Observations of spermiogenesis and epididymal sperm maturation in the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia)

Acrosomal development in the early spermatid of the rufous hare wallaby shows evidence of formation of an acrosomal granule, similar to that found in eutherian mammals, the Phascolarctidae and Vombatidae. Unlike the other members of the Macropodidae so far examined, the acrosome of this species appears to be fully compacted at spermiation and extends evenly over 90% of the dorsal aspect of the nucleus. During spermiogenesis, the nucleus of the rufous hare wallaby spermatid showed evidence of uneven condensation of chromatin; this may also be related to the appearance of unusual nucleoplasm evaginations from the surface of the fully condensed spermatid. This study was unable to find evidence of the presence of Sertoli cell spurs or nuclear rotation during spermiogenesis in the rufous hare wallaby. The majority of spermatozoa immediately before spermiation had a nucleus that was essentially perpendicular to the long axis of the sperm tail. Nuclei of spermatozoa found in the process of being released or isolated in the lumen of the seminiferous tubule were rotated almost parallel to the long axis of the flagellum; complete parallel alignment occurred during epididymal maturation. At spermiation spermatozoa have characteristically small cytoplasmic remnants compared to those of other macropods. Unlike the majority of macropodid spermatozoa so far described, the spermatozoa of the rufous hare wallaby showed little evidence of morphological change during epididymal transit. There was no formation of a fibre network around the midpiece or of plasma membrane specializations in this region; the only notable change was a distinctive flattening of midpiece mitochondria and scalloping of the anterior mitochondrial sheath to accommodate the sperm head. Preliminary evidence from spermiogenesis and epididymal sperm maturation supports the classification of the rufous hare wallaby as a separate genus but also indicates that its higher taxonomic position may need to be re‐evaluated.     

Hamster sperm motility transformation during development of hyperactivation in vitro and epididymal maturation

The transformation of hamster sperm motility during capacitation in vitro and during maturation in the caudal epididymis was analyzed and compared using videomicrography. Sperm recovered from the distal portion of the caudal epididymis, as well as ejaculated sperm recovered from the uterus exhibited low amplitude, planar flagellar beating. By 3 hr of incubation under capacitating conditions, the caudal epididymal sperm were swimming in helical patterns apparently produced by significantly increased acuteness of flagellar bending and by torsion seen as abrupt, periodic turning of the head. By 4 hr, most sperm were hyperactivated, swimming in circles resulting from asymmetrical, planar flagellar bending that was significantly more acute than the preceding patterns. When motility parameters of fresh sperm were compared with those of sperm swimming in the transitional helical pattern and with hyperactivated sperm, transitional sperm had significantly higher net and average path velocities than the others, indicating that they covered space at the greatest rate. This suggests that the transitional phase plays an important role in sperm transport. Sperm recovered from the proximal region of the caudal epididymis, near the corpus, swam in either the helical or hyperactivated patterns, or a mixture of the two. The means of their flagellar curvature ratios and linear indices were intermediate between helical and hyperactivated mean values. Thus, sperm undergoing final maturation in the caudal epididymis reverse the pattern of development of hyperactivation. Also, the development of hyperactivated motility must therefore entail induction of a preexisting potential for flagellar movement, rather than a maturational process.     

Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis

Two types of filaments were observed within the subacrosomal space of rat spermatids. The first of these types was characterized as actin by demonstration of actin filament affinity for myosin S-1 subfragments. Actin filaments were noted in the subacrosomal space shortly after the acrosomal sac made contact with the nucleus. As the acrosome increased its surface area contact with the spermatid nucleus, the number of layers of subacrosomal filaments increased. Pre-treatment with detergent, which in addition to permeablizing cells to allow entry of S-1, also caused the acrosome to vesiculate and the subacrosomal space to widen. In such preparations filaments were more easily visualized and appeared to extend between the nuclear and acrosomal membranes, indicating, but not proving, attachment to these membranes. During spermatid clongation, the number of actin filaments in the subacrosomal space increased greatly, especially over the dorsal convex region of the spermatid head. The polarity of the majority of filaments was not ascertainable since filaments were tightly packed within the narrow subacrosomal space. In late spermiogenesis (steps 18 and 19), actin filaments were no longer detected within the subacrosomal space. A second and much thicker type of filamentous structure was observed in the subacrosomal space of spermatids at steps 14-17 of spermiogenesis. About 14 nm in diameter (10-15 nm measurement range depending on fixation protocol utilized), these filaments did not decorate with myosin S-1 subfragments and were found in subacrosomal regions not containing actin. Fourteen nanometer filaments were seen in parallel array along the ventral folded portion of the nuclear membrane and extended partially around the nucleus. Like actin filaments. 14 nm filaments were not seen in the subacrosomal space during late spermiogenesis.     

Calmodulin,a calmodulin acceptor protein,and calcimedins: Unique antibody localizations in hamster sperm

A calmodulin acceptor protein has been identified in isolated hamster caudal sperm by immunofluoresence and Western transfer techniques. The protein shows a localization in sperm heads identical to calmodulin. Fluorescence of both calmodulin and the acceptor protein are lost by treatment with MgCl₂, conditions which release the acrosome. These results are consistent with the proposed function of calmodulin in a sperm function.     

Ultrastructure of spermiogenesis in the American alligator,Alligator mississippiensis (Reptilia,Crocodylia, Alligatoridae)

Testicular samples were collected to describe the ultrastructure of spermiogenisis in Alligator mississipiensis (American Alligator). Spermiogenesis commences with an acrosome vesicle forming from Golgi transport vesicles. An acrosome granule forms during vesicle contact with the nucleus, and remains posterior until mid to late elongation when it diffuses uniformly throughout the acrosomal lumen. The nucleus has uniform diffuse chromatin with small indices of heterochromatin, and the condensation of DNA is granular. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. Once the acrosome has completed its development, the nucleus of the early elongating spermatid becomes associated with the cell membrane flattening the acrosome vesicle on the apical surface of the nucleus, which aids in the migration of the acrosomal shoulders laterally. One endonuclear canal is present where the perforatorium resides. A prominent longitudinal manchette is associated with the nuclei of late elongating spermatids, and less numerous circular microtubules are observed close to the acrosome complex. The microtubule doublets of the midpiece axoneme are surrounded by a layer of dense staining granular material. The mitochondria of the midpiece abut the proximal centriole resulting in a very short neck region, and possess tubular cristae internally and concentric layers of cristae superficially. A fibrous sheath surrounds only the axoneme of the principal piece. Characters not previously described during spermiogenesis in any other amniote are observed and include (1) an endoplasmic reticulum cap during early acrosome development, (2) a concentric ring of endoplasmic reticulum around the nucleus of early to middle elongating spermatids, (3) a band of endoplasmic reticulum around the acrosome complex of late developing elongate spermatids, and (4) midpiece mitochondria that have both tubular and concentric layers of cristae. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

The ultrastructure of spermatid development during spermiogenesis within the rosebelly lizard,Sceloporus variabilis (Reptilia,Squamata, Phrynosomatidae)

Several recent studies have mapped out the characters of spermiogenesis within several species of squamates. Many of these data have shown both conserved and possibly apomorphic morphological traits that could be important in future phylogenetic analysis within Reptilia. There, however, has not been a recent study that compares spermiogenesis and its similarities or differences between two species of reptile that reside in the same genus. Thus, the present analysis details the changes to spermiogenesis in Sceloporus variabilis and then compares spermatid morphologies to that of Sceloporus bicanthalis. Many of the morphological changes that the spermatids undergo in these two species are similar or conserved, which is similar to what has been reported in other squamates. There are six main character differences that can be observed during the development of the spermatids between these two sceloporid lizards. They include the presence (S. variabilis) or absence (S. bicanthalis) of a mitochondrial/endoplasmic reticulum complex near the Golgi apparatus during acrosome development, a shallow (S. variabilis) or deep (S. bicanthalis) nuclear indentation that accommodates the acrosomal vesicle, filamentous (S. variabilis) or granular (S. bicanthalis) chromatin condensation, no spiraling (S. variabilis) or spiraling (S. bicanthalis) of chromatin during condensation, absence (S. variabilis) or presence (S. bicanthalis) of the longitudinal manchette microtubules, and the lack of (S. variabilis) or presence (S. bicanthalis) of nuclear lacunae. This is the first study that compares spermiogenic ultrastructural characters between species within the same genus. The significance of the six character differences between two distantly related species within Sceloporus is still unknown, but these data do suggest that spermiogenesis might be a good model to study the hypothesis that spermatid ontogeny is species specific. J. Morphol. 275:258–268, 2014. © 2013 Wiley Periodicals, Inc.     

Boar sperm surface glycoproteins: Isolation,localization, and temporal expression during spermatogenesis

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.     

Actin and major sperm protein in spermatids and spermatozoa of the parasitic nematode Heligmosomoides polygyrus

Nematode spermatozoa are amoeboid cells. In Caernorhabditis elegans and Ascaris suum, previous studies have reported that sperm motility does not involve actin, but, instead, requires a specific cytoskeletal protein, name y major-sperm-protein (MSP). In Heligmosomoides polygyrus, a species with large and elongate spermatids and spermatozoa, cell organelles are easily identified even with light microscopy. Electrophoresis of Heligmosomoides sperm proteins indicates that the main protein band has a molecular weight of about 15 kDa, as MSP in other nematodes, and is specifically labelled by an anti-MSP antibody raised against C. elegans MSP. A minor band at 43 kDa was specifically labelled by an anti-actin antibody. Reaction of anti-actin and anti-MSP antibodies is specific to, and restricted to, their respective targets. Actin and MSP localisation, studied by indirect immunofluorescence in male germ cells of Heligmosomoides polygyrus, are similar: spermatids show rows of dots, corresponding to the fibrous bodies, around an unlabelled central longitudinal core; spermatozoa are labelled strictly in an anterior crescent-shaped cap, at the opposite pole to the nucleus, which contains fibres of the MSP cytoskeleton. Phalloidin labelling shows that F-actin is present in spermatids, but absent in spermatozoa. Tropomyosin shows a distinct pattern in spermatids, but is located in the MSP and actin-containing cap in spermatozoa. It is hypothesized that actin plays a role in the shaping of the cell and in the arrangement of its organelles during nematode spermiogenesis, when MSP is present, in an inactive state, in the fibrous bodies. The concentration of actin and tropomyosin in the anterior cap is not compatible with previous theories about the MSP cytoskeleton which is supposed to act in the absence of actin. © 1996 Wiley-Liss, Inc.     

Nuclear protein transitions in cuttle-fish spermiogenesis: Immunocytochemical localization of a protein specific for the spermatid stage

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.     

Differential expression of lysosomal associated membrane protein (LAMP-1) during mammalian spermiogenesis

The mammalian acrosome is a secretory vesicle of mature sperms that plays an important role in fertilization. Recent evidence had pointed out that some components found at endosomes in somatic cells are associated with the developing acrosome during the early steps of spermiogenesis. Moreover, the mammalian acrosome contains many enzymes found within lysosomes in somatic cells. In this work, we studied the dynamics of some components of the endosome/lysosome system, as a way to understand the complex membrane trafficking circuit established during spermatogenesis. We show that the cation independent-mannose-6-phosphate receptor (CI-MPR) is transiently expressed in the cytoplasm of mid-stage spermatids (steps 5-11). On the other hand, gamma-adaptin, an adaptor molecule of a complex involved in trafficking from the Golgi to lysosomes, was expressed in cytoplasmic vesicles only in pachytene and Cap-phase spermatids (steps 1-5). Our major finding is that the lysosomal protein LAMP-1 is differentially expressed during spermiogenesis. LAMP-1 appears late in spermatogenesis (Acrosome-phase) contrasting with LAMP-2, which is present throughout the complete process. Both proteins appear to be associated with cytoplasmic vesicles and not with the developing acrosome. None of the studied proteins is present in epididymal spermatozoa. Our results suggest that the CI-MPR could be involved in membrane trafficking and/or acrosomal shaping during spermiogenesis.     

Effects on spermiogenesis in the mouse of a male sterile neurological mutation,Purkinje cell degeneration

Purkinje cell degeneration (pcd) is a neurological mutation in the mouse that causes male sterility, but not female sterility. In order to assess the effects of this mutation on spermiogenesis, the structure of the testis and of epididymal spermatozoa was examined by transmission and scanning electron microscopy. In the mutant males, the sperm count was reduced, sperm were nonmotile, and 93% of the sperm were characterized by structural abnormalities of the head, the tail, or both. In the testes of mutant mice, Sertoli cell structure was normal, as were also the early stages of spermiogenesis. However, the elongating and maturing spermatids were characterized by abnormally shaped heads and tails with extraneous and ectopic outer dense fibers. These defects were common in the testes of the mutant mice and rare in the testes of the littermate control mice. It was concluded that the structural abnormalities of the pcd sperm occurred during spermiogenesis and were not due to degeneration of the sperm in the epididymis. These structural abnormalities are similar to those found in all other reported male sterile mutants of the mouse; therefore, although they are caused by the expression of the pcd gene, they are not unique to the expression of this gene.     

Evaluation of assay conditions for the zona-free hamster ova bioassay of boar sperm fertility

The ability to penetrate zona-free hamster ova may be a very useful test of fresh and frozen boar sperm fertility. These studies were designed to optimize assay conditions prior to evaluation of the accuracy of the bioassay in predicting boar sperm fertility. The ability to penetrate zona-free hamster ova was greater in sperm washed on a Percoll gradient than in sperm washed by dilution and centrifugation. Penetrating ability was greater in sperm from the sperm-rich fraction than from the whole ejaculate but did not differ among different aliquots of the sperm-rich fraction and did not decrease when the prewashing interval was increased from 15 to 85 min. Frequency of collection of ejaculates (1, 3, or 5 times per week) did not affect the penetrating ability of the sperm. Penetration rate was greater when sperm were coincubated with zona-free hamster ova at 39°C compared to 37°C. Sperm from an infertile boar had reduced penetrating ability compared to sperm from fertile boars (11% vs 93%, P < .001). These studies suggest that the zona-free hamster ova bioassay may be a useful assessment of fresh boar sperm fertility.     

Assessing the efficacy of protein farnesyltransferase inhibitors in mouse models of progeria

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of a farnesylated form of prelamin A (progerin). Previously, we showed that blocking protein farnesylation with a farnesyltransferase inhibitor (FTI) ameliorates the disease phenotypes in mouse model of HGPS (Lmna^HG/+). However, the interpretation of the FTI treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin (Lmna^nHG/+) develop progeria-like disease phenotypes. The fact that Lmna^nHG/+ mice manifest disease raised the possibility that the beneficial effects of an FTI in Lmna^HG/+ mice were not due to the effects of the drug on the farnesylation of progerin, but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins. To address this issue, we compared the ability of an FTI to improve progeria-like disease phenotypes in both Lmna^HG/+ and Lmna^nHG/+ mice. In Lmna^HG/+ mice, the FTI reduced disease phenotypes in a highly significant manner, but the drug had no effect in Lmna^nHG/+ mice. The failure of the FTI to ameliorate disease in Lmna^nHG/+ mice supports the idea that the beneficial effects of an FTI in Lmna^HG/+ mice are due to the effect of drug on the farnesylation of progerin.     

Immunohistochemical localization of prolactin cells of the pars distalis in the fetal and neonatal hamster

Summary Using the immunoperoxidase technique, a small number of prolactin cells were first detected in the pars distalis of the hamster near developing sinusoids at 131/2 days gestation. Little change in number or distribution of immunoreactive cells was noted until the first few days after birth when a dramatic increase in number of immunoreactive cells was demonstrated throughout the pars distalis. Electron microscopy revealed cells in the fetal and neonatal anterior pituitary which had immunoreactive granules smaller in diameter than those seen in adult pituitary cells.Submitted by the senior author to the Graduate School at Louisiana State University, Baton Rouge, Louisiana in partial fulfillment of the requirements for the Ph.D. degree     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

P26h and dicarbonyl/L-xylulose reductase are two distinct proteins present in the hamster epididymis

We have previously identified a 34 kDa protein (P34H) on the human sperm surface covering the acrosome. Using the hamster, we have also described a sperm protein, P26h, which is acquired by spermatozoa during epididymal transit. Both P34H and P26h belong to the carbonyl reductase family. Using molecular tools derived from P34H, we searched in the hamster epididymis for another protein related to the human sperm protein. Cloning and sequencing of P31h cDNA revealed 100% homology with the kidney DCXR (Dicarbonyl/L-Xylulose reductase). Northern Blot experiments revealed a single mRNA that was more expressed in the caput than in the corpus and cauda segment of adult epididymides. In situ hybridization was performed on sexually mature hamsters showing that the mRNA was localized in the principal cells throughout the epididymis. Using an anti-P34H antibody we have identified a P34H related protein named P31h (for 31 kDa). This protein showed 2D-electrophoretic behavior different from P26h and was detectable all along the epididymis (caput, corpus, and cauda) by Western Blot analysis. Immunohistochemistry techniques showed that P31h was localized in the perinuclear region of the principal cells of the epididymal epithelium within the three sections, both in sexually mature and immature animals. Results are discussed with regards to the potential function of DCXR in the epididymis.