Molecular control of rabbit follicular testosterone production. Role of protein and RNA after stimulation with luteinizing hormone.

The time and dose dependence of the relationship between uptake of labelled precursors into protein and RNA and production of testosterone by rabbit follicles was examined. Although testosterone production was stimulated by luteinizing hormone at concentrations between 0.1 and 10 microgram/ml, the uptake of [3H]leucine into protein was significant only when the concentration of luteinizing hormone was greater than 2.5 microgram/ml. Increased production of testosterone was observed within 15 min of stimulation with luteinizing hormone whereas uptake of [3H]leucine was only significant at 90 min. Puromycin (40 microgram/ml) and cycloheximide (10 microgram/ml) in the presence of luteinizing hormone inhibited the synthesis of both testosterone and protein. However, lower concentrations of puromycin (0.1, 1 and 10 microgram/ml) and cycloheximide (1 microgram/ml) had no effect on luteinizing hormone-induced testosterone production but significantly inhibited protein synthesis by 58, 37, 31 and 71%, respectively. Actinomycin D (20, 80 and 160 microgram/ml) alone and in combination with 5 microgram luteinizing hormone/ml severely inhibited uptake of [3H]uridine into RNA without affecting testosterone production. However, with 1 microgram actinomycin/ml, testosterone production was significantly (P less than 0.01) greater than in the presence of luteinizing hormone alone. These results cast doubt on the obligatory role of RNA and protein synthesis in rabbit ovarian follicular steroidogenesis.     

Increased ornithine decarboxylase activity during meiotic maturation in xenopus laevis oocytes

The activity of ornithine decarboxylase (ornithine carboxylyase E.C. 4.1.1.17) was studied during meiotic maturation induced in vitro by progesterone in follicle cell-free oocytes. Enzyme activity increased 4–6 fold during maturation, preceding germinal vesicle breakdown. The increase in ornithine decarboxylase activity was inhibited by cholera toxin, an agent that blocks meiotic maturation and increases cAMP levels within the cell. It was also prevented by cycloheximide but not by actinomycin D. Treatment of oocytes with D,L-α-difluoromethyl-ornithine, an irreversible inhibitor of ornithine decarboxylase and of putrescine synthesis, effectively abolished enzyme activity without preventing germinal vesicle breakdown. These observations show that the progesterone-induced increase in ornithine decarboxylase activity is not required for completion of meiotic division of the oocyte.     

Binding of progesterone to cell surfaces of human granulosa-lutein cells

Progesterone is produced by granulosa cells under the influence of luteinizing hormone. Nuclear progesterone receptors have been found in rat granulosa cells. Human granulosa-lutein cells rapidly respond to progesterone with an increase in intracellular calcium suggesting the existence of a nongenomic mechanism. This study was conducted to determine whether binding of progesterone to granulosa cells could occur at the membrane. Granulosa cells were obtained from an in vitro fertilization program and examined immunohistochemically with an antiserum to membrane progesterone receptors. Approximately 14-70% of freshly harvested or cultured granulosa cells of six patients showed a positive reaction to the antiserum, limited to the cell membrane. Western blot analysis of homogenates of granulosa cells and a granulosa cell tumour confirmed the presence of progesterone receptors A, B and C and low amounts of a putative membrane receptor. These results demonstrate that the plasma membranes of human granulosa cells possess binding components for progesterone which may be involved in its nongenomic mechanism of action.     

Plasma progesterone levels during pregnancy in the little brown bat Myotis lucifugus (Vespertilionidae)

Plasma progesterone was measured by radioimmunoassay in individual female Myotis lucifugus throughout pregnancy and lactation. Progesterone levels, which averaged 6.7 +/- 0.7 ng/ml in late hibernation, rose to a mean of 18.9 +/- 6.7 ng/ml in unimplanted bats collected in the first two weeks after arrival at a maternity roost. Analysis of progesterone levels in bats in which the developmental stage of the embryo was known revealed two sharp, transient increases in plasma progesterone during the preimplantation period. The first, with values of 30-45 ng/ml, occurred at ovulation. The second, with values of 20-30 ng/ml, coincided with blastocyst formation. Progesterone levels increased exponentially from a mean of 7.4 +/- 1.0 ng/ml during early implantation to peak values of 100-200 ng/ml (means = 136.2 +/- 15.6) in the last two weeks of pregnancy, and showed no evidence of either a midpregnancy or prepartum decline. Despite involution of the corpus luteum at the end of pregnancy, progesterone levels averaged 9.0 +/- 1.0 ng/ml during lactation and did not decline until the end of lactation. In bats undergoing abortion, mean levels of plasma progesterone were already less than 6 ng/ml, equivalent to levels in nonbreeding females. The results indicate that the progesterone profile of pregnant M. lucifugus, though generally resembling those of other bats, exhibits several distinctive features. The sharp rise in plasma progesterone coinciding with blastocyst formation has not been reported in other mammals and suggests a possible role of progesterone in the cavitation process. In addition, peak values of plasma progesterone in late pregnancy were conspicuously higher than levels reported in other verpertilionid bats. The levels did not appear to fall before parturition, although such falls have been reported in other bats.     

Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment

The objective of this study was to investigate the relationship between oocyte morphology and follicular fluid steroid concentrations in patients being treated with intracytoplasmic sperm injection. A total of 82 IVF cycles were evaluated in patients aged 24-40 years. Oocytes at metaphase II were graded into four groups according to the status of the first polar body and the size of the perivitelline space. The proportion of oocytes at the germinal vesicle and germinal vesicle breakdown stages, and the proportion of degenerated oocytes and oocytes with a large polar body were compared with different concentrations of oestradiol, progesterone and testosterone in the follicular fluid. The association between these oocyte characteristics and the ratio of oestradiol:testosterone and oestradiol:progesterone was also analysed. The results showed that oocyte morphology, as assessed by the status of the first polar body and the size of the perivitelline space, is associated with the ratio of oestradiol:testosterone and oestradiol:progesterone but not with the absolute concentrations of oestradiol, progesterone and testosterone in the follicular fluid. A ratio of oestradiol:testosterone > 200 is the best indicator for a small proportion of grade 1 and 2 oocytes (poor quality), a large proportion of grade 3 and 4 oocytes (good quality), and a small proportion of oocytes with cytoplasmic inclusions. These results will be of clinical use in evaluating oocyte quality.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.