Prolactin concentrations in follicular fluid following ovarian hyperstimulation for in vitro fertilization

Prolactin (PRL) and sex steroid concentrations were measured in follicular fluids of women treated either with (1) clomiphene/hCG or with (2) clomiphene + hMG/hCG. Method 1 of ovarian stimulation resulted in lower follicular PRL and higher oestradiol-17 beta (E2) and progesterone (P) concentrations than method 2. There was no difference in the PRL and sex steroid concentrations of follicles with fertilized and of those yielding unfertilized ova, but in both stimulation types, follicles from which no oocytes were obtained had high PRL and low E2 and P levels. Significant positive correlations were evident for PRL and T and E2 and P, respectively, while PRL and P were negatively correlated.     

Differential steroid and gonadotropin response by individual tertiary porcine follicles in vitro. Possible physiologic role of atretic follicles

Since atretic follicles contain significant amounts of androgen and/or progesterone in their follicular fluid, we examined whether they also contribute to ovarian steroid secretion. Steroid secretion by atretic porcine follicles and their responsiveness to FSH was assessed by a perifusion system that allows for separate dynamic incubation of whole follicles in vitro. Identically treated nonatretic follicles of comparable size served as a reference group. The extent of granulosal pyknosis, on which the staging of atresia was based, was inversely related to follicular estradiol (E2) secretion and its responsiveness to FSH. Both basal and FSH-stimulated secretion of testosterone (T), androstenedione (A), and progesterone (P) were maintained by follicles in all stages of atresia. Secretion of A by late atretic follicles was greater than that in earlier stages or by nonatretic follicles. Atretic follicles may therefore release comparable or larger amounts of androgen and P into their intraovarian environment than do nonatretic follicles. We examined whether steroids secreted by atretic follicles in vitro could be utilized by nonatretic follicles. A static incubation system was used that allows for simultaneous incubation of a number of individual follicles. When nonatretic follicles were exposed to A, T, or P in physiologic concentrations (10(-7)-10(-5) M), their secretion of E2 increased 2-8-fold. Doses of FSH or LH that stimulated follicular steroid in vitro had no additional stimulatory effect when combined with A or P treatment, respectively. In conclusion, atretic follicles may contribute significantly to intraovarian levels of androgen and P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inverse relationships between steroid concentration and volume in preovulatory follicles of the golden hamster

In order to investigate variations in the microenvironment of oocytes within a cohort of maturing follicles the follicular volumes as well as the intrafollicular concentrations of oestradiol (E2) and progesterone (P) were measured in the golden hamster. At 10 h before ovulation the follicular volumes varied from 0.009 to 0.037 mm3 (mean +/- SD: 0.0187 +/- 0.0071 mm3; n = 36). Large follicles (greater than 0.025 mm3; n = 8) contained statistically significantly lower E2 and P levels (30.1 +/- 10.4 and 517 +/- 113 mumol/l, respectively) than the medium sized group (less than 0.025 and greater than 0.015 mm3; n =20): 46.9 +/- 16.0 (P less than 0.02) and 919 +/- 264 (P less than 0.0001) mumol/l, respectively. Small follicles (less than 0.015 mm3) showed the highest steroid levels: 97.0 +/- 33.3 and 1590 +/- 517 mumol/l for E2 and P (P less than 0.001 versus the medium sized group values). Correlation coefficients for the steroid concentrations and the follicular volumes appeared to be -0.674 for E2 and -0.612 for P (P less than 0.001). At the time studied a positive correlation between E2 and P concentrations in the follicles was found: r = 0.655 (P less than 0.001). The mean ratios of intrafollicular over serum steroid concentrations appeared to be approx 36 x 10(3) in the case of E2 and about 17 x 10(3) in the case of P. These results clearly show that there is an inverse relationship between follicular volume and intrafollicular steroid concentrations. The presence of a fine regulatory mechanism for a collective maturation of follicles is hypothesized.     

Characterization of parameters for in vitro culture of isolated ovarian follicles of greenback flounder Rhombosolea tapirina

Isolated ovarian follicles of greenback flounder Rhombosolea tapirina were incubated with a variety of gonadotropins (GtHs) and steroid precursors for periods of up to 42 h, and levels of free and glucuronated testosterone (T) and 17beta-estradiol (E(2)) in the medium, and free T and E(2) from inside follicles were measured by RIA. Short incubations (6 h) generated increases in T and E(2) in response to steroid precursors, but not human chorionic GtH (hCG), or salmon or carp GtH. At incubation times of 18 h, all GtHs stimulated T and, or E(2) production, whereas after 42-h incubation, GtH effects on E(2) production had disappeared. Steroid precursors remained effective at 18 and 42 h. T and E(2) glucuronides were formed in small quantities but did not account for loss of treatment effects at long incubation times. Instead, this could be explained by accumulation of E(2) in controls as a result of continued basal steroid production. Follicles absorbed substantial amounts of both endogenous and exogenous steroid from the medium, however, this did not appear to have any influence on changes in treatment effects with incubation time. Flounder follicles were most sensitive to hCG, followed by salmon and carp GtH at approximately 10-fold higher concentrations. Ovarian segments were not sensitive to any GtH but did convert exogenous steroid precursors indicating that tissue access by GtH may be a limiting factor under certain in vitro conditions. HCG augmented the conversion of 17-hydroxyprogesterone (17P) to T but not T to E(2), consistent with the relative GtH-insensitivity of aromatase in other species. Follicles converted a range of steroid precursors with equal competence, indicating that no step in the cleavage pathway is strongly rate-limited, and that choice of precursor is unlikely to affect the assessment of steroidogenic activity.     

Steroid concentrations in follicular fluid aspirated repeatedly from transitional and cyclic mares

The objectives of the present study were to determine follicular progesterone (P4) and estradiol-17beta (E2) in transitional mares and to compare follicular steroid concentrations between transitional and cyclic mares. Follicles > 8 mm were aspirated under transvaginal ultrasound-guidance 4 times at 3 to 4 day intervals (T1-T4) in Norwegian pony mares during vernal transition. During the breeding season, follicular aspirations were conducted in each mare on Day 6, Day 14 and Day 18 after ovulation of 3 separate estrous cycles (Day of ovulation = Day 0). Plasma and follicular fluids were analyzed for P4 and E2 with ELISA and RIA, respectively. Plasma P4 concentrations remained below 1 ng/mL throughout T1-T4, while the follicular P4 concentrations increased significantly to cyclic levels after the first transitional aspiration. Plasma E2 concentrations similarly remained at low levels during the course of the transitional aspirations, while the follicular E2 concentrations increased gradually over the 4 aspirations to cyclic concentrations. The mares ovulated on average 9.8 +/- 1.6 (mean +/- SEM) days after the last transitional aspiration, and 16.6 +/- 0.2, 11.3 +/- 1.5 and 23.2 +/- 4.4 days after aspirations conducted on Day 6, 14 and 18, respectively. The present study demonstrates that in the transitional mare newly developing follicles exhibit biosynthesis of P4 and E2. Furthermore, an increase in follicular steroid concentrations is not necessarily reflected in the peripheral steroid concentrations.     

Effect of glutamate on basal steroidogenesis by ovarian follicles of rainbow trout (Oncorhynchus mykiss)

The effects of glutamate on the in vitro basal steroid production of three maturational stages of rainbow trout (Oncorhynchus mykiss) ovarian follicles were investigated. Radioimmunoassays were used to measure the rates of synthesis of testosterone (T) and 17-estradiol (E2). High performance liquid chromatography (HPLC) was used to examine the steroid metabolites produced from a tritium labeled precursor, pregnenolone (P5). The glutamate agonist, N-methyl-d,l-aspartate (NMA) had a dose-dependent suppressive effect on T and E2 synthesis in mid-vitellogenic (MV) follicles, but had no significant effect on early- (EV) and late-vitellogenic (LV) follicles. l-glutamic acid (GA) had a dose-related suppressive effect on T synthesis by MV follicles, suppressing both T and E2 synthesis by LV follicles, but having no effect on EV follicles. HPLC separation of the steroid metabolites synthesized from P5 showed clear evidence of differences in rates of overall steroidogenesis of the three follicular stages, but no effect of either NMA or GA on the nature or the amount of the metabolites produced by the three developmental stages examined. The findings suggest that glutamate may act via a reduction in the production of P5, which is the principal rate-limiting step in the steroidogenic cascade, and not via modulation of steroidogenic enzyme activities.     

In vitro steroid production by isolated ovarian follicles of the striped trumpeter

Ovarian follicles from striped trumpeter Latris lineata were incubated in L15 medium alone, or medium supplemented with gonadotropin (GtH) preparations (human chorionic GtH, carp maturational GtH or partially purified salmon GtH), testosterone (T) or 17-hydroxyprogesterone (17P). Levels of oestrone (E₁), 17 β -oestradiol (E₂), T, and 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β P) in the medium after incubation were measured by radioimmunoassay. Basal production of E₂ was high from previtellogenic follicles, whereas little T was produced. Both T and E₂ production increased in response to treatment with GtH or steroid precursors. Vitellogenic follicles showed basal production of both T and E₂, and T but not E₂ levels generally increased in response to hormone treatment. Preparations containing follicles nearing final maturation showed low basal production of E₂ but high production of T. Treatment with steroids resulted in little change in E₂ but often very large increases in T production, whereas GtH stimulated lesser increases. 17,20 β P production was detectable from incubations of maturing follicles from two out of five fish, and in those two incubations, increased in response to treatment with 17P. E₁ was not detectable in any incubations. The results indicate that there is a shift in steroidogenesis from E₂ to T production during oocyte development, and provide further evidence that steroid biosynthesis in non-salmonids is principally regulated by substrate availability.     

Frequency of luteinizing hormone pulses in cattle influences duration of persistence of dominant ovarian follicles,follicular fluid concentrations of steroids,and activity of insulin-like growth factor binding proteins

The objectives of the present study were to determine how varying frequency of LH pulses as controlled by varying treatments with progesterone (P4) in cattle would affect: (1) concentration of steroid hormones and activity of insulin-like growth factor binding proteins (IGFBPs) in the ovarian follicular fluid and blood plasma, and (2) duration of persistence of largest ovarian follicles. There were four treatment groups (n=7 per group) and a control group (n=5) of mature, non-lactating beef cows. Treatments were: (1) two progesterone releasing intravaginal devices (PRIDs) for 16 days (2PRID); (2) a half PRID for 16 days (0.5PRID); (3) two PRIDs for 8 days, then a half PRID for 8 days (2-0.5PRID); or (4) a half PRID for 8 days, then two PRIDs for 8 days (0.5-2PRID). Treatment was initiated on the fifth day of the estrous cycle, which was designated as Day 0, and continued for 16 days. All P4-treated females were administered prostaglandin F2alpha on Day 0 and 1 to regress their corpora lutea. Frequency of LH pulses was greater during treatment with the smaller dose of P4 compared with treatment with the larger dose of P4 and the control group. Ovarian follicles were classified into five categories based on ultrasonographic observations: growing (G); atretic (A); growing dominant (GD); growing persistent (GP); or atretic persistent (AP). At ovariectomy on Day 16, the largest and second largest follicles collected were re-classified into five categories based on follicular concentration of steroids. Classification of the largest follicle collected on Day 16 was influenced by treatment (P<0.005), with the 2PRID group having A follicles, the 2-0.5PRID group GP follicles, the 0.5-2PRID group AP follicles, and the 0.5PRID group GD and GP follicles. Concentrations of 17beta-estradiol (E2) were greatest in GD and GP follicles (P<0.05). There was less (P<0.05) activity of IGFBP-2 in GD follicles and less (P<0.05) activity of IGFBP-3 in GD and GP follicles than other follicles. Activity of IGFBP-4 and -5 was greater (P<0.05) in A and AP follicles than G, GD, and GP follicles. Maintenance of a frequent release of LH pulses over a 16-day period did not result in maintenance of persistent follicles throughout this period indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia is associated with greater activity of IGFBP-2, -4, -5, and greater concentrations of P4 in follicles, whereas growing dominant and persistent follicles contained greater concentrations of E2, androstenedione (A4), and less IGFBP-2 activity than follicles of other classes. Follicle classifications based on ultrasonography or follicular concentration of steroids did differ (P<0.05) for the largest follicles from the 2PRID group. Two follicles in this group appeared as GD follicles by ultrasonography, but these were atretic based on follicular steroid contents. Objective 1 of the present study yielded the conclusion that concentrations of steroid hormones in follicular fluid and blood plasma could be predictably controlled by regulating the frequency of LH pulses with varying doses of P4. Objective 2 yielded the conclusion that maintain frequent release of LH pulses over a 16-day period could not maintain persistent follicles throughout this period, indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia in the present study was associated with increased follicular fluid activity of IGFBP-2, -4, -5, and P4, whereas growing dominant and persistent follicles contained greater concentrations of E2, A4, and less IGFBP-2 activity than follicles of other classes.     

Influence of maternal environment on the number of transferable embryos obtained in response to superovulatory FSH treatments in ewes

In a first experiment, embryo viability was estimated after recovery in the uterus or the oviduct of 70 Manchega ewes following a treatment of superovulation with decreasing doses of OVAGEN. Fewer viable embryos (5.6 +/- 0.9 vs. 8.3 +/- 0.8, P < 0.05) and more degenerative embryos (31.3% vs. 6.8%, P < 0.005) were obtained from the uterus than from the oviduct respectively. In a second experiment performed on 14 ewes, embryo viability was analyzed in relation to the follicular population estimated by ultrasonography (follicles > or = 2 mm) at the first FSH administration. Progesterone (P4) and oestradiol 17beta (E2) concentrations were also determined from the beginning of the superovulation treatment to the recovery of the embryos. The number of viable embryos (4.3 +/- 1.4) was positively correlated (r = 0.824) with of 2-4 mm diameter follicles (P < 0.05), and with E2 concentrations at -12 h (r = 0.891, P < 0.01) , 0 h (r = 0.943, P < 0.0001) and +24 h (r = 0.948, P < 0.05) from estrus detection. Prolonged high levels of E2 up to 72 h with low levels of P4 on days 3 and 4 after estrus had a negative (P < 0.05) effect on embryo viability. These results indicate that ovarian response to superovulatory protocols is related to the individual variations in the number of follicles of 2-4 mm at the start of FSH treatment, and that embryo viability is conditioned by the steroid patterns during the time spent in the genital tract of the super-ovulated ewes.     

Effects of follicle-stimulating hormone with and without luteinizing hormone on serum hormone concentrations, follicle growth, and intrafollicular estradiol and aromatase activity in gonadotropin-releasing hormone-immunized heifers

To evaluate the roles of FSH and LH in follicular growth, GnRH-immunized anestrous heifers (n = 17) were randomly assigned (Day 0) to one of three groups (n = 5 or 6). Group 1 received i.m. injections of 1.5 mg porcine FSH (pFSH) 4 times/day for 2 days; group 2 received i.v. injections of 150 microg pLH 6 times/day for 6 days; group 3 received both pFSH and pLH as described for groups 1 and 2. After slaughter on Day 6, measurements were made of follicle number and size, and follicular fluid concentrations of progesterone (P(4)), estradiol (E(2)), and aromatase activity. Injection of pFSH increased (P: < 0.01) the serum concentrations of FSH between 12 and 54 h. Infusion of pLH increased (P: < 0.05) mean and basal concentrations of LH and LH pulse frequency. Serum E(2) concentrations were higher (P: < 0.05) for heifers given pFSH + pLH than those given either pFSH or pLH alone. There was no difference (P: > or = 0.24) between treatments in the number of small follicles (<5 mm). Heifers given pFSH or pFSH + pLH had more (P: < or = 0.02) medium follicles (5.0-9.5 mm) than those that were given pLH alone (none present). Heifers given pFSH + pLH had more (P: = 0.04) large follicles (> or =10 mm) than those given either pLH or pFSH alone (none present). Overall, only 1 of 35 small follicles and 2 of 96 medium follicles were E(2)-active (i.e., E(2):P(4) >1.0), whereas 18 of 21 large follicles (all in the pFSH + pLH treatment) were E(2)-active; of these, 8 of 18 had aromatase activity. Concentrations of E(2) and E(2) activity in follicular fluid were correlated (r > or = 0.57; P: < 0.0001) with aromatase activity in heifers given pLH + pFSH. In conclusion, pLH failed to stimulate follicle growth greater than 5 mm; pFSH stimulated growth of medium follicles that were E(2)-inactive at slaughter and failed to increase serum E(2) concentrations; whereas pFSH + pLH stimulated growth of medium follicles and E(2)-active large follicles, and a 10- to 14-fold increase in serum E(2) concentrations.     

Growth hormone but not prolactin concentrations in the fluid of bovine ovarian cysts are related to the cystic stage of luteinization

Regulation of follicular growth and ovulation as well as steroid production by the ovary depends principally on gonadotropins. However nonsteroid systemic hormones and autocrine and paracrine factors contribute to the regulation of ovarian function. The objectives of the present work were 1) to asses the presence of growth hormone (GH) and prolactin (PRL) in fluid drawn from normal bovine ovarian follicles, cysts or cystic corpora lutea; 2) to relate the stage of luteinization of the cyst with the GH and PRL concentrations in fluids; and 3) to asses the feasibility of providing a defined nonsteroid hormone marker to distinguish between normal and pathological ovarian structures. Cysts were classified according to histological and morphological appearance as follicular or luteal. Concentrations of GH, PRL, estrogens (E2), progesterone (P4) and testosterone (T) were measured in follicular and cystic fluids. On the basis of the E2 to P4 ratio, ovarian formation classes were further divided into two subclasses (E2 dominant and P4 dominant). The results provide evidence of 1) the presence of immunoreactive GH and PRL in all the follicular and cystic fluids assayed, 2) an increasing concentration of GH correlated to the stage of luteinization of the cyst and a direct correlation between GH and P4 concentrations, 3) a significant variability of intraovarian fluid PRL concentration not related to the histological class of the cyst nor to the concentrations of steroid hormones examined, and 4) the possibility of distinguishing 6 different ovarian formation classes by merely measuring GH, P4, E2 and T concentrations in fluids. These data contribute to a better understanding of the endocrine milieu of bovine ovarian cystic degeneration.     

Concentrations of steroids and expression of messenger RNA for steroidogenic enzymes and gonadotropin receptors in bovine ovarian follicles of first and second waves and changes in second wave follicles after pulsatile LH infusion

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase Delta(4), Delta(5) isomerase (3beta-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump (n=5-6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 microg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at -80 degrees C. Follicular fluid (FF, 50 microl) was collected for determination of progesterone (P4), oestradiol-17beta (E2) and androstenedione (A4) concentrations. Frozen sections (14 microm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3beta-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3beta-HSD. In conclusion, follicular fluid concentrations of E2 and A4 were lower in W2S than in W1S and E2 and A4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3beta-HSD.     

正常月经周期妇女卵泡液中激素水平的变化

本文测定了24例正常月经妇女在不同时相、不同大小卵泡的卵泡液中雌二醇(E_2)、孕酮(P_0)、雄烯二酮(A)、睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)和催乳素(PRL)的含量,并分析其与外周血中相应激素浓度的关系。测定结果显示:小卵泡的卵泡液中E_2、Po,FSH,LH水平低于大卵泡中水平,而A和T水平则相反。排卵前大卵泡中E_2(9815nmol/L),P_0(3316nmol/L),FSH(1.34IU/L)和LH(3.9lIU/L)达最高值。A(280nmol/L)和T(137nmol/L)却较小卵泡中水平低(相应为692nmol/L和176nmol/L)。PRL水平在大小卵泡中无显著性差异。卵泡液中甾体激素水平高于外周血7—20.000倍,FSH、LH水平为外周血的10—80％,PRL水平为60％—3倍。     

Hormone-stimulable adenylyl cyclase and steroid concentration of follicles of the pregnant mare's serum gonadotropin-treated hen

Pregnant mare's serum gonadotropin (PMSG) treatment of the hen disrupts the follicular hierarchy and causes cessation of ovulation. We measured serum progesterone (P4) and estradiol (E2) concentrations and follicular steroid levels and adenylyl cyclase (AC) activity of PMSG-treated hens. Serum P4 and E2 levels were elevated (P less than 0.01 and P less than 0.05, respectively) in PMSG-treated hens compared to controls. There was no significant difference in P4 and E2 concentrations in granulosa and theca layers, respectively, between follicles from PMSG-treated hens and the largest (F1) follicles from control hens. Basal, luteinizing hormone (LH)-, and follicle-stimulating hormone (FSH)-stimulable AC activity was measured in granulosa layers of the largest follicles from PMSG-treated hens and the F1 and second largest (F2) follicles from control hens. Basal AC activity was increased in follicles from PMSG-treated hens (P less than 0.05) compared to F1 control follicles. There was no difference in LH- and FSH-stimulable AC of PMSG-treated hens compared to F1 controls. Control F2 follicles had lower LH- (P less than 0.001) and FSH-stimulable (P less than 0.005) AC activity than follicles from control F1 or PMSG-treated hens. Relative LH- and FSH-stimulable AC (hormone stimulable vs. basal) for follicles from PMSG-treated hens did not differ statistically from the relative AC activity of vehicle-injected F1 or F2 follicles. Therefore, in spite of the high serum P4 and E2 levels in the PMSG-treated hens, there was no change in the hormone-stimulable AC system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stage-Specific Effects of Androgens and Estradiol-17beta on the Development of Late Primary and Early Secondary Ovarian Follicles of Coho Salmon (Oncorhynchus kisutch) In Vitro

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.     

Steroidogenesis in Fundulus heteroclitus. IV. Dichotomous effects of a phorbol ester on ovarian steroid production and oocyte maturation.

The possible role of protein kinase C (PKC) activation in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) on ovarian steroidogenesis and oocyte maturation was investigated. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), alone slightly increased basal 17 alpha-hydroxy,20 beta-dihydroprogesterone (DHP) and 17 beta-estradiol (E2) synthesis and significantly stimulated germinal vesicle breakdown (GVBD). Addition of FPE promoted synthesis of DHP, testosterone (T), and E2, and initiated GVBD. Phorbol ester inhibited FPE-induced steroidogenesis but increased the number of oocytes that underwent GVBD. Phorbol ester also markedly impeded induction of steroidogenesis by dibutyryl cAMP and differentially affected the conversion of 25-hydroxycholesterol, pregnenolone, or progesterone to DHP, T, and E2: DHP production was not affected; T production diminished; and E2 synthesis increased (T aromatization also increased). These results suggest an inhibitory role for the PKC pathway on FPE-induced ovarian steroid production, with PMA appearing to affect various steroidogenic steps. The stimulatory action of PMA on oocyte maturation seems to be independent of follicular steroid production since aminoglutethimide, an inhibitor of steroidogenesis, did not block PMA-induced GVBD. Moreover, PMA had a marked stimulatory effect on GVBD in denuded oocytes. Thus, in contrast to the inhibitory role found for the PKC pathway on ovarian follicular steroidogenesis, activation of PKC in the oocyte may serve as a signal-transducing mechanism leading to GVBD.     

Dose-related effects of luteinizing hormone on the pattern of steroidogenesis and cyclic adenosine monophosphate release in superfused preovulatory rat follicles

The secretion of steroids and the release of cAMP in response to repeated luteinizing hormone (LH) stimulation were examined during superfusion of isolated preovulatory rat follicles. A high dose of ovine LH (1 microgram/ml for 20 min) caused a prolonged increase in the secretion of progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OHP) and a transient increase in the secretion of testosterone (T) and estradiol-17 beta (E2), and was accompanied by a peak of cAMP release. A single pulse of LH at a low dose level (10 mg/ml for 20 min) gave a limited increase in T secretion, but no clear change in P, 20 alpha-OHP and E2 secretion or cAMP release. When the follicles were challenged with a second pulse of LH (at 1 microgram/ml), the response varied according to the dose of LH delivered in the preceding pulse. Following exposure to the high dose of LH, the follicles were partially refractory to the second LH challenge in terms of cAMP and P and the secretion of T and E2 remained low. The low dose of LH, however, had a conditioning effect on the follicles since the response to the second LH challenge was amplified in terms of P, 20 alpha-OHP and cAMP. In this case a secondary increase in T and E2 secretion was found. The differential response to varying doses of LH are likely to reflect the physiological control of steroidogenesis during final follicular maturation.     

In vitro steroidogenesis by primary to antral follicles in the hamster during the periovulatory period: effects of follicle-stimulating hormone, luteinizing hormone, and prolactin

Hamster ovarian follicles at Stages 1 to 10 (Stages 1-4: follicles with 1-4 layers of granulosa cells (GC); Stages 5-7: 5-10 layers GC plus theca; Stages 8-10: antral follicles) were isolated on the morning of proestrus or estrus and incubated for 2 h in the absence or presence of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (Prl), progesterone (P4), 17 alpha-hydroxyprogesterone (17OHP), or androstenedione (A). Steroid accumulations in the media were measured by radioimmunoassay (RIA). On proestrus, without any hormonal stimulus, consistent accumulation of P4 through estradiol-17 beta (E2) occurred in low amounts only from Stage 6 and on; both FSH (5-25 ng) and LH (1-25 ng) significantly stimulated steroidogenesis by Stage 6-10 follicles, and the effects of FSH, except for Stage 10, were largely attributable to LH contamination. However, 25 ng FSH significantly stimulated A production by Stages 1-4, whereas 1-25 ng LH was ineffective. On estrus, follicles at all stages, especially 1-6, showed significant and dose-dependent increases in P4 production in response to FSH; both FSH and LH significantly stimulated P4 and 17OHP accumulation from Stage 5 onwards; however, there was no increase in A and E2 compared to controls. Even the smallest estrous follicles showed a shift to predominance of P4 accumulation. On proestrus, Prl had a negative influence on LH-induced accumulation of P4 and 17OHP by Stages 7-9 and 6-8, respectively, without affecting A or E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles

The objective of this study was to investigate the levels of expression of steroid biosynthetic enzymes and steroidogenic acute regulatory protein (StAR) at different stages of ovarian follicular development in zebrafish (Danio rerio), and to investigate the sites within the steroid biosynthetic pathway that may be regulated by gonadotropins. Ovarian follicles of sexually mature fish were separated into primary, previtellogenic, vitellogenic, and mature stages and the expression of StAR, P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 hydroxylase/lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), and P450 aromatase (P450aromA) was determined by Real time RT-PCR. The expression of all genes changed significantly as follicles grew, with a decrease in the expression of StAR, P450scc, 3beta-HSD and P450c17 with maturation, and an increase in the expression of 17beta-HSD3 during vitellogenesis and 17beta-HSD1 and P450aromA during previtellogenesis. In vitro incubation of vitellogenic follicles demonstrated that the expression of StAR, 17beta-HSD3, and P450aromA increased in response to hCG, and decreased in the absence of hCG. In contrast, the expression of P450scc, 3beta-HSD, P450c17, and 17beta-HSD1 remained constant between treatments and over time. Testosterone and estradiol production in the culture medium was stimulated by human chorionic gonadotropin (hCG). These experiments aid in the characterization of the roles and regulation of steroids throughout ovarian development, and suggest that gonadotropins play a key role in the regulation of StAR, 17beta-HSD3, and P450aromA in zebrafish.     

Endotoxin-induced changes in sex steroid hormone levels in male rats

Intravenous administration of Escherichia coli endotoxin (ENDO) was found to induce profound time and dose dependent changes in the serum steroid hormones, oestrone (E1), oestradiol (E2), corticosterone (B), progesterone (P4), 17 alpha-OH progesterone (17 alpha OHP4), and testosterone (T) of intact male rats. These changes were rapid, with a maximal response at 2 h and a return to close to normal values by 4 h. Non-lethal doses (0.01-2 mg/kg) of ENDO induced large increases in oestrogens (3-9-fold), P4 (4-fold) and B (2-3-fold) and decreased serum T (2-fold). The greatest increase in E2 level was seen with an ENDO dose of 2 mg/kg. Serum E1, E2 and T did not change in response to lethal ENDO doses (4-8 mg/kg); B, P4 and 17 alpha OHP4 levels alone were moderately elevated. Systemic mean arterial pressure was unchanged, except at the highest ENDO dose used. Thus, the hormonal responses are unlikely to be the result of hemodynamic changes. Low doses of ENDO did not produce an increase in serum E1 and E2 in adrenalectomized or orchidectomized rats. These results indicate that oestrogens are largely produced in the testis. The aromatization of the testicular and adrenal androgens can be stimulated by glucocorticoid.