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PubMed1987

Alternative solutions to hCG induction of ovulation in the mare.

Injection of hCG (2000-2500 i.u., i.v.) to mares when a follicle reaches 35 mm induces ovulation between 24 and 48 h. However, repeated injections induce antibodies against hCG. We report attempts to induce ovulation without this inconvenience. We called 'response' an ovulation between 24 and 48 h after treatment. The typical response to hCG was obtained in 73% (N = 145) of treated mares. After immunization against hCG, the response (0%, N = 10) was less than in nonimmunized controls (100%, N = 9). Simultaneous injection of dexamethasone and hCG resulted in induction of ovulation (71%, N = 14). However, simultaneous hCG + dexamethasone resulted in antibody formation similar to that induced by hCG alone, when injected repeatedly every 21 days. Neither GnRH (2 mg i.m.) nor partly purified pig LH (26 mg i.v. or s.c.) induced ovulation consistently (40%, N = 30 and 31%, N = 16). Crude horse gonadotrophin (60 mg i.v. or s.c.) induced ovulation (86%, N = 14). Fertility was not different from control (61%, N = 13 vs 40%, N = 10). Crude horse gonadotrophin also induced ovulation in mares previously immunized against hCG (78%, N = 9): 50 or 25 mg gave satisfactory response (86%, N = 29 and 57%, N = 40). We conclude that crude horse gonadotrophin is a good alternative to hCG for the induction of ovulation in mares.

Animals↗
PubMed1987

Reproductive characteristics of spontaneous single and double ovulating mares and superovulated mares.

For embryos collected from mares 7 days after ovulation, embryo recovery for single-ovulating mares was 53% compared to 106% for double-ovulating mares. Pregnancy rates 50 days after surgical transfer were 68 and 129%, respectively. Concentrations of LH were similar during the periovulatory period for cycles which included single or double ovulations. Horse pituitary extract given for 5.5 days resulted in greater than or equal to 2 ovulations (mean 3.8) in 26 of 28 mares and 2.0 embryos were recovered per donor compared to 0.65 for controls. Non-surgical pregnancy rates for embryos collected from superovulated mares 6 and 8 days after ovulation (18 of 46; 39%) were identical to those obtained for untreated controls (12 of 31; 39%). The overall pregnancy rate for superovulated donors, which provided 2-4 embryos, was 120%. Since the viability of multiple embryos collected from spontaneous and induced multiple-ovulating mares was equal to that of embryos from single-ovulating mares, collection and transfer of embryos from these mares will increase the efficiency of horse embryo transfer.

Animals↗
PubMed1979

[Mechanisms of ovulation in mammalian females].

The aim of this review was to briefly recapitulate the most important mechanisms involved in ovulation in the Mammals. The rabbit served as a model for the study of reflex ovulation. The triggering of ovulation by coitus was shown to be dependent on the activation of the hypothalamo-pituitary axis by sensory signals of multiple origin. The fundamental aspects of the hormonal and nervous machinery that governs spontaneous ovulation have been envisaged. The timing of LH ovulatory release and the mechanisms of action of this hormone at the ovarian level have been defined. Evidence was given that steroid hormones from ovarian and/or adrenal origin could evoke or modulate ovulatory processes. The structures responsible for both the tonic and clonic secretion of LH in subprimate and in primate mammals have been localized in the hypothalamus. The nervous endocrine mechanisms involving interactions between LHRH, neurotransmitters, prostaglandins and steroid hormones have been elucidated. Short loop feed back effects of pituitary hormones were shown to control LHRH secretion. Several examples were given attesting that the limbic system, the thalamus and the neocortex on one hand, and the environmental factors, on the other hand, were capable of modulating the activity of the hypothalamic structures implicated in the control of either ovulation or estrous rhythm regulation. An unitarian conception of the ovulatory mechanisms, based on the fact that coital-induced ovulation and estrogen-induced ovulation could occur in spontaneous and reflex ovulators respectively, has been proposed.

Adrenal Cortex↗
PubMed1979

Ovulation induction in amenorrheic women.

Seventy-six patients with primary or secondary amenorrhea who wished to conceive were treated with clomiphene citrate, 2-Br-alpha-ergocryptine, and/or human menopausal gonadotropins (hMG). Of these 71 patients who received clomiphene citrate, 39 (55%) ovulated. Of these 71 patients, 52 had withdrawal uterine bleeding following IM progesterone, and 38 (73%) ovulated; only 1 of the 19 who did not bleed ovulated (P less than 0.001). Ovulation occurred in the former group of patients whether or not they had galactorrhea. Of the 32 patients who failed to ovulate despite treatment with the maximal dose of clomiphene, 250 mg/day for 5 days, 26 received hMG-hCG. All 26 ovulated and 15 conceived. All 8 patients with amenorrhea-galactorrhea who were treated either primarily or secondarily with bromergocryptine ovulated, and 4 conceived. Therefore, the drug of choice for ovulation induction in amenorrheic patients depends on 1) the presence of withdrawal bleeding after progesterone and 2) the presence of galactorrhea. In all patients with progesterone withdrawal bleeding with or without galactorrhea, the initial treatment of choice is clomiphene citrate. In the absence of withdrawal bleeding, hMG should be administered if galactorrhea is absent, and bromergocryptine should be administered if galactorrhea is present.

Amenorrhea↗
PubMed1995

[Effect of progesterone on ovulation length and duration of the ovarian cycle induced by the male effect in the Barbarine ewe and the local Tunisian goat].

Two experiments were conducted on 30 Barbarine ewes (Exp 1) and 40 local Tunisian goats (Exp 2) in seasonal anoestrus (May). In both experiments, half of the females received 20 mg of progesterone intramuscularly just before male introduction (day 0). Blood samples were taken at 4 h intervals from the time of introduction of the ram, for the determination of the time of the preovulatory LH surge in response to the 'ram effect' (Exp 1) and once a day from days 1 to 8 to determine plasmatic progesterone concentrations (Exp 1, Exp 2). The ovulation rate was determined by coelioscopy at days 4 and 9, and oestrus behaviour was monitored twice daily in each experiment. In the ewe, progesterone delayed the time of the preovulatory LH surge (58.8 +/- 10.1 vs 20.5 +/- 10.7 h, P < 0.001), and suppressed hypofunctional corpora lutea (0/15 vs 7/14, P < 0.001). The induced ovulation rate was not significantly different between control and treated females (1.50 +/- 0.52 vs 1.26 +/- 0.46 respectively). Only 1 female in the control group and 2 in the treated group showed oestrus behaviour at the induced ovulation. Suppression of short cycles by progesterone treatment allowed synchronization of oestrus between days 17 and 20 compared to days 14 and 23 in the control group (P < 0.001). In the goat, all induced ovulations in the control group were followed by a short cycle. In the progesterone-treated group, 3 out of 20 goats developed abnormal induced corpora lutea (P < 0.001). First oestrus occurred between days 1 and 8 in control group and between days 2 and 3 in treated goats. Treatment with progesterone increased the percentage of females showing oestrus at induced ovulation (100 vs 35%, P < 0.001). The ovulation rate at first oestrus was increased in treated goats (1.85 vs 1.35, P < 0.001). In conclusion, 20 mg progesterone administered at day 0 is efficient at preventing the occurrence of short ovarian cycles. After ovulation induced by the male effect, the percentage of females in oestrus, and the induced ovulation rate are increased by progesterone treatment.

Anestrus↗
PubMed2004

Characterization of HoMADS 1 and its induction by plant hormones during in vitro ovule development in Hyacinthus orientalis L.

To understand the molecular mechanism of ovule development, a MADS box gene, HoMADS 1 , has been isolated from the ovule tissues of Hyacinthus . Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.

Amino Acid Sequence↗
PubMed1989

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 1. Variation within and between flocks.

Observations were made by repeated laparoscopy to determine the time of ovulation in seven flocks of South Australian Merino ewes treated with pregnant mare serum gonadotropin (PMSG, 400 IU per ewe) and an intravaginal pessary containing 60 mg medroxy-progesterone acetate (MAP). Ovulation most often commenced within 57 h of pessary removal and was completed in all flocks within 81 h. There was, however, significant variation in the time of onset of ovulation; it did not commence until 69 h in one of three observations of Flock 1 and in two of three observations of Flock 2. On the other hand, in a flock of two-year-old nulliparous ewes (Flock 7), ovulation commenced significantly (P < 0.001) earlier than in most observations of other flocks, with 18 39 ewes ovulating within 57 h of pessary removal. The effect of the progestagen (MAP vs flugestone acetate) and the gonadotropin preparation (Pregnecol vs Folligon) on the time of ovulation were observed in separate studies. The characteristic time of ovulation was not influenced in either comparison. However, ovulation occurred significantly (P < 0.001) earlier in ewes treated with progesterone released from a controlled internal drug release dispenser (CIDR). The implications of these findings to artificial insemination and embryo transfer are discussed.

Journal Article↗
PubMed1989

Seasonality of ovulation and estrus in Border Leicester ewes.

The number of Border Leicester ewes ovulating and their ovulation rate at 4 to 6 week intervals over 1 year and again after a further year were determined. The proportion of these ewes expressing estrus from autumn to spring and then in late summer/autumn was also determined. None of the ewes ovulated from October to early February, but for the remainder of the year, ovulation rate was always above 1.60, peaking in April to June at over 2.0. Eighty-four percent of the ewes had commenced ovulating by early March. The breeding season continued to September, when 10% of the ewes ovulated, with 25% expressing estrus in August and September. There was no significant effect of age, ewe liveweight or liveweight change on the proportion of ewes ovulating and their ovulation rate. The variability in the length of the breeding season gives scope for further selection.

Journal Article↗
PubMed1992

Initiation of superovulation in mares 5 or 12 days after ovulation using equine pituitary extract with or without GnRH analogue.

Cyclic mares were assigned to 1 of 3 treatments (n=15 per group): Group 1 received equine pituitary extract (EPE; 25 mg, i.m.) on Day 5 after ovulation; Group 2 received EPE on Day 12 after ovulation; while Group 3 received 3.3 mg of GnRH analogue (buserelin implant) on the day of ovulation and 25 mg, i.m. EPE on Day 12. Mares in each group were given 10 mg PGF2alpha on the first and second day of EPE treatment. The EPE treatment was continued daily until the first spontaneous ovulation, at which time 3,300 IU of human chorionic gonadotropin (hCG) were given to induce further ovulations. Mares in estrus with a >or=35 mm follicle were inseminated every other day with pooled semen from 2 stallions. Embryo recovery was attempted 7 days after the last ovulation. Follicular changes and embryo recovery during 15 estrous cycles prior to treatment were used as control data. During treatment, the number of follicles>or=25 mm was higher (P<0.05) for Day 5 than for Day 12 or control mares, but the number for Day-5 mares was similar (P>0.05) to that of mares treated with buserelin implants (Group 3). Initiation of EPE treatment on Day 5 resulted in a greater (P<0.05) number of ovulation (2.9) than on Day 12 (1.1) or in the control mares (1.3) but not in the buserelin-treated mares (1.8). The number of embryos recovered from mares in the Day 5 (1.2), Day 12 (1.0), buserelin (0.9) and control (0.9) groups was similar (P>0.05). The conclusions were 1) EPE initiated in early diestrus increased follicular development and ovulation and 2) treatment with GnRH analogue marginally improved response to EPE treatment.

Journal Article↗
PubMed1992

The effects of low level of feeding on response to synchronization of estrus, ovulation rate and embryo loss in goats.

Mature nonlactating British Saanen and Toggenburg does with a body score 2 were fed 25% (n=24) and 100% (n=16) maintenance rations from about 19 days before mating until slaughter at approximately 60 days after mating. Estrus was synchronized using PGF2alpha, and the ovulation rate was determined by laparoscopic examination of the ovaries once between Days 6 and 10 after mating. Pregnancy rate, potential kidding rate and embryo loss were determined by counts of viable fetuses at slaughter. The proportion of does in estrus within 96 hours of PGF2alpha administration was not different (P<0.5) between the feed-restricted and the maintenance groups (71.0% and 87.5%, respectively); however, the time of onset of estrus after PGF2alpha tended to be longer (P=0.12) in the feed-restricted group. Ovulation rate, incidence of multiple ovulations and proportion of does pregnant at 60 days were significantly lower (P=0.0004, P=0.025, P=0.05, respectively) in the restricted group. More embryos from single than multiple ovulations were lost in the restricted group (P=0.01). There was no difference in the overall ovulatory activity between right and left ovaries in the 2 groups. Transuterine migrations were observed in all does that had unilateral multiple ovulations. No migration was observed in does which had single ovulations. These data indicate that restricted feed intake in goats tended to delay the onset of estrus and lowered the ovulation rate, incidence of multiple ovulations, and pregnancy rate.

Journal Article↗
PubMed1993

Progesterone and estradiol in biodegradable microspheres for control of estrus and ovulation in mares.

Progesterone and estradiol 17-beta in poly (DL-lactide) microspheres were used to control estrus and ovulation in mares after luteolysis was induced by prostaglandin F(2)infinity. Mares were given a single intramuscular injection of biodegradable poly (DL-lactide) microspheres, 1 day following prostaglandin treatment, containing no hormones (control), 0.625 g progesterone and 50 mg estradiol (low dose), 1.25 g progesterone and 100 mg estradiol (medium dose), or 1.875 g progesterone and 150 mg estradiol (high dose; n=15 mares per group). Mares treated with the low dose had significantly longer intervals (P<0.05) to estrus and ovulation than the control mares; however, low dose mares had shorter intervals (P<0.05) to estrus than high dose mares and shorter intervals to ovulation than medium and high dose mares. Regression analysis indicated that the medium dose was sufficient for maximizing interval to ovulation while the high dose maximized interval to estrus. All groups of mares exhibited similar (P>0.05) post-treatment estrus lengths. A clinical response scoring system based on synchrony of both estrus and ovulation within a treatment group was also used to measure the effectiveness of treatments on control of estrus and ovulation. Clinical response scores did not differ (P>0.05) among treatment groups. Mares were randomly assigned for insemination at the beginning of the first post-treatment estrus. Rates for embryo recovery performed by uterine lavage 7 days post-ovulation did not differ (P>0.05) among groups. Concentrations of serum progesterone increased in mares receiving progesterone and estradiol microspheres. At 10 to 14 days post-injection of microspheres, progesterone concentrations were higher (P<0.05) and remained above 1 ng/ml in the mares receiving the high dose. Progesterone concentrations were also higher (P<0.05) on Days -3 to -1 (Day 0 = day of post-treatment ovulation) in mares receiving the high dose when compared to control mares. Gonadotropin concentrations were suppressed (P<0.05) in the medium and high dose groups.

Journal Article↗
PubMed2004

Pattern formation during early ovule development in Arabidopsis thaliana.

Ovules of higher plants are the precursors of seeds. Ovules emerge from placental tissue inside the gynoecium of flowers. Three elements, funiculus, chalaza, and nucellus, can be distinguished along the proximal-distal axis of the outgrowing radially symmetrical ovule primordium. The asymmetric initiation of the outer integument marks the switch to adaxial-abaxial development, which leads to the formation of a bilaterally symmetrical ovule. The putative transcriptional regulator NOZZLE (NZZ) plays a role in mediating this transition by controlling the timing of expression of the putative transcriptional regulator INNER NO OUTER (INO) in an abaxial domain of the chalaza, from where the outer integument initiates. Integument formation depends on the homeobox gene WUSCHEL (WUS), which is expressed in the nucellus and is sufficient to induce integuments non-cell autonomously from a region adjacent to its expression domain. In this study, we describe the expression pattern of the homeobox-leucine zipper gene PHABULOSA (PHB) during ovule development, demonstrating that adaxial-abaxial polarity is established from the very beginning of ovule development. Furthermore, we examined the expression pattern of PHB, INO, and WUS in ovules of plants, which are affected in integument initiation and thus defective in the transition from proximal-distal to adaxial-abaxial development. We found that NZZ is required to restrict PHB expression to the distal chalaza, from where the inner integument initiates. PHB expression is not established in the distal chalaza of two mutants, aintegumenta (ant) and wus, which fail to form integuments. Furthermore, we suggest that one mechanism by which WUS controls integument formation is by establishing the chalaza and that outer and inner integument identity determination depends on additional region-specific factors. In addition, we present evidence that NZZ is essential for the normal nucellar expression pattern of WUS. Thus, both WUS and PHB affect processes downstream of NZZ action during the transition from proximal--distal to adaxial--abaxial ovule development.

Arabidopsis↗
PubMed1999

BELL1 and AGAMOUS genes promote ovule identity in Arabidopsis thaliana.

Molecular and genetic analyses have demonstrated that the Arabidopsis thaliana gene BELL1 (BEL1) is required for proper morphogenesis of the ovule integuments. Several lines of evidence suggest that BEL1 may act, at least in part, to repress the function of the organ identity gene AGAMOUS (AG) during ovule development. To study the relative roles of BEL1 and AG, plants homozygous for ag, bel1 or both were constructed in an ap2 mutant background where ovules form even in the absence of AG function. The loss of either BEL1 or AG led to a decrease in the number of mature ovules, accompanied by an increase in primordial outgrowths. These data suggest that BEL1 and AG gene products act early in ovule development in a partially redundant manner to direct ovule identity. Development of the abnormal integuments characteristic of the Bel1- mutant phenotype was found to be dependent on AG function. Finally, BEL1 appears to be required for embryo sac development independent of both other aspects of ovule morphogenesis and AG function. This study therefore suggests that both BEL1 and AG are required for several distinct aspects of ovule morphogenesis.

AGAMOUS Protein, Arabidopsis↗
PubMed1997

Interactions among genes regulating ovule development in Arabidopsis thaliana.

The INNER NO OUTER (INO) and AINTEGUMENTA (ANT) genes are essential for ovule integument development in Arabidopsis thaliana. Ovules of ino mutants initiate two integument primordia, but the outer integument primordium forms on the opposite side of the ovule from the normal location and undergoes no further development. The inner integument appears to develop normally, resulting in erect, unitegmic ovules that resemble those of gymnosperms. ino plants are partially fertile and produce seeds with altered surface topography, demonstrating a lineage dependence in development of the testa. ant mutations affect initiation of both integuments. The strongest of five new ant alleles we have isolated produces ovules that lack integuments and fail to complete megasporogenesis. ant mutations also affect flower development, resulting in narrow petals and the absence of one or both lateral stamens. Characterization of double mutants between ant, ino and other mutations affecting ovule development has enabled the construction of a model for genetic control of ovule development. This model proposes parallel independent regulatory pathways for a number of aspects of this process, a dependence on the presence of an inner integument for development of the embryo sac, and the existence of additional genes regulating ovule development.

Arabidopsis↗
PubMed2005

Seed parasitism redirects ovule development in Douglas fir.

Many parasitic species of insects complete their entire development in seeds. They feed off storage reserves within the ovule. These reserves only normally accumulate in fertilized ovules. Consequently, female insects that oviposit their eggs directly into the plant ovule need to be able to select correctly, as unfertilized ovules of conifers normally become so-called empty seed. We provide clear evidence that in conifers, seed-parasitizing insects do not need to discriminate between fertilized and unfertilized plant ovules when ovipositing their eggs. A host-specific insect, the chalcid Megastigmus spermotrophus Wachtl (Hymenoptera: Torymidae), lays its eggs in ovules of Douglas fir (Pseudotsuga menziesii (Mirbel) Franco) before fertilization has taken place in the plant. Oviposition not only prevents the expected degeneration and death of unfertilized ovules, but it induces energy reserve accumulation. Ovules that would otherwise develop as empty seed are redirected in their development by the insect to provide food for the developing larvae. Instead of the insect exploiting normal events during seed development, the insect manipulates seed development for its own reproductive advantage.

Animals↗
PubMed2005

Local versus systemic effect of ovulation-inducing factor in the seminal plasma of alpacas.

BACKGROUND: Camelids are induced (reflex) ovulators. We have recently documented the presence of an ovulation-inducing factor (OIF) in the seminal plasma of alpacas and llamas. The objective was to test the hypothesis that OIF exerts its effect via a systemic rather than a local route and that endometrial curettage will enhance the ovulatory response to intrauterine deposition of seminal plasma in alpacas. METHODS: Female alpacas were assigned randomly to 6 groups (n = 15 to 17 per group) in a 2 x 3 factorial design to test the effect of seminal plasma versus phosphate-buffered saline (PBS) given by intramuscular injection, by intrauterine infusion, or by intrauterine infusion after endometrial curettage. Specifically, alpacas in the respective groups were given 1) 2 ml of alpaca seminal plasma intramuscularly, 2) 2 ml of PBS intramuscularly (negative control group), 3) 2 ml of alpaca seminal plasma by intrauterine infusion, 4) 2 ml of PBS by intrauterine infusion (negative control group), 5) 2 ml of alpaca seminal plasma by intrauterine infusion after endometrial curettage, or 6) 2 ml of PBS by intrauterine infusion after endometrial curettage (negative control group). The alpacas were examined by transrectal ultrasonography to detect ovulation and measure follicular and luteal diameters. RESULTS: Intramuscular administration of seminal plasma resulted in a higher ovulation rate than intrauterine administration of seminal plasma (93% versus 41%; P < 0.01), while intrauterine seminal plasma after endometrial curettage was intermediate (67%). None of the saline-treated controls ovulated. The diameter of the CL after treatment-induced ovulation was not affected by the route of administration of seminal plasma. CONCLUSION: We conclude that 1) OIF in seminal plasma effects ovulation via a systemic rather than a local route, 2) disruption of the endometrial mucosa by curettage facilitated the absorption of OIF and increased the ovulatory effect of seminal plasma, and 3) ovulation in alpacas is not associated with a physical stimulation of the genital tract, and 4) the alpaca represents an excellent biological model to evaluate the bioactivity of OIF.

Absorption↗
PubMed1990

Effect of GnRH treatment during the anovulatory season on multiple ovulation rate and on follicular development during the ensuing pregnancy in mares.

Seasonally anovulatory mares were injected, i.m., twice daily with a GnRH analogue (GnRH-A), and hCG was given when the largest follicle reached 35 mm in diameter. In Exp. 1, treatment was initiated on 23 December when the largest follicle per mare was less than or equal to 17 mm. An ovulatory response (ovulation within 21 days) occurred in 17 of 30 (57%) GnRH-A-treated mares on a mean of 15.8 days. The shortest interval to ovulation in control mares (N = 10) was 57 days. The diameter of the largest follicle first increased significantly 6 days after start of treatment. In Exp. 2, treatment was begun on 15 January and mares were categorized according to the largest follicle at start of treatment. The proportion of mares ovulating within 21 days increased significantly according to initial diameter of largest follicle (less than or equal to 15 mm, 9/25 mares ovulated; 15-19 mm, 13/21; 20-24 mm, 20/24; greater than 25 mm, 3/3). The multiple ovulation rate was greater (P less than 0.01) for treated mares (27/86 mares had multiple ovulations) than for control mares (2/35). Treated mares in which the largest follicle at start of treatment was greater than or equal to 25 mm had a higher (P less than 0.01) multiple ovulation rate (9/14) than did mares in which the largest follicle was less than 25 mm (18/72). The pregnancy rate for single ovulators was not different between control mares (26/30 pregnant mares) and treated mares (43/54).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

An analysis of methods for permanently mounting ovules cleared in four-and-a-half type clearing fluids.

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the cleaning fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical saggittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes--its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 14 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.

Cytological Techniques↗