Acrosome Biosynthesis in Spermatocytes and Spermatids Revealed by HPA Lectin Cytochemistry

The origin of the acrosome is controversial, because of both its lysosomal nature and at the moment of its appearance, which seems to be species‐specific. Considering the amazing organization shown by the acrosome of some urodele amphibians, HPA‐colloidal gold cytochemistry was used to analyze the biogenesis of the acrosome in the urodele Pleurodeles waltl at electron microscopy level. The results showed that HPA‐labeling is useful to label the acrosome and its precursor vesicles and, consequently, HPA‐histochemistry could be used as a marker of acrosomal content. Labeling of the Golgi apparatus and precursor vesicles was seen in primary spermatocytes and round (stage I) spermatids, thus contributing solid evidence for the beginning of acrosome biogenesis before meiosis. In both primary spermatocytes and round spermatids, an enigmatic vesicle, probably related to the biosynthesis of the neck piece or the tail, was also labeled. Labeling in elongating spermatids (stage II–IV), showed a homogeneous distribution of colloidal gold particles in the acrosomal cap, but the perforatorium was not positive to the lectin. However, in mature (stage V–VI) spermatids, a regional distribution of labeling in the acrosome was seen, with the apical knob showing a stronger labeling than the lateral barb, and the lateral barb showing a stronger labeling than the principal piece of the acrosomal cap. This regional distribution of the labeling suggests that the acrosome develops several domains with different glycoconjugate compositions. Anat Rec, 291:1097‐1105, 2008. © 2008 Wiley‐Liss, Inc.     

Incomplete nuclear transformation of human spermatozoa in oligo-astheno-teratospermia: characterization by indirect immunofluorescence of chromatin and thiol status

BACKGROUND Sperm heterogeneity in the human, as observed in oligo-astheno-teratozoospermia (OAT), is associated with hypospermatogenesis. METHODS The chromatin of sperm from OAT and normospermic males was characterized with antibodies specific for nucleosomes, the histone H3.1/H3.2 isoform, histone TH2B, apoptosis-associated H4 acetylation (KM-2) and protamines. Subsequently, sperm samples were stained with the thiol-specific fluorochrome monobromobimane (mBBr) before and after reduction with dithiotreitol (DTT) as most thiol groups reside in the cysteine-rich protamines. We also used fluorescence-activated cell sorter (FACS) for sperm histograms and sorting for high or low free and total thiol levels. These fractions were further analysed for DNA damage with the TdT-UTP nick end-labelling (TUNEL) assay. RESULTS OAT sperm nuclei stained higher for nucleosomes and KM2-epitopes, and lower for TH2B. For free, and total, thiol groups, OAT sperm were characterized by biphasic distributions, reflecting incomplete thiol oxidation as well as overoxidation, and possibly reduced protamine contents. The TUNEL assay on sperm subfractions, for both control and OAT sperm, revealed that a lower level of free thiol groups is associated with a higher TUNEL incidence, and this relationship was also found for total thiol levels. Hence, both overoxidation and a low total number of thiol groups predestine for sperm apoptosis. CONCLUSIONS Chromatin characteristics reflecting an incomplete nucleosome to protamine remodelling were found in subfertile males. Sperm apoptosis is related to both incomplete remodelling and protamine overoxidation.     

Hominoid-specific SPANXA/D genes demonstrate differential expression in individuals and protein localization to a distinct nuclear envelope domain during spermatid morphogenesis

Human sperm protein associated with the nucleus on the X chromosome consists of a five-member gene family (SPANXA1, SPANXA2, SPANXB, SPANXC and SPANXD) clustered at Xq27.1. Evolved from an ancestral SPANX-N gene family (at Xq27 and Xp11) present in all primates as well as in rats and mice, the SPANXA/D family is present only in humans, bonobos, chimpanzees and gorillas. Among hominoid-specific genes, the SPANXA/D gene family is considered to be undergoing rapid positive selection in its coding region. In this study, RT-PCR of human testis mRNA from individuals showed that, although all SPANXA/D genes are expressed in humans, differences are evident. In particular, SPANXC is expressed only in a subset of men. The SPANXa/d protein localized to the nuclear envelope of round, condensing and elongating spermatids, specifically to regions that do not underlie the developing acrosome. During spermiogenesis, the SPANXa/d-positive domain migrated into the base of the head as the redundant nuclear envelope that protrudes into the residual cytoplasm. Post-testicular modification of the SPANXa/d proteins was noted, as were PEST (proline, glutamic acid, serine, and threonine rich regions) domains. It is concluded that the duplication of the SPANX-N gene family that occurred 6-11 MYA resulted in a new gene family, SPANXA/D, that plays a role during spermiogenesis. The SPANXa/d gene products are among the few examples of X-linked nuclear proteins expressed following meiosis. Their localization to non-acrosomal domains of the nuclear envelope adjacent to regions of euchromatin and their redistribution to the redundant nuclear envelope during spermiogenesis provide a biomarker for the redundant nuclear envelope of spermatids and spermatozoa.     

The DNA-repair Ku70 protein is located in the nucleus and tail of elongating spermatids in grasshoppers

Fluorescence immunostaining for the phosphorylated H2AX histone (γH2AX) in the grasshopper Eyprepocnemis plorans has shown abundance of γH2AX in the nuclei of round and elongating spermatids, suggesting that DNA double-strand breaks (DSBs) occur regularly during spermiogenesis. Immunofluorescence patterns for Ku70, a DNA-repair protein participating in the non-homologous end-joining (NHEJ) pathway, showed that this protein is present in round and elongating spermatids, implying that the NHEJ DNA-repair pathway operates during chromatin compaction in spermiogenesis. In addition, during the final stages of spermiogenesis, the Ku70 protein concentrates on the region forming the sperm tail. Since Ku70 was also abundant in spermatid tails, it is reasonable to assume that Ku70 might play a novel function in sperm-tail formation. The analysis of Ku70 immunofluorescence patterns in 13 other grasshopper species also showed the presence of this protein in the nucleus and tail of elongating spermatids, indicating that this is a general characteristic in grasshoppers.     

Aging deteriorates quality of sperm produced by male mice with partial Yq deletion

The aim of the study was to assess the cumulative effects of aging and Y-chromosome long arm deletion on sperm quality parameters. Motility, mitochondrial activity, and head morphology were evaluated for sperm of 3- and 12-month-old males from B10.BR-Y^del and B10.BR congenic mouse strains. The study revealed that quality and fertilizing potential of sperm produced by younger and older B10.BR males persist on similar levels, but worsen significantly with age of B10.BR-Y^del males. The findings imply that partial Yq deletions might be more harmful for spermiogenesis in advancing age and may be applicable to other species including humans.

Abbreviations: AZF: azoospermia factor; MSYq: male-specific region of the Y-chromosome long arm     

Molecular biological features of male germ cell differentiation

Somatic cell differentiation is required throughout the life of a multicellular organism to maintain homeostasis. In contrast, germ cells have only one specific function; to preserve the species by conveying the parental genes to the next generation. Recent studies of the development and molecular biology of the male germ cell have identified many genes, or isoforms, that are specifically expressed in the male germ cell. In the present review, we consider the unique features of male germ cell differentiation. (Reprod Med Biol 2007; 6 : 1–9)     

Cloning and characterization of a mouse spergen-1 localized in sperm mitochondria

Development of spermatozoa is a complex process involving specific morphological formation of flagella, nucleus and mitochondria. Although detailed morphological observations of these events are available, the molecular mechanisms remain to be fully elucidated. We report here the molecular cloning and characterization of mouse spergen-1 encoding a sperm specific mitochondrial protein, from a haploid germ cell-specific subtracted cDNA library of mouse testis. Isolated cDNA is c. 0.7 kb and contains a 465 bp ORF that encodes mouse spergen-1, a sperm mitochondrial protein consisting of 154 predicted amino acids. Antibodies raised against mouse Spergen-1 identified a testis-specific c. 18 Mr x 10(3) band in Western blot analysis. The protein was localized to the mitochondria of mouse sperm. Comparison of the mouse and human genomic sequences showed that 55 bps of the 5'-upstream region containing a CAAT box and binding sequence for NF-kappa B is conserved and could be important for specific expression of mouse spergen-1 in haploid germ cells.     

Isolation and characterization of a novel cDNA encoding a DNA-binding protein (Hils1) specifically expressed in testicular haploid germ cells

A cDNA encoding a protein homologous with histone H1 has been cloned from a haploid germ cell specific cDNA library. Deduced amino acid sequence (170 amino acids) showed 40% identity with histone H1 globular domain. Messenger RNA of the gene was observed exclusively in the testis, and was accumulated after post-natal day 23. Western blotting analysis showed that the protein encoded by this gene is about 19 kDa in molecular weight, and it was exclusively recovered from the nuclei of testicular germ cells. Immunohistochemical analysis showed that the protein was localized to the nuclei of round and elongating spermatids, consistent with the results of immunoblot analysis. Thus, the gene product was named Hils1 (histone H1 like protein in spermatids 1). In vitro DNA-binding experiments using DNA-cellulose mini-columns showed that Hils1 was able to bind to both double and single stranded-DNAs in a non-sequence-specific manner. These findings suggest that Hils1 may play an important role in the structural changes of spermatid nuclei, such as nuclear condensation, and gene regulation of haploid germ cell differentiation.     

Aquaporins in spermatozoa and testicular germ cells： identification and potential role

Mammalian spermatozoa have relatively high water permeability and swell readily, as in the hypo-osmotic swelling test used in the andrology clinic. Physiologically, spermatozoa experience changes in the osmolality of the surrounding fluids in both the male and the female tracts on their journey from the testis to the ovum. Sperm volume regulation in response to such osmotic challenges is important to maintain a stable cell size for the normal shape and function of the sperm tail. Alongside ion channels for the fluxes of osmolytes, water channels would be crucial for sperm volume regulation. In contrast to the deep knowledge and numerous studies on somatic cell aquaporins （AQPs）, the understanding of sperm AQPs is limited. Among the 13 AQPs, convincing evidence for their presence in spermatozoa has been confined to AQP7, AQP8 and AQP 11. Overall, current findings indicate a major role of AQP8 in water influx and efltux for sperm volume regulation, which is required for natural fertilization. The preliminary data suggestive of a role for AQP7 in sperm glycerol metabolism needs further substantiation. The association of AQP 11 with the residual cytoplasm of elongated spermatids and the distal tail of spermatozoa supports the hypothesis of more than just a role in conferring water permeability and also in the turnover and recycling of surplus cellular components made redundant during spermiogenesis and spermiation. This would be crucial for the maintenance of a germinal epithelium functioning efficiently in the production of spermatozoa.