Monitoring sexual steroids and cortisol at different stages of the ovarian cycle from two capuchin monkey species: use of non- or less invasive methods than blood sampling.

Endocrine monitoring of non-human primates (NHP) via faecal metabolites of steroid hormones appears as a useful non-invasive alternative to evaluate the reproductive status of free living NHP, as well as of those kept in captivity but of difficult handling. However, validation is needed with plasma values before its application in the field. The aim of the present study was to monitor the different phases of the menstrual cycle from the new world NHP Sapajus apella and S. libidinosus. For this, hormonal and faecal plasma levels of E2, P4 and cortisol were assessed during different days of the menstrual cycle, together with colpocitology. The mean duration of the menstrual cycle according colpocitology was of 21.7 and 21.0 days for S. apella and S. libidinosus, respectively. These values were similar to those observed via plasma analysis, i.e. 22.7 and 20.3 days for S. apella and S. libidinosus, respectively. The day of plasmatic E2 peak was set as Day -1 and the estimated day of ovulation was set as Day 0 and occurred two days earlier in S. libidinosus than in S. apella females. In both species, it was observed a delay in faecal E2 peak of six days for S. apella and of 11 days for S. libidinosus when compared with the plasma peak. A maximum P4 plasma concentration was observed in the middle of luteal phase in S. apella and in S. libidinosus, both at around day 5. However, faecal P4 peaks were detected at days 9 and 8 in S. apella and S. libidinosus, respectively. Mean plasma and faecal cortisol levels were variable during all ovulatory cycle of S. apella and S. libidinosus females. Although no exact correlation was observed between plasmatic and faecal profile of steroid hormone, faecal samples were able to indicate ovarian cycle phase, being important to assess the reproductive status of the females applying a non-invasive method.

l-arginine alters the proteome of frozen-thawed bovine sperm during in vitro capacitation.

The aim of this work was to evaluate the proteomic changes that occurred in the frozen-thawed bovine spermatozoa after the addition of l-arginine (L-arg) during in vitro sperm capacitation. Aspects related to the sperm capacitation pattern like membrane integrity, mitochondrial activity, sperm motility and vigor, and the sperm proteome were determined. These were respectively assessed by chlortetracycline staining, H342/PI, JC-1, light microscopy, and the proteomic abundance by nUPLC-MS/MS analysis. Frozen-thawed sperm from three Nellore bulls were capacitated in vitro for 3 h in sp-TALP medium supplemented with 20 µg/mL heparin (Control) or with 20 µg/mL heparin plus 1 mM L-arg (L-arg group). Data were subjected to analysis of variance and means compared by SNK test at 5% probability. When compared to Control, the percentage of sperm motility was higher in the L-arg group (P < 0.05). For test data after 3 h of incubation, sperm capacitated with L-arg showed higher membrane integrity and mitochondrial potential when compared to Control (P < 0.05). Moreover, we observed an increase in the percentage of capacitated sperm pattern (P < 0.05). Protein abundance analysis identified 367 proteins. Forty proteins were differentially abundant between Control and L-arg group (P < 0.05), of which 11 were up-regulated, and 29 were down-regulated in L-arg group. In addition, we observed that one protein was uniquely abundant in the L-arg group. Our findings indicate that the addition of L-arg to the culture medium presented a differential protein abundance pattern and increased the bovine frozen-thawed sperm quality and the percentage of capacitated sperm. The proteomic changes observed may be linked to the molecular mechanisms involved in the action of L-arg on the in vitro sperm capacitation of cattle.

L-arginine affects the IVM of cattle cumulus-oocyte complexes.

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17ß-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control -ve). The addition of 5.0-mM L-arg increased (P < 0.05) the percentage of oocytes at the germinal vesicle breakdown stage after 7 hours of cultivation compared with control -ve. All concentrations of L-arg (2.5, 5.0, and 10.0 mM) increased the percentage of oocytes that reached the metaphase I (MI) at 15 hours (P < 0.05) but do not affect the progression from MI to metaphase II (P > 0.05) at 22 hours. All concentrations of L-arg tested increased (P < 0.05) the percentage of cumulus cells with plasma membrane integrity at 22 hours of cultivation. L-arginine did not change (P > 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control -ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control -ve and treatment with 5-mM L-arg progressively decreased (P < 0.05) after the first hour of cultivation; however, COCs treated with 5.0-mM L-arg had higher concentrations of cGMP at 1 hour of cultivation (P < 0.05). The cAMP concentration of COCs supplemented or not with 5.0-mM L-arg progressively increased until 3 hours of cultivation and at, 6 hours, decreased (P < 0.05). The results show, in using this system, that (1) the mechanisms that give the oocyte the ability to restart the meiosis until MI after adding 5.0-mM L-arg do not involve changes in the concentration of nitrate/nitrite, P4, and E2 in the culture medium and (2) L-arg acts on a pathway that involves changing the cGMP concentration but does not involve changing cAMP concentration. More studies are needed to assess whether the observed effects of L-arg during IVM using this system are via NO or not and what the role is in increasing the viability of cumulus cells in the resumption and progression of meiosis until MI.

Efeito da inibição da óxido nítrico sintase induzível na capacitação in vitro de espermatozoides bovinos / Effect of inhibition of inducible nitric oxide synthase on in vitro capacitation of bovine spermatozoa

Avaliaram-se o papel do óxido nítrico (NO) por meio da inibição da enzima óxido nítrico sintase induzível (iNOS), após a adição da aminoguanidina (AG), na motilidade, no vigor e na integridade da membrana plasmática nos tempos de 15, 60, 120, 180, 240 e 300min e a atividade mitocondrial e a capacitação de espermatozoides bovinos após 300min de cultivo. Adicionaram-se diferentes concentrações (0,001, 0,01 e 0,1M) de AG durante a capacitação induzida pela heparina e 500μM de nitroprussiato de sódio (SNP, doador de NO) à concentração deletéria. A adição de 0,1M de AG diminuiu a motilidade e o vigor espermático e a integridade da membrana (P<0,05). A adição de SNP ao meio de cultivo com 0,1M de AG somente reverteu a integridade da membrana após 300min. A inibição da síntese de NO pela adição de AG não alterou a atividade mitocondrial. A percentagem de oócitos penetrados com espermatozoides tratados com 0,01 e 0,1M de AG diminuiu 20,3 e 100 por cento, respectivamente, em relação aos não tratados (controle) (P<0,05), contudo houve aumento de 15 por cento na percentagem de oócitos desnudados penetrados com espermatozoides capacitados em presença de 0,1M de AG. Conclui-se que a inibição da síntese de NO pela AG diminuiu a qualidade espermática durante a capacitação de espermatozoides bovinos in vitro, exceto a atividade mitocondrial. Somente a integridade da membrana foi revertida após adição de NO, sugerindo diferentes vias de ação do NO na qualidade espermática ao longo da capacitação in vitro de espermatozoides bovinos.

The role of nitric oxide (NO) was evaluated by inhibition of inducible nitric oxide synthase (iNOS), with aminoguanidine (AG) on motility, vigor, and plasmatic membrane integrity of bovine spermatozoa culture after 15, 60, 120, 180, 240, and 300min and on mitochondrial activity and capacitation after 300min, respectively. Different concentrations, 0.001, 0.01, and 0.1M of AG were added during the heparin induced capacitation and sodium nitroprusside (SNP, NO donor-500μM) to the deleterious concentration. The addition of 0.1M of AG diminished progressive motility, spermatic vigor, and membrane integrity (P<0.05). SNP addition to the 0.1M of AG did revert only plasmatic membrane integrity after 300min. Mitochondrial activity was not influenced by addition of AG. Percentage of penetrated oocytes after addition of 0.01 and 0.1M of AG diminished, 20.3 and 100 percent, respectively, in relation to the control oocytes (P<0.05). However, an increase of 15 percent was observed when denuded oocytes were used with 0.1M AG treated sperm (P<0.05). It was concluded that the inhibition of NO synthesis with aminoguanidine diminished sperm quality during in vitro capacitation of bovine spermatozoa, except the mitochondrial activity. Only membrane integrity was reverted with the addition of NO to culture medium, suggesting different pathways of NO action on bovine sperm quality during in vitro capacitation.

Role of nitric oxide on quality of freshly ejaculated bull spermatozoa during heparin-induced in vitro capacitation.

The objective of this study was to assess the effects of nitric oxide (NO) on heparin-induced capacitation in vitro of fresh bull sperm, through the addition of Nomega-nitro-l-arginine methyl ester (L-NAME, a NO-synthesis inhibitor) and l-arginine (L-Arg, a NO-synthesis precursor) to the capacitation medium. In Experiment 1, different concentrations of L-NAME (0.1, 1, 10mM) and of L-Arg (10mM) were added to the capacitation medium. Sperm motility and vigor were subjectively appraised using direct light microscopy; sperm membrane integrity was examined using a 2% Trypan blue solution while the concentration of nitrate/nitrite (NO(3)(-)/NO(2)(-)) was determined by using the Griess method over a 5h capacitation period. The addition of 10mM L-NAME has inhibited NO synthesis, sperm progressive motility, sperm vigor and sperm membrane integrity (P<0.05) as compared to control. The addition of 10mM L-Arg to the capacitation medium increased all variables evaluated in comparison to the control (P<0.05). In Experiment 2, mitochondrial activity was assessed through the MTT test (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and sperm capacitation was assessed through the test of penetration in homologous oocytes after addition of the 10mM L-NAME, and of the 10mM L-Arg. The addition of 10mM L-NAME caused mitochondrial activity (40%) and the percentage of oocytes penetrated (77%) to decrease in relation to the control (P<0.05). After addition of 0.6mM L-Arg+10mM L-NAME, partial reversal of mitochondrial activity did occur (only 20%). The addition of 10mM L-Arg increased the percentage of oocytes penetrated as compared to control (21%) (P<0.05). These results indicate that: (1) NO is involved in control of progressive sperm motility, vigor, membrane integrity, and mitochondrial activity along the period of heparin-induced capacitation of fresh bovine sperm via NOS/NO; (2) adequate L-Arg/NO concentrations into the capacitation medium can potentiate heparin action or act independently for increasing the number or the quality of capacitated sperm.

Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium.

Nitric oxide (NO) in bovine ovary has been characterized as one of the controllers of granulosa cells' (GC) steroidogenesis and apoptosis. One of the pathways used by NO to have these effects is cGMP. The objectives of the present study were to verify the effect of sodium nitroprusside (SNP), a NO donor, on steroidogenesis, cell viability (mitochondrial activity) and GC cell cycle distribution and if this effect occurs by the NO-cGMP signaling pathway with the addition of SNP with or without 1H-[1,2,3] oxadiaziolo[4,3a]quinoxaline-1-one (ODQ), a selective soluble guanylate cyclase inhibitor. The antral GC from 3 to 5mm diameter cattle follicles was cultured without treatment (control), with ODQ (10(-4)M) and 10(-5), 10(-3) and 10(-1)M SNP with or without ODQ for 24h. Nitrate/nitrite (NO(3)(-)/N0(2)(-)) concentrations were evaluated by Griess method, progesterone (P(4)) and 17beta-estradiol (E(2)) concentrations by chemiluminescence, viability and cell cycle stage by MTT method (3-[4,5-dimethylthiazol-2yl]-2,3 dipheniltetrazolium bromide) and flow cytometry, respectively. Nitrate/nitrite concentration in culture medium increased (P<0.05) in a dose-dependent manner according to SNP concentration added to the culture medium. The GC cultured without treatment, with ODQ and with 10(-5)M SNP in the presence or absence of ODQ developed into cell aggregates and did not vary in cell viability (P>0.05), while GC cultured with 10(-3) and 10(-1)M SNP with or without ODQ presented disorganized GC aggregates or did not develop into cell aggregates and also had substantially decreased cell viability (mitochondrial activity inhibition) and steroids synthesis (P<0.05), and effects were not reversed with us of ODQ. Most GC cultured without treatment (control) or with ODQ, 10(-5) and 10(-3)M SNP with or without ODQ were in the G0/G1 (80-75%) stage and in a lesser proportion (20-25%) in the S+G2/M stage of the cell cycle, while the 10(-1)M SNP treatment resulted in GC in G1 phase arrest. The treatment with 10(-5)M SNP increased (P<0.05) E(2) synthesis and inhibited (P<0.05) progesterone synthesis. The addition of ODQ reversed (P<0.05) the stimulatory effect of 10(-5)M SNP treatment on E(2), but not on P(4) synthesis (P>0.05). These results demonstrated that E(2) synthesis by antral GC from small follicles is modulated by lesser NO concentrations via the cGMP pathway, but not P(4) while steroids inhibition cGMP pathway independent, mitochondrial damage and the interference on cell cycle progression caused by greater NO concentration can lead to cell death.

Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte-cumulus complexes of cattle.

The aim of the present study was to investigate the effects of inhibition of the enzyme inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on the in vitro maturation of oocyte-cumulus cell complex(es) (COC) of cattle. COC were cultured with different concentrations of AG (0, 1, 10, and 100mM) for 24h. In Experiment 1, the extent of cumulus complex expansion, nuclear maturation status and plasma membrane integrity of oocytes and cumulus cells from each treatment were assessed. Nitrate/nitrite (NO(3)(-)/NO(2)(-)) concentrations were determined in culture medium by the Griess method. Addition of different concentrations of AG to maturation medium promoted a dose-response inhibitory effect on cumulus expansion (P<0.05). Addition of 1 and 10mM AG to IVM medium did not affect plasma membrane integrity of oocytes or nuclear maturation rates (P>0.05), but it did reduce plasma membrane integrity in cumulus cells. One hundred millimolar inhibited pre-metaphase I (pre-MI) to metaphase II (MII) transition, promoted plasma membrane damage in oocytes (P<0.05), and increased NO(3)(-)/NO(2)(-) concentration when compared to controls (P<0.05). To evaluate if this effect was reversible, 10(-5)M sodium nitroprusside (SNP, NO donor) was added, only in the treatment with 100mM AG that inhibited the nuclear maturation. However, association of 10(-5)M SNP to 100mM AG did not reverse the effects of AG, but increased NO(3)(-)/NO(2)(-)concentration (P<0.05). In Experiment 2, the effect of different AG concentrations on cytoplasmic maturation in vitro was assessed based on cortical granule migration, and embryonic development. There was a dose effect on cortical granule migration rate, in which 1mM AG (83.9+/-6.2%) did not differ from control oocytes (83.6+/-8.2%; P>0.05), but 10mM partially inhibited migration (3.8+/-6.4%) and 100mM totally inhibited migration (P<0.05). SNP (10(-5)M) did not revert this inhibitory effect on cortical granules migration in oocytes treated with 100mM AG. Only those concentrations that did not inhibit IVM were used to assess cleavage and blastocyst development. Addition of 10mM AG to IVM medium reduced (73.0+/-8.1%, 15.0+/-8.9%; P<0.05) cleavage and blastocyst development, respectively when compared with controls (89.1+/-3.4%, 37.6+/-7.3%, respectively), but did not differ, (P>0.05), from the group treated with 1mM AG (80.9+/-8.4%, 41.5+/-10.5%, respectively). The results from the present study demonstrate that NO derived from iNOS affects the in vitro maturation of bovine COC, modulating the viability of cumulus cells and of oocyte, the progression of meiosis after GVBD, the migration of cortical granules, and cleavage and blastocyst development.

Ultrasonographic imaging of the reproductive tract and surgical recovery of oocytes in Cebus apella (capuchin monkeys).

Two-dimensional real-time and Doppler ultrasonography are valuable non-invasive methods to assess reproductive anatomy and physiology. In adult, postpubertal female Cebus apella (capuchin monkeys), the objectives were to determine (1) uterine and ovarian dimensions, ovarian follicular dynamics, day of ovulation, and arterial blood flow of uterus and utero-ovarian ligament during the follicular phase of the menstrual cycle and (2) the number of oocytes aspirated from antral follicles at laparotomy. Based on two-dimensional, transabdominal B-mode ultrasonography, mean (+/- S.E.M.) length, height, width, and volume of the uterus were 17.9+/-0.4, 12.4+/-0.3, 13.6+/-0.3 mm, and 1.55+/-0.08 mL, respectively, and of the ovary were 13.4+/-0.2, 8.2+/-0.1, 7.7+/-0.1 mm, and 4.5+/-0.2 mL. Ovarian follicles were monitored for 6 days before ovulation, which occurred on day 9.3+/-0.5 (range, days 7-11; day 1=start of menses), with 10 of 12 ovulations in the right ovary. Diameter and volume of the preovulatory follicle were 10.1+/-0.2 mm and 0.55+/-0.03 mL (on the estimated day of ovulation) and of the CL were 8.1+/-0.4 mm and 0.3+/-0.05 mL. Resistivity and pulsatility indices were 0.86+/-0.02 and 2.15+/-0.11 for uterine arteries, and were 0.69+/-0.04 and 1.63+/-0.15 for the utero-ovarian ligament (UOL) artery; just prior to ovulation, both indices peaked (P<0.05) in the uterine artery ipsilateral to the side of ovulation, but both reached a nadir (P<0.05) in the UOL artery. In the absence of ovarian stimulation, 31 oocytes (diameter, 137+/-10 microm) were aspirated (average of 2 oocytes/(female attempt)) on days 5, 7, and 9. In conclusion, transabdominal ultrasonography facilitated assessment of reproductive anatomy and physiology in C. apella adult females. Resistance and pulsatility indices of uterine and UOL arteries changed near the time of ovulation. Dominant follicles were easiest to aspirate at 8-9 mm in diameter ( approximately day 9), with intact cumulus-oocyte complexes recovered from ovarian follicles 2-9 mm in diameter.

Aspectos histopatológicos da adenomiose em úteros bovinos nas diferentes fases do ciclo estral / Histophatological aspects of adenomyosis in bovine uteri in different phases of the estrous cycle

Relacionaram-se as características da adenomiose com as fases do ciclo estral em 61 peças de úteros de bovinos colhidas em matadouros. A adenomiose foi classificada em superficial e profunda. A fase do ciclo estral foi estimada pela morfologia, pela coloração e pela vascularização do corpo lúteo e presença ou não de folículos ovarianos maiores que 8mm. Os animais que estavam em anestro (n=11) apresentaram a menor ocorrência de adenomiose (8,2 por cento), e os que estavam na fase lútea média (n=21), a maior (31,0 por cento). Nas fases lútea inicial (n=13) e folicular (n=16) as ocorrências foram semelhantes, 18,0 e 22,9 por cento, respectivamente. A maior porcentagem de adenomiose profunda ocorreu nas fases lútea inicial e média, 45,0 e 47,4 por cento, respectivamente, e durante o anestro e a fase folicular foram de 20,0 e 14,3 por cento, respectivamente. Os resultados sugerem que a fase do ciclo estral influencia na ocorrência de adenomiose e no grau de infiltração miometrial das glândulas endometriais

The relationship of the adenomyosis characteristics and the phases of the estrus cycle in 61 cows bovine's uteruses collected in slaughterhouses was studied. The adenomyoses were classified as superficial and deep. The morphology, staining and vascularization of the corpus luteum and the presence or not of larger ovarian follicles than 8mm helped to estimate tthe estrus cycle. The cows in anestrus (n=11) showed the least occurrence of adenomyosis (8.2 percent) and the animals in the medium luteal phase (n=21) the largest one(31,0 percent). In the initial luteal phase (n=13) and the follicular phase (n=16) the occurrences of adenomyosis were similar and equals to 18.0 and 22.9 percent, respectively. The largests percentage of deep adenomyosis were found in the initial and in the medium luteal phases, 45.0 and 47.4 percent, respectively, and during the anestrus and the follicular phase they were 20.0 and 14.3 percent, respectively. The data suggest that the cycle phase influences in adenomyosis occurrence and in the degree of miometrial infiltration of the endometrial glands

Effect of exercise on occurrence of diurnal rhythms of plasma ions and metabolites in Thoroughbred racehorses

Records of plasma calcium (Ca++), phosphorus (Pi), potassium (K+), sodium (Na+), chloride (Cl-), magnesium (Mg++), iron (Fe++), glucose, cholesterol, triglycerides and total protein levels were measured to determine the effects of exercise on occurrence of diurnal rhythms in Throughbred racehorses (n=7) under physical training. Physical activities consisted of gallop on the track and walking. Blood samples were collected from jugular vein every 4h over a 48h period. Plasma Ca++, K+, Mg++ and Na+ levels were obtained by flame photometry; and, Pi, Cl-, Fe++, glucose, cholesterol, triglycerides and total protein levels were measured by colorimetric tests using visible UV spectrophotometry. The data were analyzed using a 24h period to each exercise performed. Diurnal rhythm of Pi was observed when walking was the physical activity performed, and its acrophase occurred at the light period. Plasma triclycerides showed significant diurnal rhythms, with their acrophases occurring at the dark period, even when walking or gallop were performed. High intensity exercise (gallop) decreased triglycerides amplitude. No significant diurnal rhythms of other variables were found. Gallop, as physical activity, masked phosphorus diurnal rhythm. However, physical training did not influence triglycerides diurnal rhythm occurrence. High intensity exercise (gallop) just declined triglycerides amplitude

Mensuraram-se as concentrações plasmáticas de cálcio (Ca++), fósforo (Pi), potássio (K+), sódio (Na+), cloreto (Cl-), magnésio (Mg++), ferro (Fe++), glicose, colesterol, triglicerídeos e proteínas totais para determinar os efeitos do exercício sobre a ocorrência dos ritmos diários em cavalos de corrida da raça Puro Sangue Inglês (n=7), em treinamento. A atividade física consistiu de galope na raia e passo. Amostras de sangue foram coletadas da veia jugular a cada 4h durante um período de 48h. As concentrações plasmáticas de Ca++, K+, Mg++ e Na+ foram obtidas por espectrofotometria de absorção atômica com chama, e as concentrações de Pi, Cl-, Fe++, glicose, colesterol, triglicerídeos e proteína total foram mensuradas por testes colorimétricos utilizando-se a espectrometria de luz UV visível. Os dados foram analisados utilizando-se um período de 24h para cada tipo de exercício desenvolvido. Ritmo diário de Pi foi observado quando o passo foi a atividade física desenvolvida, apresentando sua acrofase no período diurno. A concentração plasmática de triglicerídeos mostrou significante ritmo diário com a acrofase ocorrendo no período noturno, independente se foi desenvolvido o galope ou passo. O exercício de alta intensidade (galope) diminuiu a amplitude dos triglicerídeos. Nenhum ritmo diurno foi achado nas outras variáveis. Estes resultados mostram que o galope como atividade física, mascara o ritmo diário do fósforo. Entretanto, o treinamento físico não apresentou nenhum efeito na ocorrência do ritmo diário de triglicerídeos, somente na sua amplitude

Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes.

Nitric oxide (NO) is a highly reactive free radical involved in intra- and intercellular signaling in various stages of reproduction. The objective of the present study was to evaluate the effect of the addition of sodium nitroprusside (SNP), a NO donor, on nuclear and cytoplasmic in vitro maturation of bovine oocytes. Analysis of variance was conducted and the means were compared by t test at a level of 5%. Low (10(-7) and 10(-9)M) and intermediate (10(-5)M) concentrations of SNP had no significant effect on nuclear maturation, however, when a greater concentration of SNP (10(-3)M) was added, oocytes remained in metaphase I (MI) after 24 h culture (P<0.05) and did not show cumulus expansion. To evaluate if this effect was reversible and if a retardation or inhibition had occurred in the progression from MI to MII, oocytes were cultured in presence of 10(-3)M of SNP for 24 h followed by culture for an additional 24 h in medium with or without SNP. After 48 h, the oocytes remained in MI even when the medium was changed at 24 h with or without SNP. The kinetics of nuclear maturation was assessed to evaluate if there had been or not a retardation in the progression of meiosis with the concentration of 10(-3)M SNP. This concentration delayed germinal vesicle breakdown (VGBD) at 8 h of culture (P<0.05), and at 12 h there was no significant difference between the control and the treated group. The concentrations that did not induce alterations in nuclear maturation were evaluated for cytoplasmic maturation. The concentration of 10(-5)M improved the percentage of peripheral cortical granules (P<0.05), and significantly increased the percentage of blastocysts. These results demonstrate that SNP at greater concentrations (10(-3)M) has a cytotoxic effect, but at intermediate (10(-5)M) concentrations it increases blastocyst rates. NO exhibits a dual effect on bovine oocytes, inhibits (10(-3)M of SNP) nuclear and cytoplasmic maturation or stimulates (10(-5)M of SNP) cytoplasmic maturation, depending on concentration in the culture medium.

Testosterona e gonadotrofina coriônica humana estimulam a esteroidogênese em células da granulosa de folículo pré-ovulatório de égua? / Do testosterone and human chorionic gonadotropin stimulate steroidogenesis in granulosa cells of preovulatory follicle in mare?

Avaliou-se o papel da gonadotrofina coriônica humana (hCG) e da testosterona na produção de progesterona (P4) e 17ß-estradiol (E2) pelas células da granulosa cultivadas in vitro de folículo antral de égua. Os tratamentos usados foram: 1- controle (nenhum hormônio adicionado), 2- 1UI hCG (0,3µg/ml) e 3- 10UI hCG (3,0µg/ml). O tratamento com hCG foi realizado na presença ou não de testosterona (144ng/ml). O meio foi coletado e substituído com 0,25, 3, 6, 12, 24 e 144h de cultivo. As concentrações de P4 e E2 foram mensuradas por radioimunoensaio. Não se observou diferença entre os tratamentos 1 e 3 quanto à produção de P4 e E2; o tratamento 1 resultou em aumento da concentração de progesterona após 24h de cultura (P<0,01), mas somente em presença de testosterona. A concentração de estradiol aumentou em presença de testosterona, alcançando concentração máxima com 6h de cultura (P<0,01), e diminuiu gradativamente, até atingir a concentração observada com 0,25h de cultura. A adição de hCG não influenciou a síntese do estradiol. A testosterona desempenhou importante efeito estimulador na síntese/secreção doe E2 pelas células da granulosa e modulou a ação do hormônio luteinizante na diferenciação e luteinização das células da granulosa de folículo antral presumidamente pré-ovulatório de égua in vitro.