Outer dense fibres of human spermatozoa: partial characterization and possible physiological functions

The outer dense fibres are accessory fibres in the spermatozoon. They represent up to 30% of the protein portion in human spermatozoa and are involved in sperm progressive motility. If outer dense fibres are missing or developed poorly, spermatozoa are only locally motile. For isolation of the outer dense fibres, human spermatozoa were sonicated at 25 kHz and the flagella were separated by density gradient centrifugation in Percoll. Thereafter, membranes and fibrous sheath were dissolved under reducing conditions in the cationic detergent cetyltrimethylammonium bromide for 30, 60 and 90 min, respectively. The isolation steps were monitored by phase contrast microscopy and electron microscopy. After SDS-polyacrylamide gel electrophoresis and silver staining of isolated outer dense fibres, two protein bands at 55 and 67 kDa could be detected. By means of rhodamine B staining, no phosphorus could be detected in the outer dense fibre proteins.     

Ultrastructure of spermiogenesis and mature spermatozoon of Skrjabinoporus merops (Cyclophyllidea, Metadilepididae)

The ultrastructure of the mature spermatozoon and the spermiogenesis of a cestode belonging to the family Metadilepididae is described for the first time. The mature spermatozoon of Skrjabinoporus merops is characterized by twisted peripheral microtubules, the presence of a single crested body, periaxonemal sheath and electron-dense rods, and the absence of intracytoplasmic walls and inclusions (glycogen or proteinaceous granules); no peripheral microtubules where nucleus contacts the external plasma membrane. Four morphologically distinct regions of the mature spermatozoon are differentiated. The proximal part (Region I) contains a single crested body, periaxonemal sheath is absent in some (proximal) sections and is present in others situated closer to the nucleus. The central Region II is nucleated, and is followed by Region III that contains a periaxonemal sheath. The distal pole, Region IV, is characterized by disintegration of the axoneme. Spermiogenesis follows the type III pattern (Ba and Marchand 1995) although in S. merops a slight flagellar rotation is observed. The differentiation zone is characterized by the absence of striated roots and intercentriolar body; two centrioles are present, one of which gives rise to a free flagellum. The latter rotates and undergoes proximodistal fusion with the cytoplasmic protrusion of the differentiation zone. Spermiological characters of S. merops are similar to those of the families Taeniidae and Catenotaeniidae. The mature spermatozoon differs from those of the Dilepididae (where the metadilepidid species have previously been classified) by the lack of glycogen.     

Assessment of HBV persistent infection in an adult population in Taiwan

In order to study the prevalence of hepatitis B virus (HBV) in the adult population of Taiwan, we screened for the presence of HBV DNA in 205 blood samples from adult (20-59-year-old) volunteers. According to the serological markers of HBV, samples were divided into three groups: group I (173 subjects) was negative for both HBsAg and HBeAg; group II (14 subjects) was positive for both HBsAg and HBeAg; and group III consisted of 18 subjects who were HBsAg-positive but HBeAg-negative. Plasma HBV DNA was not detected in group I, but it was found in 85.7% and 11.8% of samples in group II and group III, respectively. A free-form HBV DNA was found in 14.3% of the leukocyte samples in group II. Furthermore, an integrated form of HBV DNA was detected in the leukocytes of two cases of group I who remained healthy based on clinical data. HBV DNA was also detected in the spermatozoa and liver cells of one of the cases.     

Highly sensitive quantitative telomerase assay of diagnostic testicular biopsy material predicts the presence of haploid spermatogenic cells in therapeutic testicular biopsy in men with Sertoli cell-only syndrome

The role of a telomerase assay in the recognition of Sertoli cell-only syndrome with testicular foci of haploid cells was evaluated. Men with Sertoli cell-only syndrome (n = 23) were given a new diagnostic testicular biopsy. Part of the biopsy was stained and the remainder was processed for the quantitative telomerase assay. After 3-13 months, a therapeutic testicular biopsy was performed. This material was minced and then examined using confocal laser scanning microscopy and fluorescent in-situ hybridization. Histology of diagnostic testicular biopsy material confirmed the diagnosis of Sertoli cell-only syndrome in all the participants. All seven men with a telomerase assay value in their diagnostic testicular biopsy of >42 total product generated (TPG) U/microg protein had haploid cells (i.e. spermatozoa and/or spermatids) in their therapeutic testicular biopsy. Among participants with telomerase assay values <42 TPG U/microg protein, only one man had haploid cells in his therapeutic testicular biopsy. Thus, telomerase assay values >42 TPG U/microg protein in the diagnostic biopsy identified 87.5% of the Sertoli cell-only syndrome men with haploid cells in their therapeutic testicular biopsy. Significantly higher values of the telomerase assay were found in men with testicular foci of haploid cells than in men without these foci. The use of a quantitative telomerase assay biopsy appears to be important for identifying those men with Sertoli cell-only syndrome who have foci of haploid cells and can be candidates for assisted reproduction techniques.     

Morphogenesis of the fibrous sheath in the marsupial spermatozoon

The spermatozoon fibrous sheath contains longitudinal columns and circumferential ribs. It surrounds the axoneme of the principal piece of the mammalian sperm tail, and may be important in sperm stability and motility. Here we describe its assembly during spermiogenesis in a marsupial, the brush-tail possum, and compare its structural organization with that of eutherian mammals, birds and reptiles. Transmission electron microscopy showed that possum fibrous sheath assembly is a multistep process extending in a distal-to-proximal direction along the axoneme from steps 4 to 14 of spermiogenesis. For the most part, assembly of the longitudinal columns occurs before that of the circumferential ribs. Immunohistochemical and immunogold labelling showed that fibrous sheath proteins are first present in the spermatid cytoplasm; at least some of the proteins of the sheath precursors differ from those in the mature fibrous sheath. That immunoreactivity develops after initiation of chromatin condensation suggests that fibrous sheath proteins, or their mRNAs, are stored within the spermatid cytoplasmic lobule prior to their assembly along the axoneme. These findings are similar to those in laboratory rats, and thus suggests that the mode of fibrous sheath assembly evolved in a common ancestor over 125 million years ago, prior to the divergence of marsupial and eutherian lineages.     

Quantified ultrastructural study of spermatozoa in unexplained failure of in vitro fertilization

Failure of in vitro fertilization or very low cleavage rates may occur even though oocyte and semen parameters seem satisfactory. Quantified ultrastructural study of spermatozoa was performed in such cases of failure (n =6) or low cleavage rate (<20%; n =4). Through 1 to 11 retrievals, the number of inseminated oocytes ranged from 14 to 145. The results were compared to those of six fertile men. Quantification was achieved by cataloguing cell defects of the spermatozoon heads and mid-/principal pieces of the flagella. Using the data from each specimen, the percentages of total cellular abnormalities in the head/mid-/principal pieces were established. At the level of the head overall percentages for six groups of defects were determined. The overall percentage of combined head abnormalities, defined as the presence of at least three of these six defects on the same spermatozoon head, was established. Statistical differences among control and patient groups were analyzed by nonparametric Mann—Whitney Utest. The percentages of anomalies of the midpiece and of the principal piece were not significantly different between patients and controls. Motility assessed by spermogram was considered functionally uncompromised. In eight patients the percentage of cell alterations of the head (93–100 vs 77.3 ± 6.4%) and the percentage of combined anomalies of the head (78.1–100 vs 60.8 ± 8.5%) were significantly different between patients and controls. In two cases, the percentages established for all head parameters considered were not globally different from those observed in controls. Thus in 8 cases of 10, electron microscopy with quantified analysis supplied valuable evidence about the poor quality of these sperm samples judged as normal under light microscopy and may provide an explanation for their impaired fertilizability. In the two other cases the fact that the sperm appeared to be ultrastructurally normal does not rule out functional sperm pathology. Alternatively, defects in the oocyte may also account for unexplained failures of in vitro fertilization.     

Proper ubiquitination effect on the fertilisation outcome post‐ICSI

Ubiquitin is an 8.5‐kDa protein that tags outlived proteins for degradation by the proteasome. It also marks defective spermatozoa during epididymal passage and has been proposed as a biomarker of sperm quality. This study evaluates the relationship between sperm ubiquitination, protamine deficiency, semen parameters and fertilisation rate in infertile individuals undergoing the intracytoplasmic sperm insemination (ICSI) procedure. Semen samples from 73 ICSI candidates were collected and analysed according to World Health Organization criteria. A portion of each sample was evaluated for sperm ubiquitination using the sperm ubiquitin tag immunoassay (SUTI) with flow cytometry, and protamine deficiency by chromomycin A3 (CMA3) staining. In addition, the relationship between the fertilisation rate and sperm ubiquitination was calculated in ICSI candidates. The intensity of ubiquitination showed a significant negative correlation with sperm concentration (r = ?0.255, P = 0.032) and a positive correlation with fertilisation rate (r = 0.384, P = 0.013) post‐ICSI. No correlation was observed between protamine deficiency and the percentage of ubiquitination or ubiquitination intensity. The results of this study suggest that sperm ubiquitination prior to capacitation may be considered as a marker of defective spermatozoon. Spermatozoa that undergo proper ubiquitination may have a higher chance for fertilisation, because they are made redundant by the ubiquitin–proteasome pathway in the epididymis compared to hypo‐ubiquitinated spermatozoa.     

Pre-decondensed sperm head injections into female pronuclei result in chromosomal mingling, zygotic cleavage, and adequate embryonic and fetal development up to delivery of healthy offspring: a novel method of assisted syngamy

Investigation of the developmental potential post-injection of a pre-decondensed or non-pre-decondensed sperm head into the female pronucleus of a pre-activated oocyte. Rat pre-activated oocytes were treated with intrapronuclear pre-decondensed sperm head injections (IPSHI) (n = 133) or intrapronuclear non-pre-decondensed sperm head injections (INPSHI) (n = 138). All injected oocytes were transferred to pseudopregnant female recipients. Rat IPSHI techniques resulted in the delivery of five healthy offspring. Rat INPSHI techniques did not result in any pregnancies. Rat IPSHI techniques can result in delivery of healthy offspring. Successful performance of human IPSHI techniques might serve as a novel method to manage cases of intracytoplasmic sperm injection failure due to lack of development of male pronucleus or due to failure in pronuclei fusion.     

Ultrastructure of the spermatozoon of <Emphasis Type="Italic">Calliobothrium verticillatum</Emphasis> (Cestoda,Tetraphyllidea, Oncobothriidae)

The ultrastructure of the spermatozoon of Calliobothrium verticillatum (Cestoda, Tetraphyllidea, Oncobothriidae) parasite of the smoothhound shark, Mustelus mustelus L. (Pisces, Carcharhiniformes), was studied by transmission electron microscopy. This spermatozoon presents five regions characterized by several ultrastructural elements: an apical cone, a crested body, two axonemes of 9 + “1” pattern, electron-dense granules, a nucleus and cortical microtubules. In the present study, three of these features were the subject of a detailed attention. The first is the presence of two axonemes, which confirms that the Tetraphyllidea, Oncobothriidae possess two axonemes whereas the Tetraphyllidea, Phyllobothriidae possess only one axoneme. The second is the presence of one crested body, a criterion homogeneous in the Tetraphyllidea but heterogeneous among the different orders of Cestoda. The third is the number and the disposition of cortical microtubules. These three criteria seem to be interesting for phylogeny.