M. Forni

Vascular Endothelial Growth Factor Production in Growing Pig Antral Follicles

Abstract Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4–5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.

Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer

UMAN organs for transplantation are insufficient in quantity, and for every organ transplant undertaken there is a need for an additional five to ten organs. Waiting lists are constantly growing and many patients die before an organ can be found. Researchers continue to experiment with alternatives. One of these is xenotransplantation, which uses animals as donors and which presents an entirely new set of challenges. The pig is presently considered the most likely source of organs for human xenotransplantation because it is easy to breed, has compatibly sized organs, and offers the possibility of genetic manipulation. However, organ transplantation between distantly related species, such as pigs and humans, results in hyperacute rejection (HAR), involving the complement system. It may be possible to avoid rejection reactions by genetically engineering donor animals, so that the recipient’s immune system does not act on the graft. Thus, a number of research teams, including our group, have embarked on programs to produce pigs transgenic for the human regulators of complement activation (RCAs) genes, in the attempt to produce pigs whose organs may be suitable for transplantation into humans. 1‐3 The present study reports on the production of pigs transgenic for the human regulator of complement activation human decay accelerating factor (hDAF) by spermmediated gene transfer (SMGT), a highly efficient and reproducible alternative to microinjection, presently the most widely used system for generating transgenic animals.

Efficiency of transgenesis using sperm-mediated gene transfer: generation of hDAF transgenic pigs.

SINCE the beginning of this century, replacement of failing human organs with their animal counterparts has been an interesting topic of debate for writers and scientists. In the 1960s, prolonged survival after kidney transplantation from chimpanzee to human was obtained in the United States and Europe. Nevertheless, both the progressive improvement in surgical technique and in immunosuppressant therapy and the availability of cadaveric organs and living donation have reduced the interest in xenotransplantation. Because of the increasing requests for organs and the lack of donors to meet that need, xenotransplantation has become a reliable option again for temporary organ replacement (eg, of heart or liver) before definitive transplant. However, primates such as chimpanzees and baboons are expensive, can carry important zoonoses, and their use is burdened by ethical implications. A better choice for xenotransplantation might be offered by swine, which are closer to humans for anatomic and metabolic features. Discordant transplantation is associated with humoral hyperacute rejection, due to preformed antibodies and complement system activation (both classical and alternative pathway). This results in massive and irreversible vascular damage and cellular necrosis. Expression of the species-specific complement activation inhibitors could prevent this kind of rejection. Organs harvested from pigs transgenic for human decay accelerating factor (hDAF or CD55), membrane cofactor protein (MCP or CD46), and CD59 could be more efficiently grafted into human recipients. Therefore, a number of research teams, including our group, have generated pigs transgenic for human negative regulators of the complement cascade. This research has been aimed to generate hDAF transgenic swine with high efficiency and reproducibility. This goal has been reached through the innovative method of sperm-mediated gene transfer (SMGT), which was developed about 10 years ago by our group. Data presented in this paper show that hDAF-positive individuals can also be used as founders of stable lines of F1 generation transgenic pigs. MATERIALS AND METHODS hDAF Transgenic F1 Offspring Generation

hDAF expression in hearts of transgenic pigs obtained by sperm-mediated gene transfer.

TRANSPLANTATON has been the choice option to treat successfully an increasing number of acute and chronic human pathologies with declining morbidity and mortality. However, availability of organs from human donors is limited and dramatically inadequate with respect to patient requests. Xenotransplantation from large-sized mammals has thus been reconsidered as a tool to overcome the present unbalance between organ offers and requests. Pigs have been chosen because they can be easily and cheaply bred; they do not raise ethical questions—their use as alimentary resources is generally admitted; and they possess organs largely human compatible for size, anatomical organization, and physiology. Nevertheless, this option is usually compromised by the occurrence of an irreversible hyperacute vascular rejection of the animal transplanted organ as soon as it is perfused with human blood, mediated by natural preformed antibodies and by the activation of the complement cascade. Downregulating the complement system by expressing on the surface of the swine organs one or more species-specific negative modulators of the complement complexes, such as human decay accelerating factor (hDAF), could prevent the rejection of the graft. Our group has obtained a number of transgenic pigs for hDAF using sperm-mediated gene transfer (SMGT), an innovative and highly efficient and reproducible method. In SMGT, spermatozoa are employed as natural vectors of foreign DNA and are bound, captured, and internalized into recipient egg cells to generate a genetically modified progeny. Revealing the expression of the protein on the surface of the organs is an important confirmation for successful transgenesis. The aim of this report is to show, by immunohistochemical detection, the expression of hDAF in transgenic hearts, organs maximally eligible for xenotransplantation.

Alteration of constitutive heat shock protein 70 (HSC70) production by in vitro culture of porcine preimplanted embryos.

C. Bernardini, P. Fantinati, G. Castellani, M. Forni, A. Zannoni, E. Seren and M.L. Bacci Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Ozzano Emilia, Italy *Correspondence: Dipartimento di Morfofisiologia Veterinaria e Produzioni Animali, Facoltà di Medicina Veterinaria, via T olara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy E-mail: cbernardini@vet.unibo.it

In vitro mucolytic activity of recombinant human deoxyribonuclease on equine tracheobronchial mucus.

The viscosity of the mucus, its DNA concentration and the size range of the DNA were determined on tracheobronchial samples from 11 horses with lower airway diseases before and after incubation with recombinant human deoxyribonuclease (rhDNase). The horses were divided into two groups on the basis of the cytology of the samples: group A (five horses) with more than 60 per cent neutrophils and group B (six horses) with fewer than 50 per cent neutrophils. The mean mucus viscosity and DNA concentration in the preincubation samples were significantly higher in group A than in group B, and there was a correlation between DNA concentration and mucus viscosity in the preincubation samples from group A. Incubation with rhDNase significantly reduced the viscosity of the samples only in group A.

Endonuclease activity in swine ovarian cysts.

The biochemical hallmark of apoptosis is the degradation of genomic DNA into discrete oligonucleosome fragments by the action of a Ca2+/Mg2+-dependent endonuclease (Peitsch et al., 1993). This endonuclease is localized within the nucleus and could either be constitutively expressed or newly synthesized after apoptotic stimulus. At the ovarian level the endonuclease has been demonstrated to be present in rat granulosa and luteal cells (Zeleznik et al., 1989; Boone et al., 1995). We showed the presence of enzyme activity in swine luteal tissue as early as 2 hours after ovulation (Forni et al., 1996) and in swine gonadotropin-stimulated follicles presumably destined to ovulate (Zannoni et al., 1997), suggesting a role of this enzyme in follicular rupture during the ovulation process. Fully developed follicles sometimes do not ovulate and become cystic; the incidence of cysts in cyclic sows is about 3.3% (Gherpelli and Tarocco, 1996). There is no clear evidence about the origin of this multifactorial pathology (endocrine imbalance, seasonality, diet). Recently the cystic persistence in the bovine was correlated with a low level of apoptosis in follicular cells (Isobe and Yoshimura, 2000). The aim of our investigation was to assess the relationship between the persistence of swine cysts and onset of apoptosis in relation to Ca2+/Mg2+-dependent endonuclease activity. To do this we evaluated the level of spontaneous apoptosis as well as the presence of Ca2+/Mg2+-dependent endonuclease spontaneous cysts in swine.

Gonadotropin suppression of apoptosis in follicle somatic cells

In each oestrous cycle only a limited number of follicles are selected for ovulation whereas the remaining majority undergo atresia. The earliest and most prominent feature of atresia is the death of granulosa cells. Recent biochemical evidence has demonstrated that granulosa cell death during follicular atresia in swine (Tilly et al. , 1992), bovine (Jolly et al. , 1994) and rodent (Tilly et al. , 1991) ovaries occurs by apoptosis, a process whereby cells die in a controlled manner. A biochemical event considered to be characteristic of apoptotic cell death is the intranucleosomal cleavage of genomic DNA into fragments 180–200 bp in size, which separate into a distinctive ladder-like pattern on agarose gel electrophoresis. Detection of this pattern of oligonucleosomes in DNA provides a marker of apoptotic cell death.