Studies On The Fertilization Biology Of Gobiocypris Rarus

Rare minnow (Gobiocypris rarus) which belonging to order of Cypriniformes, family of Cyprinidae, genus Gobiocypris, is a small freshwater fish endemic to China. Because there are some biological characteristics are similar to Zebrafish, it also an very important experimental animal.The parent fish were obtained from the Key Laboratory of Aquatic Organism Reproduction and Development (Southwest University), all of them were one years' old. Prior to collecting gametes,22 pairs of them were kept together in a small aquaria, respectively. The aquaria equipped for continuous aeration and a constant temperature of 25℃. For fertilization experiments, eggs and sperm were obtained by gently pressing the abdomen of a fish, dry fertilization, activation with filtered tap water. At selected times after the mixing gametes, some eggs were removed and placed in a test tube containing fixative solutions. Morphological studies on the testis and ovary, entry of the sperm into the egg, cortical reaction, the migration and integration of the male and female pronucleus of G. rarus, were conducted with histological methods, scanning electron microscopy and transmission electron microscopy. The results showed:The G. rarus's testis is lobular type and the mature sperm consists of head, mid-piece and tail. The head like a globe, is (1.427±0.03)μm in width, there is no acrosome at the anterior end of the nucleus. The mid-piece consists of the centriolar complex and the sleeve, the centriolar complex contains proximal centriole and basal body, they are arranged like a "L".The main structure of the tail is axoneme, which is the structure of a typical "9+2". There are no lateral fins in the tail.The mature egg of G. rarus is global and demersal, with the diameter of (1.024±0.03) mm. There is a single micropyle on the animal pole and there are a lot of villiform structures in the egg surface except the (0.247±0.01) mm areas around the micropylar apparatus. The micropylar apparatus which consists of vestibule and micropylar tube is deeply sunken with a short tunnel. The micropylar apparatus like a "sperm catchment area", it enhances the chances of fertilization by as much as 99.9% once sperm were in the immediate vicinity of it. The vestibule is funnel-like with it's surface is smooth, the largest diameter is (159.178±0.608)μm and (39.299±0.607)μm in depth. The micropylar tube is (8.318±0.055)μm in depth. The diameter of the external of micropylar tube is (8.318±0.055)μm, but (5.905±0.095)μm of the external of micropylar tube, which is large enough to permit the concomitant penetration of two sperm. According to the characteristics and morphological of contents, the cortical alveoli can divided into three types. According to the release intensity of the cortical alveoli, the author divided cotical reaction into the latent period, developmental period, climactic period and declining period.Observation of the mature egg which the egg membrane was removed, there is a small cytoplasmic process at the central of micropylar apparatus. This small cytoplasmic process is the site of sperm penetration into egg.Os after fertilization, the micropylar tube is pluged up by microplyar cell and there are two sperm move toward the micropylar apparatus. We suggested that the microplyar cell might attract the sperms penetrate into the egg.3s after fertilization, the sperm began to enter the micropylar tube.10s, the micropylar tube is blocked, the sperm enter the cortex. 25s after fertilization, there formed a spermaster around the sperm.30s after fertilization, fertilization cone comes to being.50s after fertilization, the cortical reaction enter the climactic period, a large amount of cortical alveoli was released and there are a lot of sperm adhere to the egg surface.120s after fertilization, the sperm in the micropylar apparatus started to break up, and the boundary between them become vague.150s after fertilization, the fertilization cone has decomposed.270s, the plasma membrane was repaired.5min after fertilization, the egg is at telophase in the second meiosis.20min after fertilization, the male pronucleus can be seen.30min after fertilization, both the pronucleuses are conjugated into zygote.45min after fertilization, the chromosomes are situated at metaphase of the first karyokinesis, and 80min at anaphase.Observation by TEM, the egg envelope of G. rarus is composed of primary and secondary envelope. The primary envelope is (7.167±0.281)μm in thickness, which consists of affiliated membrane and zone radiate. The affiliated membrane is (0.414±0.015)μm in thickness, consists of two layers. The zone radiate consists of seven layers, which change significantly during fertilization. 40-70s after fertilization, lots of cortical alveoli were released, the egg membrane outspeading. the egg membrane rich the largest thickness. In the end, the ovum lacuna enlarges, soft egg membrane are transformed into rigid fertilization membrane. The egg membrane's outsperding can be divided into four periods:latent period, development, climactic period and declining period. Changing of egg membrane's width after fertilization is "thin-thick-thin" trend.For monospermic eggs, the mechanism of the polyspermy block in G. rarus eggs consist of the main three steps:(1) A polyspermy block related to the structure of the micropyle to reduce the number of sperm reaching the oocyte plasma membrane; (2) Formation of a fertilization cone derived from the sperm and oocyte plasma membranes to plug the inner opening of the micropyle; (3) Modification of the egg envelope by the contents released from the cortical alveoli(including the hardening of egg membrane).