Acephalic spermatozoa and abnormal development of the head–neck attachment: a human syndrome of genetic origin

H.E. Chemes1, E.T. Puigdomenech, C. Carizza, S. Brugo Olmedo, F. Zanchetti and R. Hermes

Laboratory of Testicular Physiology and Pathology, Endocrinology Division, Buenos Aires Children's Hospital, Buenos Aires, Argentina


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A series of 10 young sterile men with acephalic spermatozoa or abnormal head–mid-piece attachments is presented. Nine of these patients had 75–100% spermatozoa with minute cephalic ends and 0–25% abnormal head–middle piece attachments. Loose heads ranged between 0–35 for each 100 spermatozoa and normal forms were rare. Two patients were brothers. On ultrastructural examination, the head was generally absent and the middle piece was covered by the plasma membrane. When present, heads implanted at abnormal angles on the middle piece. A testicular biopsy showed abnormal spermiogenesis. The implantation fossa was absent and the flagellar anlage developed independently from the nucleus, resulting in abnormal head–middle piece connections. In one patient azoospermia was induced with testosterone to attempt to increase the normal sperm clone during the rebound phenomenon, but all newly formed spermatozoa were acephalic. In another patient with high numbers of defective head–mid-piece connections, microinjections of spermatozoa resulted in four fertilized oocytes, but syngamy and cleavage did not take place, suggesting an abnormal function of the centrioles. The findings indicate that acephalic spermatozoa arise in the testis as the result of an abnormal neck development during spermiogenesis. The familial incidence and the typical phenotype strongly suggest a genetic origin of the syndrome.

Key words: acephalic spermatozoa/genetic origin/infertility/pin heads/sperm pathology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
`Microcephalic' spermatozoa have been occasionally reported as the main seminal abnormality in sterile men. Earlier studies (LeLannou, 1979Go; Perotti et al., 1981Go; Baccetti et al., 1984Go) reported individual patients with headless flagella in semen and identified them as `decapitated spermatozoa'. More recently (Chemes et al., 1987bGo; Baccetti et al., 1989Go), others have reported five more cases, including familial incidence, and introduced the term `acephalic spermatozoa'. We have gathered a large series of 10 patients, including two brothers among them, with this sperm anomaly and report herein a complete ultrastructural study of spermatozoa and a testicular biopsy, as well as a detailed clinical and laboratory profile of all patients.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This report is based on the study of 10 patients consulting after various years of primary sterility. There were no records of pregnancies in any of their partners. They were young adult men in the 3rd or 4th decades of life. Two of the patients were brothers (not twins). There were no previous records of andrological illnesses and the testes, epididymis and vas deferens were normal in most of them. One of the patients had a left varicocoele, which was surgically treated, and each of the two brothers had an epididymal cyst. Another patient had hypotrophic testes (15 and 10 ml in volume). Sperm characteristics were studied according to standard methods (WHO, 1987). Numerous previous semen samples showed oligo- or normozoospermia (range 6–65, median 42x106 spermatozoa/ml), and normal motility or variable asthenozoospermia. In all cases, `microcephalic' spermatozoa and/or abnormal head–middle piece connections were recorded as the predominant anomalies in their ejaculates.

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., 1987bGo). 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 2–4 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Light microscopy of semen samples
Recently ejaculated semen samples were studied under phase contrast optics. Sperm motility ranged from normal to severe asthenozoospermia in the 10 patients (3–60% total motility values). Most spermatozoa showed small globular cephalic thickenings. These structures did not correspond to sperm heads, as shown by the negative results with haematoxylin and Feulgen stains. Characteristically, some acephalic spermatozoa displayed very active movements with extremely fast directional progression, while others moved slowly or were immotile. There were three main types of spermatozoa with abnormal head–neck configurations. The first type showed normal flagella ending cranially in a very minute cephalic end, no more than 1–2 µm in diameter. Middle pieces were usually missing. The second type had a normally structured middle piece sometimes surrounded by a cytoplasmic droplet 3–5 µm in diameter. These two were the predominant types, amounting to 75–100% of the spermatozoa in nine patients. The third type consisted of spermatozoa with heads abnormally implanted in the middle piece. The heads attached either to the tip or to the sides of the middle piece without a linear alignment with the sperm axis. The angles between heads and tails were up to 90–180°. In nine of the 10 patients, normally formed spermatozoa amounted to no more than 1% (see Clinical studies).

Ultrastructural observations
Normal development of the head–flagellar 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 1Go).



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Figure 1. Configuration of the neckpiece in normal spermatozoa. The shallow concavity of the nucleus corresponds to the implantation fossa (curved arrow), where the nuclear envelope (NE) is devoid of pores and shows narrowing of the perinuclear cistern. The basal plate (arrowheads) appears as a dense layer applied to the outer leaflet of the NE at the level of the implantation fossa. The proximal centriole (asterisk), the segmented columns and the beginning of the flagellum can be seen to the left. Scale bar 0.1 µm.

 
Ultrastructure of acephalic spermatozoa and organization of the neck piece
When studied with the electron microscope, sperm pellets from the patients showed spermatozoa with very small cranial ends devoid of any nuclear material (Figure 2AGo). These features were dramatically evident when these spermatozoa were studied by scanning electron microscopy (Figure 2B, GoC).



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Figure 2. (A) Panoramic view of the sperm pellet. Various spermatozoa show cephalic thickenings with no heads and variable degree of mid-piece organization. (B) An acephalic spermatozoon with full development of the mid-piece (MP). (C) An acephalic spermatozoon with a minute cephalic thickening (asterisk) and a loose head (H). Scale bars 1 µm.

 
The neckpiece of acephalic spermatozoa showed a variable degree of development (Figure 3Go). In the simplest variety (type I), one or two centrioles were present at the tip of the flagellum. These centrioles were embedded in a very small droplet of residual cytoplasm that contained a few scattered mitochondria not assembled in a middle piece. The second kind of acephalic spermatozoon (type II) showed a further step of development with full organization of the connecting and mid-pieces in a larger cytoplasmic droplet. In the third and less frequent variety (type III), a sperm head was present, which was abnormally aligned with the middle piece. This misalignment ranged from complete lack of connection between both structures, to a lateral positioning of the nucleus at a 90–180° angle to the mid-piece. In some of these spermatozoa, a nuclear envelope derived vesicle was present at the attachment site of the middle piece in the nucleus. This vesicle was identical to that present at the tip of some acephalic spermatozoa, suggesting that they derive from the fracture of these abnormal head–mid-piece junctions (Figure 3Go).



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Figure 3. Different kinds of acephalic spermatozoa or faulty head–neck attachments in semen. (A) A type I acephalic spermatozoon depicting a cytoplasmic droplet with few mitochondria not assembled in a mid-piece. There is no connecting piece. (B) A type II acephalic spermatozoon with a fully developed connecting piece and mid-piece embedded in a cytoplasmic droplet. (C) A similar spermatozoon to that depicted in Figure 2BGo. Notice the presence of a nuclear envelope derived vesicular structure in the most cranial region (V). The basal plate (curved arrow) and the nuclear pores (arrowheads) are clearly visible. (D) Abnormal head–tail alignment with defective head–mid-piece connection. (E and F) The head and mid-piece form a 90° and 75° angle respectively. There is a nuclear envelope derived vesicular structure at the head–mid-piece junction (V). Note the similarity between these vesicles and the one seen at the cephalic end of the sperm depicted in Figure 2CGo. Scale bars 0.5 µm.

 
The three varieties (minute cephalic end with no mitochondrial sheath, variable degree of development of the middle piece in a globular residual cytoplasm, and abnormal head–middle piece connection), presented with variable prevalence in different patients. In patients 1, 2 and 3, types I and II were seen with similar frequency, while in patients 4, 5, 6 and 7 acephalic spermatozoa with fully developed mid-pieces (type II) were clearly predominant. Loose heads were exceedingly rare in these seven patients (0–3%). When present, they were devoid of any tail structure (Figure 2CGo) and consistently lacked centrioles, basal plates or implantation fossae at their caudal pole. The last three patients (8, 9 and 10) had an admixture of mismatched head–mid-pieces (type III, Figure 3D, GoE and F), acephalic spermatozoa and variable numbers of loose heads.

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 4AGo). In succeeding steps of spermiogenesis, nuclear elongation–condensation and acrosomal development proceeded normally, but the flagellar anlage developed independently from the nucleus and failed to establish contact with it (Figure 4BGo–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



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Figure 4. Spermatid maturation in testicular biopsy from patient 8. The earliest stage (A) shows the two centrioles and the flagellum (arrowhead) not attached to the nucleus. The acrosomal vesicle (asterisks) is developing at the anterior pole of the nucleus. (B), (C), (D) and (E) show progressive nuclear elongation and chromatin condensation. The flagellar anlage is either absent from the caudal pole or shows an abnormal attachment to the nucleus (C, arrowhead). Compare this last configuration with the normal spermatozoon in Figure 1Go. (F) An acephalic spermatozoon (asterisk) and a residual body (RB) still embedded in the germinal epithelium just before spermiation. Scale bars 1 µm.

 
occasions, abnormal insertions of the flagellum in the nucleus could be documented (Figure 4CGo), which later probably resulted in misaligned nuclear middle piece connections. These findings described in the testicular biopsy were similar to those observed in maturing spermatids exfoliated in semen in the other subjects.

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 IGo shows the seminal variables in different semen analysis. Sperm concentration, that ranged ~40x106 spermatozoa/ml, declined to very low values around 4–6 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.


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Table I. Semen variables in patient 8 before, during and after TP treatment
 
Patient 9 presented a very interesting example of pathological head–mid-piece attachment. While in the other individuals most spermatozoa had suffered head–mid-piece separations resulting in acephalic spermatozoa, patient 9 had an admixture of normal forms, spermatozoa with abnormally aligned head–mid-pieces, loose heads and acephalic spermatozoa. The percentages of these different forms were determined in the fresh sample before any manipulations and in semen smears after washing and centrifugation. It was observed that normal spermatozoa decreased after centrifugation, while acephalic forms, loose heads and misaligned head–mid-pieces increased (Table IIGo). In this patient, four good quality metaphase II oocytes obtained from his wife after ovarian stimulation were microinjected with spermatozoa that showed tail movements. All of them fertilized and two pronuclei were clearly recognizable in each at 17 h post-microinjection. However, when these zygotes were observed 24 h later (41 h post-injection) cleavage had not occurred in any of them, the pronuclei remained widely separated and syngamy had not taken place. The zygotes were left in the culture media, and at 72 h post-injection degeneration had occurred with widespread fragmentation (Figure 5Go).


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Table II. Percentages of different spermatozoa in patient 9, before and after centrifugation
 


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Figure 5. Two fragmented zygotes, photographed at 72 h post-sperm injection. There is no cleavage. On the right, the two pronuclei can be seen in different cytoplasmic fragments (arrows, original magnification x400).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Teratozoospermia has been frequently reported as the main anomaly responsible for deficient fertility in males. However, this diagnosis is a poorly understood seminal condition defined solely on the basis of the alterations observed in sperm shape, and does not provide an understanding of the physiopathological mechanisms responsible for low fertility in these patients. In the last decade, we have undertaken detailed ultrastructural studies of different sperm abnormalities in >1000 infertile individuals to clarify the nature of these anomalies and the morphogenic mechanisms involved. Two main types of teratozoospermia could be identified. In the first and most frequent variety, morphological examination discloses non-specific and variable anomalies in different sperm components. This heterogeneous pattern shows no common defect and results from random alterations in sperm organelles. The second variety shows a very homogeneous microscopic pattern with a systematic sperm defect present in most spermatozoa. To this variety belong `round head acrosomeless spermatozoa' (Holstein et al., 1973Go; Nistal et al., 1978Go), the `miniacrosome sperm defect' (Baccetti et al., 1991Go), the `dysplasia of the fibrous sheath or stump tail defect' (Chemes et al., 1987aGo, 1998Go), and the dynein deficient axonemes of the `immotile cilia syndrome' (Azfelius et al., 1975). In the present communication, we report on the clinical and ultrastructural characteristics of acephalic spermatozoa, another systematic sperm defect that can be broadly included among the alterations in the head–neck attachment. This defect has been referred to as `pin heads' (Zaneveld and Polakovski, 1977Go, a misnomer that implies that the heads are very small, when they are in fact absent. Acephalic spermatozoa can be observed in very small numbers in seminal samples from numerous men. However, in the patients presented here they constitute the predominant kind of spermatozoa in the ejaculate, and define a syndrome with distinctive clinico-pathological manifestations. Characteristically, these are young adult infertile males, without any detectable andrological condition, who present with `microcephalic' spermatozoa and sometimes refer similar problems in their families.

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., 1987bGo). A somewhat similar condition has also been described in bulls (Bloom and Birch Andersen, 1970Go) 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, 1969Go; Fawcett, 1975Go). 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; Go; Perotti and Gioria, 1981;GoChemes et al., 1987bGo, aGond 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., 1986Go; Baccetti et al., 1989Go) 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 head–neck 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 head–middle piece connections, indicating that these two morphological variants are related and express a different degree of abnormality of the head–neck 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 head–middle 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., 1978Go), 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, 1992Go). 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, 1978Go). 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 head–neck 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., 1997Go). 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 head–neck 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 depletion–repopulation has been induced, and the familial incidence here documented and previously reported by others in men and bulls (Bloom and Birch Andersen, 1970Go; Baccetti et al., 1989Go) indicate that this characteristic phenotype is a centrosome-related primary sperm defect of genetic origin.


    Acknowledgments
 
H.E.C. is indebted to Raquel Berezovsky MD for her continuing support and encouragement. The technical work of Yolanda Castaño de Mansilla and Oscar Rodriguez is fully acknowledged. This work has been supported by Grants PMT-PICT 0090 from CONICET and PICT 0450 from ANPCyT, Argentina.


    Notes
 
1 To whom correspondence should be addressed at: Endocrinology Division, Hospital de Niños R. Gutierrez, Gallo 1330, 1425 Buenos Aires, Argentina Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 8, 1999; accepted on March 22, 1999.