David G. Whittingham

Influence of genetic background and media components on the development of mouse embryos in vitro

One‐cell embryos from some inbred and random‐bred mice, but not those derived from certain F1 hybrids, suffer from a block during in vitro development known as the two‐cell block. This two‐cell block can be overcome by removing glucose or inorganic phosphate from the culture system or by altering the ratio of other medium components such as sodium, potassium, or bicarbonate. This issue is made more complex by the fact that the rate of development is different for each strain of mouse and this rate of development is invariably slowed under in vitro culture conditions. This study investigated the role of glucose and inorganic phosphate, individually or in combination, in relation to the two‐cell block, and rate of development in vitro of two random‐bred strains (CF‐1 and CD‐1) and an F2 hybrid derived from a nonblocking F1 hybrid cross (C57B1/6NCr × C3H/HeNCr). Results were compared with in vivo data for each strain, and between media. There was a significant difference in the rate of preimplantation development in vivo of the three strains chosen, which was mirrored in vitro, regardless of the medium. The two random‐bred strains suffered from a glucose‐related two‐cell block which was primarily mediated by inorganic phosphate. Inorganic phosphate was detrimental to embryo development regardless of strain or the presence of glucose. Although glucose, in the absence of inorganic phosphate, resulted in some blocking in development in the inbred strains initially, its presence in media was associated with increased rates of development at later stages in embryos that did not block. Glucose, but not inorganic phosphate, was beneficial but not essential to the development of the F2 embryos. The results of this study demonstrated that mouse embryos from different strains have differential rates of development in vivo and in vitro, and different sensitivities to glucose and inorganic phosphate. The two‐cell block was primarily induced in the combined presence of glucose and inorganic phosphate. Glucose was beneficial in the absence of inorganic phosphate, and inorganic phosphate was detrimental to the rate of in vitro development.

PREIMPLANTATION DIAGNOSIS OF DEFICIENCY OF HYPOXANTHINE PHOSPHORIBOSYL TRANSFERASE IN A MOUSE MODEL FOR LESCH-NYHAN SYNDROME

Male mice embryos deficient in hypoxanthine phosphoribosyl transferase (HPRT), derived from heterozygous (carrier) females and normal males, were diagnosed by biochemical microassay of HPRT activity in a single cell isolated from the eight-cell preimplantation embryo. The sampled embryos were transferred to recipient mothers and examined on the 14th day of gestation to confirm the accuracy of the preimplantation diagnosis. The diagnosis was sufficiently rapid that freezing of the embryos before transfer was not necessary. Of the embryos diagnosed as HPRT negative all 4 that grew into fetuses were correctly identified as HPRT-deficient males.

Capacitation-like changes occur in mouse spermatozoa cooled to low temperatures

Previously we showed that > 70% of mouse spermatozoa cooled slowly from 37°C to 4°C and warmed have undergone capacitation‐like changes as examined by a chlortetracycline staining assay. These membrane changes are reflected in the ability of cooled spermatozoa to achieve fertilization rates in vitro similar to those of uncooled controls when added to oocytes immediately upon warming. The aim of this study was to determine the nature of these membrane changes. We found they were not dependent upon the rate of cooling to 4°C and similar changes were observed when spermatozoa were cooled to higher temperatures (10° and 20°C), but it took longer for 50% of the spermatozoa to undergo such changes (3, 18, and 27 min for spermatozoa held at 4°, 10°, and 20°C, respectively). Mixing cooled spermatozoa with oocytes immediately upon warming produced fertilization rates similar to fresh spermatozoa capacitated in vitro for 90 min before the oocytes were added. The rate of sperm penetration as determined by the fluorescent DNA stain Hoescht 33258 was also similar. However, the penetration time for cooled spermatozoa was significantly shortened when they were preincubated for 90 min before being added to oocytes. We conclude that membrane changes resembling capacitation (1) occur during cooling to temperatures above freezing, (2) are independent of cooling rate, (3) proceed faster at lower temperatures, and (4) obviate the need for prior capacitation in vitro before mixing with oocytes. Mol. Reprod. Dev. 46:318–324, 1997.

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: A study with various mouse strains

Abstract Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J ;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOMAA for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.

Genome cryopreservation: a valuable contribution to mammalian genetic research.

Mouse embryo banking has become an important asset to geneticists. Individual laboratories can now maintain a far greater diversity of stocks than by conventional breeding alone. Also, many mutations that in the past would have been discarded due to lack of space, can now be preserved for future use. Recent advances in cryopreservation techniques have simplified procedures and, in certain cases, resulted in increased rates of survival.

Spontaneous induction of an homologous Robertsonian translocation, Rb(11.11) in a murine embryonic stem cell line

Karyotype analysis of a series of established mouse embryonic stem cell (MESC) lines showed that the majority were aneuploid by the 7th and 9th passage and that all lines contained a single Robertsonian (Rb) translocation chromosome with a symmetrical, homologous, arm composition Rb(11.11). Although the chromosomal imbalance makes these MESC lines unsuitable for genetic manipulation in vitro and hence for subsequent production of transgenic animals, the spontaneous occurrence and stable retention of the homologous Rb(11.11) as the only metacentric chromosome in an otherwise all acrocentric karyotype, provides potentially useful cell lines for gene assignment and recombinant DNA studies.

Chapter 9 – Reproductive Physiology

Publisher Summary This chapter discusses the reproductive physiology of laboratory mice. It also discusses the sex-determining process and the process of gametogenesis in laboratory mice. The production of gametes involves a period of mitotic multiplication of the stem cells derived from the primordial germ cells before entry into meiosis. The timing of these processes differs between the sexes and profoundly affects the total number of gametes produced during the reproductive life of each sex. In most strains of mice, the fetal testis is recognizable by 13 days of gestation. The formation, development, and maturation of the female gametes begin early in embryonic life; however, it is not completed until the time of ovulation. The primordial germ cells give rise to a definitive population of oocytes, which remains in the prolonged resting (dictyate) stage of the prophase of the first meiotic division until about 12 h before ovulation. The physiological and behavioral changes associated with the estrous cycle depend upon a complex integration of pituitary, ovarian, and probably adrenal hormones under the integrative control of the hypothalamus. The alternation of light and dark controls the timing of events in the cycle.