Fertilization environment of the non-copulating marine sculpin, Hemilepidotus gilberti

To clarify the extracellular environment for external fertilization in the non-copulating marine sculpin Hemilepidotus gilberti, sperm motility was measured in NaCl, KCl, mannitol solutions, seawater, and ovarian fluid. Spermatozoa of H. gilberti actively moved in seminal plasma the moment they were removed from the genital papilla. Spermatozoa showed higher motility in NaCl solution at osmolalities between 300–400 mOsmol kg^-1. In KCl and in mannitol solutions, spermatozoa actively moved at osmolalities between 500 and 800 mOsmol kg^-1, and at osmolality 300 mOsmol kg ^-1, respectively. The ovarian fluid was a transparent and viscous gelatinous material, rich in sodium with an osmolality of 340 mOsmol kg^-1. Sperm motility in the ovarian fluid lasted more than 90 min, which was six times longer than in seawater. This sperm motility under conditions isotonic to body fluid is similar to that of copulating marine sculpins rather than to other non-copulating marine fishes. In addition, eggs of H. gilberti could be fertilized in the ovarian fluid. This suggests that external fertilization takes place under physiological conditions similar to the internal conditions of the ovary provided by the ovarian fluid, which isolates the eggs from sea water for several hours after spawning. This manner of fertilization is thought to be one of the evolutionary pre-adaptations allowing copulation among marine sculpins.     

The little dragon sculpinBlepsias cirrhosus,another case of internal gametic association and external fertilization

In the copulating cottid species,Blepsias cirrhosus, the point at which fertilization occurred was determined experimentally. Ovulated eggs were obtained directly from the ovary of impregnated females. Eggs maintained in ovarian fluid did not show any signs of development, whereas most of the eggs which had been placed in seawater had developed to the 4-cell stage after 24hrs. The eggs kept in ovarian fluid initiated segmentation when later transferred into seawater. Histological examination showed that a number of spermatozoa had entered the micropyle, but that penetration did not occur in eggs that were not immersed in seawater. In these eggs, the metaphase spindle of the second meiotic division was observed in the ooplasm, at the animal pole. These results indicate that, in impregnated females, the spermatozoa associate with the eggs in the ovarian cavity, but that fertilization occurs externally when the eggs are spawned.     

A 4-year summary of the nonsurgical recovery of baboon embryos: A report on 498 eggs

A nonsurgical embryo recovery procedure, developed to allow the economical acquisition of cleavage stage baboon embryos, has been successfully used for 4 years. With this technique, 498 eggs have been recovered from 979 uterine flushes (50.9%) on 71 baboons. Of 467 eggs recovered from mated baboons, 290 (62.1%) were fertilized. Papio anubis females provided a higher percentage of fertilized eggs (75.3%) than did Papio hamadryas (47.8%) or Papio cynocephalus (44.3%) females following exposure to males during estrus, although sexual preference may be responsible for the reduced fertilization rate in the P. cynocephalus females. Recovery rates from individual baboons ranged from 0% (n = 11) to between 66% and 93% for ten baboons from each of which 12–33 eggs have been recovered. Fertilized eggs were at the two-cell (n = 23) to blastocyst (n = 53) stage at recovery 1–6 days postdeturgescence (PD) of the sex skin, with morulae (n = 84) being the most frequent cell stage recovered (30%). The optimum time for performing the procedure was the third day PD, when 113 (40%) embryos were recovered. The abilities of baboons to become pregnant and to provide fertilized embryos were significantly related (P < 0.005), allowing the embryo recovery technique to be used as a screening procedure for evaluating baboon fertility.     

Differentiation and function of the ovarian somatic cells in the pseudoscorpion,Chelifer cancroides (Linnaeus, 1761) (Chelicerata: Arachnida: Pseudoscorpionida)

Pseudoscorpion females carry fertilized eggs and embryos in specialized brood sacs, where embryos are fed with a nutritive fluid produced and secreted by somatic ovarian cells. We used various microscopic techniques to analyze the organization of the somatic cells in the ovary of a pseudoscorpion, Chelifer cancroides. In young specimens, the ovary is a cylindrical mass of internally located germline cells (oogonia and early previtellogenic oocytes) and two types of somatic cells: the epithelial cells of the ovarian wall and the internal interstitial cells. In subsequent stages of the ovary development, the oocytes grow and protrude from the ovary into the hemocoel (opisthosomal cavity). At the same time the interstitial cells differentiate into the follicular cells that directly cover the oocyte surface, whereas some epithelial cells of the ovarian wall form the oocyte stalks – tubular structures that connect the oocytes with the ovarian tube. The follicular cells do not seem to participate in oogenesis. In contrast, the cells of the stalk presumably have a dual function. During ovulation the stalk cells appear to contribute to the formation of the external egg envelope (chorion), while in the post-ovulatory phase of ovary function they cooperate with the other cells of the ovarian wall in the production of the nutritive fluid for the developing embryos.     

In vivo fertilization and development of medaka eggs initiated by artificial insemination

Fertilization and development in the ovarian cavity of oviparous fish, Oryzias latipes, were examined using the S-rR strain in which the sex genotype can be easily distinguished by the body color of the fish. Mature eggs were fertilized within the ovarian cavity after a sperm suspension was artificially introduced with a small bore-pipette through the urinogenital opening. Three batches of eggs ovulated within 48 hrs were fertilized and began to develop in the ovarian cavity, while eggs ovulated 72 hrs post-insemination (PI) were no longer fertilized. These observations indicate that ovulation occurs irrespective of the existence of developing embryos within the ovarian cavity. All embryos developing in the ovarian cavity were, however, retarded and ceased development before the stage of initiation of blood circulation at room temperature. These embryos developed normally and hatched after they were transferred from the ovarian cavity into regular saline 48 or 72 hrs PI. When these individuals matured sexually, their sex differentiation was found to be normal, and sex reversal was not observed.     

From oocyte to embryo: a model, deduced from in vitro fertilization, for natural selection against chromosome abnormalities

A cytogenetical analysis was performed on 151 unfertilized oocytes, 22 fertilized eggs at the pronuclear stage, and 108 cleaved embryos obtained in the course of in vitro fertilization (IVF). Thirty-two per cent of unfertilized oocytes were abnormal, carrying nullisomies or disomies, mainly of D and G chromosomes, and a structural anomaly (Gq-) in one case. Fertilized eggs showed frequent asynchronism in the development of pronuclei and only 2 out of 8 karyotyped pronuclei were normal. Cleaved embryos were classified according to the number of pronuclei observed 17 hours after insemination. One per cent displayed a single pronucleus, and haploid chromosome complements were found in the corresponding cleaved embryos which were considered to be parthenotes. The rate of chromosome abnormalities of diploid eggs depended on their morphological aspect. Healthy cleaved embryos carried 12.5% of anomalies while this rate reached 37% in fragmented embryos (p less than 0.05). Lastly, 6% of fertilized eggs displayed three pronuclei or more. Only 41% of the corresponding embryos were triploid. Diploidy or diploidtriploid mosaicism were often encountered. This leads to a 21% rate of abnormalities in the preimplantation embryos. Parental karyotyping and HLA typing were carried out in a series of eight couples with in vitro idiopathic infertility or recurrent embryo degeneration in vitro. No abnormality was noted. According to these results, a model of natural selection of normal conceptuses is proposed.     

Developmental arrest of fertilized eggs from the B6.YDOM sex-reversed female mouse

When the Y chromosome of a Mus musculus domesticus mouse strain is placed onto the C57BL/6J (B6) inbred background, the XY progeny develop ovaries or ovotestes but never normal testes during fetal life. While some of the hermaphroditic males become fertile, none of the XY females produces litters. Here, we examined the fertility and development of oocytes derived from the XY female mouse. With or without preceding injection of gonadotropins, female mice were mated with normal B6 males, and their embryos were recovered at various developmental stages. In vitro fertilization was performed with the eggs recovered from the oviduct after treatment with go-nadotropins. Development of embryos was examined by both light and electron microscopy. The results indicate that the oocytes released from the B6.Y^DOM ovary were efficiently fertilized and often initiated the first cell cleavage, but all embryos died during early preimplantation periods. Even when oocytes were fertilized in vitro, minimizing their exposure to the XY oviduct/uterus environment, most embryos died at the 1- or 2-cell stage. A few exceptional embryos reached the 4- or 8-cell stage, but abnormalities were evident in both nuclear and cytoplasmic structures of all embryos. After cleavage, neighbouring blastomeres were only loosely associated, and microvilli were abundant at the intercellular interfaces. We postulate that oocytes of the B.6.Y^DOM female mouse become defective during XY ovarian differentiation, and, hence, fail to proceed through normal embryonic development. © 1994 Wiley-Liss, Inc.     

Fecundity and fertility of ladybird beetle Harmonia axyridis after prolonged cold storage

Some species of ladybird beetles (Coleoptera: Coccinellidae) mate both before and after overwintering. The purpose of the pre-diapause mating was studied in the alien invasive ladybird Harmonia axyridis (Pallas 1773). Our study demonstrates the persistence of high fecundity (daily oviposition rate of 21 eggs per fertilized female during the first month of reproduction) and fertility (85 % of eggs hatching) of females of H. axyridis after long storage (up to eight months) at low temperature (6 °C). The females were not mated after activation in spring and had to rely on the sperm supply maintained from the pre-winter period (58 % of females were fertilized). Unfertilized females also laid eggs but in low numbers (an average of 345 eggs by virgin females during an individual’s lifetime, 1,174 eggs by females fertilized before winter) and after a longer pre-oviposition period (2–5 weeks in comparison to 7–8 days for fertilized females). We show that the unfertilised eggs were not trophic eggs. The high sperm survival ability observed questions the need for the high levels of sexual activity generally observed in Coccinellidae. Fertilized females of H. axyridis may found large colonies after dispersal to new areas even without males, which contributes to the striking invasive ability of this species.     

Delayed sperm injection and fertilization in parthenogenetically activated insect egg (Athalia rosae,Hymenoptera)

Summary Mature eggs dissected from the ovary of unmated females of Athalia rosae ruficornis Jakovlev (Hymenoptera, Tenthredinidae) can be activated to develop (into haploid parthenogenetic males) simply by exposing them to distilled water. These eggs, which are primary oocytes arrested at the first meiotic metaphase, resume meiosis upon activation and reach the first meiotic telophase in 20 min. Mature eggs immediately upon dissection have previously been shown to complete karyogamy and develop as fertilized diploid females if injected with sperm. We show here that the eggs activated in water for 20 min have a much higher rate of successful fertilization if injected with sperm, and that the eggs activated for 40 min, upon sperm injection, though at a reduced frequency still develop as diploid fertilized females. Eggs left in water for 60 min, however, are no longer fertilized upon sperm injection and develop as haploid males.     

Effect of continuous intrauterine administration of prostaglandin F2 alpha and indomethacin on fertilization of rabbits

The effect of continuous intrauterine administration of prostaglandin F2 alpha (PGF2 alpha) or indomethacin or indomethacin together with PGF2 alpha and PGE2 or vehicle on fertilization of rabbits was studied. These substances and vehicle were delivered into the cornua of the uterus via an Alzet minipump for 11 days. The animals were inseminated vaginally. Compared with controls (104 eggs of which 88.5% were fertilized) a reduction of the fertilization rate was observed with indomethacin (74 eggs of which 70% were fertilized). Exogenously added PGF2 alpha did not change the fertilization rate. The administration of indomethacin together with PGE2 raised the fertilization rate to 86% (63 eggs of which 54 were fertilized). The application of PGF2 alpha together with indomethacin showed a fertilization rate of 85% (59 eggs of which 50 were fertilized). The indomethacin application was associated with a reduction of prostaglandin production in several tissues from the female genital tract, showing that indomethacin is taken up by the endometrium of the rabbit. The ovary, oviduct, cervix and vagina were mainly affected by this treatment. The route of transport of this drug is not known, however. The reduction of the total number of eggs together with the decrease of the fertilization rate after indomethacin administration point towards multiregional sites of interference of prostaglandins in reproductive functions. PGF2 alpha seems to be the more important factor since PGE2 may be converted to PGF2 alpha in reproductive tissues.     

A direct effect of some rifamycin derivatives on the morphology of mammalian mitochondria

Protein synthesis has been investigated in cell-free preparations from mature ovarian oocytes, unfertilized and fertilized eggs, and early embryos of Drosophila melanogaster. Preparations from unfertilized eggs have a specific activity that is 5- to 6-fold higher than the activity of fractions from ovarian oocytes. There is an additional small increase in activity of preparations from fertilized eggs. The specific activity that is rapidly attained in the fertilized egg remains essentially constant for 2 to 2.5 h after fertilization, decreases sharply during blastoderm formation, and again increases during gastrulation. The activities of unfertilized eggs decline slightly during the first 2 h after oviposition, and then decrease more sharply. About 35 % of the ribosomes in preparations from both unfertilized and fertilized eggs sediment in the polyribosome region of sucrose density gradients, whereas no polyribosomes could be detected in preparations from ovarian oocytes. In both ovarian oocytes and fertilized eggs, less than 1 % of the ribosome populations were present as subunits. Additional ribonucleoprotein material of buoyant densities different from those of ribosomal subunits or ribosomes was found throughout the sucrose gradients. About 3.5 % of the ribosomes were found to be membrane-bound in preparations from both unfertilized and fertilized eggs.     

Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.     

Osmotic changes during the development of eggs and larvae of the lumpsucker, Cydopterus lumpus L.

The demersal eggs of Cyclopterus appear to osmoregulate like the pelagic eggs of cod and plaice. Unfertilized eggs in ovarian fluid exhibited ovoplasm osmolarities similar to those of adult blood and ovarian fluid (356–359 mosmol kg⁻¹). Yolk osmolarities remained virtually constant from fertilization and during development (356–366 mosmol), with a slight decrease near hatching (to 332 mosmol). Yolk and body fluids of larvae (338 mosmol) had osmoconcentrations similar to egg yolk values near hatching. Yolk osmoconcentration of unfertilized eggs remained unchanged during the first 12 h in sea water, with a slow increase thereafter. Fertilized eggs of bad quality cultures exhibited higher yolk osmoconcentrations than eggs of good quality. Cyclopterus eggs were found to develop normally and survive in 20–34%o salinity, larvae seemed to have the same salinity range.     

Conditions for initiation of fertilization of eggs in the copulating elkhorn sculpin

The reason for failure to initiate fertilization internally was examined in a cottid fish, the elkhorn sculpin, Alcichthys alcicornis which has internal gametic association and external fertilization. While eggs could be activated in calcium free hypertonic media but not be fertilized, fertilization occurred in isotouic media rich in calcium ions. The rate of fertilization was dependent on calcium concentration, and eggs were not fertilized in solutions with a calcium ion concentration of less than 0·57 mmol kg⁻¹. Calcium ions could be replaced to some extent by magnesium ions, but the former were the more effective in fertilization. Since calcium ion concentration of ovarian fluid of A. alcicornis was 0·41 mmol kg⁻¹, it was inferred that low calcium concentration in the ovarian fluid was the cause of the failure of A. alcicornis eggs to fertilize internally.     

Intracellular pH shift leads to microtubule assembly and microtubule-mediated motility during sea urchin fertilization: correlations between elevated intracellular pH and microtubule activity and depressed intracellular pH and microtubule disassembly

The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.     

Ultrastructural study of polyspermy during early embryo development in pigs, observed by scanning electron microscope and transmission electron microscope

Polyspermy is generally considered a pathological phenomenon in mammals. Incidence of polyspermy in porcine eggs in vivo is extremely high (30-40%) compared with other species, and polyspermy rate in the in vitro fertilized eggs in pigs can reach 65%. It is still unknown whether polyspermy to a certain degree is a physiological condition in pigs, and whether porcine eggs have any capability with which to remove the accessory sperm in the cytoplasm. The objectives in the present study are to observe the ultrastructural changes of accessory sperm during early embryonic development in pigs. A total of 58 normal, early embryos at one-, two, three-, and four-cell and morular stages were collected from gilts and were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface ultrastructure showed that sperm fusion with the zona pellucida was a continuous process during one-, two-, three-, and four-cell and morular stages, as observed by the SEM. Accessory sperm were present in the cytoplasm of cleaved embryos. The sperm heads in the cytoplasm of cleaved embryos did not decondense. TEM revealed the presence of a condensed sperm head within a lysosome (or phagolysosome) in a three-cell embryo. These observations suggest that polyspermy may be a physiological condition in pigs and that early embryos may develop to term if accessory sperm do not interrupt the embryo genome. Furthermore, lysosome activity could be another physiological mechanism for removing accessory sperm in the cytoplasm of fertilized eggs and cleaved embryos after fertilization in pigs.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

Protein synthesis in enuleated fertilized and unfertilized mouse eggs

Summary Fertilized and unfertilized C57BL/6J eggs were microsurgically enucleated and then analyzed for their capacity to synthesize proteins using 2-dimensional polyacrylamide gel electrophoresis. In both types of enucleated eggs (cytoplasts), protein synthesis continued and was still detected up to three days in culture. Shortly after enucleation, the pattern of polypeptides remained similar to the respective non-operated control eggs but it later became gradually reduced in intensity and complexity. After two days of culture the appearance of some new proteins typical for 2-cell embryos was observed in enucleated fertilized eggs only. Our findings suggest that maternal mRNA stored during oogenesis is utilized during the preimplantation period.     

Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucius) eggs incubated in buffer with and without dimethyl sulfoxide: An X-ray microanalysis study

Fertilized and unfertilized eggs from the northern pike (Esox lucius) were incubated 2 hr in buffer with 0 and 10% (v/v) dimethyl sulfoxide and then quickly frozen in the wells of aluminum blocks submerged in liquid nitrogen. Control eggs and ovarian fluid were similarly frozen immediately after collection. The frozen eggs were sectioned, freeze dried, mounted on stubs, and carbon coated. X-ray microanalysis was used to determine changes in element levels and dimethyl sulfoxide (Me₂SO) penetration in the zona radiata, cytoplasm, cortical alveoli, and egg yolk. Unfertilized eggs incubated without Me₂SO showed decreased levels of Na, Cl, and K in the zona radiata; fertilized eggs, incubated without Me₂SO showed decreased levels of Na, P, and Cl in the zona radiata and increased levels of K in the cytoplasm; unfertilized eggs, incubated with 10% Me₂SO showed decreased Na and Cl in the zona radiata, decreased K in the cytoplasm and increased K in the cortical alveoli; fertilized eggs incubated in buffer with 10% Me₂SO showed decreased levels of Na, P, Cl, and K (zona radiata), P, Cl, and K (cytoplasm), Na (yolk), and increased Cl in the yolk (all P<.01). Me₂SO (v/v) levels reached 1.5-3.1% in the zona radiata, 0-3.2% in cytoplasm, 2.3-8.7% in cortical alveoli, and 0-1.6% in the yolk. Unfertilized eggs showed more Me₂SO penetration than fertilized eggs.