The Formation of the Second Maturation Spindle in the Eggs of Limnaea, Limax, and Agriolimax

Development ◽  
1958 ◽  
Vol 6 (1) ◽  
pp. 28-51
Author(s):  
Chr. P. Raven ◽  
Frouke C. M. Escher ◽  
W. M. Herrebout ◽  
J. A. Leussink

Egg maturation in Limnaea stagnalis has been described by Raven (1945, 1949). It exhibits some peculiarities, by which the Limnaea egg seemed to stand apart from all other animal eggs in which the maturation processes have been accurately studied so far. In the first place it appeared that the second maturation spindle arises by a direct and gradual transformation of the centrosphere at the inner end of the first maturation amphiaster, remaining in the egg after the extrusion of the first polar body. Secondly, the above-mentioned investigations revealed the still more astonishing fact that the inner aster of the second maturation spindle is none other than the sperm aster, which arises quite independently round about the time of the extrusion of the first polar body, grows considerably in size while the second maturation spindle is being formed, and then fuses secondarily with the latter's deep end.

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P < 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P < 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P < 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.


Development ◽  
1964 ◽  
Vol 12 (4) ◽  
pp. 805-823
Author(s):  
Chr. P. Raven

In a number of papers (Raven, 1949; Raven et al., 1958; Raven, 1959) I have reported on egg maturation, especially the formation of the second maturation spindle, in various pulmonate snails. It appeared that this early and quite fundamental process of development exhibits a surprising variation. In broad outline, three main modes of formation of the second maturation spindle can be distinguished among the species studied up to the present. The first of these can be observed most clearly in the eggs of Agriolimax reticulatus and Limax flavus. After the extrusion of the first polar body, the centrosphere of the inner aster of the first maturation spindle contains a pair of dark bodies (‘centrioles’) which move apart to opposite poles of the centrosphere. The latter elongates and is directly transformed into the second maturation spindle. It develops an aster at both ends, taking their origin from the ‘centrioles’.


1946 ◽  
Vol 7 (1) ◽  
pp. 91-121 ◽  
Author(s):  
Chr P. Raven

AbstractThe structure and physico-chemical composition of the egg of Limnaea stagnalis and the changes occurring during the uncleaved stage of the egg were studied by a variety of methods. A. Composition of egg: I. The cytoplasm of the egg consists of 2 parts: ectoplasm and endoplasm,which differ in their staining reactions. Immediately after oviposition, the ectoplasm occupies a sector at the vegetative pole of the egg, the endoplasm the rest of the egg. 2. Three sorts of granules can be distinguished: α-granules (probably mitochondria), β-granules and γ-granules. Moreover, fat droplets and Golgi bodies are present in the egg. 3. The α-granules, which are small, are accumulated especially in the endoplasm. Probably, glycogen, phenolases and peroxydases are bound in some way or other to these granules. 4. The β-granules, of medium size, form a major part of the ectoplasm. They consist of albumen and contain pentosenucleic acids. 5. The γ-granules are coarse, probably albuminous in nature; they lie mostly in the endoplasm. 6. Both fat droplets and Golgi bodies are distributed rather evenly in the cytoplasm, leaving free only the spindle and asters. "Praesubstances" and "Golgi systems" can be distinguished. 7. The hyaloplasm contains pentosenucleic acids in small quantity and, probably, bound sulfhydril components, especially in its central part. 8. The freshly laid egg contains the first maturation spindle in metaphase. The spindle area is free of fat droplets and Golgi bodies, but is characterized by the presence of free glutathion in reduced form. The chromosomes contain thymonucleic acid. B. Changes during uncleaved stage: 9. The course of the maturation divisions and the formation of the polar bodies are described. The egg shows amoeboid movements shortly after the extrusion of either of the polar bodies. This is accompanied with a distinct drop of the tension at the surface. 10. The α- and γ-granules of the endoplasm are attracted by the maturation spindle and asters, forming a halo surrounding the amphiaster. 11. The ectoplasm spreads beneath the egg cortex to the animal side. At first, a gap remains at the animal pole; after the completion of the maturation divisions, the ectoplasm surrounds the whole egg. 12. A fine chorion is formed, which lies inside the first, but outside the second polar body. 13. The spermastcr makes its appearance shortly before the extrusion of the first polar body; during the maturation divisions, it grows slowly. This is accompanied with a gradual rise of the viscosity of the protoplasm. No division of the spermaster with formation of an amphiaster takes place. 14. The sperm-head remains in a subcortical position till shortly after the extrusion of the second polar body; then, it migrates to the spermaster and develops into the male pronucleus. 15. The chromosomes left in the egg after the maturation divisions swell into karyomeres which fuse to the female pro-nucleus. 16. The copulation of the pronuclei takes place immediately beneath the egg cortex at the animal pole. 17. About one hour before cleavage, the animal pole plasm is formed by a local accumulation of substances attracted, probably, by the egg cortex at the animal pole. The centripetal flow of protoplasm in the dilating maturation aster may aid in this localisation process. Many α-granules are transported by it to the animal pole plasm. 18. From the time of oviposition till first cleavage the egg swells considerably, probably by the intake of water. This is accompanied with a decrease in density. At the same time, the γ-granules of the egg give rise to the formation of vacuoles, by an attraction of water from the neighbourhood. Eventually, the egg protoplasm has a vacuolated appearance throughout, with the exception of the animal pole plasm. 19. The viscosity, which is high one hour before cleavage, has a minimum 30 minutes later, then it rises again. The tension at the surface reaches a minimum immediately before the beginning of cleavage.


Author(s):  
Bruce R. Carr ◽  
Victor E. Beshay

The complex and coordinated set of events leading to sperm and egg maturation and transport in the female genital tract that culminates in fertilization is one of the most remarkable phenomena in nature. This set of events is followed by the equally important unique processes of implantation, fetal maturation, and parturition. The hormonal changes that regulate these events are dependent on the close interaction of the fetal-placental-maternal unit. Just before ovulation, the egg, which has been arrested in the diplotene stage, completes the first meiotic division and forms the first polar body. The second meiotic division starts at the time of ovulation but ends only after fertilization by a sperm. The process of egg maturation is regulated through a closely interrelated set of hormonal events, most notably involving follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estrogen. At the time of ovulation the fimbria of the oviduct are closely applied to the surface of the ovary. The extruded oocyte and adherent granulosa cells, known as the cumulus oophorus, is collected by the ciliated fimbrial end of the fallopian tube. The transport of the egg into the end of the fallopian tube occurs within minutes and is regulated primarily by ciliary action. The cumulus cells are able to communicate with one another via a network of intercellular bridges through the zona pellucida to the perivitelline space. The cumulus cells have also been reported to play a role in nutrition and maintenance of the ovum. There are three different stages of passage of the ovum through the fallopian tube. The first stage includes the transfer of the ovum from the fimbriated end of the fallopian tube until the egg reaches and is retained at the ampullary-isthmic junction. The ampullary-isthmic junction is a functional block but is not a clearly defined anatomical structure. The ovum remains at this junction for 1–2 days, during which time fertilization occurs.


2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Xiao Liang ◽  
Xue Tong ◽  
Hui-lan Du ◽  
Ming He ◽  
Yu Zhang ◽  
...  

Background. Bushen Tiaojing Decoctions (BSTJ-II-D and BSTJ-III-D) are used to assist pregnancy in clinical practice. In this study, we explored the ability of sequential administration of BSTJ-II-D and BSTJ-III-D to promote cumulus cell (CC) expansion and its underlying mechanisms in controlled ovarian hyperstimulation (COH) mice. Methods. Kunming mice were randomly divided into three groups. The normal group was injected intraperitoneally with saline, and distilled water was administered orally by gavage. As the COH model, mice were injected with GnRHa, eCG, and hCG. Subsequently, the BSTJD group received BSTJ-II-D and BSTJ-III-D orally by gavage, while the control group received distilled water. We evaluated CC expansion and oocyte first polar body (PB1) extrusion under a stereomicroscope. Serum levels of follicle-stimulating hormone (FSH) were detected by radioimmunoassay. The expression of the CC expansion-related factors PTX3 and PTGS2 was detected by immunofluorescence, western blot, and quantitative real-time-polymerase chain reaction analyses (qRT-PCR). Expression of p-MAPK14, p-MAPK3/1, MAPK14, and MAPK3/1 was detected by western blot analysis. Results. Sequential administration of BSTJ-II-D and BSTJ-III-D promoted cumulus expansion and oocyte PB1 extrusion and upregulated PTX3 and PTGS2 expression at the mRNA and protein levels. Furthermore, the levels of p-MAPK14/MAPK14, p-MAPK3/1/MAPK3/1 proteins, and serum FSH in the BSTJD group were higher than those in the normal and control groups. Conclusions. Sequential administration of BSTJ-II-D and BSTJ-III-D promotes cumulus expansion and oocyte maturation in COH mice by increasing FSH expression and activating the MAPK14 and MAPK3/1 signalling pathways, thereby increasing expression of PTX3 and PTGS2.


2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
Author(s):  
Yu-Ting Shen ◽  
Yue-Qiang Song ◽  
Xiao-Qin He ◽  
Fei Zhang ◽  
Xin Huang ◽  
...  

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.


Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.


Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 1995-2003 ◽  
Author(s):  
G.L. Russo ◽  
K. Kyozuka ◽  
L. Antonazzo ◽  
E. Tosti ◽  
B. Dale

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1–2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 × 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.


Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Yue-Liang Zheng ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.


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