Light and electron microscopic analysis of human testicular spermatozoa and spermatids from frozen and thawed testicular biopsies

D. Nogueira1,3, C. Bourgain2, G. Verheyen1 and A.C. Van Steirteghem1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The morphological changes caused by freezing and thawing human testicular spermatozoa have been assessed here. Retrieval of testicular biopsies was carried out on six patients with obstructive azoospermia preparatory to intracytoplasmic sperm injection (ICSI). Light microscope analysis was carried out on testicular cells and ultrastructural analysis was carried out on spermatozoa and different spermatid stages before and after the freezing procedure. Upon examination under light microscopy, all germ cells presented increased vacuolization in their cytoplasm and shrinkage or swelling of the nuclei and cytoplasmic membranes. These altered structures were accentuated in the spermatocyte I cell which often presented disrupted membranes. The ultrastructural findings under transmission electron microscopy demonstrated that after freezing and thawing the major types of cryoinjury were the swelling and rupture of inner and outer acrosomal and plasma membranes. The acrosome material often appeared as dispersed material or as condensed spots or was even lost. Such damage was observed mainly at the spermatozoa and late spermatid stages. We conclude that the freezing and thawing of testicular biopsies causes similar morphological damage to testicular spermatozoa and frozen–thawed ejaculated spermatozoa. It is still unclear whether these changes in testicular spermatozoa after freezing and thawing may compromise its use in the ICSI procedure.

Key words: cryopreservation/spermatids/testicular spermatozoa/ultrastructure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the introduction of spermatozoa extracted from the testicles in the microinjection programmes (Craft et al., 1993Go), high fertilization and pregnancy rates have been reported (Schoysman et al., 1993Go; Silber et al., 1995Go) even though testicular spermatozoa may not be fully mature. This treatment is restricted to couples where the male partner suffers from azoospermia due to obstruction of the epididymis, to total blockage or absence of vasa deferentiae, or to testicular failure (i.e. partial Sertoli cell-only syndrome or maturation arrest).

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 frozen–thawed testicular biopsies have already been used in clinical practice but data on the outcome are still limited (Fisher et al., 1996Go, 1998Go; Hovatta et al., 1996Go; Romero et al., 1996Go; Khalifeh et al., 1997Go; Oates et al., 1997Go). Cryopreservation of testicular spermatozoa is difficult because of their low concentration and motility as compared to ejaculated spermatozoa (Silber et al., 1995Go). 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., 1997Go). 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., 1949Go), 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., 1988Go; Gao et al., 1993Go). In frozen–thawed 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., 1997Go) 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., 1996Go; Crabbé et al., 1997Go). Other studies describe different attempts to preserve testicular spermatozoa, such as freezing of seminiferous tubules (Allan and Cotman, 1997Go) or single spermatozoa in empty zonae of human and hamster oocytes (Cohen et al., 1997Go). Histological evaluation of spermatogenesis after thawing requires freezing of the whole biopsy (Salzbrunn et al., 1996Go).

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 freezing–thawing procedure (Mahadevan and Trounson 1984Go; Heath et al., 1985Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testicular biopsy procedure
Testis biopsy specimens were obtained from six infertile patients with obstructive azoospermia (age range: 26–57 years) as sources of spermatozoa in the ICSI programme. By a small horizontal incision in the scrotal skin and through the peritoneal tunica vaginalis, a 0.5 cm opening was made in the tunica albuginea (Silber et al., 1995Go) and a small piece of extruding testicular tissue was removed. An extra biopsy specimen was removed for this research purpose. The biopsy was placed immediately into a 10 ml Falcon tube (Falcon Plastics, Becton-Dickinson, Aalst, Belgium), filled with 3 ml HEPES-buffered Earle's medium supplemented with 0.5% human serum albumin (HSA) (Red Cross, Brussels, Belgium). Each research tissue sample (6–8 mm3) was cut by sterile scissors into two equal pieces in a Petri dish containing the same Earle's medium as described above. One biopsy piece was prepared for freezing while the other was fixed immediately for histology and ultrastructural analysis.

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 TEST–yolk 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 frozen–thawed 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 freezing–thawing 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 freezing–thawing procedure of each sample.

Ultrathin sections and ultrastructural assessment parameters
Ultrathin sections of 75 nm from the fresh and frozen–thawed 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, 1963Go) 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, 1981Go). 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 frozen–thawed cells from the same original sample were taken on a Zeiss transmission microscope type EM 109. About 15–25 early-stage spermatids and about 10–15 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 freezing–thawing procedure were compared by Student's paired t-test; P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Light microscopy
After thawing, the following changes in the structure of the seminiferous tubules were demonstrated: (i) the diameter of the seminiferous tubules (mean 262.6 µm; SD ± 30.4) was similar to that in fresh tissue (mean 258.0 µm; SD ± 27.5) (difference not significant) (Table IGo); (ii) the LP of the tubules was significantly enlarged (P = 0.042) (mean 13.7 µm, SD ± 2.2) compared to the fresh tissue (mean 9.9 µm, SD ± 2.5), probably due to rupture of the layers of the connective tissue fibres. Some of the seminiferous tubules presented irregularities and shrinkage in the region between the inner layer (myofibroblastic) of the LP and the basal layer of the germinal epithelium (Table IGo).


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Table I. Mean values of seminiferous tubules (ST) and lamina propria (LP) from each biopsy specimen before and after the freezing–thawing procedure
 
Table IIGo summarizes the aspect of Sertoli cells and germ cells before freezing and after thawing. Sertoli cells showed an increased presence of translucent vacuoles in their cytoplasm, as compared to their structure before freezing. Spermatogonia, spermatocytes I and II, and early spermatids presented shrinkage or swelling of the nuclei and the cytoplasm after thawing. In spermatocytes I, the nuclear and cytoplasmic membranes were often disrupted. All the above cells presented vacuoles in the cytoplasm which were not observed in the fresh tissue.


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Table II. Aspects of nucleus and cytoplasm of spermatogenic cells before and after freezing–thawing procedure in the semithin sections
 
Late spermatids and spermatozoa did not show visible changes at this magnification after thawing. Some of these cells were found among other desquamated cells of the seminiferous epithelium, while others were normally positioned in the adluminal part of the tubules before and after freezing.

Transmission electron microscopy
Early spermatid stage
Table IIIGo summarizes the observations in spermatids and spermatozoa after the freezing and thawing procedure. In the frozen–thawed tissue, some of the early-stage spermatids including round (Figure 1bGo) 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 3aGoGoGo). The damage to the nuclear envelope was more often observed in the region not covered by the acrosomal vesicle (Figure 1bGo).


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Table III. Ultrastructure of spermatids and spermatozoa before and after freezing–thawing procedure in the ultrathin sections
 


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Figure 1. Electron micrographs of early-stage (round) spermatids. (a) Early-stage (round) spermatid before the freezing procedure illustrating characteristic morphological features at this stage. Note normal aspect of the nucleus; the NE is thickened over the whole area of the contact with the acrosomal vesicle by a thin electron-dense layer of PT (arrow). The AG is present as a small electron-dense mass; the Golgi complex appears shrunken as the acrosomal vesicle enlarges (arrowhead). Mitochondria (small arrowheads) are found at the cell periphery; cisterns of endoplasmic reticulum are visible in the cytoplasm, close to electron-dense granules. Note the formation of annulate lamellae (*) (Holstein et al., 1981). Bar = 1.1 µm. (b) Early-stage (round) spermatid from the same biopsy specimen as after the freezing and thawing procedure. The nucleus presents a degenerative aspect showing clumping of chromatin and the nuclear membrane is disrupted (arrowheads). The region in contact with the acrosomal vesicle seems to be intact. Note vesicular granules in the acrosomal vesicle. The AG is not visualized in this section. Mitochondrial cristae are damaged. Bar = 1.1 µm. AG = acrosomal granule; AM = acrosomal matrix; E = endoplasmic reticulum; ES = equatorial segment; IAM = inner acrosomal membrane; M = mitochondrial cristae; N = nucleus; NE = nuclear envelope; OAM = outer acrosomal membrane; PM = plasma membrane; PT = perinuclear theca; V = acrosomal vesicle.

 


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Figure 2. Electron micrographs of early-stage (elongating) spermatids. (a) Early-stage (elongating) spermatid before the freezing procedure. At the tip of the nucleus (N) the acrosomal granule (AG) starts to spread out to fill the acrosomal cap. The IAM (left arrowhead), OAM (middle arrowhead) as well as the PM (right arrowhead) are intact over all their extension. Bar = 0.4 µm. (b) Early-stage (elongating) spermatid from the same biopsy specimen as a after the freezing and thawing procedure. The chromatin is homogeneously distributed showing normal morphology of the nucleus as a common appearance at this cell stage (N). The AG is still present. All the membranes, IAM and OAM, as well as PM seem to be present but with a ruptured aspect (arrowheads) and the PT appears swollen (arrows). Bar = 0.4 µm. See Figure 1Go legend for abbreviations.

 


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Figure 3. Electron micrographs of more advanced early-stage (elongating) spermatids. (a) A more advanced early-stage (elongating) spermatid before the freezing procedure. The chromatin is more electron-dense and condensing, the head becomes pear-shaped. At this stage, the acrosomal cap covers one-third of the nuclear surface (arrowheads). The NE, as well as IAM, OAM and PM are present. Bar 0.4 = µm. (b) Early-stage spermatid (elongating) after the freezing and thawing of the same biopsy specimen as a. The tip of the nucleus lacks acrosomal material (*). In this case, the NE and IAM are completely disrupted. The OAM and PM are lost. Bar = 0.4 µm. See Figure 1Go legend for abbreviations.

 
The inner acrosomal membranes of most early-stage spermatids remained intact. The acrosomal granule was more preserved in frozen–thawed round spermatids than in frozen–thawed elongating spermatids. In a few round spermatid cells some vesicles were observed in the acrosomal vesicle (Figure 1bGo). Severe vacuolization was also observed in the cytoplasm of these cells after the freezing–thawing procedure.

In the elongating spermatids, compared to the spermatids in the fresh tissue (Figure 3aGo), loss of acrosomal material occurred more often in the frozen–thawed tissue (Figure 3bGo). The outer acrosomal membranes as well as the plasma membranes revealed ruptures and frequently appeared swollen (Figure 2bGo), as compared to those in the fresh tissue (Figure 2aGo).

The mitochondria in the frozen–thawed tissue (Figure 1bGo) showed increased degeneration as compared to fresh tissue (Figure 1aGo). In intact mitochondria, the shape and cristae were altered. The endoplasmic reticulum in the frozen–thawed tissue appeared swollen in comparison with fresh tissue.

Late spermatid stage and spermatozoa
The late-stage (elongated) spermatids and spermatozoa in frozen–thawed 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 bGo), 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 dGo). The nuclear envelope seemed to be conserved to the same extent as in the fresh tissue, covering the nucleus homogeneously.



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Figure 5. Electron micrographs of testicular spermatozoa. (a, b) Spermatozoa within the biopsy specimen before the freezing procedure. The chromatin is fully condensed. The acrosomal matrix is clearly present covering almost all the sperm head (arrowheads). The NE and IAM are preserved. Some small gaps are visible at OAM and PM. (a) Bar = 0.25 µm. (b) Bar = 0.6 µm. (c, d) Spermatozoa from the same biopsy specimen as in a,b after the freezing and thawing procedure. (c) The AM is present as spots distributed in the V (arrows); the IAM, OAM and PM are swollen (arrowheads) as well as the ES (*). (d) Note the absence of the AM and the disintegration of the IAM, OAM and PM (*). (c) Bar 0.25 µm. (d) Bar = 0.6 µm. See Figure 1Go legend for abbreviations.

 
Compared to those in the fresh tissue (Figure 4a and bGo), the spermatids in the frozen–thawed tissue (Figure 4c and dGo) frequently showed rupture of the inner membrane of the acrosome. The acrosomal matrix was dispersed (Figures 4c and 5dGoGo) or, as shown in Figure 5cGo, displayed condensed dots of acrosomal matrix covering the head of the spermatozoon. In a few cells (Figure 4dGo), formation of vesicles as a sign of false acrosome reaction and degeneration was observed after the freezing–thawing procedure. Lack of acrosomal matrix in the sperm head was observed more often in frozen–thawed tissue than in fresh tissue (Figure 5a and bGo).



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Figure 4. Electron micrographs of late-stage (elongated) spermatids. (a, b) Spermatids before the freezing procedure. The nuclei present more electron-dense granules of chromatin. (a) The NE, IAM and OAM as well as PM are intact. Microtubules of the manchette are present (arrow); the ES at both sides are intact (small arrowheads). The acrosomal cap covers two-thirds of the head (large arrowhead) (b). A single vacuole is seen in the nucleus (V). The acrosomal cap is present (arrowheads). (a) Bar = 0.6 µm. (b) Bar = 0.4 µm. (c, d) Late-stage (elongated) spermatids from the same biopsy specimen as a and b after the freezing and thawing procedure. The chromatin is evenly distributed over the nucleus; no degenerative morphology is seen. (c) The AM is lost (*) as well as the OAM and PM; the NE and IAM are disrupted and swollen. C = proximal centriole. (d) Vesiculation occurs as if an acrosome reaction has taken place (*); the ES is swollen on both sides (arrows). (c, d) Bar = 0.4 µm. See Figure 1Go legend for abbreviations.

 
The region of the equatorial segment of spermatids and spermatozoa (Figure 5c and dGo) in the frozen–thawed tissue showed the same disturbed morphology as the anterior acrosome region. When the region of the acrosome was swollen or represented as vesicles, the equatorial segment also showed this aspect. The postacrosomal region was also swollen, with the granular material more dispersed between the nuclear envelope and plasma membranes than in the fresh tissue. This granular material most likely corresponds to the spermatozoon's perinuclear theca.

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 frozen–thawed tissue.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From our results it appears that the freezing and thawing procedure of testicular biopsy specimens changes the structures of germ cells and damages the fine structure of human testicular spermatozoa. In the present study using semithin sections, the germ cells (spermatogonia, spermatocyte I, spermatocyte II and early spermatids) presented shrinkage and swelling of the nucleus and cytoplasm with rupture of their membranes. When certain animal cells are stressed by being placed in hypertonic media, the cells shrink as water passes out by osmotic flow. The cells can modulate their internal osmotic strength to keep their volume constant. This results in an increase in cellular salt concentration and osmotic strength, and finally solute (e.g. cryoprotectant) flows in. Eventually, the initial volume of the cell is restored, and the cells should maintain membrane integrity after a return to isotonic conditions. However, in the present study, it is believed that cryoinjury is related to osmotic stress (Gao et al., 1989Go). It has been shown (Critser et al., 1988Go) that, due to the presence of cryoprotectant agents, such osmotic stress may also provoke sperm cell injury. Further it has also been shown (Gao et al., 1993Go) that osmotic damage resulting in a loss of membrane integrity is related to severe shrinkage of the sperm cells in hyperosmotic solution. It may be followed by serious membrane damage when the cells are returned to isotonic conditions, i.e. during rehydration. The ultrastructural observations after thawing of the spermatogonia and spermatocytes were ignored due to the use of cryoprotectant medium that was probably not optimal for these types of cells. The conditions under which a particular compound will protect cells will also vary and depend on factors such as the molecular weight and the permeability of the cell to the compound. Thus, an optimal cryoprotectant agent should be defined for each cell type separately. It will be difficult to find the optimal cryopreservation conditions for all testicular cells in a testicular biopsy.

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 freezing–thawing 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., 1988Go) demonstrated that the freezing and thawing procedures induced chromatin alterations in ejaculated spermatozoa. They suggested that the chromatin of frozen–thawed 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., 1973Go; Sutovsky et al., 1996Go; Sutovsky and Schatten, 1997Go). 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 freezing–thawing 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., 1985Go), ejaculated spermatozoa was cryopreserved either with glycerol alone or with TEST–yolk 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 TEST–yolk 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, 1984Go). 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 freezing–thawing 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., 1990Go). 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, frozen–thawed testicular spermatozoa have been used successfully in clinical ICSI trials with positive results (Fisher et al., 1996Go, 1998Go; Hovatta et al., 1996Go; Romero et al., 1996Go; Khalifeh et al., 1997Go; Oates et al., 1997Go). 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., 1986Go). 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., 1998Go). Moreover, recent ultrastructural studies have demonstrated that the acrosome reaction may be found within the oocyte cytoplasm after ICSI (Sathananthan et al., 1997Go; Bourgain et al., 1998Go). 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., 1999Go).

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., 1997Go). 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, 1994Go; Van Blerkom and Davis, 1995Go; Sutovsky et al., 1996Go, 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.


    Acknowledgments
 
The authors wish to thank the clinical, paramedical and laboratory staff at the Centre for Reproductive Medicine. Furthermore, we are grateful to Frank Winter of the Language Education Centre at our University for correcting the manuscript. This work was supported by grants from the Belgium Fund for Medical Research.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 25, 1999; accepted on May 20, 1999.