Hannah Galantino-Homer

Cholesterol Efflux-mediated Signal Transduction in Mammalian Sperm β-CYCLODEXTRINS INITIATE TRANSMEMBRANE SIGNALING LEADING TO AN INCREASE IN PROTEIN TYROSINE PHOSPHORYLATION AND CAPACITATION

Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-β-cyclodextrin and OH-propyl-β-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these β-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different β-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the β-cyclodextrins with cholesterol- SO4 − to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The β-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The β-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, β-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.

Fertility and Germline Transmission of Donor Haplotype Following Germ Cell Transplantation in Immunocompetent Goats

Abstract Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.

Cryopreservation of boar semen and its future importance to the industry

Whereas AI has arguably been the most important management tool leading to improved herd productivity, long-term storage of semen brings forth additional advantages to producers of agriculturally important animals and the AI industry. Semen cryopreservation greatly facilitates the distribution of agriculturally desirable genes, rapidly increasing herd productivity. Of particular importance to the pig industry, the use of frozen semen would help to control transmission of certain pathogens, thereby protecting the health status of the herd. Moreover, a reserve of cryopreserved semen would minimize the effects of a sudden outbreak of a contagious illness or a natural disaster. Successful cryopreservation of boar semen is necessary for international sales. Finally, effective gene banking depends on the availability of functional, cryopreserved germplasm. Despite these potential advantages of long-term semen storage, porcine sperm are notoriously sensitive to cold temperatures, and frozen-thawed semen is not routinely used by the industry. The objective of our laboratories is to develop protocols for efficient long-term storage of porcine semen using cryopreservation. We hypothesize that since the sperm plasma membrane is the primary site of cold-induced damage, reinforcing the membranes with molecules having particular properties, such as cholesterol, will improve the ability of boar sperm to withstand cold temperatures and cryopreservation protocols. Based on our data, such approaches should help alleviate the problems with sperm function after cooling, thereby resulting in better survival and motility characteristics, and reduced non-regulated capacitation and spontaneous acrosome reactions.

Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation

We explored whether exposure of mammalian germ line stem cells to adeno‐associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell‐depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild‐type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV‐transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV‐mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research.—Honaramooz, A., Megee, S., Zeng, W., Destrempes, M.M., Overton, S.A., Luo, J., Galantino‐Homer, H., Modelski, M., Chen, F., Blash, S., Melican, D. T., Gavin, W. G., Ayres, S., Yang, F., Wang, P. J., Echelard, Y., Dobrinski, I. Adeno‐associated virus (AAV) ‐mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J. 22, 374–382 (2008)

Capacitation of the Mammalian Spermatozoon

Publisher Summary The acquisition of progressive motility and fertilization competence are acquired in many mammals during transit through the epididymis but complete fertilization capacity in vivo is conferred during residence in the female reproductive tract. The molecular and physiological events comprising this extratesticular maturational process that lead to the fertilization-competent state are referred to collectively as sperm capacitation. Sperm do not have the ability to fertilize eggs but require a limited time of residence in the female tract in order for sperm to gain the capacity for fertilization. The chapter provides a framework with which to develop new approaches to regulate fertility. It focuses on some of the recent developments in the field and provides a discussion of future experimental approaches to understand this important sperm maturational event. Capacitation has been defined as the time interval between sperm deposition in the female reproductive tract during natural mating and the time during which fertilization occurs.

Current Concepts of Molecular Events During Bovine and Porcine Spermatozoa Capacitation

Spermatozoa are required to undergo the processes of capacitation before they obtain fertilizing ability. The molecular changes of capacitation are still not fully understood. However, it is accepted that capacitation is a sequential process involving numerous physiological changes including destabilization of the plasma membrane, alterations of intracellular ion concentrations and membrane potential, and protein phosphorylation. There are no known morphological changes that occur to the spermatozoon during capacitation. The purpose of this review is to summarize current evidence on the molecular aspects of capacitation both in vivo and in vitro in bovine and porcine spermatozoa. For the purpose of this review, the process of sperm capacitation will encompass maturational events that occur following ejaculation up to binding to the zona pellucida, that triggers acrosomal exocytosis and initiates fertilization.

Decreased expression of p63, a regulator of epidermal stem cells, in the chronic laminitic equine hoof

REASONS FOR PERFORMING STUDY Abnormal epidermal stem cell regulation may contribute to the pathogenesis of equine chronic laminitis. OBJECTIVE To analyse the involvement of p63, a regulator of epidermal stem cell proliferative potential, in chronic laminitis. METHODS Epidermal tissues from skin, coronet and lamellae of the dorsal foot were harvested from 5 horses with chronic laminitis and 5 control horses. Tissues were analysed using histopathology, immunofluorescence microscopy and quantitative immunoblotting. RESULTS Hoof lamellae of laminitic horses had a lower frequency of p63 positive cells than control lamellae, particularly in the distal region. Quantitative immunoblotting confirmed reduced p63 expression in the laminitic distal lamellar region. The decreased p63 expression in laminitic epidermal lamellae was most apparent in the abaxial region adjacent to the hoof wall and highly associated with the formation of terminally differentiated, dysplastic and hyperkeratotic epidermis in this region, whereas lamellae from control horses maintained high p63 expression throughout the axial-abaxial axis. CONCLUSIONS Expression of p63 in equine skin resembles that reported in other species, including man and rodents, suggesting that p63 can serve as a marker for the proliferative potential of equine epidermal stem cells. p63 expression was significantly lower in the chronic laminitic hoof than in that of control horses, suggesting laminitic hoof epithelium has more limited proliferative potential with a shift towards differentiation. This may reflect reduced activity of epidermal stem cells in laminitic hoof. It is proposed that p63 contributes to the maintenance of hoof lamellae and that misregulation of p63 expression may lead to epidermal dysplasia during lamellar wedge formation. POTENTIAL RELEVANCE This study suggests that loss of epidermal stem cells contributes to the pathogenesis of equine laminitis. Autologous transplantation of p63-positive epidermal stem cells from unaffected regions may have regenerative therapeutic potential for laminitic horses.

Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue.

The dermo-epidermal interface that connects the equine distal phalanx to the cornified hoof wall withstands great biomechanical demands, but is also a region where structural failure often ensues as a result of laminitis. The cytoskeleton in this region maintains cell structure and facilitates intercellular adhesion, making it likely to be involved in laminitis pathogenesis, although it is poorly characterized in the equine hoof lamellae. The objective of the present study was to identify and quantify the cytoskeletal proteins present in the epidermal and dermal lamellae of the equine hoof by proteomic techniques. Protein was extracted from the mid-dorsal epidermal and dermal lamellae from the front feet of 5 Standardbred geldings and 1 Thoroughbred stallion. Mass spectrometry-based spectral counting techniques, PAGE, and immunoblotting were used to identify and quantify cytoskeletal proteins, and indirect immunofluorescence was used for cellular localization of K14 and K124 (where K refers to keratin). Proteins identified by spectral counting analysis included 3 actin microfilament proteins; 30 keratin proteins along with vimentin, desmin, peripherin, internexin, and 2 lamin intermediate filament proteins; and 6 tubulin microtubule proteins. Two novel keratins, K42 and K124, were identified as the most abundant cytoskeletal proteins (22.0 ± 3.2% and 23.3 ± 4.2% of cytoskeletal proteins, respectively) in equine hoof lamellae. Immunoreactivity to K14 was localized to the basal cell layer, and that to K124 was localized to basal and suprabasal cells in the secondary epidermal lamellae. Abundant proteins K124, K42, K14, K5, and α(1)-actin were identified on 1- and 2-dimensional polyacrylamide gels and aligned with the results of previous studies. Results of the present study provide the first comprehensive analysis of cytoskeletal proteins present in the equine lamellae by using mass spectrometry-based techniques for protein quantification and identification.

Osteopathology in the Equine Distal Phalanx Associated With the Development and Progression of Laminitis

Although the equine distal phalanx and hoof lamellae are biomechanically and physiologically integrated, bony changes in the distal phalanx are poorly described in laminitis. The aims of this study were (1) to establish a laminitis grading scheme that can be applied to the wide spectrum of lesions seen in naturally occurring cases and (2) to measure and describe changes in the distal phalanx associated with laminitis using micro–computed tomography (micro-CT) and histology. Thirty-six laminitic and normal feet from 15 performance and nonperformance horses were evaluated. A laminitis grading scheme based on radiographic, gross, histopathologic, and temporal parameters was developed. Laminitis severity grades generated by this scheme correlated well with clinical severity and coincided with decreased distal phalanx bone volume and density as measured by micro-CT. Laminitic hoof wall changes included progressive ventral rotation and distal displacement of the distal phalanx with increased thickness of the stratum internum–corium tissues with lamellar wedge formation. Histologically, there was epidermal lamellar necrosis with basement membrane separation and dysplastic regeneration, including acanthosis and hyperkeratosis, corresponding to the lamellar wedge. The changes detected by micro-CT corresponded to microscopic findings in the bone, including osteoclastic osteolysis of trabecular and osteonal bone with medullary inflammation and fibrosis. Bone changes were identified in horses with mild/early stages of laminitis as well as severe/chronic stages. The authors conclude that distal phalangeal pathology is a quantifiable and significant component of laminitis pathology and may have important implications for early detection or therapeutic intervention of equine laminitis.

Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof

BACKGROUND The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. HYPOTHESIS/OBJECTIVES To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. METHODS Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC transition to transit-amplifying (TA) cell] in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). RESULTS Expression of K14 was restricted to the basal layer of epidermal lamellae and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. CONCLUSIONS To the best of the authors knowledge, this is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses.