic spindle formation after parthenogenetic

activation in mammalian eggs. Rather, microtubule

crosslinking and oppositely oriented motor activ-

ity, as well as spindle matrix proteins like NuMA

and dynactin, are critical components required for

assembling these spindles.

However, the centro-

some is required to complete male and female

pronuclear apposition and serves as the dominant

MTOC and spindle pole organizing entity when

present.



HPE_Chapter26.qxp 7/13/2007 5:32 PM Page 347

HUMAN PREIMPLANTATION EMBRYO SELECTION

CENTROSOMES DURING NUCLEAR

TRANSFER IN NON-HUMAN PRIMATES

Nuclear transfer (NT) in mammals challenges fun-

damental tenets of developmental biology, includ-

ing requirements for exactly two parents of opposite

sexes during natural reproduction.

Whereas two

haploid genomes unite within the activated egg’s

cytoplasm during fertilization, as directed by

the introduced sperm centrosome, the maternal

spindle–chromosome complex is removed during

‘enucleation’ and a diploid nucleus is inserted. After

a period of reprogramming, the NT construct is acti-

vated as during parthenogenesis and ultimately trans-

ferred to a pseudopregnant recipient for production



of live young. NT is sporadically successful in a few

mammals (mice, domestic species), but its mecha-

nisms still remain largely unexplained. In non-human

primates, however, application of NT protocols

proven successful in other mammals have identified

unexpected problems, including the extranuclear

inheritance of the centrosome.

67,73

Imaged live, non-human primate NT constructs

appear normal, yet no pregnancies resulted from 140

transfers at the 2–8-cell stage into 25 surrogates.

Microtubule and DNA imaging of interphase NT

constructs showed abnormal microtubule patterns,

most likely the result of dysfunctional somatic cen-

trosomes, and inappropriate nuclear reconstitution

(Figure 26.8A–C) compared with fertilized controls,

androgenotes (ICSI fertilization of enucleated

oocytes), and bovine clones produced by similar NT

techniques (Figure 26.8D–F).

66,67,73

Furthermore,

somatic centrosomal dysfunction in the non-human

primate NT constructs appears correlated to the pri-

mate ooplasm, as evident by a focused microtubule

astral array when a monkey fibroblast nucleus is

transferred into a bovine enucleated cytoplast

(Figure 26.8G). At first mitosis, multipolar spindles

with misaligned chromosomes assembled after

somatic or embryonic donor cell nuclear transfer

(Figure 26.9A),

are distinctly different from NT

bipolar mitotic spindles in cows produced by similar

methods (compare with Figure 26.8F).

66,67

Many

resultant NT embryos that underwent cleavage were

aneuploid with limited developmental potential,

though strict adherence to the timing of enucle-

ation and method of artificial activation suggested

improved cloned blastocyst development rates.

The

high rate of incorrectly assembled bipolar mitotic

spindles in non-human primate constructs is sur-

prising, given the routine ability of polyspermic and

parthenogenetic eggs to assemble bipolar spindles

with aligned chromosomes (Figure 26.7). Without

the sperm centrosome, unanticipated obstacles

prevent proper non-human primate NT construct

development, including chromosome imbalances,

inaccurate spindle pole numbers, and the dysfunc-

tional acquired somatic cell centrosome.

Non-human primate NT constructs demand two

critical structures missing during cloning – the

Eg5/Mt/DNA

Figure 26.7 Mitotic spindle assembly after parthenogenetic

activation of monkey oocytes. (A) Parthenogenetically activated

rhesus oocytes at mitosis assemble anastral, bipolar spindles

with aligned chromosomes at metaphase. Maternal centrosomes

are absent at the spindle poles based on the lack of foci contain-

ing conserved centrosomal constituent proteins like ␥-tubulin,

pericentrin, or centrin. Oppositely oriented motor proteins like

Eg5 (green) and HSET along with spindle pole organizing

components like NuMA probably direct bipolar spindle

assembly after artificial activation. (B) Telophase stage

parthenogenetically activated monkey oocyte demonstrating

organized chromosome separation during cell division. All

images triple-labeled for Eg5 kinesin (green), microtubules

(red), and DNA (blue). Bars ⫽ 10 ␮m. Reprinted with

permission from Simerly et al.

HPE_Chapter26.qxp 7/13/2007 5:32 PM Page 348

THE SPERM CENTRIOLE: ITS EFFECT ON THE DEVELOPING EMBRYO

zygotic centrosome formed from the sperm’s proxi-

mal centriole and egg’s cytoplasmic constituents,

and the oocyte’s meiotic spindle–chromosome

complex. Microtubule minus-end residing proteins

like the M-phase kinesin HSET

and the structural

matrix proteins NuMA are present at the spindle

poles in unfertilized meiotic and fertilized mitotic

oocytes (Figure 26.9B–C). However, these vital

spindle components are not detected in NT mitotic

spindles after nuclear transfer (Figure 26.9D and E).

Conversely, the oppositely oriented kinesin motor

Eg5 binds centromere pairs in meiotic and mitotic

spindles, including those on NT-cloned mitotic

spindles (Figure 26.9F). Why NuMA and HSET are

disrupted from participating in NT mitotic bipolar

spindle assembly is not entirely clear, although

removal of the meiotic spindle during the initial enu-

cleation step may perhaps remove a large majority of

these proteins from the cytoplasmic pool.

Support-

ing evidence for this theory is found in experiments



Figure 26.8 Abnormal microtubule patterns after nuclear transfer (NT) in monkey (Rh), but not bovine, constructs. (A)–(C)

Disarrayed microtubules (Mt) (green) assembled near the transferred somatic cell nucleus (blue) indicate a dysfunctional somatic

centrosome following intracytoplasmic nuclear injection (ICNI) into enucleated monkey oocytes and artificial activation. Some

constructs demonstrate cortical microtubule patterns (C) similar to parthenogenetically activated oocytes suggesting no functional

microtubule organizing activity by the transferred somatic cell centrosome. (D) and (E) Tightly focused microtubule arrays (green)

emanating from the transferred nucleus (blue) in activated bovine enucleated cytoplasts following either embryonic (D) or somatic

cell (E) nuclear transfer. (F) Normal, anastral, bipolar spindles (green) with aligned chromosomes assemble at metaphase (blue) in

activated bovine nuclear transfer constructs. (G) A focused microtubule array (green) from a rhesus fibroblast cell (blue) transferred

into a bovine enucleated oocyte suggests that normal somatic centrosomal function at interphase is dependent on the maternal

cytoplasmic source more than the donor nuclear source. All images double-labeled for microtubules (green) and DNA (blue).

Bars ⫽ 10 ␮m. Reprinted with permission from Simerly et al.

HPE_Chapter26.qxp 7/13/2007 5:33 PM Page 349

HUMAN PREIMPLANTATION EMBRYO SELECTION

combining fertilization with nuclear transfer, where

the resulting tetraploid mitotic spindles assemble

with properly aligned chromosomes on bipolar

spindles and NuMA is detected at each spindle pole

(Figure 26.9G).

Primate NT is challenged by molecular require-

ments in assembling the first mitotic spindle that

appear to be stricter than in other mammals in

which NT succeeds. In cattle, the somatic centro-

some transferred during NT organizes a large, well-

formed microtubule aster within the recipient’s

cytoplasm,

67,74

while mice rely on the maternal

centrosome within the ooplasm.

Successful repro-

ductive cloning is achieved routinely only in those



well-studied species that provide vast numbers of

both oocytes and surrogates, systems often capable

of multiple deliveries and amenable to transfers of

supernumerary embryos.

FUTURE RESEARCH CHALLENGES FOR

THE CENTROSOME BIOLOGISTS

The specialized field of NT, although controversial,

is aimed at determining the scientific feasibility of

‘therapeutic cloning’.

As shown in relevant animal

studies, NT cloning fails owing to a number of

defects, including nuclear reprogramming, cell cycle

10 µm 10 µm

10 µm

10 µm10 µm10 µm

EF G

Figure 26.9 Faulty mitotic spindles produce aneuploid embryos after primate nuclear transfer. (A) Defective nuclear transfer (NT)

mitotic spindle with misaligned chromosomes. Centrosomal NuMA at meiosis (B) and mitosis (C), but not in mitotic spindles after

NT (D). The centrosomal kinesin HSET is also missing after NT (E), but not centromeric Eg5. (F) Bipolar mitotic spindles with

aligned chromosomes and centrosomal NuMA after NT into fertilized eggs (G). Blue, DNA; red, ␤-tubulin; green, NuMA in (B),

(C), (D), and (G); green, HSET in (E); green Eg5 in (F). Reprinted with permission from Simerly et al.

HPE_Chapter26.qxp 7/13/2007 5:33 PM Page 350

THE SPERM CENTRIOLE: ITS EFFECT ON THE DEVELOPING EMBRYO

asynchrony, gene mis-expression during develop-

ment, genomic imprinting errors, placental dysfunc-

tion, and technical damage, to name but a few.

Additionally, epigenetic obstacles like cytoplasmic

incompatibilities in mitotic spindle assembly are

noteworthy hurdles. Primate NT constructs appear

to be normal when imaged live and non-invasively,

but they demonstrate limited reproductive potential

due to severe aneuploidy. Understanding these abnor-

malities will facilitate the production of genetically

identical non-human primates for characterizing

human diseases in a relevant preclinical model. Such

investigations will be invaluable for assessing the

promise of innovative embryonic stem cell therapies

for clarity on the root causes of diseases, the produc-

tion of novel disease cures through new drug dis-

covery, and exploring early human developmental

origins, including the onset of fetal disorders.

75,77

Current approaches of NT cloning in non-human

primates cannot meet these goals due to chromosome

imbalances resulting from spindle defects. A key

element to resolving such defects will be in fully

characterizing somatic cell centrosome inheritance

and reconstitution following NT.

Reproduction, typically error prone, is clinically

successful in fewer than a quarter of natural concep-

tions.

Knowledge of the cellular and molecular

events of fertilization is, therefore, critical for the

reproductive and developmental biologists. For the

centrosomal biologists, progress in understanding

the molecular and cellular underpinnings of the cell’s

MTOC remains an exciting research area. Already,

fundamental discoveries have been made in centro-

somal inheritance, composition, assembly, duplica-

tion, and separation in a variety of systems. Future

discoveries in understanding fertilization and early

development hinge on a more complete knowledge

underlying this crucial cellular structure, and will

greatly facilitate modern molecular medicine in

addressing a multitude of clinical problems in infer-

tility, contraception, and birth defects.

ACKNOWLEDGMENTS

We thank Dr Gerald Schatten for his deep inspira-

tion, unbridled enthusiasm, and incredible support

of cell and developmental biologists. We also thank

all of our wonderful scientific colleagues, both past

and present, for stimulating scientific collaborations

in centrosomal biology, including our current

members: Ahmi Ben-Yehudah, Carlos Castro, Kevin

Grund, Laura Hewitson, Ethan Jacoby, Chih Cheng

Lin, Dave McFarland, Jodi Mich-Basso, Cindy

Oberley, Kyle Orwig, Hina Qidwai, Carrie Redinger,

Mario Rodriguez, Meena Sukhwani, and Jamie

Tomko. We thank Dr Diane Carlisle for editorial

assistance with the manuscript, and the continued

support of the National Institute of Health (non-

human primates, rodents, and domestic species). All

protocols performed in this review were approved

by the appropriate University’s Research Animal

Review and Human Subjects Institutional Review

committees.

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acidified Tyrode’s solution 137, 139–40

microneedle application 137, 138–9

adaptive immune system 155, 156

age see maternal age

alanine 186

Alzheimer’s disease 260

amino acids 185, 193

endogenous pool

factors affecting 195

in older females 197

essential amino acids 195–6

sulfur amino acids 196–7

amino acid metabolism 185–6, 193–8

ammonia toxicity 197

analysis 181

glutamine degradation 197–8

ammonia

elimination 197

toxicity 197

anaphase promoting complex (APC) 267, 268

androgenetic embryos 236

aneuploidy 309

chromosome-specific 205

and embryonic competence 215

FISH, discovered by 205

and fragmentation 57–8

frequency in cleavage stage 209

after nuclear transfer 350

polyspermy 343, 345

reduction due to PGD 288

specific chromosome rates 214

testing in IVF 206

Angelman syndrome 240, 326, 345

angiogenic factors in follicular fluid 311–13

anti-Mullerian hormone (AMH) 314–15

antioxidants 191–2

antrum formation 302

apoptosis

causing fragmentation 66–8

SNP-induced 172

in spermatogenesis 327–8

marker proteins 328

ART see assisted reproductive technologies (ART)

ascorbic acid 198

assisted hatching (AH) technique 16

in cryopreservation 130

embryo selection 138–9

general considerations 135–8

meta–analyses 136–7

monozygotic twinning 141

patient selection 138

usefulness 136

zona opening

acidified Tyrode’s solution 139–40

laser 140

assisted reproductive technologies (ART)

current trends 1

HLA-G in see assisted reproductive technologies

(ART)

in large animals 238–9

success rate 228–9

asters see cytasters; sperm-derived centrosomes

(sperm asters)

asymmetric blastomeres

blastomere size during cleavage 96

and chromosomal abnormalities 212

Barker hypothesis 164

basic fibroblast growth factor (bFGF) 265

Bax proteins 67–8, 73

Bcl-2 proteins 67–8, 73

Beckwith-Wiedemann syndrome 240

binucleated MNEs 47–8

implantation/pregnancy rates 49

bioinformatics 247–8

biomarker discovery with metabolomics 250–1

biopsy see embryo biopsy; polar body biopsy

biospectroscopy-based metabolomics 249, 253

bipolarization 6

blastocysts 2, 79–87, 156

abnormal 12, 13

conversion 112, 114

culture 9, 79–81

development 108–9, 109

Index

N.B. Page numbers in italic denote figure or table legends

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