Lack of a head in human spermatozoa from sterile patients: a syndrome associated with impaired fertilization*

Transcription

1 FERTILITY AND STERILITY Copyright 1987 The American Fertility Society Printed in U.8A. Lack of a head in human spermatozoa from sterile patients: a syndrome associated with impaired fertilization* Hector E. Chemes, M.D.t Carlos Carizza, M.D. Fernando Scarinci, M.D. Santiago Brugo, M.D. Nicolas Neuspiller, M.D. Luis Schwarsztein, M.D. Laboratory of Testicular Physiology and Pathology, Endocrine Unit, Buenos Aires Children's Hospital, Buenos Aires, Argentina Three patients with primary sterility in whom the majority of spermatozoa lacked a normally implanted head are presented. A small cephalic knob was evident in most of them by routine colo-rimetric techniques, and the Feulgen reaction failed to show any deoxyribose nucleic acid. The morphologic features of the tails was normal. Few loose sperm heads were observed in the ejaculates. Even though motility was decreased, there were numerous acephalic sperms with different degrees offorward motility. Electron microscopy showed a well-organized structure of the centrioles and connecting piece, which were located in the neck region within a small cytoplasmic mass, but no chromatin was detected in any case. Studies on immature spermatids present in semen evidenced an independent anomalous development of heads and tails and suggested that they became separated at the end of spermatid maturation. This anomaly, of probable genetic origin, is interpreted to be due either to an alteration in the mechanism of migration and positioning of the tail on the caudal pole of the nucleus or to an interference with the formation of the implantatio-n fossa of the head, which normally accommodates the connecting piece. Fertil Steri147:310, 1987 Numerous recent investigations have clarified the ultrastructure of mammalian spermatozoa (for review see Fawcett l ). Application of electron microscopy to human material has yielded accurate information on the morphologic features of normal human sperm and on the alterations that can be observed in infertile patients. 2-8 Notably a Received June 16, 1986; revised and accepted October 24, *Supported by grant 0934 from tlle Consejo Nacional de Investigaciones Cientificas y Tecnicas. treprint requests: Hector E. Chemes, M.D., Division de Endocrinologia, Hospital de Niiios, Gallo 1330, 1425 Buenos Aires, Argentina. 310 Chemes et al. Acephalic spermatozoa in men syndrome characterized by immotile spermatozoa and chronic airway infections with or without situs inversus has been characterized to be due to the lack of dynein arms, an essential part ofaxonemal ultrastructure considered to be responsible for ciliary movements. 9 During a systematic ultrastructural examination of semen in men with asthenozoospermia or teratozoospermia, we have recently found three patients with a rare sperm defect. The purpose of this study was to provide a detailed morphologic description of spermatozoa in these patients and to offer some considerations regarding the morphogenesis of this anomaly. The existence of a specific syndrome of probable genetic origin is also proposed.

2 MATERIALS AND METHODS The material for this report comes from three patients consulting for primary sterility. Subject 1 is a 33-year-old man with 3 years of primary sterility. A left varicocele was diagnosed and surgically treated in August At physical examination, both testes, epididymis, and ductus deferens were normal. A previous semen analysis (August 1981) revealed 42 x 10 6 spermatozoa/ml and 55% motile forms with 15% fast forward progression. The eosin test showed 5% of nonviable forms. Morphologic examination yielded 30% spermatozoa with normal configuration, 60% with head anomalies, and 10% with alterations of the middle piece or flagellum. Most spermatozoa showed minute heads. Some of these forms had good forward motility. Subject 2 is a 33-year-old man with 4 years of primary sterility. No personal history of previous andrologic illness was recorded. At physical examination, both testes, epididymis, and vas deferens were normal. Two previous semen analyses (September and November 1982) revealed 19 x 10 6 and 65 x 10 6 spermatozoa/ml with 35% and 20% motile forms. The eosin test revealed < 5% of nonviable forms. Seventy-five percent and 87% of sperm showed abnormalities of the head and were described as "microcephalus." Subject 3 is a 36-year-old man with 5 years of primary sterility. There was no previous record of andrologic illness. Physical examination was normal. A previous semen sample (August 1984) showed 65 x 10 6 spermatozoa/ml with 0% fast forward progression, 10% of nonviable sperm, and 82% of "microcephalic" elements in the semen smear. LABORATORY METHODS For the current study a new semen sample was obtained from each patient. These samples were examined within 30 minutes of ejaculation. A small drop was placed on a glass slide and examined under phase-contrast microscopy. Supravital staining was also performed with 1% yellow eosin to assess the viability of spermatozoa. Another portion was smeared, allowed to dry for a few minutes, fixed with 96% ethyl alcohol, and subsequently stained with Harris hematoxylin and Papanicolaou's stain, or the nuclear reaction of Feulgen was used to stain deoxyribose nucleic acid (DNA). A third portion was washed free of seminal plasma with saline and centrifuged, and the pellet fixed by immersion in 2.5% glutaraldehyde buffered with 0.2-M Na cacodylate (ph, 7.4), postfixed in 1.3% osmium tetraoxide, and embedded in Epon Araldite (Ernest F. Fullam Inc., Schnectady, NY). Sections 1 /Lm thick were obtained in a Reichert OM U2 ultramicrotome (C. Reichert AG, Vienna, Austria) and stained with 1 % toluidine blue in 1% sodium borate solution. Thin sections displaying pale golden to silver interference colors were obtained with glass knives, double stained with uranyl acetate and lead citrate, and examined on a Siemmens Elmiskop IA electron microscope (Siemmens, West Berlin, Germany). RESULTS LIGHT MICROSCOPIC EXAMINATION The semen samples from the three patients showed a high percentage of immotile forms, with few elements displaying fast forward progression. Patient 1 had only 16% normal spermatozoa. The rest showed different kinds of anomalies, with great predominance of abnormal heads totaling 66%: micro- or macrocephalic, multinucleated or amorphous elements, and, the most predominant anomalous form, very small heads, no bigger than 1 /Lm in diameter. They appeared as opaque small dots lacking the brightness characteristic of bigger spermatozoal heads. When examined after hematoxylin-papanicolaou's stain, they had a pale pink color sharply contrasting with the deep violet color characteristic of spermatozoal nuclei. Also there was a negative Feulgen reaction, indicating the absence of detectable DNA material. The sperm tails appeared normal. Spermatozoa from patients 2 and 3 had more than 90% of these abnormal forms in their ejaculates, some with a cylindrical thickening in the proximal end, which corresponded to the middle piece. Other sperms ended in a rounded small mass or were devoid of any cephalic structure but showed a very small dense dot at the cephalic end. When present, the cephalic thickenings stained negative with the Harris hematoxylin or with the Feulgen reaction. Normal forms with well-formed heads were extremely scarce, as were isolated heads. Elements with two flagella and no heads were also identified. Immature "round cells" corresponding to different steps of spermatid differentiation were observed (near 20% of the total elements). The more immature round spermatids Chemes et ai. Acephalic spermatozoa in men 311

3 had normally formed nuclei, but later spermatids evidenced abnormalities consisting of alterations in nuclear shape, condensation, and number. ELECTRON MICROSCOPIC EXAMINATION Figure 1 depicts the structure of the head and middle piece of a normal human spermatozoon. The sperm nucleus appears as a highly condensed structure in which chromatin granules are densely packed and cannot be discerned individually. Some small nuclear vacuoles can be seen. In its anterior aspect, the nucleus is covered by the acrosome. The cell membrane is closely applied to the acrosomal membrane, and more caudally to the nuclear envelope, forming the postacrosomal dense lamina. Caudally to this structure, the nuclear envelope shows numerous pores and detaches from the chromatin mass to form the redundant nuclear envelope. The caudal pole of the nucleus shows a shallow concave area, the implantation fossa, in which the nuclear envelope is covered by the basal plate. This is in contact with the capitulum and segmented columns of the neck piece, which enclose the nine triplets of the proximal centriole, seen here in cross section. The narrow interspace between the basal plate and capitulum is bridged by a system of fine filamentous densities. Caudally to this, the first part of the axoneme and outer dense fibers in longitudinal section are seen surrounded by the mitochondrial sheath. Longitudinal sections of "anucleated spermatozoa" (Figs. 2 and 3) show that the cranial poles lack completely the nucleus and acrosome, which are replaced by small globular structures. In Figure 2 this globular mass shows the two centrioles at right angles and a few dense masses of granular material closely resembling the remains ofthe chromatoid body. These organelles lie in a cytoplasmic droplet with microgranular and vesicular structures and are covered by the cell membrane. From the distal centriole, the initial part of the flagellum can be seen running through the spindle-shaped body and continuing in the tail region. The sperm head, which would normally be implanted cranially to the proximal centriole, and the mitochondrial sheath of the middle piece are completely lacking. The presence of the spindleshaped body and the lack of a mitochondrial sheath probably indicate that this is an immature spermatid (no more mature than step 6 of Holstein and Roosen Runge 8 ). The anucleated form in Figure 3 shows a drop of cytoplasm and lacks detailed structures in its cephalic end. Two distinct tail capitula can be discerned with the presence of the centriolar complexes, the beginning of the axonemes and the mitochondrial sheaths of the middle pieces. Also in this case there is no nuclear material. Figure 4A shows an example of anucleated spermatozoa with a well-organized flagellum and middle piece ending in its anterior aspect with the two centrioles positioned at right angles. When the proximal centriole is transver- Figure 1 Normal spermatozoon from a fertile control subject. The condensed nucleus (N) is covered by the acrosome (A). The redundant nuclear envelope (R) is located in the caudal pole. The first part of the axoneme is surrounded by the mitochondrial sheath (M) (x 21,750), The inset depicts an enlargement of the neck region: basal plate curved arrow), capitulum and striated columns (arrowheads), and the nine cross-sectioned triplets of proximal centriole (asterisk) (magnification, x 47,700). 312 Chemes et al. Acephalic spermatozoa in men

4 Figure 2 The globular cephalic mass to the right contains the two centrioles (arrows) and the remains of the chromatid body (CB ). The nucleus is completely lacking. The first part of the axoneme runs through the spindle-shaped body (SB). There is not a mitochondrial sheath (magnification, x 29,150). sally sectioned, the nine triplets can clearly be seen (Fig. 4B). In these last two examples, the cranial parts of the proximal centrioles are covered by the plasma membrane, and the lack of a head is evident. Many immature round and elongated spermatids were also identified in the ejaculate. The flagellum was not observed in a normal relation with the nucleus in any of the specimens studied. Frequently, the condensing spermatids (Fig. 5) showed the flagellum-middle piece complex completely divorced from the nucleus. The few loose spermatozoal heads that could be identified in these sections showed a small cytoplasmic drop devoid of a connecting piece in the neck region (Fig. 6). No implantation fossa or basal plate was identified in these nuclei. DISCUSSION Abnormalities of the sperm head are a common finding in sperm smears. Virtually all kinds of morphologic variations have been described: tapering, pyriform, amorphous, and duplicate heads, as well as large and small ones. Among the category of small heads a variety exists that has been identified with the light microscope as ~~pin heads." Zaneveld and Polakoski 10 describe them as spermatozoa that have virtually no head at all and merely possess a knob at the end of the midpiece. However, they do not fully characterize this anomaly. Bergman et al. ll reported that they constitute a small percentage of the total number of sperm and are presumably without biologic importance. They added that pin-headed spermatozoa penetrated the cervical mucus normally. Figure 3 A double-tailed anucleated spermatozoon. Two centriolar c,omplexes (arrows) and axonemes surrounded by mitochondrial sheaths (M ) are clearly seen (magnification, x 25,300). Chernes et al. Acephalic spermatozoa in men 313

5 Figure 4 (a), The two centrioles (arrowheads), middle piece, and principle piece of the tail ( x 21,950). (b), The neck region of an anucleated spermatozoon. The nine triplets of the proximal centr\ole (T) and the striated columns (curved arrow) are directly covered by the plasma membrane (slight arrows). Note the absence of the head (compare with inset in Fig. 1) (magnification, x 55,100). In 1981 Perotti et a1. 7 reported an infertile patient in whom most spermatozoa exhibited complete separation of the head and tail and called them decapitated spermatozoa. Perotti and Gioria12 found numerous early and elongated spermatids in which the heads and tails developed independently and became separated by the development of a furrow between them.12 We previously found very low numbers of individual spermatozoa bearing this abnormality in semen samples from astheno- or teratozoospermic patients.13 However, in a small percentage of patients pinheaded spermatozoa were the predominant anomaly and were as frequent as 90% to 95% of all spermatozoa. The three patients reported here belong to this category and constitute a defined syndrome associated with sterility. We propose that anucleated spermatozoa are of testicular origin and that the head is lost during the last steps of spermatid maturation as a consequence of some morphogenetic anomaly. There are two indirect pieces of evidence supporting this interpretation. Patient 1 showed, in addition to anucleated spermatozoa, a high percentage of sperms with various alterations of the head. Moreover, patients 2 and 3 had abundant immature spermatids in the ejaculate, and whereas the more immature round forms had normal configurations, the more mature spermatids evidenced various kinds of head abnormalities (e.g., binucleation, abnormal condensation, amorphous heads). These two observations suggest some anomaly in the morphogenesis of the head-middle piece that results in the head disattachment late in spermiogenesis. Both motile and immotile forms could be observed in anucleated spermatozoa, which indicates that the lack of a head does not preclude sperm motility, an observation that coincides with reports showing that segments of sperm tails experimentally dissected away from the head possess the ability to initiate and coordinate waves of movement. 14 Electron microscope studies of the connecting piece have shown that the basal plate is connected with the capitulum and segmented columns by a system of fine filaments bridging the gap between both structures. Fawcete has proposed that the synthesis of these filaments could be defective in genetically infertile bulls with acephalic spermatozoa. In agreement with Perotti and Gioria,12 we have found that the connecting piece evolves independent from the nucleus in immature spermatids. This observation indicates that there is a defect in the mechanisms ofmigration or positioning of the flagellum in relation to the nucleus. This rules out the possibility that anucleated forms could originate by a late separation of normally developed heads and tails as a consequence of an abnormal fragility of the headconnecting piece attachment, as reported for acephalic spermatozoa in bulls. 1, 7 Fawcett and Phillips15 have demonstrated that the formation of the neck structures begins very early, before the centrioles establish contact with the nucleus. The formation of the implantation fossa and basal plate seems to occur as a conse- 314 Chemes et al. Acephalic spermatozoa in men

6 Figure 5 An immature spermatid. There is no connection between the condensing nucleus (N) and the middle piece (arrow). A, acrosome (magnification, x 7950). Figure 6 A free head in semen. Note absence of flagellar anlage and lack of a basal plate (arrow) (magnification, x 15,900). quence of the proximity of the capitulum to the nucleus. The capitulum and striated columns of the neck are well formed in anucleated spermatozoa, but the few loose sperm heads observed by us and Perotti et al. 7 do not show a basal plateimplantation fossa. Therefore, we propose that anucleated sperms result either from a defect in the migration of the tail anlage to the caudal pole of the nucleus or as a consequence of some nuclear defect that interferes with the formation of the implantation fossa and basal plate. In either case, the nucleus and the flagellum would develop separately within the spermatid cytoplasm and would become separated at the end of spermiogenesis. It is not known why many tails but only a few heads are found in the ejaculates. Possibly, the heads are phagocytized by Sertoli cells, epididymal cells, or luminal macrophages, but the tails are not. The observation that most spermatozoa from our three patients were of this anomalous kind and were also present in numerous semen samples obtained over a period of time indicates that it was a chronic condition and responsible for the patients' primary sterility. Acknowledgments. We appreciate the skillful collaboration of Mr. Oscar Rodriguez in the preparation of the photographs and the editing of the manuscript by Ms. Lidia Evans and Ms. Lucia Chemes. REFERENCES 1. Fawcett DW: The mammalian spermatozoon. Dev BioI 44:394, Pedersen H, Rebbe H, Hammen R: Human sperm fine structure in a case of severe asthenospermia-necrospermia. Fertil Steril 22:156, Ross A, Christie S, Kerr MG: An electron microscope study of a tail abnormality in spermatozoa from a subfertile man. J Reprod Fertil 24:99, Ross A, Christie S, Edmond P: Ultrastructural tail defects in the spermatozoa from two men attending a subfertility clinic. J Reprod Fertil 32:243, 1973 Chemes et al. Acephalic spermatozoa in men 315

7 5. Chemes HE, Fawcett DW, Dym M: Unusual features of the nuclear envelope in human spermatogenic cells. Anat Rec 192:493, Holstein AF, Schrirren C: Classification of abnormalities in human spermatids based on recent advances in ultrastructure research on spermatid differentiation. In The Spermatozoon, Maturation, Motility, Surface Properties and Comparative Aspects, Edited by DW Fawcett, M Bedford. Baltimore-Munich, Urban and Schwarzenberg, 1979, pp 86, Perotti ME, Giarola A, Gioria M: Ultrastructural study of the decapitated sperm defect in an infertile man. J Reprod Fertil 63:543, Holstein AF, Roosen-Runge EC: Atlas of human spermatogenesis. Berlin, Grosse Verlag, Eliasson R, Mossberg B, Camner P, Azfelius BA: The immotile cilia syndrome. N Engl J Med 297:1, Zaneveld LJD, Polakoski KL: Collection and physical examination of the ejaculate. In Techniques of Human Andrology, Edited by ESE Hafez. Amsterdam, Elsevier North Holland Biomedical Press, 1977, p Bergman A, Yedwab G, David MP, Homonnai ZT, Paz F: A method of improvement of sperm quality of an ejaculated human specimen using bovine cervical mucus as a biological filter. J Androl 3:199, Perotti ME, Gioria M: Fine structure and morphogenesis of "headless" human spermatozoa associated with infertility. Cell BioI Int Rep 5:113, Chemes HE: Unpublished data 14. Lindeman CB, Rikmoenspoel R: Sperm flagella: autonomous oscilations of the contractile system. Science 175:337, Fawcett DW, Phillips DM: The fine structure and development of the neck region of the mammalian spermatozoon. Anat Rec 165:153, Chemes et al. Acephalic spermatozoa in men