Alban Massip

Recent progress in cryopreservation of cattle embryos

Abstract Two cryopreservation methods adapted to field use were investigated with cow embryos. Firstly, a freezing method combining 1.36 M glycerol and 0.25 M sucrose in PBS. The use of sucrose, by its osmotic properties, predehydrates the embryo allowing slow cooling at 0.3°C/min to be interrupted at −25°C. Direct transfer, after thawing from liquid nitrogen, gave a pregnancy rate of 51.8% ( 14 27 ). Secondly, a vitrification method involving a mixture of glycerol and 1,2 propanediol in PBS at concentrations giving an amorphous state on plunging in liquid nitrogen. Pregnancy rate after transfer of vitrified late morulae very early blastocysts was 39.1% ( 9 23 ).

Glucose metabolism during bovine preimplantation development: analysis of gene expression in single oocytes and embryos.

Glucose metabolism of the bovine embryo is low during the first cleavages and increases sharply after the major resumption of the genome (8–16 cells). The mRNA level for genes involved in glucose metabolism was tested by RT‐PCR on individual oocytes and embryos at different stages of development. These genes were: glucose transport GLUT‐1, hexokinase (HK), glucose‐6‐phosphate‐dehydrogenase (G6PDH), and glucose‐phosphate‐isomerase (GPI); actin was used as a reference transcript. RT‐PCR results revealed three types of oocytes or embryos: positive with a PCR signal for each transcript considered, nul with no signal for any transcript, and heterogeneous with a PCR signal for some transcripts and none for others. The number of nul and heterogeneous samples was higher for slow than for fast‐cleaving embryos (81% vs. 36%), and the proportion of positive embryos increased significantly at the 16‐cell and morula stages (P < 0.002), suggesting a correlation between mRNA content and developmental capacity. In positive embryos, GLUT‐1 mRNA level was reduced by half during maturation and fertilization. Actin and hexokinase mRNA levels decreased during the first cleavages, but significantly increased at the 16‐cell and morula stages, respectively. GPI transcript remained stable throughout development, whereas there was a significant rise for G6PDH at the 4‐cell stage, perhaps due to a polyadenylation process. Finally, the absence or decrease in intensity of several transcripts at the blastocyst stage suggests suboptimal culture conditions. Mol. Reprod. Dev. 48:216–226, 1997.

Cell Cycle Duration at the Time of Maternal Zygotic Transition for In Vitro Produced Bovine Embryos: Effect of Oxygen Tension and Transcription Inhibition

Abstract Early embryonic cleavages are mostly regulated by maternal components then control of development progressively depends on newly synthesized zygotic products. The timing of the first cleavages is a way to assess embryo quality. The goal of this study was to evaluate the duration of the fourth cell cycle, at the time of maternal-to-zygotic transition (MZT) in in vitro-produced bovine embryos by means of cinematographic analysis. We found that 75% of the embryos displayed a long fourth cycle (43.5 ± 5.4 h) whereas the remaining embryos had a very short fourth cell cycle (8.9 ± 2.9 h). Both groups did not differ in cleavage rhythm up to the eight-cell stage and timing of cavitation and blastocyst expansion was identical. However, embryos with a short fourth cell cycle had a better blastocyst rate than embryos with a long cycle (59% versus 38%, P < 0.01). Total cell number, inner cell mass (ICM):total cell ratio, and hatching rate were identical for blastocysts produced from embryos with either a long or a short fourth cell cycle. In a second experiment, we showed that increasing the oxygen tension, from 5% to 20%, decreased the percentage of embryos with a short fourth cell cycle, from 25% to 11% (P < 0.01), indicating that suboptimal culture conditions can influence the length of this cycle. Finally, we investigated whether fourth cell cycle duration could be influenced by transcription inhibition. With alpha-amanitin added at 18 h postinsemination (HPI), cleavage was reduced (66% versus 79%) and, at 70 HPI, the 9- to 16-cell rate increased (50% versus 25%) concomitantly with a 5- to 8-cell rate decrease (16% versus 47%). A similar pattern was observed when the drug was added at 6 HPI or 42 HPI but not at 0 HPI. Cinematographic analysis revealed that alpha-amanitin increased the first cell cycle duration whereas the second and third cell cycles were not affected. With the drug, one third of the embryos could develop up to the 9- to 16-cell stage and they all had a short fourth cell cycle (11.2 ± 3.7 h) with a good synchrony of cleavage between blastomeres. These results suggest that duration of the fourth cell cycle of bovine embryo, during the MZT, is under a zygotic transcriptional control that can be affected by oxidative conditions.

Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage

Immature bovine oocytes were matured and fertilized in vitro, and the resulting zygotes were cultured to the blastocyst stage in droplets of tissue culture medium 199 (TCM 199) conditioned by oviduct cells in the absence of serum. In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing 1, 4 or 40 zygotes in 40 microl of culture medium. The cleavage rate was low with a single embryo (36%) but increased with the number of embryos, to reach 50% with 4 embryos/40 microl and 59% with 40 embryos/40 microl. Blastocyst formation was nil with 1 embryo per 40 microl, reaching 2.5% with 4 embryos/40 microl and 18% with 40 embryos/40 microl. The effect of the size of the drop was assessed in Experiment 2, the concentration of embryos remaining constant (1 embryo/1 microl). The volumes tested were 10, 20, 30 and 40 microl. Development into blastocysts increased gradually from 12% in the 10 10 group to 20% in the 40 40 group. Experiment 3 was designed to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. The minimum tested volume was 5 microl and was not successful. These results show that bovine embryos cultured in oviduct-conditioned TCM 199 need to cooperate to reach the blastocyst stage. The mechanism of this cooperation is not known, but some autocrine/paracrine factors, probably growth factors, could promote embryo development as was demonstrated in mice. From Experiment 2 we can hypothesize that the surface volume ratio of the droplets could play a role in the culture conditions by interfering with the exchanges between the culture medium and the surrounding environment.

Effect of Conventional Controlled-Rate Freezing and Vitrification on Morphology and Metabolism of Bovine Blastocysts Produced In Vitro

Abstract This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.

EXPRESSION OF CU/ZN AND MN SUPEROXIDE DISMUTASES DURING BOVINE EMBRYO DEVELOPMENT: INFLUENCE OF IN VITRO CULTURE

Temporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase–polymerase chain reaction (RT‐PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5‐ to 8‐cell embryos while no transcript was found at the 9‐ to 16‐cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo–derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro‐derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions. Mol. Reprod. Dev. 58:45–53, 2001.

Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos.

The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H2O2 as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H2O2 degrading ability. Essential and nonessential amino acids also significantly reduced the H2O2 concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9–16‐cell stage embryos and blastocysts to 0 M; 10−7 M ; 10−6 M, and 10−5 M H2O2 in pyruvate‐free mSOF was evaluated. Developmental rates of the H2O2‐treated zygotes to the 5–8‐cell or blastocyst stages and survival of H2O2‐treated blastocysts were reduced in a dose‐dependent manner whereas the 9–16‐cell embryos were unaffected by those treatments. Blastocysts treated with H2O2 also tended to have lower numbers of bisbenzimide‐stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10−5 M H2O2 pulse highly reduced the H2O2 concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H2O2, the 9–16‐cell embryos being more resistant than zygotes and blastocysts and that H2O2 and its toxic effects can be attenuated by including pyruvate in the medium. Mol. Reprod. Dev. 52:149–157, 1999.

Embryo production by ovum pick up in unstimulated calves before and after puberty.

The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty.

Calving outcome following transfer of embryos produced in vitro in different conditions

A survey of calving outcome from embryos produced in vitro by three laboratories under different culture conditions is presented. A total of 572 embryos were transferred after culture and 170 calves were born: 9 calves from 47 (19%) embryos produced in coculture with bovine oviduct cells (BOCC) or medium conditioned by these cells (BOCM+); 5 from 16 (31%) embryos produced in serum free oviduct conditioned medium (BOCMS-); 8 from 24 (33%) embryos derived from BRL conditioned medium (BRLCM-); 0 from 9 embryos cultured in SOF (20% O-2); 132 from 427 (31%) cocultured with granulosa cells (GCC); 16 from 49 (33%) embryos cultured in SOF (5% O-2). Frozen embryos (n = 120) were transferred soon after thawing and 25 calves were obtained: ii from 59 (19%) embryos in the first condition (BOCC); 6 from 19 (32%) in the second condition (BOCMS+); 6 from 34 (18%) in the third (BOCMS-) and 2 from 8 (25%) in the fourth condition (BRLCMS-). Gestation length and birth weight were in the normal range except for 5 calves that were exceptionally large. Sex ratio was in favour of males. Neo and perinatal mortality ranged from O to 19%. Three cases of malformations were observed, Growth rate was normal.

Comparative immunohistochemical distribution of connexin 37 and connexin 43 throughout folliculogenesis in the bovine ovary

Among gap junctional proteins previously identified in the mouse ovary, connexins (Cx) Cx37 and Cx43 appeared to be essential for normal follicular growth. The aim of this work was to detect Cx37 expression in the bovine ovary, then to quantify and compare its follicular distribution pattern with that of Cx43 using quantitative analysis of immunofluorescently labeled ovary sections viewed with a confocal laser scanning microscope. Cx37 immunoreactivity was detected in bovine ovarian follicles and was predominantly localized at preantral stages. Unlike follicular Cx43 expression which was restricted to granulosa cells, Cx37 staining was observed in both oocyte and granulosa cell compartments. While no changes were seen during early follicular growth, the level of Cx37 expression decreased significantly at the onset of antral cavity formation (P ≤ 0.01). On the contrary to what was found for Cx37, Cx43 was weakly expressed in preantral follicles. Concomitant with antrum formation, the level of Cx43 expression increased significantly (P ≤ 0.01). A further increase was correlated with antral follicular size (P ≤ 0.01). Cx43 immunoreactivity declined significantly in morphologically atretic follicles (P ≤ 0.01). A comparative analysis showed that Cx37 and Cx43 expression patterns were differentially regulated and could reflect specific physiological roles for each gap junction protein throughout folliculogenesis in cow. Mol. Reprod. Dev. 57:60–66, 2000.