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PubMed2000

In vitro culture of brushtail possum (Trichosurus vulpecula) epididymal epithelium and induction of epididymal sperm maturation in co-culture.

A medium modified from eutherian systems was used to culture epididymal epithelial cells of the brushtail possum (Trichosurus vulpecula) for more than 2 months. Epididymal tubule fragments from the caput, corpus and cauda epididymides were used to generate cell monolayers. All three epididymal cell culture systems supported maturational changes in marsupial spermatozoa and enabled immature possum spermatozoa to differentiate from a T-shape to a streamlined shape, accompanied by the development of progressive motility after co-culture with 7-day-old cultured epididymal cell monolayers. This epididymal cell and sperm co-culture system for marsupial species may facilitate the identification of specific epithelial factors that affect sperm maturation, particularly in a species in which morphological maturation is readily visible.

Animals↗
PubMed1986

Colocalization of sulfogalactosylacylalkylglycerol (SGG) and its binding protein during spermatogenesis and sperm maturation. Topology of SGG defines a new testicular germ cell membrane domain.

By use of double-labelling indirect immunofluorescence, we have shown that the major mammalian testicular glycolipid sulfogalactosylacylalkylglycerol (SGG) and a membrane protein, previously shown to bind specifically to SGG in vitro, are colocalized on the surface of rat testicular germ cells during spermatogenesis. SGG is restricted to convoluted membrane domains within these cells. Thus, the binding affinity in vitro is reflected in the cell surface topology. The topological relationship between these two antigens was also studied during epididymal sperm maturation. Whereas these antigens were colocalized in caput spermatozoa (on the middle and principal piece of the tail and on the concave surface of the head), the distribution of the binding protein was altered for cauda sperm in that the convex surface of the sperm head was now strongly labelled. These studies illustrate the dynamic nature of protein-glycolipid interactions during germ cell differentiation.

Animals↗
PubMed1995

Intra-acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: a study using a monoclonal antibody MC101.

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis.

Acrosome↗
PubMed1996

Epididymal sperm maturation proteins.

Mammalian spermatozoa are not motile when they leave the testis. They have to undergo a complex maturation process to be able to fertilize in vivo. The maturation changes of mammalian sperm membrane have been extensively studied using lectins and antibodies. Some of these antigens have been purified and cloned. The interaction of secreted proteins with sperm membranes and acquisition of sperm motility as essential steps for spermatozoa to be fertile are well documented. The role of these epididymal maturation proteins in infertility and the possibility of using these antigens for immunocontraception are discussed in this review.

Animals↗
PubMed1996

Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest.

This is the first reported delivery following intracytoplasmic sperm injection (ICSI) of mature live testicular sperm cells collected in a case of hypergonadotrophic azoospermia with maturation arrest. The 30 year old couple presented with primary infertility of 11 years duration, the man being submitted in childhood to five orchidopexy operations for the treatment of cryptorchism. He had elevated serum follicle stimulating hormone (FSH; 18.8 IU/I), an atrophic left testis and a normal sized right testis, the biopsy of which diagnosed maturation arrest and focal scarring. The couple refused donor insemination for religious reasons and the only option was an attempt at testicular sperm collection. Multiple testicular and epididymal fine needle aspirations were performed, using an aspiration handle loaded with 20 ml syringe and 21-23 gauge butterfly needles. The mature spermatozoa recovered were used to inseminate the oocytes by ICSI. Prior to this procedure, the patient's wife underwent ovulation induction using a long protocol of mid-luteal gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG). At oocyte retrieval, ten oocytes were recovered. Eight live sperm cells were recovered from the aspirates of the right testis. Following ICSI into four metaphase II and two metaphase I oocytes, one mature oocyte was fertilized, cleaved and was transferred to the uterus 48 h after oocyte retrieval. The patient conceived and delivered a 3300 g boy at term. In conclusion, our results demonstrate that this novel approach should be considered in cases with hypergonadotrophic azoospermia due to testicular failure. Further experience is needed to establish the exact criteria for its use.

Adult↗
PubMed1986

Sperm maturation associated with subfertility following hyperbaric exposure of mice.

Mice exposed to 50 ATA heliox pressure during one spermatogenic cycle subsequently exhibit subfertility which can recover over an 8-wk period. This study was designed to investigate the changes in spermatid and testicular histology associated with these phenomena. Electron microscopy of testicular tissue from hyperbaric-exposed animals revealed a significant effect on spermatogenesis including disorganization and patchy necrosis of the epithelium. Two weeks after exposure to pressure, abnormalities were more difficult to find and tissues taken from animals 8 wk after exposure were indistinguishable from normal. These data directly confirm our earlier hypothesis that chronic hyperbaric exposure has a deleterious effect on sperm maturation, which is subsequently manifest as a failure in fertilizing capacity.

Animals↗
PubMed1996

Cell-to-cell transfer of glycosylphosphatidylinositol-anchored membrane proteins during sperm maturation.

In spermatozoa, as in other eukaryotic cells, integral membrane proteins may be anchored by a hydrophobic protein domain, or by a glycosylphosphatidylinositol (GPI) lipid anchor. Contrary to the current understanding of sperm membrane biogenesis, recent evidence shows that some of the GPI-anchored proteins are not synthesized by the spermatozoa themselves, but by cells of the male genital tract. They are transported to the fluid secretions, possibly associated with membrane vesicles, and then incorporate into the sperm membrane. This novel mechanism, by which proteins can move from the membrane of one cell to that of another in vivo, may account for a significant proportion of the surface changes occurring during sperm maturation. The function of these GPI-anchored molecules is largely unknown. However, they are remarkably abundant, and the phenomenon of cell-to-cell transfer seems to be well conserved across mammalian species. All of the post-testicularly acquired GPI-anchored proteins identified thus far have also been found on cells of the immune system (CD62, CD55, CD59, CD73), and we speculate that they may have a role in protecting spermatozoa from immune attack in the male and female reproductive tracts.

Amino Acid Sequence↗
PubMed1984

Ultrastructure of the epididymis of the tammar, Macropus eugenii, and its relationship to sperm maturation.

The ductus epididymidis of the tammar is lined by an epithelium composed of principal, mitochondria-rich, apical and basal cells, and intraepithelial leucocytes. The epithelium is structurally differentiated into 6 zones referred to as the initial segment, middle segment (3 subdivisions) and terminal segment (2 subdivisions). The occurrence of the initial, middle and terminal segments corresponds quite closely to the anatomical differentiation of the epididymis into a head, body and tail. The initial segment epithelium in the tammar is lower and has shorter and more slender stereocilia than in other mammals which have been described. Otherwise, the structure of the epithelium has similar characteristics in the tammar to that described in other mammals. Spermatozoa begin to develop the capacity for motility within the initial segment, but only show structural signs of maturation in the middle segment. The sperm head rotates through 90 degrees in the proximal subdivision of the middle segment. The cytoplasmic droplet is detached and spermatozoa develop the capacity for motility in the middle subdivision of the middle segment. The cytoplasmic droplets are phagocytosed by the epididymal epithelium of the middle segment. Sperm storage appears to be the main function of the terminal segment.

Animals↗
PubMed1988

How is the flagellar length of mature sperm determined? I. Comparison of flagellar growth in spermatids between newt and Xenopus in vitro.

The kinetics of flagellar growth in round spermatids were compared between Xenopus laevis and Cynops pyrrhogaster in vitro, the latter of which has about 13 times longer flagella in mature sperm than the former. In both species, more than 90% of the spermatids derived from marked primary spermatocytes grew flagella. In Xenopus the average flagellar length increased to 28 microns by the 6th day and then stopped growth, while in the newt, flagellar growth did not stop until reaching 107 microns in average on the 10th day. Maximal length was 36-38 microns in Xenopus and 187 microns in the newt. Two major differences in kinetics of flagellar growth were found between the two species. First, the initial rate of growth in the newt was about double the rate in Xenopus. Second, the period of flagellar growth in the newt (10 days) was also about double the period in Xenopus (5-6 days). Actinomycin D (10 micrograms/ml) had no inhibitory effect on flagellar growth in either species, whereas cycloheximide (10 microM) inhibited flagellar growth by more than 80% in both species. These results indicate that translational control presumably of flagellar protein synthesis plays an important role in flagellar growth in both species and in the difference in flagellar length in spermatids between Xenopus and newt.

Animals↗
PubMed1984

Insect radiosensitivity: dose curves and dose-fractionation studies of dominant lethal mutations in the mature sperm of 4 insect species.

Males of 4 species of insects: Musca domestica L. (housefly) (Diptera), Oncopeltus fasciatus (Dallas) ( milkweed bug) (Hemiptera), Anagasta kuhniella ( Zeller ) ( mealmouth ) (Lepidoptera) and Heliothis virescens (Fab.) (tobacco budworm ) (Lepidoptera) were irradiated as adults. Dose--response curves for the induction of dominant lethal mutations in the mature sperm were constructed. The curves were analyzed mathematically and compared with theoretical computer simulated curves requiring 1, 2, 4, 8 and 16 'hits' for the induction of a dominant lethal mutation. The 4 species belonging to 3 different orders of insects showed a wide range in radiation sensitivity and vastly different dose--response curves. The house fly was the most sensitive and displayed a '1-hit' curve. The milkweed bug was intermediate in sensitivity and the curve exhibited '4-hit' kinetics. The mealmoth and tobacco budworm were most radioresistant and the response curves resembled 8-16--hit simulated curves. When the data were analyzed by several mathematical models we found that a logistic response curve gave reasonably good fit with vastly different parameters for the 4 species. Dose-fractionation experiments showed no reduction in the frequency of lethal mutations induced in any species when an acute dose was fractionated into 2 equal exposures separated by an 8-h period. Various reasons for large differences in radiosensitivity are discussed, e.g., (1) differences in repair mechanisms, (2) variation in oxygen concentration, (3) holokinetic versus monokinetic chromosomes and (4) intrinsic differences in mitotic cell cycles which affect the behavior of radiation-induced chromosome breaks.

Animals↗
PubMed1995

Persistence of protamine precursors in mature sperm nuclei of the mouse.

During mouse spermiogenesis, two protamines, mP1 and mP2, are synthesized in replacement of histones. One of them (protamine mP2, 63 residues) appears at first in elongating spermatid nuclei as a protamine of 106 residues (pmP2) with an amino-terminal extension that is progressively excised. The two protamines were previously described as the only proteins associated with DNA in sperm chromatin. This paper shows that the nuclear proteins of mouse spermatozoa are indeed heterogeneous: at least six minor polypeptides in addition to protamines can be identified. The primary structure of four of them has been established. They are intermediate in the maturation of the precursor of protamine mP2 and correspond to polypeptides pmP2/11, pmP2/16, pmP2/20, and pmP2/32, characterized previously in mouse testis. Therefore, these intermediates of proteolysis generated from pmP2 inside spermatid nuclei persist in mature sperm, whereas the largest precursors, pmP2 and pmP2/5, disappear. These findings clearly indicate that limited proteolysis events still occur outside of the testis.

Amino Acid Sequence↗
PubMed1999

A C-terminal-truncated PrP isoform is present in mature sperm.

PrP(C), the normal isoform of the prion component PrP(Sc), is a 33-35-kDa glycophosphatidylinositol-anchored glycoprotein expressed in the plasma membrane of many cells and especially in the brain. The specific role of PrP(C) is unknown, although lately it has been shown to bind copper specifically. We show here that PrP(C) is present even in mature sperm cells, a polarized cell that retains only the minimal components required for DNA delivery, movement, and energy production. As opposed to PrP(C) in other cells, PrP in ejaculated sperm cells was truncated in its C terminus in the vicinity of residue 200. Sperm PrP, although membrane-bound, was not released by phosphatidylinositol phospholipase C as well as not localized in cholesterol-rich microdomains (rafts). Although no infertility was reported for PrP-ablated mice in normal situations, our results suggest that sperm cells originating from PrP-ablated mice were significantly more susceptible to high copper concentrations than sperm from wild type mice, allocating a protective role for PrP in specific stress situations related to copper toxicity. Since the functions performed by proteins in sperm cells are limited, these cells may constitute an ideal system to elucidate the function of PrP(C).

Animals↗
PubMed1982

Dumpy mutations following X-irradiation of Drosophila melanogaster mature sperm in oxygen or in nitrogen.

A comparison was made of the oxygen enhancement pattern among the different kinds of dumpy mutations (olv, ov, ol, lv, o and v types), yellow mutations on the scute-8 chromosome, white, miniature and forked mutations, and the marker losses in the doubly marked Y chromosome [BS Y sc8 (y+)], all of which were induced by X rays in mature sperm of Drosophila melanogaster. The results indicate that (1) an essential difference does not exist in the oxygen enhancement pattern between the different kinds of dumpy mutations, except for the ov exceptions. For these exceptions, relatively high enhancement by oxygen is elucidated; (2) a similarity exists in the oxygen enhancement pattern among the different kinds of dumpy mutations (except for the ov exceptions), yellow, miniature and forked mutations, and BS and y+ marker losses; and (3) the oxygen enhancement pattern elucidated for the ov exceptions is similar to that for the white mutations. These findings suggest that the nature of the different kinds of dumpy mutations is not different from one another, except for the ov exceptions, and that except for these ov exceptions and the white mutations, there seems to be some kind of similarity in the nature of mutation among the different kinds of mutations studied.

Animals↗
PubMed1991

Studies on egg zona pellucida-binding molecule (ligand) of mouse sperm. I. Sperm maturation and zona-binding ability.

The epididymis has three major regions; the caput, corpus and cauda. Sperm were obtained from each region, and their binding ability to egg zona pellucida was assayed. In the case of intact sperm: cauda sperm bound in high number, corpus sperm bound at an intermediate level (an average of 30% of cauda sperm binding level), and caput sperm bound rarely (1% of cauda sperm binding level). In the case of sperm immobilized by pretreatment with LaCl3, when eggs and sperm were shaken in order to allow them to come into collision with each other, the caput and corpus sperm were able to bind to zona pellucida as well as the caudal sperm could. These results suggest that the difference in binding efficiency of the native caput and corpus sperm to bind to the zona pellucida was not due to a defect in a ligand molecule on the plasma membrane but rather to their lack of motility. When a sperm-egg binding assay was done in the presence of a binding inhibitor of caudal sperm, the binding of caudal sperm was significantly inhibited by fetuin or N-acetylgalactosamine, but the binding of caput and corpus sperm was only slightly inhibited by them. We prepared a plasma membrane fraction from each epididymal sperm, and solubilized it with sodium dodecyl sulfate (SDS). Each SDS-solubilizable fraction inhibited the binding of corresponding epididymal sperm to egg zona pellucida but didn't inhibit the binding of sperm from different regions of epididymis. These results suggested that a ligand molecule of caudal sperm was different from that of caput and corpus sperm.

Animals↗
PubMed1989

Role of epididymis in sperm maturation.

One hundred ninety patients with obstructive azoospermia caused by bilateral epididymal blockage have been followed up for four years or longer after undergoing "specific tubule" vasoepididymostomy. When anastomosis was required in the corpus epididymis, the "patency" rate was 78 percent, and the overall pregnancy rate was 56 percent. The pregnancy rate for "patent" cases was 72 percent, indicating that a high fertility rate can be obtained with sperm that have not transited the full length of corpus epididymis. By contrast, with vasoepididymostomy to the caput epididymis there was a 73 percent "patency" rate, but the overall pregnancy rate was only 31 percent. The pregnancy rate for "patent" cases was 43 percent. Sperm from the corpus epididymis have a higher rate of fertility than sperm from the caput epididymis, but sperm from proximal areas of the corpus have no less fertility than sperm from the distal corpus epididymis. The most remarkable observation is that in almost half the cases sperm that have never journeyed beyond the caput epididymis seem to be capable of causing pregnancy.

Anastomosis, Surgical↗