The relationship among ovarian condition, steroid hormones, and estrous behavior in Anolis carolinensis

The lizard Anolis carolinensis ovulates a single egg alternately from each ovary every 14 days. The ovulated egg enters the ipsilateral oviduct, acquires a shell, and is oviposited 18 or 19 days later. Thus, there is only a single egg in one of the oviducts (one-egg condition) during most of the estrous cycle, but there is a 4-5 day period when a mature, shelled egg is in one oviduct while a recently ovulated egg is in the contralateral oviduct (two-egg condition). Analysis of ovarian follicular diameters indicates that the largest follicle in a single ovary exhibits a growth spurt about halfway through its 28-day vitellogenic growth phase. The corpus luteum, once formed from the ovulated follicle, exhibits a linear decrease in weight, becoming fully regressed just before oviposition of the egg in the ipsilateral oviduct. Plasma concentrations of estradiol-17 beta and progesterone are high in two-egg animals. After oviposition, plasma concentrations of progesterone are still high in one-egg animals whereas those of estradiol-17 beta are much lower. These ovarian and hormonal changes during the estrous cycle are discussed in relation to the probable sources of estradiol-17 beta and progesterone, the control of the alternating pattern of ovulation and oviposition, and the role of steroid hormones in female sexual receptivity in this species.     

Identification of maturation-inducing steroid in a freshwater perch Anabas testudineus and differential responses of intact follicles and denuded oocytes to cyclic AMP in oocyte maturation

Postvitellogenic follicles of freshwater perch Anabas testudineus incubated with [(3)H]pregnenolone as exogenous precursor produced several metabolites, including 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and 5 beta-pregnane-3 alpha, 17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P). These were identified by chromatography, microchemical reactions, and crystallization to constant specific activity. Following stimulation with fish (perch) pituitary extract (FPE) there was significant high production of DHP and 5 beta-3 alpha,17 alpha,20 beta-P, concomitant with a high percentage of germinal vesicle breakdown (GVBD). Inhibitor of steroidogenesis (trilostane) and inhibitors of protein synthesis (cycloheximide and actinomycin-D) completely blocked FPE-induced pregnenolone metabolism and oocyte maturation. The effectiveness of various C(21) steroids in inducing GVBD was examined. Results indicate that DHP was the most potent inducer of GVBD than other structurally related C(21) steroids. In intact follicles, FPE-stimulated production of DHP was shown to be mediated through the adenylate cyclase-cAMP pathway. Addition of IBMX or forskolin, which increases the endogenous cAMP level, as well as directly supplementing dbcAMP to the incubation medium, had no inhibitory effect on DHP-induced GVBD in the intact follicles. But all these agents were shown to inhibit GVBD in fully denuded oocytes. This study provides evidence that DHP, produced by postvitellogenic follicles through the adenylate cyclase-cAMP pathway, is the maturation-inducing steroid in freshwater perch and that the role played by cAMP in the induction of GVBD in intact follicles is different from that in the denuded oocytes. J. Exp. Zool. 287:294-303, 2000.     

Evidence for Follicle Wall Involvement in Ovulation and Progesterone Production by Frog (Rana pipiens) Follicles in vitro

Involvement of different cellular investments of the amphibian ovarian follicle wall in the ovulatory process, progesterone production, and oocyte maturation was investigated. Following microdissection, to selectively remove one or more layers (surface epithelium, theca, follicle cells) of the follicle wall, dissected and undirected ovarian follicles were treated with frog pituitary homogenate (FPH) or progesterone. Intact follicles ovulated in response to pituitary homogenate and this was associated with contractions of the follicle wall. Ovulation and follicular contractions were not observed following removal of the surface epithelium without removing the thecal layer. Oocyte maturation occured in response to FPH following removal of the surface epithelium alone or together with the theca, but not in the absence of the follicle cells. Intact follicles were most responsive to FPH with respect to progesterone production, and removal of all somatic cells from oocytes obliterated FPH stimulated progesterone production. Oocytes, regardless of wether any or all follicular wall layers were removed, matured but did not ovulate following exposure to progesterone. The results suggest that the surface epithelium, but not the theca, is required for FPH-induced extrusion (ovulation) of the oocyte from ovarian follicle wall. Additionally, the somatic tissue rather than the oocyte appears to be the cells producing progesterone following FPH treatment. The results indicate that separate cellular layers (individually and/or as a result of interactions) of the follicle wall carry out different functions during follicular differentiation and mediation of ovulation. Data provide functional evidence for a role of the surface epithelium in controlling the process of ovulation and follicular contraction.     

Effects of high and low preovulatory concentrations of progesterone on ovulation from the isolated perfused rabbit ovary

Rabbit ovaries were isolated surgically before the ovulatory gonadotrophin stimulation and perfused in vitro. Untreated, control ovaries never ovulated. Ovaries treated in vitro with ovine LH ovulated 10-14 h later and the oocytes had undergone germinal vesicle breakdown (GVB). LH induced increases in progesterone secretion from the treated ovaries. A 3 beta-hydroxysteroid dehydrogenase blocker ('Compound A') effectively reduced progesterone secretion into the perfusate and follicular fluid to very low levels but had no effect on ovulation rate or on oocyte maturation. Excessively high progesterone levels were obtained artificially in perfusates by addition of exogenous steroid; the number of ovaries ovulating was markedly reduced but there was no effect on oocyte maturation. It is concluded that the rise in progesterone that normally occurs immediately after the LH surge is not a prerequisite for ovulation in the rabbit. However, progesterone may have a modifying effect on LH-induced follicle rupture when at a pharmacologically high level.     

Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

Background  

Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.     

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

Response of the 1-5 day-aged ovine corpus luteum to prostaglandin F2alpha

The hypothesis that, in the ewe, prostaglandin (PG) F2alpha administration on day 3 after ovulation is followed by luteolysis and ovulation was tested using 24 animals. The ewes were treated with a dose of a PGF2alpha analogue (delprostenate, 160 microg) on days 1 (n=8), 3 (n=8) or 5 (n=8) after ovulation, was established by transrectal ultrasonography. Daily scanning and blood sampling were performed to determine ovarian changes and progesterone serum concentrations by radioinmunoassay. The treatment induced a sharp decrease of progesterone concentrations followed by oestrus and ovulation in all ewes treated on days 3 and 5 and in one ewe treated on day 1 (8/8, 8/8, 1/8; P<0.05). Seven ewes treated on day 1 did not respond to PGF2alpha treatment and had an inter-ovulatory cycle of normal length (17.4 +/- 0.5 days). However, the profile of progesterone concentrations during the cycle of these ewes was delayed 1 day (P<0.05) compared with a control cycle. The overall interval between PGF2alpha and oestrus for the 17 responding ewes was 42.4 +/- 2.3 h. In 15 of these ewes the ovulatory follicle was originated from the first follicular wave and the ovulation occurred at 60.8 +/- 1.8 h after PGF2alpha treatment. The other two responding ewes ovulated an ovulatory follicle originated from the second follicular wave between 72 and 96 h after treatment. These results support the hypothesis and suggest that refractoriness to PGF2alpha of the recently formed corpus luteum (CL) may be restricted to the first 1-2 days post-ovulation.     

Regulation of steroid synthesis and metabolism in isolated binucleate cells of the placenta in sheep and goats.

Binucleate cells of sheep and goat fetal placentae comprise about one-fifth of the trophectodermal layer at the feto-maternal interface. When isolated and incubated in vitro they produce the steroids that are synthesized by the placenta in vivo (progesterone in sheep, 5 beta-pregnane-3 alpha,20 alpha diol in goats). This study demonstrates that progesterone synthesis in binucleate cell preparations in sheep was increased by prostaglandin (PG) E-2, nordihydroguaiaracetic acid (NDGA) and methylisobutylxanthine, but reduced by indomethacin, whereas in goats only NDGA produced any effect (an increase). None of the other compounds tested (luteinizing hormone, follicle stimulating hormone, prolactin, dibutyryl cAMP, A23187 or phorbolmyristic acetate) had any effect. Sheep binucleate cells also produced PGE-2 from arachidonic acid. These results suggest that, in sheep, products of both the cyclooxygenase (producing PGE-2) and lipoxygenase (inhibited by NDGA) pathways of arachidonic acid metabolism have regulatory roles in placental steroid synthesis, but only the lipoxygenase pathway is relevant in goats.     

Induction of maturation of Atlantic croaker oocytes by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one in vitro: consideration of some biological and experimental variables

We characterized the in vitro control of germinal vesicle breakdown (GVBD) by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) in intact ovarian follicles of gonadotropin-primed Atlantic croaker. 20 beta-S-induced GVBD was determined in relation to ovarian (oocyte) morphology, duration of incubation, steroid metabolism, and interaction with other steroids. The rate of GVBD in vitro in the absence of exogenous steroid was positively correlated with initial stage of ovarian morphological development. Maximal responsiveness to 20 beta-S was seen in ovaries with oocytes showing the first signs of morphological maturation. Dose-response experiments with 20 beta-S and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) over a range of incubation times yielded similar results for both steroids, suggesting that conversion of 17 alpha,20 beta-P to 20 beta-S is not required for 17 alpha,20 beta-P-induced GVBD. The ED50 of these steroids markedly decreased with increasing incubation times. Comparisons between patterns of follicular transformation of various radiolabelled steroids to 20 beta-S and their respective activities (using unlabelled steroids) in the GVBD bioassay suggested that, in addition to 17 alpha,20 beta-P, progesterone has some intrinsic maturational activity. However, the maturational effects of 11-deoxycortisol and pregnenolone may be explained by their conversion to 20 beta-S. For the first time in any vertebrate, we showed that the proposed maturation-inducing steroid (20 beta-S) is not significantly transformed to any extractable, potentially active metabolite by intact, maturing ovarian follicles. These findings strongly suggest that 20 beta-S is the terminal product of the MIS biosynthetic pathway in Atlantic croaker ovaries. Estradiol had no acute effects on 20 beta-S-induced GVBD. However, testosterone decreased and cortisol augmented the maturational activity of 20 beta-S. Excess progesterone reduced the activity of a maximally effective dose of 20 beta-S, but pregnenolone was without effect. The effects of these steroids on 20 beta-S-induced GVBD are discussed in relation to their possible interactions with 20 beta-S at the MIS receptor level.     

Ovarian function in Nelore (Bos taurus indicus) cows after post-ovulation hormonal treatments

Maternal recognition of pregnancy in the cow requires successful signaling by the conceptus to block luteolysis. Conceptus growth and function depend on an optimal uterine environment, regulated by luteal progesterone. The objective of this study was to test strategies to optimize luteal function, as well as prevent a dominant follicle from initiating luteolysis. Nelore (Bos taurus indicus) beef cows (n=40) were submitted to a GnRH/PGF(2alpha)/GnRH protocol. Cows that ovulated from a dominant ovarian follicle (ovulation=Day 0) were allocated to receive: no additional treatment (G(C); n=7); 3000IU of hCG on Day 5 (G(hCG); n=5); 5mg of estradiol-17beta on Day 12 (G(E2); n=6); or 3000IU of hCG on Day 5 and 5mg of estradiol-17beta on Day 12 (G(hCG/E2); n=5). Ultrasonographic imaging of the ovaries, assessment of plasma progesterone concentration, and detection of estrus were done daily from Day 5 to the day of subsequent ovulation. Treatment with hCG induced an accessory CL, increased CL volume, and plasma progesterone concentration throughout the luteal phase (P<0.01). Estradiol-17beta induced atresia and recruitment of a new wave of follicular growth; it eliminated a potentially estrogen-active, growing ovarian follicle within the critical period for maternal recognition of pregnancy, but it also hastened luteolysis (Days 16 or 17 vs. Days 18 or 19 in non-treated cows). In conclusion, the approaches tested enhanced luteal function (hCG) and altered ovarian follicular dynamics (estradiol-17beta), but were unable to extend the life-span of the CL in Nelore cows.     

Granulosa cell steroidogenesis before in vitro fertilization

Endocrine and gametogenic functions of the ovulatory follicle may be linked. To verify this, we studied granulosa cell steroidogenesis in relation to oocyte fertilization and preimplantation embryo development in vitro. Multiple follicles were stimulated in in vitro fertilization patients with clomiphene citrate and ovulation was induced with human chorionic gonadotropin (hCG). Oocytes were fertilized with husband's sperm and normal embryos were replaced 48 h later. Granulosa cells were separated from follicular fluid from 64 follicles and incubated for 3 h with and without aromatase substrate (1 microM testosterone). Progesterone and estradiol levels were measured in follicular fluid and incubation medium. Follicular fluid steroid levels and granulosa cell steroidogenesis showed no significant differences for oocytes which cleaved normally and those which did not. Granulosa cell aromatase activity was high in all follicles, suggesting that the low periovulatory follicular fluid estradiol level is not explained by a fall in granulosa cell aromatase after hCG. High granulosa cell progesterone production and follicular fluid progesterone were consistent with advanced granulosa cell luteinization. Oocytes undergoing polyspermic activation were from larger follicles with elevated follicular fluid progesterone levels, suggesting that follicular size and follicular fluid progesterone are correlated with "over-ripeness" and polyspermy. No simple relationship exists between oocyte function and the present indices of granulosa cell steroid metabolism.     

Efficacy of PGF(2alpha) to synchronize estrus in water buffalo cows (Bubalus bubalis) is dependent upon plasma progesterone concentration,corpus luteum size and ovarian follicular status before treatment

This study was conducted to identify factors affecting PGF(2alpha) efficacy to synchronize estrus in water buffalo cows. After detection of a corpus luteum (CL) by rectal palpation, cows were treated (im) with dinoprost (12.5, 25 or 50mg) or D(+) cloprostenol (75, 150 or 300 microg) in a total of 66 treatments. Blood samples were collected 0, 24 and 48 h after treatment and ultrasound examinations and observations for estrus were performed daily to the day of ovulation or to 6 days after treatment. No PGF(2alpha) dose-response pattern was observed and overall rates of luteal regression (progesterone <1.0 ng/ml at 48 h), estrus, no detected behavioral estrus with ovulation occurring, and ovulation were 71.2, 36.4, 19.7 and 54.5%, respectively. To analyze plasma progesterone concentrations and ovarian dynamics, cows were divided in three groups according to their response to treatment. Cows that failed to have ovulations from a follicle after treatment (Group A, n = 30) had (P < 0.05) a lower plasma progesterone concentration (2.98 ng/ml) and smaller CL area (CLA; 187.3 mm(2)) before treatment as compared with cows that had an ovulation from a follicle (4.43 ng/ml and 223.7 mm(2), respectively; Groups B and C, n = 36). In cows that failed to ovulate, plasma progesterone concentration decreased in the first 24 h, but did not decline further and was >1.0 ng/ml 48 h after treatment. Moreover, no significant change in CLA after treatment was detected, indicating that treatment induced only partial luteolysis. In cows that ovulated, plasma progesterone concentration and CLA decreased continuously from treatment to ovulation (consistent with complete luteolysis). Threshold values of 2.8 ng/ml for plasma progesterone concentration and 189 mm(2) for CLA were identified as the best predictors of ovulation before treatment (83.3 and 80.6% sensitivity and 58.6 and 65.5% specificity, respectively, with positive and negative predictive values around 71%). When the origin of the ovulatory follicle was investigated, the interval from treatment to ovulation was shorter (91.9 versus 113.3 h; P < 0.05), and the ovulatory follicle had a slower growth rate (1.02 versus 1.55 mm per day; P < 0.005), a lesser increase in diameter from treatment to ovulation (4.7 versus 8.0 mm; P < 0.001), and a greater maximum diameter (13.2 versus 12.1 mm; P < 0.05) in cows that ovulated from the largest follicle present in the ovary before treatment (Group B, n = 27) compared with cows that ovulated from the second largest follicle present in the ovary before treatment (Group C, n = 9). In summary, the efficacy of PGF(2alpha) for causing luteolysis and synchronizing estrus and ovulation in buffalo cows was dependent upon plasma progesterone concentration, CL size and ovarian follicular status before treatment.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Stages of oocyte development in the zebrafish,Brachydanio rerio

Oocyte development has been divided into five stages in the zebrafish Brachydanio rerio, based on morphological criteria and on physiological and biochemical events. In stage I (primary growth stage), oocytes reside in nests with other oocytes (Stage IA) and then within a definitive follicle (Stage IB), where they greatly increase in size. In stage II (cortical alveolus stage), oocytes are distinguished by the appearance of variably sized cortical alveoli and the vitelline envelope becomes prominent. In stage III (vitellogenesis), yolk proteins appear in oocytes and yolk bodies with crystalline yolk accrue during this major growth stage. Ooctes develop the capacity to respond in vitro to the steroid 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) by undergoing oocyte maturation. In stage IV (oocyte maturation), oocytes increase slightly in size, become translucent, and their yolk becomes non-crystalline as they undergo final meiotic maturation in vivo (and in response to DHP in vitro). In stage V (mature egg), eggs (approx. 0.75 mm) are ovulated into the ovarian lumen and are capable of fertilization. This staging series lays the foundation for future studies on the cellular processes occurring during oocyte development in zebrafish and should be useful for experimentation that requires an understanding of stage-specific events. © 1993 Wiley-Liss, Inc.     

Oocyte maturation in the amago salmon (Oncorhynchus rhodurus): in vitro effects of salmon gonadotropin, steroids, and cyanoketone (an inhibitor of 3 beta-hydroxy-delta 5-steroid dehydrogenase)

The effect of partially purified chinook salmon gonadotropin (SG-G100) and a number of steroids on the induction of germinal vesicle breakdown (GVBD) in amago salmon (Oncorhynchus rhodurus) oocytes (with intact follicle layers) was investigated in vitro. SG-G100 was effective only at the highest concentration tested (1 microgram/ml). 17 alpha,20 beta-Dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) was the most potent maturation-inducing steroid tested, followed by 17 alpha-hydroxyprogesterone. Testosterone or deoxycorticosterone (DOC) enhanced the rate of GVBD in response to SG-G100. DOC also enhanced the response to 17 alpha,20 beta-diOHprog but testosterone was without effect, suggesting that DOC has a direct action on the oocyte while testosterone probably acts at the level of the follicle. Estradiol-17 beta had no effect on GVBD in response to SG-G100 or 17 alpha,20 beta-diOHprog. The action of SG-G100 was shown to be dependent on the synthesis of a second delta 4 steroidal mediator of maturation since cyanoketone, a specific inhibitor of 3 beta-hydroxy-delta 5-steroid dehydrogenase, completely abolished the maturational effects of the gonadotropin and pregnenolone but not delta 4 steroids. Radioimmunoassay of media in which oocytes were induced to mature in vitro with SG-G100 revealed significantly elevated levels of progesterone and 17 alpha,20 beta-diOHprog. Estradiol-17 beta levels, high in control media, were only elevated twofold by SG-G100. Levels of the two progestogens were extremely low or nondetectable in media in which oocytes were incubated with cyanoketone, while estradiol-17 beta levels remained high. These results are discussed in relation to other evidence indicating that 17 alpha,20 beta-diOHprog is the naturally occurring maturation-inducing steroid of amago salmon. The role of other steroid hormones, particularly the possible involvement of corticosteroids, in the control of final oocyte maturation in teleosts is explored.     

Urinary analysis of 16(5alpha)-androsten-3alpha-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.     

Oxygen and steroid concentrations in preovulatory follicles of lactating dairy cows exposed to acute heat stress

Maternal heat stress reduces oocyte competence for fertilization and post-fertilization development, but the mechanism is unknown. The present experiment investigated two potential mechanisms: (1) reduced oxygen delivery to the preovulatory follicle (due to increased thermoregulatory vascular perfusion of skin and respiratory tract); (2) reduced follicular steroid synthesis. These hypotheses were tested by measuring the fractional concentration of oxygen and concentrations of estradiol-17beta and progesterone in follicular fluid of the preovulatory follicle of lactating Holstein cows. Estrous cycles were synchronized using GnRH on Day -9 and PGF(2alpha) on Day -2. On Day 0, all cows without a CL and with a large preovulatory follicle were assigned to control or heat stress treatments for 1d (beginning at 1030 h). Between 4 and 6 h after treatment (1430-1630 h), follicular fluid was aspirated by transvaginal puncture, and fractional oxygen concentration in follicular fluid of the dominant follicle was determined with a fluorometric fiber-optic oxygen sensor. There was no significant effect of heat stress on follicular fluid P(O2) or concentrations of estradiol-17beta or progesterone among cows that had follicular fluid steroid concentrations considered typical of a preovulatory follicle. Follicular oxygen concentration was 6.9+/-0.4% for control cows and 7.3+/-0.3% for heat-stressed cows. Oxygen concentration tended to be inversely correlated to follicular diameter (P=0.09). In conclusion, it was unlikely that reduced oocyte competence due to acute heat stress was caused by reductions in follicular concentrations of oxygen, estradiol-17beta, or progesterone.     

Changes in ovarian follicles and in vitro sex hormone release in the lizard Podarcis sicula sicula

An in vitro superfusion method was used to test sex hormone release from different kinds of ovarian follicle (growing follicles, postovulatory follicles, and atretic follicles) in the lizard Podarcis sicula sicula. Sex hormone output changes with the stage of follicle evolution and sexual cycle. Previtellogenetic follicles prevail in early-spring quiescent ovaries and secrete mainly progesterone, which is probably utilized at that phase to delay ovarian resumption. In the active ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17β-estradiol, which is necessary for sustaining vitellogenin synthesis by the liver and oviduct growth. As follicles become ripe, progesterone production is resumed, and it increases in young postovulatory follicles. This is in line with the functions assigned to the hormone at that phase of the sexual cycle, i.e., the induction of oocyte maturation and the regulation of egg retention in the oviduct. Postovulatory follicles can also synthetize 17β-estradiol. After oviposition, this hormone, which is secreted by the old postovulatory follicles, can reinitiate vitellogenin synthesis, allowing the development of a new oocyte set. Our data confirm that active, although ephemeral, corpora lutea are also formed in oviparous species. A limited contribution to ovarian sex steroid production derives also from atretic follicles, at least at the early stages of the breeding cycle. © 1993 Wiley-Liss, Inc.