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The mechanisms that regulate the apoptosis are essential to the normal development and maintenance of homoeostasis and play an important role in placental development in mammals. During porcine pregnancy, there must be a proper cellular remodelling to achieve a normal gestational development. Knowledge of pig physiology during pregnancy will explore options to increase the productivity of this species of high economical value. The purpose of this work was to study the cell morphology and apoptosis of porcine placentas from early, mid and late pregnancy. For that purpose, high-resolution light microscopy and transmission electron microscopy were performed to the study of cell morphology. TUNEL, the apoptosis index (IAp) and the expression of c-FLIP through immunohistochemistry technique were used to the study of apoptosis. High-resolution light microscopy and transmission electron microscopy confirmed the presence of placental cells with ultrastructural apoptotic features. Apoptotic nuclei were detected by TUNEL in different placental structures and phagocytes containing apoptotic bodies. The IAp in villi was 9.34% at early, 0.82% at mid and 23.85% at late pregnancy. Statistically significant differences were found between periods (p < 0.05). In previous studies, we determined a differential induction of the apoptotic routes in the placental villi in agreement with the gestational period. A co-expression of receptors and mitochondrial proteins in placental connective tissue was detected, but the immunolocalization of c-FLIP would indicate an endogenous inhibition of the extrinsic pathway. In conclusion, in swine there exists differential activation of inducing apoptotic pathways in different placental structures according to the gestational period.

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Apoptosis is a permanent and dynamic physiological process by which an organism eliminates the undesirable cells without causing an inflammatory response. The objective of this work was to study the expression of FAS, DR4 and other members of the TNF-R1 superfamily extrinsic route apoptotic receptors the DNA fragmentation and the cellular apoptosis in placental samples at the early, mid and late pregnancy on +/- 30, +/- 55 and +/- 114 gestational days, respectively. We used placental histological sections of samples fixed in buffered saline formaldehyde. Immunohistochemical techniques were performed to detect the apoptotic receptors, whereas the DNA fragmentation was detected by TUNEL reaction and apoptotic cellular ultrastructure was detected by TEM conventional techniques. Apoptosis related receptors were immunolocalized in the early pig gestation and correlated with apoptosis, suggesting a role in the cellular remodelling of the placenta. At gestation day 55, apoptosis might be correlated to FAS route, but not by DR4-mediating pathway. At the end of gestation, increased apoptosis and both receptors markers were detected showing cellular death due to the extrinsic route through FAS and DR4 receptors. In conclusion, the immunolocalization of FAS and TNF R-1 receptors along the pig placental development correlates with TUNEL reaction and with apoptotic ultrastructure observed by TEM and seems to occur through different pathways along gestation.

La apoptosis es un proceso fisiológico, dinámico y permanente a través del cual un organismo elimina células indeseables sin provocar una respuesta inflamatoria. El objetivo del presente trabajo fue estudiar la expresión de los receptores de la vía extrínseca de apoptosis, FAS, DR4 y otros miembros de la superfamilia TNF-R1, la fragmentación del ADN y la apoptosis celular a través de TEM, en muestras placentarias del inicio, la mitad y el final de la gestación, hacia el día +/- 30, +/- 55 y +/- 114 de preñez, respectivamente. Se realizaron cortes histológicos de las muestras placentarias fijadas en formol tamponado. Para la detección de los receptores de apoptosis se realizaron técnicas inmunohistoquímicas, para el estudio de la fragmentación del ADN se utilizó el ensayo TUNEL y para el análisis de la ultraestructura celular apoptótica la técnica convencional de TEM. La inmunolocalización de los receptores de muerte celular al inicio de la preñez porcina sugiere el rol de la apoptosis en la remodelación celular placentaria. Hacia el día 55 de preñez, la apoptosis detectada ocurriría únicamente a través de la vía del receptor FAS, no del receptor DR4. Al final de la gestación, se detectó un incremento de la apoptosis y la expresión de ambos receptores, indicando que la muerte celular a través de la vía de señalización extrínseca estaría inducida por los receptores FAS y DR4. En conclusión, la inmunolocalización de los receptores FAS y otros miembros del TNF-R1, los resultados de TUNEL y la ultraestructura celular apoptótica observada en la placentación porcina, indican que la apoptosis detectada ocurre por diferentes vías de inducción a lo largo de la gestación.

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Several cytokines appear to be implicated in peri-implantation events and in maternal-fetal interaction. The majority of these molecules appear in supernatants or in extracts of placental origin. The purpose of this study was to investigate the action of human placenta conditioned medium (HPCM) on peripheral lymphocytes in vitro. Peripheral lymphocytes of women with different numbers of deliveries, of nulliparous and pregnant women, as well as of men, were cultivated in their own plasma with and without HPCM at 5%, 10% and 20%. The index of lymphocyte blast transformation (ILBT) was determined (values of > or = 0.5 indicated stimulation). In women who had had children, the highest lymphocyte blastogenesis was observed with HPCM at 10%, this finding being correlated to the numbers of deliveries (r = 0.828). In pregnant women, even though the highest answer was also obtained with HPCM at 10%, the addition of suboptimal concentration of HPCM (5%) produced blastic transformation (IBLT: x = 0.54 +/- 0.05), which was not observed in women who had had children. Lymphocytes of men and nulliparous women did not respond to any of the HPCM concentrations tested. In conclusion, HPCM at 10% was still the best concentration to produce lymphocyte blastogenesis. In addition, in the plasma of pregnant women, there may be some substance that enhanced the action in vitro of the extract, thus permitting threshold reduction necessary for lymphocyte stimulation.

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The purpose of this work was to obtain placental extracts with the capacity of producing lymphocyte blastogenesis. Placental homogenates were incubated at 37 degrees C in McCoy's 5A supplemented with 5% fetal bovine serum and 25 mM Hepes. After a 6-day incubation period, the cultures were harvested and the sterility and cellular viability were controlled. The supernatant was centrifuged at 22,000g at 4 degrees C for 30 minutes. Sterilization was achieved by filtration (0.22 microns). This medium was denominated human placenta conditioned medium (HPCM-1). The other half of the original placenta was processed by the Burgess method and the extracts thus obtained were denominated HPCM-2. The activity of the extracts thus obtained was tested in peripheral lymphocyte cultures from women after the third pregnancy; 5, 10 and 20% HPCM-1 or HPCM-2 was added to these cultures and the index of lymphocyte blast transformation (ILBT) was determined. The highest stimulation occurred at 10% HPCM. The ILBT obtained with HPCM-1 at 10% was more constant than that obtained with HPCM-2 at the same concentration. In conclusion, it can be assumed that this methodology is appropriate to obtain conditioned media from human placenta with good blastogenic activity on peripheral lymphocytes in vitro.

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