Ovarian follicles during infancy in Romanov and Ile-de-France ewe lambs

The ovaries of new born lambs (15 Ile-de-France and 19 Romanov, 34 ovaries) and of 4-week-old lambs (6 Ile-de-France and 12 Romanov, 18 ovaries) were examined histologically to compare ovarian follicular development in infant lambs of breeds differing in their prolificacy. Breed was the major factor affecting follicular population at birth. Ile-de-France lambs had a higher total number of growing follicles (P less than 0.001), and more preantral (P less than 0.001) and antral (P less than 0.005) follicles than did Romanov lambs. Furthermore, the size of the largest follicles was also reduced in Romanov compared to Ile-de-France lambs. At 4 weeks of age, most of the features of the ovarian follicular population except the mean size of the third largest follicle were similar between the two breeds. However, atresia of antral follicles had appeared only in Ile-de-France and not in Romanov lambs. When a challenge with exogenous gonadotrophins (1000 i.u. PMSG followed by 1500 i.u. hCG) was attempted, ovulation was triggered in 2/6 and 0/12 Ile-de-France and Romanov lambs respectively. Massive follicular development was noted in 3/6 Ile-de-France lambs but in none of 12 Romanov lambs. Retardation of follicular development together with retardation in the establishment of ovarian sensitivity to gonadotrophins are therefore features typical of the ovaries of Romanov lambs compared to Ile-de-France lambs during the post-natal period.     

Influence of a gonadotrophin-releasing hormone agonist and gonadotrophins on morphometric characteristics of the population of small ovarian follicles in cynomolgus monkeys (Macaca fascicularis).

Small follicles, less than or equal to 100 microns, in monkey ovaries were divided into four types based on the morphological characteristics of the granulosa cells that surrounded the oocyte: primordial, intermediary, primary and secondary follicles. The proportion of primordial follicles positively correlated, whereas those of intermediary, primary and secondary follicles negatively correlated, with the total number of follicles less than or equal to 100 microns. There was no relationship between the population of nongrowing follicles (primordial and intermediary) and that of early-growing follicles (primary and secondary). Administration of exogenous gonadotrophins did not induce significant changes in the population of small follicles, whereas there was a significant increase in the number of intermediary follicles when gonadotrophins were associated with a gonadotrophin-releasing hormone agonist, buserelin. Buserelin can therefore partly inhibit the initiation of ovarian follicular growth in monkeys.     

Effect of long-term hypophysectomy on ovarian follicle populations and gonadotrophin-induced adenosine cyclic 3',5'-monophosphate output by follicles from Booroola ewes with or without the F gene

Long-term (i.e. approximately 70 days) hypophysectomy led to a significant (P less than 0.05) reduction in ovarian weight but no reduction in the total number of antral follicles (greater than 0.1 mm in diameter). In hypophysectomized ++ Booroola ewes (N = 8) follicles were always less than or equal to 3 mm and in hypophysectomized FF Booroola ewes (N = 6) follicles were always less than or equal to 2 mm in diameter; in ewes of both genotypes follicles reached diameters which were approximately 40% of their predicted final size at ovulation. Under in-vitro conditions, follicles from the FF and ++ hypophysectomized ewes produced significant increases in cAMP within 1 h of exposure to gonadotrophins (P less than 0.05) although no genotypic differences in cAMP production were noted. We conclude that ovarian follicles in FF and ++ ewes have absolute requirements for pituitary hormone on reaching diameters of 2 mm and 3 mm respectively and that appreciable numbers of antral follicles in ewes of both genotypes remain responsive to pituitary gonadotrophins despite prolonged deprivation of these hormones.     

Studies on prostaglandin metabolism in corpora lutea of rabbits during pregnancy and pseudopregnancy

Corpora lutea and ovarian stromal tissue were analysed for prostaglandin (PG) concentrations and activities of enzymes involved in PG metabolism at 8, 10, 12, 13 and 15 days after induction of ovulation. In CL of pseudopregnant rabbits, the PGE-2-9-ketoreductase (PGE-2-9-KR) was highly active on Days 10, 12 and 15 when compared with Day 8 (P less than 0.01; P less than 0.001; P less than 0.05). In pregnant animals PGE-2-9-KR activity was only increased on Day 12 (P less than 0.05) but declined to basal levels on Days 13 and 15. Comparing PGE-2-9-KR activity of pseudopregnant and pregnant animals, a significant elevation was found on Day 15 of pseudopregnancy (P less than 0.025). Activities of PG-15-hydroxydehydrogenase did not exhibit any significant changes with time in pseudopregnant or pregnant rabbits. PGE-2 concentrations were increased on Days 12, 13 and 15 (P less than 0.025) when compared with Day 8. Changes in PGF-2 alpha concentrations paralleled those of PGE-2-9-KR. The concentrations of PG metabolites 13,14-dihydro-15-keto-PGE-2 and -PGF-2 alpha were lower than those of the primary PGs and did not show stage-specific changes in pseudopregnant and pregnant animals. These results demonstrate that the rabbit CL possesses enzymes to convert PGE-2 to PGF-2 alpha and to metabolize both PGs. PGE-2-9-KR may be involved in regulating the PGF-2 alpha/PGE-2 ratio and possibly in controlling the life-span of the corpus luteum.     

Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles 相似文献   

Relationship of macroscopic appearance of the surface of bovine ovarian follicles concentrations of steroids in follicular fluid, and maturation of oocytes in vitro

The relationship among opaqueness of the surface of bovine ovarian follicles, concentrations of follicular steroids, and capacity of oocytes to achieve nuclear maturation in vitro was examined in this study. Follicles greater than or equal to 5 mm in diameter were classified as clear (n=68) or opaque (n=72) based on their surface appearance. An oocyte and follicular fluid (FF) were removed from each follicle. Each oocyte was cultured, and the concentration of estradiol (E), progesterone (P), and testosterone (T) was determined for each sample of FF. Oocytes that extruded the first polar body by 30 h in culture were considered mature. All other oocytes were immature. More (p less than 0.05) mature oocytes came from clear (56%) than opaque follicles (29%). Clear follicles had lower concentrations of E (p less than 0.05) and P (p less than 0.10) in FF than opaque follicles. Follicles with mature oocytes had greater (p less than 0.05) concentrations of P than follicles with immature oocytes. Follicles were separated into three categories based on ratio of P:E in FF: high = P:E greater than or equal to 10, medium = P:E greater than or equal to 1 less than 10, and low = P:E less than 1. The percentage of mature oocytes from clear follicles was similar for high (64%), medium (48%), and low (57%) P:E groups; however, the percentage of mature oocytes from opaque follicles was greater (p less than 0.05) for the high (59%) than for the medium (21%) or low (19%) P:E groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.     

Prolactin in follicular fluid and intracellular store calcium in follicular cells are related to morphological signs of ovarian follicle atresia in cows: work in progress

It is known that prolactin (PRL) is the third pituitary hormone serving gonadotropic function in mammals. However, its role in the regulation of ovarian folliculogenesis and, in particular, its relationship to follicular atresia as well as the mechanism of its influence on follicular cells are poorly understood. We investigated PRL levels in follicular fluids (FFs) and intracellular store calcium ([Ca2+]is) in cell walls of bovine ovarian follicles with diameters of 10 to 20 mm and their relationship to follicular atresia. Ovarian follicles were categorized on the basis of macroscopic criteria and of microscopic examination of granulosa cell (GC) smears. Prolactin concentrations in FFs were measured by RIA and levels of [Ca2+]is in follicular cells were determined by using the fluorophore chlortetracycline. Compared to atretic follicles, morphologically normal follicles were characterized by higher concentrations of PRL in FFs (P < 0.001) and lower contents of [Ca2+]is in follicular cells (P < 0.01). Furthermore, follicles containing no more than 20% of pycnotic GCs had higher levels of PRL in their fluids than those containing over 40% of pycnotic GCs (P < 0.05). Finally, the direct effect of PRL on [Ca2+]is content in follicular cells was studied in vitro. Compared to control, PRL decreased (P < 0.001) the levels of [Ca2+]is in the cells after 24 h culture of follicular walls from morphologically normal follicles in TCM 199 supplemented by 10% fetal calf serum. Our findings suggest that the decline of PRL concentrations in FFs and the rise of [Ca2+]is contents in follicular cells are related to atresia of large bovine follicles and that there appears to be a relationship between the two biochemical parameters.     

Distribution of follicles in canine ovary – A simple and rapid method for counting follicles-

The distribution of follicles within canine ovarian cortex was evaluated to estimate follicular homogeneity. The analysis of follicular homogeneity prior to ovarian tissue transplantation limits the impact if follicular heterogeneity on experimental results. In this report, ovarian fragments from 14 immature bitches were embedded in OCT compound. Sections (5-μm-thick) were cut on a cryostat and stained with methylene blue. The mean number follicles ranged from 3.7 to 15.6/mm² in the 14 ovaries examined. The variance and distortion ranged from 2.05 to 144.30 and −2.09 and 2.01, respectively. The distribution of follicles was considered even, when the variance value was lower than 10 or between 10 and 16; and absolute value of distortion was inferior to 1. The distribution of follicles within ovarian cortex in 9 of 14 bitches was judged uneven. These results indicated that follicles were not homogeneously distributed within the ovarian cortex of the majority of bitches.     

Detection of unsuspected ovarian pregnancy by wedge resection

Five follicular ovarian implantations occurred among 200 ectopic pregnancies encountered during a 14-year period. Abortions from impregnated follicles may cause hemoperitoneum more often than is generally suspected. Wedge resection or cystectomy to ensure hemostasis provides tissue for histological examination, without which ruptured ovarian pregnancy may masquerade as rupture of a corpus luteum with hemorrhage (“ovarian apoplexy”). Including patients reported here, IUCD users have within the past five years accounted for about 10% of all ovarian pregnancies recorded in English.     

Effect of the peripubertal pattern of LH and FSH secretion on in-vitro oestradiol-17 beta secretion and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perfusion culture and exposed to (1) no gonadotrophin, (2) tonic NIH-FSH (200 ng RP-1 equiv./ml) or (3) NIH-FSH + NIH-LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml). After 3 h of perifusion, the ovaries were prepared for histological analysis and the perifusate assayed for oestradiol-17 beta. Since the NIH-FSH preparation is contaminated with LH, a second experiment was conducted using recombinant bovine LH and FSH. Ovaries were perifused for 3 h with (1) no hormones, (2) recombinant FSH (200 ng/ml) or (3) recombinant FSH plus 25 ng recombinant LH/ml. NIH-FSH alone increased the number of mid-size antral follicles (P less than 0.05) and decreased the number of small antral follicles (P less than 0.05). Pulsatile LH in the presence of FSH increased the number of mid-size antral follicles without reducing the number of small antral follicles. Studies with recombinant FSH and LH demonstrated that both FSH and LH are necessary to stimulate follicles to grow, indicating that the growth-promoting property of the NIH-FSH is due to the presence of both FSH and LH. Regardless of whether NIH or recombinant gonadotrophins were used, follicular growth was induced without increasing oestradiol secretion. These results demonstrate that enhanced oestradiol secretion is not essential for the induction of follicular growth, while both LH and FSH are necessary to stimulate small antral follicles to grow into mid-size antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactive roles of progesterone, prostaglandins, and collagenase in the ovulatory mechanism of the ewe

Interrelationships between production of progesterone (P4), prostaglandin (PG) E2 and PGF2 alpha, and collagenase by periovulatory ovine follicles and their possible involvements in the ovulatory process were investigated. Follicles were isolated from ovaries at intervals (0 to 24 h) after the initiation of the preovulatory surge of luteinizing hormone (LH). Progesterone and PGs within follicles were determined by radioimmunoassay. Digestion of radioactive collagen during coincubation with tissue homogenates was used to assess the production of a bioactive follicular collagenase(s). Follicular accumulation of PGs and P4 increased at 12 and 16 h, respectively, after the onset of the surge of LH; PGE2 then decreased at 20 h. Collagenolytic activity of follicular tissue increased at 20 h and was maximal at 24 h (during the time of follicular rupture). An inhibitor of synthesis of P4 (isoxazol) or PGs (indomethacin) was injected into the follicular antrum at 8 h. Isoxazol did not prevent the initial rise in PGs, but inhibited synthesis of PGF2 alpha at 16 h and therafter. Isoxazol negated the decline in PGE2 and increase in collagenolysis. Indomethacin did not influence synthesis of P4; however, it suppressed collagenolytic activity of follicular tissue. Ovaries with treated follicles were left in situ and observed for an ovulation point at 30 h. Isoxazol or indomethacin was a potent inhibitor of ovulation. The blockade of ovulation by isoxazol was reversed by systemic administration of P4 or PGF2 alpha, but not by PGE2. Reversal of the blockade by indomethacin was accomplished with PGE2 or PGF2 alpha. Collagenolytic activity of follicular tissue was likewise restored by such treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the interval of serial sections of ovarian tissue in the tissue chopper on the number of isolated caprine preantral follicles.

The present work investigated the effect of the interval of serial sections of ovarian tissue on the number of isolated preantral follicles in the goat. Goat ovaries were cut in the tissue chopper at eight different intervals. The quality of isolated follicles were evaluated by histology and transmission electron microscopy. Best results were obtained when the ovaries were cut in the tissue chopper at intervals of 75.0 microm (9664 preantral follicles per ovary). Histochemical and ultrastructural analysis showed that the follicular morphology was preserved after mechanical isolation as demonstrated by the normality of oocytes and granulosa cells as well as by preservation of basement membrane. The percentages of isolated primordial, primary and secondary follicles were 96.3%, 2.5%, and 1.2% and their average diameters were 21.5, 34.7 and 65.3 microm, respectively. It was concluded that the interval of serial sections of ovarian tissue in the tissue chopper affects the number of isolated preantral follicles, and that the follicles remained intact after mechanical isolation in goats.     

Effect of gonadotrophin environment on growth and development of isolated mouse primary ovarian follicles

Collagen gels containing isolated primary follicles devoid of other ovarian tissue were transferred beneath the kidney capsule of 3 types of female recipients: cycling, ovariectomized and hypogonadal, known to have different circulating concentrations of gonadotrophins. After 10 days the gels were recovered and processed for histology or the oocytes were recovered and their diameters measured and their ability to resume meiosis was determined. The growth of isolated primary follicles was positively correlated with the concentrations of circulating gonadotrophins in the recipient mice, but the numbers of oocytes recovered, the rate of oocyte growth and resumption of meiosis did not differ in the 3 types of recipient studied. This indicates that, in the conditions provided, oocyte growth was not related to the extent of follicular development.     

Frequency of luteinizing hormone pulses in cattle influences duration of persistence of dominant ovarian follicles,follicular fluid concentrations of steroids,and activity of insulin-like growth factor binding proteins

The objectives of the present study were to determine how varying frequency of LH pulses as controlled by varying treatments with progesterone (P4) in cattle would affect: (1) concentration of steroid hormones and activity of insulin-like growth factor binding proteins (IGFBPs) in the ovarian follicular fluid and blood plasma, and (2) duration of persistence of largest ovarian follicles. There were four treatment groups (n=7 per group) and a control group (n=5) of mature, non-lactating beef cows. Treatments were: (1) two progesterone releasing intravaginal devices (PRIDs) for 16 days (2PRID); (2) a half PRID for 16 days (0.5PRID); (3) two PRIDs for 8 days, then a half PRID for 8 days (2-0.5PRID); or (4) a half PRID for 8 days, then two PRIDs for 8 days (0.5-2PRID). Treatment was initiated on the fifth day of the estrous cycle, which was designated as Day 0, and continued for 16 days. All P4-treated females were administered prostaglandin F2alpha on Day 0 and 1 to regress their corpora lutea. Frequency of LH pulses was greater during treatment with the smaller dose of P4 compared with treatment with the larger dose of P4 and the control group. Ovarian follicles were classified into five categories based on ultrasonographic observations: growing (G); atretic (A); growing dominant (GD); growing persistent (GP); or atretic persistent (AP). At ovariectomy on Day 16, the largest and second largest follicles collected were re-classified into five categories based on follicular concentration of steroids. Classification of the largest follicle collected on Day 16 was influenced by treatment (P<0.005), with the 2PRID group having A follicles, the 2-0.5PRID group GP follicles, the 0.5-2PRID group AP follicles, and the 0.5PRID group GD and GP follicles. Concentrations of 17beta-estradiol (E2) were greatest in GD and GP follicles (P<0.05). There was less (P<0.05) activity of IGFBP-2 in GD follicles and less (P<0.05) activity of IGFBP-3 in GD and GP follicles than other follicles. Activity of IGFBP-4 and -5 was greater (P<0.05) in A and AP follicles than G, GD, and GP follicles. Maintenance of a frequent release of LH pulses over a 16-day period did not result in maintenance of persistent follicles throughout this period indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia is associated with greater activity of IGFBP-2, -4, -5, and greater concentrations of P4 in follicles, whereas growing dominant and persistent follicles contained greater concentrations of E2, androstenedione (A4), and less IGFBP-2 activity than follicles of other classes. Follicle classifications based on ultrasonography or follicular concentration of steroids did differ (P<0.05) for the largest follicles from the 2PRID group. Two follicles in this group appeared as GD follicles by ultrasonography, but these were atretic based on follicular steroid contents. Objective 1 of the present study yielded the conclusion that concentrations of steroid hormones in follicular fluid and blood plasma could be predictably controlled by regulating the frequency of LH pulses with varying doses of P4. Objective 2 yielded the conclusion that maintain frequent release of LH pulses over a 16-day period could not maintain persistent follicles throughout this period, indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia in the present study was associated with increased follicular fluid activity of IGFBP-2, -4, -5, and P4, whereas growing dominant and persistent follicles contained greater concentrations of E2, A4, and less IGFBP-2 activity than follicles of other classes.     

A potential role for insulin-like growth factor binding protein-4 proteolysis in the establishment of ovarian follicular dominance in cattle.

A critical transition in ovarian follicular development is the selection of a dominant follicle, capable of ovulating, from a cohort of synchronously growing antral follicles. However, little is known about mechanisms and factors that regulate the selection and growth of dominant ovarian follicles. We have investigated whether a component of the insulin-like growth factor (IGF) system, namely IGFBP-4 protease, is associated with the establishment of follicular dominance in cattle. IGFBP proteases degrade IGFBPs, freeing IGFs to interact with their receptors. In experiment 1, follicular fluid from preovulatory follicles (n = 4) degraded about 80% of the added recombinant human (rh) IGFBP-4 within 18 h of incubation. The IGFBP-4 protease exhibited optimal activity at neutral/basic pH and its sensitivity to various protease inhibitors suggested a metalloprotease. The decline in the intensity of the band corresponding to intact rhIGFBP-4 was accompanied by the appearance of immunoreactive fragments of molecular weights approximately 18 and 14 kDa, which were not detectable by ligand blot analysis. In experiment 2, follicular fluid samples were collected from dominant and subordinate follicles on Day 2 or 3 of the first follicular wave, after ovariectomy (experiment 2a, n = 3/day) or by ultrasound-guided follicular aspiration (experiment 2b, n = 4-5/day). Estradiol concentrations in follicular fluid from dominant vs. subordinate follicles confirmed their identities and indicated that the dominant follicle had been selected by Day 2 of the follicular wave. In both experiments 2a and 2b, IGFBP-4 proteolytic activity was 2- to 3.5-fold (P < 0.05) and 5-fold (P < 0.01) higher in follicular fluid from dominant than subordinate follicles on Days 2 and 3 of the follicular wave, respectively. The finding that IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the follicular wave, strongly suggests a role for IGFBP-4 protease in the establishment of ovarian follicular dominance.     

Concentrations of insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins

Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2 alpha (PGF2 alpha). Blood and follicular fluid were collected 48-50 h post-administration of PGF2 alpha (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RIA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p less than 0.05) number of large (greater than or equal to 4 mm) follicles and 47% greater (p less than 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p less than 0.05) concentrations of IGF-I (+/- SE) in the two largest follicles than control (unselected) cows (327 +/- 28 vs. 243 +/- 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p less than 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (p less than 0.05) in small follicles than in large follicles, and was greater (p less than 0.05) in large follicles of control (93.2 +/- 5.3%) than twin-producing (76.2 +/- 4.4%) cattle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotrophins, fecundity genes and ovarian follicular function

The Booroola Merino is a sheep breed having a major gene(s) (F) influencing its ovulation-rate. Homozygous (FF), heterozygous (F+) and non-carriers (++) of the gene have ovulation-rates of greater than or equal to 5, 3 or 4 and 1 or 2 respectively with the durations of each oestrous cycle and oestrous behaviour being similar in all genotypes. Although the principal site(s) of gene expression are obscure, FF genotypes have mean plasma concentrations of FSH and LH which are higher than in the F+ ewes, which in turn are higher than in the ++ animals. Thus, the FF and F+ animals provide a unique system in which to examine ovarian function under continual exposure to elevated gonadotrophin concentrations. At the ovarian level, F gene-specific differences in follicular development and function were noted. In small follicles (0.1-1.0 mm dia.), the basal levels of cAMP and the in vitro synthesis of cAMP, progesterone, androstenedione and oestradiol-17 beta in response to LH and FSH were significantly influenced by genotype (FF greater than F+ greater than ++; P less than 0.05). In larger follicles (1-4.5 mm dia.) the granulosa cells from FF and F+ ewes were more responsive to FSH and/or LH than in ++ ewes with respect to cAMP synthesis and they also had higher levels of aromatase activity. In vivo, the ovarian secretion-rates of oestradiol from greater than or equal to 5 ("oestrogenic") follicles in FF ewes, 3-4 such follicles in F+ ewes, and 1-2 such follicles in ++ animals during the follicular phase were similar. In FF and F+ ewes, the preovulatory follicles ovulated at a smaller diameter (i.e. 3-5 mm) than in ++ ewes (greater than 5 mm diam.) and also produced smaller corpora lutea. Thus, after continual exposure to elevated levels of gonadotrophins, follicles may synthesize steroid and mature at smaller diameters compared to those exposed to normal levels of FSH and LH.     

Production of tissue inhibitors of metalloproteinases (TIMPs) by pig ovarian cells in vivo and the effect of TIMP-1 on steroidogenesis in vitro

Precisely which ovarian cells produce tissue inhibitors of metalloproteinases (TIMPs) is unclear. Although granulosa cells are reported to produce TIMPs, thecal TIMP production has not been investigated nor has the influence of TIMPs on theca cells. Furthermore, although periovulatory follicles have been examined, little is known about smaller ovarian follicles. Follicles >/= 2 mm in diameter were collected from Large White hybrid gilts on the day before predicted oestrus (n = 3) or after hCG treatment (n = 3) and divided into 1 mm size classes. Small (2 to < 5 mm) follicles were kept intact, whereas follicles >/= 5 mm were separated into follicular fluid, granulosa and theca cell compartments. After homogenization, TIMP-1, -2 and -3 were detected by reverse zymography. Theca cells (50 x 10(3) per well) were cultured with TIMP-1 (10, 100 or 200 ng ml(-1) with or without long-R3 insulin-like growth factor I (IGF-I)) in a serum-free system to investigate the effect on steroidogenesis and the number of cells. Both large and small pig follicles produced TIMPs and TIMP-1, -2 and -3 were detected in follicular fluid, granulosa and theca cell samples. There was a phase x tissue type interaction for the presence of both TIMP-1 and -2 (P < 0.03, P < 0.05, respectively), and TIMPs were detected in more granulosa and theca cell samples after hCG than during the follicular phase. The concentrations were influenced by the type of tissue (TIMP-1, P < 0.005; TIMP-2, P < 0.005, TIMP-3, P > 0.05), and the highest concentrations occurred in the theca tissue. There were tissue type x follicle size interactions for the presence of both TIMP-1 and -2 (P < 0.001). In vitro, TIMP-1 increased thecal steroidogenesis after 144 h (oestradiol, P < 0.05, progesterone, P < 0.001) but reduced the number of viable cells (P < 0.001). In conclusion, TIMP-1, -2 and -3 were present in large and small pig follicles and were produced by both granulosa and theca cells, although concentrations differed with the type of tissue. Production was regulated by factors including follicle size and phase of the oestrous cycle. In addition to controlling tissue remodelling, TIMP-1 may also regulate steroidogenesis.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)