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Changes in mitochondrial membrane potential and accumulation of reactive oxygen species precede ultrastructural changes during ovule abortion.

In many species, environmental stress reduces plant fertility. In Arabidopsis thaliana, a significant fraction of this reduction in plant fertility results from ovule abortion and embryo senescence. In this species, environmental conditions were identified that induced 94% of the developing ovules to either undergo stress-induced ovule abortion or embryo senescence (Sun et al. Plant Physiol 135:2358-2367, 2004). Following salt stress, physiological and anatomical changes were first detected in the female gametophyte of an aborting ovule. Two to four hours after a period of salt stress that induces most ovules to abort, the mitochondrial membrane potential dissipated. Subsequently, cells in the gametophyte accumulated reactive oxygen species, which are known to be molecules that promote programmed cell death (PCD). Because mitochondria often play an important role in PCD, these organelles were closely examined for changes in structure. Although the anatomy of mitochondria varied, reproducible changes in mitochondria structure were not observed. Nonetheless, other changes in ultrastructure were found. In some aborting gametophytes, concentric rings of endoplasmic reticulum were formed. In a fraction of the aborting ovules, cytoplasmic contents and organelles were invaginated into the vacuole. Even in cryofixed sections, many of these bodies appeared indistinct, which is consistent with the degradation of their contents.

Arabidopsis↗

Gypsy embryo specifies ovule curvature by regulating ovule/integument development in rice.

The embryo position in a seed is stable in most plant species, indicating the existence of a strict regulatory mechanism that specifies the embryo position in the seed. To elucidate this mechanism, we analyzed the gypsy embryo (gym) mutant of rice, in which the position of the mature embryo in the seed is altered at a low frequency. Analyses of early embryogenesis and ovule development showed that the ectopic embryo was derived from an ill-positioned egg cell, which resulted from the incomplete curvature of the ovule. Although the development of both the inner and outer integuments was impaired, the ovule curvature was associated closely with the extent of inner integument growth. Therefore, inner integument development controls ovule curvature in rice. The expression patterns of OSH1 and OsMADS13 indicated that, in gym, a small number of indeterminate cells are maintained on the style side of the ovule and then in the integument primordium at a low frequency. The prolonged survival of these indeterminate cells disturbs normal integument development. The gym fon2 double mutant suggests that GYM and FON2 are involved redundantly in floral meristem determinacy. Possible functions of the GYM gene and the ovule developmental mechanism are discussed.

Gene Expression Profiling↗

Cycloheximide treatment of cotton ovules alters the abundance of specific classes of mRNAs and generates novel ESTs for microarray expression profiling.

Fibres of cotton (Gossypium hirsutum L.) are single elongated epidermal cells that start to develop on the outer surface of cotton ovules on the day of anthesis. Little is known about the control of fibre initiation and development. As a first step towards discovering important genes involved in fibre initiation and development using a genomics approach, we report technical advances aimed at reducing redundancy and increasing coverage for anonymous cDNA microarrays in this study. Cotton ovule cDNA libraries (both normalised and un-normalised) from around the time of fibre initial formation have been prepared and partially characterised by sequencing. Re-association-based normalisation partially reduced library redundancy and increased representation of novel sequences. However, another library generated from in vitro cultured cotton ovules treated with the protein synthesis inhibitor, cycloheximide, showed a significantly altered gene representation including a greater proportion of protein phosphorylation genes, transport genes and transcription factors and a much reduced proportion of protein synthesis genes than were identified in the conventional types of libraries. Over 10,000 expressed sequence tag (EST) clones randomly selected from the three libraries were printed on microarray slides and used to assess gene expression in tissue cultured ovules with and without cycloheximide treatment. The microarray results showed that cycloheximide had a dramatic effect in modifying the pattern of the gene expression in cultured ovules, affecting the same types of genes identified in the preliminary analysis on relative EST abundance in the different ovule cDNA libraries. Cycloheximide clearly provided a simple and useful method for enriching novel gene sequences for genomic studies.

Base Sequence↗

Induction of multiple ovulations during the ovulatory season in mares.

The efficacy of an equine pituitary extract for induction of multiple ovulations during the ovulatory season was studied in 112 horse mares in four experiments. Combined for all experiments, 70% of the mares (78/112) had multiple ovulations for an average of 3.0 ovulations per mare. The interval between first and last ovulation was decreased (P<0.01) when human chorionic gonadotropin (hCG) was included in the treatment regimen (0.0+/-0.0 versus 1.6+/-0.4 days). Ovulation rate was lower (P<0.01) when extract treatment was initiated at day 19 (1.3+/-0.2) than when initiated at day 15 post-ovulation (2.9+/-0.5). Initiation of extract treatment at day 11 was as effective as initiation at day 15, with or without an injection of prostaglandin F(2alpha) on the first day of extract treatment. Increasing the dose of extract from 750 to 1500 or 2250 units per day did not significantly increase the ovulatory response.

Journal Article↗

Postpartum ovarian activity in dairy cows: Correlation between behavioral estrus, pedometer measurements and ovulations.

The efficacy of pedometers as estrus detection aids was tested in a group of 47 Holstein cows during the postpartum period. Pedometers were strapped on the left front leg of each cow on the day of parturition and pedometer measurements were recorded twice daily for 60 days. The cows were also observed twice a day for behavioral signs of estrus. Daily blood samples were collected during Days 1-30, and thereafter once every two days from Days 30-60 after parturition for progesterone assay. Rectal palpations of the ovaries were carried out once every four days during the first month after parturition. Based on the progesterone profiles and rectal palpation findings, 91 ovulations were recorded in 42 cows and 5 cows remained acyclic during the observation period. Seventy-six percent (69 91 ) of the ovulations were associated with pedometer indications of estrus, while only 35% (32 91 ) of the ovulations were associated with observed estrus. Fifty-seven percent (24 42 ) of the first postpartum ovulations were associated with pedometer measurements, but only 19% (8 42 ) of the first postpartum ovulations were associated with behavioral signs of estrus. Similarly, pedometers were superior to observations as a method of detection of estrus associated with the second and third postpartum ovulations (91% vs 37%, 92% vs 78% respectively). It is concluded that pedometers are useful aids for estrus detection, particularly in the immediate postpartum.

Journal Article↗

Induction of ovulation in the acyclic postpartum ewe following continuous, low-dose subcutaneous infusion of GnRH.

Pituitary and ovarian responses to subcutaneous infusion of GnRH were investigated in acyclic, lactating Mule ewes during the breeding season. Thirty postpartum ewes were split into 3 equal groups; Group G received GnRH (250 ng/h) for 96 h; Group P + G was primed with progestagen for 10 d then received GnRH (250 ng/h) for 96 h; and Group P received progestagen priming and saline vehicle only. The infusions were delivered via osmotic minipumps inserted 26.6 +/- 0.45 d post partum (Day 0 of the study). Blood samples were collected for LH analysis every 15 min from 12 h before until 8 h after minipump insertion, then every 2 h for a further 112 h. Daily blood samples were collected for progesterone analysis on Days 1 to 10 following minipump insertion, then every third day for a further 25 d. In addition, the reproductive tract was examined by laparoscopy on Day -5 and Day +7 and estrous behavior was monitored between Day -4 and Day +7. Progestagen priming suppressed (P < 0.05) plasma LH levels (0.27 +/- 0.03 vs 0.46 +/- 0.06 ng/ml) during the preinfusion period, but the GnRH-induced LH release was similar for Group G and Group P + G. The LH surge began significantly (P < 0.05) earlier (32.0 +/- 3.0 vs 56.3 +/- 4.1 h) and was of greater magnitude (32.15 +/- 3.56 vs 18.84 +/- 4.13 ng/ml) in the unprimed than the primed ewes. None of the ewes infused with saline produced a preovulatory LH surge. The GnRH infusion induced ovulation in 10/10 unprimed and 7/9 progestagen-primed ewes, with no significant difference in ovulation rate (1.78 +/- 0.15 and 1.33 +/- 0.21, respectively). Ovulation was followed by normal luteal function in 4/10 Group-G ewes, while the remaining 6 ewes had short luteal phases. In contrast, each of the 7 Group-P + G ewes that ovulated secreted progesterone for at least 10 d, although elevated plasma progesterone levels were maintained in 3/7 unmated ewes for >35 d. Throughout the study only 2 ewes (both from Group P + G) displayed estrus. These data demonstrate that although a low dose, continuous infusion of GnRH can increase tonic LH concentrations sufficient to promote a preovulatory LH surge and induce ovulation, behavioral estrus and normal luteal function do not consistently follow ovulation in the progestagen-primed, postpartum ewe.

Journal Article↗

Effects of LH administration at the end of an FSH superovulatory regimen on ovulation rate and embryo production in three breeds of sheep.

In 3 experiments, 168 ewes of Manchega (n = 72), Churra (n = 62), and Merina (n = 34) breeds were used to test the hypothesis that administration of pure LH, coincident with progestogen removal during superovulation with FSH, causes an increase in the ovulation rate and number of embryos. This administration of LH can further interact with genotype, resulting in breed differential response. In each experiment, the animals were randomly assigned to 1 of 3 treatments. Estrus in all sheep was synchronized with intravaginal sponges of 30 mg of FGA for 12 d, then 270 microg of FSH were administered in 6 injections at 12-h intervals in decreasing doses, starting 48 h before sponge removal. The FSH/LH ratio of the original preparation was 3, and remained constant throughout the treatment in the control group (C). In Treatment 1, (T1) and Treatment 2, (T2), pure LH was administered coincident with progestogen removal-5th FSH injection, and with the 6th FSH injection, at 2 dose levels: 60 and 120 microg, (T1), and 120 and 240 microg (T2). Mating occurred 36 and 48 h after the progestogen removal, and the embryos were surgically collected and morphologically evaluated on Days 7 and 8 after sponge withdrawal. Overall, the results showed that LH administration at the end of the FSH treatment did not increase the ovulation rate and number of embryos in Merino (5.9 +/- 1.4 and 5.6 +/- 1.4, respectively, T1; 7.0 +/- 1.0 and 5.7 +/- 1.2, T 2; 4.9 +/- 1.1 and 2.6 +/- 0.7, C), Churra (6.8 +/-1.4 and 5.2 +/- 1.4, T1; 8.1 +/- 1.5 and 6.3 +/- 1.4, T2; 6.1 +/- 1.5 and 5.4 +/- 1.3, C) and Manchega (6.0 +/- 1.0 and 4.4 +/- 1.0, T1; 5.0 +/- 0.8 and 4.2 +/- 0.8, T2; 4.8 +/- 1.5 and 3.8 +/- 1.0, C). Administration of LH induced a significant (P < 0.05) increase in the frequency of multiple ovulations (72.3 +/- 4.3 %, T1; 74.1 +/- 11.5 %, T2; 55.6 +/- 5.9 %, C) paralleled to a decrease in the occurrence of ewes with no ovulations (8.7 +/- 2.6 %, T1; 7.6 +/- 4.6 % T2; 17.3 +/- 3.2 %, C) or 1 to 2 ovulations (18.7 +/- 4.6 %, T1; 18.1 +/- 7.5 %, T2; 26.8 +/- 5.8 % C), regardless of breed or dose of LH. No increase in the mean number of viable embryos was observed, probably due to both the high individual variability and the lower fertilization rates observed in sheep showing multiple ovulations.

Journal Article↗

Functional analysis of MADS-box genes controlling ovule development in Arabidopsis using the ethanol-inducible alc gene-expression system.

In Arabidopsis, different combinations of ABC organ identity proteins interact in the presence of SEPALLATA (SEP) proteins to regulate floral organ differentiation. Ectopic expression of SEP3 in combination with class A and B or B and C genes is sufficient to homeotically convert vegetative leaves into petal-like organs and bracts into stamen-like structures, respectively. Recently, it has been shown that the three MADS-box genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2 act redundantly to control ovule identity. Protein interaction assays performed in yeast in combination with genetic studies demonstrated that these MADS-box factors only interact in the presence of SEP proteins to form complexes that determine ovule differentiation. Here, we address the question whether the ectopic co-expression of ovule identity proteins is sufficient to induce the homeotic conversion of vegetative leaves into carpel-like structures bearing ovules. We present the phenotypic characterization of Arabidopsis plants that ectopically express ovule identity factors under the regulation of the ethanol inducible gene expression system. These experiments indicate that the ectopic co-expression of SEP3 and SHP1 and/or STK is probably not sufficient to homeotically transform vegetative tissues into carpels with ovules. However, comparing the phenotypes obtained by ectopic expression of STK and/or SHP1 with or without SEP3 shows that co-expression of factors that are able to form complexes in yeast cause more extreme homeotic transformations, confirming the functional role of these complexes in vivo.

Arabidopsis↗

The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster.

In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from specific regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being "movers" to "fertilizers." Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. We review the processes of ovulation and sperm storage for Drosophila melanogaster, whose requirements for gamete maturation and sperm storage as well as powerful molecular genetics make it an excellent model organism for study of these processes. Within the female D. melanogaster, both processes are triggered by male factors during and after mating, including sperm and seminal fluid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization.

Animals↗

An unusual, structurally preserved ovule from the Permian of Antarctica.

Anatomically preserved ovules are described from silicified peat of Late Permian age collected from Skaar Ridge in the central Transantarctic Mountains, Antarctica. The small ovules are significant in possessing fleshy apical appendages and a funnel-shaped micropylar extension formed by the sarcotestal layer of the integument, by which they differ from all other Permian ovules described to date. The apical modifications may have functioned in pollination and/or seed dispersal. Similarity with the apical organization of earlier Paleozoic ovules is shown to be superficial, since the analogous structures are developmentally derived from different tissues. Although the ovules occur in rocks in which glossopterids are the only gymnosperms represented, there is insufficient evidence to assign them to a taxonomic group. These ovules are of particular importance because there are so few anatomically preserved gymnosperm reproductive structures known from the Permian and thus provide new data on the diversity of late Paleozoic gymnosperms.

Journal Article↗

Comparison of three methods of acute administration of progesterone on ovarian follicular development and the timing and synchrony of ovulation in Bos indicus heifers.

The aim of this study was to induce the formation of a persistent dominant ovarian follicle and to compare the effects of 3 methods of acute administration of P4 on ovarian follicular development and on the timing and synchrony of ovulation. Stage of the estrous cycle was initially synchronized in Bos indicus heifers with a norgestomet implants (3 mg) for 10 d and with an analogue of PGF2 alpha (15 mg) on the first and last day of norgestomet treatment. Eight days after removal of the implants, heifers were randomly assigned to 4 groups. All heifers received a norgestomet implant (Day 0), which was removed 17 d later (Day 17); PGF2 alpha was administered on Days 0 and 4. Heifers in the control group (n = 5) received no other treatment. On Day 10 heifers in Group P4C (n = 5) were treated with a CIDR for 24 h; heifers in Group P4O (n = 5) were administered 100 mg i.m. of P4 in oil, while heifers in Group P4S (n = 5) were administered 100 mg i.m. of P4 in saline/alcohol. Data were analyzed using bootstrap estimates of location (mean) and spread (standard deviation; SD). Compared with the control heifers, day of emergence of the ovulatory follicle was delayed, and age and duration of dominance of the ovulatory follicle were reduced in the P4C and P4O heifers (P < 0.05) but not in the P4S heifers (P > 0.05). In all groups treated with P4 both the mean and variability (SD) in the timing of ovulation did not differ with that of the control group (P > 0.05) but there was less variability in the day of emergence, age, duration of dominance and diameter of the ovulatory follicle than in the control group (P < 0.05). Delayed timing and reduced synchrony (SD) of ovulation and greater age of the ovulatory follicle (P < 0.05) occurred in P4S heifers than in P4C heifers. We conclude that administration of 100 mg of P4 in oil is as effective as treatment with a CIDR for synchronizing emergence and ovulation of a newly recruited dominant follicle. However, reduced synchrony of ovulation, greater age of the ovulatory follicle and delayed timing of ovulation occurred following administration 100 mg of P4 in saline/alcohol compared with the CIDR device.

Administration, Intravaginal↗

A note on the effect of continuous ram presence on estrus onset, estrus duration and ovulation time in estrus synchronized ewes.

During fall season, 18 multiparous Corriedale ewes were divided into two equal groups for the continuous (CON) and intermittent (INT) presence of a ram. Estrus was synchronized with fluorgestone acetate intravaginal sponges that were left 14 days, plus an injection of 200&mgr;g of a prostaglandin F-2alpha analog at sponge removal. Estrus was detected three times a day (at 6 a.m., 2 p.m. and 10 p.m.) by using rams with harnessess and markers. Ovulation time was determined by laparoscopy, starting 24h after estrus detection. Estrus onset was (mean+/-S.E.M.) 32.9+/-1.6 and 45.3+/-4.4h for the CON and INT groups, respectively (P<0.01). Estrus duration was 31.1+/-0.9 and 30.2+/-1.2h, for the same groups, respectively (P>0.05). Ovulation time and the interval from sponge removal to ovulation (ISRO) for the CON and INT groups was 29.0+/-1.5, 62.0+/-2.0, 26.7+/-1.3 and 72.0+/-4.2h, respectively. Ovulation time was not different (P>0.05), but ISRO was shown to be different between treatments (P<0.05). It is concluded that the continuous presence of a ram after sponge removal hastens estrus onset and reduces the interval between sponge removal and ovulation, without modifying estrus duration and time between estrus onset and ovulation.

Journal Article↗

Arabidopsis floral homeotic gene BELL (BEL1) controls ovule development through negative regulation of AGAMOUS gene (AG).

Ovules are the developmental precursors of seeds. In angiosperms the ovules are enclosed within the central floral organs, the carpels. We have identified a homeotic mutation in Arabidopsis, "bell" (bel1), which causes transformation of ovule integuments into carpels. In situ hybridization analysis shows that this mutation leads to increased expression of the carpel-determining homeotic gene AGAMOUS (AG) in the mutant ovules. Introduction of a constitutively expressed AG transgene into wild-type plants causes the ovules to resemble those of bel1 mutants. We propose that the BEL1 gene product directs normal integument development, in part by suppressing AG expression in this structure. Our results allow expansion of the current model of floral organ identity to include regulation of ovule integument identity.

Arabidopsis↗

The phenotype of Arabidopsis ovule mutants mimics the morphology of primitive seed plants.

In seed plants, the ovule is the female reproductive structure, which surrounds and nourishes the gametophyte and embryo. This investigation describes the PRETTY FEW SEEDS2 (PFS2) locus, which regulates ovule patterning. The pfs2 mutant exhibited developmental defects in the maternal integuments and gametophyte. This mutation was inherited as a maternal trait, indicating that gametophyte defects resulted from ovule patterning aberrations. Specifically, the boundary between the chalaza and the nucellus, two regions of the ovule primordia, shifted towards the distal end of pfs2 ovule primordia. Results indicated that the PFS2 locus could: (i) be involved in the development of either the nucellus or the chalaza; or (ii) establish a boundary between these two regions. Examination of genetic interactions of the pfs2 mutation with other well-characterized ovule loci indicates that this locus affects integument morphogenesis. Interestingly, the pfs2 inner no outer and pfs2 strubbelig double mutants had inner integuments that appeared similar to their ancestral precursor. The fossil record indicates that the inner integument evolved by fusion of sterilized sporangia or branches around a central megasporangium. The question of whether the structures observed in these double mutants are homologous or merely analogous to the ancestral precursors of the inner integument is discussed.

Arabidopsis↗

Interaction of boron with components of nucleic Acid metabolism in cotton ovules cultured in vitro.

Cotton (Gossypium hirsutum L.) ovules grown in a defined nutrient medium undergo normal morphogenesis, including fiber production. In identical medium lacking boron, ovules callus and accumulate brown substances. Boron deficiency-like symptoms were induced by 6-azauracil and 6-azauridine in ovules growing in boron-sufficient media. Other nucleoside base analogs either reduced or had no effect on over-all growth, but did not cause typical boron-deficient callus growth of cotton ovules. Orotic acid and uracil countered the effects of 6-azauracil. Actinomycin D, fluorodeoxyuridine, and ethidium bromide reduced not only fiber production on ovules growing in boron-sufficient media but also callusing of ovules in boron-deficient media.Similarities between symptoms of boron deficiency and 6-azauracil injury, and the ability of uracil to suppress both, suggest that boron deficiency symptoms are related to reduced activity in the pyrimidine biosynthetic pathway. Growth inhibition by most nucleoside base analogs tested, actinomycin D, fluorodeoxyuridine, and ethidium bromide, as compared to callusing brought on by boron deficiency and 6-azauracil, indicates that boron deficiency symptoms are not related to a reduction in nucleic acid biosynthesis. Based on this information, a discussion of the possibility that boron deficiency causes reduced synthesis of UDP-glucose is presented.

Journal Article↗

Independent control of fiber development and nitrate reduction in cultured cotton ovules.

Several lines of evidence implicate ammonium as an important factor in the growth and development of cotton (Gossypium hirsutum L.) ovules cultured in vitro. For example, ovules cultured at 28 C require indoleacetic acid (IAA) and either ammonium or gibberellic acid (GA(3)) in the medium for fiber development, whereas ovules cultured at 34 C require only IAA. Because of this effect of ammonium supply, it seemed possible that hormones or increased temperature were also promoting the availability of reduced nitrogen by induction of increased nitrate reductase activity in the ovules. This possibility was tested.In vivo, where ovules received mostly reduced nitrogen and very little nitrate, they did not display appreciable nitrate reductase activity even when nitrate was forced into the ovary wall by transpiration. After initiation of culture, nitrate became freely available to ovules and their nitrate reductase activity increased rapidly. Treatment with ammonium, GA(3), IAA, or increased temperature had no effect upon this induction. It is concluded that ammonium, hormone, and temperature effects on fiber development are independent of the availability of reduced nitrogen as a general substrate for growth.

Journal Article↗

Ovule Development in Wild-Type Arabidopsis and Two Female-Sterile Mutants.

Ovules are complex structures that are present in all seed bearing plants and are contained within the carpels in flowering plants. Ovules are the site of megasporogenesis and megagametogenesis and, following fertilization, develop into seeds. We combined genetic methods with anatomical and morphological analyses to dissect ovule development. Here, we present a detailed description of the morphological development of Arabidopsis ovules and report on the isolation of two chemically induced mutants, bell (bel1) and short integuments (sin1), with altered ovule development. Phenotypic analyses indicated that bel1 mutants initiate a single integument-like structure that develops aberrantly, sin1 mutants initiate two integuments, but growth of the integuments is disrupted such that cell division continues without normal cell elongation. Both mutants can differentiate archesporial cells, but neither forms a normal embryo sac. Genetic analyses indicated that bel1 segregates as a single recessive mutation, and complementation tests showed that the two mutants are not allelic. The phenotypes of the mutants indicate that normal morphological development of the integuments and proper embryo sac formation are interdependent or are governed in part by common pathways. The ovule mutants that we describe in Arabidopsis represent novel genetic tools for the study of this stage of reproductive development.

Journal Article↗

A novel class of MADS box genes is involved in ovule development in petunia.

We isolated and characterized two ovule-specific MADS box cDNAs from petunia, designated floral binding protein (fbp) genes 7 and 11. The putative protein products of these genes have approximately 90% of their overall amino acid sequence in common. In situ RNA hybridization experiments revealed that both genes are expressed in the center of the developing gynoecium before ovule primordia are visible. At later developmental stages, hybridization signals were observed only in the ovules, suggesting that these genes are involved in ovule formation. To test this hypothesis, we raised transgenic petunia plants in which both fbp7 and fbp11 expression was inhibited by cosuppression. In the ovary of these transformants, spaghetti-shaped structures developed in positions normally occupied by ovules. These abnormal structures morphologically and functionally resemble style and stigma tissues. Our results show that these MADS box genes belong to a new class of MADS box genes involved in proper ovule development in petunia.

Amino Acid Sequence↗