1 Centre for Reproductive Medicine and 2 Department for Pathology, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan, 101, 1090 Brussels, Belgium
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Abstract |
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Key words: cryopreservation/spermatids/testicular spermatozoa/ultrastructure
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Introduction |
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In order to avoid successive surgery in this category of azoospermic patients, with the risk of causing irreparable damage to the testes, testicular biopsies may be frozen for subsequent intracytoplasmic sperm injection (ICSI) trials. As a recent advance in assisted reproductive technology, spermatozoa from frozenthawed testicular biopsies have already been used in clinical practice but data on the outcome are still limited (Fisher et al., 1996, 1998
; Hovatta et al., 1996
; Romero et al., 1996
; Khalifeh et al., 1997
; Oates et al., 1997
). Cryopreservation of testicular spermatozoa is difficult because of their low concentration and motility as compared to ejaculated spermatozoa (Silber et al., 1995
). For this reason, comparative studies to optimize tissue preparation, freezing protocols and freezing media are rare and few attempts have been made to define the effects of cryopreservation on testicular spermatozoa (Verheyen et al., 1997
). No comparative studies on the efficiency of different media are available. Since the first report describing the cryoprotectant properties of glycerol for freezing spermatozoa (Polge et al., 1949
), this has been confirmed as the most effective agent in lowering the freezing point of intracellular water. However, this does not mean that glycerol may not have toxic effects on spermatozoa: the presence of hyperosmotic injury in ejaculates has been extensively studied (Critser et al., 1988
; Gao et al., 1993
). In frozenthawed testicular spermatozoa, the investigation of possible damage has been restricted to basic sperm parameters such as vitality and motility. In a recent study (Verheyen et al., 1997
) 24% of the initial motility and 32% of the initial vitality was recovered after thawing of cryopreserved testicular spermatozoa.
In clinical practice, essentially mechanical methods are used to retrieve spermatozoa from the testicular tissue, and biopsies are usually frozen as shredded suspensions. If the tissue is frozen as whole biopsies it may either be shredded after thawing, or enzymatic digestion (collagenase type IA or IV) may be used as an alternative method of sperm recovery (Fisher et al., 1996; Crabbé et al., 1997
). Other studies describe different attempts to preserve testicular spermatozoa, such as freezing of seminiferous tubules (Allan and Cotman, 1997
) or single spermatozoa in empty zonae of human and hamster oocytes (Cohen et al., 1997
). Histological evaluation of spermatogenesis after thawing requires freezing of the whole biopsy (Salzbrunn et al., 1996
).
Conventional light microscopic assessment of sperm morphology for routine semen analysis allows visualization of the entire head but no details of the subcellular entities. The fine structure of the spermatozoa may, however, be evaluated by electron microscopy. In ejaculated spermatozoa, damage to the plasma membranes and alterations to the acrosomal content have been demonstrated by ultrastructural analysis as a result of the freezingthawing procedure (Mahadevan and Trounson 1984; Heath et al., 1985
). However, it is not clear so far whether rather immature testicular spermatozoa show effects of freezing and thawing similar to those for ejaculated spermatozoa. The routine use of this type of spermatozoa in current clinical practice necessitates a better understanding of their ultrastructural features before freezing and an assessment of possible subcellular changes after thawing.
The aim of the present study was to investigate the morphological aspects of testicular spermatozoa and spermatids before freezing and after thawing by light and transmission electron microscopy.
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Materials and methods |
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Cryopreservation
The biopsy piece designated for freezing was transferred into an ampoule containing 200 µl of HEPES-buffered Earle's medium with 0.5% HSA. The cryoprotectant TESTyolk buffer medium with glycerol (12% v/v) (TYB; Irvine Scientific, Santa Ana, CA, USA) was thawed at room temperature. The medium containing the sample was diluted 1:1 (v/v) by dropwise addition of cryoprotectant. The freezing procedure was carried out by a computer-controlled slow freezing programme (Planer Products, Mettler Toledo, Brussels, Belgium) using the following procedure: from room temperature to +5°C at a cooling rate of 1°C/min, from +5°C to 80°C/min at a cooling rate of 10°C/min, from 80°C to 130°C at a cooling rate of 25°C/min and a final plunge into liquid nitrogen (196°C).
Thawing
The ampoule was taken from the liquid nitrogen tank and thawed at room temperature for 10 min. The removal of the cryoprotectant was carried out by placing the sample in a tube to which 10 ml of HEPES-buffered Earle's medium containing 0.5% HSA was added slowly drop by drop. The supernatant was removed by one washing step after centrifugation performed at 750 g for 6 min. The biopsy specimen was placed in fixative for preparation of semithin and ultrathin sections.
Fixation, postfixation and embedding
The fresh and frozenthawed tissue was fixed on a resin plate containing a 50 µl droplet of 2.5% glutaraldehyde (in a 0.1 mol/l cacodylate buffer) and further cut into pieces of about 1 mm3. The pieces were then immersed in a tube containing 1.5 ml of 2.5% glutaraldehyde and kept at 4°C (for at least 2 days) until the time of postfixation and embedding.
Postfixation was performed by transferring the samples into 1% OsO4 (in water) and incubating them for 90 min, followed by staining in uranyl acetate 2% (in veronal buffer) for 120 min; dehydration was carried out in ascending alcohol concentration steps. Afterwards, the samples were embedded by incubation in 1:1 propylene/Spurr resin (Taab; Bodson, Liège, Belgium) for 30 min and then immersed twice in undiluted Spurr for 60 min. Finally, polymerization was performed overnight in an oven at 70°C.
Semithin sections and light microscopy assessment parameters
In order to observe the aspect of the seminiferous tubules after the freezingthawing procedure, 20 semithin sections from three to five tubules (section thickness: 0.5 µm) were cut serially from each sample, using an LKB Bromma 2128 ultramicrotome (Bromma, Stockholm, Sweden) equipped with a glass knife. The sections were collected on a slide, dried and stained with permanganate and methylene blue at 70°C, a coverslip was applied and finally observations made on a light microscope (Olympus) at a magnification of x400.
The diameters of seminiferous tubules as well as the thickness of the lamina propria were recorded. The aspect of the Sertoli cells was observed. The shape of the nucleus and cytoplasm of each germ cell and its membrane integrity were recorded. All these observations were performed before and after the freezingthawing procedure of each sample.
Ultrathin sections and ultrastructural assessment parameters
Ultrathin sections of 75 nm from the fresh and frozenthawed tissue were cut using a diamond knife. The sections (five from each biopsy) were placed on single slot copper grids (Agar Scientific Ltd, Stansted, UK) and subsequently stained with Reynold's lead citrate. The ultrastructural classification of different spermatid stages, early-stage (round and elongating), late-stage (elongated) and spermatozoa (Clermont, 1963) was performed using electron microscopy. The nucleus of round spermatids has a spherical shape with a finely granular karyoplasm. The elongating spermatids have an ovoid nucleus containing denser chromatin aggregation by comparison with earlier stages. Finally, the nucleus of elongated spermatids has further condensed granules and transparent areas remaining in the karyoplasm in which finely granular material is seen (Holstein and Roosen-Runge, 1981
). The abnormal or degenerative aspect of the nucleus was considered when dots of dispersed chromatin were distributed in the nucleus. The aspect of intact, disrupted or swollen nuclear envelope (NE) as well as the inner (IAM) and outer acrosomal (OAM) and plasma membranes (PM) were recorded. The presence of vesiculation in the acrosomal vesicle as well as the presence or absence of an acrosomal matrix were recorded. The normal or swollen aspect of the equatorial segment (ES) and the postacrosomal region (PR) were examined in the late spermatids and spermatozoa.
In the cytoplasm of early-stage (round) spermatids, flat cisternae of endoplasmic reticulum (ER) are frequently found embedded in dense material. The ER membranes often are fused in small areas, e.g. annulate lamellae, which may serve for discarding the redundant nuclear pore complexes during NE reduction. Mitochondria are found predominantly in the cell periphery. The swollen aspect of ER as well as the degeneration and the abnormal integrity of mitochondrial cristae were recorded. Electron micrographs from fresh and frozenthawed cells from the same original sample were taken on a Zeiss transmission microscope type EM 109. About 1525 early-stage spermatids and about 1015 late-stage spermatids per section were examined.
Statistics
The mean diameters as well as the mean thicknesses of the lamina propria of seminiferous tubules before and after the freezingthawing procedure were compared by Student's paired t-test; P < 0.05 was considered significant.
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Results |
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Transmission electron microscopy
Early spermatid stage
Table III summarizes the observations in spermatids and spermatozoa after the freezing and thawing procedure. In the frozenthawed tissue, some of the early-stage spermatids including round (Figure 1b
) and elongating spermatids (images not shown) presented unusual morphology of the nucleus, such as sparse dots of condensed granules of chromatin aggregation, probably as a sign of degeneration. This nuclear aspect was rarely present in spermatids from the fresh tissue (Figures 1a, 2a and 3a
). The damage to the nuclear envelope was more often observed in the region not covered by the acrosomal vesicle (Figure 1b
).
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In the elongating spermatids, compared to the spermatids in the fresh tissue (Figure 3a), loss of acrosomal material occurred more often in the frozenthawed tissue (Figure 3b
). The outer acrosomal membranes as well as the plasma membranes revealed ruptures and frequently appeared swollen (Figure 2b
), as compared to those in the fresh tissue (Figure 2a
).
The mitochondria in the frozenthawed tissue (Figure 1b) showed increased degeneration as compared to fresh tissue (Figure 1a
). In intact mitochondria, the shape and cristae were altered. The endoplasmic reticulum in the frozenthawed tissue appeared swollen in comparison with fresh tissue.
Late spermatid stage and spermatozoa
The late-stage (elongated) spermatids and spermatozoa in frozenthawed tissue showed a proportion of nuclei with a degenerative morphology of the chromatin similar to that in the fresh tissue. Compared to the fresh tissue (Figure 5a and b), the number of vacuoles in the centre of the head was not increased and the karyoplasm was conserved and compact after freezing and thawing (Figure 5c and d
). The nuclear envelope seemed to be conserved to the same extent as in the fresh tissue, covering the nucleus homogeneously.
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After freezing and thawing, the outer acrosomal membranes were disintegrated and frequently disrupted and revealed more damage than the inner membranes of the acrosome. The plasma membrane was also severely damaged and it was not uncommon to observe swollen plasma membranes in the region of the head. Moreover, disintegration and disruption of the plasma membrane was also observed more often in the cells of the frozenthawed tissue.
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Discussion |
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Attention was focused on the effects of freezing and thawing on early and late-spermatid stages and on spermatozoa. In view of their possible future application for ICSI, such as in-vitro differentiation of spermatogenic cells, the quality of spermatids after the freezing and thawing procedure needs to be assessed, but light microscopic resolution was too low. On the basis of ultrastructural analysis, no shrinkage or swelling of the nuclear membranes and the nucleus of these more mature cells was observed. A homogeneous chromatin content and conserved shape of the sperm nucleus were observed after thawing, as for fresh tissue.
In some early-stage spermatids, however, a degenerative morphology of the chromatin has been observed, which suggests that the nucleus of such cells is less protected during the freezingthawing procedure than that of more differentiated cells. These morphological observations suggested that late-stage spermatids and spermatozoa are more resistant to the cryopreservation procedure than other germ cells. This cryopreservation resistance may be due to the presence of the sperm perinuclear theca, a cytoskeletal element described in mammalian spermatozoa, which covers the whole sperm nucleus at these stages of spermiation (Oko et al., 1988, 1994).
The increased resistance of the late-stage spermatids to freezing and thawing may also be due to their small quantity of cytoplasm and more condensed chromatin. However, it is possible that chromatin changes occur in testicular spermatozoa as a result of the cryopreservation procedure, which could not be detected by electron microscopic observation. A cytochemical study (Royère et al., 1988) demonstrated that the freezing and thawing procedures induced chromatin alterations in ejaculated spermatozoa. They suggested that the chromatin of frozenthawed ejaculated spermatozoa is present in a state of `overcondensation'. It is possible that testicular spermatozoa are more susceptible to damage of the DNA material than ejaculated spermatozoa. Normally, nuclear maturation continues during the spermatozoon's passage through the epididymis with an increase in the formation of protamine disulphide bonds which are responsible for the packaging of sperm DNA (Bedford et al., 1973
; Sutovsky et al., 1996
; Sutovsky and Schatten, 1997
). Further studies using methods such as terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) analysis for the identification of DNA breaks may be useful to investigate whether the freezingthawing procedure causes changes in the nucleus of such immature spermatozoa.
In our study, electron microscopic analyses showed that the major cryoinjury in the sperm head occurs at the membranes and acrosomes of spermatids and spermatozoa, which appear swollen and ruptured. In a comparative study (Heath et al., 1985), ejaculated spermatozoa was cryopreserved either with glycerol alone or with TESTyolk buffer. The results showed that the most obvious change after glycerol treatment alone was acrosomal loss. In the present study, although the cryoprotectant used was glycerol diluted in TESTyolk buffer, severe damage to the acrosome content has also been demonstrated. Using the same cryoprotectant medium as in the present study, it has been shown that freezing of human spermatozoa from ejaculates caused damage to the acrosomes and membranes of the sperm cells after thawing (Mahadevan and Trounson, 1984
). Similarly, we observed swelling, vesiculation and loss of acrosomal material mostly in late-stage spermatids and spermatozoa in testicular tissue. Thus, it may be assumed that freezingthawing of whole biopsies does not optimally protect the sperm cells against cryoinjury.
The freezing protocol may also influence sperm cell quality. In the present study, a slow, computerized method with a temperature decrease in three different steps was used, before the samples were plunged into liquid nitrogen. This method has been reported to limit cryoinjury, specially for low-quality spermatozoa (Ragni et al., 1990). For this reason, it was used for freezing testicular biopsies where there is usually a lower number of progressive motile spermatozoa within the tissue.
Nowadays, frozenthawed testicular spermatozoa have been used successfully in clinical ICSI trials with positive results (Fisher et al., 1996, 1998
; Hovatta et al., 1996
; Romero et al., 1996
; Khalifeh et al., 1997
; Oates et al., 1997
). It has been shown that the freezing and thawing of human semen significantly reduces the total number of spermatozoa with normal head ultrastructure, motility and capacity to fertilize (Serafini et al., 1986
). Moreover, after cryopreservation, swelling, disruption and loss of acrosomal material results in a substantial reduction of sperm function, e.g. decreasing the capacity to penetrate zona-free hamster ova. After freezing and thawing of testicular tissue, plasma and acrosomal membranes of testicular spermatozoa appeared frequently ruptured and swollen. Furthermore acrosomes were absent or were swollen after freezing and thawing.
Acrosomal material is a prerequisite for penetration and fertilization of the oocyte. In the proximity of the ovum, the acrosome reaction proceeds and results in vesiculation of surface membranes involving the intermittent fusion of the sperm plasma membrane with the outer acrosomal membrane. However, during ICSI, all barriers to sperm penetration of the oocyte are bypassed and the spermatozoon is introduced directly into the ooplasm. Here, the question arises of whether the presence of an intact acrosome is mandatory when the ICSI procedure is used, which is the only available technique when using testicular spermatozoa. It seems that acrosome and perinuclear thecal structures must be eliminated by oocyte cytoplasm in order to facilitate male pronuclear development (Sutovsky et al., 1997) and the release of the oocyte-activating substances from the perinuclear theca into oocyte cytoplasm (Kimura et al., 1998). Moreover, recent ultrastructural studies have demonstrated that the acrosome reaction may be found within the oocyte cytoplasm after ICSI (Sathananthan et al., 1997
; Bourgain et al., 1998
). These studies suggest that acrosome deletion is a possible prerequisite for sperm incorporation after ICSI. If this hypothesis is true, it might be speculated that this observed morphological damage does not necessarily influence sperm function after ICSI, since the spermatozoon is directly injected into the ooplasm. In view of the present data, it might also be that this phenomenon corresponds to the persistence of the subacrosomal layer of the sperm perinuclear theca. This question has been addressed in a recent study (Hewitson et al., 1999
).
It might be that changes in other areas of the spermatozoon also occur after freezing and thawing. A morphological abnormality frequently seen after cryopreservation of spermatozoa is a `broken neck' (Verheyen et al., 1997). The broken neck syndrome is most likely a result of damage to the sperm tail connecting piece. Although these changes were not addressed in the present study, damage to the neck and midpiece of a spermatozoon might disturb the proximal centriole, which is an organizing structure for the beginning of embryonic development (Schatten, 1994
; Van Blerkom and Davis, 1995
; Sutovsky et al., 1996
, 1997). This hypothesis could not be answered in this study because in testicular tissue, these minute single structures are extremely difficult to find. Moreover, they are not always oriented adequately within the tissue section to allow valid interpretation of their fine structure.
We may conclude that cryopreservation of human testicular spermatozoon within its tissue structure causes similar damage to membranes and to the acrosome as that seen in ejaculated spermatozoa. Thus, a certain loss of morphologically intact spermatozoa should be taken into account when cryopreserving human testicular spermatozoa. Whether these alterations interfere with normal fertilization capacity and further embryo development in an ICSI procedure, remains under investigation.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 25, 1999; accepted on May 20, 1999.