Expression of 3 beta-hydroxysteroid dehydrogenase in early bovine embryo development.

We analyzed the presence of 3 beta-Hydroxysteroid Dehydrogenase/Delta(5-->4)-isomerase enzyme (3 beta-HSD) activity, a key enzyme of the steroid metabolic pathway, the mRNA of this enzyme, and the steroid metabolism in in vitro produced bovine embryos. 3 beta-HSD activity was detected in in vitro matured oocytes (74.4 +/- 1.4%), 1-cell (72.9 +/- 6.1%), 2-cell (61.8 +/- 7.4%), 8-cell (50 +/- 5%), morulae (50.8 +/- 2.6%), blastocysts (94.4 +/- 3%), and hatched blastocysts (100 +/- 0%) meanwhile the 4-cell stage showed a significant reduction (16.7 +/- 4.7%). When total embryonic RNA of different stages was subjected to RT-PCR assays, the mRNA of 3 beta-HSD was found to be present in all developmental stages of in vitro produced bovine embryos, from the oocyte to the blastocyst, with a marked decrease at the 4-cell stage. To determine whether the temporal pattern of enzyme activity was dependent on the maternal to zygotic transition, embryos were incubated in the presence of a transcription inhibitor, alpha-amanitin. The reappearance of the enzyme activity after the 4-cell stage was blocked in alpha-amanitin treated embryos, indicating the requirement of embryonic transcription. On the other hand, the embryonic steroid metabolism was tested by incubating blastocyst with tritiated pregnenolone. Analysis of the metabolites by TLC indicated the production of a compound with a mobility identical to progesterone. These results described the expression of the 3 beta-HSD and the activity of this metabolic enzyme in bovine oocytes and preimplantation embryos, suggesting that steroids may act as autocrine effectors on preimplantation embryo development.

Inverse complementary homologues of short cysteine signatures.

Inversions of short genomic sequences may play a central role in the generation of protein complexity. We report here the existence of an heterogeneous group of proteins (the trefoil precursors MUC-1 and MUA-1, six preproendothelins, and five classes of zinc finger knot proteins) having both cysteine signatures (Cs) and their inverse complementary sequences (Cs) in the same polypeptide chain. We have also found cases in which the (Cs) of a given signature is not present in the same protein, but elsewhere. TGEKPYK, a cysteine-free motif of the human transcription factor, Krab, coexists with its inverse complementary sequence in 31 proteins; the inverse complementary alone is present in a great number of proteins. Our findings suggest that short DNA inversions are a widespread feature of the genome.

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis.

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-beta (TGF-beta), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of 3H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGF beta (2.5 ng/ml), at concentrations as low as 5 x 10(-11) M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED50 = 1.58 +/- 0.22 x 10(-10) M) and native GnRH (ED50 = 1.4 +/- 0.3 x 10(-6) M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10(-8) M could prevent the inhibition elicited by 10(-7) M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development.

Concerted stimulation of rat granulosa cell deoxyribonucleic acid synthesis by sex steroids and follicle-stimulating hormone.

Although follicle-stimulating hormone (FSH) and estrogens are known to be the main physiological stimuli for the development of the ovarian follicle in mammals, their growth-promoting activity has not been clearly established "in vitro". Furthermore, experimental evidence indicates that FSH and estradiol can independently inhibit granulosa cell proliferation. The present study was aimed at examining the effect of sex steroids in combination with FSH, on DNA synthesis in rat granulosa cells cultured in completely defined medium. Estradiol and FSH, when added separately, produced a significant inhibition of [3H] thymidine incorporation. In contrast, a combination of a low dose of FSH (20 ng/ml) with estradiol (100 ng/ml) produced a shift in the period of maximal DNA synthesis from 96 to 48 h after plating. Dose response studies showed that estradiol effects were produced at physiological intraovarian concentrations (1-100 ng/ml), whereas the effects of FSH were biphasic, with high doses (200 ng/ml) being inhibitory. A similar biphasic dose response curve was observed with increasing concentrations of a cAMP derivative in the presence of maximally effective doses of either an aromatizable steroid (androstenedione), insulin or insulin-like growth factor I. Non-aromatizable androgens (5alpha-dihydrotestosterone, 5alpha-androstane 3alpha-17beta diol and androsterone) showed a potency comparable to that of estradiol. The effect of 5alpha-dihydrotestosterone was completely blocked by a specific antiandrogen (hydroxy-flutamide), indicating that it was mediated by the androgen receptor. The effects of estradiol and androgens were not additive. The interaction between estradiol and FSH was further amplified in the presence of a maximally effective dose of insulin. Data presented herein indicate that both estrogens and androgens are able to elicit a mitogenic response in purified granulosa cells, cultured in a completely defined medium, provided the cells are stimulated by a physiological dose of FSH. These results suggest that, during follicular development, the stimulus for granulosa cell proliferation is given by the concerted action of steroid and peptide hormones acting through different signalling pathways.

Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm.

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M(r) > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M(r) 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human IgG. A polyclonal antibody raised against the purified protein and anti-human IgG were both able to suppress the acrosome reaction-inducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human IgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies.

Hormonal regulation of rat granulosa cell deoxyribonucleic acid synthesis: effects of estrogens.

Although it is widely accepted that estrogens exert a major trophic effect on follicular growth, their mechanism of action has not been established. We examined the effect of estrogen treatment in vivo or in vitro on DNA synthesis in rat granulosa cells cultured under defined conditions (DMEM:F12, collagen-coated plastic wells). Treatment with diethylstilbestrol (DES) in vivo (silastic implants containing 5 mg DES) for at least 2 days was required to observe a significant stimulation of 3H-thymidine incorporation by insulin (1 microgram/ml) in culture. Rat thecal/interstitial cells (TI) were isolated from DES-treated rats and cultured under the same conditions as granulosa cells. Conditioned media from TI cells stimulated DNA synthesis in granulosa cell cultures (as much as twofold). This effect was markedly amplified by estradiol treatment (1 microgram/ml) of the TI cell cultures. Addition of estradiol to granulosa cell cultures enhanced the effect of conditioned medium from nontreated TI cells. Conditioned medium from estradiol-treated TI cells stimulated DNA synthesis in granulosa cells from both DES-treated and nontreated rats. Estradiol had no effect when added directly to purified granulosa cell cultures but stimulated 3H-thymidine incorporation in crude preparations of ovarian cells. The stimulatory effects of TI cell-conditioned medium and insulin were reflected in the final cell densities achieved after 9 days in culture. We conclude that the mitogenic actions of estrogens in the ovary involve sensitization of granulosa cells to locally present mitogens like insulin and a TI cell-derived growth factor.

Insulin regulation of steroidogenic activity in rat culture luteal cells.

The effect of insulin on the function of rat luteal cells in monolayer culture was examined. Cells were obtained from PMSG-hCG primed immature rats and further cultured in serum free medium with or without insulin. The hormone produced an increase of progesterone production and maximal stimulation was achieved at 0.2 nM of insulin (100% stimulation). This effect was enhanced by addition of methyl-isobutyl-xantine (MIX 0.1 mM) to the culture medium. However, the stimulation produced by LH was not augmented by the presence of insulin. The conversion of progesterone into 20 alpha-hydroxy-progesterone was also enhanced after insulin treatment. Luteal cells were also cultured in the presence of 25-hydroxy-cholesterol (10 micrograms/ml). In these conditions insulin produced a 2-fold increase in progesterone production. Aromatase activity was assessed by adding androstenedione (0.25 microM) as substrate. Insulin produced a 14-fold stimulation of estradiol production after 24 h of culture. Insulin action was tested in short time incubations of luteal cells in a glucose free medium, in these experiments the hormone was able to induce a significant increase in progesterone and 20 alpha-hydroxy-progesterone production. These data suggest that luteal cell function is regulated by insulin and that this hormone has a direct effect on the steroidogenic process.