Ultrastructural sperm tail defects associated with sperm immotility

Transcription

1 FERTILITY AND STERILITY Copyright 1984 The American Fertility Society PrintRd in U.SA. Ultrastructural sperm tail defects associated with sperm immotility Roger A. Williamson, M.D. * James K. Koehler, Ph.D.t W. Dianne Smith, M.S.:!: Morton A. Stenchever, M.D. University of Iowa Hospitals and Clinics, Iowa City, Iowa, and University of Washington School of Medicine, Seattle, Washington Four individuals with a complaint of infertility were evaluated after a semen analysis had demonstrated no motility. The semen analyses were otherwise normal, with the exception of a low count in one subject. All had normal percentages of living sperm. One of the individuals had chronic respiratory disease, and two others had a previous history of genitourinary infection and/or testicular injury. Three subjects possessed antisperm antibodies. Electron microscopy revealed a multiplicity of sperm tail structural defects seen in all specimens. Some of these data support the concept of acquired immotile sperm syndrome(s) with ultrastructural defects, as contrasted with the usual congenital forms of the immotile-cilia syndrome. Fertil Steril41 :103,1984 Sperm motility can be impaired by a number of factors, including genital infections l, 2 and antisperm antibodies. 3,4 There is also evidence of a direct effect of leukocytes in the ejaculate on sperm motility and function. 5 Complete lack of sperm motility in the absence of cell death is commonly a result of ultrastructural abnormalities ascribed to the heterogeneous spectrum of the immotile-cilia syndrome. 6 Also, individuals with immotile sperm without structural tail abnormal- Received July 5, 1983; accepted September 12, *Reprint requests: Roger A. Williamson, M.D., Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, Iowa City, Iowa tdepartment of Biological Structure, SM-20, University of Washington School of Medicine. :j:department of Obstetrics and Gynecology, RH-20, Reproductive Genetics Laboratory, University of Washington School of Medicine. Department of Obstetrics and Gynecology, RH-20, University of Washington School of Medicine. ities have been reported in which an associated biochemical defect has been identified. 7 Sperm tails and cilia elsewhere in the body have the same axonemal ultrastructure, defects of which are often genetically determined and hence generalized. 6 Several well-characterized structural variants producing ciliary immotility have been identified that produce chronic respiratory disease from childhood and subsequent infertility.b-io An exception to this has recently been seen in a fertile male patient with Kartagener's syndrome (bronchiectasis, sinusitis, and situs inversus) whose respiratory cilia showed the most characteristic lesion of the immotile cilia syndrome, missing dynein arms, but whose sperm tails were structurally normal. 11 We have studied three male patients with the converse of the above, namely, immotile sperm with structural abnormalities but no respiratory complaints. A fourth individual was also studied who presented with immotile sperm and chronic respiratory disease. The studies included electron Williamson et al. Immotile sperm with tail defects 103

2 microscopic examination of sperm tails as well as seminal fluid analyses, antisperm antibody assays, and determination of numbers~ of living sperm by trypan blue exclusion. SUBJECTS MATERIALS AND METHODS All patients were referred because of a history of infertility. At the time of the study, all were normally virilized and had normal testicular size and consistency, and none had evidence of pyospermia. Subject A, age 41, had fathered a child 4 years previously and was secondarily infertile. He had incurred a testicular injury at age 14 and had had one episode of left epididymitis 4 years previously. Two months before the testing reported herein, his semen analysis showed - 2% motility. Four female siblings are healthy. Subject B, age 27, had undergone a right orchiectomy at age 19 because of testicular trauma but was otherwise free of illness or known toxic exposures. He has fathered no children. Subject C had a 4-year history of attempting conception without success when referred at age 33. A semen analysis 3 years previously had shown 25% motility, and one performed 2 years ago revealed 5% motility. Levels of serum testosterone, follicle-stimulating hormone, and luteinizing hormone were all within normal limits. A male sibling has fathered tw~ children. A course of Bactrim (Roche Laboratories, Nutley, NJ) had not altered sperm motility. Subject D was 31 years old and presented with primary infertility. In contrast to Subjects A, B, and C, he had experienced chronic respiratory infections. In addition to bronchiectasis, he had chronic sinusitis and a conductive hearing loss thought due to middle ear scarring and had required nasal polypectomies. There was no reversal of internal organs. His only sibling, a sister, had only "sinusitis:" SEMEN ANALYSIS, LIVE DEAD DETERMINATION, AND ANTISPERM ANTIBODY ASSAYS Semen was collected by masturbation into a sterile plastic cup after at least 48 hours of absti~ nence. Twenty minutes were allowed for liquefaction, after which a standard semen analysis was performed. 12 Two separate specimens were evaluated for motility (%), count, and morphology, this latter parameter by modification of the Bryan-Leishman stain, which allows differentiation of immature sperm forms from white blood cells. 13 A portion of one specimen was stained with trypan blue, which reveals live (white) and dead (blue) cells. Antisperm antibody assays were determined by the methods of Kibrick et al. 14 and Isojima et au 5 ELECTRON MICROSCOPY Transmission electron microscopy (EM) speci mens were fixed in glutaraldehyde (1.25%) buffered with 0.15 M cacodylate at ph 7.4. They were postfixedinosmium tetroxide (1%), dehydrated in graded alcohol solutions, and embedded in Epon 812. Ultrathin sections were cut with a diamond knife on a PB II Ultratome (Ivan Sorvall, Norwalk, CT), stained with uranyl acetate and lead citrate, and examined under a Philips 201 microscope (Philips Electronics, Eindhoven, The Netherlands). RESULTS Table 1 summarizes the findings in these four subjects. With the exception of absence of motility in all four and a low count in patient A, the semen analyses were normal. Subjects A, B, and Chad evidence of antisperm antibodies, A and C with an abnormal titer of agglutinating antibodies and subject B with evidence of sperm immobilizing antibodies. All of these men had normal percentages of live sperm. Normal sperm tails possess an ordered structural arrangement that can be seen on cross section (Fig. 1a and b). EM demonstrated structural abnormalities in the majority of sperm tails of subject A and in all sperm examined from subjects B, C, and D. The predominant lesions from subjects A, B, and G included confused arrangement of the axoneme; extra coarse fiber; displaced central pair; missing, displaced, and/or supernumerary doublets; absent axoneme; and other flagellar abnormalities. Figures 2 to 5 show the spectrum of abnormalities seen in the sperm of one subject without respiratory symptoms. The EMs of the sperm tails of subject D were all abnormal and showed multiple flagellar defects (Figs. 6 to 11). Confused arrangements were seen, with the doublets rarely arranged in a configuration. Extra coarse fiber, displaced central pair, supernumerary displaced doublets, absent central pair, and absent axoneme were seen 104 Williamson et al. Immotile sperm with tail defects Fertility and Sterility

3 Table 1. Results of Seminal Fluid Analyses, Numbers of Living Sperm, Antisperm Antibody Assays, and Electron-Microscopic Studies Normal Agglutina- Patient Motility oval form Count Uvea tion(igbrick) % x 106 % Irnmobiliza- Electron mi- Probable cause(s) tion(isojima) croscopy of immotility A ' Abnormal titer 1:10 B 0 80"'-83 57"' Normal C Abnormal titer 1:30 D "' Normal "The laboratory range for 147 fertile men has been 21% to 86%. Normal Occasional normal Structural defects; forms but major- previous testicular ity abnormal; miss- injury and genitoing dynein arms; urinary infection; missing and super- antisperm antinumerary doublets; bodies absent axoneme Abnormal pa- Missing central pair; Structural defects; testient 30-40%; o.ther flagellar ticular injury; anticontrol 80% abnormalities sperm antibodies Normal Supernumerary doub- Structural defects; lets; other flagellar antisperm anti bodabnormalities ies;? Normal Multiple flagellar ab- Immotile-cilia synnormalities;. extra drome coarse fiber; displaced central pair; supernumerary displaced doublets; absent central pair; absent axoneme; confused arrangements singly and in combination. At high magnification the dynein. arms of the "A" microtubules were rarely seen or were very short. DISCUSSION The pervasive and key roles of cilia in human gestational development and an array of disease states have only recently been appreciated. In addition to their probable influence in directing normal organ placement during embryonic life,16 cilia may also playa pathogenic role in individuals who show polysplenia as a part of the immotile-cilia syndrome17 and even in individuals with the association of the polysplenia syndrome and extrahepatic biliary atresia.18 This present study expands the findings with regard to ciliary abnormalities with medical consequences. The immotile-cilia syndrome requires several criteria for diagnosis.19 This diagnosis does not apply to the three men lacking respiratory symptoms, patients A, B, and C. The observation of sperm tail abnormalities in patients without respiratory complaints had previously been demonstrated in individuals with immotile sperm that lacked one or both central microtubules.20 Respiratory cilia can show a variety of ultrastructuralabrrormalities felt to be induced by infection or injury, including disorganized axo" nemes and missing or supernumerary microtubules Although not yet supported by similar cases from the literature, it could be postulated that genitourinary infection and/or testicular injury may have been a causal factor(s) in producingthe defects of patients A and B. Thus, by analogy to the situation with respiratory tract cilia, early germ cells may respond to these insults, producing replicable changes as reflected in the EM findings. A variety of EM defects has been seen in differentiating spermatids of infertile men,24 although this latter study does not make mention of motility. Most of the defects seen involved the acrosome, and no cases with missing dynein arms were visualized. A recent study25 has shown that it is also possible for the sperm to retain motility with the absence of outer dynein arms. Therefore, some caution must be used in equating structural defects with motility loss. An explanation for the structural defects of the sperm tails of subject C is not readily apparent, because his history does not suggest an environmental insult. Also unexplained are the presence Williamson et al. Immotile sperm with tail defects 105

4 Representative flagellar cross sections of two of the patients described in this study. Figure 1a illustrates the nonnal axonemal configuration of human spermatozoa with the accompanying micrograph. (lb). Figures 2 to 5 show the spectrum of abnonnalities seen in patient C (Table 1), and Figures 6 to 11 illustrate the lesions seen in the flagellar of patient D (Table 1). Similar multiple defects were seen in the other two patients. of antisperm antibodies detected in the three subjects without respiratory complaints. Again, testicular trauma with breach of the blood-testis barrier may have played a role in subjects A and B, thus producing exposure of sperm antigens. Infection may also have been a contributing factor in subject A.1t is not likely that the antibodies are primarily related to the disease process as reflected in the EM findings, but rather are unrelated or represent a secondary phenomenon. Subject D does.fit within the category of immotile-cilia syndrome as outlined by the Swedish 106 Williamson et al. Immotile sperm with tail defects group,19 in that he has had chronic bronchitis and rhinitis since early childhood and has living but immotile spermatozoa of normal appearance. However, in contrast to most cases of the immotile-cilia syndrome with a genetically determined discrete lesion, his sperm tails demonstrated a multiplicity of findings, including confused arrangements and disorganized axonemes, felt to represent nonspecific defects. 26 In summary, three patients presented with a variety of sperm tail axonemal abnormalities in immotile sperm but without respiratory sympfertility and Sterility

5 toms. Two had previously had measurable, although markedly reduced, sperm motility. Two had a history of testicular injury and/or genitourinary infection. These three cases support the classification of acquired immotile sperm disorders with EM defects. The sperm tails of a fourth patient also showed defects, but he additionally had respiratory complaints. This latter individual fits the generally agreed-upon definition of immotile-cilia syndrome but differs from the usual cases in the multiplicity of ultrastructural abnormalities seen. All of these cases lend more credence to the heterogeneity of ciliary defects and suggest that EM be considered for selected patients presenting with sperm immotility. Acknowledgments. The authors are grateful to Ms. Jan Norbisrath for the scientific drawings, Ms. Agnpena Cerdena for her technical skills, Ms. Annmarie Daily for secretarial help, and Drs. Howard Stoll, Richard Berger, and Steve Plymate for referral of patients. REFERENCES 1. Del Porte GB, Derrick FC Jr, Bannister ER: Bacterial effect on sperm motility. Urology 5:638, Teague NS, Boyarsky S, Glenn JF: Interference of human spermatozoa motility by Escherichia coli. Fertil Steril 22:281, Ansbacher R, Manarang-Pangan S, Srivannaboon S: Sperm antibodies in infertile couples. Fertil Steril22:298, Wilson L: Sperm agglutinins in human semen and blood. Proc Soc Exp Bioi Med 85:652, Berger RE, Karp LE, Williamson RA, Koehler J, Moore DE, Holmes KK: The relationship ofpyospermia and seminal fluid bacteriology to sperm function as reflected in the sperm penetration assay. Fertil Steril 37:557, Afzelius BA: Genetical and ultrastructural aspects of the immotile-cilia syndrome. Am J Hum Genet 33:852, Gagnon C, Sherins RJ, Phillips DM, Bardin CW: Deficiency of protein-carboxyl methylase in immotile spermatozoa of infertile men. N Engl J Med 306:821, Afzelius BA, Eliasson R, Johnsen 0, Lindholmer C: Lack of dynein arms in immotile human spermatozoa. J Cell Bioi 66:225, Sturgess JM, Chao J, Wong J, Aspin N, Turner JAP: Cilia with defective radial spokes: a cause of human respiratory disease. N Engl J Med 300:53, Sturgess JM, Chao J, Turner JAP: Transposition of ciliary microtubules: another cause of impaired ciliary motility. N Engl J Med 303:318, Jonsson MS, McCormick JR, Gillies CG, Gondos B: Kartagener's syndrome with motile spermatozoa. N Engl J Med 307:1131, MacLeod J: The semen examination. Clin Obstet Gynecol 8:115, Couture M, Ulstein M, Leonard JM, Paulsen CA: Improved staining method for differentiating immature germ cells from white blood cells in human seminal fluid. Andrologia 8:61, Kibrick S, Belding DL, Merrill B: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, Isojima S, Li TS, Ashitaka Y: Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained infertility. Am J Obstet Gynecol 101:677, Afzelius BA: A human syndrome caused by immotile cilia. Science 193:317, Schidlow DV, Katz SM, Turtz MG, Donner RM, Capasso S: Polysplenia and Kartagener syndromes in a sibship: association with abnormal respiratory cilia. J Pediatr 100:401, Teichberg S, Markowitz J, Silverberg M, Aiges H, Schneider K, Kahn E, Daum F: Abnormal cilia in a child with the polysplenia syndrome and extrahepatic biliary atresia. J Pediatr 100:399, Mossberg B, Afzelius BA, Eliasson R, Camner P: On the pathogenesis of obstructive lung disease: a study on the immotile-cilia syndrome. Scand J Respir Dis 59:55, Afzelius BA, Eliasson R: Flagellar mutants in man: on the heterogeneity of the immotile-cilia syndrome. J Ultrastruct Res 69:43, Howell JT, Schochet SS, Goldman AS: Ultrastructural defects of respiratory tract cilia associated with chronic infections. Arch Pathol Lab Med 104:52, Cornillie F, Lauweryns J, Corbeel L, Boel M, Eeckels R, van de Walle J: Acquired ultrastructural abnormalities of bronchial cilia in recurrent airway infections and bronchiectases as compared with the findings in Kartagener syndrome. Pediatr Res 14:168, Ailsby RL, Ghadially FN: Atypical cilia in human bronchial mucosa. J Pathol 109:75, Holstein AF, Schirren C: Classification of abnormalities in human spermatids based on recent advances in ultrastructure research on spermatid differentiation. In The Spermatozoon, Edited by DW Fawcett, JM Bedford. Baltimore-Munich, Urban & Schwarzenberg, 1979, p Joummet P, Escalier D, Serres C, David G: Motility of human sperm without outer dynein arms. J Submicrosc Cytol 15:67, Afzelius BA: "Immotile-cilia" syndrome and ciliary abnormalities induced by infection and injury. Am Rev Respir Dis 124:107, 1981 Williamson et ai. Immotile sperm with tail defects 107