Laboratory of Testicular Physiology and Pathology, Endocrinology Division, Buenos Aires Children's Hospital, Buenos Aires, Argentina
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: acephalic spermatozoa/genetic origin/infertility/pin heads/sperm pathology
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transmission and scanning electron microscopy studies
Semen samples were obtained by masturbation and studied when liquefaction was complete, ~30 min after ejaculation, according to methods previously reported (Chemes et al., 1987b). Briefly, the samples were diluted (1:3) with phosphate buffer (0.1 M, pH 7.4) and spermatozoa were separated by centrifugation. The pellets were fixed for 24 h with 3% glutaraldehyde in the same buffer, postfixed for 2 h in 1.3% osmium tetroxide and embedded in Epon-Araldite. The blocks were cut in an RMC MT-7000 automatic ultramicrotome (RMC Inc., Tucson, AZ USA) with glass and diamond knives and the sections were double stained with uranyl acetate and lead citrate and studied on a Zeiss EM 109 electron microscope (Zeiss Oberkochen, Germany). For studies with the scanning electron microscope, the same fixatives were used. The spermatozoa were fixed in suspension with buffer washes between and after both fixatives. Sperm cells were subsequently sedimented on poly-L-lysine coated slide fragments, to assure sperm adherence to the glass, dehydrated in a graded series of ethanol followed by absolute acetone, dried in a Balzers CDP 030 critical point drying apparatus (Balzers Union Ltd, Balzers, Lichtenstein), using CO2 as transition fluid, coated with gold-palladium in a Balzers Union SCD 040, and observed in a Philips 515 scanning electron microscope (Philip Nederland BV, Eindhoven, The Netherlands).
In all cases, a small aliquot of fresh semen was studied under phase contrast microscopy, and motility, viability and light microscopic morphology were studied according to standard methods (WHO, 1987). Semen smears were stained according to Papanicolaou or with the Feulgen stain to detect DNA.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ultrastructural observations
Normal development of the headflagellar connection (neck piece or connecting piece)
The flagellar axoneme of the mature spermatozoon derives from the centrioles of the maturing spermatid. During step 1 of spermiogenesis, the distal centriole gives rise to the axoneme, and progressively approaches the nucleus to become attached to it. From an initial lateral position, the flagellar anlage migrates to the caudal pole of the nucleus in early stages of spermatid differentiation. At the site of implantation, the nucleus shows a shallow concavity, the implantation fossa, where the proximal centriole establishes close contact with the nucleus. At this concavity, the outer leaflet of the nuclear envelope develops a narrow electron dense layer on its cytoplasmic side, the basal plate, where the perinuclear cistern is narrowed and shows periodic densities bridging together the two leaflets of the nuclear envelope (Figure 1).
|
|
|
Morphogenesis of acephalic spermatozoa during spermiogenesis
In patient 8, a testicular biopsy was performed which was used to study the events leading to the formation of acephalic spermatozoa. In this biopsy early step 1 spermatids had a round euchromatic nucleus, and a normal Golgi apparatus in the process of forming the acrosome. Both centrioles could be seen away from the nucleus. The axoneme started to grow from the distal centriole (Figure 4A). In succeeding steps of spermiogenesis, nuclear elongationcondensation and acrosomal development proceeded normally, but the flagellar anlage developed independently from the nucleus and failed to establish contact with it (Figure 4B
F). The caudal pole of the nucleus of elongating spermatids did not show an implantation fossa, and rather appeared as a convex protruding area without the differentiation associated with the implantation site of the middle piece. In general, nucleus and flagellum developed independently, never establishing contact with each other and became separated at the end of spermiogenesis. In rare
|
Clinical studies
In patient 8 (the same individual in whom the testicular biopsy was performed), it was possible to follow the evolution of this sperm abnormality in several semen samples obtained over an 18 month period, before, during and after testosterone suppression of spermatogenesis. This patient originally showed a very small percentage of normally formed spermatozoa (~1%). Testosterone propionate treatment was instituted to achieve spermatogenic regression in an attempt to promote the expansion of the clone of normal spermatozoa during the rebound phenomenon that follows testosterone-induced oligo-azoospermia. Table I shows the seminal variables in different semen analysis. Sperm concentration, that ranged ~40x106 spermatozoa/ml, declined to very low values around 46 months of TP administration. However, azoospermia was never achieved. Sperm concentration returned to pretreatment values 6 months after testosterone discontinuation. There were no significant changes in sperm morphology along the course of spermatogenic regression or after recovery, and the percentage of normal spermatozoa remained very low and unchanged throughout the observation period.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Various authors have previously reported small numbers of patients with this anomaly (see Introduction), the largest series being our previous report of three men (Chemes et al., 1987b). A somewhat similar condition has also been described in bulls (Bloom and Birch Andersen, 1970
) and suggestions have been made that the defect is the consequence of an abnormal fragility of the neck piece leading to the sperm tail separation in the seminal pathway (Fawcett and Phillips, 1969
; Fawcett, 1975
). However, even though the final result is similar, acephalic spermatozoa in men are of testicular origin and result from a failure of the tail anlage to establish a proper attachment to the caudal pole of the spermatid nucleus during spermiogenesis. This concept is supported by studies (Le Lannou, 1979;
; Perotti and Gioria, 1981;
Chemes et al., 1987b
, a
nd the present results), that demonstrate an abnormal spermiogenic development. In our biopsy material we have found increased phagocytic activity of Sertoli cells. This, and the rare appearance of loose heads in semen, indicates that the latter remain attached to the residual body at spermiation and undergo phagocytosis by Sertoli cells. Earlier studies (Holstein et al., 1986
; Baccetti et al., 1989
) have reported a patient and two brothers in whom the cleavage takes part between the proximal and distal centriole or along the mid-piece, but we have found the separation to occur at the headneck interface in all our patients. These non-coincident reports suggest that there are various mechanisms responsible for the formation of acephalic spermatozoa, yet the present material and other reports from the literature indicate that the most frequent mechanism is the separation at the neck as a consequence of an independent development of heads and tails.
Three of our patients present an admixture of acephalic spermatozoa and abnormal headmiddle piece connections, indicating that these two morphological variants are related and express a different degree of abnormality of the headneck junction with acephalic forms representing the most extreme situation due to separation of heads and tails. In cases of abnormal insertion of the head in the mid-piece, the normal configuration of the neck region is replaced by a nuclear envelope-derived vesicle joining heads and tails. This explains why some headless flagella have a basal plate and a fraction of the nuclear envelope in their cranial pole, as if the caudal-most part of the head had broken free from the main body of the nucleus. In these cases, the separation of heads and tails can occur at spermiation or in the seminal pathway due to an increased instability of the headmiddle piece junction. This last possibility is supported by our observation that in one of these patients the percentage of loose heads and acephalic spermatozoa increased after centrifugation, probably because of the separation of heads from tails in these fragile spermatozoa.
Very early during human spermiogenesis the spermatid nucleus differentiates a cranial pole where the Golgi complex attaches to form the acrosome (Chemes et al., 1978), and later on the centrioles and flagellum approach the nucleus and migrate towards its caudal pole. Acephalic spermatozoa derive from the failure of this caudal migration, while some acrosomeless spermatozoa result from the lack of proper attachment of the Golgi complex to the anterior aspect of the spermatid nucleus (Zamboni, 1992
). The unusual case described by Aughey and Orr (1978), with round acrosomeless heads and acephalic spermatozoa in the same patient indicate that these two abnormal mechanisms have combined (Aughey and Orr, 1978
). These observations suggest that there are different pathologies derived from an abnormal differentiation of the bipolar nature of the spermatid nucleus.
The alterations of the headneck attachment described here are due to an abnormal migration of the tail anlage, which includes the centrioles as its main component. It is particularly significant that in patient 9 an intracytoplasmic sperm injection resulted in four fertilized oocytes in which syngamy and cleavage never occurred. Similar alterations have been attributed to a failure in the microtubular systems in the zygote, which are derived from the sperm centriole (Hewitson et al., 1997). We propose that the lack of syngamy and cleavage in our patient is probably a consequence of an abnormal centriole in spermatozoa carrying alterations in the headneck region. This represents a pathology with characteristic abnormalities in sperm structure and the corresponding abnormal behaviour during fertilization.
Acephalic spermatozoa present very similar characteristics within a given semen sample and between different patients. The most obvious example of this uniformity is its presence in brothers, as is the case in two men in our population. This is also a condition very stable in time as seen in our clinical studies, and does not respond to pharmacological interventions leading to severe spermatogenic regression. The similar characteristics of the semen after testosterone withdrawal and the failure of the small clone of normal spermatozoa to expand during spermatogenic recovery, imply that this condition is also unmodifiable.
The uniform pathological phenotype, its origin as a consequence of a systematic alteration during spermiogenesis, the fact that seminal characteristics remain constant along clinical evolution even when a pharmacological germ cell depletionrepopulation has been induced, and the familial incidence here documented and previously reported by others in men and bulls (Bloom and Birch Andersen, 1970; Baccetti et al., 1989
) indicate that this characteristic phenotype is a centrosome-related primary sperm defect of genetic origin.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aughey, E. and Orr, P.S. (1978) An unusual abnormality of human spermatozoa. J. Reprod. Fertil., 53, 341342.[Abstract]
Baccetti, B., Selmi, M.G. and Soldani, P. (1984) Morphogenesis of `decapitated spermatozoa' in a man. J. Reprod. Fertil., 70, 395397.[Abstract]
Baccetti, B., Burrini, A.G., Collodel, G. et al. (1989) Morphogenesis of the decapitated and deccaudated sperm defect in two brothers. Gamete Res., 23, 181188.[ISI][Medline]
Baccetti, B., Burrini, A.G., Collodel, G. et al. (1991) A `miniacrosome' sperm defect causing infertility in two brothers. J. Androl., 12, 104111.
Bloom, E. and Birch Andersen, A. (1970) Ultrastructure of the `decapitated sperm defect' in Guernsey bulls. J. Reprod. Fertil., 23, 6772.[Medline]
Chemes, H.E., Fawcett, D.W., Dym, M. (1978) Unusual features of the nuclear envelope in human spermatogenic cells. Anat. Rec., 192, 495512.
Chemes, H.E., Brugo Olmedo, S., Zanchetti, F. et al. (1987a) Dysplasia of the fibrous sheath: an ultrastructural defect of human spermatozoa associated with sperm immotility and primary sterility. Fertil. Steril., 48, 664669.[ISI][Medline]
Chemes, H.E., Carizza, C., Scarinci, F., et al. (1987b) Lack of a head in human spermatozoa from sterile patients: a syndrome associated with impaired fertilization. Fertil. Steril., 47, 310316.[ISI][Medline]
Chemes, H.E., Brugo Olmedo, S., Carrere, C. et al. (1998) Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men. Hum. Reprod., 13, 25212526.[Abstract]
Fawcett, D.W. (1975) The mammalian spermatozoon. Dev. Biol., 44, 394436.[ISI][Medline]
Fawcett, D.W. and Phillips, D.M. (1969) The fine structure and development of the neck piece of the mammalian spermatozoon. Anat. Rec., 165, 153184.[ISI][Medline]
Hewitson, L., Simerly, C. and Schatten G. (1997) Inheritance defects of the sperm centrosome in humans and its possible role in male infertility. Int. J. Androl., 20(Suppl. 3), 3543.[ISI][Medline]
Holstein, A.F., Schirren, C. and Schirren, C.G. (1973) Human spermatids and spermatozoa lacking acrosomes. J. Reprod. Fertil., 35, 489491.[Medline]
Holstein, A.F., Schill, W.B. and Breucker, H. (1986) Dissociated centriole development as a cause of spermatid malformation in a man. J. Reprod. Fertil., 78, 719725.[Abstract]
LeLannou, D. (1979) Teratospermie consistant en l'absence de tete spermatique par defaut de connexion tete-col, chez l'homme. J. Gynecol. Obstet. Biol. Reprod. Paris, 8, 4345.[Medline]
Nistal, M., Herruzo, A. and Sanchez Corral, F. (1978) Teratozoospermia absoluta de presentación familiar: espermatozoides microcéfalos irregulares sin acrosoma. Andrologia, 10, 234240.[ISI][Medline]
Perotti, M.E. and Gioria, M. (1981) Fine structure and morphogenesis of `headless' human spermatozoa associated with infertility. Cell. Biol. Int. Rep., 5, 113.[ISI][Medline]
Perotti, M.E., Giarola, A., Gioria, M. (1981) Ultrastructural study of the decapitated sperm defect in an infertile man. J. Reprod. Fertil., 63, 543549.[Abstract]
World Health Organization (1987) Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction. Cambridge University Press, Cambridge.
Zamboni, L (1992) Sperm structure and its relevance to infertility. Arch. Pathol. Lab. Med., 116, 325344.[ISI][Medline]
Zaneveld, L.J.D. and Polakoski, K.L. (1977) Collection and physical examination of the ejaculate. In Hafez, E.S.E. (ed.), Techniques of Human Andrology. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 147172.
Submitted on January 8, 1999; accepted on March 22, 1999.