Induction of lordosis behavior in female rats by intravenous administration of progestins

Progesterone is rapidly metabolized by neural cells in the rat. Progesterone could, therefore, act as a “prohormone,” stimulating lordosis behavior in estrogen-primed rats only after metabolic conversion. Were such the case, one might expect one or more of the naturally occurring metabolites of progesterone to be more potent than the parent compound. Estradiol benzoate-primed rats were therefore administered intravenously 200 μg of progesterone or one of five immediate metabolites of progesterone. The steroid 20α-dihydroprogesterone was found to be more potent than progesterone. Both 20α-hydroxy-5α-pregnan-3-one and 3α-hydroxy-5α-pregnan-20-one were less potent than progesterone, but more potent than the vehicle propylene glycol. Neither 5α-pregnane-3α, 20α-diol nor 5α-pregnane-3,20-dione (dihydroprogesterone, DHP) differed from the vehicle in potency. The data suggest that 20α-dihydroprogesterone, which is secreted at high levels during the estrous cycle, could play a role in the regulation of sexual receptivity. The data also suggest that 5α-reduction is probably not crucial for progesterone's action.     

Circulating levels of pregnanolone isomers during the third trimester of human pregnancy

This study addresses the question of whether changes in the biosynthesis and metabolism of neuroactive pregnanolone isomers (PIs) might participate in the timing of parturition in humans. The time profiles of unconjugated allopregnanolone (3-hydroxy-5-pregnan-20-one, P35), pregnanolone (3-hydroxy-5β-pregnan-20-one, P35β), isopregnanolone (3β-hydroxy-5-pregnan-20-one, P3β5) and epipregnanolone (3β-hydroxy-5β-pregnan-20-one, P3β5β), pregnenolone, their polar conjugates, progesterone, 5-dihydroprogesterone (P5), and 5β-dihydroprogesterone (P5β) were monitored in the plasma of 30 healthy women during the third trimester of pregnancy, at 1-week intervals from the 30th week of gestation using GC–MS. Changes in the steroid levels were evaluated by two-way ANOVA with gestational age and subject as independent factors. The mean concentrations of free PIs ranged from 2 to 50 nmol/L, while the mean levels of their polar conjugates were 40–100× higher. The ratio of 5-PIs to progesterone significantly but inconspicuously culminated in the 35th week. The decelerating biosynthesis of free 5β-PIs from the 31st week and their escalating sulfation was found from the 30th week. The changes were particularly evident in the second most abundant PI pregnanolone that may, like the allopregnanolone, sustain the pregnancy via attenuation of hypothalamic GABA_A-receptors and prevent uterine contractility via binding to nuclear pregnane X receptor.     

Cooperative erythropoietic assay of several steroid metabolites in polycythemic mice

A blinded cooperative assay of several androstane and pregnane steroid metabolites has been carried out in order to determine whether 5β-H derivatives are as active as testosterone in stimulating in vivo erythropoiesis. The steroids tested were: testosterone, 5-dihvdrotestosterone, 5β-dihydrotestosterone, 5β-pregnane-3,20-dione, 3-dihydroxy-5β-pregnàne-11,20-dione and 3β-hydroxy-5β-pregnan-20-one. The incorporation of radioactive iron into newly formed red cells in exhypoxic polycythemic mice was used to compare the effects of the steroids. Testosterone and 5-dihydrotestosterone both produced significant increases in ⁵⁹Fe incorporation. 5β-dihydrotestosterone, 5β-pregnane-3,20-dione, 3-hydroxy-5β-pregnane-11,20-dione and 3β-hydroxy-5β-pregnan-20-one were all devoid of significant erythropoietic activity in polycythemic mice in almost all instances. Thus, under the conditions chosen, this study failed to demonstrate that 5β-steroids increase radioactive iron incorporation in red cells of exhypoxic polycythemic mice.     

Adrenergic influence on progesterone metabolism and cyclicity in the rat ovary: autotransplantation and chemical sympathectomy

Elimination of adrenergic nerve endings by chemical sympathectomy with 6-hydroxydopamine of normally cycling rats produced no differences in the weights of body, uterus, ovaries or adrenals, but suppressed significantly proestrus/estrus stages. Unilateral fully denervated (autotransplanted) ovaries showed the following changes in [¹⁴C]progesterone metabolism: the formation of 20-hydroxy-4-pregnen-3-one increased, whereas 5-pregnane-3,20- and 3ß,20-diol, 3- and 3ß-hydroxy-5-pregnan-20-one, 20-hydroxy-5-pregnan-3-one, an unidentified metabolite Y and a group of hydrophobic metabolites decreased dramatically. Enzyme activities could not be restored with epinephrine. Sympathectomy changed the spectrum of [¹⁴C] progesterone metabolites in the same direction, but only at diestrus and metestrus. Autotransplantation suppressed 5-reductase, 3- and 3ß-hydroxysteroid dehydrogenase activities (-HSD) measured by the sum of all 5-, 3, and 3ß-metabolites, respectively. Sympathectomy suppressed significantly 5-reductase and 3-HSD at metestrus. 20-HSD was not changed in any experiment. These studies provide evidence that 5-reductase depends on adrenergic input in ovaries of rats at metestrus, a stage of nadir of gonadotropins. During the estrous cycle 5-reductase may be a regulatory enzyme for progesterone metabolism and also influence estradiol biosynthesis.     

Interaction of progesterone with all or isolated portions of the amphibian (Rana pipiens) oocyte surface : Physical and biological characteristics

Progesterone induces the resumption of meiotic maturation of fully grown oocytes of Rana pipiens both in vivo and in vitro. The nature of the interaction of progesterone with the oocyte was investigated using a technique which allowed the application of steroid to a portion of the oocyte surface. Uptake of [³H]progesterone from the incubation media with time and with varying concentrations of steroid was approximately proportional to the surface area exposed. After 1.5 or 24 hr of continuous exposure of a portion of the oocyte surface to [³H]progesterone, greater than 90% of the radioactivity was associated with the hemisphere exposed. Restriction of the portion of oocyte surface exposed reduced the biological potency of progesterone in the induction of maturation as assessed by germinal vesicle breakdown. Decrease in hormone effectiveness was not due to direct physical effects of the technique. Removal of the surface restriction resulted in an increase in biological activity of the steroid; this change in steroid potency was correlated with an increase in steroid distribution over the cell. Oocytes continuously exposed over a restricted part of their surface to high levels of progesterone (10 μg/ml) matured to a limited extent. After 24 hr of incubation, 55% of the oocytes exposed to 10 μg/ml of progesterone over the animal pole matured as compared to 0% of those oocytes exposed over the vegetal pole. Using [³H]progesterone, no difference was detected in the amount of steroid taken up or retained by the two polar regions. These investigations suggest that the amount of progesterone required to induce maturation is related to its distribution over the oocyte and that the animal and vegetal hemispheres differ in their ability to respond to progesterone.     

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.     

Metabolism of progesterone by Dioscorea deltoidea suspension cultures

Dioscorea deltoidea plant tissue suspension cultures are capable of metabolizing progesterone to 5α-pregnan-3-β-ol-20-one and 5α-pregnan-3β,20β-diol. The latter product has not previously been reported as a metabolic product of progesterone by plant systems. Both transformation products are present as conjugates in this plant tissue culture.     

The in vitro biosynthesis and metabolism of progesterone and estrogens by chimpanzee placental tissue

The biosynthesis and metabolism of progesterone and estrogens have been studied in chimpanzee placental tissue in vitro. The conversion of androstenedione-4-14C to estrone and estradiol-17β and of pregnenolone-7α-3H to progesterone has been demonstrated. In addition, the following metabolites were isolated following incubation of either pregnenolone-7α-3H or progesterone-4-¹⁴C: 20α-dihydroprogesterone, 20β-dihydroprogesterone, 6β-hydroxyprogesterone, 5α-pregnane-3,20 dione. The compound 5α-pregnan-3β o1-20-one was identified only after incubation with pregnenolone-7α-3H, while 5β-pregnane-3, 20 dione was identified only after incubation with progesterone-4-¹⁴C. No estrogens could be demonstrated following the incubation of placental preparations with either of the C₂₁ substrates.     

Involvement of steroid hormones in bovine oocytes maturation in vitro.

Influences of steroid hormone additions or of their binding by specific antisera on nuclear maturation and subsequent fertilization and cleavage of bovine oocytes were studied in vitro. It was found that progesterone in doses of 50 ng/ml, 250 ng/ml, 1 μg/ml or 5 μg/ml stimulates reinitiation and in doses of 1 or 5 μg/ml stimulates further development of meiosis. Antiserum to progesterone had opposite effects on nuclear maturation, but has no influence on the ability of matured oocytes to subsequent fertilization and cleavage. Testosterone additions (10 ng, 100 ng, 1 μg or 5 μg/ml) did not influence nuclear maturation, but antiserum to this hormone inhibited both meiosis reinitiation and completion, as well as lowered the rate of oocytes fertilized and embryos obtained. Estradiol (5, 50, 100 or 500 ng or 5 μg/ml) treatment stimulated reinitiation, but not nuclear maturation. Antiserum to estradiol activated both reinitiation, development and completion of meiosis, but the cells matured by estradiol deficit were as a rule uncapable of fertilization and further cleavage. Estradiol addition (1 μg/ml) to maturation medium together with FSH (10 μg/ml) (but not of FSH alone) lead to a significantly higher rate of fertilization and cleavage of matured cells.

Results obtained suggest (1) relative independence of reinitiation, further development of nuclear maturation and cytoplasmic maturation regulation in bovine oocytes as well as (2) the involvement of steroid hormones in these three processes.     

The effects of progesterone and its metabolites on the induction of sexual receptivity in rats

Estrogen-primed female rats were administered progesterone, 5 a-pregnan-3,20-dione, 5a-pregnan-3a-ol-20-one, 5a-pregnan-3β-ol-20-one, 4-pregnen-20a-ol-3-one, and 5a-pregnan-20a-ol-3-one in oil or oil alone and tested for the display of lordosis behavior 3 and 5 hr after progestin treatment. Progesterone was the most effective progestin in facilitating lordosis behavior. 5a-Pregnan- 3,20-dione and 5a-pregnan-3a-ol-20-one were partially effective, while the other progestins were no more effective than oil. The data suggest that the neuronal system which controls lordosis behavior is not entirely specific for progesterone.     

Steroid production in toads

In Bufo arenarum, androgen biosynthesis occurs through a complete 5-ene pathway, including 5-androstane-3β,17β-diol as the immediate precursor of testosterone. Besides, steroidogenesis changes during the breeding period, turning from androgens to C₂₁-steroids such as 5-pregnan-3,20-diol, 3-hydroxy-5-pregnan-20-one and 5-pregnan-3,20-dione. In B. arenarum, steroid hormones are not involved in hCG-induced spermiation, suggesting that the steroidogenic shift to C₂₁-steroids during the breeding be not related to spermiation. The activity of 17-hydroxylase-C_17–20 lyase (CypP450_c17) decreases during the reproductive season, suggesting that this enzyme would represent a key enzyme in the regulation of seasonal changes. However, the increase in the affinity for pregnenolone of 3β-hydroxysteroid dehydrogenase (3HSD)/isomerase could also be involved. Moreover, the reduction in CypP450_c17 leading to a reduction in C₁₉-steroids, among them dehydroepiandrosterone (DHE), would contribute to the conversion of pregnenolone into progesterone, avoiding the non-competitive inhibition exerted by DHE on this transformation. Additionally, CypP450_c17 possesses a higher affinity for pregnenolone than for progesterone, explaining the predominance of the 5-ene pathway for testosterone biosynthesis. Animals in reproductive condition showed a significant reduction in circulating androgens, enhancing the physiological relevance of all the in vitro results. The in vitro effects of mGnRH and hrFSH on testicular steroidogenesis revealed that both hormones inhibited CypP450_c17 activity. In summary, these results demonstrate that, in B. arenarum, the change in testicular steroidogenesis during the reproductive period could be partially due to an FSH and GnRH-induced decrease in CypP450_c17 activity.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Identification and quantification of unconjugated neutral steroids in adrenal and liver tissue of early and mid-term human fetuses

In order to study further the metabolism of neutral steroids in human fetal adrenal and liver tissue the fractions of unconjugated neutral steroids isolated from these tissues were analyzed by gas-liquid chromatography and gas chromatography — mass spectrometry. In the adrenals, pregnenolone and 17-hydroxypregnenolone, but no corticoids, were detected. In the liver, pregnenolone, 3α-hydroxy-5β-pregnan-20-one, 5β-pregnane-3α, 20α-diol and 3β, 16α-dihydroxy-5β-pregnan-20-one were found. Thus, all the free steroids detected were C₂₁ compounds. From these results and those obtained earlier by the analysis of the sulfate-conjugated steroids present in these tissues it is concluded that in the fetal adrenals $\text{in situ}$ both sulfated and unconjugated steroids are actively metabolized. Regarding the liver it is obvious that the conjugated metabolites of progesterone are rapidly eliminated from this tissue. Here, pregnenolone is present both in the free and sulfate conjugated form, whereas its metabolites are found only as sulfate conjugates.     

Progesterone synthesis,secretion and metabolism by human teratoma-derived cell-lines

The synthesis, secretion and metabolism of progesterone have been examined in six human teratoma-derived cell lines with the objective of determining if they exhibit trophoblast-related or other specific steroidogenic functions. Progesterone was synthesised in nanogram amounts (per 10⁶ cells/day) by the cell line SuSa, as measured by radioimmunoassay, and in lesser amounts by line LICR-LON HX-39. Lines Tera 1, Tera 2, T₃B₁ and PA-1 did not secrete detectable progesterone. All teratomas, however, metabolized added progesterone in microgram amounts (per 10⁶ cells/day). In all cases the major metabolite was a polar compound, identified by reversed phase HPLC, TLC and GC-MS as 3β, 6α-dihydroxy-5α-pregnan-20-one. This pattern of metabolism was not confined to the teratomas as equivalent amounts of this polar metabolite were formed by cultures of adult differentiated human epithelial and fibroblast cells. When progesterone and its metabolites, separated by HPLC, were included in the estimation, the Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity of SuSa was equivalent to 47ng pregnenolone (3β-hydroxy-5-pregnen-20-one) metabolised/mg protein/day, that of HX-39 to 9ng/mg protein/day and those of other teratomas to <3.5ng/mg protein/day.     

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.     

Energy Metabolism and Pyridine Nucleotide Levels during Xenopus Oocyte Maturation*, 1

The rate of oxygen consumption increased in maturing Xenopus oocytes within 2 hr after progesterone addition, well before GVBD. This suggested an early requirement for energy metabolism during maturation, similar to the situation in sea urchin eggs during fertilization. Yet, the absence of similar increases in glucose-6-phosphate levels, glucose-6-phosphate dehydrogenase activity, glucose conversion to CO₂, and the conversion of NAD(H) to NADP(H), indicated that carbohydrate metabolism was not being stimulated in Xenopus oocytes during maturation. The oxidation of other energy yielding substrates is discussed which might account for the finding that, within 5 min of progesterone addition, both reduced forms of the pyridine nucleotides increased 20% over control levels. This was later followed by a drop in NADH levels and a rise in NAD relative to controls. The significance of these changes in pyridine nucleotide levels and their relationship to a number of maturation events are discussed.     

Comparative facilitation and inhibition of lordosis in the guinea pig with progesterone, 5-alpha-pregnane-3,2-dione, or 3-alpha-hydroxy-5-alpha-pregnan-20-one

The major 5α-reduced metabolites of progesterone tentatively identified in neural tissue of the guinea pig were evaluated in this species for their ability to facilitate and inhibit lordosis responses of spayed females after estradiol benzoate (EB) pretreatment. 5α-Dihydroprogesterone was found to be an effective facilitative agent, but at doses of 0.05-0.3 mg administered at time intervals from 12–60 hr after estradiol, it was not as potent as progesterone. The steroids 3α-hydroxy-5α-pregnan-20-one and 5β-pregnane-3,20-dione, evaluated at only one dose level (0.18 mg) and at one time interval after estradiol (36 hr), were found to have moderate facilitative effects, but they were not as effective as 5α-dihydroprogesterone.The inhibitory influences of the metabolites studied were found to be weak relative to progesterone when given at doses of 0.6 mg 1 hr after EB. However, when 5α-dihydroprogesterone was given at a higher dose (3.6 mg) it was then found to be an effective inhibitor of the lordosis response. The results indicate that this metabolite has behavioral influences similar to those of progesterone for both facilitation and inhibition of estrus. It was suggested that the superior potency of injected progesterone may be due to mechanisms of bioavailability, including relative solubility differences of the two steroids when administered subcutaneously.     

Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP3-, are involved in oocyte maturation was investigated. Microinjection of IP3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP3 concentration of 1 microM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.     

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001–1 μg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01–10 μg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01–10 μg/ml), estradiol output by rabbit granulosa cells (at 1 μg/ml) and porcine ovarian follicles (at 10 μg/ml), as well as cAMP production by bovine (at 0.001–1 μg/ml) and rabbit (at 1 μg/ml) granulosa cells. No effects of genistein (at 10 μg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 μg/ml), as well as the preimplantation development of rabbit zygotes (at 1 μg/ml). Lavendustin A (0.001–1 μg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 μg/ml). Lavendustin (at 1 μg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 μg/ml). Inhibitory actions of lavendustin (at 10 μg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.