Michael K. Holland

Comprehensive mapping of the bull sperm surface proteome

While the mechanisms that underpin maturation, capacitation, and sperm–egg interactions remain elusive it is known that these essential fertilisation events are driven by the protein complement of the sperm surface. Understanding these processes is critical to the regulation of animal reproduction, but few studies have attempted to define the full repertoire of sperm surface proteins in animals of agricultural importance. Recent developments in proteomics technologies, subcellular fractionation, and optimised solubilisation strategies have enhanced the potential for the comprehensive characterisation of the sperm surface proteome. Here we report the identification of 419 proteins from a mature bull sperm plasma membrane fraction. Protein domain enrichment analyses indicate that 67% of all the proteins identified may be membrane associated. A large number of the proteins identified are conserved between mammalian species and are reported to play key roles in sperm–egg communication, capacitation and fertility. The major functional pathways identified were related to protein catabolism (26S proteasome complex), chaperonin‐containing TCP‐1 (CCT) complex and fundamental metabolic processes such as glycolysis and energy production. We have also identified 118 predicted transmembrane proteins, some of which are implicated in cell adhesion, acrosomal exocytosis, vesicle transport and immunity and fertilisation events, while others have not been reported in mammalian LC‐MS‐derived sperm proteomes to date. Comparative proteomics and functional network analyses of these proteins expand our systems level of understanding of the bull sperm proteome and provide important clues toward finding the essential conserved function of these proteins.

Species-Specific Differences in the Activity and Nuclear Localization of Murine and Bovine Phospholipase C Zeta 1

Injection of mammalian sperm extracts or cRNA of the sperm-specific phospholipase C zeta 1 (PLCZ1) has been shown to trigger repetitive oscillations in the concentration of free calcium ([Ca2+]i), leading to oocyte activation and embryo development in all mammals studied to date. While PLCZ1 has cross-species activity, it has also been observed that species-specific differences may exist in the frequency and pattern of the resulting [Ca2+]i oscillations following PLCZ1 cRNA injection into oocytes of different species. Accordingly, we used a crossover design strategy to directly investigate the activity of murine and bovine PLCZ1 in both murine and bovine oocytes. In murine oocytes, injection of murine Plcz1 cRNA induced [Ca2+]i oscillations at 10-fold lower concentrations than bovine PLCZ1, although in bovine oocytes bovine PLCZ1 was more effective than murine Plcz1 at inducing [Ca2+]i oscillations. Investigation of ITPR1 (IP3R1) down-regulation in bovine oocytes by PLCZ1 cRNA also showed that bovine PLCZ1 was more active in homologous oocytes. To determine whether these PLCZs exhibited similar cellular distribution, Venus-tagged PLCZ1 cRNA was injected into oocytes, and PLCZ1 was overexpressed. Bovine PLCZ1 failed to accumulate in the pronucleus (PN) of bovine or murine zygotes, despite possessing a putative nuclear localization signal. Conversely, murine PLCZ1 accumulated in the PN of both murine and bovine zygotes. These results demonstrate that murine PLCZ1 and bovine PLCZ1 possess species-specific differences in activity and suggest potential differences in the mode of action of the protein between the two species. Variation in sperm PLCZ1 protein content among species, along with oocyte-specific differences in the localization and availability of PLCZ1 substrates, may further contribute to optimize the activation stimulus to enhance embryo development.

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

Mitochondrial DNA transmission and transcription after somatic cell fusion to one or more cytoplasts.

Following fertilization, mitochondrial DNA is inherited from the oocyte and transmitted homoplasmically. However, following nuclear transfer, mitochondrial DNA can be transmitted from both the donor cell and recipient oocyte, resulting in a state of heteroplasmy. To determine whether the genetic diversity between donor cell and recipient cytoplast mitochondrial DNA influences development, we generated bovine embryos by fusing a donor cell to one or more enucleated cytoplasts. Analysis of mitochondrial DNA from embryos, fetal tissues, and blood samples from offspring revealed that early preimplantation embryos from two or three cytoplasts had significantly more mitochondrial DNA variants than fetal tissues. Phylogenic analysis of embryos generated using single cytoplasts divided the mitochondrial DNA sequence variants into three separate groups with various amounts of genetic divergence from the donor cell line. In heteroplasmic tissue and blood samples, the predominant mitochondrial DNA population was significantly more divergent from the donor cell than the less frequent allele. Furthermore, analysis of the mitochondrially encoded cytochrome B gene showed that two heteroplasmic alleles encoded for different amino acids, and the ratios of mitochondrial DNA/mRNA for each allele differed significantly between tissues. The degree of evolutionary distance between the donor cell and the cytoplast and the variability in heteroplasmy between tissues may have an impact on more divergent intergeneric nuclear transfer and the use of this approach for the generation of embryonic stem cells.

Cloning and expression of recombinant rabbit fertilin

Fertilin is a sperm surface protein complex which is reported to play an essential role in sperm‐egg fusion in mammals. It is comprised of two related subunits, α and β, both of which are glycosylated and have cytoplasmic and extracellular domains. This protein has been reported to play an essential role in sperm‐egg fusion in mammals. We report on the cloning and sequencing of the complete cDNA sequences of both subunits from rabbit testis, and the production of recombinant proteins for testing their potential as antigens for use in an immunocontraceptive vaccine to control wild rabbit populations. The cDNAs for rabbit fertilin α and β (Genbank accession numbers, U46069 and U46070) are predicted to encode proteins of 9l9 and 751 amino acids, respectively, and to show significant levels of homology to fertilin subunits isolated from other species. Analysis of the predicted protein sequences of fertilin α but not β reveals the presence of 21 direct repeats of the hexameric sequence A/PPPPEA at the extreme carboxy terminus, similar to what has been described for a fertilin α gene isoform in the monkey. DNA sequences corresponding to the predicted mature α and β fertilin subunits were individually cloned into a bacterial expression system, and the recombinant proteins were used to raise polyclonal antibodies in mice. These antibodies detect components of the native fertilin complex from rabbit sperm.

Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos.

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation.

Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss.

In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.

Restricted Entry of IgG into Male and Female Rabbit Reproductive Ducts Following Immunization with Recombinant Rabbit PH-20

PROBLEM: Successful immunocontraception using sperm antigens is dependent on achieving sufficient sperm‐specific antibody in the reproductive ducts to prevent fertilization. The blood : luminal barrier of the male and female reproductive ducts must be overcome for this to occur. We have, therefore, investigated the relative titers of antigen‐specific immunoglobulin G (IgG) in luminal fluids collected from male and female rabbit reproductive ducts following immunization with recombinant rabbit PH‐20 (rPH‐20).

Bovine Sertoli Cells Colonize and Form Tubules in Murine Hosts Following Transplantation and Grafting Procedures

The contribution of somatic cells to nonrodent male germ cell transplantation success has not been well established due to lack of cell type-specific markers to distinguish donor cells from host cells. In the present study, we first screened antibodies and a lectin to identify markers suitable for unequivocal distinction between germ cells and Sertoli cells in bovine testes compared with mouse testes. Anti-vimentin and the Dolichos biflorus agglutinin (DBA) lectin detected only bovine Sertoli cells and spermatogonia, respectively; anti-NONO and anti-GCNA1 detected only mouse Sertoli and germ cells, respectively. The outcome of transplanting bovine testis cells into nude mouse testes was then studied using these markers. Our results clearly showed that immature bovine Sertoli cells survive and colonize mouse testes at 2.5 months after transplantation and that tubular structures composed of donor Sertoli cells formed adjacent to murine tubules within the host mouse testis. Bovine germ cell colonization and survival in mouse testes after transplantation were confirmed, but this was restricted to areas of bovine Sertoli cell colonization. In addition, ectopic grafts of intact bovine testis tissue and cell aggregates from hanging drop cultures were placed under the back skin and testis capsule of nude mice. Bovine Sertoli cells in ectopic grafts and aggregates were able to form tubular structures, and some bovine germ cells were observed around 2 months after implantation. This study therefore identifies a practical strategy to assess the outcome of testicular cell transplantation using different antibodies and a lectin to distinguish bovine cells from mouse cells. It identifies an approach that can readily be adapted to study other nonrodent species.

The Efficient Generation of Cell Lines from Bovine Parthenotes

The generation of embryonic stem cell (ESC) lines from parthenogenetically activated oocytes can provide transplantable cells, which are immunocompatible for the oocyte donors as well as an invaluable tool for genetic engineering and epigenetic studies. We report the efficient isolation of eight putative bovine parthenogenetic embryonic stem cell (bpESC) lines from 15 in vitro produced parthenotes. Five of these cell lines were maintained for more than 15 passages (>140 days) and analyzed. The cells displayed typical ESC morphology, stained positive for alkaline phosphate by histochemical staining, expressed Oct4, Nanog, and either stage-specific embryonic antigens, SSEA1, or SSEA4, detected by immunofluorescence staining. RT-PCR analysis of the cells demonstrated expression of Oct4, Rex1, SSEA1, and ALP. All the cell lines except one had a normal karyotype of 60, XX. The cells differentiated in suspension culture to form embryoid bodies (EBs) expressing markers of the three embryonic germ layers as assessed by RT-PCR. In conclusion, we report efficient derivation of putative ESCs from bovine parthenogenetic embryos. The cells express pluripotent markers, have the ability to form EBs, and differentiate into cells of the three embryonic germ layers. This is the first report of characterized putative parthenogenetic bovine ESC lines.