Human Reproduction vol.12 no.12 pp.2713–2719, 1997
The early haploid male gamete develops a capacity for
fertilization after the coalescence of the proacrosomal
granules
Nikolaos Sofikitis1, Yasuhisa Yamamoto,
Tadahiro Isoyama and Ikuo Miyagawa
Reproductive Physiology and IVF Center, Department of Urology,
Tottori University School of Medicine, 36 Nishimachi, Yonago 683,
Japan
1To
whom correspondence should be addressed
We compared the fertilizing capacity of rabbit testicular
round spermatids (i) before appearance of the proacrosomal
granules (stage –1), (ii) with two to three proacrosomal
granules attached to the nucleus (stage –2) and (iii) with
one granule attached to the nucleus (stages –3 to –5).
Testicular samples were minced and observed via confocal
scanning laser microscopy, which allows intracellular observation at high magnification and the subsequent identification of acrosomal granules and round spermatids. Nuclei
of round spermatids of stage –1 (group A), –2 (group B),
and –3 to –5 (group C) were isolated and injected into
mature rabbit oocytes. At 24 and 72 h post-injection
the number of cleaved oocytes and morulae/blastocysts,
respectively, were significantly lower in groups A and B
than group C. These results suggest that the rabbit round
spermatid develops fertilizing potential after the stage at
which the coalescence of the acrosomal granules occurs.
Key words: acrosome/fertilization/infertility/round spermatid
Introduction
Male gamete alterations during spermiogenesis can be grouped
into formation of the acrosome, nuclear changes, development
of the flagellum and reorganization of the cytoplasm. Alterations in the shape/size of the acrosomal vesicles/granules can
be used as a marker to define different stages in the early
spermiogenesis. Stage –1 begins with the formation of a new
group of spermatids as a result of the second meiotic division
(Oakberg, 1956). These spermatids lack specific characteristics.
They may be most commonly confused with secondary
spermatocytes but they are 30–40% smaller than secondary
spermatocytes (Russel et al., 1990). The appearance of two or
three proacrosomal granules marks stage –2. Fusion of the
proacrosomal granules into a single granule adjacent to the
nucleus defines stage –3 (Oakberg, 1956). The acrosomal
granule subsequently becomes larger, flattens onto and extends
over the nucleus.
Presented at The 92nd Annual Meeting of The American Urological
Association in New Orleans, LA, from April 12 to 17, 1997.
© European Society for Human Reproduction and Embryology
Round spermatid nuclei injections (ROSNI) into the
ooplasm have resulted in fertilization and pregnancies in
the rabbit (Sofikitis et al., 1994a), mouse (Kimura and
Yanagimachi, 1995a, b), and human (Hannay, 1995; Sofikitis
et al., 1995a). Blastocyst formation has been demonstrated
after ooplasmic injections of round spermatids into bovine
oocytes (Goto et al., 1996). In addition, intact round
spermatid injections (ROSI) into human oocytes have been
followed by delivery of two healthy children (Tesarik et al.,
1995, 1996; Tesarik and Mendoza, 1996). Additional
pregnancies achieved with ROSI techniques or ooplasmic
injections of elongating spermatids have been reported by
Antinori (1997a, b), Fishel and co-workers (1995), and
Araki and co-workers (1997). However, in the above studies,
the exact stage of the round spermatids injected into oocytes
was not defined. It appears that human ROSNI/ROSI
procedures can be used as experimental techniques for the
treatment of non-obstructive azoospermia with maturation
arrest at the round spermatid stage (Edwards et al., 1994;
Fishel et al., 1996; Sofikitis et al., 1997). Furthermore,
Yamanaka and co-workers (1997) have shown that ROSNI
procedures can be occasionally applied in the treatment of
men with arrest in spermatogenesis at the primary spermatocyte stage. It appears that in patients whose routine diagnostic
testicular biopsy has demonstrated primary spermatocytes
only, a more thorough search of therapeutic testicular biopsy
material may reveal few round spermatids (Yamanaka
et al., 1997).
Our objective was to compare the reproductive capacity of
the round spermatid prior to appearance of the proacrosomal
granules (stage –1), prior to coalescence of two or three
proacrosomal granules (stage –2), and after fusion of the
proacrosomal granules and before the spreading acrosome
reaches an angle of 90° (stages –3 to –5) (Russel et al., 1990).
Such a study may be clinically important because it could
define the round spermatid stage with the maximal probability
for fertilization after ooplasmic injections.
Materials and methods
Recovery and preparation of oocytes
Mature New Zealand white female rabbits were superovulated as
previously described (Sofikitis et al., 1996a, b). Mature oocytes were
collected, washed three times in modified RD medium (Sofikitis
et al., 1996a), and freed from cumulus cells by treatment with 0.1%
bovine testicular hyaluronidase (Type VIII; 320 units/mg solid; Sigma
Co., St Louis, MO, USA) in modified RD medium. Cumulus free
oocytes were rinsed and kept in modified RD medium at 37°C under
5% carbon dioxide in air.
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N.Sofikitis et al.
Figure 1. Round spermatid of stage –1. Observation via confocal
scanning laser microscopy (originally 32500) with a computer
assisted system (originally 33).
Isolation of round spermatids
Spermatogenic cells were recovered from male New Zealand white
rabbits (8 months old) and resuspended in modified Dulbecco’s
phosphate buffered saline (DPBS) as previously described (Sofikitis
et al., 1994a, 1996a,b). Then the cells were observed by confocal
scanning laser microscopy (1LM06; Lasertec, Yokohama, Japan) with
a computer assisted system (Apple Computers Inc., Cupertino, CA,
USA) attached to a micromanipulator (MO-204 Narishige, Tokyo,
Japan). The confocal scanning laser microscope has the capacity to
provide three-dimensional images of live cells under high magnification without requiring the staining of materials or damaging the cells
(Hyodo et al., 1991; Sofikitis et al., 1994b). Thus, live cells could
be used for a variety of further in vitro procedures after observation.
Furthermore, the confocal scanning laser microscope allows selective
intracellular observation at various depths. Round spermatids of
stages –3 to –5 could be identified by the presence of a single
acrosomal granule adjacent to the nucleus (Sofikitis et al., 1996b).
Careful observation also allowed the identification of round spermatids
of stage –2 (i.e., two or three proacrosomal granules adjacent to the
nucleus; Figure 2). Round spermatids of stage –1 could be identified
by the absence of proacrosomal/acrosomal granules and their size
(,10 µm; Figure 1). Cells of similar shape and intracellular characteristics (i.e., absence of proacrosomal granules) with a diameter of 11–
12 µm were considered to be secondary spermatocytes. Round
spermatids of stage –1 (group A), stage –2 (group B), and stages –3
to –5 (group C) were selected via the micromanipulator pipette, after
confocal imaging.
When the angle subtended by the single acrosomal granule in
group C cells extended to a maximum of 90°C (ø90°; Figure
3)(Russel et al., 1990) the spermatids were considered to be stages
–3 to –5 (Russel et al., 1990). The internal diameter of the pipette
was 20 µm. Selected round spermatids were transferred to SOF
medium (Yamanaka et al., 1997). Before use, 2.85 µg of bicarbonate
was added/ml of SOF medium. Our methodology has been proven
to be accurate enough to select round spermatids of the first stages
of spermiogenesis (Sofikitis et al., 1996a, b). However, taking into
consideration that the identification of round spermatids of stage –1
was based mainly on their size, the probability that a small number
of secondary spermatocytes was included in group A cells could not
be ruled out.
2714
Figure 2. Round spermatid of stage –2. Observation via confocal
scanning laser microscopy (originally 32500) with a computer
assisted system (originally 33.2). A large and a small proacrosomal
granules are indicated by arrows.
Figure 3. Round spermatid of stage ù3. Observation via confocal
scanning laser microscopy (originally 32500) with a computer
assisted system (originally 33). The angle ‘b’ subtended by the
acrosomal granule is smaller than 90°.
Transmission electron microscopy (TEM) of selected cells
To confirm that the confocal scanning laser microscopy methodology
applied was sufficient to distinguish different stages of round spermatids, a large number of round spermatids of stages –1 to –5 were
selected using the micromanipulator pipette. These fractions of round
spermatids were observed via confocal scanning laser microscopy
and subsequently were processed for TEM as previously described
(Figure 4)(Sofikitis et al., 1996a). Percentages of round spermatids
of stage –1, stage –2, and stages –3 to –5 were recorded during
confocal scanning laser microscopy and TEM. The mean percentage
of round spermatids of stage –1, stage –2, or stages –3 to –5 recorded
Spermatid of stage –2 and fertilization
Figure 4. Round spermatid of stage ù3. Observation via
transmission electron microscopy (32000) with a computer assisted
system (33).
via confocal scanning laser microscopy was similar to that estimated
via TEM.
Nuclei isolation
Each group of selected round spermatids was transferred from SOF
medium to a hypersonic sucrose solution (Sofikitis et al., 1996a, b)
and resuspended in the same hypertonic solution containing Triton
X-100 (0.035%; v/v). After gentle pipetting for 20 s and further
centrifugation at 31000 g for 9 min, the nuclei in the pellet were
collected and transferred into modified DPBS. Isolated nuclei were
washed three more times in modified DPBS and finally resuspended
in the same medium supplemented with 1 mM MgCl2 and 10%
polyvinylpyrrolidone (molecular weight 360 000, PVP K 90; ICN
Biochemicals, Costa Mesa, CA, USA) and kept at 34°C.
ROSNI procedures
Cumulus free mature oocytes were preincubated at 10°C in an
atmosphere of 5% carbon dioxide in air for 2 h. They were then
incubated in the presence of A23187 (10 µM; C7522; Sigma) at 37°C
under 5% carbon dioxide in air for 8 min. The oocytes were
subsequently transferred to modified RD medium for injections. A
single round spermatid nucleus was injected into each oocyte. The
time interval between treatment of oocytes with A23187 and ooplasmic
injections was 30–45 min. The technique for ooplasmic injections
was different from the one previously applied (Sofikitis et al., 1996a).
Each nucleus was aggressively compressed by the tip of the injecting
micropipette 8–10 times. Preliminary experiments have demonstrated
that the above action on nuclear surface before ooplasmic injections
facilitates spermatid nucleus decondensation and male pronucleus
formation. The internal diameter of the injecting pipette was 7–8 µm.
Each oocyte was positioned with the first polar body at 0600 h or
1200 h and punctured directly at 1500 h by the injecting micropipette.
During ooplasmic injections a great deal of care was taken to introduce
as little medium as possible into the oocyte by placing the nucleus
close to the tip of the micropipette before entry into the oocytes.
After the tip of the injecting micropipette had penetrated the vitelline
membrane and approached the centre of the oocyte, ooplasmic content
and the nucleus were drawn into the micropipette to a distance equal
to the oocyte diameter 32 approximately, the aspirated ooplasm was
then expelled into the cytoplasm of the oocyte, and the nucleus was
placed again at the tip of the micropipette. The above ooplasmic
aspirating/expelling manoeuvres were repeated 35 without moving
the injecting micropipette and finally the aspirated cytoplasm of the
oocyte, together with the spermatid nucleus and a small amount of
medium were expelled into the oocyte. Then the nucleus was observed
within the ooplasm. Forty-nine, 38, and 41 mature oocytes were
successfully injected with spermatid nuclei from groups A, B and C,
respectively. An additional 51 oocytes (group D) were injected in a
similar fashion with an amount of medium equivalent to that injected
into oocytes during nuclear injections (,1.2 pl; calculated by the
computer service department of Tottori University). This amount of
medium is smaller than is commonly injected into the oocytes during
human ooplasmic sperm injections (Hoshi et al., 1995) because of
the smaller size of the round spermatid nucleus and subsequently the
smaller volume it occupies within the injecting micropipette. The
injected oocytes were transferred to modified RD medium without
antioxidants and incubated at 37°C under 5% carbon dioxide in air
for 72 h. Oocytes were observed at 5, 9 and 24 h post-injection.
Previous studies have shown that the optimal time for observation of
two pronuclei after ROSNI procedures is 9 h post-injection (Sofikitis
et al., 1995b; Yamanaka et al., 1997). Medium was changed at 24 h
post-injection. An activated oocyte was defined as one with first and
second polar bodies and at least one female pronucleus present (Ogura
and Yanagimachi, 1993; Sofikitis et al., 1996a).
Observations of the oocytes and embryos were performed in a
blinded fashion. Statistical analysis was performed using χ2 test. A
probability ,0.05 was considered to be statistically significant.
Results
Oocyte activation
There were no significant differences in the proportion of
activated oocytes to injected oocytes among groups A, B and C.
Fertilization
There was no significant difference in the proportion of
fertilized oocytes to injected oocytes between groups B and
C. However, the proportion of fertilized oocytes to injected
oocytes was significantly lower in group A (Table I).
Zygote cleavage
The proportion of cleaved oocytes to injected oocytes was
significantly lower in groups A and B than in group C. In
contrast, the difference in the proportion of cleaved oocytes
to injected oocytes between groups A and B was not significant.
Embryonic development
The proportion of morulae/blastocysts to injected oocytes was
significantly lower in groups A and B than in group C.
However, the proportion of morulae/blastocysts to injected
oocytes was not significantly different between groups A and B.
Parthenogenetic activation
Approximately one third of the oocytes injected with medium
were activated. One and three of them showed two and
three female pronuclei, respectively. The parthenogenetically
activated oocytes did not complete the first cleavage.
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N.Sofikitis et al.
Table I. Injections of round spermatids at different stages into rabbit
oocytes
Spermatid groups
A
B
C
(stage–1) (stage–2) (stage –3 to –5)
No. of injected oocytes 49
Activated oocytes (%) 41 (83)a
Oocytes with two (%) 18 (36)a
pronuclei (fertilized)
Cleaved oocytes (%)
8 (16)a
Morulae or (%)
2 (4)a
blastocysts
Control
group D
(medium)
38
33 (86)a
24 (63)b
41
37 (90)a
29 (70)b
51
18 (35)b
1(2)c
9 (23)a
1 (2)a
23 (56)b
11 (26)b
0 (0)c
0 (0)a
Within each line, values not sharing the same superscript a, b or c are
significantly different (P , 0.05).
Discussion
It has been shown that round spermatid nuclei injected into
hamster oocytes form pronuclei and participate in syngamy
(Ogura and Yanagimachi, 1993). However, the developmental
potential of the zygotes obtained was not evaluated in that
study. In another study, intact round spermatids were injected
into the perivitelline space of mature hamster or mouse
oocytes and a fusion pulse was applied to fuse the spermatids
with the oocytes (Ogura et al., 1993). It was found that
the nuclei commonly failed to develop into large pronuclei.
In one additional study it was found that when mouse intact
round spermatids were successfully fused with the oocytes,
some of the zygotes developed into normal offspring (Ogura
et al., 1994). The overall success rate of the electrofusion
of intact spermatids with oocytes was low due to the
difficulty of fusing large cells like oocytes with small cells
like spermatids without lysis of the larger cells (Ogura
et al., 1994). To avoid oocyte damage due to the fusion
process we chose a microsurgical approach to transfer round
spermatid nuclei into oocytes and achieved the first
pregnancies in mammalian species via microsurgical ROSNI
techniques (Sofikitis et al., 1994a). In that study the
proportion of implanted embryos to the number of injected
oocytes and the ratio of offspring to the number of injected
oocytes were low. The low values of these parameters may
be attributable to low developmental potential of the injected
oocytes due to inadequate mechanical stimulation applied
to activate oocytes prior to ROSNI procedures. For this
reason we designed another study, the objective of which
was to evaluate the effects of electrical stimulation of
oocytes before ROSNI procedures on oocyte activation and
subsequent embryonic development (Sofikitis et al., 1996a).
That study provided information on the optimal stimulation
required for oocyte activation, fertilization, and normal
embryonic development when rabbit ROSNI-embryo transfer
procedures are scheduled. It was found that electrical
stimulation of oocytes prior to ROSNI techniques had
beneficial effects on oocyte activation, fertilization, and
subsequent embryonic development. In the present study we
applied a combination of chemical and multiple, vigorous
mechanical stimulation to activate the rabbit oocytes. Within
2716
the group of round spermatids of stages –3 to –5 the
percentage of activated oocytes was higher than that we
previously reported when applying a minor mechanical
stimulation only (Sofikitis et al., 1996a). Furthermore, the
combination of mechanical and chemical stimulation applied
was sufficient to support/induce activation in a high
percentage of oocytes injected with less mature cells (i.e.,
round spermatids of stages –1 or –2). Preliminary experiments
have indicated that the combination of chemical plus multiple
mechanical stimulation during ROSNI procedures results in
a larger activation rate (90% in the present study; 92% in
preliminary experiments) than a mild/minor mechanical
stimulation alone (85%; Sofikitis et al., 1996a), a single,
vigorous mechanical stimulation alone (76%), or a multiple,
vigorous mechanical stimulation alone (87%). Oocytes are
incubated in the presence of ionophore after intracytoplasmic
sperm injections in several assisted reproduction programmes
(Hoshi et al., 1995; Tesarik and Sousa, 1995a).
We applied the chemical stimulus prior to ROSNI
techniques in the current study since previous studies and
preliminary experiments showed better activation rates for
rabbit oocytes when the electrical (Sofikitis et al., 1996a)
or chemical stimulation was applied before ROSNI procedures
than after these techniques. Furthermore, Kimura and
Yanagimachi (1995b) reported that mouse ROSNI procedures
were most effective when oocytes were activated first and
injection was performed at the telophase of the second
meiotic division of the oocyte. Oocytes had been incubated
at 10°C before the activating stimulus was applied because
we previously showed that incubation of rabbit oocytes at
5–10°C prior to electrical stimulation plus ROSNI techniques
had beneficial effects on oocyte activation and fertilization
process (Sofikitis et al., 1996a). Mouse round spermatids
lack the oocyte-activating factor (OAF) (Kimura and
Yanagimachi, 1995a, b). However, there is much controversy
over whether the rabbit OAF is expressed at the round
spermatid stage. The authors believe that rabbit OAF is
expressed at/before the round spermatid stage since (i)
healthy offspring were born after ROSNI into oocytes treated
with a minor mechanical stimulation (Sofikitis et al., 1994,
1996a), (ii) the percentage of activated oocytes after minor
mechanical ooplasmic stimulation plus ROSNI techniques
was relatively high in a previous study (Sofikitis et al.,
1996a) and (iii) the proportion of activated oocytes to
injected oocytes in the current study was significantly higher
in the groups of oocytes injected with round spermatids
than in the group of oocytes injected with medium
(parthenogenetic activation) in a similar fashion. Electrical
(Sofikitis et al., 1996a) or chemical stimulation of the
oocyte prior to ROSNI techniques may support the functionality/action of the OAF and subsequently have beneficial
effects on oocyte activation and fertilization. This thesis is
consistent with the suggestion of Tesarik and Sousa
(1995b) that a vigorous mechanical stimulation during
intracytoplasmic sperm injection making the oocyte undergo
a substantially high Ca12 load may support the action of
the human OAF supposed to trigger oocyte activation.
Recently, Yamanaka and co-workers (1997) and Sousa and
Spermatid of stage –2 and fertilization
co-workers (1996) suggested that the OAF is present at the
human round spermatid.
After the encouraging messages from the above
experimental studies an attractive challenge was to apply
ROSNI procedures for the treatment of non-obstructed
azoospermic men. The first human pregnancies with ROSNI
techniques were achieved in 1994 and reported in April
1995 (Hannay, 1995; Sofikitis et al., 1995a). However, these
pregnancies resulted in abortions. A few months later,
Tesarik et al. (1995) reported two human pregnancies. The
latter pregnancies resulted in deliveries of healthy children
(Tesarik and Mendoza, 1996; Tesarik et al., 1996). Fishel
and co-workers (1995) reported a pregnancy after an
elongated spermatid injection into an oocyte. Vanderzwalmen
and co-workers (1995), Chen et al. (1996), and Yamanaka
and co-workers (1997) fertilized human oocytes with
late-stage spermatids, intact round spermatids, and round
spermatid nuclei, respectively. Additional ROSI pregnancies
have been reported by Antinori and co-workers (1997a, b).
However, in all of the above studies the exact stage of the
round spermatids injected into oocytes was not defined. We
used the rabbit as an experimental model to evaluate whether
there are alterations in the reproductive capacity of the early
haploid male gamete during the first stages of spermiogenesis.
We compared the fertilizing capacity of round spermatids
without proacrosomal/acrosomal granules (stage –1), round
spermatids with two or three proacrosomal granules (stage –
2), and round spermatids with a single acrosomal granule
rounded on the nuclear surface (stage –3) (Russel et al.,
1990) or a single acrosomal vesicle flattened/extended on
the nuclear membrane to an angle smaller than 90° (stages
–4 and –5) (Russel et al., 1990; the mouse classification of
spermiogenesis was applied). Although there were no
significant differences in the activation rate among the
groups of oocytes injected with various stages of round
spermatids, injections of spermatids of stages –3 to –5
resulted in a significantly higher cleavage rate and capacity
for embryonic development. In addition, the fertilization rate
was significantly higher after injections of stage –2 than
stage –1 spermatids. However, differences in the cleavage
rate were not significant. The superior outcome of ROSNI
procedures using spermatids of stages –3 to –5 may not be
attributed to a biophysical/biochemical maturation of the
cytoplasmic contents of the round spermatid because the
methodology for nuclear extraction applied in the present
study has been proven to yield nude nuclei with defects in
the nuclear membrane and subsequently without cytoplasmic
layers (Sofikitis et al., 1996a). The enhanced round spermatid
nuclear capacity to fertilize oocytes and initiate early
embryonic development at/after stage –3 may be due to
biochemical alterations in the nucleus. Coalescence of
chromatin granules, replacement of lysine-rich histones by
arginine-rich, and accumulation of lysine-rich proteins in
the sphere chromatophile are nuclear changes known to
occur during the early stages of spermiogenesis (De Kretser
and Kerr, 1988). However, nuclear changes during the first
stages of spermiogenesis have not been described extensively
and more studies are in process in our laboratory to correlate
early alterations in the protein profiles of the round spermatid
nucleus with events occurring during nucleus-ooplasm
interaction (i.e., nuclear decondensation, speed of extrusion
of second polar body, and male pronucleus formation). It
is interesting that although there was no significant difference
in the fertilization rate between oocytes injected with round
spermatids of stage –2 and spermatids of stage –3 to –5,
the cleavage rate was significantly higher in the latter group.
The development of most of the oocytes fertilized by
stage –2 spermatids was arrested at the 2-pronuclei stage.
Furthermore, one cleaved oocyte only developed up to
morula stage. We may speculate that the capacity of the
nucleus of the stage –2 spermatid to participate in syngamy
and initiate early embryonic development is impaired. Recent
studies have shown that there is a paternal contribution to
the early embryonic development (Janny and Menezo, 1994).
More studies are necessary to clarify whether paternal
factors contributing to the initiation of the early embryonic
development are expressed/activated at stage –1 or –2 of
spermiogenesis.
The results of the present study are consistent with a
previous report (Sofikitis et al., 1996a) showing that rabbit
oocytes treated with an activation stimulus plus medium
injection have low potential for cleavage and further development. In contrast, the proportion of morulae/blastocysts to the
oocytes injected with stages –3 to –5 round spermatids was
smaller in the current study than that previously reported
(Sofikitis et al., 1996a). This difference may be attributed to
the different stimulus applied for support/induction of oocyte
activation in the present study or to the absence of antioxidants
(i.e., taurine) in the embryo culture medium. Li and co-workers
(1993) have demonstrated that addition of antioxidants to
rabbit embryo culture medium results in faster embryonic
development.
Centrosomic components (i.e., the reproducing element of
the centrosome, the microtubule organizer and a γ-tubulinbinding protein) of the rabbit zygote are considered to be
paternally inherited (Longo, 1976; Schatten, 1994). In three
previous studies (Sofikitis et al., 1994a, 1996a, b) and the
current study normal embryonic development was achieved
both in vitro and in vivo by introducing an early haploid
nucleus without any cytoplasmic coverings and subsequently
without centrosomic material within a rabbit oocyte. The
above observations, the artificial parthenogenesis in several
mammalian species, and the observations of Ozil (1990)
that rabbit oocytes electrically stimulated repeatedly could
implant and further develop up to the day –10 of pregnancy,
could be interpreted as a challenge to the classical theory
of centrosomes (Schatten, 1994) and tend to support the
speculation of Mazia (1984) that when paternal centrosomic
material is absent, novel maternal spindle organizing centres
can develop or previously denatured or non-functional female
centrosomic material can undergo resuscitation after oocyte
activation. Alternatively, the idea that centrosomic material is
firmly attached to the male gamete nucleus and subsequently
introduced into the oocyte via nuclear injections (Navara
et al., 1994) could explain the development of oocytes
injected with round spermatid nuclei to embryos, fetuses,
2717
N.Sofikitis et al.
and offspring (Sofikitis et al., 1996a). The present study
reveals that the rabbit round spermatid of stages –3 to –5
has larger capacity to fertilize oocytes and initiate embryonic
development than round spermatids of earlier stages. If the
results of the present study could be transferred to the
human, recovery of round spermatids of stages ù3 would
be recommended in assisted reproduction programmes
applying ROSNI procedures for the treatment of nonobstructive azoospermia. Although human pregnancies have
been achieved by ROSNI or ROSI techniques (Sofikitis et
al., 1995a; Tesarik et al., 1995; Antinori et al., 1997a, b)
genetic implications of ROSNI procedures should be carefully
considered. To evaluate the genetic risks of assisted
reproductive technologies, one has to consider the genetic
risk inherent in the procedure performed and the genetic
risk inherent to treatment population. Genetic risks inherent
to ROSNI/ROSI techniques may involve (i) centrosomic
abnormalities resulting in an aberrant spindle formation and
subsequently in an increased risk of mosaicism, (ii) injection
of diploid or disomic genetic material which could give rise
at fertilization to a triploid or trisomic embryo/fetus
(the presence of diploid spermatids/spermatozoa is well
documented in the mammalian testis with severe spermatogenic impairment) (Levy and Burgoyne, 1986) and (iii)
genomic imprinting abnormalities. A genetic risk of ROSNI/
ROSI procedures inherent to a population of non-obstructed
azoospermic men is transferring an azoospermia-related/
causing gene to the next generation. An additional problem
concerning the application of human ROSNI/ROSI techniques
in centres without a confocal scanning laser microscope is
the identification of the human round spermatid. To identify
human round spermatids quantitative (Yamanaka et al.,
1997) or qualitative criteria (Mendoza and Tesarik, 1996)
can be used. However, qualitative criteria are highly
subjective. More experimental studies are necessary to
overcome the above drawbacks to human ROSNI procedures.
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Received on March 19, 1997; accepted on August 8, 1997
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