Equine Ovary Research Articles

Expression and localization of relaxin in the ovary of the mare.

Immunoreactive, chromatographic and molecular techniques were used to study the expression of relaxin in mare ovaries at different stages of the oestrous cycle. Relaxin in follicular fluid ranged from 1.6 to 2.5, from 1.4 to 5.2, from 1.2 to 6.7 and from 1.0 to 3.5 ng ml-1 in small (< or = 2 cm), medium (> 2 < or = 3 cm), medium-large (> 3 < or = 4 cm) and large (> 4 cm) follicles, respectively, and total content of fluid relaxin per follicle increased (P < 0.05) with follicular size. When subjected to reverse phase HPLC analysis, follicular fluid yielded absorbance profiles corresponding closely to those of purified relaxin, and immunoreactive peaks in follicular fluid fractions measured by radioimmunoassay matched peaks of the relaxin standard. While relaxin was localized immunocytochemically to granulosa and theca cells of preovulatory follicles, northern blot and reverse transcriptase-PCR followed by Southern blot analysis failed to detect a relaxin transcript in these tissues. A single relaxin transcript (428 bp) corresponding to mRNA encoding relaxin was identified in early, mid- and late stage corpora lutea but not in corpora haemorrhagica or albicantia. Northern blot analysis revealed a weakly expressed 1 kb transcript in total cellular RNA from mature corpora lutea. In situ hybridization studies localized the mRNA to the large luteal cells of mature corpora lutea and relaxin protein was detected by immunocytochemistry in the same tissue. This is the first report demonstrating relaxin in the equine ovary and its expression by luteal cells, thereby suggesting a role for relaxin in follicular or corpus luteum function in cyclic mares.

Large-scale isolation of equine zonae pellucidae

A simple and rapid method is described for the large-scale isolation of cumulus cell-free, zona pellucida-intact equine oocytes. Aspiration of palpable antral follicles present on frozen-thawed equine ovaries was accomplished using a constant vacuum source. The resultant follicular fluid, oocytes, and particulate matter were then filtered through a series of nylon screens of alternating mesh openings in combination with sodium citrate-containing buffer to a final volume of approximately 20 ml. This fluid was transferred to scored Petri dishes and a stereomicroscope was used to locate the oocytes for futher processing or storage. The methodology described is inexpensive, time-efficient, and the recovery rate is similar to or better than other methods previously described for equine oocyte recovery. Collected oocytes are adequate for biochemical evaluation of the equine zona pellucida (EZP) as well as sperm-egg binding assays.

In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries

The aim of this study was to examine 2 techniques for oocyte recovery from equine ovaries at slaughter: by aspiration of follicles and by additional slicing of ovaries. The morphology and nuclear configuration of oocytes recovered with either technique, and the time course of nuclear maturation during in vitro maturation were evaluated. Recovery rates were 1.75 and 4.14 oocytes per ovary for aspiration and slicing (total 145 and 344 oocytes from 83 ovaries), respectively. The oocytes were classified according to their cumulus/ooplasm morphology into 4 groups: compact/circular(A), compact/semicircular(B), expanded(C) and others(D). The percentages of oocytes in Groups A, B, C and D were 34, 38, 25 and 3% (aspiration) and 55, 26, 17 and 3% (slicing), respectively. The proportions of oocytes with a germinal vesicle in Groups A, B, C and D were 28 29 (97%), 23 35 (66%), 11 23 (48%) and 2 4 (50%) in oocytes from aspiration and 91 100 (91%), 52 65 (80%), 15 29 (52%) and 1 2 (50%) in oocytes from slicing, respectively. Group A and B oocytes recovered by aspiration (n=212) and slicing (n=312) were cultured in TCM199 supplemented with 10% fetal bovine serum, 1 μg/ml estradiol-17β, and 0.02 AU/ml FSH at 38.5°C in 5% CO 2 in air (5 to 10 oocytes per 50- μ l microdrop). At 8, 16, 24, 32 and 40 h of culture, the oocytes were fixed and stained. There were no significant differences in the percentages of Metaphase II stage (MII)-oocytes between recovery techniques at any time points examined. The proportions of MII-oocytes were 1 42 (2%), 4 43 (9%), 21 42 (50%), 28 45 (62%), and 28 40 (70%) at the respective time point in oocytes from aspiration and 0 51 (0%), 3 54 (6%), 22 59 (37%), 43 72 (60%), and 51 76 (67%) in oocytes from slicing, respectively. In most of the oocytes, resumption of meiosis occurred between 8 and 16 h of culture. The proportions of MII-oocytes increased significantly between 16 and 24 h and between 24 and 32 h of culture.

Evaluation of three techniques (follicular aspiration, follicular aspiration and flushing and slicing of the ovaries) for recovery of equine oocytes from excised ovaries

Summary The objective of this study was to compare three different methods of oocyte recovery from equine ovaries obtained from an abattoir. The techniques used were a) aspiration of follicles, b) aspiration plus flushing of the follicles and c) slicing of the ovaries in a grid pattern. The number of oocytes recovered from each of the three methods was different (P

Characterization of equine zona pellucida glycoproteins by polyacrylamide gel electrophoresis and immunological techniques.

This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubilized, and then analysed by 1D- and 2D-PAGE. Silver stained 2D-PAGE of the EZP revealed the presence of three EZP glycoprotein families of apparent molecular mass ranges of 93-120 kDa, 73-90 kDa and 45-80 kDa. Immunoblot analysis of EZP glycoproteins resolved by 2D-PAGE using rabbit antisera against pig zonae pellucidae (R alpha HSPZ) confirmed the presence of three EZP glycoprotein families and established the existence of common epitopes between equine and porcine ZP glycoproteins. Further immunodetection using 2D-PAGE-separated glycoproteins illustrated that the 45-80 kDa family is recognized by the monoclonal antibody R5, developed against the porcine ZP glycoprotein of molecular mass 55-120 kDa. Guinea-pig antiserum against endo-beta-galactosidase-treated rabbit ZP 55 kDa glycoprotein (R55K), which specifically recognizes the rabbit ZP glycoprotein with the lowest molecular mass, also recognized the EZP 45-80 kDa glycoprotein family. Guinea-pig polyclonal antisera developed against total heat-solubilized rabbit ZP (GP alpha HSRZ) recognized the 73-90 kDa EZP glycoprotein family exclusively. After heat solubilization and treatment of EZP with endo-beta-galactosidase to remove polylactosaminoglycans, silver stained 1D-PAGE again demonstrated the presence of three glycoproteins with apparent molecular masses of 60, 75 and 90 kDa. The partially deglycosylated 60 kDa equine glycoprotein is recognized on immunoblot by the monoclonal antibody R5; the 75 kDa EZP glycoprotein is recognized by GP alpha HSRZ; and all three EZP glycoproteins separated by 1D-PAGE are recognized by R alpha HSPZ. These data add further support to the concept of cross-species zona pellucida glycoprotein antigenicity.

Steroid 21-hydroxylase activity in equine ovarian follicles evidenced by isotope dilution-mass spectrometry

Steroid 21-hydroxylase activity of the microsome-enriched fraction of follicular linings from equine ovaries has been demonstrated by gas chromatography-mass spectrometry. The 21-hydroxylated metabolites were quantified by isotope dilution with deuterated analogues. The two most abundant potential substrates for follicular steroid 21-hydroxylase, progesterone (P) and 17-hydroxyprogesterone (17OHP), were converted respectively to 11-deoxycorticosterone (DOC) and 11-deoxycortisol with corresponding apparent specific activities of 308 and 24 pmol/mg protein/h and apparent K m values of 1.1 and 6.4 μM. Competitive inhibition of the P-to-DOC conversion was exerted by 17OHP and pregnenolone. Hence, the ovarian follicle of the mare is an extraadrenal site of preferential DOC biosynthesis by an enzyme having steroid 21-hydroxylase activity.

A Congenital Interstitial Cell Hamartoma of the Equine Ovary

4 Nesbitt MN, Skamene E: Recombinant inbred mouse strains derived from A/J and C57BL/6J: a tool for the study of genetic mechanisms in host resistance to infection and malignancy. J Leukoc Biol36:357-364, 1984 5 Pelletier M, Forget A, Bourassa D, Gros P, Skamene E: Immunopathology of BCG infection in genetically resistant and susceptible mouse strains. J Immunol129:21792185, 1982 6 Silver J: Confidence limits for estimates of gene linkage based on analysis of recombinant inbred strains. J Hered 76:436-440, 1985 7 Skamene E: Genetic control of resistance to mycobacterial infection. Curr Top Microbiol Immunol 124:4966, 1986 8 Skamene E, Gros P, Forget A, Kongshavn PA, St. Charles C, Taylor BA: Genetic regulation of resistance to intracellular pathogens. Nature 297:506-509, 1982 9 Stach JL, Gros P, Forget A, Skamene E: Phenotypic expression of genetically controlled natural resistance to Mycobacteriurn bovis (BCG). J Immunol 132:888-892, 1984 10 US Department of Agriculture: Laboratory methods in veterinary mycobacteriology, revised ed. National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, pp. 34-39. US Department of Agriculture, Ames, IA, 1974 11 Zneimer SM, Womack JE: Regional localization of the fibronectin and gamma crystallin genes to bovine chromosome 8. Genomics 5 2 15-220, 1989

Ultrasonic anatomy of equine ovaries

A linear-array ultrasound scanner with a 5-MHz transducer was evaluated for studying follicular and luteal status in mares, and the ultrasonic properties of equine ovaries were characterized. Follicular diameters were estimated in vivo and after removing and slicing six ovaries. Correlation coefficients between the two kinds of determinations were 0.91 for number of follicles ≥2 mm in diameter and 0.95 for diameter of largest follicle. The ovaries of five mares were examined daily until all mares had been examined from three days before an ovulation to three days after the next ovulation. There was a significant difference among days for diameter of largest follicle and second largest follicle and for number of follicles 2–5 mm, 16–20 mm, and >20 mm. Differences seemed to be caused by the presence of many 2- to 5-mm follicles during early diestrus, initiation of growth of large follicles at mid-cycle, selective accelerated growth of an ovulatory follicle beginning five days before ovulation, and regression of large nonovulatory follicles a few days before ovulation. In one of the five mares, the corpus luteum was identified throughout the interovulatory interval, and the corresponding corpus albicans was identified for three days after the second ovulation. In the other four mares, the corpus luteum was last identified an average of 16 days after ovulation or five days before the next ovulation. In a blind study, the location of the corpus luteum (left or right ovary) as determined by ultrasonography agreed with a previous determination of side of ovulation by palpation in 88% of 40 mares on days 0–14. In the remaining 12% and in all of 12 estrous mares, the location was recorded as uncertain. The ultrasound instrument was judged effective for monitoring and evaluating follicles and corpora lutea.

Ultrasonic evaluation of the reproductive tract of the mare: Ovaries

Ultrasonography is useful for monitoring the dynamic follicular and luteal changes of equine ovaries, since it permits rapid, visual, non-invasive access to the reproductive tract. A 5 MHz transducer has greater resolving power and is far more suitable for evaluation of ovaries than a 3–3.5 MHz transducer. Follicles as small as 2–3 mm can be seen and the corpus luteum can usually be identified throughout its functional life. In a study involving daily ovarian examinations, there was a pronounced change in shape of the preovulatory follicle from a roughly spherical to a pear-shaped or oblong form in 66% of the ovulatory periods, This change usually occurred on the day preceding ovulation. The occurrence of ovulation was detectable by the disappearance of a large follicle. In addition, the ovulation site on day 0 was characterized by an intense echogenic area in 88% of 32 ovulations. The developing corpus luteum retained the echogenicity for a mean of 2.4 days. In a blind study, the location of the corpus luteum, as determined by ultrasound, agreed with a previous independent determination of the side of ovulation by palpation in 88% of the 40 bred mares on days 0–14. In all of the 12 mares that were in estrus, the location of the corpus luteum could not be ascertained. In another study, the corpus luteum was identified for a mean of 16 days in 14 estrous cycles. One or more days before the corpus luteum became ultrasonically unidentifiable, it developed increased echogenicity in 36% of the mares, indicating greater tissue density. It is concluded that ultrasonic evaluation of the corpus luteum is superior to digital evaluation by rectal palpation. Some of the potential applications of ultrasonic examination of the ovaries include: 1) obtaining important, sometimes definitive, information by a single examination for judging whether a mare has entered the ovulatory season, 2) aiding in estimating the stage of the estrous cycle, 3) detecting double preovulatory-sized follicles which are in close apposition and difficult to discern by palpation, 4) detecting failure of ovulation or anovulatory estrus by the absence of a corpus luteum, 5) differentiating a persistent corpus luteum from anovulatory or anestrous conditions, 6) diagnosing certain pathological conditions such as peri-ovarian cysts and ovarian tumors, and 7) diagnosing anovulatory hemorrhagic follicles.

Seasonal effects on ovarian follicular development in pony mares.

To define ovarian follicular kinetics in the equine ovary during anestrus and the breeding season, the follicular population of pony mares was investigated at mid-anestrus and at the beginning and end of the breeding season. There was a clear effect of season on the exit of reserve (primordial and initiated) follicles since at the beginning of the breeding season we noticed a higher mitotic index for the smaller preantral follicles, leading to an accumulation of small and medium antral follicles. In contrast, the ovaries sampled during anestrus or at the end of the breeding season were very similar; only preovulatory development was lacking in anestrous ovaries. However, atresia was unaffected by season.

The steroidogenic ability of various cell types of the equine ovary

The fully developed follicular wall consisting of theca interna and granulosa cells after antrim formation, granulosa cells alone, corpus hemorrhagicum, corpus luteum, corpus albicans and stromal tissue were collected from ovaries of different mares. All cell preparations were incubated with progesterone-4- 14C and some were also incubated with 17-hydroxyprogesterone-4- 14C androstenedione-4- 14C or testosterone-4- 14C to study the relative activities of the different enzymes involved in the biosynthesis of estrogens from progesterone. Because of the differences in amount of extracellular fluid and connective tissue the yields of the different metabolites were expressed per mg DNA as well as per unit weight of tissue. The follicular walls showed active 17α-hydroxylase, 17,20-lyase, 17β-dehydrogenase and aromatizing enzyme systems. Granulosa cells alone showed a similar pattern except that the aromatizing system did not seem to be proportionately as active. The recently ovulated corpus hemorrhagicum showed high 17α-hydroxylase, 17,20-lyase and 17β-dehydrogenase activities but little aromatizing activity, while the corpora lutea showed 17α-hydroxylase and (17-hydroxyprogesterone)-20-oxido-reductase activities but low 17,20-lyase and no measurable formation of estrogens. When androstenedione or testosterone were substrates, both follicular walls and corpora lutea produced estrogens in similar amounts; the decreased lyase activity in the luteal cells, therefore, appears to account for the lack of estrogen production from C 21 precursors.

A histological study of non-follicular cysts in the ovulation fossa region of the equine ovary.

AbstractForty‐two unselected ovaries from adult mares were examined histologically, and with histochemical methods for mucins. A considerable part of the surface of the ovulation fossa was directly covered by columnar epithelium, with many ciliated cells. This epithelium, which was distributed mainly on the anterior side of the ovulation fossa, closely resembled the contiguous epithelium of the infundibulum of the oviduct, was frequently folded, and gave rise to short clefts projecting into the ovarian substance. The remainder of the ovulation fossa was covered by non‐ciliated, low cuboidal or squamous epithelium, lacking folds or clefts.“Fossa cysts,” up to 6.5 mm in diameter, were observed in the ovarian tissue around the ovulation fossa in 27 (64%) of these ovaries. Both simple and branched, tubular and vesicular forms were present, and all were blind‐ending. Their epithelial lining cells, which varied from simple squamous to columnar in type, were frequently ciliated. Many fossa cysts contained secretions histochemically similar to those of the columnar epithelium of the ovulation fossa and infundibulum. Both sialic acidcontaining and neutral mucins were present. It is suggested that these cysts were probably derived by ingrowth from the columnar epithelium of the ovulation fossa. This epithelium may be of müllerian duct origin.

Cholesterol formation by granulosa and thecal cells of equine follicle.

Granulosa and thecal cells were separated from the estrous follicle of an equine ovary. The individual cell types were determined to be essentially homogeneous by microscopic examination, although the thecal layer contained both theca interna and externa cells. The 2 classes of cells were incubated with acetate-l-C in vitro and labeled cholesterol was isolated and crystallized to constant specific activity. (Endocrinology 78: 214,1966) T in vitro metabolism of acetate-1-C by isolated granulosa and thecal cells of the equine ovarian follicle was studied in an effort to evaluate the ability of these individual cell types to form steroid hormones. The conversion of radioactive acetate to steroids and cholesterol by the perfused porcine ovary (1) and by the human ovarian follicle in vitro (2) has been described but the activities of the individual cells that comprise the follicle have not yet been reported. Techniques for the separation of granulosa and thecal cells have been developed (3) and the conversion of acetate-1C to labeled cholesterol by these cells is the subject of the present communication. Materials and Methods The ovaries were obtained from a mare at the time of sacrifice and a ripe follicle was dissected from one ovary. The appearance of the follicle and the old corpus luteum in the contralateral ovary indicated that the animal was in estrus. This was further substantiated by hormone assay of the follicular fluid (Table 1) carried out as previously described (4). The follicle was 4X4.5X5 cm and contained 40 ml of follicular fluid. The follicle was incised and the granulosa (184 mg) separated from the thecal layers (268 mg) as previously described (3). The isolated granulosa cells were essentially homogeneous on microscopic examination and the thecal layer consisted of both theca interna and externa cells denuded of the granulosa. The Received June 28,1965. This work was supported in part by Grant AM 05739 from the USPHS. thecal layer was minced with scissors prior to use. The separated cells were incubated with 50 nc of acetate-l-C (278 jic/mg) in Krebs phosphosaline buffer at pH 7.4 for 3 hr with 10 /M DPN and 10 MM ATP. The incubation was stopped by the addition of 10 volumes of hot acetone and the mixture was filtered. The acetone-aqueous mixture was evaporated under reduced pressure and the aqueous residue was extracted 3 times with 6 volumes of CHC13. The remainder of the extraction was carried out as previously described (2). A pentane-90% methanol partition separated the more polar steroids from the sterols and lipids. The 90% methanol extract was partitioned into neutral and phenolic fractions which were subjected to column and paper chromatography. The pentane fraction was subjected to digitonin precipitation. The digitonin-precipitable material was mixed with carrier cholesterol, the dibromide formed and free cholesterol regenerated (1). The cholesterol was crystallized to constant specific activity from methanol. Radioactivity was measured in duplicate in a Nuclear-Chicago low background counter with an efficiency for C of 36 %, and cholesterol was measured by the Lieberman Burchard reaction (5).