Human Reproduction Vol.16, No.8 pp. 1575–1582, 2001
Live human germ cells in the context of their
spermatogenic stages
Larry Johnson1,4, Christophe Staub1, William B.Neaves2, and Ryuzo Yanagimachi3
1Department
of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843, 2Stower’s Institute for
Medical Research, Kansas City, Missouri 64110 and 3The Institute for Biogenesis Research, Department of Anatomy and
Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822, USA.
4To
whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: Various types of live, dispersed, human testicular cells in vitro were previously compared with
the morphologic characteristics of human spermatogenic germ cells in situ within seminiferous tubules. The current
study extends those observations by placing live human germ cells in the context of their developmental steps and
stages of the spermatogenic cycle. METHODS: Live human testicular tissue was obtained from an organ-donating,
brain-dead person. A cell suspension was obtained by enzymatic digestion, and dispersed cells were observed live
with Nomarski optics. Testes from 10 men were obtained at autopsy within ten hours of death, fixed in glutaraldehyde,
further fixed in osmium, embedded in Epon, sectioned at 20 µm, and observed unstained by Nomarski optics.
RESULTS: In both live and fixed preparations, Sertoli cells have oval to pear-shaped nuclei with indented nuclear
envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. For germ cells,
size, shape, and chromatic pattern of nuclei, the presence of meiotic metaphase figures, acrosomic vesicles/structures,
tails, and/or mitochondria in the middle piece are characteristically seen in live dispersed cells and those in the
fixed seminiferous tubules. These lead to identification of live germ cells in man and placement of each in the
context of their developmental steps of spermatogenesis at corresponding stages of the spermatogenic cycle.
CONCLUSIONS: This comparative approach allows verification of the identity of individual germ cells seen in vitro
and provides a checklist of distinguishing characteristics of live human germ cells to be used in clinical procedures
or by scientists interested in studying live cells at known steps in spermatogenic development characteristic of germ
cells in specific stages of the spermatogenic cycle.
Key words: spermatogenic stage/live germ cell/human spermatogenesis/ICSI/ROSI
Introduction
New methods developed by scientists and fertility clinicians
have given to many infertile couples new hope for producing
their own biological children. Intracytoplasmic sperm injection
(ICSI) is a technique which successfully treats some forms of
male infertility by microinjection of a spermatozoon into an
unfertilized oocyte (Palermo et al., 1992; Schoysman et al.,
1993; Van Steirteghem et al., 1993a,b; Devroey et al., 1994,
1995; Nagy et al., 1995; Silber et al., 1995; Kahraman et al.,
1996). In some patients, however, spermatozoa may not exist
in the ejaculate or may not be obtained from the epididymis.
Round spermatid injection (ROSI), round spermatid nucleus
injection (ROSNI) or elongated spermatid injection (ELSI) are
new techniques in which younger germ cells—spermatozoan
precursors—are obtained by testicular biopsy or from the
ejaculate and then either injected or electrofused into the
oocyte to serve as the source of the male genome for fertilization
to occur.
Following the encouraging work on animal models such as
© European Society of Human Reproduction and Embryology
hamster (Ogura and Yanagimachi, 1993), mouse (Ogura et al.,
1993, 1994; Kimura and Yanagimachi, 1995), rabbit (Sofikitis
et al., 1994), and experimentation in the human (Sousa et al.,
1996), conception by ROSI was proposed for the treatment of
sterility in humans (Edwards et al., 1994). The first human
spermatid-derived pregnancies have been achieved by ROSNI
(Hannay, 1995), and the first births have resulted from ELSI
(Fishel et al., 1995) and ROSI (Tesarik et al., 1995). These
works were followed by a large number of reports on ELSI
and ROSI treatment cycles (Aslam et al., 1998a, review).
However, the results obtained after ROSI remain disappointingly low. The birth of only a few babies has been reported after
ROSI (Fishel et al., 1995, 1996; Tesarik et al., 1995, 1996;
Vanderzwalmen et al., 1997; Gianaroli et al., 1999). An
important known problem in the use of ROSI is the difficulty
in identifying round spermatids within the heterogeneous
population of testicular cells in a wet preparation
(Vanderzwalmen et al., 1998a,b; Verheyen et al., 1998).
The lack of accurate selection methods may partly explain
the low success rate of ROSI (Yamanaka et al., 1997; Silber
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and Johnson, 1998; Vanderzwalmen et al., 1998a,b; Verheyen
et al., 1998). Notwithstanding, current protocols for correct
live selection of round spermatids have been presented (Sousa
et al., 1998, 1999; Cremades et al., 1999), and the low clinical
outcome of ROSI has been attributed to other causes, such as
genetic anomalies or epigenetic disorders of male germ cells,
capability of round spermatids to activate oocytes, and quality
of oocytes (Sousa et al., 1998; Tesarik et al., 1998).
To improve the identification of various types of live,
dispersed, human testicular cells in vitro and place them in
the context of their developmental steps at corresponding stages
of the spermatogenic cycle, this comparative morphologic study
used both live in-vitro cells and fixed cells within the context
of the embedded testicular tubules. Our laboratory has used
this approach to characterize identifying characteristics of
spermatogonia, spermatocytes and spermatids and it was found
that the size of nuclei were comparable whether fixed or live
germ cells (Johnson et al., 1999).
This study places the live germ cells in the context of
specific stages of the human spermatogenic cycle that is
comparable with the conventional description (Clermont, 1963;
Holstein and Roosen-Runge, 1981) of human germ cells in
each spermatogenic stage. This more detailed description of
live cells, of small changes in spermatocytes and spermatids
as they progress through subsequent stages of the cycle, will
facilitate a more accurate comparison of ROSI results of
different laboratories by this detailed description of the
spermatids that are injected into unfertilized oocytes. Also the
stage specific identity of germ cells will facilitate evaluation
of treatment effects or toxicant effects that might influence
specific steps of germ cell development in given stages of the
cycle. Isolation of a group of germ cells at specific steps of
development, identified by this detailed comparison, will
facilitate molecular biological studies as different kinds of
spermatids might differ in specific types of gene expression.
The live testicular cells were obtained from a brain-dead
human male who was an organ donor. The cells were dispersed
enzymatically and incubated in vitro (Johnson et al., 1999).
The fixed tissue was originally obtained from 10 control men,
representing varied spermatogenic potential (Johnson et al.,
1981). The higher production levels represented the live cell
donor, and the lower ones represented that of typical clinical
patients. It was found that distinct morphologic characteristics
of germ cells at different developmental steps in the spermatogenic cycle could be distinguished in live human testicular
cells in vitro viewed with Nomarski optics. Hence, this
generated a checklist of distinguishing characteristics to allow
identification of various types of human germ cells in situ
for scientific investigation at various developmental steps
throughout spermatogenesis and specific cells in defined stages
of the human spermatogenic cycle.
Materials and methods
Testes were obtained at autopsy from ten human males between 26
and 53 years of age (Johnson et al., 1981). Tissues were fixed
with 2% glutaraldehyde in 0.1 mol/l cacodylate buffer by vascular
perfusion, embedded in Epon, and cut into 20 µm sections as
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described (Johnson et al., 1981; Johnson, 1995) for observation with
Nomarski optics.
Live, dispersed human germ cells were obtained from an individual
in his early twenties listed as an organ donor and diagnosed as braindead after a car accident (Johnson et al., 1999). Fragments of testicular
tissue were removed and subjected to enzymatic digestion according
to a modified method (Bellve et al., 1977). Testicular parenchyma
fragments where placed in HEPES-TC199 medium containing
1.0 mg/ml collagenase for 15 min at 32°C in a shaking water bath.
Tubules were separated from the dispersed interstitial cells by
unit gravity sedimentation for 3–4 min, and the supernatant was
decanted. This step was repeated 3 times. The tubular fragments were
then placed in HEPES-TC199 medium with 0.5 mg/ml trypsin and
1 µg/ml DNase-I for 15 min at 32°C. The tubular fragments were
pipetted vigorously to separate spermatogenic cells and were washed
in HEPES-TC199 medium with 0.5 mg/ml BSA. The resulting chunks
of cells were filtered through a 40 µm mesh wire screen. The resulting
cell suspension was washed by centrifugation for 5 min at 400 g in
a large volume of HEPES-TC199 medium. The suspended cells were
examined unstained on a glass slide using Nomarski’s differentialinterference optics.
Morphological characteristics of live, dispersed testicular cells as
described in our previous paper (Johnson et al., 1999) of this
individual were compared with those of previously identified cell
types (Clermont, 1963; Heller and Clermont, 1964; Johnson et al.,
1992) in fixed, embedded, and thick-sectioned seminiferous tubules
in various developmental steps in the six stages of the spermatogenic
cycle for the 10 control men.
Results
The isolation of seminiferous tubules by unit gravity sedimentation from fragments of human testicular tissue yielded cell
preparations that contained numerous live dispersed human
germ cells. These cells retained their morphological characteristics after enzymatic dissociation that allowed direct comparison with germ cells of similar characteristics within the
context of fixed seminiferous tubules during spermatogenesis.
In this study, Normarski optics was used to identify live germ
cells in suspension at their different steps of differentiation.
The germ cells were sorted according to the size, shape,
chromatin pattern of the nuclei; the presence and shape of the
acrosome and flagella; and the presence of mitochondria in
the middle piece of the tail (Figures 1 and 2, Table I).
Stage I of the spermatogenic cycle begins with the appearance of a new group of spermatids at the end of the second
meiotic division of spermatocytes. The stage is characterized
by the presence of two generations of spermatids. The first
generation is composed of newly formed Sa (Golgi phase)
spermatids with small spherical nuclei. These cells may not
yet have acrosomic structure (Figures 1t and 2t). The older
second generation of spermatids is composed of Sd1 (maturation phase) spermatids. These cells have a mature shaped head
and an annulus that has migrated to the distal position with
mitochondria around the middle piece of the tail (Figures 1z
and 2z). Seminiferous tubule epithelium also contains early
pachytene primary spermatocytes, which have just entered the
long pachytene step of meiotic prophase. Their large nuclei
show dense aggregates (Figures 1m and 2m). Type A and type
B spermatogonia are present. Type A spermatogonia have well-
Human germ cells in the spermatogenic cycle
Figure 1. Composition of germ cells throughout human spermatogenesis viewed by Nomarski optics in unstained 20 µm Epon sections.
These images were selected from various profiles of the six stages of the spermatogenic cycle in humans in a group of 10 men.
(a–f) Various profiles of A spermatogonia with distinct nuclear envelopes characterize the six stages, but not all types of profiles are
indicated. (g) Type B spermatogonia noted by a less defined nuclear envelope and nucleoli away from the nuclear envelope are found in
stages I and II. (h, i) In stage II, B spermatogonia divide to produce preleptotene primary spermatocytes which are also found in stage III.
(j, k) Leptotene primary spermatocytes in stages IV and V, and (l) zygotene primary spermatocytes in stage VI are present as meiosis
progresses. (m–q) These cells give rise to pachytene, then diplotene, primary spermatocytes which are the largest germ cells and have
thickened chromatin threads. In stage VI, (r) secondary spermatocytes result from the first meiotic division which divides at the second
meiotic division and separate at telophase to produce spermatids (s). (t) Newly formed Sa spermatids of stage I have the smallest spherical
nuclei of all germ cells and may not yet have acrosomic structures. (u) The acrosomic vesicle (solid arrow) next to the nucleus is present in
stage II. (v) Sb1 spermatids which nucleus is still spherical, but the acrosome has developed a cap (solid arrow) over the nucleus. Flagella
(arrowhead) can sometimes be observed. (w, x) In stage IV and V, the manchette (solid arrow) has developed causing the elongation and
tapering of the nucleus of Sb2 (w) and Sc (x) spermatids. (y) The nucleus of Sc spermatids in stage VI has shortened to the typical length of
mature spermatozoa. (z) Sd1 spermatids of stage I have the annulus (solid arrow) migrated to the distal position in preparation of
mitochondria migration around the middle piece. (aa) Sd2 spermatids of stage II have enlarged middle pieces (solid arrow) with
mitochondria placement and the cytoplasm droplet still attended. Bar length equals 10 µm.
defined nuclei which contain homogeneous, finely granular,
chromatin and one or more distinctive nucleoli (Figures 1a
and 2a). Type B spermatogonia are smaller than type A
spermatogonia and are characterized by a less defined nucleus
with the nucleoli away from the nuclear envelope (Figures 1g
and 2g).
Stage II of the cycle begins with the appearance of the
acrosomic vesicle flattened over the nuclei of young Sa
spermatids (Figures 1u and 2u). The flagellum has developed
sufficiently to be seen extending beyond the cell diameter and
can sometimes be observed (not shown). Before being released
from the seminiferous epithelium, Sd2 spermatids have small
spear-shaped heads and enlarged middle pieces with mitochondria in place around the tail. A cytoplasmic droplet is often
present, located at the anterior end of the middle piece, in the
proximal position of the tail (Figures 1aa and 2aa). The
spermatocytes associated with these two generations of
spermatids are in the pachytene step of meiosis. These cells
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L.Johnson et al.
Figure 2. Composition of live human germ cells placed into the context of the different developmental steps throughout the six stages of
the spermatogenic cycle. These nuclear profiles characterize germ cell development throughout spermatogenesis as viewed by Nomarski
optics in cultured dispersed live human germ cells. Open arrows indicate the cell of interest among other nuclear profiles. (a–f) Various
nuclear profiles of A spermatogonia characterize some, but not all, of the spermatogonial profiles found in specific stages. Spermatogonia
are identified based on their obvious nuclear envelope, nucleoli patterns, size of chromatin granules, and often there appears to be an empty
space (less heterochromatin) in the nucleoplasm. (g) Type B are smaller and have a less distinct nuclear envelope than A spermatogonia.
(h, i) Type B spermatogonia divide to produce preleptotene primary spermatocytes first seen in stage II, but typical of stage III. Nuclear
size, chromatin distribution, and chromatin clump size distinguish the (j, k) leptotene, (l) zygotene and (m–q) pachytene primary
spermatocytes. In stage VI, diplotene primary spermatocytes (not shown) divide to produce (r) secondary spermatocytes which are slightly
larger than Sa spermatids, but much smaller than pachytene primary spermatocytes. Meiotic telophase figures also characterize stage VI as
secondary spermatocytes and divide to produce spermatids (s). (t) Newly developed Sa spermatids are the smallest of male germ cells and
do not yet have signs of acrosomic development. (u) In stage II, the acrosomic vesicle (solid arrow) is visible identifying the nuclear
envelope of Sa spermatids. (v) The acrosomal cap (solid arrow) characterizes the Sb1 spermatid which still has a spherical nucleus.
(w) Elongation of the spherical nucleus starts in Sb2 spermatids of stage IV. (x) Sc spermatids of stages V are characterized by further
elongation of the nucleus and condensation of chromatin. The annulus (solid arrow) is distinct in its location in the proximal position (near
the head) of the developing tail. (y) Sc spermatids have a shorter tapered nucleus profile and distinct tail formation (solid arrow) in stage
VI. (z) Sd1 spermatids have a mature shaped head and mitochrondria moving around the middle piece of the tail (solid arrow). (aa) Sd2
spermatids have enlarged middle pieces by the presence of mitochondria in place around the tail (solid arrows). Also, a cytoplasmic droplet
(solid arrow) in the proximal position of the tail may be present in Sd2 testicular spermatids to be spermiated in stage II. Bar length equals
10 µm.
show slow, progressive growth (Figures 1n and 2n). Type A
and type B spermatogonia are also present at this stage (Figures
1b, 2b, and 1h, 2h, respectively).
Stage III of the cycle includes only one generation of
spermatids, the older ones having spermiated from the seminiferous epithelium as spermatozoa. The remaining generation
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of spermatids is composed of Sb1 (cap phase) spermatids.
These cells still have a spherical nucleus, but the acrosome
has developed a cap over the nucleus (Figures 1v and 2v).
Two generations of spermatocytes are found. One is obviously
at the midpachytene step of the meiotic prophase (Figures 1o
and 2o) while the second is a new generation of so-called
Human germ cells in the spermatogenic cycle
Table I. Description of live and fixed germ cells at different developmental steps in each of six stages of the human spermatogenic cycle
Stage Description
I
Type A spermatogonia have well-defined nuclei which contain a homogeneous, finely granular, chromatin and one or more distinctive nucleoli. These
cells are present in all stages. Type B spermatogonia are smaller than type A spermatogonia and are characterized by a less defined nucleus with the
nucleoli away from the nuclear envelope. Type B spermatogonia are also present in stage 2. Early pachytene primary spermatocytes have large nuclei
showing dense aggregates. Two generations of spermatids: Newly formed Sa (Golgi phase) spermatids show small spherical nuclei, without acrosomic
structure. Sd1 (maturation phase) spermatids have mature shaped head and an annulus that has migrated to the distal position.
II
The spermatocytes are in the pachytene step of meiosis. These cells show slow, progressive growth. Sa spermatids show acrosomic vesicle flattened
over the nuclei. The flagellum can sometimes be observed. Sd2 spermatids have small spear-shaped heads and enlarged middle pieces by the presence
of mitochondria in place around the tail. A cytoplasmic droplet is often present, located at the anterior end of the middle piece, in the proximal
position of the tail.
III
Two generations of spermatocytes are found: One is at the midpachytene step of the meiotic prophase while the second is a new generation of socalled resting primary spermatocytes resulting from the division of type B spermatogonia. The nuclear envelope of these resting spermatocytes is not
well defined and chromatin flakes are fine. Their nuclear size is slightly larger than that of type B spermatogonia. One generation of spermatids, as the
older ones have spermiated from the seminiferous epithelium as spermatozoa. The remaining generation of spermatids is composed of Sb1 (cap phase)
spermatids. These cells still have a spherical nucleus, but the acrosome has developed a cap over the nucleus.
Whereas the older generation of spermatocytes is always at the midpachytene step of meiosis, the younger generation of spermatocytes is just entering
meiotic prophase. The nucleus of these leptotene spermatocytes is finely granular with evenly distributed areas of chromatin condensation. Sb2
spermatid have nuclei showing initial signs of elongation and the presence of the manchette. The acrosomal cap is a predominant feature attached to
the nucleus opposite to that of the attached flagellum.
IV
V
The older generation of primary spermatocytes has entered the late pachytene step of meiotic prophase. These cells have the largest spherical nuclei of
all germ cells, well-defined nuclear envelopes, and one or more large nucleoli. The younger generation of spermatocytes is still at the leptotene step of
meiosis. Elongation and condensation of the Sb2 spermatid nucleus form Sc (acrosome phase) spermatids. The annulus is distinct in its location in the
proximal position of the developing tail near the nucleus. Mitochondria have not yet wrapped around the middle piece of the flagellum.
VI
The younger generation of primary spermatocytes has just entered the zygotene step of meiotic prophase. These cells have larger nuclei, larger evenly
dispersed chromatin clumps, and well-defined nuclear envelopes in comparison to leptotene primary spermatocytes. Diplotene primary spermatocytes
result from the differentiation of late pachytene spermatocytes. They divide to produce secondary spermatocytes. These are slightly larger than Sa
spermatids, but much smaller than primary spermatocytes. The interphasic nucleus of secondary spermatocytes shows a homogeneous, finely granular
chromatin and usually several nucleoli. Meiotic telophase figures also characterize this stage as secondary spermatocytes divide to produce round
spermatids. Newly formed spermatids have the smallest spherical nuclei of all germ cells. The maturing Sc spermatids show a shorter tapered nuclear
profile and distinct tail formation.
resting primary spermatocytes resulting from the division of
type B spermatogonia. The nuclear envelope of these resting
spermatocytes is not well defined and chromatin flakes are
fine. Their nuclear size is slightly larger than that of type B
spermatogonia (Figures 1i and 2i). Type A spermatogonia are
also present at this stage (Figures 1c and 2c).
Stage IV of the cycle is characterized by Sb2 spermatids
which have nuclei showing initial signs of elongation and the
presence of the manchette. The acrosomal cap is a predominant
feature attached to the nucleus opposite to that of the attached
flagellum (Figures 1w and 2w). Whereas the older generation
of spermatocytes is always at the midpachytene step of meiosis
(Figures 1p and 2p), the younger generation of spermatocytes
is just entering meiotic prophase. The nucleus of these leptotene
spermatocytes is finely granular with evenly distributed areas
of chromatin condensation (Figures 1j and 2j). Type A spermatogonia are also present (Figures 1d and 2d).
Stage V of the cycle is characterized by further elongation
and condensation of the spermatid nuclei to form Sc (acrosome
phase) spermatids. The annulus is distinct in its location in
the proximal position of the developing tail near the nucleus.
Mitochondria have not yet wrapped around the middle piece
of the flagellum (Figures 1x and 2x). The older generation of
primary spermatocytes has entered the late pachytene step of
meiotic prophase. These cells have the largest spherical nuclei
of all germ cells, well-defined nuclear envelopes, and one or
more large nucleoli (Figures 1q and 2q). The younger generation of spermatocytes is still at the leptotene step of meiosis
(Figures 1k and 2k). Type A spermatogonia are also present
at this stage (Figures 1e and 2e).
Stage VI of the cycle has diplotene primary spermatocytes
(not shown), resulting from the differentiation of late pachytene
spermatocytes, that divide to produce secondary spermatocytes.
These are slightly larger than Sa spermatids, but much
smaller than primary spermatocytes. The interphasic nuclei of
secondary spermatocytes show a homogeneous, finely granular
chromatin and usually several nucleoli (Figures 1r and 2r).
Meiotic telophase figures also characterize this stage as secondary spermatocytes divide to produce spermatids (not shown).
Newly formed spermatids have the smallest spherical nuclei
of all germ cells (Figures 1s and 2s). The maturing Sc
spermatids show a shorter tapered nuclear profile and distinct
tail formation (Figures 1y and 2y). The younger generation
of primary spermatocytes has just entered the zygotene step
of meiotic prophase. These cells have larger nuclei, larger
evenly dispersed chromatin clumps, and well-defined nuclear
envelopes in comparison to leptotene primary spermatocytes
(Figures 1l and 2l). Type A spermatogonia are also seen at
this stage (Figures 1f and 2f).
Discussion
Although a few pregnancies and births (Fishel et al., 1995,
1996; Hannay, 1995; Tesarik et al., 1995, 1996; Antinori et al.,
1997a,b; Vanderzwalmen et al., 1997; Gianaroli et al., 1999)
have been reported to result from ROSI into human oocytes,
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L.Johnson et al.
the efficiency of this procedure in terms of fertilization rates and
pregnancy rates remains very low (Vanderzwalmen et al.,
1997, 1998a,b; Silber and Johnson, 1998; Verheyen et al.,
1998; Balaban et al., 2000; Levran et al., 2000; Silber et al.,
2000). A major problem, which might be related to the
technique of ROSI, lies in the identification of live round
spermatids from other types of cells present in the ejaculate
or among a dispersed population of testicular cells. In fact,
identification of round spermatids in wet preparations is not
as easy as in stained preparations (Vanderzwalmen et al.,
1998a,b).
There has been a great deal of effort towards improving the
recognition of spermatids within a heterogeneous population
of testicular cells in a wet preparation. Tesarik and Mendoza
described how to recognize a round spermatid under an inverted
microscope with Hoffman modulation (Tesarik and Mendoza,
1996). However, Hoffman modulation contrast optics may not
allow reliable identification of the round spermatid
(Vanderzwalmen et al., 1997; Verheyen et al., 1998). Even the
difference between the acrosomal vesicle and a vacuole often
seems unclear (Tesarik and Mendoza, 1996). Several other
techniques have been investigated to identify round spermatids,
such as immunocytochemistry methods (Mendoza and Tesarik,
1996), vital staining coupled with morphological features and
fluorescence in-situ hybridization (Angelopoulos et al., 1997),
confocal scanning laser microscopy and computer-assisted
image analysis (Yamanaka et al., 1997), fluorescent-activated
cell sorting (FACS) (Aslam et al., 1998b), and epifluorescence
microscopy after incubation of dispersed male germ cells with
a vital mitochondrion-specific fluorescent probe (Sutovsky
et al., 1999). All these studies provide good methods for round
spermatid identification and evaluation, but they are often
expensive and cannot be introduced in every laboratory.
Moreover, the question should be asked whether these cells
remain suitable for therapeutic ICSI after these treatments. As
pointed out by Tesarik, it is urgent to define as exactly as
possible the developmental stage of the spermatids used in
each injection (Tesarik, 1997). In fact, without a clear terminology for each stage of spermiogenesis, it is difficult to compare
the results of the different laboratories as they could be
injecting spermatids representing different stages of the cycle.
More recently, however, criteria for the correct diagnosis and
selection of live round spermatids (Sousa et al., 1998; Cremades
et al., 1999) and the correct recognition and classification of
spermatids along the different spermiogenic steps (Sousa et al.,
1999) have been published.
We offer here a simple, rapid, objective and reliable way to
identify male germ cells at each stage of their differentiation
from spermatogonia to mature elongated spermatids using
Normarski optics. In fact, Normarski differential interference
contrast microscopy allows identification of cellular morphology inside whole cells, as it uses a small depth of focus. This
enables one to optically section through cells or tissues such
as testicular specimens (Saacke and Marshall, 1968; Johnson
et al., 1976, 1981, 1983, 1984a,b, 1987, 1999; Bellve et al.,
1977; Johnson and Neaves, 1981; Neaves et al., 1984; Sutovsky
et al., 1999).
In this current study, we provide, with the help of more
1580
recent tools, a detailed organized presentation of live human
germ cells in the context of their developmental steps and
stages of the spermatogenic cycle, as described (Clermont,
1963; Holstein and Roosen-Runge, 1981), and convincingly
considered. This comparative approach allows verification of
the identity of individual germ cells seen in vitro and provides
a checklist of distinguishing cytologic and morphologic characteristics of live human germ cells to be used by scientists
interested in studying live cells at known steps in spermatogenic
development characteristic of germ cells in specific stages of
the spermatogenic cycle. The most important morphological
characteristics noted in this comparative study were size;
shape; chromatin pattern within nuclei; number and shape of
nucleoli; definition of the nuclear membrane; presence of
meiotic metaphase figures; the Golgi apparatus; the acrosomic
vesicles or cap; presence of the tail; location of the annulus;
and/or mitochondria around the middle piece of the tail.
Previous published studies using spermatids for ROSI
procedures have not defined the level of spermatogenic development of injected spermatids, because a systematic evaluation
of the conception potential of spermatids in different developmental steps of spermiogenesis could not be made. The
influence of the immaturity of the early round spermatids on
the development rate of the embryos resulting from ROSI is
important. The problem does not appear to be imprinting since
it has been shown that expression of imprinted genes in mouse
embryos derived by ROSI do not differ from normallyproduced animals (Shamanski et al., 1999). This indicates that
paternal genes have undergone proper imprinting by the
first step of round spermatid development. However, not all
postmeiotic cells have the same chances of producing a high
quality embryo (Sousa et al., 1999). A classification (Figure
2, Table I) is essential to know if the development rate of
ROSI embryos correlates with the development step of the
round spermatids injected.
Spermatogenesis is accomplished by an amazing array of
gene regulation by hormonal stimulation and local controlling
factors (Parvinen, 1982; Skinner, 1991; Jegou, 1993; Carreau
et al., 1994; Kierzenbaum, 1994; Lejeune et al., 1996, 1998;
Mauduit and Benahmed, 1996). To study this complex process
in humans, methods need to be developed to isolate and
identify individual germ cells in each developmental step
throughout spermatogenesis including spermatocytes and
spermatogonia. To this end, this study allows an easy identification of human germ cells in the context of their developmental steps and stages of the spermatogenic cycle. This
could be useful to study stage specific events of human
spermatogenesis. Moreover, after isolating germ cells at the
same stage, it allows the study of the expression of genes of
germ cells at specific stages. It makes it possible to determine
germ cell specific gene expression patterns for each germ cell
at any step of its differentiation.
In conclusion, comparison of live and fixed human germ
cells in the context of development step and stage of the
spermatogenic cycle provides a detailed description of
spermatogonia, spermatocytes and spermatids as they progress
through subsequent stages. This description facilitates a more
precise comparison of ROSI results among laboratories; facilit-
Human germ cells in the spermatogenic cycle
ates molecular biological studies using a few germ cells
identified to be at steps in a specific stage; and facilitates
evaluation of treatment effects and toxicant effects on specific
development steps of live human germ cells in specific stages
of the spermatogenic cycle.
Acknowledgements
The authors wish to thank Rebecca S.Heck and Steven W.Brown for
technical assistance. This work was supported in part by NIH contract
N01-HD-8–3281.
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Received on November 17, 2000; accepted on April 25, 2001