Elder K. Human preimplantation embryo selection

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HPE_Chapter25.qxp 7/14/2007 5:52 PM Page 336

26. The sperm centriole: its effect on the

developing embryo

Calvin R Simerly and Christopher S Navara



Centrosomes are classically defined as a pair of cen-

trioles, structures of nine triplet microtubules with-

out a central microtubule pair, surrounded by the

pericentrosomal matrix (PCM). The PCM is the

amorphous material that nucleates the microtubules

and defines their intrinsic polarity (i.e. minus ends

(slower turnover) anchored at each centrosome;

plus ends (faster turnover) radiating outward).

1,2

The past 25 years have seen an explosion in under-

standing basic centrosomal biology with regards to

molecular constituents and the minimal structure

required to promote microtubule nucleation from

this structure. Rapid advances in understanding

centrosomal inheritance, assembly, duplication, and

segregation in a variety of cell types provide crucial

clues for understanding how the centrosome medi-

ates intracellular motility, cytoplasmic organization,

and the many other cellular processes linked to cen-

trosomal activities.

3–5

These advances are beginning

to translate to modern molecular medicine, where

clinical challenges including infertility treatments

and contraception require a greater understanding

of this crucial cellular organelle.

Within the fields of reproductive and develop-

mental biology, the cellular and molecular events

during fertilization are intrinsically grounded in

understanding the role of the centrosome. Boveri

recognized more than a century ago that at fertiliza-

tion the sperm contributes the centrosome, the cell’s

major microtubule organizing center (MTOC) and

the structure that organizes the mitotic spindle poles

(cleavage centers). However, the process of centro-

some reduction and restoration during meiosis,

fertilization, and mitosis has remained perplexing

(Figure 26.1). In somatic cell cycles, the chromo-

somes, cytoplasm, and centrosome double during

interphase, with each daughter cell receiving one

diploid chromosome set, one centrosome, and half

of the cell volume following division (Figure 26.1).

During fertilization, however, there is ambiguity

about the inheritance and reconstitution of the func-

tional zygotic centrosome (Figure 26.1). Although

each gamete contributes a haploid genome at insem-

ination, the egg provides most of the cytoplasmic

contribution. Boveri first recognized that the egg

typically loses the centrosome during oogenesis and

that the sperm typically introduces this structure at

fertilization.Yet, understanding centrosome behavior

has remained a persistent problem in cell biology

and an especially enigmatic one during mammalian

development.

The foundations for the fields of cell, molecular,

developmental, and reproductive biology, provided

by the study of fertilization in lower animal species

(i.e. sea urchins, frogs) is being greatly extended by

enhanced methods for performing in vitro fertiliza-

tion (IVF) in many mammals, including humans.

A century after the discovery of the centrosome,

human IVF was achieved

– more than one million

IVF babies have now been born.

The otherwise dis-

carded gametes and embryos from IVF clinics are

providing a precious and unique research resource

for the modern-day centrosomal physician/scientist.

As a result, reproductive errors like polyspermy

(fertilization by more than one sperm) and partheno-

genesis (development beginning in an activated egg

without any sperm) have provided insights that

subtly challenge aspects of the unipaternal centro-

some inheritance theory. Promising investigations

in non-human primates appear to align closely to

humans, and avoid the many complexities in work-

ing with fertilized human oocytes for research.

Such advancements have direct implications in clin-

ical reproductive medicine, which in many ways has

pioneered amazing clinical achievements in assisted

reproductive technologies (ART) such as intracyto-

plasmic sperm injection (ICSI) that have produced

thousands of human babies.

Although fundamental

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HUMAN PREIMPLANTATION EMBRYO SELECTION

research still lags in efforts to understand these

clinical accomplishments, biomedical researchers

are now poised to begin investigations on the

cellular and molecular events that underlie ART

advances.

This review focuses on the role of the centro-

some during fertilization, with special attention to

human reproduction and development. We also

consider the centrosome in devastating disease dis-

orders that arise in human development resulting

from aberrant genomic imprints. These new research



frontiers represent unique challenges for the next

generation of centrosome biologists, owing in large

part to the ethical, moral, political, and religious hur-

dles that accompany working with human gametes.

Consequently, experimental results are obtained

and/or corroborated by studying non-human pri-

mate development. This is essential, ironically,

because fertilization in outbred mice, hamsters,

and rats – while vital for fundamental research

investigations – represent rare exceptions to Boveri’s

theory on centrosome inheritance.

Somatic cells cycles Gametes at fertilization

1N + 1N

2(2N)

100%

zygote

100%

egg

>99%

sperm

>1%

200%

100%

Cytoplasm CytoplasmChromosome ChromosomeCentrosomes Centrosomes

Figure 26.1 The problem of centrosome inheritance. During each cycle in somatic cells (A), the chromosomes (2N1→2N2),

cytoplasm (100→200%), as well as the centrosomes (·→··), duplicate during interphase. During cell division, the chromosomes,

cytoplasm, and centrosomes all split in two. Each of the gametes during fertilization (B) contributes a haploid chromosome set

to the zygote (1N

⫹ 1N

→ 2N

), and while the egg contributes the vast bulk of the cytoplasm (⬎99%), the relative contributions

of the sperm and the egg to the zygotic centrosome are not yet understood (*). Reprinted with permission from Schatten.

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THE SPERM CENTRIOLE: ITS EFFECT ON THE DEVELOPING EMBRYO

CENTROSOMES DURING HUMAN

FERTILIZATION

Centrosome activity in spermatogenic cells centers

around the generation of the sperm axoneme required

for sperm motility. With the exception of rodents,

where the paternal centrioles are completely disman-

tled, mammalian sperm reduce the pair of centrioles

to a single inactive structure during spermatogenesis,

the proximal centriole.

The vast majority of the

proteins of the pericentriolar material are shed in the

cytoplasmic droplet during sperm maturation during

migration through the epididymal tract. Conversely,

oocytes lose both centrioles during oogenesis as

investigated by electron microscopy, but retain a sig-

nificant pool of pericentriolar proteins as observed by

immunocytochemistry and Western blotting.

Centrosomal inheritance during human fertil-

ization

is depicted from the organization of micro-

tubules and DNA observed in discarded human

oocytes (Figure 26.2) and mirrors the inheritance

pathway found in most animals, except rodents.

7,15,16

Microtubules are only observed in the metaphase-

arrested second meiotic spindle in the unfertilized

human oocyte (Figure 26.2A), unlike the rodent

that retains multiple cytoplasmic microtubule asters

(‘cytasters’).Within 6 hours postinsemination, a small

microtubule aster emanates from the introduced

sperm proximal centriole (Figure 26.2B and C). The

activated oocyte extrudes the second polar body,

observed here attached to the developing female

pronucleus by the microtubule based midbody

structure (Figure 26.2B and C). The enlarged sperm

centrosome (the ‘zygote centrosome’) nucleates

microtubules that assemble the first microtubule-

based structure in the fertilized egg – the sperm

aster. The sperm aster in fertilized human oocytes is



Figure 26.2 Microtubule and DNA organization in normal

inseminated human oocytes. The meiotic spindle in mature,

unfertilized human oocytes is anastral, oriented radially to the

cell surface, and asymmetric, with a focused pole abutting the

cortex and a broader pole facing the cytoplasm (A). No other

microtubules are detected in the cytoplasm of the unfertilized

human oocyte. Shortly after sperm incorporation (3–6.5 hours

postinsemination), sperm astral microtubules assemble around

the base of the sperm head, as the inseminated oocytes complete

second meiosis and extrude the second polar body (B)–(E). M,

male pronucleus; F, female midbody. The close association of

the meiotic midbody identifies the female pronucleus (B)–(E).

Short, sparse, disarrayed cytoplasmic microtubules can also be

observed in the cytoplasm following confocal microscopic

observations of these early-activated oocytes (C). As the male

pronucleus continues to decondense in the cytoplasm, the

microtubules of the sperm aster enlarge, circumscribing the

male pronucleus (D) and (E). By 15 hours postinsemination,

the centrosome splits and organizes a bipolar microtubule array

that emanates from the tightly apposed pronuclei (F). The

sperm tail is associated with an aster (arrow) (F). At first mitotic

prophase (16.5 hours postinsemination), the male and female

chromosomes condense separately as a bipolar array of micro-

tubules marks the developing first mitotic spindle poles (G). By

prometaphase, when the chromosomes begin to align on the

metaphase equator, a barrel-shaped, anastral spindle forms in

the cytoplasm (H). The sperm axoneme remains associated with

one small aster found at one of the spindle poles (arrow) (H).

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

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HUMAN PREIMPLANTATION EMBRYO SELECTION

the typical radially arrayed monaster juxtaposed to

the sperm nucleus (Figure 26.2D and E) (which is

called the ‘male pronucleus’) after the sperm chro-

matin has decondensed within the egg cytoplasm.

These microtubules elongate throughout the cyto-

plasm during early development, some contacting

the female pronucleus and initiating pronuclear

migration (Figure 26.2E and F). As in most animal

eggs, the sperm tail enters the egg (Figure 26.2F).

Often, one or two punctate foci are found at the cen-

ter of the sperm aster exactly at the junction between

the sperm axoneme and the male pronuclear surface

which correspond to the sperm centriole(s).

The

reconstituted zygotic centrosome duplicates and splits

during late interphase, presumably under the control

of cell cyle regulatory machinery

17,18

(Figure 26.2F).

Mitotic prophase commences with the separate

chromosomal condensation of the male and female

pronuclei, as the zygotic centrosomes nucleate the

microtubules of the bipolar mitotic spindle apparatus

(Figure 26.2G). By late prometaphase, the condensing

parental chromosomes align along the equator of

the bipolar, anastral mitotic spindle (Figure 26.2H),

completing the fertilization process in humans.

These data confirm that humans inherit their

centrosomes from their fathers.

Evidence supporting the sperm centriolar complex

as the foundation for zygotic centrosome formation in

humans comes from studies on polyspermic fertiliza-

tion and parthenogenesis.

16,19

As shown in Figure 26.3,

when two sperm enter the oocyte, each paternal cen-

triolar complex organizes a sperm aster at the base of

the sperm head (Figure 26.3A and B). Conversely,

parthenogenetic (artificial) activation of oocytes, in

which no contribution of the paternal centrosome

is provided, causes random, disarrayed interphase

microtubule patterns (Figure 26.3C and D). Collec-

tively, these data reinforce the observation that the

centrosome is paternally inherited in humans.

7,14

CENTROSOME DYSFUNCTION AND HUMAN

INFERTILITY

Human gametes discarded from infertility clinics

performing ART and donated for research provide

important insights into the diagnosis of novel forms

of human infertility, especially male factor infertil-

ity.

14,20,21

Centrosome dysfunction after the sperm

enters the oocyte is being recognized as a new cause

of male factor infertility during human reproduc-

tion (Figure 26.4).

14,21–26

For instance, when the

assembled sperm centrosome after incorporation is

dysfunctional in microtubule assembly or organiza-

tion, the sperm aster may fail to form or be so

poorly organized as to be inconsistent with the abil-

ity to conduct pronuclear apposition. Sperm that

fail to activate the oocyte and initiate exit from

metaphase arrest often have underdeveloped micro-

tubule asters adjacent to incorporated sperm heads

(Figure 26.4A and B). Other sperm may nucleate

multiple asters (Figure 26.4C and D), or can fail to

correctly organize the sperm astral microtubules



DNA

Figure 26.3 Microtubules (Mt) and DNA organization during

human polyspermic fertilization and parthenogenesis. (A) and

(B) During dispermic fertilization, a microtubule-based sperm

aster assembles at the base of each incorporated sperm head

(M) (B) from the site of the paternal proximal centriole

(arrows) (A). (C) and (D) Activation of the human oocyte

without sperm penetration (parthenogenesis) results in

random, disarrayed cortical microtubule assembly during

interphase. F, female pronucleus. Bar ⫽ 10 ␮m. Reprinted

images with permission from Simerly et al.

The white bar

dividing A and B indicates the two focal planes.

HPE_Chapter26.qxp 7/13/2007 5:32 PM Page 340

THE SPERM CENTRIOLE: ITS EFFECT ON THE DEVELOPING EMBRYO

after incorporation resulting in oocytes that fail the

fertilization process and arrest in early development

(Figure 26.4E and F). About 25% of the human

oocytes classified as ‘failed to fertilize’ demonstrated

successful sperm incorporation, but with the oocytes

failing to properly assemble sperm astral micro-

tubules and/or undergo pronuclear formation.

14,25

These data show that the formation and functioning

of the sperm aster is essential during human

fertilization, and that naturally occurring defects are

causes of fertilization failures.

A study on the phenotypic expression of bull

centrosomes further illustrates the clinical, agricul-

tural, and fundamental importance of the centro-

some during fertilization.

Sperm from bulls were

selected and classified as superb, average, or subfer-

tile based on estrous non-return rates of 2500 cows

who underwent artificial insemination in field

studies, as well as blastocyst production rates



Bull A

100

Sperm aster size (µm)

Sperm aster quality

120

n = 100

n =91

n =81

Bull B Bull C

Figure 26.4 Microtubule (A), (C) and (E) and DNA (B), (D)

and (F) organization in ‘fertilization failures’ and phenotypic varia-

tions among paternal centrosomes. Common examples of the

stages at which human fertilization arrests after sperm incorpo-

ration include incomplete sperm aster assembly (A) and (B);

inappropriate detachment of the sperm aster and tail from the

male pronucleus (M), as well as androgenesis (C) and (D); and

disarrayed sperm astral organization (E) and (F). (A) and (B) A

truncated aster at the base of the sperm head has formed, but

not enlarged, by 24 hours postinsemination and the oocyte

remains arrested at a late meiotic phase. The incorporated

sperm tail is observed (arrow) (A). (C) and (D) The insemi-

nated oocyte displays a single pronucleus separated from the

nearby sperm tail (arrow) (C), indicating the presence of a male,

but not female, pronucleus. This example of androgenesis may

have occurred after the loss of the maternal chromosomes dur-

ing first or second polar body extrusion. Two small asters are

visible; one in association with the incorporated sperm tail

(arrow), while the other is detected at the male pronucleus

(arrowhead) (C). (E) and (F) A defect in microtubule aster

organization around the decondensing male pronucleus (E).

(G) Comparison of sperm aster size and quality in three bulls of

known fertility. Diameter of the sperm aster at its largest plane

was measured using the confocal microscope. A quality score

was also given to each aster. The bull with the highest field fertil-

ity and in vitro fertility (bull A) also had the largest and best-

organized sperm asters with averages of 101.4 ␮m and 1.8,

respectively. Bull B had an average sperm aster diameter of

78.2 ␮m and an aster quality score of 1.4. The bull with the

worst in vitro fertility (bull C) had the smallest (77.9 ␮m) and

most poorly organized sperm asters (1.2). These data represent

five repetitions: comparisons were made using the protected

means of the least squares method. Different letters indicate

significant differences (p ⫽ 0.025); bars indicate standard error.

Bar ⫽ 10 ␮m. Images (A)–(F) reprinted with permission from

Simerly et al.

Graph reprinted from Navara et al.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

following IVF. Using randomized bovine oocytes,

each bull sperm set was used for IVF and zygotes

fixed at a selected timepoint. As shown in Figure

26.4G, the organization and size of the sperm aster

varies according to the bull sperm, suggesting that

the quality or quantity of the sperm centrosome

directly affects the success and speed of fertilization,

and is correlated with the frequency of live births.

Perhaps variations in centrosomal vigor occur as

is found in other inherited components, a view

strengthened by recent observation of impaired cat

embryonic development in vitro correlated with

poor centrosomal function of cat testicular sperma-

tozoa following ICSI.

Collectively, these investigations have the

potential to be developed into novel screens for

male fertility.

7,19,22

Most sperm assays examine

parameters – motility, morphology, and counts –

that are factors more geared to successful fusion of

the sperm and egg plasma membranes. Yet, fertiliza-

tion is not successfully concluded until the sperm

and egg genomes align at metaphase of first mitosis,

and this requires the proper formation and func-

tioning of the zygotic centrosome and sperm aster.

DIAGNOSING MALE INFERTILITY BY

CENTROSOME FUNCTION ASSAYS

Zygotic centrosome formation as an early critical

step towards the accurate completion of the fertil-

ization process is a multistep pathway occurring

between the end of second meiosis and the transi-

tion into interphase of the first cell cycle. Central to

this process is assembling microtubules in the proper

organization to form the sperm aster that can

quickly direct proper pronuclear migration. Later,

the zygotic centrosome, duplicated under cell cycle

control, will define the site of first bipolar mitotic

spindle assembly within the activated cytoplasm

and participates in spindle organization by serving

as a dominant MTOC at the spindle poles.

Understanding centrosome reconstitution during

fertilization is inherently important for exploring

the molecular components necessary for determin-

ing centrosome parental origin and function.

7,19

Cell-free cytoplasmic extracts obtained from cyto-

static factor (CSF)-arrested Xenopus laevis oocytes

have effectively explored centriole formation and

microtubule assembly in vitro.

30–32

These pioneering

studies demonstrated that the crucial constituents

necessary for centrosome construction and reproduc-

tion reside in the oocyte. With regards to the relative

parental contributions to the zygote’s centrosome, the

Xenopus studies demonstrate that the sperm centro-

some contains conserved centrosomal constituent

proteins like centrin (a ubiquitous Ca

2⫹

-binding

protein of centrosomes) and pericentrin (a 220 kDa

component of the centrosomal matrix),

30,33

but

undetectable amounts of ␥-tubulin, a rare, invariant

constituent of the centrosome required for micro-

tubule nucleation and for defining the intrinsic

polarity of assembled microtubules from the cen-

trosome.

After exposure to Xenopus egg extracts,

both ␥-tubulin and phosphorylated epitopes are

detected on the sperm centrosome. Careful experi-

mental analysis of sperm in egg extracts suggests that

the Xenopus sperm contributes a structure capable

of binding maternal ␥-tubulin, does not require

assembled microtubules or microfilaments, but

does require egg extract and is ATP-dependent.

30–32

The sperm centrosome thus becomes competent for

nucleating microtubule growth into sperm asters in

vitro.

Analysis of human and bovine sperm in X. laevis

CSF-arrested extracts provides a basis for studying

the assembly of a zygotic centrosome capable of

nucleating and organizing microtubules in vitro.

19,34

Mammalian sperm exposed to increased calcium

levels, plasma membrane destabilization, and disul-

fide bond reduction unveils paternal ␥-tubulin and

other centrosomal protein binding sites, concomi-

tant with the onset of pronuclear decondensation.

This ‘procentrosome’ structure is thus primed to

attract and bind maternal ␥-tubulin from the egg’s

cytoplasmic pool. Conversely, other paternal cen-

trosomal proteins predicted to be critical for the

reorganization of the sperm centrosomal complex

following insemination (i.e. centrin) are modified

following exposure to the egg’s cytoplasm.

33,35

Exposure to an elevated kinase activity within the

meiotic cytoplasm then shifts the microtubule



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THE SPERM CENTRIOLE: ITS EFFECT ON THE DEVELOPING EMBRYO

useful prospective centrosomal assay system.

This test permits observation of pronuclear apposi-

tion mediated by a functional human sperm centro-

some within a living egg, as opposed to just

microtubule assembly in egg extracts. Research has

shown that the recipient oocyte must be from a

species other than rodents, i.e. species that follow

the paternal inheritance of the centrosome.

The

zona-free hamster oocyte sperm penetration test,

for assaying human male infertility, is a uniquely

inappropriate model for the investigation and diag-

nosis of impaired sperm centrosome function of

human sperm, since hamster oocytes retain their

maternal centrosomes from oogenesis.

Instead,

oocytes from animals like the rabbit or cow, which

support paternal centrosomal functioning, are

more relevant models to investigate human centro-

some reconstitution, sperm aster formation, and

sperm-mediated pronuclear apposition.

Centrosome microinjection therapy could theo-

retically overcome defective centrosomes responsi-

ble for fertilization arrest in certain types of male

infertility.

7,19

However, research suggests that only

centrosomes introduced from intact sperm will

complete the entire fertilization process and cor-

rectly segregate their chromosomes at first cell

division,

indicating that centrosome numbers and

positioning at the pronuclear surface is a critical

parameter for completion of fertilization. While still

speculative, if successful as a therapeutic treatment,

the resulting embryo would have two fathers: a

genomic father and a centrosomal one.

POLYSPERMY AND THE ‘DISPERMY

HYPOTHESIS’ FOR THE ORIGINS

OF GENOMIC IMPRINTED

DISORDERS IN HUMANS

Polyspermy is invaluable for testing the relative

parental contributions of the centrosome, since the

paternal contribution is multiplied. In most animals,

dispermic insemination introduces two centrosomes

that duplicate and separate at mitosis to form

tetrapolar spindles. The resultant embryos, however,

are aneuploid because the triploid chromosome



dynamics to a state conducive to nucleation and

polymerization.

A model for mammalian sperm reconstitution is

presented in Figure 26.5.

In the mature human

sperm, centrin is found on the proximal centriole

(Figure 26.5A), with the doublet microtubules of

the sperm tail anchored to the triplet microtubules

of the centriole(s). The centrosome is probably

not phosphorylated, but extensively crosslinked by

disulfide bonds that mask the presence of paternal

␥-tubulin. After sperm incorporation, a functional

zygotic centrosome is quickly assembled (Figure

26.5B). Paternal centrin is lost, perhaps caused by

the binding of calcium ions released during oocyte

activation, and this triggers a calcium-induced trans-

formation of the proximal centriole, with its axial

microtubules, into a structure resembling a func-

tioning centrosome that can participate in the

organization of radial arrays of microtubules

post-insemination. The sperm centriolar complex is

predicted to be a coiled-coil structure,

3,7

and the lat-

tice structure is unravelled by the reduction of

disulfide bonds through the reducing environment

of the oocyte’s cytoplasm. This exposes minute

amounts of paternal ␥-tubulin and probably addi-

tional binding sites onto which maternal ␥-tubulin

complexes can attach. These events, along with

centrosome phosphorylation, lead to the nucleation

of the microtubules that can assemble the sperm

aster.

Clinical assays for the initial molecular charac-

terization of the human sperm centrosome have

been proposed (Figure 26.5).

Examining micro-

tubule assembly in vitro from the disulfide-reduced

(i.e. ‘primed’) sperm centrosome can be assayed

using Xenopus egg extracts in combination with

polymerization-competent rhodamine-tagged tubu-

lin protein.

This prospective assay may measure

the ability of a population of a patient’s sperm to

nucleate microtubules after sperm incorporation,

identifying men who are poor candidates for ART

procedures, prior to couples’ undergoing arduous

procedures of ART such as ICSI.

Examining microtubule assembly and centrosome

function after microinjecting human sperm into

mature bovine or rabbit oocytes is also a potentially

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HUMAN PREIMPLANTATION EMBRYO SELECTION



Sperm centrosome

Centrin

γ-Tubulin

Pericentrin, etc.

Phosphorylation

Disulfide bonds

Sulthydryl groups

Zygote centrosome

Human

+ Frog extract

No extractHuman

Figure 26.5 Molecular dissection of the human sperm centrosome and its reconstruction in the zygote leading to assembly of sperm

astral microtubules. (A) The human sperm centrosome has centrin concentrated in one or two focal sites, corresponding to the

centrioles. ␥-Tubulin is not apparent in mature human sperm, but becomes detectable after ‘centrosomal priming’ of the sperm with

disulfide reducing agents; this is a novel type of cytoplasmic capacitation. ␥-Tubulin is also detectable on Western blots with intact or

sonicated human sperm. The centrosome is not phosphorylated and the sperm tail microtubules extend from a centriole. The coiled-

coil infrastructure of the centrosome probably anchors the centrosome to the sperm nucleus and regulates the exposure of, and bind-

ing sites for, ␥-tubulin. In the zygote centrosome (B), after permeabilization and incubation in extracts from Xenopus oocytes, the

human sperm becomes phosphorylated and heavily immunoreactive with antibodies to ␥-tubulin. The ␥-tubulin found in the

human sperm is probably a combination of some paternal (light) and largely maternal protein (dark). The binding of calcium ions,

released during the transient increase during egg activation, to centrin is predicted to result later in a centrin-induced severing of the

doublet sperm tail microtubules from the triplet microtubules of the centriole. Perhaps the severing of the tail microtubules from the

basal body frees the basal body complex so that it can bind additional ␥-tubulin and undergo transformation into a centriole. The

coiled-coil domains of the centrosome are drawn as unraveling, expanding, and averting in the zygote; this exposes paternal ␥-tubulin and

also exposes binding sites for maternal ␥-tubulin. The halo of ␥-tubulin nucleates the microtubules, which assemble into the sperm

aster. (C) and (D). Human sperm exposed to 5 ␮mol/l ionomycin and primed with 5 ␮mol/l dithiothreitol, (DTT) demonstrate

assembly of microtubules in vitro from the centrosomal region after 40–60 minutes of incubation in CSF-arrested Xenopus

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THE SPERM CENTRIOLE: ITS EFFECT ON THE DEVELOPING EMBRYO

complement is abnormally divided into the result-

ant four blastomeres at the end of cell division. Such

experimental evidence clearly supports the hypoth-

esis that only one centrosome – duplicated under

cell cycle control – is required for correctly forming

a bipolar spindle that can accurately segregate the

chromosomes.

2,17,18

In mammals, however, polyspermy experiments

argue against this central dogma on the universal

contribution of the sperm centrosome. Rodents vio-

late the notion of the paternal centrosome contribu-

tion at fertilization, as both the distal and proximal

sperm centrioles degenerate during spermatogene-

sis

and no sperm aster assembles at the base of the

sperm head in the cytoplasm following incorpora-

tion. Furthermore, di- or trispermic mouse zygotes

divide from one to two,

indicating no dominant

sperm centrosomal contribution during mouse

fertilization. In most other mammals, including

marsupials, cows, sheep, pigs, rabbits, monkeys, and

humans, supernumerary sperm asters form after

polyspermy.

7,39–42

However, as shown in human

fertilization, these dispermic zygotes can divide

from one-cell into two-, three-, or even four-cell

embryos.

42,43

As shown in Figure 26.6,

dispermic

human zygotes at mitosis assemble bipolar metaphase

spindles in the presence of supernumerary centro-

somes. At prophase, a disorganized multipolar

spindle with four foci of ␥-tubulin assembles first

(Figure 26.6A–C), which surprisingly resolves into

a bipolar spindle with aligned chromosomes at

mitotic metaphase (Figure 26.6D–F) and divides

one to two cells. These observations in humans may

reflect requirements of non-centrosomal components

(i.e. molecular motors and spindle matrix proteins)

necessary for bipolar spindle assembly in somatic

cells.

Analysis of parthenogenetic development in

primate oocytes supports this view (discussed

below).

Dispermic fertilizations in humans are observed

frequently.

42,43

Diandric triploidy is one of the con-

sequences, although other mis-segregations leading

to chromosome mosaicism can be found. 2N/3N

mixoploidies can give rise to live births but with

a variety of developmental disorders.

44,45

Diploid

sperm, the result of male meiotic error, can also

produce triploidy.

Studies of postzygotic diploidization in triploid

embryos

have suggested the origins of disorders

arising from genomic imprinting errors. Mammals

have the paternal and maternal chromosomes specif-

ically modified by DNA-methylation of ‘imprinted’

genes, so that certain maternal genes are silenced, as

are complementary paternal genes. Uniparental

disomy disorders (UPD) like Angelman (AS) and

Prader-Willi syndromes (PWS)

47,48

result from the

loss of either two maternally (AS) or two paternally

(PWS) expressed genes. Since centrosomes in

human zygotes form bipolar spindles even after

dispermic insemination, the possibility exists that

daughter blastomeres after cell division inherit two

or more paternal or maternal chromosome sets.

While the exact origins for uniparental disomies

remain elusive, understanding the molecular conse-

quences of these imprinting errors in humans

would be enormously useful in resolving develop-

mental, neurological, and behavioral consequences

of these devastating diseases.

MATERNAL CENTROSOME ANOMALIES AND

BIRTH DEFECTS

A primary cause of reproductive failure in older

women may reflect aging of crucial maternal factors

relevant to genetic fidelity and/or assembling a func-

tional zygotic centrosome.

Aneuploidy rates as high

as 52–61% have been reported in preimplantation



cytoplasmic extract and subsequent exposure to rhodamine-conjugated bovine brain tubulin (C). Primed sperm not exposed to

CSF extract do not nucleate microtubules after incubation in rhodamine-conjugated bovine brain tubulin (D). Bars ⫽ 1 ␮m.

(A)–(C) Reprinted with permission from Schatten.

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