Determination of the ultrastructural pathology of human sperm by atomic force microscopy

Transcription

1 FERTILITY AND STERILITY VOL. 75, NO. 5, MAY 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Determination of the ultrastructural pathology of human sperm by atomic force microscopy Narahari Joshi, Ph.D., Honorio Medina, B.Sc., Ibis Crúz, B.Sc., and Jesus Osuna, M.D. Department of Physiology, Faculty of Medicine, and Laboratory of Andrology, Los Andes University Hospital, Mérida, Venezuela Objective: To investigate ultrastructural changes, particularly at the surface, that occur in pathological spermatozoa by using atomic force microscopy (AFM) and to examine the morphological alterations responsible for infertile sperm. Design: Normal fertile and pathological spermatozoa were examined by using a conventional AFM in a noncontact mode. Setting: Andrology clinic at Los Andes University Hospital. Patient(s): Sperm of patients with oligoasthenoteratozoospermia (OAT) and asthenozoospermia were obtained from the Los Andes University Hospital, Mérida, Venezuela. Intervention(s): Main Outcome Measure(s): Morphological details, topological information, and three-dimensional images of the head, neck, and flagellum are presented for both normal and pathological sperm. Result(s): The obtained images clearly show dramatic alterations in the morphology of the head, neck, and flagellum of pathological sperm. Even the ultrastructure at the top of the flagellum and the region of the acrosome cap are clearly distinguishable. Conclusion(s): This study has significant importance not only for identifying spermatozoa alterations but also for understanding morphological defects and their effects on infertility. If properly exploited, this technique could be an important research tool. (Fertil Steril 2001;75: by American Society for Reproductive Medicine.) Key Words: Ultrastructure, human fertile sperm, infertile sperm, atomic force microscopy, oligoastenoteratozoospermic (OAT) Received June 5, 2000; revised and accepted January 23, Reprint requests: Narahari Joshi, Ph.D., Departamento de Fisiologia, Laboratorio de Fisiologia de Conducta, University of Los Andes, Mérida, Venezuela (FAX: ; joshi@ciens.ula.ve) /01/$20.00 PII S (01) Usually, human sperm are analyzed on fixed and stained specimens by optical microscopy, which provides valuable information about the morphology and corresponding pathological features of spermatozoa. If alterations are gross and on the order of a micron or fraction thereof, then this method is an adequate and useful tool (1). However, if the morphological defects are of the order of a few nm, then the method certainly fails and optical microscopy needs to be replaced by electron microscopy (EM; transmission or scanning) (2). Zamboni (2) applied this technique to study the pathology of sperm and evaluate its quality. Some of the pathological changes at the nm scale in the head and flagellum have already been reported (2, 3). This approach, however, does not provide topographical information, which can be of great interest in examining three-dimensional alterations. Very recently, the atomic force microscopic approach (4) has been employed to examine the sperm in its natural environment, which permits viewing of the morphological details as three-dimensional images with precise topographical details. Earlier EM investigation revealed the presence of nano-grooves or channels on the top of the flagellum (3, 5) of healthy spermatozoa, and atomic force microscopy (AFM) obtained precise topographical information (4). Such studies, along with studies of the head morphology, have been carried out to examine defective sperm. These defects cannot be directly correlated functionally with loss of fertilizing capacity. Nevertheless, they may provide, in certain cases, clues to the abnormal 961

2 behavior. For example, swimming capacity originates from the flagellum and a defect in it will certainly have effects on motility. The cause(s) of male sterility or infertility is very complex; among many other factors, it may be due to alterations of the hypothalamus-hypophyseal-testicular axis and morphological alterations in sperm. Gross defects such as bending and coiling and the relative length of flagellum can be observed with the help of a conventional optical microscope, and ultrastructural defects are studied with an EM, which is suitable for examining the morphological fine details of the interior parts of the sperm. The ultrastructure pathology of sperm has been extensively investigated in the last three decades (2, 3, 5). It is known that in this case, the procedures used for examination such as fixation, dehydration, and sectioning do not noticeably alter the morphological details and thus have no substantial effect on the information obtained about morphological details. Scanning and transmission EM in surface replica form does obtain some information in the Z direction but does not allow three-dimensional imaging. This situation can be improved by using AFM, which allows investigators to carry out measurements in natural environments and in some cases even in living conditions (6). The applicability of AFM has been tested for human sperm, and excellent results have been reported (4). The purpose of the present investigation, therefore, is to extend the use of the AFM technique for examining the pathology of infertile spermatozoa. It is known that AFM is a powerful technique for investigating the ultrastructure of biological materials such as tissues, living cells, and organisms (7). This approach is particularly useful for investigating the structure near the upper surface of the membrane, and, therefore, this method is suitable for studying the morphology of the groove located at the upper part of the flagellum, the acrosome situated at the tip of the head, and the protein alterations at the surface. In this report, we will not focus on the ultrastructural details of the axoneme, the connecting pieces and midpieces. Such details have been reported in the literature elsewhere (4, 5). MATERIALS AND METHODS Fresh samples of semen from both infertile patients and fertile controls were obtained from the Andrology Clinic at the Los Andes University Hospital. The healthy spermatozoa were selected by using the conventional swim-up procedure. For this purpose, conventional GIBCO 199 culture (as obtained, without adding any kind of antibiotics) was used. Five hundred microliters of semen were mixed with 1 ml of culture media and incubated at 37 C for 30 min. Then 500 L of the upper part of the mixture, where only healthy spermatozoa had been able to swim, was extracted by pipette and mixed in an equal volume of cultured medium. After the mixture was homogenized, a drop of it was spread on the FIGURE 1 Head of a normal sperm. Dimensions of the head can be appreciated with the help of the topography given below. The acrosome cap is clearly visible. glass plate and examined with an optical microscope. The normal spermatozoa were followed by optical microscopy until they became very sluggish, up to the point that they became practically immobilized. Then AFM studies were carried out. To examine morphological alterations, we selected four patients with oligoasthenoteratozoospermia (OAT) and asthenozoospermia. Our earlier clinical work showed that sperm concentration from OAT patients varies between 3 and 8 million per ml and that the abnormal head concentration lies between 72% and 83%; this means that the detection of abnormal spermatozoa is not a difficult task and is generally carried out with an optical microscope with 1,000 magnification. This approach revealed that the majority of spermatozoa were sluggish, indicating that defects exist in the flagellum region also. For this investigation, an AMF unit attached to a conventional Olympus microscope (BX 60) was used. The system is known as SIS-ultraobjective and is obtained from the Surface Imaging System (Germany). We used silicon cantilevers that were 450 m in length and 50 m in width. The spring constant of the cantilever was 1 nm 1. The unit was installed on a vibration-free table, and the complete system guarantees reproducibility. The system can be used either in 962 Joshi et al. Ultrastructure of sperm studied AFM Vol. 75, No. 5, May 2001

3 FIGURE 2 Head of a pathologic sperm. The thickness of the head is considerably increased. See topographic table given below. FIGURE 3 Flagellum of a normal sperm. Middle pieces and groove are well marked. matter is concentrated at the center of the head instead of being gradually spread out. The most interesting feature is that in a normal sperm there is a narrow region about 1.6 m in length where the acrosome cap is attached. The topogra- FIGURE 4 Flagellum of an abnormal sperm. The difference between Fig. 3 and this figure is striking. contact or noncontact mode, and for this investigation we tried both. More informative results were obtained when the experiment was performed in noncontact mode, and the results are shown in Figures 1 5. RESULTS AND DISCUSSION Figures 1 and 2 show the images obtained for the heads of normal and pathological spermatozoa. The first change is in the length of the head. The normal length is about 7,000 nm, while for a pathological sperm the length is about 6,000 nm. Similarly, the height is also altered. For a normal sperm it is 846 nm, and for an abnormal sperm it is 1,418 nm. Optical microscopy does indicate the increase in height, but in the present investigation we have quantified it. In this case, the FERTILITY & STERILITY 963

4 FIGURE 5 Aerial view of normal (top) and pathologic (bottom) sperm. FIGURE 6 Globozoospermic sperm showing head, neck, and flagellum. phy clearly shows how the presence of the cap alters the shape of the head. A defective acrosome or partial or total absence of it is one of the causes for infertility. The presence and distribution of the acrosome and DNA material is generally determined with the laser scanning confocal microscopic technique using proper dyes. FITC-Psium sativum lectin for acrosome and TOTO-3 iodine for DNA are used. Membrane lipid is identified with Nile red. Earlier investigation (8) using this approach clearly revealed the presence of an acrosome cap at the front of the head. However, the information about the height of the cap cannot be obtained with this method. The AFM technique is suitable to examine this type of defect as compared with conventional EM. The major advantage of this technique permits the evaluation of the position and form of the acrosome. With this view, we have focused our attention on the front region of the head. AFM provides detailed structural information. In the healthy sperm, the maximum height is about 200 nm. It is clear that in a pathological sperm, there is a complete absence of the acrosome. It seems that this cap does not have uniform thickness; at the tapered end of the head it is much thicker than at the center of the head. Moreover, the variation in the thickness is not uniform but decreases rather exponentially. The detailed variation can be appreciated with the help of topography. The absence of the acrosome reduces the head size and makes it look round, as seen in Figure 2. When the sample is examined under a normal microscope, the lack of the acrosome could be interpreted as a round head defect, frequently characterized as globozoospermia, which leads to infertility (1, 9). This has been known for several years, but images obtained in the present investigation directly confirm total or partial absence of the acrosome and its effect on the morphology of the head. Defects in the flagellum, which cause immobility in the sperm, have been extensively investigated by EM methods (2, 3, 5). Examination of numerous abnormal cross-sectioned tails revealed the presence of alteration in singlets, absence of peripheral doublets, changes in dense fibers, and selective and focal absence of outer dynein arms. These factors are responsible for defective/absence of motility. The most im- 964 Joshi et al. Ultrastructure of sperm studied AFM Vol. 75, No. 5, May 2001

5 portant aspect is that changes do not only occur in the central part of the flagellum but also at the outer part of it, which can be detected very easily and opportunely by AFM. Figures 3 and 4 show the surface morphology of healthy and abnormal flagella, respectively. The obtained results for a healthy sperm can be compared with the data reported by Holstein and Roosen Rough (5), and they are in agreement. Figure 3 shows the well-organized structure of the middle and neck. A well-marked groove runs from the end of the neck to the end of the flagellum. Figure 4 shows the absence of organized structure over the entire length and uneven height of the flagellum. Patches of conglomerated proteins are randomly distributed. The difference is dramatic and can be appreciated with the help of an aerial view of the flagellum as shown in Figure 5. In the case of a normal sperm, a well-marked groove is seen throughout the entire length, whereas in abnormal cases the groove is absent. In addition to this, the lack of uniformity is also a well-known feature. It is worth mentioning that the movement of the same sperm was examined before AFM by optical microscopy and it found that the sperm was moving the tail, but was not advancing enough. These defects observed with AFM are not visible by optical microscopy. Figure 6 shows a sperm of an OAT patient along with its longitudinal topography. The head has a normal morphology, but the tail is defective. This type of sperm is characterized by a slow and disoriented motion and has no chance of overcoming obstacles like cervical mucus, uterotubal junction, and oviduct walls. In short, the present technique provides information about ultrastructural details and three-dimensional imaging of spermatozoa in their natural environment. Even though this method is not intended to examine the quality of sperm motility, it gives some indication of motility as a consequence of defects in a specific region such as the flagellum. Acknowledgements: The authors are thankful to CONICIT (No. G ) and CDCH for financing the research project (No. M B) under which the present work was carried out. References 1. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. Cambridge: Cambridge University Press, Zamboni L. The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril 1987;48: Schaller M, Panhans-Gross A, Benzold G, Korting HS, Wolff H. Ultrastructural defects in acquired immotile sperm flagella. Fertil Steril 2000;73: Joshi N, Medina H, Colasante C, Osuna A. Ultrastructural investigation of human spermatozoon by using atomic force microscope. Arch Androl 2000;44: Holstein HG, Roosen Rough EC. Atlas of human spermatogenesis. Berlin: Groose Verlag, Joshi NV, Medina H, Urdaneta H, Barrueta L. In vivo nano imaging and ultrastructure of Entamoeba histolitica by using atomic force microscope. Exp Parasit 1999;93: Nagao E, Dvorak JA. An integrated approach to the study of living cells by atomic force microscopy. J Microscopy 1997;191: Wu C. New spermicides stop cells gently. Science News 1998(153)23: Zamboni L. Sperm ultrastructural pathology and infertility. In: Pathology of infertility. B. Gondons and D. Riddicks, eds. New York: Thieme Medical Publishers, 1987:281. FERTILITY & STERILITY 965