Advanced Embryo Research Articles

In vitro maturation and artificial activation of donkey oocytes

Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.

In Vitro Compaction of Germinal Vesicle Chromatin is Beneficial to Survival of Vitrified Cat Oocytes

The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro. Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8-16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes.

Toxic effects of an oil spill on fish early life stages may not be exclusively associated to PAHs: Studies with Prestige oil and medaka ( Oryzias latipes)

Polycyclic aromatic hydrocarbons (PAHs) are assumed to be the primary determinant of oil petroleum toxicity. Since the PAH content in Prestige oil was relatively high, we investigated the effects of different oil fractions (crude or weathered oil −0.05 to 50 g/L, and shaken or sonicated water accommodated fractions, WAFs, 25–100%, v/v) on the embryo–larval development of medaka ( Oryzias latipes). Concentrations of ∑16PAHs analyzed in the incubation medium were highest in the shaken WAF followed by the crude oil, the sonicated WAF and the weathered oil. Both oils (≥0.25 g/L) induced developmental abnormalities whereas no significant effects were seen in the WAF exposures. In vivo morphometric analysis of the surface of the gallbladder during advanced embryo organogenesis (192 h post-fertilization, hpf) revealed significant dilation in both WAF exposures (>3 × 10 4 μm 2 at ≥25%, v/v, compared to <1.7 × 10 4 μm 2 at 0%, v/v) followed by the crude oil (>2.2 × 10 4 μm 2 at ≥0.05 g/L). Fluorescent aromatic compounds were observed in the gallbladder and the yolk sac of 168-hpf embryos exposed to all oil fractions. Results suggest the presence of components in both oils capable of penetrating the chorion and inducing a toxicity not observed in the WAFs. Hence, the hazard and risk assessment of Prestige oil should not be based solely on the presence of PAHs since proximity or direct contact may induce toxicity not associated exclusively to these compounds. This research offers a new hypothesis for explaining the reported biological observations, which could be correlated to direct oil exposure rather than the traditional mechanism of waterborne PAH exposure. Further research is needed to identify those oil components responsible for toxicity.

Light and electron microscopy study of late embryonic development in the land snail Limicolaria flammea (Müller) (Pulmonata, Achatinidae)

The late stages of embryogenesis in the achatinid land snail Limicolariaflammea (Müller, 1774) were described using light and electron microscopy. Embryos at various stages of development were present in the eggs during the first hour after they were laid, from 4-cell blastulae to morulae and fairly advanced stages. The advanced embryo which was fully developed on the second day bears a long cephalic sac, first to be developed, attached to the embryo and a podocyst which is attached to the foot of the embryo. Both of these structures are reduced in size as embryogenesis progresses until they finally disappear at about the 7th day after the egg was deposited. The embryonic shell was apparent on the second day and spiral coiling was apparent at about day 5. The spiral shell had one whorl when formed and more spirals were added so that at hatching the young snails had three whorls.

State-of-the-art embryo technologies in cattle

Over the past 30 years, basic and applied studies on classical and advanced embryo technologies have generated a vast literature on factors regulating oocyte and embryo development and quality. In addition, over this period, commercial bovine embryo transfer has become a large international business. It is well recognised that bovine embryos derived in vivo are of superior quality to those derived from in vitro maturation, fertilization and culture. Relatively little has changed in the techniques of producing embryos in vivo although there is increasing evidence of the importance of, for example, peripheral and follicular endocrine profiles for the subsequent developmental competence of the embryo. The in vitro production of ruminant embryos is a three-step process involving oocyte maturation, oocyte fertilization and in vitro culture. Only 30-40% of such oocytes reach the blastocyst stage, at which they can be transferred to a recipient or frozen for future use. We know now that the quality of the oocyte is crucial in determining the proportion of immature oocytes that form blastocysts while the post-fertilization culture environment has a major influence on the quality of the blastocyst. Use of sexed-sorted sperm in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of the desired sex. Concerns regarding the use of sexed semen technology include the apparent lower fertility of sorted sperm, the lower survival of sorted sperm after cryopreservation and the reduced number of sperm that could be separated in a specified time period. Assessment of embryo quality is a challenge. Morphological assessment is at present the most popular method for embryo selection prior to transfer. Other non-invasive assessment methods include the timing of the first cleavage division which has been linked to developmental ability. Quantitative examination of gene expression is an additional valuable tool to assess the viability of cultured embryos. A substantial amount of evidence exists to demonstrate that the culture conditions to which the embryo is exposed, particularly in the post-fertilization period, can have perturbing effects on the pattern of gene expression in the embryo with potentially important long-term consequences. Collectively, in vivo and in vitro studies support the notion that the environment of the embryo is critical for its future. The identification and characterization of the short-term effects of in vitro culture raises the question about long-term consequences and safety of assisted reproductive technologies. The impact of some of these technologies on animal production will be the subject of this review.

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: efficiency and kinetics of oocyte fertilization and embryo development

The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower (p < or = 0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower (p < or = 0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower (p < or = 0.01 or p < or = 0.05) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower (p < or = 0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.

Efectos sobre la fertilidad bovina de las técnicas de manipulación de embriones

During the past thirty years, basic and experimental studies on classical (superovulation; non-surgical recovery and transfer of cattle embryos) and advanced embryo technologies (in vitro embryo production; cloning by somatic cell nuclear transfer) have generated structural and functional information on oocyte development and quality, fertilisation and conceptus development. This information has provided new insight, not only into these technologies per se but also into the factors contributing to fertility in cattle. It is now known that the peripheral and follicular endocrine profiles have a profound influence on the subsequent developmental competence of the embryo. It is also well established that manipulation of the oocytes or embryos may adversely affect embryonic and foetal development, leading to the so-called 'large offspring syndrome'. Information from such studies has alerted scientists to the importance of epigenetics in cattle reproduction.

Ghrelin inhibits the development of mouse preimplantation embryos in vitro.

Although ghrelin acts as a modulator of feeding behavior and energy metabolism in the central nervous system, recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we investigated the expression of ghrelin and its receptor (GHS-R) in mouse oocyte and preimplantation embryos, and we examined the role of ghrelin in the regulation of early embryo development. Both ghrelin and GHS-R mRNAs were detected in morula or more advanced embryo stages. As for the origin of ghrelin, both ghrelin mRNA and protein were identified in the uterine endometrium. The levels of ghrelin in uterine fluid as well as plasma were significantly increased in fasting mice compared with animals with free access to foods. Addition of ghrelin to culture media inhibited the development of two-cell embryos to the hatched blastocysts, and the inhibitory effects of ghrelin were abolished by an antagonist for the GHS-R. In addition, ghrelin significantly decreased the number of total cells, inner cell mass, and trophectoderm cells in blastocysts. These observations suggest that ghrelin could inhibit the development of preimplantation embryos during fasting. Thus, ghrelin may act as a peripheral factor to avoid the excess metabolic demands imposed by pregnancy during malnutritional states.

Study of Cynops pyrrhogaster notochord differentiation using a novel monoclonal antibody.

Two monoclonal antibodies which reacted specifically with the notochord of the early Cynops pyrrhogaster embryo were screened. The antigen molecules were detected within and around the notochord. They were first found mostly between the neural plate and the dorsal part of the notochord in the early neurula (stage 15). They were subsequently detected between the notochord and the somite in the advanced embryo, and they were last detected between the notochord and the underlying endoderm. Whole-mount labeling indicated that the antigen molecules were first detected in the anterior half of the notochord in the early neurula (stage 15). The signals gradually spread along the anterior-posterior axis, especially towards the posterior region. This fact suggests that notochord differentiation progresses from the anterior region which first receives the dorsal mesoderm-inducing signals released horizontally from the lower dorsal marginal zone during early gastrulation. The present study suggested that: (i) notochord differentiation proceeds from the anterior region; and (ii) secretion of the antigen molecules results in the drawing of a boundary between the adjacent tissues.

Advanced embryo development during extended in vitro culture: Observations of formation and hatching patterns in non-transferred human blastocysts

Human embryos not chosen for fresh transfer or cryopreservation were maintained in extended in vitro culture for up to 9 days after fertilization to observe blastocyst formation and hatching features. These non-transferred embryos were derived from 64 consecutive IVF cycles, and were not cryopreserved either because of compromised morphology or because the patients did not consent to cryopreservation for personal reasons. Embryos were cultured individually to monitor daily growth until developmental arrest, and differential blastocyst formation and hatching were analysed among groups of patients and embryos. In the population studied, hatching occurred most commonly on day 7 after fertilization (range 5–9 days). A total of 301 blastocysts was observed, of which 116 (38.5%) eventually hatched in vitro irrespective of day of formation. A trend towards earlier blastocyst formation and a greater likelihood of hatching was noted in this population. Both blastocyst formation and hatching appeared negatively correlated with increasing maternal age and higher basal serum FSH concentrations on day 3 of development, although these trends did not reach statistical significance. Comparison of intracytoplasmic sperm injection (ICSI) (n = 25) and conventional insemination (n = 39) cycles showed a similar rate of blastocyst formation in both groups (54 and 52%, respectively; P > 0.05), but hatching patterns varied significantly between these groups (4.1 versus 61.6%, respectively; P < 0.0001). The discovery of marked impairment of hatching among non-transferred ICSI embryos supports the case for reconsideration of the appropriateness of assisted blastocyst hatching in selected cases.

High Pregnancy Rate in “Abandoned” Blastocyst Cycles

Objectives: The ability of ART laboratories to grow embryos to the blastocyst stage in vitro has increased the chance that viable embryos will be selected and therefore reduced the need to transfer more than 2–3 embryos. However, it is not clear 3 days after oocyte retrieval, when most early stage embryo transfers are performed, which, if any, embryos will develop to the blastocyst stage by Day 5. The objective of this study was to determine if advanced embryo cell number on Day 3 was predictive of blastocyst development and increased pregnancy rates. Design: A retrospective review of 77 ART cycles from January 1999 through January 2000 was performed. All patients had desired development of their embryos to the blastocyst stage prior to transfer. Materials and Methods: We decided to use the presence of at least 3 embryos at the ≥8-cell stage on the morning of Day 3 as the minimum requirement for continuation of the blastocyst cycle to Day 5 in our patients. Embryo quality on Day 3 was not a criterion for deciding whether patient cycles were continued to the blastocyst stage in vitro prior to embryo transfer. Patients in whom the blastocyst cycle was “abandoned” on Day 3 had their embryos transferred on Day 3 after assisted hatching. A regimen of sequential media was utilized to optimize embryo growth to the blastocyst stage. Statistical analysis was performed using a t-test and chi-square analysis. Results: The results of this study are presented in the table below. Despite a similar average age in both groups, the patients in B group had more oocytes retrieved (14.7 vs. 10.5) and more embryos to choose from on Day 3 (9.8 vs. 4.9) than those in the D3 group. In the D3 group, 43/45 patients had an embryo transfer and the mean # of cells in the transferred embryos was 5.3 cells. In the B group, 31/32 (97%) patients had a transfer on Day 5, although only 23/31 (74%) had 1 or more actual blastocysts at the time of transfer. The remaining 8 patients had embryos only at the early cavitation stage when transferred. Nevertheless, 2 pregnancies occurred in these 8 patients. ∗Group#Age# Eggs# Embryos Day 3# Cells Day 3# Trans- ferred(+) hCGClinical pregnanciesBlastocyst (B)3236.314.7∗p<0.5 vs. D3.9.8∗p<0.5 vs. D3.6.5∗p<0.5 vs. D3.1.615 (46.8%)12 (37.5%)Day 3 (D3)4536.210.54.95.34.318 (40%)17 (37.8%)∗ p<0.5 vs. D3. Open table in a new tab Conclusions: (1) Patients without multiple embryos at or beyond the 8-cell stage on the morning of Day 3 still have a good prognosis for clinical pregnancy when Day 3 embryo transfer is performed after assisted hatching. (2) The presence of at least 3 embryos at or beyond the 8-cell stage on Day 3 results in a high percentage of patients having at least 1 blastocyst to transfer and good pregnancy rates. (3) Clinical pregnancies can occur in patients who have no embryos at the blastocyst stage on Day 5. (4) Patients without multiple embryos at advanced cell stages on Day 3 may benefit more from early transfer with assisted hatching rather than continued growth to the blastocyst stage.

The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras.

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid<-->8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid<-->8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell<-->8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid<-->8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

Threatened Fishes of the World: Carcharias taurus (Rafinesque, 1810) (Odontaspididae)

Threatened Fishes of the World: Carcharias taurus (Rafinesque, 1810) (Odontaspididae)

Perifollicular blood flow doppler indices, but not follicular pO 2, pCO 2, or pH, predict oocyte developmental competence in in vitro fertilization

Objective: To assess the relationships among perifollicular blood flow; follicular fluid pO 2, pCO 2, and pH; oocyte developmental capacity; preimplantation embryo quality. Design: Prospective study. Setting: Academic, tertiary care institution. Patient(s): Unselected, gonadotropin-stimulated IVF cycles. Intervention(s): Color, pulsed Doppler analysis of perifollicular blood flow, and follicular pO 2, pCO 2, and pH determinations of randomly designated, mapped ovarian follicles. Main Outcome Measure(s): Fertilization and day 3 embryo cleavage and morphology. Result(s): Perifollicular vascularity indices were significantly and negatively correlated with day 3 embryo cleavage. Pulsatility index and S-D ratio also were significantly and negatively correlated with follicular pO 2. The same correlation was found between resistance index and the fertilization rate of preovulatory oocytes. No relationship existed between follicular metabolic analysis and fertilization or embryo quality. The resistance index had a sensitivity of 0.57 and a specificity of 0.71 for the prediction of advanced embryo cleavage status. Conclusion(s): Results confirm and extend previous reports demonstrating that color, pulsed Doppler ultrasound analysis of individual preovulatory follicles during IVF therapy may provide an indirect index of the developmental competence of the corresponding oocyte. Although these methods may provide means to select embryos for transfer with the highest implantation potential, the moderate predictive power showed so far may limit their clinical applicability.

SOMATIC EMBRYOGENESIS IN ANISE (PIMPINELLA ANISUM L.): THE EFFECT OF PROLINE ON EMBRYOGENIC CALLUS FORMATION AND ABA ON ADVANCED EMBRYO DEVELOPMENT

Somatic embryos of anise (Pimpinella anisum) are an excellent system to investigate the biosynthesis of food-related phenolic metabolites like anethol which is linked to differentiation. Somatic embryogenesis in anise is a two part system. The first is a 2,4-D (2,4-dichlorophenoxy acetic acid)-induced embryogenic callus formation which requires optimal levels of endogenous auxin and cytokinin levels. The second is the advanced embryo development stage in the absence of 2,4-D which requires abscisic acid to stimulate embryo growth. Our work has shown that proline and the precursors of proline (ornithine and arginine), in combination with the proline analog, azetidine-2-carboxylate (A2C) significantly stimulated 2,4-D-induced embryogenic callus formation and subsequent somatic embryo development in hormone-free medium. Further, abscisic acid stimulated somatic embryo development in anise in the second stage. Total phenolic assays done on embryogenic tissue during 2,4-D-induced embryogenic callus, formation, and during abscisic acid-mediated embryo development showed that levels were highest in embryogenic tissue induced in the presence of proline plus A2C. Total phenolic levels of developing embryos in the absence of 2,4-D and in the presence of abscisic acid were higher than that of the control. A hypothesis for further investigation is proposed based on the potential role of the proline-linked pentose phosphate pathway in stimulating the biosynthesis of purines and phenolic metabolites during anise somatic embryogenesis.

Intracytoplasmic sperm injection of in vitro-matured equine oocytes.

Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and processed for light and transmission electron microscopy. Single pronucleus formation was observed in 2 out of 12 presumptive zygotes 10 h postinjection, at which time abundant cortical granules were observed in the subplasmalemmal region. Twenty hours postinjection, however, 2 pronuclei were observed in 6 of 12 injected oocytes (fertilization rate 50%), and almost all cortical granules were released. The cleavage rate in vitro was 16% after 72 h in culture, and the most advanced embryo stages obtained were 6- to 8-cell embryos. The cleavage rate in vivo was very low since only 1 of 10 recovered had cleaved to the 2-cell stage. Thus, in conclusion, ICSI fertilization of equine oocytes did result in fertilization, pronucleus formation, and cortical granule release. However, the observed fertilization rate and oocyte activation was not paralleled by substantial cleavage of the zygotes.

Beneficial effects of cysteine and β-mercaptoethanol on development of bovine IVM/IVF embryos in a cell-free, serum-free culture system

Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.

DNA methyltransferase activity in the early stages of a sea urchin embryo Evidence of differential control

The specific activity of DNA methyltransferase increases in the nuclei of Sphaerechinus granularis sea urchin embryos at increasing stages of development. The activity reaches maximal value at about 20 h of growth, when embryos are at the mesenchyme blastula stage, then abruptly decreases and is essentially zero at about 35 h of development, when embryos are at the early gastrula stage. Both the increase and the drop of the activity are faster than embryonic cell duplication indicating that the enzyme is under strict control during development and that, in the more advanced embryo, a mechanism is activated to specifically block its activity.

Physiologically Inactive Nucleoli Contain Nucleoplasmic Ribonucleoproteins: Immunoelectron Microscopy of Mouse Spermatids and Early Embryos

Physiologically inactivating or reactivating nucleoli represent a good model to investigate modifications of the nucleolar activity, which influence to a great extent the morphology of this organelle. We have studied the nucleoli of mouse spermatids during gradual inactivation, as well as those of early mouse preimplantation embryos, which undergo reactivation. In the 2-cell systems, inactive nucleoli are represented by homogeneously, finely fibrillar spherical bodies. With the aim of clarifying the composition of these nucleoli, we have analyzed them by means of immunoelectron microscopy using specific antibodies directed against nucleoplasmic snRNPs, hnRNPs, ribosomal proteins, or fibrillarin as well as by cytochemical methods for visualizing DNA and RNA. Our results indicate that RNA is present in zygote and 2-cell nucleolus precursor bodies (NPBs) as well as in 4- to 8-cell or more advanced embryo nucleoli, but not in inactive spermatid nucleoli. DNA is absent from inactive spermatid nucleoli and NPBs but is present within the nucleolus-associated chromatin and the nucleolonema of active nucleoli. The dense masses constituting the NPBs of the zygote and 2-cell embryos contain ribosomal proteins and fibrillarin but also hnRNPs and nucleoplasmic snRNPs. Of these, only fibrillarin is present in the spermatid residual nucleoli.

In vitro production of bovine embryos: A progress report and the consequences on the genetic upgrading of cattle populations

In vitro bovine embryo production (IVP) has improved tremendously during the past decade. Repeatable and reliable systems for mass production of embryos for use in cattle breeding schemes are now available in many laboratories all over the world. The different steps involved in IVP are discussed in the present review. In vitro maturation of oocytes (collected at the abattoir or by transvaginal ultrasound-guided aspiration from live animals) for 24–27 h results in a maturation rate of 82%. In vitro fertilization with heparin capacitated spermatozoa leads to a normal penetration rate of 56%, an incidence of polyspermia and parthenogenetic activation of 6% and 12%, respectively. By in vitro culture in a cell-free medium or a medium containing bovine oviduct epithelial cells (or conditioned medium) of presumptive zygotes it is possible to obtain compact morula and blastocyst rates of 33% and 26%, respectively. The quality of the embryos is inferior to in vivo produced and even to zygotes cultured in sheep oviducts. This is reflected in their morphology (darker, early blastoceol formation, fewer cells per embryo) and their increased susceptibility to freezing. A higher incidence of embryonic loss is likewise registered after non-surgical transfer. Possible causes are unknown as of today, but placental defects may be suspected. This is a serious obstacle to a more efficient use of IVP in cattle breeding schemes, which includes: twinning, cloning, transgenesis, other research purposes, studies on embryonic mortality; use of oocytes from calves and fetuses; alleviation of certain problems in heifers and cows (genital problems, poor superovulatory response) and bulls (treatment of certain types of infertility) and finally more efficient use of semen. In the final part of the paper a model for an advanced embryo technology center is presented. This center will be based on the conventional embryo transfer methodology, but will also include newer techniques such as in vitro production of embryos (recovered at slaughter or by ultrasonography), cloning and preimplantation diagnosis of embryos (sexing). The future of cattle breeding depends heavily on these new technologies and it is the responsibility of the scientist to address one essential issue: to improve the viability of in vitro produced embryos in order to be able to achieve satisfactory survival rates after freezing and transfer of both fresh and frozen in vitro produced embryos.