Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature.

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN(2)) from -10, -20, or -33 degrees C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN(2) from -33 degrees C, very rapid thawing (10 s in 30 degrees C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30 degrees C for 10 s). By contrast, oocytes plunged into LN(2) from -10 or -20 degrees C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (-20 to -33 degrees C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to -33 degrees C could be plunged to -196 degrees C with little or no further damage.

Cryopreservation of activated mouse oocytes and zygote reconstitution after thaw.

A new approach to cryopreservation of unfertilized oocytes is proposed using techniques of artificial egg activation combined with nuclear transplantation. Matured mouse oocytes were released from metaphase II arrest by brief exposure to alcohol, allowed to progress to the pronuclear stage and then frozen according to a standard freezing protocol in propandiol. After thaw the female pronuclei were enucleated and fused with a male karyoplasts that were divided from in-vivo fertilized zygotes. Reconstituted zygotes, fresh and cryopreserved culture control zygotes were cultured to the blastocyst stage and transferred to pseudopregnant recipients. The rate of blastocyst formation was 75.8, 91.6 and 44.1% respectively. A total of 110, 215 and 70 blastocysts were transferred to pseudopregnant females respectively. The implantation rates were 36.4, 72.0 and 75.7% while the rates of fetal viability at mid-gestation were 15.5 (P < 0.0001), 51.1 and 37.1% respectively.

Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stage in vitro. Specifically, the effects of cooling to subzero temperatures, thawing rate, LN2 plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN2 from -10, -20, or -33 degreesC at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, -0.3 C/min, plunging at -33 degreesC) rapid thawing by direct submersion in 30 degreesC water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.

Cloning of sheep and cow embryos.

The results of experiments aimed at cloning of sheep and cattle embryos are described. Two experimental approaches were used to study the developmental potential of blastomeres from sheep and cow embryos: (i) blastomere separation followed by culture and (ii) fusion of isolated blastomeres with enucleated eggs followed by culture. Both approaches allow embryos to be cloned, but whereas blastomere separation allows only a relatively small number of genetically identical animals to be produced, nuclear transplantation will probably open the way for large-scale cloning of livestock.

Transfer of immunity to Ostertagia circumcincta and IgA memory between identical sheep by lymphocytes collected from gastric lymph.

Sheep rendered immune to Ostertagia circumcincta were challenged with 50,000 larvae and lymphocytes were collected from the gastric lymph up to eight days after challenge. The cells were transferred intravenously to genetically identical worm-free sheep which, together with controls, were challenged with 50,000 larvae and killed nine days later. Cells obtained during the donors lymphoblast response to challenge transferred partial immunity, measured either as stunting or loss of worms. Significantly less immunity was transferred by cells collected either before or after this response. Thus the responding cells can mediate protective immunity to O circumcincta. On the other hand the donor sheep remained immune to their challenge infection despite being depleted of these functional cells, showing that their presence was not essential for immunity to be maintained. Comparison of the immunoglobulin A (IgA) concentrations in the gastric lymph of recipient and control sheep showed that a local IgA response had also been transferred. Enumeration of mucosal mast cells suggested that a mastocytosis had been transferred to the two recipients which were most immune to challenge.

Nuclear transplantation in sheep embryos.

Nuclear transplantation and cell fusion techniques have proved valuable for embryological studies in several non-mammalian animal species. More recently these procedures have been used successfully in small laboratory mammals, notably the mouse, to investigate the ability of nuclei and cytoplasm from various sources to produce viable embryos when combined. The use of a similar approach to study the developmental biology of large domestic animals presents a number of technical and practical difficulties, and so far there has been no report of attempts to perform nuclear transplantation in sheep embryos. Here I describe such a procedure and its use to investigate the development of embryos in which whole blastomeres from 8- and 16-cell embryos were combined with enucleated or nucleated halves of unfertilized eggs. The procedure involves bisection of single-cell eggs in a medium containing cytochalasin; fusion of egg halves with single blastomeres, induced using Sendai virus or an electrofusion apparatus; and embedding in agar, followed by culture of the reconstituted embryos in the ligated oviducts of ewes in dioestrus. I show that fully viable embryos may be obtained by this procedure.

Cytogenetic and blood group studies of sheep/goat chimaeras.

Aggregation chimaeras were composed of quarter (or 1 cell) contributions from 4-cell blastocysts of sheep or goats, or of an 8-cell blastocyst of one species enveloped in three 8-cell blastocysts of the other. Gestation was in sheep or goat recipient females. Of the 10 living animals born, 3 were identified as interspecific chimaeras by body conformation and coat type among the 7 quarter/quarter aggregations and 1 among the 3 giant aggregates. Interspecific chimaerism was identified by cytogenetic study of umbilicus and blood lymphocytes respectively of 2 of these, one from each type of aggregate. Intraspecific sex chimaerism was found in 3 other animals; 2 were of giant aggregate origin, but the 1 of quarter/quarter origin must have acquired it by placental anastomosis with a twin conceptus. Tests using species-specific monoclonal antibodies and electrophoretic separation of haemoglobins and isoenzymes demonstrated sheep and goat erythrocytes in one giant aggregate chimaera; their relative proportions and those of the blood lymphocytes changed over a period of 31 months from approximately 60% goat and 40% sheep to more than 90% sheep. The plasma transferrins and amylases did not show similar relative changes from their predominantly goat-like character and, by implication, neither did their tissues of origin.

Resistance to Haemonchus contortus transferred between genetically histocompatible sheep by immune lymphocytes.

The pattern of faecal egg counts after infection with 10,000 Haemonchus contortus larvae was similar within but not between, four pairs of monozygous twin sheep. Transfer of whole lymph or washed lymph cells from three immune donor sheep to their identical co-twin recipients reduced the susceptibility of the recipients to challenge with 10,000 larvae as measured by faecal egg counts. Cells from a nonimmune donor did not have this effect. In a final experiment, washed cells from an immune triplet sheep, which was actively responding to a repeated challenge infection, transferred a secondary local IgA response to a second recipient triplet and resulted in a marked reduction in worm count when compared with that of a third infectivity control triplet.

A simple procedure for the production of identical sheep twins.

Eggs were collected surgically on day 6, 7 or 8 from 18 Jacob ewes mated to a Welsh mountain ram. Thirty one (86 per cent) of the 36 eggs ovulated were recovered and of these 27 (87 per cent) had developed normally. All ovulated eggs were collected from 14 of the ewes. One (or more) normally developing morula or blastocyst was collected from 16 of the ewes. While the ewes remained under general anaesthesia each embryo was divided into two 'half' embryos with a thin glass needle. One monozygotic pair of 'half' embryos was retransferred to the embryo donor. The two ewes from which no normal embryos had been recovered were used as recipients for surplus bisected embryos from two other donors. Two of the 18 ewes returned to oestrus. The remaining 16 went to term producing, in all, eight pairs of identical twins, one pair of non-identical twins and seven single lambs.

Interspecific chimaerism between sheep and goat.

In rodents, chimaeric blastocysts produced by combining embryonic cells of two different species have been used in investigations of cell lineage and interaction during development (Mus musculus-Rattus norvegicus, M. musculus-Clethrionomys glareolus, M. musculus-Mus caroli). However, interspecific chimaerism also offers new approaches to the study of reproductive incompatibilities between species and may even allow such incompatibilities to be neutralized, thus improving the chances of successful hybridization and interspecific embryo transplantation. We report here the production of sheep-goat chimaeras by embryo manipulation and the use of interspecific chimaerism to allow successful interspecific embryo transplantation in sheep and goats.

Experimental chimaerism in sheep.

Composite sheep embryos (N = 110) were produced by aggregation of blastomeres from 2-, 4- or 8-cell embryos. Each composite embryo consisted of equal numbers of blastomeres from 2-8 parent embryos, the total cell number ranging from one quarter of the normal cell number to 8 times the normal cell number. The embryos were embedded in agar and transferred to ligated sheep oviducts to allow development up to the early blastocyst stage. Of the 101 embryos subsequently recovered, 77 had formed normally organized blastocysts and 74 of these were transferred to 51 recipients. Thirty-eight recipients went to full term, producing a total of 53 lambs. Of the 48 lambs which survived to be blood typed at 60 days of age, 36 were judged to be chimaeric on the basis of their blood type and/or on the basis of external features. The proportion of chimaeras was larger amongst the lambs produced from composite embryos of the normal number of cells or more (25/26) than amongst lambs produced from composite embryos of less than the normal cell number (11/22).

Attempts to produce monozygotic quadruplets in cattle by blastomere separation.

The blastomeres of eight-cell cow embryos were separated by micromanipulation into four pairs, inserted in foreign zonae pellucidae, embedded in agar and cultured for approximately four days in ligated sheep oviducts. Of 44 "quarter" embryos (11 monozygotic groups) transferred to sheep, 91 per cent had continued to develop at a normal rate and 77 per cent had formed small blastocysts with a single inner cell mass. Twenty-six blastocysts freed from the agar were transferred to heifers, each heifer receiving two monozygotic embryos, one to the tip of each uterine horn. Nine recipients were diagnosed pregnant by rectal palpation on day 50, six carrying twins. Thus 15 of the embryos had continued to develop including two sets of monozygotic quadruplets and one set of monozygotic triplets. Eight fetuses developed to full term, one set of monozygotic triplets, two sets of monozygotic twins (one set born dead) and one single.

The viability of early cleavage stages containing half the normal number of blastomeres in the sheep.

The developmental capacity of embryos produced by injection of single blastomeres of 2-cell embryos, pairs of blastomeres of 4-cell embryos or 4 blastomeres of 8-cell embryos into foreign zonae pellucidae was studied. The micromanipulated embryos were embedded in tiny agar cylinders, transferred to ligated oviducts of ewes on Days 2-8 of their oestrous cycle and recovered when the total age of the embryos was 5 1/2 to 6 1/2 days. Of 78 embryos transferred, 69 (88.5%) were recovereod, and of the latter 65 (94.2%) had developed at a normal rate. Thirty of these 'half' embryos were transferred to ewes on Day 6 of the oestrous cycle: 24 (80%) developed into lambs. There were no differences apparent between embryos derived from the 2-, 4- or 8-cell embryos with respect to developmental capacity. A further 12 'half' embryos were stored by deep-freezing for 1 or 2 months. After thawing, 9 of these were selected for transfer to ewes on Day 6: 3 developed into lambs, each of which was the monozygotic twin of a lamb resulting from the first series of transfers.

Effect of follicular steroids on the maturation and fertilization of mammalian oocytes.

Pronuclear development was used to measure the effects on ovine oocytes of altering follicular steroidogenesis during maturation in vitro. Follicular steroid secretion was altered using enzyme inhibitors and exogenous steroid supplementation. Abnormalities induced during maturation were measured 24 h after tranfer of oocytes to the oviducts of inseminated hosts. The presence throughout maturation of aminoglutethimide, an inhibitor of the conversion of cholesterol to pregnenolone, reduced steroid secretion to 7% of that in controls and decreased from 77% to 33% the number of normal oocytes. Abnormalities were substantially reduced by the addition of aminoglutethimide during the final 8 h of maturation only. The inhibition of 17 alpha-hydroxylase enzymes with SU10603 reduced oestrogen and testosterone secretion to about 10% of control levels but had no effect on progestin secretion. Only 13% of oocytes matured in the continual presence of SU10603 underwent normal fertilization. The number of oocytes undergoing normal fertilization was increased to about 50% by (i) delaying the addition of SU10603 until the last 8 h of the maturation period or (ii) adding exogenous steroids to follicles cultured with inhibitor from explantation. It is concluded that oocytes require a specific intra-follicular steroid environment for the completion of the full maturation process. Alterations to the steroid profile during maturation induce changes in the oocyte which are expressed as gross abnormalities at fertilization.