Human protamines and the developing spermatid: their structure, function, expression and relationshipwith male infertility

<abstract>During spermiogenesis, the protamine proteins play an integral role in spermatid chromatin compaction. Recent research has focused on many facets of protamine biology, including protamine gene and protein structure/ function relationships, mechanisms of protamine expression regulation and involvement of the protamines in male fertility. In this paper, we review our current understanding of the structure and function of the protamine-1 (P1) and protamine-2 (P2) proteins and genes, the expression and regulation of these genes and the relationship between the protamines and male fertility. In addition, we offer a brief outlook on future investigation into protamine proteins.

Evidence for the role of KIF17 in fish spermatid reshaping:expression pattern of KIF17 in Larimichthys polyactis spermiogenesis

The homodimeric kinesin-2 protein KIF17 functions in intracellular transport and spermiogenesis in mammals.However,its role in fish spermiogenesis has not been reported.Here,we aimed to clone full-length kif17 cDNA and determine the molecular characteristics and expression patterns of KIF17 in Larimichthys polyactis spermiogenesis.The full-length cDNA of L.polyactis kif17(Lp-kif17)was sequenced and found to contain a 332-bp 5′untranslated region,480-bp 3′untranslated region,and 2433-bp open reading frame encoding 810 amino acids.Bioinformatics analyses showed that L.polyactis KIF17(Lp-KIF17)shared high sequence similarity with homologs in other animals and possessed an N-terminal motor domain with microtubule-binding sites and adenosine triphosphate(ATP)hydrolysis sites,a stalk domain containing two coiled-coil regions,and a C-terminal tail domain.The Lp-kif17 mRNA was widely expressed in various tissues,with the highest level in the brain,followed by that in the testis.Fluorescence in situ hybridization(FISH)analysis revealed that Lp-kif17 was continuously expressed in spermiogenesis,showing that it had potential functions in this process.Using immunofluorescence(IF)analysis,we found that Lp-KIF17 colocalized with tubulin and was transferred from the perinuclear cytoplasm to the side of spermatid where the tail forms during spermiogenesis.These findings suggested that KIF17 is involved in L.polyactis spermiogenesis.In particular,it may participate in nuclear shaping and tail formation by interacting with perinuclear microtubules during spermatid reshaping.In addition to providing evidence for the role of KIF17 in fish spermatid reshaping,this study provides important data for studies of reproductive biology in L.polyactis.

Two New Selenoproteins Found in the Prostatic Glandular Epithelium and in the Spermatid Nuclei

After labeling of rats in vivo with 75Se and protein separation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis more than 25 Se-containing bands could be distinguished.Of those proteins which were detected only in certain compartments and might therefore have tissue-specific functions, two were chosen for detailed investigation.A 15 kDa-protein was found in the prostatic epithelium where it accounted for about two thirds of the protein-bound 75Se. It was mainly present in the cytosol but was not released into the prostatic secretion. After gel chromatography it was found in the fraction which contained proteins with molecular masses of about 300 kDa. Using two-dimensional electrophoresis a plvalue of about 4. 5 was determined.In the testis a specific Se-containing 34 kDa-protein was observed which appeared after the onset of puberty. It was localized in the spermatid nuclei where it contained about 80% of the Se tracer present and was found to be bound to the DNA. After extraction it partly disintegrated into a 20 kDa-protein.Both compounds contain Se in the form of selenocysteine. The fact that their formation had priority over that of glutathione peroxidase during insufficient Se intake is an indication of their biological significance. Special interest in the prostatic epithelial selenoprotein derives from a possible inverse relationship between the Se status and the incidence of prostate cancer observed in epidemiological studies, whereas with the 34 kDa-selenoprotein its appearance during the condensation phase of the spermatid nuclei might suggest its participation in some processes of sperm maturation

The Effect of Tripterygium Wilfordii Monomer T4 on Rat Spermatid Nuclear Protein Transition

Rat testis elongating spermatids and epididymal sperms were collected after 7 weeks of treatment with Tripterygium wilfordii monomer T4.Total nuclear basic protein(TNBP)was extracted from the elongating spermatid nuclei and the sperm nuclei isolated by sonication.Polyacrylamide gel electrophoresis has been used to separate the TNBP and individual proteins were quantified by scanning microdensitometry.It was found that the content of protamine was reduced and the TH(Total Histones)/RP(Rat Protamine)ratios were increased following treatment in the testis elongating spermatids, and same result was found in the epididymal sperms.These results suggest that the interruption of nuclear protein transition of testis spermatids induced by T4 might cause aberrant epididymal sperm nuclear protein and lead to infertility.The relationship between protamine and fertility was discussed.

Immortalized Sertoli cell lines sk11 and sk9 and binding of spermatids in vitro

Aim： To determine the effectiveness of the ski 1, sk9 and ski 1 TNUA5 Sertoli cell lines in binding germ cells in vitro. Methods： The immortalized Sertoli cell lines sk9, ski 1 and ski 1 TNUA5 were used in co-culture experiments with germ cells in media with or without reproductive hormones and incubated for 44 h at 32℃. The number of germ cells bound to Sertoli cells was then determined and statistically analyzed. Western blot analysis and reverse transcriptasepolymerase chain reaction （RT-PCR） studies were employed to investigate the presence of cell adhesion proteins and follicle stimulating hormone （FSH） receptor, respectively. Results： No statistical difference between the number of bound step-8 spermatids and bound pre-step 8 spermatids on Sertoli cells from any of the cell lines existed. After the addition of germ cells, Sertoli ceils showed more lipid accumulation in their cytoplasm, indicating active phagocytosis. Western blot analysis in the ski I TNUA5 line indicated the expression of N-cadherin. FSH-only and testosterone-only treatments increased N-cadherin expression, regardless of germ cell addition. The addition of germ cells to the ski l TNUA5 Sertoli cells increased the expression of espin, as did the addition of FSH with germ cells. RT-PCR studies of the ski I TNUA5 cells indicated that the mRNA for FSH receptor decreased with successive passages. Conclusion： In vitro binding between isolated germ cells and sk9, skll or skll TNUA5 Sertoli cells is not feasible, and therefore these cell lines are not useful for the in vitro investigation of Sertoli-germ cell interactions and primary Sertoli cell isolates must still be used.

Characterization,expression dynamics,and potential function of OPA1 for regulation of mitochondrial morphology during spermiogenesis in Phascolosoma esculenta

Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular characteristics,expression dynamics and subcellular localization of optic atrophy protein 1(OPA1),a mitochondrial fusion and cristae maintenance-related protein,to reveal the possible regulatory mechanisms underlying mitochondrial morphology in Phascolosoma esculenta spermiogenesis.The full-length cDNA of the P.esculenta opa1 gene(Pe-opa1)is 3743 bp in length and encodes 975 amino acids.The Pe-OPA1 protein is highly conservative and includes a transmembrane domain,a GTPase domain,two helical bundle domains,and a lipid-interacting stalk.Gene and protein expression was higher in the coelomic fluid(a site of spermatid development)of male P.esculenta and increased first and then decreased from March to December.Moreover,their expression during the breeding stage was significantly higher than during the non-breeding stage,suggesting that Pe-OPA1 is involved in P.esculenta reproduction.The Pe-OPA1 protein was more abundant in components consisting of many spermatids than in components without,indicating that Pe-OPA1 mainly plays a role in the spermatid in coelomic fluid.Moreover,Pe-OPA1 was mainly detected in the spermatid mitochondria.Immunofluorescence experiments showed that the Pe-OPA1 are constitutively expressed and co-localized with mitochondria during spermiogenesis,suggesting its involvement in P.esculenta spermiogenesis.These results provide evidence for Pe-OPA1's involvement in the regulation of mitochondrial morphology during spermiogenesis.

圆头精子注射临床疗效的Meta分析

目的探讨圆头精子注射(ROSI)应用于圆头精子症患者的临床疗效。方法检索Medline、Embase、Cochrane Library以及ClinicalTrials.gov数据库,纳入从建库至2022年10月关于圆头精子症患者应用ROSI治疗的研究,采用荟萃分析与亚组分析评估ROSI的临床疗效。结果共纳入29项研究,共计1363例患者进行分析。ROSI后的总受精率(2PN)为37.9%(95%CI:30.7%~45.4%),总妊娠率为3.8%(95%CI:0.8%~8.1%),最终分娩率为1.8%(95%CI:0%~5.6%)。改进圆头精子的鉴别方法之后,ROSI后的妊娠率(21.2%)和分娩率(13.1%)有所提高;亚洲地区的受精率(41.5%)高于非亚洲地区(33.4%),但其妊娠率和分娩率(2.8%、0.9%)低于非亚洲地区(6.4%、5.9%)。结论ROSI对圆头精子症患者的生育具有积极意义,不同地区间ROSI治疗结局有差异。改善圆头精子的鉴别方法之后,ROSI的临床疗效得到提高。

BET bromodomain inhibitor JQ1 regulates spermatid development by changing chromatin conformation in mouse spermatogenesis

As a BET bromodomain inhibitor, JQ1 has been proven have efficacy against a number of different cancers. In terms of male reproduction, JQ1 may be used as a new type of contraceptive, since JQ1 treatment in male mice could lead to germ cell defects and a decrease of sperm motility, moreover, this effect is reversible. However, the mechanism of JQ1 acting on gene regulation in spermatogenesis remains unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse testes treated with JQ1 or vehicle control to determine the transcriptional regulatory function of JQ1 in spermatogenesis at the single cell resolution. We confirmed that JQ1 treatment could increase the numbers of somatic cells and spermatocytes and decrease the numbers of spermatid cells. Gene Ontology (GO) analysis demonstrated that differentially expressed genes which were down-regulated after JQ1 injection were mainly enriched in “DNA conformation change” biological process in early developmental germ cells and “spermatid development” biological process in spermatid cells. ATAC-seq data further confirmed that JQ1 injection could change the open state of chromatin. In addition, JQ1 could change the numbers of accessible meiotic DNA double-stranded break sites and the types of transcription factor motif that functioned in pachytene spermatocytes and round spermatids. The multi-omics analysis revealed that JQ1 had the ability to regulate gene transcription by changing chromatin conformation in mouse spermatogenesis, which would potentiate the availability of JQ1 in male contraceptive.

Subzonal fertilization of mouse round spermatids

Mouse round spermatids were electrofused with homologous mature oocytes to examine the be-haviour of their nuclei within the ooplasm and the abilities of development. A single spermatid was injected in the perivitelline space of a mature oocyte and an electron fusion pulse was given. The best round spermatid-oocyte pairs (RS-O) fusion took place at 20-30 s AC (1 MHz, 50V/cm) followed by a single fusion DC pulse (3 700-3 800 V/cm, 25 μs) and another 30 s AC current. The total survival rate and fusion rate of RS-O were 89.0% (575/646) and 61. 9% (356/575), respectively. 49.2% (175/356) of fused oocytes developed to 2PN stage . The concentration of Ca2+ in the fusion medium produced no significant effect on the above targets. The 2PN development rate of the fused RS-O from the oocytes collected 14-16 h after hCG injection was higher than others. 32.6% (57/175) of the 2PN oocytes had fully developed spermatid (male) and oocyte (female) pronuclei. The rest spermatid-derived pronu-clei remained small in size throughout the pronuclear stage. 192 fertilized eggs were transferred surgically into the oviducts of the pseudopregnant female mouse. 12 offspring were produced.

小鼠睾丸精子细胞的分离与鉴定

目的 :建立一种简单、可有效分离小鼠睾丸精子细胞的方法。 方法 :用组合酶消化成年小鼠睾丸制备睾丸细胞悬液 ,然后经 6层非连续Percoll梯度 (15 %、2 2 %、30 %、4 0 %、5 0 %和 6 0 % )离心法分离 ,通过形态学和流式细胞术鉴定各个Percoll梯度中精子细胞的含量 ,并以伊红Y排斥试验测定细胞的存活率。 结果 :组合酶消化后获得的睾丸细胞悬液中 ,97%以上细胞仍然存活。经Percoll梯度离心 ,共形成 6条细胞带 ,其中 2 2 %梯度中主要为精子细胞 (平均 86 .7% ,P <0 .0 5 ) ,85 .5 %以上细胞存活 ;而 30 %梯度中细胞密度最高 (P <0 .0 5 ) ,含有各级未成熟生精细胞 ,存活细胞超过 92 %。 结论 :采用组合酶消化结合非连续Percoll梯度离心法 。

小鼠精子细胞变态成形过程中manchette的免疫荧光染色分析

目的通过观察manchette结构在小鼠精子细胞中的定位和形态变化,探讨其在小鼠精子细胞变态成形过程中的作用和意义。方法利用免疫荧光FITC/DAPI共染技术显示manchette结构在小鼠精子细胞变态成形各阶段的细胞内定位,观察精子细胞成熟过程中manchette结构的形态学改变。结果manchette结构紧密附着在精子细胞核表面;该结构在圆形精子细胞核变形和延伸的起始阶段形成,并随着精子细胞核的浓缩和变长逐渐向精子细胞尾部移位,直至精子变态成形后消失;在精子细胞变态成形过程中,manchette随着精子细胞核形态的改变逐渐从"帽状"结构变形为"微管样"结构。结论Manchette结构的形成和消失与精子细胞核的浓缩及延伸同步,其形态变化和位置改变与精子细胞核的形态学变化相吻合,在小鼠精子细胞变态成形过程中具有重要意义。

中药二仙汤及其拆方对老年大鼠精子细胞和精子的亚微结构和SDH的作用

本文观察了二仙汤及其温肾药和泻火药两个拆方对老年(20月龄)大鼠睾丸精子细胞、精子的亚微结构,以及精子尾部中段琥珀酸脱氢酶(SDH)的作用。结果证明,老年对照组大鼠精子细胞和精子退化显著,精子细胞顶体形成障碍,高尔基体多消失;精子顶体薄窄,尾部中段线粒体鞘多变性、缩窄,SDH 反应颗粒减少,甚至消失。二仙汤及其拆方具有不同程度地改善老年大鼠精子细胞和精子的亚微结构,使SDH 反应颗粒增多。

褐菖■精细胞晚期的变化及精子结构研究

本文研究卵胎生硬骨鱼褐菖（Ｓｅｂａｓｔｉｓｃｕｓｍａｒｍｏｒａｔｕｓ）精细胞的成熟变化和精子结构。褐菖精细胞发育晚期已具有硬骨鱼类精子的结构雏形：细胞核的背面较平坦，腹面稍外鼓，呈弧面；染色质浓缩成团块状，核的腹侧和后端的染色质较致密；中心粒复合体由近端中心粒和基体组成，近端中心粒和基体排成“Ｌ”形；近端中心粒向细胞核的背侧伸出中心粒附属物，中心粒附属物由９条微管组成，９条微管围成一筒状结构，类似轴丝。在晚期精细胞形成精子的过程中，中心粒附属物和近端中心粒相继退缩以至消失不见，同时细胞核后端的形状也随着发生变化。中心粒附属物和近端中心粒的相继消失可以看作是成熟的最后标志。精子的中心粒复合体由基体及其上方的基体帽组成，袖套接于核的后端，其中约有３０～４０个线粒体；鞭毛从袖套腔中伸出，鞭毛的中心结构是轴丝；轴丝外方为细胞质形成的侧鳍，在鞭毛的近核段，轴丝两侧的侧鳍较宽且不对称。

大鼠精核蛋白的纯化

用大鼠睾丸制备长形精子细胞核及用附睾制备的附睾精子核，用盐酸胍法提取总碱性蛋白（Ｔｏｔａｌ ｂａｓｉｃ ｐｒｏｔｅｉｎ，ＴＢＰ）。通过Ｓｅｐｈａｄｅｘ Ｇ－１００分子筛层析，其第三峰用５％ＴＣＡ沉淀，得到纯大鼠精核蛋白（Ｒａｔ ｐｒｏｔａｍｉｎｅ，ＲＰ）。ＲＰ也可用ＴＢＰ通过ＨＰＬＣ反相柱得到纯化，ＲＰ位于２６％－２８％乙腈梯度。

水泥池养殖香鱼性腺发育的观察

对水泥池养殖的香鱼卵巢、精巢发育做组织学观察,并与洄游型、陆封型香鱼的性腺发育做比较:养殖香鱼卵母细胞的结构和洄游型香鱼、陆封型香鱼基本一致;受精孔和精孔细胞出现的时间早于洄游型香鱼和陆封型香鱼。养殖香鱼卵巢发育略早于野生香鱼,但发育速度比野生香鱼慢,尤其慢于陆封香鱼。精巢结构属叶型壶腹型结构。雄鱼发育成熟早于雌鱼,能自然排精。雌鱼不用激素诱导即可获得游离卵。

小鼠精子和球形精子细胞显微注射受精研究

目的研究动物受精机理。方法以昆明白鼠为实验动物，利用显微注射技术，对精子和球形精子细胞的显微注射受精进行了研究。结果１．带下注射受精，注射４～６个精子的卵的受精率（３２．５％）和卵裂率（２５．０％）显著高于注射单个精子的卵的受精率和卵裂率（分别为１２．５％和６．３％）（Ｐ＜０．０５）。２．带下注射４～６精子后，有第１极体卵母细胞的存活率（６９．６％）、受精率（３２．５％）和卵裂率（２５．０％）分别高于无第１极体卵母细胞的存活率、受精率和卵裂率（分别为６４．４％、２０．７％和１３．８％），但两者无显著差异（Ｐ＞０．０５）。３．胞质内精子注射卵的存活率（２１．４％）极显著低于带下注射卵的存活率（６９．６％）（Ｐ＜０．０１）；而胞质内精子注射卵的受精率（３５．５％）、卵裂率（３２．３）％和囊胚率（２０．０％）则高于带下注射卵的受精率（３３．３％）、卵裂率（２５．０％）和囊胚率（１０．０％），但无显著差异（Ｐ＞０．０５）。４．将球形精子细胞注入经电活化处理的卵母细胞的透明带下，注射卵的存活率为９０．５％，电融合率为３４．６％，２－细胞分裂率为２８．３％。结论１．注射精子的数量对小鼠带下注射的受精率和卵裂率有显著影响?

近日节律基因Clock对雄性小鼠生殖功能的影响

目的通过干扰小鼠睾丸节律基因Clock的表达,研究Clock对雄性小鼠生殖能力的影响。方法通过RNA干扰技术干扰雄性小鼠睾丸节律基因Clock的表达,以Western blot检测干扰效果,研究干扰Clock表达后对小鼠体内胎仔数和精子数量、精子活力、体外受精率的影响。结果Clock干扰质粒使小鼠睾丸Clock蛋白的表达明显下调。在体内实验不但影响了雄性小鼠的胎仔数,而且其相对应的精子体外受精能力也明显下降。结论节律基因Clock与雄性小鼠的生殖功能有密切的关系。

小鼠精原细胞体外分化为精子细胞的研究

目的探讨在辅以外源生殖激素混合培养的小鼠睾丸细胞中，精原细胞向精子细胞的转化。方法采用7～8d龄小鼠睾丸组织，以组合酶消化法制备睾丸细胞，在含重组卵泡刺激素（r-FSH）和睾酮的培养基中进行体外培养；定期观察细胞的生长和形态变化；用分子生物学和流式细胞技术对生长各阶段细胞进行分析。结果培养5d即可观察到形态与大小类似于圆形精子细胞，7d后可见带有鞭毛与变形的精子细胞；逆转录聚合酶链反应（RT-PCR）结果显示，培养前细胞的睾丸特异蛋白激酶（TESK1）与鱼精蛋白2（Prm2）mRNA表达阴性，培养9与17d细胞TESK1与Prm2 mRNA表达均阳性；DNA倍体分析显示，培养5d细胞有单倍体峰出现，并随培养天数增加而增加。结论在体外混合培养的小鼠睾丸细胞中加入外源生殖激素可以使精原细胞转化为精子细胞。

羊睾丸提取液对铅损伤小鼠精子顶体形成的影响

目的观察羊睾丸提取液对铅损伤小鼠精子顶体形成的影响。方法成年健康清洁级ICR雄性小鼠30只,随机分为对照组、模型组和给药组,每组10只。模型组采用醋酸铅(30mg/kg)灌胃建立睾丸损伤模型,给药组在醋酸铅灌胃的同时腹腔注射羊睾丸提取液0.5ml,对照组采用等体积蒸馏水灌胃及腹腔注射,每天1次,共21d。经心脏行甲醛灌流固定,制备睾丸标本,PAS染色,光镜下观察圆形精子细胞期顶体的形态学变化。结果与对照组相比,模型组精子顶体形成障碍,顶体囊泡不完整,膜皱缩,较粗糙,结构模糊;给药组精子顶体形成接近对照组,顶体囊泡形状较规则,膜平滑。结论羊睾丸提取液可保护小鼠精子顶体在形成过程中免受铅的损害,从而维持其正常的结构及功能。

阿维菌素对小鼠早期精细胞的微核效应

阿维菌素是北京农业大学研制的一种新型、安全、广谱、高效的抗寄生虫药物。本文以小鼠早期精细胞微核试验方法研究其对雄性生殖细胞的诱变性。将昆明系雄性小鼠随机分为５组，每组５只，第１、２和３组为低、中和高３个剂量组，分别以１．９２ｍｇ／ｋｇ、４．８０ｍｇ／ｋｇ和９．６０ｍｇ／ｋｇ（相当于１／１０ＬＤ５０、１／４ＬＤ５０和１／２ＬＤ５０）剂量的阿维菌素丙二醇溶液给小鼠一次灌胃。第４组为阳性对照组，以８０ｍｇ／ｋｇ环磷酰胺给小鼠一次腹腔注射。第５组为阴性对照组，给小鼠灌服溶剂，于给药后第１４天杀鼠制片。各组小鼠早期精细胞的微核率分别为：阴性对照组１．２±０．４５‰，阳性对照组５．６±１．１４‰，阿维菌素低剂量组１．８±０．８４‰，中剂量组１．８±０．４５‰和高剂量组２．０±１．００‰。生物统计结果表明，环磷酰胺阳性对照组与阴性对照组相比，差异极显著（Ｐ＞０．０１）；阿维菌素各剂量组与阴性对照组比较，差异均不显著（Ｐ＞０．０５），试验结果均为阴性。本研究表明阿维菌素对于精母细胞前细线期不具诱变作用。本研究对小鼠早期精细胞微核试验方法进行了一些改进。试验结果表明，早期精细胞微核试验是一种有前途的评价兽药雄性生殖毒性的试验?