In vitro fertilization and early embryo culture was undertaken in the South American marsupial, the grey short-tailed opossum, Monodelphis domestica. Adult females were induced into oestrus by a system of pairing with an unfamiliar male and mature oocytes were recovered from the ovary 15–18 h after mating and placed in pre-warmed modified MEM medium at 33°C (basal body temperature) or 37°C. Spermatozoa recovered from the cauda epididymides of adult males were preincubated in medium for 2 h during which time paired spermatozoa separated and initiated hyperactivated motility. Oocytes were transferred to 0.4 ml drops of spermatozoa containing 0.5–1.0 × 106 spermatozoa ml−1. Only single spermatozoa bound to the zona pellucida, and fertilization occurred within 1–2 h as indicated by a breach in the zona and confirmed by electron microscopy. At 37°C, 95 of 152 (62.5%) oocytes were fertilized and 64 (67%) developed to two-cell stage or beyond. At 33°C, 5 of 28 (18%) oocytes were fertilized. This is the first report of complete in vitro fertilization in a marsupial.
H. D. M. Moore and D. A. Taggart
J. R. Parratt, M. J. Taggart and S. Wray
Changes in intracellular pH have differing effects on contraction in different types of muscle, and between species. Because of the large number of women requiring a caesarean section due to failure to progress in labour, it is important to know how the human myometrium responds to the changes in intracellular pH that may occur during labour. The pH-sensitive dye, carboxy-SNARF, was used to measure intracellular pH in small strips of human myometrium. Intracellular pH and tension were simultaneously recorded in pregnant and nonpregnant tissue. Intracellular pH was altered by the addition of weak acids and bases. Intracellular alkalinization caused an increase in the frequency and amplitude of contractions. Intracellular acidification led to an initial increase in the frequency and/or the amplitude of the contractions, followed by abolition of contractions. Alterations in intracellular pH had profound effects on contraction in human uterine smooth muscle. Possible mechanisms are discussed whereby pH could influence force production, and changes in contraction are related to the speed and extent of the change in intracellular pH.
R R, R Speed, M Taggart and HJ Cooke
Dazl knockout male mice are infertile because their germ cells are unable to complete the first meiotic prophase in the first wave of spermatogenesis and thereafter decrease in number due to a block at the A-aligned to A1 transition. The ability of the surviving somatic components of the testes to retain their function in the absence of mature germ cells was tested by injecting marked wild-type germ cell suspensions containing spermatogonial stem cells. Comparison of the frequency and extent of colonization of Dazl knockout testes with that of testes chemically depleted of germ cells showed little if any difference. It was concluded that Dazlko testes seem unimpaired in their ability to support spermatogenesis. Therefore, Dazlko testes provide a useful and reliable recipient in which to evaluate spermatogonial stem cells. The results furthermore demonstrate that the somatic compartment of the testis of these animals retains functionality.
PT Saunders, JM Turner, M Ruggiu, M Taggart, PS Burgoyne, D Elliott and HJ Cooke
The autosomal gene DAZL is a member of a family of genes (DAZL, DAZ, BOULE), all of which contain a consensus RNA binding domain and are expressed in germ cells. Adult male and female mice null for Dazl lack gametes. In order to define more precisely the developmental stages in germ cells that require Dazl expression, the patterns of germ cell loss in immature male and female wild-type (+/+, WT) and Dazl -/- (DazlKO) mice were analysed. In females, loss of germ cells occurred during fetal life and was coincident with progression of cells through meiotic prophase. In males, testes were recovered from WT and DazlKO males obtained before and during the first wave of spermatogenesis (days 2-19). Mitotically active germ cells were present up to and including day 19. Functional differentiation of spermatogonia associated with detection of c-kit positive cells did not depend upon expression of Dazl. RBMY-positive cells (A, intermediate, B spermatogonia, zygotene and preleptotene spermatocytes) were reduced in DazlKO compared with WT testes. Staining of cell squashes from day 19 testes with anti-gamma-H2AX and anti-SCP3 antibodies showed that germ cells from DazlKO males were unable to progress beyond the leptotene stage of meiotic prophase I. It was concluded that in the absence of Dazl, germ cells can complete mitosis, and embark on functional differentiation but that, in both sexes, progression through meiotic prophase requires this RNA binding protein.
W. G. Breed, D. A. Taggart, V. Bradtke, C. M. Leigh, L. Gameau and J. Carroll
The effect of different cryopreservation methods on the development and ultrastructure of preimplantation embryos of Sminthopsis crassicaudata, a small carnivorous marsupial and member of the family Dasyuridae, was investigated. Females were primed with 1 iu pregnant mares' serum gonadotrophin to induce oestrus and ovulation. Mating generally ensued and, approximately 6 days after priming, embryos were collected and cultured in 5% CO2 in air at 35°C for 18–22 h in either Dulbecco's modified Eagles medium (DMEM) with high glucose or human tubal fluid medium (HTF), both supplemented with 10% fetal calf serum. Cleavage rates were higher in DMEM than in HTF. One slow and two ultrarapid freezing methods were used. Two out of 12 (17%) embryos cleaved in culture after freezing and thawing using the slow regimen, compared with six of 16 (38%) non-frozen controls. In addition, two of 11 (18%) embryos cleaved in culture following ultrarapid freezing and thawing by one of the two methods, compared to 31 of 41 (76%) non-frozen controls. Most of the embryos appeared morphologically normal under the light microscope after freezing and thawing by the slow regimen, but considerable variation in the degree of ultrastructural damage to the cellular organelles was evident with the transmission electron microscope. The rather low rate of cleavage after freezing and thawing was probably due, at least in part, to ultrastructural damage of the cells.
D B B P Paris, D A Taggart, G Shaw, P D Temple-Smith and M B Renfree
Changes in semen quality and morphology of the male reproductive tract were studied throughout the year in the highly promiscuous tammar wallaby. Body size, semen quality and gross morphology of the reproductive organs were assessed in adult males each month from January to November. The mean weight of males was similar in most periods sampled, but males were slightly heavier in the minor (P < 0.05) than the non-breeding season. Since body weight was correlated with weights of the testes, epididymides and accessory sex glands, organ weights were adjusted for body weight in subsequent analyses. In the major breeding season (late January/early February), when most females go through a brief, highly synchronized oestrus, the testes, prostate, Cowper’s glands, crus penis and urethral bulb were heaviest, volume and coagulation of ejaculates were greatest, and sperm motility had increased. Semen samples collected by electroejaculation at this time contained low numbers of spermatozoa, possibly as a result of dilution and entrapment by the seminal coagulum or depletion of epididymal stores during intense multiple mating activity. In the non-breeding season (late May–July), when mating does not normally occur in the wild, there was a significant decrease in the relative weight of nearly all male reproductive organs and a decline in most semen parameters. In the minor breeding season (September–November), when pubertal females undergo their first oestrus and mating, the weights of testes, epididymides and most accessory sex glands had significantly increased similar to those of males in the major breeding season. The total number and motility of ejaculated spermatozoa were highest during this period, but the volume and coagulation of ejaculates and weight of the prostate had only increased to levels that were intermediate between the major and non-breeding seasons. Ejaculate volume was strongly correlated with prostate weight, and % motile spermatozoa was strongly correlated with epididymis weight. Semen quality thus varied seasonally with changes in androgen-dependent reproductive organs in the male tammar wallaby and appeared to be influenced by the seasonal timing of oestrus in females. Semen quality may also improve in response to an increase in the number of available oestrous females.