Regulation Of Female Reproduction Research Articles

Ecdysteroid regulation of ovarian growth and oocyte maturation in the red flour beetle, Tribolium castaneum

Previous studies from our laboratory showed the involvement of juvenile hormone (JH) and ecdysteroid signaling in the regulation of female reproduction in the red flour beetle, Tribolium castaneum. JH regulates vitellogenin (Vg) synthesis in the fat body but the role of ecdysteroid signaling is not known. Here, we report on ecdysteroid regulation of ovarian growth and oocyte maturation. Microarray analysis of RNA isolated from ovaries showed the up-regulation of several genes coding for proteins involved in ecdysteroid signaling on the 4th day after female adult eclosion. The functional analyses of genes coding for proteins involved in ecdysteroid and JH signaling pathways by RNA interference (RNAi) revealed that ecdysteroids but not JH regulate ovarian growth and primary oocyte maturation. Ultrastructural studies showed the temporal sequences of key events in oogenesis including the development of primary oocytes, the differentiation and development of follicle epithelial cells, and the formation of intercellular spaces to facilitate uptake of Vg protein. RNAi studies showed that ecdysone receptor (EcR) and ultraspiracle (USP) are required for the ovarian growth, primary oocyte maturation and the growth and migration of the follicle cells. These studies suggest important roles for ecdysteroids in the regulation of oocyte maturation in the beetle ovaries.

The members of bHLH transcription factor superfamily are required for female reproduction in the red flour beetle, Tribolium castaneum

Proteins containing the basic Helix–Loop–Helix (bHLH) domain function as transcription factors and play important roles during the development of various metazoans including insects, nematodes and vertebrates. Insect genomes contain more than 50 bHLH transcription factors, but the function of only a few of these proteins in regulation of female reproduction is known. Using RNA interference, we have tested knock-down in the expression of genes coding for bHLH transcription factors in newly emerged adult females to determine their function in regulation of female reproduction in the red flour beetle, Tribolium castaneum. Knock-down in the expression of genes coding for four bHLH transcription factors (TcSRC, TcSim1, TcAsh and TcDaughterless) caused mortality in the female beetles. In addition, knocking-down the expression of 16 bHLH genes affected oogenesis and knock-down in the expression of 13 genes affected embryogenesis. Two genes TcSide1 and TcSpineless are required for both oogenesis and embryogenesis. Thus, the data reported here showed that 31 bHLH transcription factors are required for female survival, reproduction and embryogenesis.

249 THE DIRECT AND CUMULUS-MEDIATED EFFECTS OF PROLACTIN ON BOVINE OOCYTES

Prolactin (PRL) is an important regulator of female reproduction, as indicated using PRL receptor knockout mice (Bole-Feysot C et al. 1998 Endocrinol. Rev. 19, 225–268). The presence of PRL receptors or their mRNA has been shown in different follicular compartments of various mammalian species, including oocytes of sheep and mice (Picazo RA et al. 2004 Reproduction 128, 545–553; Kiapekou E et al. 2005 Reprod. Biomed. Online 10, 339–346). The aim of the present study was to characterize the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes. The expression of PRL receptor mRNA in follicular cells was detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. For oocytes, nested PCR was used. In addition, a total of 707 COC and 689 denuded oocytes (DO) from follicles 2 to 8 mm in diameter were cultured for 6 h (COC), 8 h (DO), or 24 h (COC and DO) in the presence or absence of 50 ng mL–1 bovine PRL (20 IU mg–1; Research Center for Endocrinology, Moscow, Russia). The following systems for COC and DO culture were used: (1) TCM-199 containing 10% fetal calf serum (system 1) and (2) DMEM containing 10 U mL–1 pregnant mare’s serum gonadotropin (PMSG), 5 U mL–1 hCG (Intervet Scandinavia, Copenhagen, Denmark), and 5% estrous cow serum (system 2). The nuclear status of oocytes was evaluated by Tarkowski’s cytogenetic method. After IVM in system 2, a portion of the oocytes (348 COC and 311 DO) underwent IVF and IVC, and the embryo development was tracked until the blastocyst stage. All treatments were repeated 4 to 7 times. Results were expressed as mean ± SEM. Arcsine-transformed data were analyzed by two-way ANOVA. Messenger RNA expression of long and short isoforms of PRL receptor was revealed in both bovine oocytes and cumulus cells. In system 1, PRL raised the rate of DO remaining at the diplotene stage by 8 h of culture (from 32.6 ± 3.0 to 47.5 ± 4.7%, P < 0.05), whereas there was no effect of PRL on the meiotic resumption in COC. By contrast, the hormone exerted a stimulatory action on the meiotic progression in the presence of cumulus cells by increasing the proportion of oocytes reaching the telophase I or metaphase II stages during 24 h of maturation (from 61.9 ± 1.2 to 73.5 ± 0.9%, P < 0.05). In system 2, PRL did not affect nuclear maturation of either cumulus-enclosed or cumulus-free oocytes, with the maturation rate varying between 93.9 ± 2.7 and 95.2 ± 2.4% (COC) and between 85.6 ± 3.0 and 83.0 ± 4.0% (DO). When added to IVM system 2, PRL raised the cleavage and blastocyst rates only in the case of COC (from 72.2 ± 2.4 to 80.2 ± 2.1%, P < 0.05, and from 20.5 ± 3.6 to 40.9 ± 4.2%, P < 0.01, respectively). These findings suggest functioning of the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes, with the hormone affecting meiosis via both pathways. We thank A. D. Roed for expert technical assistance with IVF and IVC. This research was supported in part by RFBR (project no. 07-04-01485). I.L. was the recipient of an Erasmus Mundus fellowship.

CDC25 protein expression and interaction with DAZL in human corpus luteum

To determine whether the human corpus luteum exhibits CDC25 protein expression and its expression pattern, and identify the interaction with DAZL (deleted in azoospermia-like) in human luteal cells. Experimental study. Medical research laboratory in a university hospital. Twelve human corpus luteum (CL), four in the early stage, four in the midstage, and four in the late stage of the luteal phase, respectively, and 10 granulosa-lutein cells from IVF patients. Immunohistochemical stain and Western blot were used to characterize the expression of CDC25 protein, and protein immunoprecipitation was used to identify the interaction of CDC25 with DAZL in human luteal cells. CDC25 protein expression pattern in different stages of luteal phase and interaction with DAZL. In this study, we show evidence that CDC25 protein is express in granulosa-lutein cells from different stages of CL, and identified only the CDC25A interaction with DAZL in luteal cells. The CL is the final form of a developing follicle and is the major endocrine component of the ovary in maintaining early successful pregnancy. Expression of CDC25 protein throughout different stages of the ovarian cycles implies important functional roles in the regulation of female reproduction.

Reduced fertility and inability of oocytes to resume meiosis in mice deficient of the Lxr genes

Cholesterol precursors act as activators of the nuclear hormone receptor, liver X receptor (LXR). One of these LXR-activating ligands is meiosis activating sterol (MAS), which also induces resumption of meiosis in oocytes from mice in vitro. Whether LXR participates in the regulation of oocyte maturation and whether the expression of either one of the two paralogues of LXR (α and β) affect fertility of mice has, however, not yet been clarified. Female mice lacking Lxra, Lxrb or both genes ( Lxra −/−, Lxrb −/− and Lxrab −/−, respectively) conceive less frequently and have significantly fewer pups per litter as compared to wild type mice. Both Lxra and Lxrb mRNA were found to be expressed in mouse oocytes. The relative expression of, in particular, Lxrb was almost two orders of magnitude higher than in liver, brain and testis. A water-soluble LXR agonist caused naked oocytes, but not cumulus enclosed oocytes (CEO), from wild type mice to resume meiosis significantly more often than control oocytes. Follicle stimulating hormone (FSH) is a potent stimulator of meiosis in CEO from wild type mice, but was without effect in mice lacking both Lxr genes. Zymosterol, a MAS active substance, induced resumption of meiosis in oocytes from Lxrab −/− mice, but significantly less effectively than in oocytes from wild type mice. Taken together, LXRs seem to affect ovarian function, suggesting specific roles of cholesterol precursors in regulation of female reproduction.

Estrogen Receptors and Estrogen-Metabolizing Enzymes in Human Ovaries during Fetal Development

Estrogen action plays a crucial role in many processes throughout the human life span, including development. Estrogens are pivotal in the regulation of female reproduction, but little is known about their role during ovarian development. To better understand estrogen action during ovarian development, the expression of estrogen receptors (ERs)-alpha and -beta and key enzymes regulating estradiol production, 17beta-hydroxysteroid dehydrogenases (17HSDs) types 1, 2, and 7, were analyzed in human fetal ovaries. The expression of ERs was related to the development of ovarian follicles. Before the 26th week of fetal life ERalpha was only occasionally detected, but from then onward, its expression was detected in ovarian follicles. Consistent expression of ERbeta was seen from the 20th week until term. Both ERalpha and ERbeta were localized to the granulosa cells and oocytes. Expression of 17HSD1 and 17HSD7 enzymes, catalyzing the conversion of estrone to more active estradiol, was detected as early as at the 17th week of fetal life. The expression of 17HSD1 displayed a pattern similar to that of ERs and increased toward term, whereas that of 17HSD7 decreased and was negative by the 36th week. 17HSD1 was localized to the granulosa cells, whereas 17HSD7 expression was more diffuse and was found in both granulosa and stromal cells. 17HSD2, converting estradiol to less potent estrone, was negative in all samples studied. The simultaneous appearance of estrogen-converting enzymes and ERs at the time of follicle formation indicates that the machinery for estrogen action exists in fetal ovaries and suggests a possible role for estrogens in the developing ovary.

Nonsteroidal Progesterone Receptor Modulators: Structure Activity Relationships

Progesterone, acting primarily via the progesterone receptor (PR), plays an essential role in the regulation of female reproduction. Steroidal progestins (i.e., PR agonists) are commonly used in women's health, such as in contraception and hormone therapy and for the treatment of gynecological disorders. Recent studies in women and in nonhuman primates also indicate that PR antagonists may have potential applications in contraception and for the treatment of reproductive disorders such as fibroids and endometriosis. Currently, all clinically available PR agonists and antagonists are steroidal compounds. They often cause various side effects due to their functional interactions with other steroid receptors or because of effects associated with their steroidal metabolites. In an effort to identify more receptor-selective and structurally diverse compounds that may render clinical advantages over steroidal PR ligands, numerous receptor-selective novel nonsteroidal PR agonists and antagonists have been discovered. This review focuses on the structure activity relationships and the biological profile of the nonsteroidal PR modulators discovered in the last decade.

Progesterone receptor mRNA levels during pregnancy, labor, lactation and the estrous cycle in rat uterus.

Progesterone plays important roles in the regulation of female reproduction. In this study, progesterone receptor (PR) mRNA levels in rat uterus during pregnancy, labor, lactation and the estrous cycle were examined by competitive RT-PCR. During pregnancy and lactation, PR mRNA levels had decreased on day 20 of pregnancy (P20) and P21 compared with P15 but increased during labor. After a decline on day 1 of lactation (L1), PR mRNA levels had increased again on L3 and L14 compared with P15, P18, P20, P21 and P21pm (at 2200-2300 h on P21). There was no significant change in the PR mRNA level during the estrous cycle. The PR mRNA level did not change during 1 week of progesterone treatment or afterwards. Injection of 17beta-estradiol did not affect PR mRNA levels in rats treated with progesterone or those without any injections. In rats on P18, 17beta-estradiol injection did not change PR mRNA levels after sham-operation but induced an increase in PR mRNA levels of rats ovariectomized 6 h before the treatment. These results suggest that uterine PR mRNA levels are differently regulated during late pregnancy, labor and lactation, and during labor estrogen is one of the essential factors for the increase in PR mRNA levels.

In search of a vitellogenesis-inhibiting hormone from the eyestalks of the South African spiny lobster, Jasus lalandii

Summary Investigations into the control of reproduction in crustaceans have mostly concentrated on the regulation of female reproduction, specifically, the process of vitellogenesis which involves the accumulation of yolk proteins within the oocytes. The lipoglycoprotein, vitellin (VT), is a chief component of the yolk. In the current study, an attempt was made to identify a vitellogenesis-inhibiting hormone (VIH) from the sinus glands (SGs) of the spiny lobster, Jasus lalandii, by utilising a heterologous in vitro assay in which the incorporation of radiolabeled amino acids into proteins of shrimp (Penaeus semisulcatus) ovaries was assessed. Crude extracts of SGs, as well as purified neuropeptides from J. lalandii, were tested for VIH activity. We found that the crude extracts from the spiny lobster elicit a biological effect in penaeid shrimp ovaries at a physiological dose. Not all the purified eyestalk peptides inhibited de novo protein synthesis in the shrimp ovaries. VIH activity was not associated with a unique peptide but was ascribed to peptides with a hyperglycaemic effect in the spiny lobster. Moreover, all the ovarian proteins were affected by the active peptides. These results suggest that the term gonad-inhibiting hormones (GIH) is more appropriate for describing the biological function of cHH-family peptides than the term VIH. Furthermore, C-terminally shortened hyperglycaemic peptides did not have a significant effect on the inhibition of protein synthesis, indicating that the C-terminal portion of the cHH peptides is important for eliciting a biological function.

Atrophy and degeneration of peptidergic neurons and cessation of egg laying in the aging pond snail Lymnaea stagnalis.

The morphology of the neuroendocrine caudodorsal cells (CDCs), which are involved in the regulation of female reproduction in the pond snail Lymnaea stagnalis, was studied in young (200 to 234 days of age) and old (400 to 500 days) animals. Lucifer Yellow fills of ventral CDCs showed that in young animals ventral CDCs branch ipsilaterally as well as contralaterally in the cerebral commissure. In old animals these branches were reduced at different degrees and in some cases even lacking completely, leaving only an axon crossing the commissure. Immunocytochemical stainings with antibodies against CDC peptides (CDCH-I and alpha CDCP) corroborated the finding that ventral CDCs degenerate. Among the other types of CDCs (dorsal, lateral), degeneration was found as well. The immunocytochemical findings showed that in old animals the axon terminals of the CDCs were strongly stained, indicating that they are packed with secretory vesicles containing peptides. It was also found that these darkly stained, peptide-containing axon terminals protruded into the perineurium. These findings suggest that accumulation of peptides in the terminals of the CDCs of old animals may be due to the impaired release. The relationship between atrophy and degeneration of CDCs and cessation of egg-laying activity in Lymnaea is discussed.

The Role of Steroid Modulation of Amino Acid Transmitters in the Regulation of Female Reproduction

The amino acid transmitters can be placed in two general categories, excitatory and inhibitory. This discussion focuses on the role of the inhibitory transmitter GABA and the excitatory amino acids aspartate and glutamate in the control of gonadotropin secretion and reproductive behavior. GABAergic neurotransmission in the preoptic area inhibits gonadotropin secretion via direct synaptic contact with LHRH neurons and possibly through presynaptic inhibition of noradrenergic fibers that stimulate LH release. In the arcuate-median eminence, GABA acting at GABA A receptors increases gonadotropin release by inhibiting a currently unidentified inhibitory interneuron

Inhibiting and stimulating neuropeptides controlling reproduction in Crustacea

Summary Studies on the control of reproduction in crustaceans have, for the most part, dealt with the regulation of female reproduction. However, not a great deal of attention has been paid to cellular interactions of the neurohormones involved in this regulation. In this contribution recent data on inhibitory and stimulatory neuropeptides are discussed and reflections are made on their importance for further application-directed research. Attention is paid to: histophysiological events in the oocytes during vitellogenesis; biochemical aspects of the regulating neuropeptides and cytological localization of these neuropeptides in the neuroendocrine system. Opportunities for further research are identified and appraised in light of the knowledge reviewed.