Intervention of Xuduan Zhongzi Formula on spermatogenesis epididymal morphological changes in a mice model of oligospermia

Objective:To investigate the intervention of Xuduan Zhongzi Formula on the epididymis structure of model of oligospermia mice.Methods:Ten of the 45 male mice were labeled as the normal group,and the remaining 35 mice were injected with chloral hydrate for five consecutive days,and the randomly selected five mice were anesthetized with chloral hydrate.The left epididymis was removed,and a few sperm were found in the epididymis under the optical microscope,indicating successful construction of the model.They were randomly divided into three groups:the normal group,the L-carnitine group and the Xuduan group with 10 rats in each group.The the L-carnitine group and the Xuduan group were administrated with equivalent dose of human solvent,and the normal group and the model group were administrated with normal saline.After 8 weeks of intragastric administration,all experimental mice were killed,and the left epididymis was extracted for sperm detection,seminal plasma biochemical detection,HE staining,and electron microscopy.Results:In the model group,enlarged epididymal epithelial cells,vacuolar degeneration of primary and basal cells,and edema of interstitial cells and vascular dilation were found.Compared with the normal group,semen concentration,activity,SOD,A-glucosidase and fructose in the model group were significantly decreased,and the difference was statistically significant(P<0.01).After eight weeks of drug intragastric treatment,the semen concentration,activity,SOD,A-glucosidase and fructose in the Xuduan group were significantly increased compared with the model group,and the difference was statistically significant(P<0.01).Conclusion:Xuduan Zhongzi Formula can significantly improve the epididymal structure,the microenvironment of epididymal sperm maturation and the stability of epididymal epithelial structure of oligospermia model mice,creating conditions for sperm maturation in the epididymis.

Epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit

During epididymal transit, spermatozoa acquire new proteins. Some of these newly acquired proteins behave as integral membrane proteins, including glycosylphosphatidylinositol （GPI）-anchored proteins. This suggests that the secreted epididymal proteins are transferred to spermatozoa by an unusual mechanism. Within the epididymal lumen, spermatozoa interact with small membranous vesicles named epididymosomes. Many proteins are associated with epididymosomes and the protein composition of these vesicles varies along the excurrent duct and differs from soluble intraluminal proteins. Some epididymosome-associated proteins have been identified and their functions in sperm maturation hypothesized. These include P25b, a zona pellucida binding protein, macrophage migration inhibitory factor, enzymes of the polyol pathway, HE5/CD52, type 5 glutathione peroxidase, and SPAM 1 or PH-20. The electrophoretic patterns of proteins associated to epididymosomes are complex and some of these proteins are transferred to defined surface domains of epididymal spermatozoa. Epididymosomes collected from different epididymal segments interact differently with spermatozoa. This protein transfer from epididymosomes to spermatozoa is timedependent, temperature-dependent and pH-dependent, and is more efficient in the presence of zinc. Some proteins are segregated to lipid raft domains of epididymosomes and are selectively transferred to raft domains of the sperm plasma membrane. Some evidence is presented showing that epididymosomes are secreted in an apocrine manner by the epididymal epithelial cells. In conclusion, epididymosomes are small membranous vesicles secreted in an apocrine manner in the intraluminal compartment of the epididymis and play a major role in the acquisition of new proteins by the maturing spermatozoa. （Asian J Androl 2007 July; 9： 483-491）

Sperm maturation in the epididymis： a new look at an old problem

The osmotic challenges facing maturing spermatozoa and their responses to them are discussed in relation to the concept of sperm maturation, defined as the increased ability of more distally recovered epididymal spermatozoa to fertilize eggs when inseminated into the female tract. One explanation could be that the more distal cells are better able to regulate their volume, and reach the oviducts, as a consequence of uptake of epididymal osmolytes. Increased motility, zona binding and oolemma fusion capacities are also acquired within the epididymis and are necessary for those cells that finally arrive at the site of fertilization. （Asian J Androl 2007 July; 9： 533-539）

Human ribonuclease 9,a member of ribonuclease A superfamily,specifically expressed in epididymis,is a novel sperm-binding protein

To explore the functions of human ribonuclease 9(RNase 9),we constructed a mammalian fusion expression vector pcDNA-hRNase9,prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences.According to the determined mature protein,recombinant human RNase 9 was prepared in E.coli.Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected,and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay.The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA,but exhibited antibacterial activity,in a concentration/time dependent manner,against E.coli.Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis,but not present in other tissues examined,and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa.These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.

The ＇omics revolution and our understanding of sperm cell biology

The foundations of proteomics are to study gene products and their regulatory roles within cells. Paradoxically, the only evidence that sperm cells make new proteins is through mitochondrial protein synthesis. Yet despite this, spermatozoa are the perfect candidates for mass spectrometry and hence, proteomic analysis. These enterprising cells use a plethora of post-translational modifications in order to gain functionality following their production within the testis. By using a combination of two-dimensional polyacrylamide gel electrophoresis （2D-PAGE）, and more recently liquid chromatography-mass spectrometry （LC-MS）/MS, recent advances in sperm cell biology, through the use of proteomics, is making unparalleled progress. The protein inventory lists being generated have shed light on transmembrane proteins, kinases and chaperones never previously recognized. In addition, the ability to isolate either phosphopeptides or glycopeptides and quantify the differences between cells of two different populations make proteomic analysis of spermatozoa a real chance to finally answer some age old questions.

Deletion or underexpression of the Y-chromosome genes CDY2 and HSFY is associated with maturation arrest in American men with nonobstructive azoospermia

Maturation arrest （MA） refers to failure of germ cell development leading to clinical nonobstructive azoospermia. Although the azoospermic factor （AZF） region of the human Y chromosome is clearly implicated in some cases, thus far very little is known about which individual Y-chromosome genes are important for complete male germ cell development. We sought to identify single genes on the Y chromosome that may be implicated in the pathogenesis of nonobstructive azoospermia associated with MA in the American population. Genotype-phenotype analysis of 132 men with Y-chromosome microdeletions was performed. Protein-coding genes associated with MA were identified by visual analysis of a genotype-phenotype map. Genes associated with MA were selected as those genes within a segment of the Y chromosome that, when completely or partially deleted, were always associated with MA and absence of retrievable testicular sperm. Expression of each identified gene transcript was then measured with quantitative RT-PCR in testicular tissue from separate cohorts of patients with idiopathic MA and obstructive azoospermia. Ten candidate genes for association with MA were identified within an 8.4-Mb segment of the Y chromosome overlapping the AZFb region. CDY2and HSFYwere the only identified genes for which differences in expression were observed between the MA and obstructive azoospermia cohorts. Men with obstructive azoospermia had 12-fold higher relative expression of CDY2transcript （1.33__.0.40 vs. 0.11＋_0.04; P=O.O003） and 16-fold higher expression of HSFYtranscript （0.78__.0.32 vs. 0.05_0.02; P=O.O005） compared to men with MA. CDY2 and HSFYwere also underexpressed in patients with Sertoli cell only syndrome. These data indicate that CDY2and HSFYare located within a segment of the Y chromosome that is important for sperm maturation, and are underexpressed in testicular tissue derived from men with MA. These observations suggest that impairments in CDY2 or HSFYexpression could be implicated in the pathogenesis of MA.

Maturation of sperm volume regulation in the rat epididymis

Sperm maturation in the epididymis may involve differences between mature and immature spermatozoa in their volume regulatory osmolyte response. Spermatozoa obtained from the rat caput and cauda epididymidis were examined for their ability to regulate volume after transfer from in situ epididymal osmolality （measured to be 343 ± 13 and 365 ± 19 mmol kg^-1, respectively） to that of the female tract in single- and multiple-step protocols. Cells withstood the single-step treatment better than the multistep protocol. Sperm volume estimates by flow cytometric measure- ments of forward scatter of cells with intact head membranes was more sensitive than those by assessing cell coiling microscopically. At osmolalites below 210 mmol kg l both caput and cauda cells ruptured, limiting the use of flow cytometry. Above this critical value, the use of quinine showed that both caput and cauda cells could regulate volume, but cauda cells were the more effective. Of several organic osmolytes studied, myo-inositol, glutamate and KCl caused only temporary and slight swelling of spermatozoa cells in hypotonic medium. Spermatozoa of both maturities seemed to use potassium as the preferred osmolyte for regulating volume.

Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling （TUNEL） assay, the comet assay, the sperm chromatin structure assay （SCSA） and the sperm chromatin dispersion （SCD） test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay.

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.

Fatty acid content in epididymal fluid and spermatozoa during sperm maturation in dogs

Background： During sperm maturation, there is a reorganization of fatty acids from plasmatic membrane of the spermatozoa, which allows higher membrane integrity and acquisition of sperm motility. However, the fatty acid profile during sperm maturation remains unclear in dogs. Thus, the aim of this study was to identify the fatty acids from the epididymal spermatozoa and plasma during the sperm maturation, and observed changes in the motility and plasmatic membrane parameters. Twenty one adult dogs were used, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy, computer-assisted motility analysis, sperm plasma membrane permeability and the fatty acid analysis（lipids were extracted, transmethylated and analyzed by chromatography）.Results： Caput and corpus sperm showed lower values for the motility variables evaluated and plasmatic membrane integrity, indicating different levels of the fatty acids organization. Saturated, monounsaturated and polyunsaturated fatty acids were in higher concentrations in the spermatozoa from epididymis cauda. Highlighting the presence of caprylic, stearic and docosahexaenoic acids.Conclusions： These findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.

90d Exposure to Nonylphenol has Adverse Effects on the Spermatogenesis and Sperm Maturation of Adult Male Rats

This study was conducted to elucidate the reproductive effect of NP on testis,epididymis and epididymal sperm in vivo.Adult male SpragueDawley rats were gavaged with NP at 0,40,100,or 250 mg/kg body weight（bw） on alternate days for 90 d.The results showed that oral administration of NP may damage the structure and function of testis,induce apoptosis and oxidative stress in epididymis or even have cytotoxic effects on epididymal sperm.

Role of the epididymis in sperm competition

Although it is generally understood that the testes recruited kidney ducts for reproductive function during the evolution of vertebrates, little is understood of the biological significance of the adaptation. In the context of the evolution of the mammalian epididymis, this report provides evidence that a major role of the epididymis is to enhance a male＇s chance of achieving paternity in a competitive mating system. A unique example of sperm cooperation in monotremes is used as evidence that the epididymis produces sperm competition proteins to form groups of 100 sperm into bundles that have a forward motility nearly thrice that of individual spermatozoa. As it required 3-h incubation in vitro under capacitation conditions to release motile sperm from the bundles, it is suggested that the monotremes provide an example of capacitation that is quite different from capacitation in higher mammals. It is suggested that variation between species in the intensity of sperm competition could explain the variation that occurs between species in the amount of post-testicular sperm maturation and storage in the epididymis, an explanation of why the human epididymis does not play as important a role in reproduction as the epididymis of most mammals. （Asian J Androl 2007 July; 9： 493-499）

The epididymis, cytoplasmic droplets and male fertility

The potential of spermatozoa to become motile during post-testicular maturation, and the relationship between the cytoplasmic droplet and fertilizing capacity are reviewed. Post-testicular maturation of spermatozoa involves the autonomous induction of motility, which can occur in vivo in testes with occluded excurrent ducts and in vitro in testicular explants, and artefactual changes in morphology that appear to occur in the testis in vitro. Both modifications may reflect time-dependent oxidation of disulphide bonds of head and tail proteins. Regulatory volume decrease （RVD）, which counters sperm swelling at ejaculation, is discussed in relation to loss of cytoplasmic droplets and consequences for fertility. It is postulated that： （i） fertile males possess spermatozoa with sufficient osmolytes to drive RVD at ejaculation, permitting the droplet to round up and pinch off without membrane rupture; and （ii） infertile males possess spermatozoa with insufficient osmolytes so that RVD is inadequate, the droplet swells and the resulting flagellar angulation prevents droplet loss. Droplet retention at ejaculation is a harbinger of infertility caused by failure of the spermatozoon to negotiate the uterotubal junction or mucous and reach the egg. In this hypothesis, the epididymis regulates fertility indirectly by the extent of osmolyte provision to spermatozoa, which influences RVD and therefore droplet loss. Man is an exception, because ejaculated human spermatozoa retain their droplets. This may reflect their short midpiece, approximating head length, permitting a swollen droplet to extend along the entire midpiece; this not only obviates droplet migration and flagellar angulation but also hampers droplet loss.

Molecular aspects of mammalian fertilization

Mammalian fertilization is a highly regulated process, much of which are not clearly understood. Here we presentsome information in order to elaborate a working hypothesis for this process, beginning with the sperm modifications inthe epidydimis up to sperm and egg plasmalemma interaction and fusion. We also discuss the still poorly understood ca-pacitation process, the phenomenon of sperm chemo-attraction that brings the capacitated sperm to interact with theoocyte vestments and certain aspects of the acrosome reaction. (Asian J Androl 2001 Dec; 3 : 243 - 249)

Protein profiles in various epididymal segments of normal and castrated rats

Aim: Epididymal proteins are known to play an important role in the maturation of spermatozoa, we ougnt to deter-mine if there are regional differences in androgen-dependent epididymal proteins. Methods: A group of adult rats wascastrated and epididymides were removed three days following castration. The epididymides were dissected into caput,corpus and cauda segments, homogenized, and proteins were fractionated by anion exchange HPLC. Proteins in select-ed fractions were resolved by SDS-PAGE and visualized by silver staining. Results: It was observed that the levels ofmultiple proteins drastically reduced in the various regions of epididymis of the orchiectomized rats. Conclusion: Theepididymal proteins appear to be useful markers to study androgenic action in the epididymis.

Molecular cloning and identification of mouse epididymis-specific gene mHongl, the homologue of rat HongrES1

Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHongl. The mHongl gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHongl mRNA shows similarity with HongrES1 in the expression patterns： （i） specific expression in epididymal tissue, especially in the cauda region; and （ii） androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES 1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHongl with high specificity and sensitivity was raised. Like HongrES1, the mHongl protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHongl protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHongl is localized to the equatorial segment, which is different from that of HongrES 1. The mHongl protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHongl is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHongl.

Development of Newer Approaches to Male Contraception: Prospects of Availability for Mass Application in the Near Future

Progress in development of a simple, effective reversible male contraceptive has been difficult due to the indispensable role of testosterone in spermatogenesis and maintenance of secondary sexual characters. Efforts are in progress to develop methods, which do not interfere with testosterone production. These include blockade of FSH action by immunization against FSH or FSHR, and interfere with sperm maturation by interfering with estrogen action or by immunization against epididymal or sperm specific proteins. An evaluation of these various approaches for practical application is also presented.

In silico docking analysis of beta-defensin 20 against cation channel sperm-associated protein 1–4 to predict its role in the sperm maturation

Beta-defensin 20(DEFB20)is widely expressed in the epididymis with gene features involved in epididymal sperm maturation.However,the action mechanism and function of DEFB20 in sperm maturation are still unclear.One of the important roles of beta-defensin is the ion channel activity.The cation channel sperm-associated protein(CatSper)alpha is an ion channel protein found on the sperm surface.This study aimed to investigate the interaction between DEFB20 and CatSper1–4 protein in relation to the sperm maturation process.Protein sequences were obtained from the National Center for Biotechnology Information(NCBI).Protein modeling and validation were carried out by using the Robetta modeling server and the Ramachandran plot method.Rosetta web server was used for the docking analysis.The results revealed a natural interaction between DEFB20 and CatSper1–4.The interaction occurred at the cation channel(close to the casein kinase II),ion transport protein,and kinase c phosphorylation of the CatSper1–4 active site.The DEFB20 region interacting with CatSper2–4 was the beta-defensin domain,while with CatSper1 was the non-beta-defensin domain.Based on the analysis,DEFB20 may interact with CatSperαsubunits,particularly CatsSper1,to affect ion channel activity during sperm maturation.

Interaction between basigin and monocarboxylate transporter 2 in the mouse testes and spermatozoa

Basigin is a member of the immunoglobulin superfamily and plays various important roles in biological events including spermatogenesis. To examine the basigin molecular variants during spermatogenesis and sperm maturation in the mouse, immunoprecipitated basigin samples from testis and epididymal spermatozoa were analyzed by liquid chromatography-mass spectrometry/mass spectrometry （LC-MS/MS）. The results demonstrated that basigin molecules from the testis and spermatozoa were separable into two major bands and that the differences in the molecular sizes were possibly because of an endoproteolytic cleavage. Since basigin is known to be a chaperone for the monocarboxylate transporter 1 （MCT1）, the localization of basigin, MCT1 and MCT2 was examined during postnatal testicular development. Immunohistochemical studies showed different expression patterns of MCT1 and MCT2. MCT1 was localized on the surface of spermatogonia, spermatocytes, and spermatids. In contrast, MCT2 appeared on the principal piece of spermatozoa in the testis, where basigin was also observed. In mature epididymal spermatozoa, MCT2 was located on the midpiece, where basigin co-localized with MCT2 but not with MCT1. Furthermore, MCT2 was immunoprecipitated with basigin in mouse testes and sperm. These results suggest that basigin has a functional role as a binding partner with MCT2 in testicular and epididymal spermatozoa.

Dependence of sperm structural and functional integrity on testicular calcineurin isoform PPP3R2 expression

After leaving the testis,mammalian sperm undergo a sequential maturation process in the epididymis followed by capacitation during their movement through the female reproductive tract.These phenotypic changes are associated with modification of protein phosphorylation and membrane remodeling,which is requisite for sperm to acquire forward motility and induce fertilization.However,the molecular mechanisms underlying sperm maturation and capacitation are still not fully understood.Herein,we show that PPP3R2,a testis-specific regulatory subunit of protein phosphatase 3(an isoform of calcineurin in the testis),is essential for sperm maturation and capacitation.Knockout of Ppp3r2 in mice leads to male sterility due to sperm motility impairment and morphological defects.One very noteworthy change includes increases in sperm membrane stiffness.Moreover,PPP3R2 regulates sperm maturation and capacitation via(i)modulation of membrane diffusion barrier function at the annulus and(ii)facilitation of cholesterol efflux during sperm capacitation.Taken together,PPP3R2 plays a critical role in modulating cholesterol efflux and mediating the dynamic control of membrane remodeling during sperm maturation and capacitation.