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In some species, including swine, cattle and sheep, the placenta synthesizes all the enzymes needed to convert cholesterol into progestagen and estrogen. However, in goat reproduction the processes specific to and the knowledge of goat placental steroidogenesis is unclear. Therefore, the objective of the present study was to investigate the steroidogenic pathways used by the goat placenta to synthesize androgens and estrogens, with special emphasis on androgen production. Five postpartum placentas obtained immediately after expulsion were processed for the study. A cell suspension was prepared from excised cotyledons and aliquots incubated in DMEM and antibiotics, and 3 H-progesterone, 3 H-dehydroepiandrosterone, 3 H-androstenedione or 3 H-testosterone. Steroids were extracted from the culture media and analyzed by thin layer chromatography (TLC). Testosterone and androstendione were then recrystallized from the placenta incubation media. Placental tissue was processed for in situ -RT-PCR in order to investigate the expression of 17α-hydroxylase/17,20 lyase. Results showed the synthesis of tritiated androgens and estrogens when cells were incubated with 3 H-progesterone. The incubation with 3 H-testosterone or 3 H-androstenedione yielded 3 H-17β-oestradiol and 3 H-oestrone. Placental cells also transformed 3 H-DHEA to the androgens 3 H-androstenedione, 3 H-androstenediol and 3 H-testosterone and also to 3 H-estrogens. Androstendione and testosterone synthesis were re-confirmed by recristallization. The results of this study show that goat placental cells may use both the Δ-4 and the Δ-5 steroidogenic pathways to synthesize androgens and estrogens. These data show the steroidogenic pathways used by the goat placenta to yield androgens, and the ability of both androstendione and testosterone to be converted into estrogens for the first time.

In most mammals, the corpus luteum (CL) and placenta are the major sources of progesterone. The goat pregnancy depends on the presence of CL after mid-gestation, while sheep pregnancy does not. The expression and distribution of P450-aromatase (P450-Aro) mRNA throughout gestation has not been investigated in the goat CL and partially in the sheep CL. The present research was designed to characterize the expression of P450-Aro mRNA in small ruminant CL with emphasis in the goat. For this purpose, ovaries from Criollo goats and Pelibuey sheeps were analysed using in situ reverse transcription-polymerase chain reaction (RT-PCR) for the histological detection of P450-Aro transcripts. In addition, P450-Aro expression was determined by in vitro RT-PCR. In situ RT-PCR studies showed that the goat and sheep CL were rich in cells positive for P450-Aro mRNA. We have also found in vitro RT-PCR expression of P450-Aro mRNA in goat CL at 1, 3 and 4 months of gestation. This study shows that the goat CL expresses P450-Aro mRNA along gestation, suggesting that this structure is capable to produce oestrogens up to the end of gestation.

Sphingosine-1-phosphate (S1P) is a bioactive polar sphingolipid which stimulates proliferation, growth and survival in various cell types. In the ovary S1P has been shown protect the granulosa cells and oocytes from insults such as oxidative stress and radiotherapy, and S1P concentrations are greater in healthy than atretic large follicles. Hence, we postulate that S1P is fundamental in follicle development and that it is activated in ovarian granulosa cells in response to FSH and VEGF. To test this hypothesis we set out: i) to evaluate the effect of FSH and VEGF on S1P synthesis in cultured bovine granulosa cells and ii) to analyse the effect of S1P on proliferation and survival of bovine granulosa cells in vitro. Seventy five thousand bovine granulosa cells from healthy medium-sized (4-7mm) follicles were cultured in 96-well plates in McCoy's 5a medium containing 10ng/mL of insulin and 1ng/mL of LR-IGF-I at 37°C in a 5% CO2/air atmosphere at 37°C. Granulosa cell production of S1P was tested in response to treatment with FSH (0, 0.1, 1 and 10ng/mL) and VEGF (0, 0.01, 0.1, 1, 10 and 100ng/mL) and measured by HPLC. Granulosa cells produced S1P at 48 and 96h, with the maximum production observed with 1ng/mL of FSH. Likewise, 0.01ng/mL of VEGF stimulated S1P production at 48, but not 96h of culture. Further, the granulosa cell expression of sphingosine kinase-1 (SK1), responsible for S1P synthesis, was demonstrated by Western blot after 48h of culture. FSH increased the expression of phosphorylated SK1 (P

The formation of a triple helix by a third N3′→P5′ phosphoramidate GT-rich strand 5′-d(T9G5)NP-3′ has been studied by FTIR, CD and UV absorbance spectroscopies. We show that in presence of sodium counterions, in conditions of molecular crowding induced by high DNA concentrations, the self-association of the phosphodiester analog into a tetrameric structure is favored and prevents the formation of the triple helix. The use of a phosphoramidate third strand in the same conditions allows to minimize self-association and to form the triple helix with a 5′-d(T9G5)NP-3′ third strand. Characteristic signatures of T*A·T and G*G·C base triplets have been obtained by FTIR spectroscopy. Formation of a triple helix with the unmodified GT third strand can be observed in dilute solution, in presence of lithium and divalent magnesium ions (1 M LiCl, 50 mM MgCl2). In that case a biphasic UV melting profile (TM at 40 and 60 °C) and a characteristic CD spectrum of a parallel stranded GT triple helix have been obtained.

Formation of a pyrimidine motif triple stranded structure containing a N3′→P5′ phosphoramidate 5′-d(TTC-TCC-TTT-CTT)-3′ third strand targeting the 5′-d(AAG-AGG-AAA-GAA)-3′ sequence has been followed by Fourier transform infrared (FTIR) spectroscopy and UV spectroscopy. The use of a N3′→P5′ phosphoramidate d-C,T third strand is aimed at increasing triplex stability at neutral pH. FTIR spectroscopy measurements at neutral pH show a biphasic melting profile ( T m at 25 and 54 °C). The triple helix is stabilized by the formation of T * A–T base triplets, in spite of the presence of four unprotonated cytosines in the 12mer third d-C,T phosphoramidate strand and therefore of the absence of C + * G C base triplets. All N3′→P5′ phosphoramidate nucleoside sugars in this triple helix adopt an S-type (C2′ endo) conformation. No triple helix has been detected at neutral pH when a natural isosequential phosphodiester third strand was used. By decreasing the pH, the FTIR spectra show the formation of C + * G C base triplets in addition to the already formed T * A T base triplets. The melting of this stabilized triple helix is observed at a temperature higher than that of the initial Watson–Crick duplex. The existence of N-type sugars is then detected. When the concentration is decreased, at neutral pH, UV spectroscopy measurements show that the intermolecular triple helix formed by three short 12mer strands is no longer stable. In dilute solution at acidic pH the triplex is more stable than the initial Watson–Crick duplex.

Formation of intramolecular tetraplex structures by the thrombin-binding DNA aptamer (TBA) in the presence of K(+), Pb(2+), Ba(2+), Sr(2+) and Mn(2+) has been studied by vibrational spectroscopy. All tetraplex structures contain G-G Hoogsteen type base pairing, both C2'endo/anti and C2'endo/syn deoxyguanosine glycosidic conformations and local B like form DNA phosphate geometries. Addition of Pb(2+) ions modifies the structure by interacting at the level of the guanine carbonyl groups. The very important downshift of the guanine C6=O6 carbonyl vibration mode in the TBA spectrum induced by the addition of one Pb(2+) ion per TBA molecule is in agreement with a localization of the metal ion between both guanine quartets. FTIR melting experiments show an important stabilization of the tetraplex structure upon addition of Pb(2+) ions (DeltaT = 15 degrees C). This strong interaction of lead cations may be correlated with a change in the geometry of the cage formed by the two guanine quartets. A similar but weaker effect is observed for barium and strontium cations.

FTIR spectroscopy has been used to follow the formation of parallel stranded DNA duplexes incorporating isoG or m5isoC bases and determine their base pairing scheme. The results are discussed in comparison with data concerning anti-parallel duplexes with comparable base composition and sequence. In duplexes containing A–T and isoG–C or m5isoC–G base pairs shifts of the thymine C2 O2 and C4 O4 carbonyl stretching vibrations (to lower and higher wavenumbers, respectively, when compared to their positions in classical cis Watson–Crick (WC) base pairs) reflect the formation of trans Watson–Crick A–T base pairs. All carbonyl groups of cytosines, m5isocytosines, guanines and isoguanines are found to be involved in hydrogen bonds, indicative of the formation of isoG–C and m5isoC–G base pairs with three hydrogen bonds. Molecular modeling shows that both structures form regular right handed helices with C2′endo sugar puckers. The role of the water content on the helical conformation of the parallel duplexes has been studied by FTIR and CD. It is found that a conformational transition similar to the B → A transition observed for anti-parallel duplexes induced by a decrease of the water content of the samples can occur for these parallel duplexes. Their helical flexibility has been evidenced by FTIR studies on hydrated films by the emergence of absorption bands characteristic of A type geometry, in particular by an S-type → N-type repuckering of the deoxyribose. All sugars in the parallel duplex with alternating d(isoG–A)/d(C–T) sequence can adopt an N-type geometry in low water content conditions. The conformational transition of the parallel hairpin duplex with alternating d(isoG–A)/d(C–T) sequence was followed by circular dichroism in water/trifluoroethanol solutions and its free energy at 0 °C was estimated to be 6.6 ± 0.3 kcal mol−1.