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.     

Kit ligand actions on ovarian stromal cells: effects on theca cell recruitment and steroid production

Factors that control recruitment of theca cells from ovarian stromal-interstitial cells are important for early follicle development in the ovary. During recruitment, theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. Recruitment of theca cells from ovarian stromal stem cells is proposed to involve cellular proliferation, as well as induction of theca cell-specific functional markers. Previously, the speculation was made that a granulosa cell-derived "theca cell organizer" is involved in theca cell recruitment. Granulosa cells have been shown to produce kit-ligand/stem cell factor (KL). KL is known to promote stem cell proliferation and differentiation in a number of tissues. Therefore, the hypothesis was tested in the current study that granulosa cell-derived KL may help recruit theca cells from undifferentiated stromal stem cells during early follicle development. The actions of KL were examined using adult bovine ovarian fragment organ culture and isolated ovarian stromal-interstitial cells. In organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal-interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production). Bovine ovarian stromal cells produced low levels of androstenedione (5-40 ng/microg DNA) and progesterone (5-30 ng/microg DNA) in vitro that were approximately 20-fold lower than theca cells under similar conditions. Treatment with KL did not affect ovarian stromal cell androstenedione or progesterone production. Interestingly, hormones such as estrogen and hCG did stimulate stromal cell steroid production. The results in this study suggest that granulosa cell-derived KL appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles. KL directly stimulated ovarian stromal cell proliferation but alone did not induce functional differentiation (i.e., high steroid production). Therefore, KL is proposed to promote early follicle development by inducing proliferation and organization of stromal stem cells around small follicles. Observations suggest that KL may act as a granulosa-derived "theca cell organizer" to promote stem cell recruitment of ovarian stromal cells in a manner similar to the way that KL promotes hematopoietic and lymphoid stem cells in bone marrow and the thymus.     

Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells

To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.     

Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes

We have previously demonstrated that activin is likely an ovarian mediator of pituitary gonadotropin(s) and local epidermal growth factor in their stimulating oocyte maturation and maturational competence in the zebrafish. However, the downstream events controlled by activin remain unknown. One possible mechanism is that activin may directly work on the oocytes to promote the development of oocyte maturational competence. To substantiate this hypothesis, we performed the present study to demonstrate the expression of the activin system in different compartments of zebrafish follicles, namely, the follicle cells and oocytes. The proteins examined include activin subunits (betaA and betaB), activin-binding protein (follistatin), activin type II receptors (type IIA and IIB), the type I activin receptor-like kinases (ALK1-like, ALK2-like, and ALK4-like), and the intracellular activin signaling molecules (Smad2, Smad3, Smad4, and Smad7). The results showed that the entire activin signaling system is expressed by the full-grown immature zebrafish oocytes ( approximately 0.65 mm in diameter), including ALK4-like (ActRIB), ALK2-like (ActRIA), ActRIIA, ActRIIB, Smad2, Smad3, Smad4, and Smad7, therefore supporting our hypothesis that the oocytes are one of the direct targets of activin actions in the zebrafish ovary. In contrast, activin itself (betaA and betaB) and ALK1-like type I receptor are predominantly expressed in the follicle cells surrounding the oocytes. Interestingly, although follistatin is expressed in both the follicle cells and oocytes, its level of expression is significantly higher in the oocytes than the follicle cells, implying that follistatin may serve as a signal from the oocytes to modulate the activity of activin produced by the follicle cells. Taken together, the present study provides convincing evidence that although all members of the activin system are expressed in the whole follicle, they exhibit distinct spatial patterns of expression among different compartments of the follicle. It is likely that activin works directly on the oocytes in a paracrine manner to promote oocyte maturation and maturational competence. On the other hand, instead of being controlled passively by the follicle cells, the oocytes may actively participate in the regulation of follicle development by releasing various modulating molecules such as follistatin.     

Environmental hazard in the aetiology of somatic and germ cell aneuploidy

Sources of environmental exposures to potentially aneugenic agents are many and include occupational and therapeutic exposures, and exposures associated with lifestyle habits. In this present study, some of these agents and exposure scenarios are discussed that involve potentially large population targets and/or seem to affect chromosome segregation by previously unsuspected mechanisms: metals, possibly acting by epigenetic mechanisms; nano-sized particles that might directly interact with subcellular components of the mitotic and meiotic machineries; cytostatic drugs in healthcare occupations; anticancer therapies potentially affecting the genetic integrity of gametes; continuously increasing electromagnetic field exposures with some sparse evidence of aneugenic activity; endocrine disruptors and their seemingly elusive effects in mouse oocytes, including the first evidence that prenatal exposure could affect meiotic nondisjunction in adult life. Hazards are considered for both somatic cells at risk of neoplastic transformation or tumour progression by chromosome loss and gain and germ cells at risk of heritable aneuploidies associated with spontaneous abortions or genetic diseases. Finally, possible synergistic interactions between environmental exposure and ageing or genetic predisposition are considered that could influence ultimate risks.     

Further evidence in support of a short-loop feedback action of estrogen on ovarian androgen production

We have demonstrated previously an ability of estrogen to inhibit ovarian androgen production. We report here further evidence in support of this intraovarian short-loop feedback mechanism. Thecal cells from ovarian follicles of estradiol-17β (E)-treated rats demonstrated an enhanced capability of producing progesterone in response to LH $\text{in vitro}$. In contrast, testosterone production by the same thecal preparations was markedly inhibited by pretreatment with E, suggesting a selective inhibitory action of E at the level of the androgen-producing cells in the ovarian follicle. In a somewhat contrasting experiment in hypophysectomized rats, while simultaneous administration of purified follicle-stimulating hormone (FSH) antagonized an inhibitory action of E on ovarian progesterone production, treatment of the hypophysectomized rats with either E alone or concomitantly with E plus FSH still attenuated ovarian testosterone production by these animals in response to acute LH stimulation. These results are consistent with a direct inhibitory action of estrogen at the level of the ovarian C_17α-hydroxylase /C_17,20-lyase enzyme system.     

The microfilament pattern in the somatic follicle cells of mid-vitellogenic ovarian follicles of Drosophila

The microfilament pattern in the somatic follicle cells of mid-vitellogenic stage 9 to 11 follicles of Drosophila was analyzed by staining F-actin with fluorescence-labeled phalloidin. During the analyzed stages of oogenesis, the follicular epithelium differentiates morphologically and functionally. These changes are also reflected at the organization of the microfilaments. At stage 10, they show no preferred orientation in the very thin follicle cells covering the nurse cells. In contrast, the microfilaments in the basal part of the columnar follicle cells covering the oocyte become organized in parallel bundles oriented perpendicular to the long axis of the follicle. During stages 10B/11 this organization is maintained at the nurse cell/oocyte border but becomes more sloppy towards the posterior pole of the follicle. The basal part of the follicle cells containing the microfilament bundles adheres so tightly to the basement membrane that this acellular layer cannot be separated mechanically from the epithelium. Indirect evidence from inhibition studies with cytochalasins and the effects of collagenase or pronase E added to the culture medium suggest that the microfilament bundles may promote increased adhesiveness of the follicle cells to the basement membrane. The possible functional implications of the microfilaments and their orientation are discussed.     

Expression of the regulation of cAMP metabolism in somatic cell hybrids

Normal rat liver cells (BRL-1) that respond to isoproterenol (beta+2), prostaglandin E1 (PGE+1) and adenosine (Ado+) with a rise in adenosine 3':5'-monophosphate (cAMP) content have been hybridized with rat hepatoma cells (H35) which do not respond to any of these agonists (beta-2, PGE-1 and Ado-). Both the initial hybrid line (BF5) and a subclone (BF5-1-1) expressed a beta+2, PGE+1, Ado- phenotype. However, full expression of the responsive phenotype in the BF5 line was apparent only if phosphodiesterase activity was blocked, for example, by methylisobutylxanthine (MIX). Direct measurements showed the rate of degradation of cAMP to be 7 times greater in intact BF5 cells than in the BRL-1 parent. In contrast to BF5 cells, the BF5-1-1 cells did not express maximal responsiveness to any of the agonists even in the presence of MIX. The differential accumulation of intracellular cAMP observed with BRL-1, BF5 and BF5-1-1 cells in response to isoproterenol was shown not to be as a result of differential rates of excretion of cAMP. Furthermore, no differences in the apparent affinities of the beta 2-catecholamine receptors for isoproterenol were observed. It is suggested that the increased degradative capacity of BF5 cells accounts for the difference in cAMP accumulation in these cells compared with the BRL-1 parent. The reduced responsiveness of BF5-1-1 cells, however, does not appear to be solely due to increased phosphodiesterase activity. It appears that the beta 2- phenotype may not always be dominant in hybrid crosses of this type as has been reported previously.     

Development of mouse and rat oocytes in chimeric reaggregated ovaries after interspecific exchange of somatic and germ cell components

The germ cell and somatic cell compartments of newborn rat and mouse ovaries, which contain only primordial stage follicles, were completely exchanged and reaggregated to produce xenogeneic chimeric ovaries. The reaggregated ovaries were grafted beneath the renal capsules of ovariectomized SCID mice to develop for periods up to 21 days. Xenogeneic follicles developed with essentially normal morphological characteristics. Both rat and mouse oocytes with species-specific characteristics grew within follicles that were composed of somatic cells exclusively of the alternative species. Rat oocytes grown in mouse follicles became competent to resume meiosis, and progressed to metaphase II when they were removed from follicles and cultured. In addition, mouse oocytes grown in rat follicles underwent fertilization and preimplantation development in vitro, and developed to term after embryos were transferred to pseudopregnant mouse foster mothers. Therefore, despite an estimated 11 million years of divergent evolution, oocytes and somatic cells of rat and mouse ovaries can be exchanged and can produce functional oocytes. It is concluded that factors involved in oocyte-somatic cell interactions necessary to support oocyte development and appropriate differentiation of the oocyte-associated granulosa cells are conserved between rats and mice. Moreover, although granulosa cells play important roles in oocyte development, the development of species-specific characteristics of oocytes occurs without apparent modification by a xenogeneic follicular environment.     

Exercise and ovarian steroid hormones: their effects on mitochondrial respiration

The effect of exercise on mitochondria respiration was studied in gastrocnemius muscle of ovariectomized rats, pseudopregnant rats, and estrous rats. The estrous cycles were followed by vaginal smears. Rats were made pseudopregnant (PSP) by 45 s cervical stimulation with a glass rod on the day of estrous. The treadmill protocol (21 m/min, 10 grade uphill) induced a significant decrease in state 3 oxygen consumption (oxidative phosphorylation) in estrous (0.26 +/- 0.02 vs. 0.49 +/- 0.05 microatoms O min(-1) mg protein(-1)) and ovariectomized rats (0.18 +/- 0.03 vs. 0.40 +/- 0.03 microatoms O min(-1) mg protein(-1)). In contrast, pseudopregnant and progesterone-treated ovariectomized rats did not decrease state 3 nor state 4 respiratory rates. These results show that the effect of exercise on mitochondria respiration does vary according to the hormonal status.     

In-vitro effects of angiotensin II on steroid production by hamster ovarian follicles and on ultrastructure of the theca interna

Summary Angiotensin II (AII) is present in the mammalian ovary and has been correlated with atresia in follicles. Since the theca interna may be one site at which atresia is intiated, we wished to determine whether AII exerts an effect on theca interna from explanted ovarian follicles of hamsters. Hamsters were sacrified on the morning of proestrus, and ovaries were removed. Preovulatory follicles were excised from the ovaries, and cultured with one of the following components: medium alone (control); medium plus AII (1x10^-6 M); the AII-receptor antagonist [Sar¹, Ile⁸] AII (1x10^-4 M); or AII plus antagonist. After 72 h, the follicles were processed for transmission electron microscopy (to determine quantities of theca interna organelles involved in the steroid synthetic pathway) or for protein determination (to normalize steroid production rates). The incubation medium was drawn off and analyzed by radioimmunoassay for progesterone, androstenedione, or estradiol-17. There was a significant positive correlation (r=0.92, P<0.01) between follicular androstenedione secretion and area comprising theca interna smooth endoplasmic reticulum. In the theca interna, AII induced a two-fold and 1.6-fold increase in lipid droplet number and area comprising smooth endoplasmic reticulum, respectively (P<0.05). Excess antagonist negated the increase in cell or-ganelles and also reduced androstenedione secretion compared with AII alone (P<0.05). Most importantly, AII significantly augmented the ratio of androstenedione: estradiol-17 secretion by 44% over that of control. The ultrastructural changes observed in this study and the increase in the andostenedione: estradiol-17 production ratio are consistent with atresia-like changes in ovarian follicles. We believe, therefore, that AII is involved, possibly at its membrane receptor, in an aspect of the overall process of follicular atresia, operating in part at the level of the theca interna.     

Pharmacological evidence for the involvement of the cAMP cascade in sensory fatigue in Drosophila

Summary We have investigated the effect of systemic treatment with drugs that affect the cAMP cascade on the sensory response and sensory fatigue in an identified mechanosensory neuron of Drosophila. Forskolin, an activator of adenylate cyclase, decreases the sensory response of the neuron. H7, an inhibitor of protein kinase, inhibits sensory fatigue. Octopaminergic ligands facilitate sensory fatigue. These results, together with our previous neurogenetic analysis of sensory fatigue in Drosophila (Corfas and Dudai 1990), corroborate the hypothesis that the cAMP cascade is involved in the generation and modulation of sensory fatigue.Abbreviations ANP antero-notopleural (neuron) - CDMF chlordimeform - ISI interstimulus interval     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Combined action of stem cell factor,leukemia inhibitory factor,and cAMP on in vitro proliferation of mouse primordial germ cells

In the present paper we investigated the effects of stem cell factor/mastocyte growth factor (SCF/MGF), leukemia inhibitory factor/differentiating inhibitory activity (LIF/DIA) (two growth factors known to affect primordial germ cell growth in vitro) and forskolin (FRSK) (an activator of adenylate cyclase in many cell types) alone or in combination on the survival and proliferation of primordial germ cells (PGCs) obtained from 8.5, 10.5, and 11.5 days post coitum (dpc) mouse embryos and cultured without pre-formed cell feeder layers. The results showed that both at 1 and 3 days of culture the addition of 100 ng/ml SCF, 20 μM FRSK, or in some instances 20 ng/ml LIF alone caused a significant increase of PGC number as compared with controls. The highest effects were obtained when SCF and/or LIF were used together with FRSK. Moreover, we found that FRSK elevated cAMP levels in purified 11.5 dpc PGCs and that this compound, but not SCF and LIF, stimulated PGC proliferation, as assessed by 5-bromo-2′-deoxyuridin (BrdU) incorporation. These results suggest a mechanism of combined action of cAMP with SCF and/or LIF in the control of proliferation of mouse PGCs in vitro. © 1993 Wiley-Liss, Inc.     

Photoperiodic variations of somatic and germ cell populations in the Soay ram testis

Seasonal changes in the Leydig, Sertoli and stem cells (undifferentiated A0 and cyclic A1) spermatogonia and the daily spermatid production were analysed in the testes of adult Soay rams exposed to short days (8L:16D) or long days (16L:8D) for 12 weeks. The total numbers of Leydig, Sertoli and stem cells (A0 + A1) were not affected by the treatments, but the size of the Leydig and the Sertoli cells, the efficiency of spermatogenesis (i.e. the number of male gametes produced by an A1 spermatogonium) and the daily sperm production were all significantly reduced in the rams exposed to long days. There was a positive correlation between the concentration of FSH and testosterone and many of the cytological changes consistent with a causal role for these hormones in mediating the effects of photoperiod on the testicular function in the ram.     

Mechanism for amine modulation of the neurogenic Limulus heart: evidence for involvement of cAMP

The role of cyclic nucleotides as intracellular second messengers mediating the excitatory chronotropic and inotropic actions of octopamine (OCT) and dopamine (DA) on the neurogenic Limulus heart was investigated. Tissue levels of cAMP, but not cGMP, were significantly increased in isolated cardiac ganglia and cardiac muscle following 10 min exposure to 10(-5) M OCT or 10(-5) M DA. In both tissues, OCT elicited larger increases in cAMP than did DA. Amine-induced cAMP accumulation in the cardiac ganglion and in the cardiac muscle was prevented by the alpha-adrenergic blocker phentolamine. The adenylate cyclase activator forskolin and the phosphodiesterase inhibitor IBMX produced amine-like chronotropic and inotropic effects when applied to the isolated heart preparation. However, the kinetics of the responses differed for the two agents. Additional pharmacological agents (RO-20-1724, papaverine, SQ 20,009, and 8-parachloro-phenylthio cAMP) also had amine-like effects but to a lesser extent. The chronotropic, but not inotropic, effects of OCT and DA were potentiated in the presence of IBMX. These data suggest that a cAMP-dependent mechanism underlies the excitatory effects of the neuromodulators OCT and DA on the Limulus heart.     

The effects of forskolin and rolipram on cAMP,cGMP and free fatty acid levels in diet induced obesity

Obesity is a major health problem. We investigated the effects of forskolin and rolipram in the diet of animals in which obesity had been induced. We used 50 female albino Wistar rats that were assigned randomly into five groups as follows: group 1, control; group 2, high fat diet; group 3, high fat diet + forskolin; group 4, high fat diet + rolipram; and group 5, high fat diet + rolipram + forskolin. The rats were fed for 10 weeks and rolipram and forskolin were administered during last two weeks. The animals were sacrificed and blood samples were obtained. Serum cAMP, cGMP and free fatty acids (FFA) levels were measured using ELISA assays. We also measured weight gain during the 10 week period. cAMP and FFA levels of groups 3, 4 and 5 were significantly higher than those of groups 1 and 2. We found no significant differences in serum cGMP levels among the groups. The weight gain in groups 3, 4 and 5 was significantly less than for group 2. We also found that the weight gain in group 5 was significantly less than in groups 3 and 4. We found that both forskolin and rolipram stimulated lipolysis and inhibited body weight increase by increasing cAMP levels. Also, combination therapy using the two agents may be more effective in preventing diet induced obesity than either agent alone. We found also that these agents did not effect cellular cGMP levels in diet induced obesity.