Karl M. Ebert

Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes☆

To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13- to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization , in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtubule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components.

Expression of the human α1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis

Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell‐based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human α1,2‐fucosyltransferase (H‐transferase, HT). H‐transferase expression modifies the cell surface carbohydrate phenotype of the xe‐nogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Galα1,3‐Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine‐inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Galα1,3‐Gal epitope, reduced XNA reactivity, and an increased resistance to human serum‐mediated cytolysis. Transgenic pigs that express H‐transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.—Costa, C., Zhao, L., Burton, W. V., Bondioli, K. R., Williams, B. L., Hoagland, T. A., DiTullio, P. A., Ebert, K. M., Fodor, W. L. Expression of the human α1,2‐fucosyltrans‐ferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum‐mediated cytolysis. FASEB J. 13, 1762–1773 (1999)

Transgenic farm animals: Progress report

Abstract The transfer of genetic material by recombinant DNA technology is an innovative method designed to produce animals with an altered genotype. Transgenic animals may demonstrate a variety of new phenotypes through the expression of the exogenous DNA molecule. Mice developed by these methods have shown that a wide range of promoter elements result in predictable patterns of tissue-specific and hormonally regulated fusion gene products. However, only a limited number of promoter elements have been introduced into domestic farm animals. Although several experiments were initially designed to alter the phenotype through increased rate of growth and improved carcass composition, the lack of specificity and regulation of fusion genes has generally resulted in negative side effects. The commercial sector, however, has invested in this new technology with the goal of producing large amounts of valuable human pharmaceutical drugs in a more efficient manner. If this is to be successfully accomplished transgenic animals must maintain their normal physiological characteristics. The challenge we face is to apply this novel approach to the large domestic species without altering their inherent genetic competence. This report updates the research on transgenic farm animals and outlines a strategy for the production of transgenic goats.

Collection and transfer of microinjectable embryos from dairy goats.

Abstract This pilot project was designed to optimize the collection and transfer of microinjectable embryos. Does were synchronized with norgestomet (6 mg) ear implants between December 1987 and April 1988. Implants remained for at least 14 d, and does were superovulated with one of the following: Treatment 1, PMSG (1000 IU) 24 h prior to implant removal; Treatment 2, FSH-P (20 mg) over 4 d, beginning 48 h prior to implant removal; or Treatment 3, FSH-P (24 mg) over 3 d, beginning 24 h prior to implant removal. Animals in Treatment 2 were in estrus by 24 h, in Treatment 3 within 36 h, and in Treatment 1 between 24 and 48 h post implant removal. Treatment 2 had the largest number of ovulations (-x=19.8) and the largest collection of both embryos and unfertilized oocytes (-x=13.6). The collection of fertilized embryos was higher in Treatment 3 (-x=5.0) than in Treatment 2 (-x=2.6) or Treatment 1 (-x=2.7). One-cell embryo collection was optimized by collecting 72 to 79 h following implant removal. All embryos were microinjected with 2 to 3 picol of tris-EDTA buffer into the male pronucleus. Of seven recipient does receiving a total of 34 injected embryos, three (43%) maintained pregnancy and six kids were born. These data establish regimens for superovulation/ synchronization and timing of pronuclear embryo collection for dairy goats for the purpose of microinjection.

Novel repeat elements direct rat proenkephalin transcription during spermatogenesis.

The developmental program controlling sperm formation occurs in multiple stages that sequentially involve mitosis, meiosis, and spermiogenesis. The transcriptional mechanisms regulating these distinct phases are poorly understood. In particular, while a required role for the germ cell transcription factor cyclic AMP response element modulator-τ during spermiogenesis has recently been demonstrated, the transcriptional mechanisms leading to early haploid cell formation are unknown. The rat and mouse proenkephalin genes are selectively expressed from an alternate, germ cell-specific promoter in meiotic and early haploid cells. In this study, the minimal rat proenkephalin germ line promoter was localized to a 116-bp region encompassing the transcriptional start site region. Further, a proximal 51-bp sequence located in the 5′-flanking region is absolutely required for germ line promoter activity. This 51 bp sequence corresponds to a previously characterized binding element (GCP1) that forms cell-specific complexes with rat spermatogenic cell nuclear factors distinct from cyclic AMP response element binding proteins. Further, GCP1 contains novel direct repeat sequences required for factor binding and transgene expression in spermatogenic cells. These repeat elements are highly similar to sequences within the active regions of other male germ line promoters expressed during meiosis. GCP1 may therefore contain transcriptional elements that participate more generally during meiosis in the differentiation of spermatocytes and early haploid spermatids.

Porcine growth hormone gene expression from viral promoters in transgenic swine.

Abstract The production of porcine growth hormone (pGH) from novel expression vectors containing the promoter/enhancer elements of the Moloney murine leukemia virus (MLV) LTR or the human cytomegalovirus (CMV) immediate early gene was examined in transgenic swine. Both fusion genes resulted in elevated levels of serum pGH, elevation of insulin‐like growth factor 1 (IGF‐1), and a pronounced decrease in carcass fat deposition. The two viral promoter/enhancer elements were constitutively active in the transgenic swine throughout the life of the animals. In individual swine, the CMV‐pGH transgene was expressed predominantly in the pancreas while the MLV‐pGH transgene was expressed in a wide variety of tissues. These swine were infertile, had insulin resistance, and demonstrated an accelerated form of osteochondritis dissicans. Our results show that excess pGH produces a phenotype identical to that seen in swine expressing heterologous growth hormones, and provides a baseline for assessing the overall efficien...

Culture of 5-day horse embryos in microdroplets for 10 to 20 days

Embryos were recovered from the uteri of mares 5 d after ovulation. Six embryos, all morulae, were placed singly in 200-ul droplets of Hams F-12 with 10% fetal calf serum and cultured at 37 degrees C in a 5% CO(2) atmosphere. The embryos expanded to form blastocysts by the third day of culture. The blastocysts hatched from their zona pellucida, rather than the zona thinning and flaking off, as occurs in vivo. Hatching from the zona pellucida began on the third day of culture and was complete in five of six embryos by the sixth day. The embryonic capsule, normally present in equine embryos after Day 6, was not seen in the cultured embryos. The blastocysts continued to expand until 15 to 17 d of age (10 to 12 d in culture), reaching an average diameter (+/- SD) of 2052 +/- 290 um, after which time they either collapsed or contracted. These results demonstrate that equine embryos can be maintained in long-term culture in vitro, exhibiting continued growth and expansion in the absence of the embryonic capsule.

In vivo culture of embryos in the immature mouse oviduct

In-vitro culture of mammalian preimplantation embryos is associated with subsequent decreased viability. This phenomenon is more pronounced with the domestic species embryos as culture conditions are at present unable to sustain cleavage of early preimplantation embryos for more than one or two cell divisions. In this study, the immature mouse oviduct is shown to be capable of supporting cleavage and morphological development of rabbit and porcine embryos. The immature mouse oviduct was shown to be comparable to in vitro culture as 76% and 60% of the transferred zygotes developed to the morula stage after 2 and 3 d respectively. The porcine zygotes, however, failed to develop beyond the 4-cell stage in either the immature mouse oviduct or in vitro. Porcine morula showed better tolerance of the oviduct environment and when recovered after 2 d contained an average of 64 cells, which was significantly more than in in vitro cultured morulae (40 cells). Early porcine blastocysts transferred to the mouse oviduct had over a two-fold increase in cell division (104 cells) over comparable blastocysts grown in vitro (57 cells). The immature mouse oviduct is, therefore, a potential surrogate environment for short-term storage of embryos of other species.