Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
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Examinations of Sperm by Light and Electron Microscopic Levels:
Friendly Preparation Techniques
Y. Ersoy Canillioglu1, G. Erkanli Senturk1 and C. Hurdag2
1
2
Bahcesehir University, School of Medicine, Department of Histology and Embryology, Goztepe, Istanbul, Turkey
İstanbul Science University, School of Medicine, Department of Histology and Embryology, Sisli, Istanbul, Turkey
Sperm examinations may be useful in both clinical and research settings, for investigating male fertility status as well as
monitoring spermatogenesis during and following male fertility regulation and other interventions. Among the possible
causes of male infertility, defects of sperm morphology represent an important factor that may explain decreased fertilizing
potential of sperm. The limits of light microscopy can be overcome by the use of transmission electron microscopy (TEM)
permits the exploration of the ultrastructural organelles rigorously which are characterizing sperm abnormalities. Electron
microscopy allowed us to identify systematic sperm defects that affect the vast majority of sperm in a semen sample and
non-systematic sperm defects, a heterogeneous combination of randomly distributed alterations affecting the head and the
tail organelles in a varied percentage of ejaculated sperm. Correct diagnosis of specific altered sperm phenotypes is
important for the advancement of new therapies. Observing a liquid material, sectioning and visualization are more
difficult compared to the technically solid materials. The problems resulting from these may cause diagnostic problems.
Preparation techniques we tried to display in this chapter will possibly help researchers willing to study sperm and obtain
real-like visuals.
Keywords: sperm; preparation; techniques
1. Overview of Morphological Structures
1.1 Histology of Testis
Each testis is surrounded by a capsule which is called tunica albuginea. The posterior aspect of the tunica albuginea is
somewhat thickened, forming the mediastinum testis, from which connective tissue septa radiate to subdivide each testis
into approximately 250 pyramid-shaped as the lobuli testis. Each lobule has one to four blindly ending seminiferous
tubules. Seminiferous tubule is composed of a central lumen with specialized seminiferous epithelium comprising two
distinct cell populations. These cells are somatic Sertoli cells and Spermatogenic cells. Spermatozoa are produced by
the seminiferous epithelium [1].
1.2 Sperm Morphology
The mature sperm consists of two components: the head and the tail. The sperm head is flattened. It is composed of the
nucleus covered by the acrosome. The acrosomal cap that covers the anterior two thirds of the nucleus contains
hydrolytic enzymes such as hyaluronidase, neuroaminidase and acid phosphatase. The acrosome is regarded as a special
type of lysosomes. Defective sperm head shape is one of the abnormalities associated with male infertility.
The sperm tail is subdivided into the neck, the middle piece, the principle piece and the end piece. The short neck
contains the centrioles and the origin of the coarse fibers. The middle piece contains the mitochondria, helically
wrapped around the coarse fibers and the axonemal complex. These mitochondria provide the energy for movement of
the tail and thus are responsible for motility. The principal piece is the longest segment of the tail. It consists of the
central axoneme surrounded by seven outer dense fibers and a fibrous sheath. The outer dense fibers provide a scaffold
that contributes to the wave like movement pattern of the sperm tail. The end piece is very short segment of the tail, it
contains only axonemal complex [2, 3].
2. Fixatives for Light Microscopy
Fixation is very important when examining both testis tissue and semen samples by light microscope. For this reason
certain chemicals are used. The most commonly used are listed below:
2.1 Formaldehyde Fixative
Commercially available solutions are unsuitable for electron microscopy because of their content of methanol.
Paraformaldehyde powder is used to prepare methanol-free formaldehyde. Main advantage of formaldehyde is that it
has a higher rate of penetration than either glutaraldehyde or osmium tetroxide so that large blocks of tissue are well
fixed. It will penetrate most tissues at a rate of about 10 mm / hour, but will take a longer time to stabilize the tissue.
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Paraformaldehyde is a polymerized form of formaldehyde admixed with methanol; it is generally employed as a
fixative for specialized immunohistologic procedures.
2.2 Neutral Formalin Fixative
It is usually used as a 10% Neutral Buffered Formalin (NBF), that is approximately 3.7% - 4.0% formaldehyde in
phosphate buffered saline. Because formaldehyde is a gas at room temperature, formalin-formaldehyde gas dissolved in
water (~37% w/v) is used when making the former fixative 10% Neutral Buffered Formalin is a non-coagulative
additive fixative. In surgical pathology, neutral buffered formalin (NBF, aqueous solution of 4% buffered
formaldehyde) has been the “gold standard” fixative for decades. It is cheap, enables long-term storage of surgical
material, preserves morphologic features well, allows special histologic stains, and, in combination with antigen
retrieval, allows for reliable immunohistochemical analysis. However, its cross-linking masks antigens, which may
hamper immunohistochemical analysis and fragments nucleic acids, which impairs the extraction efficiency and quality
of DNA and RNA [4, 5].
It is the standard fixative used in most laboratories. Most frequently the routine fixative will be neutral buffered
formalin. Immunohistochemistry methods, that generally include an antigen retrieval step, have been optimized for
formalin-fixed tissues, and tissue specimens can be stored in formalin for extended periods without major deleterious
effects.
Biopsies of the testes are fixed routinely in NBF. Many of these formulae are based on those presented in standard
textbooks of histochemistry. They vary slightly from text to text but these variations are unlikely to cause problems.
Formalin is a more general fixative, used widely for other tissues when mitotic or meiotic cycles are not necessary to
observe. Also used as a mordant for staining procedures.
Compound fixatives with both dehydrant and cross linking actions include alcohol-formalin mixtures.
2.3 Bouin’s Fixative
Bouin’s solution is one of the picric acid fixatives. Bouin solution is a compound fixative used in histology. Bouin’s
fixative contains formaldehyde as a major component, together with picric and acetic acids in aqueous solution. The
effects of the three chemicals in Bouin solution balance each other. Formalin causes cytoplasm to become basophilic
but this effect is balanced by the effect of the picric acid. The tissue hardening effect of formalin is balanced by the soft
tissue fixation of picric acid. The tissue swelling effect of acetic acid is balanced by the tissue shrinking effect of picric
acid. Bouin’s-fixed specimens acquire a yellow color (because of the effects of picric acid) that must be removed by
postfixation washing in 70% ethanol and lithium carbonate [6].
Bouin’s fixative is generally used for testicular fixation because it preserves nuclei and chromosomes especially
observe well during meiosis. This reagent affords excellent preservation of nuclear morphology but suffers from
failings pertaining to brittleness of tissue, pigment deposition, and adverse effects on cytoplasmic polypeptides. In
addition, Bouin’s fixative is preferred for visualization of delicate mesenchymal tissues because of its superior
differentiating abilities in regard to these elements. Accordingly, some ‘‘stromal’’ special stains (such as the Masson
trichrome method) are best performed on specimens preserved in this solution [6]. It leads to partial or complete lysis of
the erythrocytes. It may cause collagen fibrils to swell. It does not lead to excessive hardening. It provides bright
staining with cytoplasmic dyes. Glycogen is well protected (especially with alcohol). It can be used both as a
micranatomical and cytological fixative. In animal testis studies in order to have good fixation the testis must be whole
and be pricket with a needle. It helps simplify fixation. Before any further procedures are conducted to tissues fixed
with Bouin the yellow color arising from the picric acid should be washed with 70% alcohol saturated with lithium
carbonate until it comes bleached [7].
Bouin Fixative (Bouin-1897)
Saturated picric acid 3000.0 ml
Formaldehyde 1000.0 ml
Glacial acetic acid 200.0 ml
3. Light Microscope Following Procedure
3.1 Testicular Sperm Extraction
Testicular sperm extraction (TESE) is the process of removing a small portion of tissue from the testicle under local
anesthesia and extracting the few viable sperm cells present in that tissue for intracytoplasmic sperm injection (ICSI).
The testicular sperm extraction process is recommended to men who cannot produce sperm by ejaculation due to
azoospermia. Some men do not have enough sperm in their ejaculate to succeed with IVF. Fortunately, with the use of
ICSI, many of these men will still have the opportunity to proceed with IVF. As long as there are some sperm in the
testicle, these sperm can usually be obtained and individually inserted in to their partner’s (or donor’s) eggs (ICSI).
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3.2 Preparation of Sperm Samples
Semen samples usually examined in smear cause of liquid samples. Smear preparation can be evaluated
morphologically with dyes such as Difquick, giemsa. Here we will focus on how we embed semen into paraffin block.
This process allows us to better examine sperm samples by using immunohistochemistry much easier in the laboratory.
It is necessary to obtain sperm sections from paraffin. Cytoblock kits can be used to obtain paraffin blocks. The
Cytoblock system can also be used to process tissue biopsies and fragments which are difficult or impossible to process
using other techniques. Preparation of cell blocks with the Cytoblock system is so simple and reliable that retention of a
paraffin-embedded block for every cytological sample is a practical laboratory procedure. Cytoblock can be used to
produce paraffin-embedded blocks from fine needle aspirates, cutting needle cores, body fluids, and residual sediment
from other cytological preparations. Cytoblock is also an ideal method for processing tissue fragments such as small
biopsies, curettings, and other specimens which are too small to be processed in standard cassette.
3.3 Light Microscopic Preperation
A viscous structure semen samples should be treated with collection fluid to have homogeneous structure before
paraffin procedures. Samples are treated with collection fluid. After centrifugation procedure, pellets are prepared for
sandwich model by using cytoblock kit (Cytoblock Kit, Thermo Scientific, USA). After one hour of 4% paraformaldyde
fixation rising alcohol series is done through dehydration process and finally embedded into paraffin. Sections from
paraffin blocks can be examined both histochemical and immunohistochemical. Parafin blokdan alınan kesitlere
histokimyasal ya da immunohistokimyasal uygulamalar yapılabilir (Figure 1) [8].
Fig. 1
Cytoblock kit sandwich model obtained from paraffin blocks eNOS immunostaining applied to sperm samples
(Magnificaiton x100).
4. Fixatives for Electron Microscopy
Electron microscopy is preferred when evaluating fertility cases where light microscopy results need to be confirmed or
when the data is insufficient to evaluate.
Infertility can now be considered a topic of general health; male infertility is a significant problem in humans and it
may be caused by different pathologies, such as anatomical problems, infections, hormonal imbalances, chromosomal
alterations or gene anomalies, although the cause of infertility remains unknown for 30% of infertile men [9]. Sperm
analysis is important to determine the fertilizing potential of sperm [10]. Therefore, altered sperm heads and tails,
highlighted at the level of LM, are the expression of anomalies of chromatin, acrosome, perinuclear theca,
mitochondria, axonemal and periaxonemal structures that cannot be evaluated by this method. The limits of LM can be
overcome by the use of electron microscopy: transmission electron microscopy (TEM) permits the exploration of the
“ultrastructure world” and the study of the different organelles rigorously characterizing sperm abnormalities, and
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scanning electron microscopy (SEM) gives a three-dimensional image of the cell. Both techniques provide a more
detailed evaluation of sperm characteristics.
TEM and SEM are the best methods for studying teratozoospermia, a heterogeneous combination of defects in the
shape of different sperm components that influence fertilizing potential. These approaches go beyond a descriptive
morphology of the "look" of sperm and they play an essential role in the definition and the study of sperm pathology,
the discipline that characterizes the structural and functional deficiencies of altered sperm [11].
The ultrastructural organization of sperm organelles plays a significant role for cell function and, therefore, for the
reproductive process is described. Also, the major abnormalities and defects of the various organellar systems and how
they impair the reproductive function and/or the viability of the cell are reviewed [12].
Human semen is heterogeneous in nature with reference to sperm count and morphology even when motility is
normal. This, coupled with limitations in measurement of structural alterations, has made the assessment of cause and
possible therapy of asthenozoospermia difficult. Different kinds of abnormalities have been reported in patients with
asthenozoospermia and/or teratozoospermia [13]. Among them the "Kartegener's" or "Immotile cilia syndrome" has
been well documented. Normally sperm tail morphology displays 9+2 microtubule aksonem structure. The detailed fine
structure of the axoneme was first described in sea urchin sperm [14]. It was shown that the nine doublets have
projections which cross-bridge neighboring doublets. The projections were named "arms" and assigned a function in
sperm movement (Figure 2). Unlike normal sperm tail aksonem structure, Kartegener’s Syndrome or Immotıle cilia
syndrome is a congential defect with either lack of dynein arms [15] and/or lack of radial spokes and central
microtubules or transposition of ciliary microtubules [16]. Analysis of sperm motility needs assessment of flagellar
substructures in addition to proportion of motile spermatozoa [17]. The most appropriate examination method of
detailed sperm tail structure is transmission electron microscopy (TEM) for it is not possible to examine it in detail at
light microscopy level. Thus the reasons of infertility related with the sperm tail structure can be exposed. Besides the
mitochondria structure which is found in the neck of sperm is responsible for the motility and the energy mechanism of
sperm. Any change in this arrangement of mitochondria may be the cause of infertility related with the man. The
examination of this structure can be realized with TEM (Figure 3). The share of electron microscopy in the
identification of specific sperm phenotypes is significant for the development of new therapies treating male factor
infertility.
Fig. 2
9+2 axonemal structure in cross section of the sperm tail.
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Fig. 3
The arrangement of mitochondria in the neck of the sperm.
Below are a number of fixation protocols that should provide good starting points for most EM studies.
4.1 Glutaraldehyde
Reacts rapidly with proteins and that, being a dialdehyde, stabilizes structures by cross-linking before there is any
opportunity for extraction by the buffer. Hence more ground substance of the cytoplasm (glycogen) and of the
extracellular matrices is preserved. Glutaraldehyde alone is not an adequate fixative, since certain cell components
especially lipids, are not fixed and may be extracted during dehydration, therefore secondary fixation is required using
osmium tetroxide. Depth of penetration 2 - 3 mm / hour.
Glutaraldehyde is similar in chemical activity to formaldehyde; both cause cross-linkage of proteins in tissue.
However, glutaraldehyde penetrates specimens very slowly, making the size of the tissue sample a critical determinant
of fixation with this reagent. Moreover, 2–4% glutaraldehyde (representing the usual working concentration) has a
propensity to cause brittleness of specimens that are immersed in it for more than 2–3 hours; transfer to a buffer
solution is absolutely necessary after this point. For these reasons, among others, glutaraldehyde is not used often for
the preservation of biopsy samples that are intended for light microscopy. However, it is the preferred fixative for
electron microscopy, wherein specimens are very small and limited ‘‘hardening’’ of tissue may actually be
morphologically advantageous [6].
4.2 Osmium tetroxide
The solution should have a yellow color, but may become colorless if stored for too long, if this occurs discard solution
and prepare fresh. Reacts with lipids and also acts as a stain, osmium will penetrate most tissues at a rate of about 1mm
/ hour. Extended times will cause extraction of many proteins, therefore keep time of immersion to a minimum.
4.3 Formaldehyde and Glutaraldehyde mixtures
Fixatives containing both paraformaldehyde and glutaraldehyde provides a much better quality of fixation, than either
aldehyde alone. Formaldehyde penetrates tissues rapidly and mildly stabilizes proteins etc. which are then permanently
fixed by the glutaraldehyde.
Uranyl acetate - (EN-BLOC stain)
Used as 2% solution in distilled water , this takes 24 hours to dissolve. The solution will keep for 1 week if stored in
dark at 4oC. En bloc stain with 2% aqueous uranyl acetate (UA) for ~2 h at 4°C IN DARK (must be carried out in the
dark as UA is photo reductive and will precipitate – this you don’t want to happen as UA crystals make sectioning
difficult and don’t look nice in the specimen!).
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5. Semen and Testis Tissue Preparation for Transmission Electron Microscopy (TEM)
5.1 Semen Preparation for TEM
After collection from donor, semen is allowed to liquefy for at least 30 minutes at room temperature. The 1 to 2 mL of
semen is transferred to a 15-mL polyethylene centrifuge tube and combined with cold 2.5% gluteraldehyde, 0.1 M
sodium cacodylate or PBS, pH 7.2 to 7.4. The tube is capped and repeatedly inverted until thoroughly mixed. The
specimen is then stored at 4°C for further fixation 2 hours on to the washing process, PBS is added and the specimen is
then centrifuged at 2000rpm for 5 minutes, and the sperm pellet is transferred to a 1.9-mL polypropylene centrifuge
tube. Sperm are then washed in cacodylate- buffered or PBS and post-fixed in cacodylate-buffered or PBS 1% osmium
tetroxide, pH 7.2 to 7.4, for 1 hour. Later spermatozoa are incubated in uranil asetate for 30 minutes. Then spermatozoa
are dehydrated in ascending graded ethanols (70% 2x5’, 96% 2x5’, 100% 2x10’) and incubated in propylenoxide
(2x10’). For embedding process, spermatozoa are held in propyleneoxide:epon (1:1) for 20 minutes and in
propyleneoxide:epon (1:2) overnight. The day after they are held in pure epon mixture for 3 hours. Then they are
embedded in epoxy resin in the same polypropylene tube. Before each processing step, spermatozoa are pelleted by
centrifugation and resuspended in the reagent. Finally sperm pellets were polymerized at 60°C for 18 to 20 hours [6].
Semi-ultra thin sections are taken from epon blocks and painted with toluidin blue. Then ultrathin sections are taken on
the grids. To obtain contrast, these sections are painted with uranil asetat (30-40 minutes) and lead citrate, finally
examined at transmission electron microscope.
5.2 Testis Tissue Preparation for TEM
Approximately 3 mm3 testis tissue blocks are fixed by immersion into 2.5% glutaraldehyde in PBS (0.1 M, pH 7.2),
postfixed in 1% osmium tetroxide in PBS (0.1 M, pH 7.2), dehydrated in ascending graded ethanols (70% 2x5’, 96%
2x5’, 100% 2x10’) and incubated in propylenoxide (2x10’). The next step, tissues are held in propyleneoxide:epon (1:1)
for 20 minutes and in propyleneoxide:epon (1:2) overnight. The day after they are held in pure epon mixture for 3
hours. Then they are embedded in epoxy resin, are polymerized at 60 °C. The epon blocks are sectioned with glass
knives on ultramicrotome. Ultrathin sections are cut at 60 nm and are collected on grids and stained with uranyl acetate
and lead citrate. The ultrathin sections are investigated using a transmission electron microscope [18]. Below is the
figure of the spermatozoon structure in the testis tissue at TEM level (Figure 4).
Fig. 4
Transmission electron microscopic photography of normal spermatozoa.
6. Semen Preparation for Scanning Electrone Microscope (SEM)
Initially, freshly ejaculated semen is allowed to liquefy at 37°C. Then it is diluted with medium and spermatozoa are
separated by centrifugation. The pellets are fixed for 2 hours with 2,5 % glutaraldehyde and postfixed for 1 hour in 1 %
osmium tetroxide. Then spermatozoa are washed with buffer. Spermatoza are transferred on poly-L-lysine coated glass
slide and are dehydrated in ascending graded ethanols (70% 2x5’, 96% 2x5’, 100% 2x10’). Later they are dried in
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critical point dying apparatus using CO2 as transmission fluid and are covered by 10 nm of Au/Pd. Finally gold covered
samples are examined under a scanning electrone microscope [19]. Spermatozoa are evaluated.
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