Rabbit Ovary Research Articles

EVIDENCE FOR THE INHIBITORY EFFECT OF 5‐HYDROXYTRYPTAMINE AT CENTRAL AND PERIPHERAL SITES ON OVULATION IN RABBITS

1. The effect of 5-hydroxytryptamine (5-HT) has been investigated on ovulation per se as well as on induced ovulation in rabbits. 2. 5-HT administered intracerebroventricularly (i.c.v.) did not induce ovulation per se. 3. The ovulation was induced by coitus, subcutaneous administration of progesterone and intravenous administration of cupric acetate. 4. Postcoital and progesterone induced ovulation was found to be blocked by i.c.v. administered 5-HT. 5. Cupric acetate induced ovulation was, however, not found to be blocked by i.c.v. administered 5-HT. 6. Intraperitoneal administration of 5-HT was found to block cupric acetate induced ovulation. 7. It is concluded that 5-HT exerts an inhibitory control over ovulation by acting at central as well as at peripheral sites in rabbits.

NAD +-dependent 15-hydroxyprostaglandin dehydrogenase: Immunochemical characterization of the lung enzyme from pregnant rabbits

A polyclonal antibody was produced in guinea pig against the lung NAD +-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000 g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.

Neuropeptide Y reduces ovarian blood flow in the rabbit

Neuropeptide Y-containing nerve fibers have previously been demonstrated to innervate the mammalian ovary. These nerve fibers innervate primarily the vasculature. In this study we have developed a method for in vivo measurement of the ovary blood flow rate by means of the 133Xe method. Using this technique we measured the ovary blood flow rate and investigated the dose-response relationship between close intraarterial-injected NPY and the ovary blood flow rate. A monoexponential washout curve for 133Xe was found for the whole washout process, ensuring that the blood flow rate at any time could be calculated from the curve. We found a mean blood flow rate in the nonpregnant rabbit ovary at 43.6±4.4 ml·(100 g) −1· min −1 (mean±SEM). Injection of NPY (20, 200, 2000 pM) in the aorta close to a. ovarica resulted in a dose-dependent decrease in the ovarian blood flow rate with a maximum reduction to 40.7±6.3% (mean±SEM) of the control blood flow rate. These findings make it likely that receptors able to interact with NPY are present in the vasculature of the rabbit ovary.

Inhibitors of lipoxygenase increase the ovulation rate in the in‐vitro perfused luteinizing hormone‐stimulated rabbit ovary

In order to determine whether leukotrienes, products of the lipoxygenase pathway, are involved in ovulation, pairs of rabbit ovaries were treated with the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) while being perfused in vitro. The control ovaries from each rabbit received luteinizing hormone (LH) (1.5-2.25 micrograms ml-1) while the contralateral ovaries were treated with LH + NDGA (100 microM) or LH + CA (100 microM). The numbers of ovulations from both the LH + NDGA- and LH + CA-treated ovaries were significantly higher (P less than 0.05) than from their respective LH-stimulated controls. Treatment with NDGA alone in the perfusate did not cause any ovulation, while CA alone caused one ovulation from one of six ovaries perfused. Ovarian tissue levels of prostaglandins after 7 h of perfusion with LH + NDGA or with LH alone showed that, in five of the six ovaries perfused in this group, the tissue levels of PGE2, 6-keto-PGF1 alpha and PGF2 alpha were higher in the presence of NDGA. The mean differences were significant (P less than 0.05) for prostacyclin but not significant (P greater than 0.05) for PGE2 and PGF2 alpha. Our interpretation of the findings is that, when used for blocking the lipoxygenase pathway, NDGA and CA increase the substrate availability for the cyclo-oxygenase pathway of arachidonic acid metabolism, resulting in a net increase in prostaglandins. The increased ovarian levels of prostaglandins, especially prostacyclin, may cause the observed increase in ovulation rate. Consequently, although the leukotrienes may be involved in the mechanism of ovulation in the rabbit, their effects appear to be less pronounced than those of prostaglandins.

Immunological and Enzymological Localization of Carbonyl Reductase in Ovary and Liver of Various Species

The tissue distribution of carbonyl reductase in ovary and liver of various animal species was investigated by measuring the reduction of 13,14-dihydro-15-keto-prostaglandin F2a, a specific substrate for rat ovarian carbonyl reductases, and by means of Western blotting analysis using anti-rat ovarian carbonyl reductase antibody. The highest ovarian carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was found in rat among ten animal species tested, followed by hamster and monkey. The immunoreactive protein was detected in hamster and monkey ovaries. Although carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was not detectable in non-pregnant rabbit ovary, pregnant rabbit ovary showed not only moderate activity but also immunoreactivity with anti-rat ovarian carbonyl reductase antibody. On the other hand, carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was detected in hepatic tissue of all the species tested, except for rat and left-eye flounder. Immunoreactive proteins were present in hepatic tissue of various species that exhibited measurable carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a.

Prolactin Inhibits Plasminogen Activator Activity in the Preovulatory Follicles

The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 10(2), or 10(3) ng/ml. PRL at 10(3) ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 +/- 0.08 to 28.4 +/- 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity.

The recovery of follicular oocytes from the rabbit ovary during prepubertal age and in vitro maturation of their oocytes.

The recovery of oocytes from minced ovarian tissues was examined as well as the ability of their oocytes to mature to Metaphase II in immature rabbits (2-, 3-and 4-month-old), treated with PMSG or not. The number of visible large follicles (over 1 mm in diameter) on the ovary, as well as ovarian weight, increased with advancing age and the increase was remarkable in 3-and 4-month-old PMSG-treated does. Blood follicle formation was marked only in PMSG-treated does 4-month-old. In histology, many antral follicles were grown in 3-and 4-month-old does; especially the number of healthy follicles >700μm in diameter was markedly increased in 3-month-old PMSG-treated does. Ratios of the number of healthy antral follicles >700μm in diameter were still higher in the PMSG-treated does than in non-treated does. In oocytes recovered by mincing ovaries using a pair of razors, only oocytes surrounded by a thick layer of cumulus cells (Type I oocyte) were selected and cultured for in vitro maturation. The number of Type I oocytes was increased by PMSG treatment at each age; the number in 3-month-old does was the highest (3 times that in non-treated does). Ratios of Type I oocytes which emitted the first polar body in in vitro culture increased with advancing age up to 4 months, though the ratios remained at lower levels than in adult does. There were no differences in the maturation ratios between PMSG-treated and non-treated groups. These results show that oocytes to undergo in vitro maturation are obtainable from the ovary in immature rabbits, especially in 3-month-old PMSG-treated does.

The Effect Of Placenta On Lactogen Receptor In Pseudopregnant Rabbits

This study examines the effect of placenta on the evolution of lactogen receptor in virgin pseudopregnant rabbit ovary, adrenal gland and mammary gland. Pseudopregnancy was induced with human chorionic gonadotropin. Does were injected with vehicle or placenta daily beginning on day six of the pseudopregnancy. Vehicle-treated rabbits during pseudopregnancy demonstrated a peak of ovarian lactogen receptor on day eight of pseudopregnancy. After treatment with placental homogenate a shift of this peak to twenty days of pseudopregnancy occurred. Lactogen receptor in adrenal and mammary gland membranes had peak receptor concentrations on day 14 of pseudopregnancy. Injection of placenta induced a shift to day 17 and days 17-20 in mammary and adrenal membranes, respectively. Serum concentrations of progesterone, estradiol, 20 alpha dihydroprogesterone and prolactin in placenta-treated groups were not significantly different from those of vehicle-treated groups. Treatment of pseudopregnant does with a composite of hormones at the concentrations found in placental homogenate produced no modulation of tissue lactogen receptor. Fractionation of 20-day pregnant rabbit placenta revealed that 80% of this activity could be found in the acetone extract while 20% was in the bicarbonate extract. These observations suggest that increases of lactogen receptor in ovary, adrenal and mammary glands occur during pseudopregnancy in rabbits and it is further concluded that placenta can alter these receptor induction patterns to ones similar to those seen in these tissues during pregnancy.

In vivo administration of allopurinol affects ovulation and early embryonic development in rabbits

The potential role of the hypoxanthine-xanthine oxidase system in human chorionic gonadotropin-induced ovulation, nuclear maturation, and preimplantation development was investigated in the rabbit by use of the xanthine oxidase inhibitor allopurinol. Allopurinol (50 mg/kg) or vehicle was injected, followed by human chorionic gonadotropin (100 IU). Administration of inhibitor or vehicle was repeated 3 hours later. Ovaries and ovulated ova were inspected 12 or 24 hours after human chorionic gonadotropin administration for follicle rupture, cumulus dispersal, and nuclear maturation. Ova were inseminated in vitro and assessed for development to the blastocyst stage, up to 96 hours after insemination. Allopurinol significantly reduced ovulatory efficiency, defined as the percent of large follicles that ovulate, and decreased the percent of ova with cumulus dispersal. Allopurinol significantly inhibited morula and blastocyst formation. These data suggest that the xanthine oxidase system may play a role in ovulation and early embryonic development.

The intraovarian progesterone modulation of follicle development in the rabbit ovary

Intraovarian progesterone levels were manipulated by surgically adjusting the number of corpora lutea (CL) present in rabbit ovaries and this model was used to study the local effect of luteal progesterone on growth of follicles. The results show that when a single CL or several CL were present, follicle growth was inhibited. However, when all CL on one ovary were removed, increased numbers of follicles grew even when a single CL was present in the contralateral ovary. These findings show that progesterone inhibits follicle growth and that at least part of its action is local, i.e., exerted within the ovary. Additionally, ovarian blood vessels and periovarian lymph ducts were cannulated, and samples were collected and analyzed for steroid and protein content. The results show that when CL were present, ovarian vein progesterone levels were elevated 10 – 30-fold over levels in ovaries without CL; this high concentration points to the blood vascular system as the principal carrier of the steroid within the ovary. Analysis of lymph showed that protein content was consistently high and that the progesterone concentration was not significantly altered with the presence of CL; these two findings show that ovarian capillaries are extremely permeable to proteins, but the unexpectedly low concentrations of progesterone in lymph may signal an intraovarian countercurrent mechanism by which it is returned to the blood.

Effects of systemic administration of indomethacin on ovulation, luteinization, and steroidogenesis in the rabbit ovary

Indomethacin blocks ovulation in human chorionic gonadotropin-stimulated rabbits. Experiments were done with an in vitro ovarian perfusion system to investigate whether indomethacin affects luteinization and steroidogenesis. Indomethacin (10 mg/kg) was administered in combination with human chorionic gonadotropin (100 IU) via a marginal ear vein, and a second dose of indomethacin was given 8 hours later. Control animals received vehicle in place of indomethacin. Laparotomy was performed 24 hours after the initial treatment. The presence of unruptured follicles and corpora lutea was recorded and the ovaries were perfused in vitro for 3 hours. Progesterone, prostaglandin F2 alpha, prostaglandin E2, and 6-keto-prostaglandin F1 alpha were measured in samples obtained at 0, 30, 60, 120, and 180 minutes from the circulating perfusion medium entering and exiting the ovary. At the end of the perfusion all ovaries (12 treated and 10 controls) were fixed for histologic analysis. Ovulation occurred in all control ovaries but in none of the indomethacin-treated ovaries. The mean number of unruptured follicles per ovary in the treated group was not significantly different from the number of corpora lutea plus unruptured follicles per ovary in the controls. Cells in both groups were qualitatively similar in ultrastructure; abundant lipid droplets, smooth endoplasmic reticulum, and mitochondria were seen. Secretion rates of progesterone and prostaglandin did not differ between the two groups during the 3-hour perfusion period. These results suggest that transformation of granulosa cells into fully functional luteal cells can occur in the absence of follicular rupture.

Developmental potential of rabbit oocyte matured in vitro: the possible contribution of prolactin.

The present study was undertaken to define hormonal conditions for in vitro maturation that support subsequent fertilization and embryonic development. Follicular oocytes were recovered from nonstimulated rabbit ovaries and cultured for 12 h in Brackett's medium supplemented with or without hormones. Matured oocytes were inseminated in vitro and transferred 12 h later to Ham's F-10 medium supplemented with 20% fetal calf serum. The initial cleavage frequency of matured oocytes in Brackett's medium was comparable to the frequency of development for in vitro-matured oocytes under various hormonal conditions. However, the addition of estradiol (E2, 1 microgram/ml) to incubation medium containing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased significantly (p less than 0.001) the percentage of embryos achieving morula or blastocyst formation (16/98, 16.3%), as compared to the mature oocytes in medium containing LH, LH plus FSH, or no hormone. The addition of prolactin (PRL) to the maturation medium increased the percentage of development to organized embryos in a dose-dependent manner. In vitro-matured oocytes in medium containing LH, FSH, and PRL exhibited a significantly (p less than 0.001) lower incidence of developmental competence (5/95, 5.3%) than oocytes matured in the presence of E2 in conjunction with pituitary hormones (43/89, 48.3%). These results demonstrate that hormonal composition in the environment of the oocyte is critical for acquisition of developmental capacity. PRL as well as E2 appears to be an important constituent in the process of oocyte maturation, promoting preimplantation embryonic development.

Ultrastructural and histochemical investigations of Call-Exner bodies in rabbit Graafian follicles.

In a histological survey of 19 mammalian species, Call-Exner bodies of conventional size and appearance were found in only 5, namely, human, rhesus monkey, rabbit, guinea-pig and sheep. Rabbit ovaries were used for characterizing these bodies using quantitative histochemistry, lectin binding and electron microscopy. Call-Exner bodies were topographically distinct lacunae of the extracellular space probably containing hyaluronic acid with proteoglycan complexes. The staining characteristics of the antrum and Call-Exner bodies were generally similar. However, in contrast to the antrum, the smaller lacunae contained suspended filaments with a distinctive peripheral membrane upon which a rosette of granulosa cells was resting. The membrane and narrow intercellular clefts probably prevent much exchange of large glycosaminoglycan complexes with the antrum. The origin and significance of Call-Exner bodies require further study, but it is clear that they are associated with secretion rather than with necrosis as sometimes suggested.

Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary

The involvement of cyclic AMP (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and in-vitro perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without dibutyryl cyclic AMP [Bu)2cAMP) at 10(-3), 10(-4) or 10(-5) M for 4-12 h. At 4 h spontaneous meiotic maturation was significantly inhibited by (Bu)2cAMP (P less than 0.025). With prolonged incubation, spontaneous maturation progressed despite exposure to (Bu)2cAMP. When ovaries were continuously perfused in vitro for 12 h with (Bu)2cAMP (10(-3) M) or medium alone, (Bu)2cAMP stimulated ovarian progesterone production, but did not affect ovulation or maturation of follicular oocytes. When ovaries were perfused in vitro with or without (Bu)2cAMP (10(-3), 10(-4) or 10(-5) M) for the first 2 h and then transferred to medium without (Bu)2cAMP for an additional 10 h, ovulation did not occur, but transient exposure to (Bu)2cAMP stimulated a dose-related increase in maturation of follicular oocytes. Degeneration of follicle-enclosed oocytes and cumulus-oocyte complexes was not affected by exposure to (Bu)2cAMP. These results suggest that transient, but not continuous, elevation of cAMP after the gonadotrophin surge may be required for the initiation of oocyte maturation.

Development of the perifollicular capillary network. Autoradiographic and morphometric studies in the rabbit ovary

The published data give a report on systematic examinations on the development of the perifollcular capillary network in the theca interna in rabbit ovaries. The follicle size and the number of capillaries were investigated by means of morphometric methods. The extent of proliferation of granulosa and endothelial cells was studied autoradiographically by determining the labelling index (LI). The results show that the perifollicular capillary network is initiated only when the stratum granulosum has developed into a multilayered cell population. After that the number of capillaries increases linearly with increasing follicle size, independently of the oestrus cycle. During follicle growth at different times in the oestrus cycle the decrease and increase in the LI of granulosa cells is followed by a corresponding decrease and increase in the LI of endothelial cells several hours later. It should be noted, however, that the proliferation of endothelial cells in follicles with a diameter of 900 μm continues, although the decreasing LI of granulosa cells already indicates a reduced follicle growth. The number of labelled endothelial cells in atretic follicles decreases only after a decrease in proliferation of granulosa cells. In conclusion, it can be noted that the follicle growth and function is not influenced by variations in the number of capillaries in the theca interna. The curve of LI of granulosa and endothelial cells suggests that the granulosa cells have a certain regulatory function with respect to the growth of the capillary network in the theca interna.

Effects of age at unilateral ovariectomy on compensatory responses of remaining ovary in rabbits.

Effects of age at unilateral ovariectomy (ULO) on compensatory responses of the remaining ovary were examined in rabbits. ULO was performed on 8 does for each group at 30 (group 30), 90 (group 90), 160 (group 160) and 230 days of age (group 230). Eight intact does were served as controls. Four does of each group were laparotomized and remaining ovaries were removed at first estrus (over 250 days of age). Numbers of large follicles on ovarian surface ( ?? 1.5 mm in diameter) per doe in all groups were nearly 10 and these values of ULO groups were twice as large as controls. Mean ovarian weight and volume of group 90 were the highest, and those of group 30 were second. The values in these ULO groups showed significant differences in comparison with controls. In histology of these ovaries, numbers of large, healthy follicles ( ?? 1 mm in diameter) per ovary in ULO groups were roughly double compared with those in controls. There was a tendency that the number of small, atretic antral follicles (< 1 mm in diameter) was increased in ULO animals operated at young age. The number of atretic antral follicles was closely correlated with ovarian weight and volume. To induce ovu-lation, the remaining 4 does of each group were injected with 20 IU of hCG. The num-bers of corpora lutea per doe of all groups were about 10 with no significant difference among groups. These results suggest that ULO in the immature period induces marked compensatory hypertrophy after puberty and that, regardless of age at ULO, the re-maining ovary in the ULO animal fulfills the function of both ovaries in the intact animal.

Effect of ovarian endometriosis on ovulation in rabbits

To study the relationship between endometriosis and ovulatory dysfunction, we induced ovarian endometriosis in the rabbit model Adipose tissue was placed in the contralateral ovary as a control. Ovulation was induced with human chorionic gonadotropin, and ovulation points were counted before and after induction of endometriosis. Periovarian adhesions were graded, and ovaries were histologically examined. A significant decrease in the number of ovulation points was observed in ovaries with endometrial tissue (p = 0.001) but not in ovaries that contained adipose tissue (p = 0.095). Periovarian adhesions decreased the number of ovulation points (p less than 0.01) in ovaries that contained adipose or endometrial tissues. Multivariate analysis demonstrated that an increase in adhesion severity was correlated with a decrease in the number of ovulation points (p less than 0.05), but endometrial tissue was not (p = 0.45). We conclude that, in the rabbit model, minimal ovarian endometriosis impairs ovulation primarily through a mechanism related to periovarian adhesions.

Long-term effects of immunization with porcine zona pellucida on rabbit ovaries

Groups of rabbits were immunized with 4 doses of porcine zona pellucida antigen along with different adjuvants. The presence of antibody after immunization was checked periodically using different assay systems. The impact of immunization on ovaries was evaluated at different time intervals up to 2 years. Results of the study showed generation of high titre antibodies which could be detected until the end of the period of observation. The antibodies specifically bound to zona pellucida in situ were demonstrated by immunoperoxidase staining, and in vitro by inhibition of enzymatic digestion of zonae. Histologically, antibodies had numerous pathological effects on ovaries including thinning of the cortex, aberrant cell clusters, stromal cell hyperplasia, perifollicular fibrosis, irregular zona formation and arrest of follicle development. These changes could be correlated with the length of exposure to antibodies. It is concluded that heat-solubilized porcine zona (HSPZ) protein is a very strong immunogen and causes infertility by arresting the maturation of developing follicles. There is a need to identify the sperm receptor so that immunization could selectively impair sperm-egg interaction.

Prostaglandin-E 2 9-ketoreductase from swine kidney. Production of antisera and application to development of a radioimmunoassay

Prostaglandin-E 2 9-ketoreductase (PGE 2-9-KR, EC 1.1.1.189), the enzyme which catalyzes the reaction from prostaglandin E 2 (PGE 2) to prostaglandin F 2α (PGF 2α), was purified 580-fold from swine kidney. The molecular mass of the enzyme determined by SDS-gel electrophoresis was 33 kDa. Antiserum against the purified enzyme was raised in three rabbits. The antiserum was able to precipitate PGE 2-9-KR from swine kidney and to crossreact with PGE 2-9-KR from several reproductive organ tissues, such as rabbit ovary, rabbit corpus luteum, rabbit endometrium and human decidua vera. When swine kidney PGE 2-9-KR was labelled with 125I and incubated with affinity-purified antiserum in the presence of increasing amounts of unlabelled enzyme, competitive binding of the unlabelled enzyme to the antibody was observed. A radioimmunoassay for the quantitation of the enzyme was developed. The standard curve was linear from 5 to 500 ng enzyme. The intra- and interassay coefficients of variation were 6.4 and 13.2%, respectively. The assay may be useful for the quantitation of PGE 2-9-KR in several tissues under various physiological conditions.

Presence of immunoreactive atrial natriuretic peptide in follicular fluid, ovary and ovarian perfusates

Immunoreactive atrial natriuretic peptide (ir-ANP) was measured in the follicular fluid of pig ovarian follicle, and rabbit ovarian homogenates and perfusates using a specific radioimmunoassay (RIA). Serial dilution curves made with the extracts of follicular fluid, ovarian homogenates and perfusates using Sep-Pak C18 cartridges were parallel with the RIA standard curve. On gel filtration chromatography and reverse phase HPLC, all extracted materials showed high and low molecular weight forms of ir-ANP. The amount of ir-ANP in rabbit ovary was 40.70±0.39 pg/mg and that in follicular fluid of pig ovarian follicle was 18.88±2.49 pg/ml.