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.

The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4-5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% +/- 0.58% vs. 1.29% +/- 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150-300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.

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In order to investigate the effect of the somatic cells of the follicle on the preparation of the oocyte for fertilization three experiments were carried out. In the first, pig oocytes, cultured for 46 h in the presence of extroverted follicles (follicle oocytes) or surrounded by the cumulus (cumulus oocytes), were exposed to sperm in an in vitro fertilization system. Follicle oocytes showed a higher rate of fertilization than that recorded in cumulus oocytes (80% vs. 47%). In addition, significantly more sperm penetrated into the ooplasm of follicular oocytes (3.77/egg) than into that of cumulus oocytes (1.42/egg). To investigate the reason for the observed fertilization difference zona-free oocytes were studied in the second experiment. Significantly more spermatozoa were recorded in the ooplasm of follicle oocytes than in that of cumulus oocytes, thus suggesting that the effect of the follicle on fertilizability may partly depend on an action on the plasma membrane of the oocyte. A further effect of the follicular tissue was on cytoplasmic maturation: only follicular oocytes were capable of consistently promoting male pronucleus formation. In cumulus oocytes, sperm that entered the cytoplasm remained in a condensed form near the female pronucleus. In the third experiment cumulus oocytes and denuded oocytes were matured in medium that had been previously used to mature follicle oocytes. This conditioned medium was alone able to affect sperm penetration and male pronucleus formation in cumulus oocytes, but it did not exert any influence on denuded oocytes. This suggests that the observed effect of the follicle is mediated by soluble factors that, however, cannot influence the oocyte without some direct cell-oocyte contract.

Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates. These proteins are also present in mature sperm cells, which, in fact, need carriers for uptake energetic sources that are important for maintaining cell basic activity as well as specific functions, such as motility and fertilization ability. Likewise, several GLUTs have been studied in various mammalian species (man, bull, rat, mouse, boar, dog, stallion, and donkey) to point out both their actual presence or absence and their localization on plasma membrane. The aim of this work is to give an overall picture of the studies available on GLUTs in mammalian spermatozoa at this moment, pointing out the species peculiarity, the possible role of these proteins, and the potential future research on this item.

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The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.