Estradiol secretion by the ovary of 19-day-old hypophysectomized and sham-operated chick embryos

The amounts of estradiol released into culture media by ovaries from 19-day-old hypophysectomized (decapitated) and sham-operated chick embryos were determined by RIA. Per single ovary, ovaries from decapitated embryos secreted slightly less estradiol than ovaries from sham-operated ones during a 4-h culture period (837 +/- 413 vs 935 +/- 378 pg), but this difference was not significant. On an ovarian weight basis, ovaries from decapitated embryos secreted slightly more estradiol than ovaries from sham-operated ones (1.15 vs 0.91 ng), but this difference was not significant either. It is concluded from these results that the hypophysis does not control estradiol secretion by the ovary in the 19-day-old chick embryo.     

Inhibin-like activity in ovarian venous serum after unilateral ovariectomy in prepubertal gilts

To examine a role for inhibin in compensatory ovarian hypertrophy after unilateral ovariectomy (ULO) of prepubertal gilts, changes in inhibin activity in ovarian venous blood were estimated by bioassay. Three groups of 130-day-old gilts were unilaterally ovariectomized after collecting blood from an ipsilateral ovarian vein (Day O); blood samples were obtained from the remaining ovary on Day 2, 4, or 8. Coetaneous gilts underwent sham ovariectomy on Day 0, and venous blood was collected from both ovaries on Day 2, 4, or 8. An assay for inhibin activity, which measured inhibition of secretion of follicle-stimulating hormone (rFSH) by rat pituitary cells in culture, was validated for serum samples. Presumptive inhibin activity was always greater in ovarian venous serum than in peripheral serum samples. In the ULO groups, inhibin activity (in terms of a house reference preparation) in ovarian venous serum was 55 +/- 13 micrograms/ml (means +/- SE, n = 13) on Day 0, 251 +/- 79 (n = 5) on Day 2, 275 +/- 111 (n = 4) on Day 4, and 68 +/- 14 micrograms/ml (n = 4) on Day 8. The five-fold increases on Days 2 and 4 were significant (p less than 0.05). In contrast, no significant differences in inhibin activity were detected between ovarian venous serum (within gilts) or between Days 2, 4, and 8: 82 +/- 29, 73 +/- 30, and 99 +/- 48 micrograms/ml (n=4/day) in control groups. These results demonstrate that, in prepubertal gilts, the remaining ovary's response to ULO includes a major increase in release of inhibin-like activity.     

Successful cryopreservation of mouse ovaries by vitrification

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.     

Time course of sympathetic denervation of the rat ovary after freezing its nerve supply

Brief freezing of the ovarian vascular pedicle in rats reduced ovarian noradrenaline concentration, measured with HPLC, by 67% (P less than 0.01). Freezing of the ovarian suspensory ligament caused a 22% reduction (P less than 0.01) and 33% reduction (P less than 0.05) in 2 experiments. The numbers of adrenergic nerve terminals detected by fluorescence microscopy after these procedures were similarly reduced (P less than 0.01). Freezing of both the pedicle and the ligament produced complete sympathetic denervation in about 50% of the ovaries. From Days 2 to 10 after operation no noradrenaline or nerve terminals were detected in 14 out of 27 ovaries. Nerve terminals were also eliminated from the oviduct. Reinnervation of the ovary began between Days 12 and 30. It is concluded that the adrenergic innervation of the ovary is predominantly through nerves that accompany the vascular supply to the ovary and the ovarian suspensory ligament. Freezing of these routes is a simple and relatively atraumatic means of denervating the ovary for experimental studies.     

Characterization of ovarian function in granulocyte-macrophage colony-stimulating factor-deficient mice

During the estrous cycle and early pregnancy, lymphohemopoietic cytokines and chemokines contribute to the regulation of ovarian function by orchestrating the recruitment and activation of leukocytes associated with the ovulatory follicle and corpus luteum. The purpose of this study was to investigate the physiological role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the ovary, utilizing mice genetically deficient in GM-CSF. Our results show that the mean duration of the estrous cycle in GM-CSF-deficient (GM-/-) mice was extended by 1.5 days (mean +/- SE, 4.9 +/- 0.3 vs. 6.5 +/- 0.5 days for GM+/+ and GM-/- mice, respectively). Similar ovulation rates were observed in immature superovulated mice (31.8 +/- 7.7 vs. 28.9 +/- 6.4 oocytes per mouse) and adult naturally cycling mice (10.4 +/- 0.8 vs. 10.3 +/- 0.8 oocytes per mouse). Furthermore, comparable numbers of oocytes were released from GM+/+ and GM-/- ovaries in an in vitro perfusion model. However, ovaries in pregnant GM-/- mice were found to comprise fewer cells and synthesize less progesterone (141.6 +/- 10.3 vs. 116.5 +/- 6 nM plasma), although the duration of pseudopregnancy was unaltered by GM-CSF deficiency (11.0 +/- 0.2 vs. 11.0 +/- 0.5 days). Immunohistochemical staining of leukocytes in the ovary during the periovulatory period indicated that the size and composition of ovarian leukocyte populations were unaltered in the absence of GM-CSF. However, an effect of GM-CSF deficiency on the activation phenotype of ovarian leukocytes was indicated by a 57% increase in mean secretion of nitric oxide in in vitro-perfused GM-/- ovaries, and diminished major histocompability complex (MHC) class II (Ia) expression in ovarian macrophages and/or dendritic cells (30.5 +/- 7. 2% vs. 9.1 +/- 1.8% positive stain in GM+/+ and GM-/- ovaries, respectively). Furthermore, ovarian macrophages and neutrophils were diminished in number after parturition, with significantly decreased CD11b+ (Mac-1) staining in the stromal region of postpartum GM-/- ovaries (6.7 +/- 0.6 vs. 3.6 +/- 0.7% positive stain). In summary, GM-CSF does not appear to be essential for ovarian function but may play a role in fine-tuning the activation status and adhesive properties of ovarian myeloid leukocytes. Aberrant activation of these cells appears to compromise the luteinization process and the steroidogenic capacity of the corpus luteum during early pregnancy in GM-CSF-deficient mice.     

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.     

Ovarian sympathectomy in the guinea pig

The influence of ovarian adrenergic nerves on follicular growth during the estrous cycle in the adult guinea pig was ascertained by comparing follicular development in control and chemically sympathectomized ovaries from the same animal. Selective ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian membranous sac (bursa) on day 2 of the cycle (day 1 = day of estrus). The contralateral surgically closed ovarian bursa was injected with solvent used for 6-hydroxydopamine. Animals were laparotomized on days 5, 10 and 14 of the cycle. Blood from the utero-ovarian vein was collected bilaterally for measurement of progesterone and androstenedione. The ovaries were processed for histologic examination, and the number of follicles in each ovary was analyzed morphometrically. Sympathectomy on day 2 caused a decrease in healthy preovulatory follicles (greater than 700 micron diameter) on day 10 of the cycle. There were no differences in ovarian weights or the total number of follicles per ovary at this time. On days 5 and 14 of the cycle, there were no differences in ovarian weights, total number of follicles per ovary or follicles in any size classification. Sympathectomy did not alter progesterone levels in the utero- ovarian vein as compared to contralateral control levels. From control ovaries, there was a significant increase in progesterone in the blood of the utero-ovarian vein on day 10 but venous levels of progesterone from sympathectomized ovaries were not significantly different at any day of the cycle. In the venous effluent from sympathectomized ovaries, androstenedione was elevated at day 5 compared to days 10 and 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of unilateral sectioning the superior ovarian nerve of rats with unilateral ovariectomy on ovarian hormones (progesterone,testosterone and estradiol) levels vary during the estrous cycle

The present study analyzed the participation of the left and right superior ovarian nerves (SON) in regulating progesterone, testosterone, and estradiol serum levels in unilaterally ovariectomized rats on each day of the estrous cycle. For this purpose, ovarian hormone concentrations in serum were measured in animals with either sham-surgery, unilateral ovariectomy (ULO), unilateral sectioning of the SON, or sectioning of the SON innervation of the in situ ovary in rats with ULO.     

Changes in rabbit corpus luteum progesterone secretion and cellular morphology following unilateral luteectomy or ovariectomy.

The objective of this study was to determine whether removal of corpora lutea (CL) from one ovary (unilateral luteectomy; ULL) or removal of the entire ovary (unilateral ovariectomy; ULO) of pseudopregnant rabbits would cause compensatory growth and progesterone production by the contralateral ovary. Pseudopregnancy was induced in rabbits with hCG (Day 0). On the first day of pseudopregnancy, one group of rabbits received a sham operation (controls), another group underwent ULL, and a third group underwent ULO. On Day 11 of pseudopregnancy, each rabbit underwent laparotomy, the ovarian artery and vein were cannulated, and the ovary(ies) was removed and perfused in vitro for 6 h. The mean CL weight increased by 33% in the ULL group and by 28% in the ULO group as compared to sham-operated controls. Peripheral estradiol and progesterone levels in sham-operated control, ULL, and ULO groups were similar. Ovarian venous estradiol levels were similar in the control and ULL groups, but were significantly increased in the remaining ovary of the ULO group. Both ovarian venous progesterone in vivo and progesterone secretion in vitro increased significantly in contralateral ovaries from ULL and ULO rabbits as compared to sham-operated controls. Progesterone secretion by ovaries perfused in vitro increased significantly in the contralateral ovary of the ULL and ULO groups. Mean number of luteal cells per CL increased significantly in the ULL group, but not in the ULO group. In contrast, luteal cell volume increased significantly in the ULO, but not in the ULL group. The stimuli responsible for increased progesterone production following ULL and ULO result in morphological changes in the remaining CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian features contributing to the variability of PMSG-induced ovulation rate in sheep

In Exp. 1, ovulation rate was measured in three groups of Romanov ewes given two injections of 600 i.u. PMSG 3 weeks apart with the ewes intact (Group I, N = 8), a similar treatment with the ewes intact at the first injection and unilaterally ovariectomized at the second (Group II, N = 8), or unstimulated ewes which were hemispayed at the same time as Group II ewes (Group III, N = 6). In Exp. 2, the follicular population of one ovary was correlated with the number of ovulations induced by 600 i.u. PMSG in the contralateral ovary (10 Romanov ewes). From 8.4 +/- 1.8 (Group I) and 8.2 +/- 3.3 (Group II) CL at the first injection, PMSG-induced ovulation rate at the second injection decreased to 3.9 +/- 1.8 and 3.7 +/- 1.2 in Groups I and II respectively, a value similar for ewes with 1 or 2 ovaries. Furthermore, despite no major changes in the number of antral follicles after the first injection, there was no correlation (r = -0.09) between the response to the two successive injections in intact ewes. Comparison of the ovarian status of the ovary removed before the PMSG injection (Group II ewes of Exp. 1, ewes of Exp. 2) to the number of CL found in the remaining ovary demonstrated that PMSG-induced ovulation rate was not correlated with the overall antral follicle population (r = 0.62 in Exp. 1, r = 0.49 in Exp. 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Unilateral sectioning of the superior ovarian nerve of rats with polycystic ovarian syndrome restores ovulation in the innervated ovary

The present study tested the hypothesis that if polycystic ovary syndrome (PCOS) results from activating the noradrenergic outflow to the ovary, unilaterally sectioning the superior ovarian nerve (SON) will result in ovulation by the denervated ovary, and the restoration of progesterone (P4), testosterone (T) and estradiol (E2) normal serum level. A single 2 mg dose of estradiol valerate (EV) to adult rats results in the development of a syndrome similar to the human PCOS. Ten-day old rats were injected with EV or vehicle solution (Vh) and were submitted to sham surgery, unilateral or bilateral sectioning of the SON at 24-days of age. The animals were sacrificed at 90 to 92 days of age, when they presented vaginal estrus preceded by a pro-estrus smear. In EV-treated animals, unilateral sectioning of the SON restored ovulation by the innervated ovary and unilateral or bilateral sectioning of the SON normalized testosterone and estradiol levels. These results suggest that aside from an increase in ovarian noradrenergic tone in the ovaries, in the pathogenesis of the PCOS participate other neural influences arriving to the ovaries via the SON, regulating spontaneous ovulation. Changes in P4, T and E2 serum levels induced by EV treatment seem to be controlled by neural signals arising from the abdominal wall and other signals arriving to the ovaries through the SON, and presents asymmetry.     

Pulmonary feedback and gestational age-dependent regulation of fetal breathing movements

Prenatal lung development requires fetal breathing movements (FBM). To investigate the dependence of FBM on feedback originating from the lung, we hypothesized that pneumonectomy stimulates FBM. Time-dated fetal sheep underwent bilateral pneumonectomy, unilateral pneumonectomy, or sham surgery at 125-130 days gestation. The incidence of FBM decreased in sham-operated fetuses at 142 days versus 130 days (p = 0.013), but was unchanged across all gestational ages in bilaterally pneumonectomized fetuses (p > or = 0.52). In unilaterally pneumonectomized fetuses, the incidence of FBM remained unchanged until 139 days and was higher than that of the bilaterally pneumonectomized fetuses at 130-136 days gestation (p < or = 0.03). The amplitude of integrated diaphragmatic electromyographic activity (integralEMG(di)) and total respiratory output (frequency of breathing x integralEMG(di)) were lower in pneumonectomized fetuses versus sham-operated fetuses at later gestational ages (p < 0.05). These decreases in integralEMG(di) and total respiratory output were most pronounced at 142 days in bilaterally pneumonectomized fetuses versus sham-operated fetuses (p = 0.006 and 0.016, respectively). Low-voltage electrocortical activity (ECoG) increased, and high-voltage ECoG decreased, in unilaterally pneumonectomized fetuses compared with sham-operated fetuses (p = 0.04). In conclusion, we provide new evidence that feedback from the fetal lung modulates the incidence and various components of phrenic nerve output, suggesting a positive feedback mechanism between FBM and lung development.     

Ultrasonographic study of the effects of the corpus luteum on antral follicular development in unilaterally ovulating western white-faced ewes

The objective of this study was to examine the local effects of the corpus luteum (CL) on ovarian antral follicle development by looking at follicle populations and dynamics in ovaries with or without CL, in unilaterally ovulating ewes, using a retrospective analysis of daily ultrasonographic records. The present report summarises the data from the first luteal phase of the breeding season (August-October; n = 4), a luteal phase in the mid-breeding season (November-December; n = 5), the last luteal phase of the breeding season (January-March; n = 5), and the luteal phase after GnRH-induced ovulations in mid-anoestrus (May-June; n = 4) of western white-faced ewes. Mean daily numbers of 3mm follicles that did not grow any larger were significantly reduced in the CL-containing ovaries of ewes at all periods of study except for the transition to anoestrus. With all scanning periods combined, daily numbers of 3mm follicles not growing further increased (P<0.05) between day 6 and 15 after ovulation in the CL-containing ovaries. Based on mean data for the whole periods of observation, the non-CL-bearing ovaries of ewes in the transition to anoestrus had fewer (P<0.05) follicles growing from 3 to > or =5mm in size before regression compared with the mid-breeding season and mid-anoestrus. The lifespan of follicles reaching > or =5mm in diameter was shorter (P < 0.05) in the CL- compared with non-CL-containing ovaries of anoestrous ewes induced to ovulate with GnRH ((6.5+/- 1.3) and (9.0+/- 1.0) days, respectively). Circulating concentrations of progesterone were lower during both transitional periods (into and out of anoestrus) and mid-anoestrus than during the mid-breeding season (P < 0.001), and were less during anoestrus than during both transitional periods (P < 0.05). It was concluded that CL/luteal structures locally suppressed the growth of ovarian antral follicles to the 3mm size-range except during the transition to anoestrus, but that there was no inhibitory effect of the CL on the growth of ovarian follicles to larger diameters. The presence of CL/luteal structures did not affect the length of the lifespan of follicles reaching > or =5mm in diameter nor the number of ovulations per ovary in cyclic ewes, but shortened large follicle lifespan in anoestrous ewes. Variations in peripheral concentrations of progesterone across the breeding season and between the breeding season and anoestrus did not alter the lifespan of large antral follicles. In the transition to anoestrus and during mid-anoestrus, the presence of the CL in an ovary appeared to maintain follicle development to ovulatory sizes and to increase the rate of turnover of large antral follicles, respectively.     

Treatment of cystic ovarian follicles in dairy cows with chorionic gonadotropin

Thirty dairy cows exhibiting both nymphomania and cystic ovaries - 15 unilateral and 15 bilateral - were injected intravenously with human chorionic gonadotropin (hCG) over a twentyfold dose range, varying from 1.1 to 22.0 IU/kg (0.5 to 10.0 IU/1b) body weight to determine its efficacy of inducing ovulation and subsequent fertilization. Ovulation of cystic and/or noncystic follicles was induced in 28 cows (93.3%), with a higher rate among those with unilateral than bilateral cysts whether based on total (32 vs 22) or mean (2.1 vs 1.5) number of ovulations. While ovulations per cow varied from zero to three, no correlation between dose level of hCG and number of ovulations was evident. Time of ovulation following hCG injection did not differ significantly between unilaterally and bilaterally cystic cows nor between cystic and noncystic follicles, ranging from 23 to 31 +/- 1 hr and averaging 27.3 +/- 1 hr postinjection. A significant difference was found between unilaterally and bilaterally cystic cows in the occurrence of fertilized (11:1), unfertilized (11:6), degenerate (2:2), and unrecovered (8:11) ova.     

Effect of unilateral ovariectomy on compensatory ovarian hypertrophy, peripheral concentrations of follicle-stimulating hormone and luteinizing hormone, and ovarian venous concentrations of estradiol-17 beta in prepuberal gilts

A study was designed to characterize the compensatory ovarian response to unilateral ovariectomy (ULO) in prepuberal gilts and to investigate further the mechanisms involved in compensatory ovarian hypertrophy (COH). Forty-eight crossbred gilts were sham ovariectomized (Sham) or unilaterally ovariectomized at 130 days of age (Day 0). Remaining ovaries in ULO gilts were removed and Sham gilts were bilaterally ovariectomized 2, 4 or 8 days later. A peripheral blood sample was taken before surgery and ovarian venous blood samples were taken before removal of each ovary. Serum estradiol-17 beta (E2) concentrations were determined. Mean wet and dry ovarian weights per ovary on Day 2 for ULO and Sham gilts were 3.4 versus 2.8 and 0.26 versus 0.24 g, respectively. Those weights on Days 4 and 8 were greater (P less than 0.01) for ULO than Sham gilts. Follicular fluid weight per ovary was greater (P less than 0.05) for ULO than Sham gilts on Days 2, 4 and 8. Ovarian venous E2 concentrations were greater (P less than 0.01) for ULO than for Sham gilts on Days 2 and 4 but were similar on Day 8. In a second experiment, 42 prepuberal gilts 130 days of-age were subjected to Sham (n = 18), ULO (n = 18) or bilateral ovariectomy (BLO; n = 6) to evaluate follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion immediately after surgical treatment. Release of FSH within the first 24 h was greater for BLO than ULO and for ULO than Sham gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian innervation develops before initiation of folliculogenesis in the rat

Summary Sympathetic neurotransmitters have been shown to be present in the ovary of the rat during early postnatal development and to affect steroidogenesis before the ovary becomes responsive to gonadotropins, and before the first primordial follicles are formed. This study was undertaken to determine if development of the ovarian innervation is an event that antedates the initiation of folliculogenesis in the rat, Rattus norvegicus. Serial sections of postnatal ovaries revealed a negligible frequency of follicles 24 h after birth (about 1 primordial follicle per ovary). Twelve hours later there were about 500 follicles per ovary, a number that more than doubled to about 1300 during the subsequent 12 h, indicating that an explosive period of follicular differentiation occurs between the end of postnatal days 1 and 2. Electron microscopy demonstrated that before birth the ovaries are already innervated by fibers containing clear and dense-core vesicles. Immunohistochemistry performed on either fetal (day 19) or newborn (less than 15h after birth) ovaries showed the presence of catecholaminergic nerves, identified by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. While some of these fibers innervate blood vessels, others are associated with primordial ovarian cells, thereby suggesting their participation in non-vascular functions. Since prefollicular ovaries are insensitive to gonadotropins, the results suggest that the developing ovary becomes subjected to direct neurogenic influences before it acquires responsiveness to gonadotropins.     

Cryopreservation of mouse ovarian tissue following prolonged exposure to an Ischemic environment.

In cases in which ovarian tissue is to be cryopreserved for tissue or gene banking it is important to maintain its integrity and viability. This study examined how delays between the death of an animal and the collection/cryopreservation of its ovarian tissue influenced follicle viability. Mouse ovaries were placed in PBS+antibiotic (in vitro) or left within the body (in situ) at room temperature for 0, 3, 6, 12, or 24 h following the death of the donor. These ovaries were cryopreserved at 1 degrees C/min on dry ice or in a -84 degrees C freezer using a passive cooling device or by conventional slow cooling (0.3 degrees C/min). The ovaries were grafted under the kidney capsule of ovariectomized recipient mice and collected 2 weeks later, and the size and number of follicles were determined. Cryopreserved ovarian tissue grafted immediately after the death of the donor contained numerous viable and healthy follicles independent of the cooling procedure (dry ice, 134 +/- 32; -84 degrees C, 165 +/- 54; slow, 214 +/- 55 follicles per half ovary). Tissues stored in vitro before cryopreservation retained viable follicles up to 12 h after death (dry ice, 30 +/- 15; -84 degrees C, 86 +/- 45; slow, 93 +/- 33), whereas tissue left in situ had significantly reduced follicle numbers within 3 h of death (dry ice, 36 +/- 12; -84 degrees C, 19 +/- 6; slow, 28 +/- 7). No significant difference was found between the cooling rates tested, indicating that a passive cooling container which cools at 1 degrees C/min is a suitable alternative to conventional slow cooling. We conclude that ovarian tissues for cryobanking should be cryopreserved as soon as possible after collection or death of the animal to ensure maximal follicular survival.     

Effects of bilateral and unilateral eyestalk ablation on vitellogenin synthesis in immature female kuruma prawns, Marsupenaeus japonicus

In penaeid shrimp species, vitellogenin (VTG) synthesis in the ovary and hepatopancreas is under the inhibitory regulation of a neuroendocrine system, the X-organ/sinus gland complex in the paired eyestalks, and eyestalk ablation (removal of the X-organ/sinus gland complex) is widely used for inducing ovarian development. However, the difference in effects of bilateral and unilateral ablation on VTG gene expression has not been clarified so far. In the present study, VTG synthesis was monitored over a 16-day period after ablation and compared between replicates of immature female kuruma prawns, Marsupenaeus (Penaeus) japonicus, that had been bilaterally or unilaterally ablated and control specimens. After bilateral ablation, ovarian development was induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary increased significantly. Significant VTG mRNA increase was detected 12 h after bilateral ablation. In contrast, after unilateral ablation, ovarian development was not induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary did not change significantly from the control. These results indicate that in immature female prawns, unilateral ablation does not induce VTG gene expression, whereas bilateral ablation induces rapid VTG gene expression (<12 h). The ineffectiveness of unilateral ablation suggests that the remaining X-organ/sinus gland complex in the unilaterally ablated female prawns may secrete sufficient VIH to suppress VTG synthesis.     

Effects of prolactin on ovarian plasmin generation in the process of ovulation.

The present study was undertaken to assess the effects of prolactin (PRL) on gonadotropin-induced plasmin generation in the in vitro-perfused rabbit ovary. The ovarian plasmin activity was determined by measuring plasmin bound to its major inhibitor, alpha 2-plasmin inhibitor (alpha 2 PI-Plm). In the first experiment, exposure to hCG enhanced ovarian alpha 2 PI-Plm generation from 1.2 +/- 0.3 ng/min/ovary in unstimulated ovaries to 2.9 +/- 0.3 ng within 2 h. The concentration of alpha 2 PI-Plm reached a maximum at 4 h and then declined. A second peak occurred 8 h after hCG administration; however, the ovarian alpha 2 PI-Plm generation without hCG was very low throughout the entire perfusion period. In the subsequent experiment, the addition of PRL(10-10(3) ng/ml) to the perfusate inhibited hCG-induced ovulation in a dose-dependent manner. Exposure to PRL at 10(3) ng/ml significantly (p less than 0.05) inhibited hCG-induced alpha 2 PI-Plm generation in ovaries throughout the entire perfusion period. Furthermore, PRL inhibited hCG-stimulated alpha 2 PI-Plm generation at 4 h after hCG administration in the perfused rabbit ovaries in a dose-dependent manner. In conclusion, PRL directly inhibits hCG-induced ovulation in rabbit ovary, at least in part, by a mechanism depending upon inhibition of the plasmin-generating system in the preovulatory follicles.