A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation

Ovulation is essential for the propagation of the species and involves a proteolytic degradation of the follicle wall for the release of the fertilizable oocyte. However, the precise mechanisms for regulating these proteolytic events are largely unknown. Work from our lab and others have shown that there are several parallels between Drosophila and mammalian ovulation at both the cellular and molecular levels. During ovulation in Drosophila, posterior follicle cells surrounding a mature oocyte are selectively degraded and the residual follicle cells remain in the ovary to form a corpus luteum after follicle rupture. Like in mammals, this rupturing process also depends on matrix metalloproteinase 2 (Mmp2) activity localized at the posterior end of mature follicles, where oocytes exit. In the present study, we show that Mmp2 activity is regulated by the octopaminergic signaling in mature follicle cells. Exogenous octopamine (OA; equivalent to norepinephrine, NE) is sufficient to induce follicle rupture when isolated mature follicles are cultured ex vivo, in the absence of the oviduct or ovarian muscle sheath. Knocking down the alpha-like adrenergic receptor Oamb (Octoampine receptor in mushroom bodies) in mature follicle cells prevents OA-induced follicle rupture ex vivo and ovulation in vivo. We also show that follicular OA-Oamb signaling induces Mmp2 enzymatic activation but not Mmp2 protein expression, likely via intracellular Ca²⁺ as the second messenger. Our work develops a novel ex vivo follicle rupture assay and demonstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in Drosophila, which is likely conserved in other species.     

Non-steroidal anti-inflammatory drugs (NSAIDs) and ovulation: lessons from morphology

Ovulation constitutes the central event in ovarian physiology, and ovulatory disfunction is a relevant cause of female infertility. Non-steroidal anti-inflammatory drugs (NSAIDs), widely used due to their analgesic and anti-inflammatory properties, consistently inhibit ovulation in all mammalian species investigated so far, likely due to the inhibition of cyclooxygenase 2 (COX-2), the inducible isoform of COX, that is the rate-limiting enzyme in prostaglandin (PG) synthesis. COX-2 inhibition has major effects on ovulation, fertilization and implantation, and NSAID therapy is likely implicated in human infertility and could be an important, frequently overlooked, cause of ovulatory disfunction in women. Although there is compelling evidence for a role of PGs in ovulation, the molecular targets and the precise role of these compounds in the ovulatory process are not fully understood. Morphological studies from rats treated with indomethacin (INDO), a potent inhibitor of PG synthesis, provide evidence on the actions of NSAIDs in ovulation, as well as on the possible roles of PGs in the ovulatory process. Cycling rats treated with INDO during the preovulatory period show abnormal ovulation, due to disruption of the spatial targeting of follicle rupture at the apex. Noticeably, gonadotropin-primed immature rats (widely used as a model for the study of ovulation) show age-dependent ovulatory defects similar to those of cycling rats treated with INDO. These data suggest that NSAID treatment disrupts physiological mechanisms underlying spatial targeting of follicle rupture at the apex, which are not fully established in very young rats. We summarize herein the ovulatory defects after pharmacologic COX-2 inhibition, and discuss the possible mechanisms underlying the anti-ovulatory actions of NSAIDs.     

Evidence for a role of the ovarian surface epithelium in the ovulatory mechanism of the sheep: secretion of urokinase-type plasminogen activator

The extent of dissolution of tissues within the apical wall of the preovulatory ovine follicle (formative site of rupture) is greater than that of the counterpart basal hemisphere. It has been hypothesized that proteolytic enzymes released from contiguous ovarian surface epithelial cells contribute to apical follicular weakening and ovulation. Ovulation occurs from the dominant ovarian follicle of proestrous ewes at approximately 24 h after administration of luteinizing hormone-releasing hormone (LHRH). Follicular rupture was inhibited in sheep in which the ovarian surface epithelium was surgically removed at 8 (but not at 16) h following LHRH. Plasminogen activator bioactivity was greater within the follicular apex compared to basal wall at 12 h; this difference was negated by prior removal of epithelium at 8 h after LHRH. A low M_r plasminogen activator of the urokinase-type (uPA) was secreted by epithelial cells recovered from the surface of preovulatory follicles (Western blot analysis). Ovarian epithelium, not associated with a preovulatory follicle, produced very little uPA. Finally, ovulation was suppressed by intrafollicular injection (8 h post-LHRH) of uPA antibodies. It is suggested that secretion of uPA by ovarian surface epithelium and consequent plasmin up-regulation within neighboring tunica albuginea and follicular theca is a contributing factor in the mechanism of ovulation.     

Matrix Metalloproteinase 2 Is Required for Ovulation and Corpus Luteum Formation in Drosophila

Ovulation is critical for successful reproduction and correlates with ovarian cancer risk, yet genetic studies of ovulation have been limited. It has long been thought that the mechanism controlling ovulation is highly divergent due to speciation and fast evolution. Using genetic tools available in Drosophila, we now report that ovulation in Drosophila strongly resembles mammalian ovulation at both the cellular and molecular levels. Just one of up to 32 mature follicles per ovary pair loses posterior follicle cells (“trimming”) and protrudes into the oviduct, showing that a selection process prefigures ovulation. Follicle cells that remain after egg release form a “corpus luteum (CL)” at the end of the ovariole, develop yellowish pigmentation, and express genes encoding steroid hormone biosynthetic enzymes that are required for full fertility. Finally, matrix metalloproteinase 2 (Mmp2), a type of protease thought to facilitate mammalian ovulation, is expressed in mature follicle and CL cells. Mmp2 activity is genetically required for trimming, ovulation and CL formation. Our studies provide new insights into the regulation of Drosophila ovulation and establish Drosophila as a model for genetically investigating ovulation in diverse organisms, including mammals.     

Generation and sequence characterization of a normalized cDNA library from swine ovarian follicles

Ovulation rate is a major factor determining litter size in swine and is, therefore, a trait of economic importance to the pork industry. The dynamics of follicle development, which in turn are dictated by a balance between follicle recruitment, maturation, selection, and atresia, are a major determining factor of ovulation rate. The role of several genes expressed in the ovaries during these processes has been described, but studies utilizing large-scale genomic approaches have yet to be conducted to examine gene expression in this tissue more globally. We have developed a normalized cDNA library from swine ovarian follicles in various stages of development, ranging from 2.0 to 10.0 mm in diameter, collected from gilts from divergent genetic lines selected for high and low ovulation rates, during the 7 initial days of the follicular phase of the estrous cycle. EST sequences were obtained from 5231 distinct clones derived from this library. In total, 3479 unique sequence clusters were obtained, of which 2661 singletons (76.5%) were observed. BLASTN searches with the primary sequences from the clusters obtained resulted in 1037 sequences not matching (E <1.0(-06) any of the sequences in the nt database (29.8% novelty rate). This resource will facilitate the use of cDNA microarrays in functional genomics studies aiming at unraveling the genetic and physiological mechanisms underlying follicle maturation and ovulation rate in swine.     

Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation

Profound hormonally controlled tissue remodelling occurs in the equine ovary for follicle growth and development, and also for the alteration in follicle shape directed towards the ovulation fossa, the site where ovulation occurs. The aim of this study was to examine the spatial and temporal regulation of matrix metalloproteinases (MMP)-2 and MMP-9, important enzymes in tissue remodelling, during follicle growth, and ovulation. Using gelatin substrate zymography, we measured these MMPs in follicular fluid of large anovulatory follicles collected during spring transition, early dominant follicles (> 23 mm), and at oestrus in follicles approximately 3 days prior to ovulation, and post-hCG treatment when ovulation was predicted in approximately 4 h. The most abundant activity detected in follicular fluid was MMP-2, although there were no changes in secretion or activation in association with ovulation. The activity of MMP-9 was detected in lower amounts, with no changes prior to ovulation, although it decreased significantly (P < 0.05) post-hCG treatment. At oestrus, when different regions of the ovary were maintained in explant culture for 24 h, there were no significant changes in either MMP-2 or MMP-9 secretion by stromal tissues collected at the ovarian fossa, adjacent to the preovulatory follicle but away from the fossa, and a further site remote from the preovulatory follicle. Over this same time period, follicular progesterone (P < 0.01) and oestradiol (P < 0.05) increased significantly, although oestradiol tended to decrease after hCG administration. These findings indicate that MMP-2 and MMP-9 are not key acute regulators for the changes in follicle shape immediately prior to ovulation.     

Interrelationships between prostaglandins, cyclic AMP and steroids in ovulation.

Ovulation is a complex phenomenon, involving a series of biochemical events within the ovary, leading to the rupture of the follicle. This paper summarizes recent studies in our laboratory of some of these biochemical changes using the rabbit as an experimental model. It has been shown in our laboratory that isolated Graafian follicles obtained from oestrous rabbits synthesize steroids and cyclic AMP when incubated in vitro. Luteinizing hormone added to the incubation medium increased steroidogenesis and cyclic AMP synthesis many fold. When follicles were isolated from rabbits at different times following the ovulatory stimulus (mating or HCG injection) it was found that the in vitro response to LH in terms of steroidogenesis and cylcic AMP synthesis was lost as ovulation approached. In contrast, when prostaglandins (PGF and PGE) were measured in rabbit Graafian follicles it was found that the PGF and PGE levels increased as ovulation approached. From these data and from reports in the literature, we have developed a hypothetical model for ovulation in the rabbit which may help in a better understanding of the ovulatory process.     

Analytical and experimental studies on the relationship between Na+, K+, and water uptake during volume increases associated with Fundulus oocyte maturation in vitro

Summary Oocytes of marine and estuarine teleosts often undergo pronounced volume increases during the maturation phase of development that precedes ovulation and fertilization. To examine the physiological correlates of these volume increases, prematuration follicles of the saltmarsh teleost, Fundulus heteroclitus, were cultured in vitro with a maturation-inducing steroid (17-hydroxy-20-dihydroprogesterone). Mean follicle volume rose significantly (75%) during a 40-h incubation period. Similar to the situation previously found in vivo, uptake of water by the maturing follicle was responsible for this volume increase in vitro, with the water content increasing from 62% to 78% of the total follicle mass. The follicle contents of two probable osmotic effectors-Na⁺ and K⁺-also rose, the increase in K⁺ being twice that of Na⁺. The influx of K⁺ even exceeded water uptake, resulting in a net increase in the concentration of this cation. It thus appears that the influx of these cations, in particular K⁺, is a major cause of the uptake of osmotically obligated water and subsequent volume increase experienced by maturing F. heteroclitus follicles. In a search for operant mechanisms, it was found that follicle hydration, but not maturation, was strictly dependent on external K⁺ in a concentration-dependent manner. The mechanism by which K⁺ accumulates in the follicle was insensitive to ouabain, so that a typical Na⁺, K⁺-ATPase mechanism does not appear to be involved. The ability of external K⁺ to promote follicle hydration was gradually lost during the maturation process as the oocyte dissociated from the surrounding granulosa cells in preparation for ovulation. Removal of all associated somatic cells prior to maturation prevented subsequent steroid-initiated hydration but not maturation. The results suggest that K⁺ may be translocated from surrounding granulosa cells to the oocyte via gap junctions during maturation.Abbreviations GVBD germinal vesicle breakdown     

Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.     

Effects of Cycloheximide,Aminoglutethimide, Cytochalasin B,and Colchicine on Ovulation and the Ultrastructure of the Ovarian Follicle Wall in the Stellate Sturgeon Acipenser stellatus Pall

The inhibitor of protein synthesis cycloheximide, inhibitor of steroidogenesis aminoglutethimide, and inhibitor of prostaglandin synthesis indomethacin, as well as the drugs affecting the cell cytoskeleton, such as cytochalasin B and colchicine, were used for studying the mechanisms of ovulation in the stellate sturgeon Acipenser stellatus Pall. Follicles were isolated from the body cavity within certain time intervals after the injection of pituitary suspension to a female and cultivated in media with the inhibitors. In the case of follicles isolated in the middle of the period from hormonal injection until ovulation, cycloheximide, cytochalasin B, and aminoglutethimide suppressed ovulation most effectively, while in the case of oocytes isolated during the last quarter of this period, aminoglutethimide and cytochalasin B were the most effective. It was shown using TEM and SEM that cycloheximide suppressed all processes related to the preparation for ovulation, except the initial ones: contraction of follicle cells and their processes and secondary flattening of these cells. In the presence of aminoglutethimide, the follicle cells underwent pathological changes. Incubation in the media containing indomethacin and colchicine prevented degradation of the outer theca layer at the follicle apex. In the presence of cytochalasin B affecting the cytoskeleton, the drawing of follicle cell processes from the jelly coat channels was blocked, the outer theca cells were strongly contracted, but the cell layer integrity was affected and it was divided in separate fragments. A relationship is discussed between the metabolic processes and morphological changes that lead to ovulation. It was proposed on the basis of the present and previous data that the preovulatory preparation of the follicle tissues comprises two contractile and two apoptotic processes distinctly coordinated in time and space.     

Apparent involvement of plasmin in early-stage follicle rupture during ovulation in medaka

Until recently, the role of the proteolytic system involving serine proteases in follicle rupture during ovulation in mammalian species has been a subject of controversy. We undertook the present study to examine whether proteases play a role in follicle rupture using the teleost medaka (Oryzias latipes) model. Various serine protease inhibitors, including a specific plasmin inhibitor, drastically reduced the rate of ovulation, as assessed by an in vitro ovulation assay, which was established for the fish. Biochemical, molecular biological, and immunological analyses demonstrated that plasminogen/plasmin was present in large follicles destined to ovulate. The active protease, plasmin, was detected in follicles approximately 3-7 h before the expected time of ovulation. Specific antibodies against the medaka plasmin light chain suppressed the ovulation rate of the follicles when antibodies were added to the medium during the period in which active plasmin was generated. This finding was an indication that a plasmin-like protease similar if not identical to plasmin plays a role in follicle rupture during ovulation in the medaka. Our data also indicate that this serine protease participates in the rupture for only a few hours prior to the activation of matrix metalloproteinase (Mmp)-mediated hydrolysis at ovulation. Based on our previous and current data, we propose a follicle rupture model involving two different proteolytic enzyme systems, serine protease and Mmp, in medaka ovulation. The current study is the first to provide evidence of the indispensable role of plasmin or a plasmin-like protease in the ovulation of a nonmammalian vertebrate species.     

Induced expression of pattern recognition receptors in cumulus oocyte complexes: novel evidence for innate immune-like functions during ovulation

Ovulation is the complex, inflammatory-like process by which the cumulus oocyte complex (COC) is released from a mature, preovulatory follicle through a rupture site at the ovarian surface and requires expression of genes that generate and stabilize the expanded extracellular COC matrix. Gene profiling analyses of COCs at selected time intervals during ovulation revealed that many genes associated with immune related surveillance functions were also induced in cumulus cells. Specifically, cell surface signaling molecules known as pattern recognition receptors that act as sensors of the external environment important for the innate immune system to detect self from nonself or altered self are induced and/or expressed in cumulus cells as well as granulosa cells. These include the complement factor q1, CD14, and the Toll-like receptors (TLRs) 4, 8, and 9 as well as mediators of TLR activation, myeloid differentiation primary response gene 88 and interferon regulatory factor 3. COCs exposed to bacterial lipopolysaccharide exhibit enhanced phosphorylation of p38MAPK, ERK1/2 and nuclear factor-kappaB and increased expression of Il6 and Tnfa target genes, documenting that the TLR pathway is functional. Cumulus cells and granulosa cells also express the scavenger receptors CD36 and scavenger receptor type B1 and exhibited phagocytic uptake of fluorescently tagged bacterial particles. Collectively, these results provide novel evidence that cumulus cells as well as granulosa cells express innate immune related genes that may play critical roles in surveillance and cell survival during the ovulation process.     

Delivery of Polynucleotides with Polyamine Lipids and Polymers

Abstract

Several non-permanent polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyethylenimines, are efficient transfection agents per se, i.e. without the addition of lysosomotropic bases, or cell targeting or membrane disruption agents. These vectors have been shown to deliver genes as well as oligonucleotides both in vitro and in vivo. Our hypothesis is that their efficiency relies on extensive endosome swelling and rupture that provides an escape mechanism for the polycation/DNA particles.     

Prostaglandin E(1) inhibits abnormal follicle rupture and restores ovulation in indomethacin-treated rats

In the presence of indomethacin, an inhibitor of prostaglandin (PG) synthesis, the gonadotropin surge induces abnormal follicle rupture at the basolateral follicle sides, thus preventing effective ovulation in rats. This study was undertaken to analyze whether exogenous prostaglandin administration can overcome the antiovulatory action of indomethacin. Cycling rats were treated with vehicle (olive oil) or indomethacin (1 mg/rat) on the morning of proestrus. Rats treated with indomethacin were injected with different doses (50, 250, or 500 micro g/rat) of PGE(1), PGE(2), PGF(2alpha), or vehicle (saline) at 1900 h in proestrus. The ovulatory response was analyzed on the morning of estrus by evaluating follicle rupture and the location of the oocytes in serially sectioned ovaries. The number of oocytes in the oviducts was also counted in rats treated with the highest prostaglandin doses. In indomethacin-treated rats, most newly formed corpora lutea showed abnormal follicle rupture at the basolateral sides. In addition, invasion of the ovarian stroma and blood and lymphatic vessels by granulosa cells and follicular fluid was observed. Prostaglandins of the E series, and especially PGE(1), inhibited abnormal follicle rupture and restored ovulation, although the number of oocytes in the oviducts were significantly decreased. PGF(2alpha) was only partially effective in inhibiting abnormal follicle rupture and restoring ovulation. These data suggest that prostaglandins of the E series, and particularly PGE(1), play a crucial role in ovulation by determining the targeting of follicle rupture at the apex, thus allowing release of oocytes to the periovarian space.