Effects of growth hormone on the ultrastructure of bovine preimplantation embryos

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.This work was supported by the Deutsche Forschungsgemeinschaft (FOR 478/1)     

Lactate dehydrogenase in preimplantation rabbit embryos

Unfertilized eggs and early embryos up to the 2-day (16-cell) cleavage stage of development in the rabbit contain predominantly the most cathodal lactate dehydrogenase isoenzyme made up of A-type subunits. Following early cleavage there is a progressing increase in total LDH activity in the embryo as development proceeds through 4- and 6-day blastocyst stages. This is accompanied by an increase in the amount of B-type subunits and a concomitant shift in the lactate dehydrogenase isoenzyme electrophoretic pattern toward the anodal isoenzyme types.     

Development retardation in cultured preimplantation rabbit embryos

Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine. In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P less than 0.001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.     

Distribution patterns of rabbit embryos during preimplantation stage

The pattern of transport and distribution of rabbit embryos in the oviduct and uterus was studied 15 to 168 hours post coitum (p. c.). The reproductive tract was frozen in liquid nitrogen, thawed, and cleared in benzyl-benzoate solution using Orsini's technique. The location of the eggs and the ampullary-isthmic junction were identified using transmitted light from a dissecting microscope. Accumulation of the eggs in the oviduct occured in two phases. In the first phase the eggs were retained above the ampullaryisthmic junction, 3–12 hours after ovulation. In the second phase, the eggs were retained 36–60 hours after ovulation, above the uterotubal junction (at a distance approximately 12 % of the oviductal length). The rate of transport of individual eggs in the oviduct, and the time of the entry of eggs into the uterus were variable. Au 78 hours p. c. most blastocysts occupied the proximal half of the uterine horn, although some appeared very close to the internal os of the cervix. Spacing of blastocysts in the uterus, 114 to 120 hours p. c., involved movement of blastocysts away from the cervix. Unfertilized eggs remained in the uterus, along with developing blastocysts 168 hours p. c. Few eggs were retained in the oviduct at 108 and 115 hours p. c.     

RNA and protein synthesis in preimplantation rabbit embryos

A phenol-sodium dodecyl sulfate procedure has been used to extract DNA, RNA, and protein from 3-, 4.5-, and 6.5-day-old preimplantation rabbit embryos that were incubated for different lengths of time in the presence of labeled precursors. The results suggest that the newly-synthesized RNA and protein are more stable in the growing blastocyst than in the trophoblast at the time of implantation.This conclusion was derived from the results of embryo incorporation kinetics. The results also suggest that the increase in protein and RNA content in preimplantation rabbit blastocysts was preceded by an increase in protein synthesis accompanied by low catabolism.     

Effect of oxygen concentration in the incubator's gas phase on the development of cultured preimplantation rabbit embryos

Development of rabbit preimplantation embryos cultured under 20, 5 or 1% oxygen was studied. Three-day-old morulae were cultured in a protein-free medium (BSM II supplemented with 5 mg PVP/ml medium) for 24 and 48 h. Embryonic development was evaluated by gross morphology and by incorporation of tritiated thymidine as an indicator of cell proliferation. The lower oxygen concentrations yielded significantly better embryo development at 24 and at 48 h than the 20% concentration. There was no significant difference in development between 5% and 1% oxygen. Addition of the radical scavanger superoxiddismutase (SOD), either alone or in combination with catalase or reduced glutathione, did not improve embryo development even in the 20% oxygen group. Our data suggest the need to reduce in vitro oxygen levels from 20% to more physiological concentrations.     

Expression of adipokines in preimplantation rabbit and mice embryos

Recent studies point to a role for adipokines in reproduction. Leptin is involved in embryo metabolism and may participate in embryo-maternal crosstalk. Little is known about potential roles of other adipokines in reproduction. We therefore studied the expression of adiponectin and pathway members during the pre- and periimplantation period in rabbits and mice. Adiponectin protein is localized in glandular epithelium of the rabbit endometrium on day 6 and 8 p.c. and in mouse endometrium on day 3.5 and 5 p.c. Rabbit, but not mice blastocysts express adiponectin mRNA. Adiponectin receptors one and two, adiponectin paralogues and PPARs were found in both species. Both, trophoblast and embryoblast were adiponectin positive. Real time PCR for adipoR1 and adipoR2 in rabbit blastocysts of different gastrulation stages at day 6 p.c. revealed a specific switch in expression: Expression was high in the trophoblast in early stages and in the embryoblast shortly prior to implantation. In conclusion, during the pre- and periimplantation period, members of the adiponectin pathway are expressed in endometrium and blastocysts, with a specific expression pattern in the embryonic disk of the gastrulating rabbit blastocyst, giving support to a role of the adipokine network in blastocyst differentiation and embryo-maternal interactions.     

Binding of IGF-I to preimplantation rabbit embryos and their coats

It is known that insulin-like growth-factor I (IGF-I) promotes early embryonic development from the morula to the blastocyst stage in rabbits (28). Therefore we used autoradiography to investigate whether IGF-I binds to preimplantation embryos and its coats. From Day 3 after mating onwards, a clear binding of IGF-I to the embryos was observed. There was no difference in binding to the embryoblast or trophoblast cells. Using ligand blot, several IGF-binding proteins (IGFBP; 31 kDa, 33 kDa, 36 kDa, three overlapping bands at 40 to 55 kDa) were obvious in the embryoblast and trophoblast. A 120 to 130 kDa protein was observed exclusively in the embryoblast. Significant binding of (125)I IGF-I to the coats of embryos older than 3 d was detected, and IGF-I was bound via a 38 kDa protein, as detected by ligand blot. To investigate the origin of this protein, the patterns of IGFBP were determined in the oviductal and uterine fluids of pregnant animals (Days 0 to 6). The following binding proteins were observed regularly in the oviductal and uterine flushings: 28 kDa, 32 kDa and 3 overlapping bands in the area of 40 to 55 kDa. In the oviduct the main IGF binding protein was the 32 kDa band (38.7% to 45.9%), while in the uterus it was the 3 overlapping bands at 40 to 55 kDa (42.5% to 24.1%). Because IGF-I is produced in the oviduct and uterus (27), IGFBPs are found in oviductal and uterine fluids, IGF-I is stored in the coats, IGF-I binds to preimplantation embryos and IGF-I promotes early embryonic development (28), the IGF system seams to have a function in the maternal-embryonic interaction.     

Development of preimplantation rabbit embryos in uterine flushing-supplemented culture media

The development of cultured rabbit preimplantation embryos grown in standard media (Ham's F-10 or BSM II supplemented with bovine serum albumin (BSA) or homologous serum) or in Ham's medium supplemented with uterine flushings was compared. The uterine flushings derived from donors of 0.5-6 years of age. Uterine flushing supplemented media were used natively or after treatments like sterilization by filtration, lyophilization, three times freezing/thawing, heat denaturation, dialysis, or ultrafiltration. Compared with in vivo controls, embryonic growth was substantially reduced during in vitro culture, demonstrably by smaller diameters and impaired cell proliferation (measured by thymidine incorporation). The growth retardation was more pronounced in blastocysts (recovered at day 4 post coitum [p.c.]) than in morulae (recovered at day 3 p.c.). Development in uterine flushing media was notably better than in standard media but did not comply with in vivo development. Highest thymidine incorporation was observed in media with increased concentrations of uterine secretions and after sequential supplementation of flushings from subsequent progestational stages. Advanced donor ages, heating up to 80 degrees C, freezing, and lyophilizing did not affect incorporation data statistically significantly, whereas sterilization by filtration, ultrafiltration, and dialysis led to a significantly reduced thymidine incorporation in the cultured embryos. The positive effects of uterine flushing supplementation are attributed to the supply of components more adjusted to the needs of the cultured embryos and/or to a reduction of pathological effects in vitro like washing out of nutritive and regulatory components from the embryo into the surrounding culture medium.     

The effect of hyperthermia in vitro on vitality of rabbit preimplantation embryos

The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 degrees C or 42.5 degrees C) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 degrees C) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 degrees C or 42.5 degrees C) or standard temperature (control 37.5 degrees C), post-incubated overnight (16-20 h) at 37.5 degrees C and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 degrees C did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 degrees C. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 degrees C) and embryos (exposed to 41.5 degrees C). Following the elevation of temperature to 42.5 degrees C embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.     

Oxidative and conjugative metabolism of diethylstilbestrol by rabbit preimplantation embryos

Five- and six-day-old rabbit preimplantation embryos were found to be capable of metabolizing [3H]diethylstilbestrol (DES) in vitro. Based on the analysis of the metabolites formed during a 24-hr incubation period we conclude that these early stage embryos do have active monooxygenase and conjugative activity. The monooxygenase seems to be specific to this early stage of embryonic development.     

Development of preimplantation rabbit embryos in vivo and in vitro

Qualitative patterns of protein synthesis in preimplantation rabbit embryos grown in vivo and in vitro were examined by SDS polyacrylamide gel electrophoresis followed by autoradiography. The results demonstrate that (1) most qualitative changes in the pattern of protein synthesis occur during cleavage, (2) the blastocyst period of development is characterized by a remarkably uniform and constant pattern of protein synthesis, and (3) the qualitative pattern of protein synthesis in embryos cultured in vitro from the 1-cell to the blastocyst stage is essentially identical to the pattern of protein synthesis in embryos grown to a comparable stage in vivo.These results indicate that no “special” maternal factors, such as uterine proteins, are required in vitro either for the qualitative changes in the pattern of protein synthesis during cleavage, or for the initial expression of a pattern of protein synthesis characteristic of the entire blastocyst period. From these studies we conclude that, once fertilized, the rabbit egg proceeds through cleavage and blastocyst formation on its own endogenous developmental program.     

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.     

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.     

Effect of superoxide dismutase on the development of preimplantation mouse embryos

The role of superoxide dismutase (SOD) was tested on preimplantation development of mouse embryos in vitro. The presence of SOD in ovarian antral follicles and in oviductal and uterine secretions was also investigated. Zygotes from superovulated ICR female mice were cultured in modified Whittingham's T6 medium supplemented with SOD (0 to 370 U) or EDTA (100 muM) at 37 degrees C under 5% CO(2) in air. Supplementation of SOD (370 U) significantly promoted the development of zygotes to the blastocyst stage (45%) as compared to that of the controls (1.4%). This favorable effect of SOD was comparable to that of EDTA and completely suppressed by anti-SOD antibody. Blastocysts cultured with SOD consisted of 78.2+/-10.4 blastomeres and possessed as many blastomeres as those (81.6+/-9.3) developing in vivo; blastocysts cultured with EDTA had significantly fewer blastomeres (42.6+/-13.7). These findings suggest that SOD protects embryos against oxidative insults and that it can be an effective substitute for EDTA for supporting mouse embryo development in vitro. The SOD activity was detected in 3 different lumina from mouse reproductive organs, and SOD was identified as a cytosolic Cu,Zn-SOD on photochemically stained polyacrylamide gels. Our results suggest that oxidative injury may be responsible for developmental retardation of preimplantation-stage mouse embryos in vitro and that Cu,Zn-SOD may play a crucial role in protecting embryos against oxygen toxicity in vivo as well as in vitro.