粗糙沼虾精巢发育的组织学

利用光镜技术,对粗糙沼虾精巢发育进行了研究,根据精子发生过程中每种生殖细胞所占的比例和发生的次序,并结合精巢的形态特征,把精巢发育过程分为五个时期,即精原细胞期,精母细胞期,精细胞期,成熟精子期及退化期,精原细胞期,精巢小,透明乳白色,生精小管内的生殖细胞以精原细胞为主;精母细胞期;精巢体积增大,半透明乳白色,主要由处于初级精母细胞的次级精母细胞阶段的生殖细胞组成;精细胞期,精巢体积继续增大,颜色加深,生精小管内的生殖细胞以精细胞为主;成熟精子期,精巢体积可达最大,紫红色,生精小管内充满着成熟的精子,退化期;精巢体积减小,半透明乳白色,生精小管内的成熟精子几乎排空。     

蓝太阳鱼第一次性周期性腺发育的组织学

蓝太阳鱼(Lepomis cyanellus)是广东省于1997年从北美引进的小型淡水鱼类,本文采用组织切片技术对蓝太阳鱼的性腺发育进行了研究。蓝太阳鱼的性腺发育程序可以分为5个时期。在1月龄,卵巢和精巢处于第Ⅰ期;2~3月龄时,卵巢和精巢发育到第Ⅱ期;4月龄时发育到第Ⅲ期;5~6月龄时发育到第Ⅳ期;7~8月龄时达到性成熟第Ⅴ期。过冬时,卵巢退化到第Ⅱ~Ⅲ期,而精巢仍停留在第Ⅴ期。成熟卵巢的成熟系数为2.80%~8.10%,成熟精巢的成熟系数为1.45%~3.45%。精巢和卵巢发育都为不完全同步型,且精巢发育比卵巢稍快。蓝太阳鱼的繁殖期在广州地区为3~11月,为多次产卵类型。本文从生殖和生长的关系上对蓝太阳鱼生长缓慢的原因进行了初步探讨,并将蓝太阳鱼作为一种鱼类实验动物的可行性进行了分析。     

褶皱臂尾轮虫的培养试验

本文是作者于1989年5—10月间,于广西防城县在锯缘青蟹人工育苗试验中进行的试验,试验用普通面粉及黄豆加工成发酵面粉与豆浆作轮虫的主饵料,用动物性饵料如小鱼汁、螺肉汁、蛤肉汁、发酵人尿等含有机质丰富的液体作辅助饵料,用7个25m~3的大池培养,共培养出了约900×10~(10)多个轮虫,供给锯缘青蟹幼体足够的活饵料,使当年培养出C_1期以上的幼蟹达22.269万只。本试验中,光照对轮虫的生长繁殖有较大影响。     

拟穴青蟹OTUB的分子克隆及其在性腺发育过程中的表达

OTUB1是哺乳动物雌激素信号通路中的一个关键因子,它可以使雌激素受体α(ERα)去泛素化并抑制其转录活性.本研究从实验室拟穴青蟹(Scylla paramamosain)转录组测序数据库中筛选出OTUB基因片段,利用SMART RACE技术克隆得到Sp-OTUB的cDNA全长.该序列全长1 261 bp,包括5’非编码区(5’-UTR)40 bp,开放阅读框(ORF)804 bp和3’非编码区(3’-UTR)417 bp.其ORF编码267个氨基酸,预测其分子量为31 ku.该蛋白序列中包含一个OTU样的半胱氨酸蛋白酶催化结构域和一个由半胱氨酸(C)、天冬氨酸(D)和组氨酸(H)组成的催化三联体.实时定量PCR结果显示,Sp-OTUB在成熟拟穴青蟹各组织器官中均有表达,其中在血淋巴中的表达量最大,精巢中表达量最低.在卵巢发育过程中,Sp-OTUB的表达量在卵巢增值期为最大,与其他各时期具有显著差异;在精巢发育各阶段,Sp-OTUB表达量在精子细胞期达到最大,显著高于精母细胞期和成熟精子期.同源系统进化分析Sp-OTUB与大多数物种的OTUB1聚为一支,与肩突硬蜱(Ixodes scapularis)和紫海胆(Strongylocentrotuspurpuratus)亲缘关系最近.实验结果初步显示Sp-OTUB在拟穴青蟹生殖调控方面可能发挥重要作用.     

浙江乐清湾锯缘青蟹渔业生物学初步研究

锯缘青蟹(Scylla serrata)系为热带性种,在我国主要分布于南方诸省沿海,是构成潮间带滩栖游泳蟹类的优势种群之一。锯缘青蟹(以下简称青蟹)对环境适应性强,具有食性杂、生长快、易养殖、营养丰富、肉味鲜美、商品价值高等优点,近年来是颇受各国重视的优良养殖品种。由于对青蟹的生物学研究缺少,尚不能开展大规模的养殖。为此,作者于1979—1983年对具有亚热带海区特点的乐清湾青蟹生物学及其增殖作了深入的研究,现整理分述如下。     

长江刀鲚与池塘人工养殖刀鲚性腺发育的初步观察

2006～2008年间采集了长江刀鲚(Coilia nasus)及池塘养殖刀鲚共104尾,对其生物学指标进行了测定,并对不同月份、不同江段的刀鲚性腺发育状况进行了初步比较观察。组织切片观察显示:在江阴段长江刀鲚从4～7月份卵巢从Ⅱ期发育到Ⅳ期;同一时期(7月份)安庆段刀鲚精巢和卵巢的成熟系数略高于江阴段,但差异不大。池塘养殖雄性刀鲚在6月、9月基本处于增殖期,精小叶腔中存在一定量的精子细胞,但未见性成熟个体。对于雌性,在12月份卵巢处于Ⅱ期,而6月份卵巢能发育至Ⅳ期晚期,此时卵巢的成熟系数显著高于同年5月份江阴长江刀鲚和7月份安庆段刀鲚。综合组织切片结果认为:长江刀鲚的性腺发育成熟度可能与所处江段关系不大,而不同洄游群体的性腺发育情况可能相同;在人工池塘养殖状态下,部分刀鲚的卵巢至少能够发育到Ⅳ期晚期。     

锯缘青蟹(Scylla serrata)Hsp70基因cDNA的克隆及序列分析

采用RT-PCR及RACE技术克隆锯缘青蟹(Scylla serrata)的热休克蛋白Hsp70基因并进行序列分析。克隆测序后拼接得到一条长2482 bp的cDNA序列,该序列ORF(Open reading frame,开放阅读框)为1950 bp,编码649个氨基酸,分子量约为71.06 kD,理论等电点为5.24。3'UTR(untranslated region,非编码区)为158 bp,5'UTR为40 bp。通过antheprot分析发现2个Hsp70家族的签名序列:IFDLGGGTFDVSIL,IVLVGGSTRIPKIQK;Dnak特征基序DLGTT-S-V;非细胞器基序:RARFEEL;核定位信号标签:KKDPSESKRALRRL;胞质Hsp70特征基序EEVD。同源性分析表明,锯缘青蟹Hsp70编码区核苷酸序列与凡纳滨对虾(Litopenaeus vannamei)、斑节对虾(Penaeus monodon)、罗氏沼虾(Macrobrachium rosenbergii)的相似性分别为84.02%、83.87%和79.60%;核苷酸序列所推导出的Hsp70氨基酸序列,与凡纳滨对虾、斑节对虾和罗氏沼虾的相似性分别为92.79%、92.17%和96.47%。本研究克隆了锯缘青蟹Hsp70基因,为进一步深入研究锯缘青蟹的抗逆机理及其遗传改良奠定了基础。     

贵州福泉的索氏桃花水母生长及 精巢发育初步研究

2017年10月中旬,在贵州省福泉县一个池塘中发现了桃花水母,经分子鉴定确认为索氏桃花水母（Craspedacusta sowerbyi）。研究了其生长发育的情况,建立了拟合优度线性相关函数,并观察了雄性索氏桃花水母精巢外形发育过程。结果表明,福泉的索氏桃花水母雌雄异体,雌性触手细长,伸向上方;雄性触手短粗,垂向下方。缘膜宽度、口径宽度、触手数量与伞径相关函数的相关系数r均大于0.75,说明3个指标与伞径之间均存在极强正相关关系。福泉的索氏桃花水母雄性个体性腺4个,淡绿色,成熟雄性个体精巢呈囊状、三角囊状、长三角囊状,4个精巢的发育不同步。研究表明,当伞径小于12 mm时,福泉的索氏桃花水母处于生长发育期,缘膜、口径宽度、触手数量、雄性精巢4个性状随之增长而变化,当伞径大于等于12 mm时,个体处于成熟期,上述4个性状相对稳定,变化不大。本文对于性腺作为桃花水母分类指标的有效性做了探讨。     

萍乡肉红鲫的性腺发育研究

萍乡肉红鲫(Pingxiang red-transparent crucian carp,Carassius auratus L.)是在江西省萍乡地区分布的天然三倍体鲫突变体经人工选育后获得的遗传性状基本稳定的后代,具有两性生殖和雌核生殖两种生殖方式.研究以F5代萍乡肉红鲫为材料,自孵化后每满1个月开始取性腺,观察了其卵巢1周年性成熟和精巢的发育过程,结果表明萍乡肉红鲫的性腺为1年成熟类型.卵巢发育进程町以分为6个时期,卵母细胞发育相应可分为6个时相.统计了卵巢成熟系数周年变化,体重为95 g左右的雌性萍乡肉红鲫,其成熟卵巢的成熟系数约为(11.73±2.8)%,成熟的卵母细胞内充满卵黄,相对怀卵量为(3018±310)粒/g.萍乡肉红鲫精巢属于小叶型,在精小叶中可观察到不同发育阶段的生殖细胞.由精原细胞分裂而来的仞级精母细胞经分裂增殖,产生次级精母细胞并最终发育成为精子.萍乡肉红鲫的精巢发育程序与普通鲫鱼和鲤鱼相似,卵巢和精巢的发育过程基本同步,孵化后50日龄内性腺分化不明显,到70日龄左右开始出现雌雄分化,3月龄发育为第1期,4-5月龄发育为第2期,6-7月龄发育至第3期,7-10月龄可见第4期卵巢,1年即可成熟产卵,精巢可排出精液.结果表明,该鲫鱼突变体的性腺发育与普通二倍体鲤(鲫)鱼的性腺发育方式类似.     

锯缘青蟹复眼的单一感受系统

锯缘青蟹视网膜电图的暗视光谱敏感曲线的峰值在500nm 左右,与视紫红(λ_max=510nm)的吸收光谱吻合得很好。其波形和振幅-强度曲线与刺激波长无关,均符合单变量原理。在蓝或绿背景光明适应时,长波段的相对光谱敏感性增高。红光明适应未能抑制这种增高,表明它并非是由于对长波敏感的感受系统的存在。在校正了屏蔽色素的选择性吸收特性后,长波段光谱敏感性与暗视时十分接近,提示该现象可能系明适应造成的色素迁移所致。本文结果表明,锯缘青蟹的复眼仅具有单一的感受系统。     

Age‐related changes in the development of oocytes and testicular follicles after emergence in Stavsolus japonicus (Plecoptera)

Abstract Female stoneflies oviposit several times during the adult stage of their life cycle. The number of eggs within the deposited egg masses decreases at successive ovipositions. To clarify the reason for this decrease and to determine the conditions of testicular follicles, the patterns of development of oocytes and testicular follicles on different days after emergence are investigated in the systellognathan species Stavsolus japonicus (Okamoto) (Perlodidae). The size of the mature oocytes in the ovariole peaks a few weeks after emergence but decreases to the lowest level by 35 days after emergence. Several maturing oocytes can be observed in the ovarioles of individuals a few weeks after emergence but only one mature oocyte is observed at 35 days after emergence. The decreased number of eggs laid per mass by older individuals may therefore be due to the lower maturation of all the ovarioles.     

Reproductive biology of the shore crab Carcinus maenas (Decapoda,Portunidae): a macroscopic and histological view

A histological study of the reproductive cycle of male and female shore crabs, Carcinus maenas (Linnaeus), was performed monthly on the South West coast of Ireland (from December 2006 to July 2008). The calculated sex ratio deviated from equality, 1:0.53, revealing a strong male bias. A system was devised, based on screening of tissue sections, to describe and stage gametogenic development. Histological examinations revealed that ovarian development occurred biannually, with a primary winter cycle in which the larger crabs reproduced and a secondary summer cycle, when smaller crabs reproduced. An association was observed where more of the larger specimens were caught in the summer months and the smaller specimens in the winter months, which inversely correlated with the segregated breeding cycles. There was strong evidence that mature male crabs could potentially copulate year round since all mature specimens, collected throughout the year, contained viable spermatozoa. Developmental stages of oogenesis and spermatogenesis were described to develop a practical gonadal index for this portunid crab, providing information on the biology of this species, which will be of benefit for fisheries management.     

Changes in the testicular acid and alkaline phosphatase activities at different durations following prostatectomy: a correlative study with spermatogenesis.

The purpose of this investigation was to make a correlative study between spermatogenesis and testicular acid and alkaline phosphatase activities in mature prostatectomized animals at different post-operative periods. The results demonstrate that there was a significant augmentation in the activity of testicular acid and alkaline phosphatases subsequent to 14 and 21 days of prostatectomy. A parallel quantitative study of spermatogenesis at stage VII of the seminiferous cycle, namely, type A spermatogonia (ASg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7sd), revealed that there was a significant reduction in the number of step 7 spermatids after 14 and 21 days. No change was observed in the above testicular enzymes and spermatogenesis after 7 days of prostatectomy. Therefore, it is concluded that prostatectomy can alter the above testicular enzyme activities and spermatogenesis in chronic prostatectomized state.     

Testis and gonopodium development in Anableps Dowi (Pisces: Anablepidae) correlated with pituitary gonadotropic zone area

Testicular development in the viviparous fish, Anableps dowi, is described from embryonic stages through sexual maturation and is correlated with development of the gonadotropic zone of the pituitary. Only isolated spermatogonia or cysts of early spermatogonia are found in embryos 1.8 to 4.4 cm in length and postnatal specimens 5.4 to 8.8 cm in length. Cysts with more advanced spermatogonia are seen in specimens from 7.9 to 10.8 cm in length. Spermatogenetic meioses are first observed in a 10.2 cm male. Specimens greater than 12.0 cm have mature spermatozoa (partial spermatozeugmata) within the main testicular ducts, indicating gonadal maturation. The morphological transformation of the anal fin into the intromittent gonopodium is also described, using both whole and cleared specimens. Even in the smallest postnatal male available (5.4 cm), the anal fin can be distinguished from that of an equivalent sized female. Anal fin changes proceed until the mature gonopodium is developed, when body lengths greater than 16 cm are reached. Testicular maturation, defined as the presence of partial spermatozeugmata in the main testicular ducts, precedes the completion of gonopodial development. Measurements of ventral gonadotropic zone area in pituitary mid-sagittal sections show a continuous increase from neonatal through sexually mature specimens. The results point to a role for testicular androgens in early testis development.     

Estrogen synthesis in relation to gonadal development of Japanese scallop, Patinopecten yessoensis: gonadal profile and immunolocalization of P450 aromatase and estrogen

Aromatase activities and estrogen contents in the gonad of Japanese scallop, Patinopecten yessoensis, were determined during gonadal development and estrogenic cells in the testis were identified immunohistochemically. Ovaries and testes developed rapidly during January and February to reach the mature stage in March and the spawning stage in April. Increases in aromatase activities of the ovary and testis preceded the onset of the ovarian and testicular development. Aromatase activities reached the highest level at the growing stage in February and the mature stage in March, and showed a striking decrease at the spawning stage in April. Contents of ovarian and testicular estradiol-17beta changed similarly to the profile of aromatase activities in the ovary and testis, although estrone showed no change. Immunoreactivities against P450 aromatase and estradiol-17beta were detected in the cells along the inside of the acinar wall of the testis, whereas in the previous reports, the cells are distributed along the outside of the acinar wall in the ovary. This study thus suggests that estrogen is synthesized in the estrogenic cells of the ovary and testis through aromatization by P450 aromatase and that testicular estrogen may play a physiological role in spermatogenesis.     

BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox.

The utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 days after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry. The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis. Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H-TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.     

The birth of mice from testicular spermatozoa retrieved from frozen testicular sections

Male gamete cryopreservation has been widely used for both human reproduction and animal breeding. We investigated whether testicular spermatozoa retrieved from frozen testicular sections (10 or 25 mum thick) could support the full-term development of normal progeny. For this purpose, frozen testicular sections were prepared from two genetic backgrounds (BDF1 or B6 GFP transgenic mice), and the functional ability of testicular spermatozoa after preservation for 1 day, 1 mo, and 3 mo was assessed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were successfully retrieved from frozen testicular sections for the use of ICSI, regardless of the preservation period. The ICSI technique revealed that oocytes (BDF1 or B6 background) injected with testicular spermatozoa prepared from frozen testicular sections developed into normal progeny, even though the sections had been cryopreserved for 3 mo at -30 degrees C. Approximately 15% and 5% of the embryos preserved for 3 mo developed to full term if the testicular spermatozoa were prepared from the 25- and 10-mum sections, respectively. These results clearly indicate that male gametes can be viably preserved in frozen testicular sections. The technique described herein will allow the preservation of male gametes in the form of a "book" or "file" by mounting the sections on a paper-thin sheet. Furthermore, this technique may be of value in the clinical treatment of severe male infertility, since testicular spermatozoa can easily be found through examination of testicular cross sections rather than by attempts to identify them in testicular cell suspension.     

Quantitative determination of sperm production from biopsy tissue through the estimation of maximum production ability of the testes in rabbits.

Male rabbits were brought to ejaculate with the artifical vagina 6 times a week and from this the weekly sperm output was calculated. Testicular biopsies were taken 3 times, once in a 4-week interval from the same rabbits. The volumetric proportion of primary spermatocytes (method 1), all round spermatids (method 2) and round spermatids of stage 1 of the cycle (method 3) were determined from the biopsied tissue and with this and the corrected testicular volume the maximal sperm production was calculated. The correlations between the calculated sperm production and the actually counted sperm output was 0.80 with method 1,0.77 with method 2, and 0.66 with method 3. The best method to determine the maximal sperm production from testicular tissue is by counting the number of spermatids at stage 1 of the cycle, but in cases with reduced spermatogenesis it is difficult to get enough of these tubular sections, method 2 is therefore sometimes easier to apply.     

Gonadal development and diandric protogyny in two populations of Dascyllus reticulatus from Madang, Papua New Guinea

Two Dascyllus reticulatus populations from Madang, Papua New Guinea exhibited diandric protogyny. In both populations, gonads began as undifferentiated, and then developed oocytes in the primary growth stage and an ovarian lumen. From this ovarian state or from more developed ovaries containing oocytes beyond the primary‐growth stage, some gonads developed into testes. The first sign of testicular development was degeneration of oocytes, degeneration of oocytes in the primary growth stage in ovarian gonads and degeneration of oocytes of all growth stages present including the primary growth stage in ovaries, which was then followed by development of spermatogenic tissue. In both populations, most of the fish that had gonads with degenerating oocytes were smaller than the smallest mature females, indicating that development towards testes was mostly initiated in immature gonads containing only pre‐vitellogenic oocytes. On some occasions, however, females as large as other mature females also had gonads with degenerating oocytes, suggesting that development towards testes may have occurred in mature ovaries as well. This latter notion is further strengthened by the discovery of a fish having a gonad that contained both degenerating vitellogenic oocytes and developing spermatogenic tissue. Taken together, these results suggest that D. reticulatus can exhibit diandric protogyny, because testes in D. reticulatus developed from juvenile gonads as well as from mature ovaries.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.