棘蛙族(Tribe Paini)精子形态结构及精巢年周期研究

棘蛙族(Tribe Paini)隶两栖纲(Amphibia)、无尾目(Anura)、蛙科(Ranidae)、叉舌蛙亚科(Dicroglossinae)，由棘蛙属(Paa)、倭蛙属(Nanorana) 和沙巴蛙属(Chaparana)构成（Dubois,1992）。由于特殊的形态特征和染色体核型，棘蛙族受到国内外学者的广泛重视和研究，但是到目前为止，棘蛙族的系统发育关系尚未明晰，族下属种的分类和归属问题还有待进一步研究和新的证据出现。本文通过光学显微镜、电子显微镜和石蜡切片对棘蛙族10 物种的精子和精巢进行研究，旨在了解棘蛙族精子的形态、量度、超微结构特征及不同季节精巢结构的变化规律，同时为棘蛙族的系统研究提供新的依据，也为棘蛙族濒危物种的保护和经济物种的繁殖提供基础资料。研究结果表明：棘蛙族各属物种精子的形态基本相似，精子整体呈线形，由头部、中片和尾部构成。精子头部呈长条状，顶体呈锥状，位于头部顶端并向前伸出，中片较长，尾部波动弯曲。棘蛙族各属物种精子量度差异较大，将各属物种精子头部、中片、尾部、头宽、尾宽的量度数据进行聚类分析，结果表明棘蛙族10 物种可分为三类：第一类包括棘侧蛙、合江棘蛙、小棘蛙、棘腹蛙和棘胸蛙，特点是精子较短，全长在72.6～103.35µm 之间；第二类包括倭蛙、高山倭蛙、腹斑倭蛙，特点是精子较长，全长在107.74～129.75µm 之间；第三类包括隆肛蛙和双团棘胸蛙，特点是精子最长，全长在145.89～165.84µm 之间。棘蛙族各属精子超微结构基本相似：精子头部由顶体、细胞核构成；中片由中心粒、线粒体构成；尾部由单根轴丝构成。精子顶体横切呈圆环状，细胞核电子密度高；线粒体为卵圆形，呈环状围绕轴丝排列，线粒体数目较多，约30层；尾部轴丝为典型的9＋2结构，即由2根中央微管和9对外周微管组成。不同季节的倭蛙精巢结构变化表明倭蛙精巢每年只有一个生精周期，生精周期始于7 月，繁殖季节从5 月到6 月，生精高峰期为9 月；根据倭蛙不同季节精巢结构的变化，可将生精周期分为3 个阶段：第一阶段从7 月到9 月，为精子形成期；第二阶段从10 月到翌年4 月，为精子的贮存阶段，也即倭蛙的冬眠期；第三阶段从5 月到6 月，为精子的排放阶段，即倭蛙的繁殖期。不同季节的隆肛蛙精巢结构变化表明5 月为隆肛蛙的繁殖高峰期。根据棘蛙族各属精子的形态、量度和超微结构特征，结合已有的棘蛙族形态学、生态学、染色体核型及系统学研究成果，本文认为：1．基于精子数据对棘蛙族的划分和基于形态学及分子系统学数据对棘蛙族的划分均有相同之处，精子形态结构可为棘蛙族的系统研究提供新的证据。2． 棘蛙族各属精子的形态、量度及超微结构不仅与蛙科其他属种有明显差异，而且在无尾类中也较为特殊，精子学研究结果支持将棘蛙族从蛙科中分离出来，归隶于叉舌蛙科的叉舌蛙亚科的系统学修正。3. 精子的顶体、细胞核、中片的形态结构及量度可作为蛙科的分类指标。On the base of unique morphological and kyrotype characters, Dubois(1992)recognized three genera Paa, Narnorana, Chaparana as tribe Paini, which is amember of Dicroglossinae, Ranidae. In present study, the sperm shape, size andultrastructure of 10 paini species were investigated through the light and electronmicroscope, and testis structure of N. pleskei and F. quadrana was also studied. Wesuppose this study could offer some spermatological evidence to phylogeny andreproduction study of tribe Paini. The results were as follows:The sperm shape of tribe paini is homologically similar, the spermatozoa arefiliform, composed of elongate head, long mid-piece and waved tail. The acrosome isapically associated with the nucleus and extend anteriorly.The sperm length of tribe paini differ remarkably among genera. Cluster for thelength of sperm head, mid-piece, tail, total length, head-width, tail-width of ten painifrogs indicated the 10 species could be separated into three groups: GroupⅠcontainsP. shini, P. robertingeri, P. spinosa, P. exilispinosa, P. boulengeri, the spermatozoa ischaracterized with short in total length, ranging from 72.6µm to 103.35µm; GroupⅡcontains N. pleskei, N. parkeri, N. ventripunctata, the spermatozoa ischaracterized with relatively long in total length, ranging from 107.74µm to129.75µm; Group Ⅲ contains F. quadrana and P. yunnanensis, the spermatozoa is characterized with longest in total length, ranging from 145.89µm to 165.84µm. thethree groups based on spermatological data is partially match the classification basedon morphological and molecular data.The ultrastructure of spermatozoa in tribe paini is also basic similar, includingacrosome vescile, nuleus of the head proper, centriole, mitochondriol of themid-pieces, axoneme of the tail. The acrosome vescle is circle in TEM transversesection, the density of nucleus is high; The mitochondrions is oval, surrounding theaxial filament with about 30 layers of mitochondria; The axoneme has the typical 9+2pattern of microtubules.The seasonal changes in testis of N. pleskei indicates it has only onespermatogenesis circle, which begin in July, the reproduction season is from May toJune, the spermatogenesis is active in September. On the base of seasonal changes intestis, the spermatogenesis circle can be separated into three stages: In stageⅠfromJuly to September, spermatids are formed; In stage Ⅱ from October to April next year,the spermatozoa are stored in testis,which is the hibernated period; In stage Ⅲ fromMay to June, mature spermatozoa were released from the testis, which is thereproduction season of N. pleskei. As to F. quadrana, reproduction is active in May.With the previous study of morphology, ecology, karyotypes and phylogenyresearch of tribe Paini, the spermatological data in present study suggests:1. The spermatological classification of tribe paini is partially consistant with themorphological and molecular classification respectively.2.The sperm morphology and ultrustructure of tribe paini is unique not only inthe family Ranida but also in Anura, which suggest the tribe paini is monophyletic andmight be transfered from the family Ranida to the family Dicroglossidae based onmolecular evidence.3. The acrosome, nuleus, shape, length and ultrastructure of mid-piece can beused as an alternative taxonomic character in Anura.

Alleviation of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma-ray on male reproductive endocrine damages induced by subsequent high-dose irradiation

Irradiation has been widely reported to damage organisms by attacking on proteins, nucleic acid and lipids in cells. However, radiation hormesis after low-dose irradiation has become the focus of research in radiobiology in recent years. To investigate the effects of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma (gamma)-ray on male reproductive endocrine capacity induced by subsequent high-dose irradiation, the brains of the B6C3F(1) hybrid strain male mice were irradiated with 0.05 Gy of C-12(6+) ion or Co-60 gamma-ray as the pre-exposure dose, and were then irradiated with 2 Gy as challenging irradiation dose at 4 h after pre-exposure. Serum pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), testosterone, testis weight, sperm count and shape were measured on the 35th day after irradiation. The results showed that there was a significant reduction in the levels of serum FSH, LH, testosterone, testis weight and sperm count, and a significant increase in sperm abnormalities by irradiation of the mouse brain with 2 Gy of C-12(6+) ion or Co-60 gamma-ray. Moreover, the effects were more obvious in the group irradiated by C-12(6+) ion than in that irradiated by Co-60 gamma-ray. Pre-exposure with low-dose C-12(6+) ion or Co-60 gamma-ray significantly alleviated the harmful effects induced by a subsequent high-dose irradiation.

Melatonin modulates acute testicular damage induced by carbon ions in mice

The present study was performed to obtain evidence of the radioprotective function of melatonin at different administration levels on carbon ion-induced mouse testicular damage. Outbred Kun-Ming strain mice were divided into six groups, each composed of eight animals: control group, melatonin alone group, irradiation group and three melatonin plus irradiation-treated groups. An acute study was carried out to determine alterations in DNA-single strand break, cell apoptosis, and oxidative stress parameters as well as histopathology in mouse testis 24 h after whole-body irradiation with a single dose of 4 Gy Tie results showed that pre-treatment and post-treatment with high-dose melatonin (10 mg/kg) both significantly alleviated carbon ion-induced acute testicular damage, a greater radioprotective effect being observed in the pre-treatment group. On the other hand, low-dose melatonin (1 mg/kg) had a limited radioprotective effect on irradiation-induced degeneration and DNA lesions in mouse testis. Taken together, the data suggest that prophylactic treatment with a higher dose of melatonin is probably advisable to protect against the effects of heavy-ion irradiation.

The immobilization of proteins on biodegradable polymer fibers via click chemistry

A facile and efficient method to immobilize bioactive proteins onto polymeric substrate was established. Testis-specific protease 50 (TSP50) was immobilized on ultrafine biodegradable polymer fibers, i.e., (1) to prepare a propargyl-containing polymer P(LA90-co-MPCIO) by introducing propargyl group into a cyclic carbonate monomer (5-methyl-5-propargyloxycarbonyl-1,3-dioxan2-one, MPC) and copolymerizing it with L-lactide; (2) to electrospin the functionalized polymer into ultrafine fibers; (3) to azidize the TSP50, and (4) to perform the click reaction between the propargyl groups on the fibers and the azido groups on the protein.

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Isolation and mapping of telomeric pentanucleotide (TAACC)(n) repeats of the pacific whiteleg shrimp, Penaeus vannamei, using fluorescence in situ hybridization

To develop genetic and physical maps for shrimp, accurate information on the actual number of chromosomes and a large number of genetic markers is needed. Previous reports have shown two different chromosome numbers for the Pacific whiteleg shrimp, Penaeus vannamei, the most important penaeid shrimp species cultured in the Western hemisphere. Preliminary results obtained by direct sequencing of clones from a Sau3A-digested genomic library of P. vannamei ovary identified a large number of (TAACC/GGTTA)-containing SSRs. The objectives of this study were to (1) examine the frequency of (TAACC)(n) repeats in 662 P. vannamei genomic clones that were directly sequenced, and perform homology searches of these clones, (2) confirm the number of chromosomes in testis of P. vannamei, and (3) localize the TAACC repeats in P. vannamei chromosome spreads using fluorescence in situ hybridization (FISH). Results for objective I showed that 395 out of the 662 clones sequenced contained single or multiple SSRs with three or more repeat motifs, 199 of which contained variable tandem repeats of the pentanucleotide (TAACC/GGTTA),, with 3 to 14 copies per sequence. The frequency of (TAACC)n repeats in P. vannamei is 4.68 kb for SSRs with five or more repeat motifs. Sequence comparisons using the BLASTN nonredundant and expressed sequence tag (EST) databases indicated that most of the TAACC-containing clones were similar to either the core pentanucleotide repeat in PVPENTREP locus (GenBank accession no. X82619) or portions of 28S rRNA. Transposable elements (transposase for Tn1000 and reverse transcriptase family members), hypothetical or unnamed protein products, and genes of known function such as 18S and 28S rRNAs, heat shock protein 70, and thrombospondin were identified in non-TAACC-containing clones. For objective 2, the meiotic chromosome number of P. vannamei was confirmed as N = 44. For objective 3, four FISH probes (P1 to P4) containing different numbers of TAACC repeats produced positive signals on telomeres of P. vannamei chromosomes. A few chromosomes had positive signals interstitially. Probe signal strength and chromosome coverage differed in the general order of P1 > P2 > P3 > P4, which correlated with the length of TAACC repeats within the probes: 83, 66, 35, and 30 bp, respectively, suggesting that the TAACC repeats, and not the flanking sequences, produced the TAACC signals at chromosome ends and TAACC is likely the telomere sequence for P. vannamei.

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

Oestrogen shuts the door on SOX9.

Oestrogen exerts a robust yet imperfectly understood effect on sexual development in vertebrate embryos. New work by Pask and colleagues in BMC Biology indicates that it may interfere with male development by preventing nuclear localization of SOX9, a master regulator of the testis differentiation pathway. See research article http://www.biomedcentral.com/1741-7007/8/113.

Field methods for capture and measurement of three monkey species in Costa Rica.

A total of 54 free-ranging monkeys were captured and marked in Santa Rosa National Park, Costa Rica, during May 1985, and an additional 17 were captured during March 1986. The animals were darted using a blowpipe or a CO2 gun. The drugs used were Ketaset, Sernylan and Telazol. Ketaset was effective for Cebus capucinus but unsuccessful for Alouatta palliata and Ateles geoffroyi. Sernylan was successful for A. geoffroyi and A. palliata but is no longer commercially available. Telazol proved to be an excellent alternative capture drug for both A. palliata and A. geoffroyi.

CYSTATIN RELATED EPIDIDYMAL SPERMATOGENIC PROTEIN RESIDES IN THE OUTER DENSE FIBRES AND LIKELY TRANSIENTLY ASSOCIATES WITH THE SURFACE OF EPIDIDYMAL MOUSE SPERMATOZOA

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm.

Effects of vasectomy on spermatogenesis and fertility outcomes after assisted conception

Abstract BACKGROUND: Each year 40,000 men have a vasectomy in the UK whilst another 2400 request a reversal to begin a second family. Sperm can now be obtained by testicular biopsy and subsequently used in assisted conception with intracytoplasmic sperm injection (ICSI). The study aims were to compare sperm yields of men post-vasectomy or with obstructive azoospermia (OA) of unknown aetiology with fertile men and to assess any alteration in the clinical pregnancy rates after ICSI. METHODS: Testicular tissue was obtained by Trucut needle from men who had undergone a vasectomy >5yrs previously, had OA from other causes and from fertile men during vasectomy. Seminiferous tubules were milked to measure sperm yields. Numbers of Sertoli cells, spermatids and thickness of the seminiferous tubule walls were assessed using quantitative computerized analysis. RESULTS and CONCLUSIONS: Sperm yields/g testis were significantly decreased in men post-vasectomy and in men with OA, relative to fertile men. Significant reductions were also observed in early (40%) and mature (29%) spermatid numbers and an increase of 31% was seen in the seminiferous tubule wall (basal membrane and collagen thickness) of vasectomised men compared to fertile men. Clinical pregnancy rates in couples who had had a vasectomy were also significantly reduced.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Advanced Glycation End Products (AGEs) accumulate in the Reproductive Tract of Men with Diabetes.

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable affects of diabetes on sperm function. Specifically, a recent study has found significantly higher sperm nuclear DNA (nDNA) fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and 9 non-diabetic subjects. CML immunoreactivity was found through out the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells (cytoplasm and nuclei) of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggests that these compounds may play a hitherto unrecognized role in male infertility.

Protein expression pattern of CDK11(p58) during testicular development in the mouse.

Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11(p58) is a p34(cdc2) related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11(p58) during postnatal development in mouse testes and examined the cellular localization of CDK11(p58) and cyclinD3, which was associated with CDK11(p58) in mammalian cells. Western blot analysis revealed that CDK11(p58) was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11(p58) was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11(p58) were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11(p58) expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11(p58) and cyclinD3 in the adult testis was revealed by immunofluorescence analysis.