Minor FSH surge,minor follicular wave,and resurgence of preovulatory follicle several days before ovulation in heifers

Blood samples were collected and follicle diameters were determined daily beginning on Day 12 (Day 0 = ovulation) in 35 interovulatory intervals (IOIs) in heifers. A minor follicular wave with maximal diameter (6.0 ± 0.3 mm) on Day −4 was detected in six of seven IOIs that were scanned for follicles 4 mm or greater. The number of IOIs with a CV-identified minor FSH surge toward the end of the IOI was greater (P < 0.03) in two-wave IOIs (10/17) than in three-wave IOIs (4/18). The 17 two-wave IOIs were used for study of the temporal relationships among preovulatory follicle, FSH, LH, and estradiol. Daily growth rate of the preovulatory follicle was maximum on Days −11 to −7, minimum (P < 0.05) on Days −7 to −4, and increased (resurged, P < 0.05) on Days −4 to −3. A transient increase in FSH was maximum on mean Day −4, and the peak of a minor FSH surge occurred on Day −4.5 ± 0.2. Concentration of LH and estradiol increased between Days −5 and −4. Results demonstrated resurgence of the preovulatory follicle apparently for the first time in any species. Resurgence seemed more related temporally to the minor FSH surge than to the LH increase, but further study is needed. Results supported the novel hypotheses that a minor FSH surge near the end of the IOI is temporally associated with (1) the emergence of a minor follicular wave and (2) the resurgence in growth rate of the preovulatory follicle.     

Hormone concentrations temporally associated with contralateral and ipsilateral relationships between the CL and preovulatory follicle during the third follicular wave in heifers

Concentrations of circulating hormones after Day 14 (Day 0 = ovulation) were determined daily in 87 interovulatory intervals (IOIs) in heifers. The IOIs were grouped into four permutations according to an ipsilateral (Ipsi) or contralateral (Contra) relationship between the CL and the preovulatory follicle and two (2W) or three (3W) follicular waves per IOI. The number of IOIs per group differed (P < 0.005) from equality among the Ipsi-2W (n = 27), Contra-2W (n = 31), Ipsi-3W (n = 9), and Contra-3W (n = 20) groups. A continuous decrease in progesterone (luteolysis) began later (P < 0.05) in the Contra-3W group (Day 18.0 ± 0.4) than in each of the Ipsi-2W (15.4 ± 0.2), Contra-2W (15.6 ± 0.2), and Ipsi-3W (16.2 ± 0.5) groups. Concentrations of LH and estradiol began to increase near the beginning of luteolysis in each group. A minor FSH surge that did not stimulate a major follicular wave developed in about 50% of the IOIs in each group, except that none were detected in the Ipsi-3W group. The minor FSH surge reached a peak about 4 days before ovulation and several days after wave 3 had emerged. The hypothesis that luteolysis begins earliest in two-wave IOIs, intermediate in three-wave IOIs with an ipsilateral CL/follicle relationship, and latest in three-wave IOIs with a contralateral relationship was supported. The hypothesis that a minor FSH surge occurs most frequently in association with three follicular waves was not supported.     

Blood flow to follicles and CL during development of the periovulatory follicular wave in heifers

The hemodynamics of the developing CL and the future dominant follicle (DF) was studied in 22 heifers during wave 1 on Days 0 to 5 (Day 0 = ovulation). Color-Doppler ultrasonography was used to determine the resistance index (RI) at the most prominent Doppler signal in an ovarian arterial branch before entry into the ovary; a decrease in RI indicates a downstream increase in vascular perfusion. The RI for each of four intraovarian patterns averaged over days was different (P < 0.05) from each of the other patterns as follows: DF–CL (DF and CL in the same ovary), 0.52 ± 0.02; CL alone, 0.60 ± 0.01; DF alone, 0.67 ± 0.01; neither DF nor CL, 0.78 ± 0.01. The differences in RI among intraovarian patterns began on Day 0 or 1, indicating that the extent of vascular perfusion on Days 0 to 5 for the various patterns may have been influenced by events that occurred before ovulation. The percentage of the DF wall with color-flow signals was greater (P < 0.05) in the DF–CL pattern than in the DF pattern on each of Days 2 to 5 and was greater (P < 0.0001) in the DF–CL pattern when the DF was adjacent to the CL (40.2 ± 2.0%) than when separated (24.5 ± 1.9%). Dimensions of DF (P < 0.01) and CL (P < 0.02) were greater when adjacent to each other. The results supported the hypotheses for wave 1 that (1) vascular perfusion is greater for the DF–CL intraovarian pattern than for the DF or CL pattern and (2) the extent of blood-flow Doppler signals in the wall of the developing DF is greater for the DF–CL pattern than for the DF pattern. Our preferred interpretation is that a change in vascular perfusion of the CL is accompanied by a similar change in perfusion of the DF when the two structures are in the same ovary especially adjacent.     

Delayed follicular development and ovulation following inhibition of FSH with equine follicular fluid in the mare

In two experiments, PGF(2alpha) was given to all mares on Day 10 (ovulation = Day 0). In experiment 1, mares received either whole follicular fluid or saline i.v. every 12 hours on Days 10 to 14. Experiment 2 was similar to experiment 1, except the follicular fluid was extracted with charcoal to remove steroids. Analysis of the FSH data for Days 10 to 21 indicated an effect of treatment (P<0.08) with whole follicular fluid, but not with charcoal-extracted follicular fluid. However, there was an effect of day (P<0.05) and an interaction (P<0.01) of treatment with day for both experiments. The interaction of treatment with day seemed primarily due to a marked post-treatment increase in FSH concentrations between Days 15 and 17 for mares treated with either whole follicular fluid or charcoal-extracted follicular fluid. Analysis of the diameter of the largest follicle for Days 10 to 18 indicated a main effect of treatment (P<0.05) and day (P<0.05) and an interaction (P<0.05) of treatment with day for both experiments. The interaction of treatment with day was attributable to the inhibition of follicular growth by Day 14 for mares treated with whole follicular fluid and by Day 15 for mares treated with charcoal-extracted follicular fluid. The length of the interovulatory interval was longer (P<0.05) in the treated group than in controls for both experiments. Results indicated that equine follicular fluid contained a proteinaceous substance that suppressed circulating concentration of FSH. The inhibited follicular growth and the delay in ovulation were attributed to the reduced concentrations of circulating FSH.     

Morphological patterns of cell death in ovarian follicles of primary and secondary growth and postovulatory follicle complex of the annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae)

The dynamics of cellular development and homeostasis of the ovary depend on the balance between proliferation and cell death throughout the reproductive cycle. Millerichthys robustus is an annual fish whose ovarian follicles develop asynchronously, allowing daily reproduction from sexual maturity until death. The objective of this research is to describe, histologically, the processes of follicular atresia and regression of postovulatory follicular complexes (POC) throughout a reproductive cycle of M. robustus. Patterns of cell death were documented by apoptosis in atretic follicles and POC, and necrosis in the POC after ovulation with an associated inflammatory response. Atretic follicles were seen from the onset of sexual maturity, during week three post-hatching (PH), both in primary growth (from the Cortical alveoli step, with folliculogenesis completed) and secondary growth Stages, with a higher prevalence in the latter. POCs were observed in different stages of regression from week four PH until the death of the fish. The apoptotic characteristics found were: (i) fragmentation of the nuclear membrane and zona pellucida, and liquefaction of the cortical alveoli and yolk; (ii) follicular cells becoming phagocytic, increasing their size, and migrating within the oocyte; and (iii) formation of an intrafollicular lumen, a product of phagocytosis of the oocyte constituents and dispersed pigments that remain after the digestion of yolk and cortical alveoli. The morphological changes of the follicular cells of the POC, from a squamous morphology after ovulation to columnar during its regression with PAS+ contents, was documented, suggesting a secretory activity.     

Repeatability of 2-wave and 3-wave patterns of ovarian follicular development during the bovine estrous cycle

Characteristics predictive of a 2-wave versus 3-wave pattern of ovarian follicular development during the interovulatory interval (IOI) were examined by ultrasonographic monitoring of 91 IOIs from 31 beef heifers. Repeatability of the wave pattern within individuals and the effects of season and age were determined using a subset of 75 IOIs from 15 heifers examined for multiple IOIs. The 2-wave pattern was detected in 62 of 91 (68%) IOIs, and the 3-wave pattern was detected in 29 of 91 (32%) IOIs. The preponderance of the 2-wave versus 3-wave pattern (P < 0.05) was not influenced by season (P = 0.61) but was even greater in the more mature age group (P = 0.02). The majority of IOIs ≤21 d was of the 2-wave pattern (88%; P < 0.05), whereas the majority of IOIs ≥22 d was of the 3-wave pattern (78%; P < 0.05). The proportion of nonalternating patterns (repeatability) was more than twofold greater than the proportion of alternating patterns (70% vs. 30%; P < 0.01). This relationship was consistent among seasons (P < 0.01) and even more marked in the more mature age group (P = 0.01). Emergence and follicular dominance of Wave 2 were delayed (P < 0.01), and the onset of corpus luteum regression was earlier (P < 0.01) in 2-wave versus 3-wave IOI. In conclusion, the duration of the IOI was predictive of the wave pattern, and the pattern was repeatable within individuals. Factors influencing the period of follicular dominance of Wave 1 in 2-wave versus 3-wave IOI may be responsible for regulating the wave pattern and may be associated with heifer maturity or relative nutritional demand during the postpubertal period. The impact of greater follicular attrition recorded in 3-wave versus 2-wave IOI on ovarian depletion and reproductive senescence is worthy of critical evaluation.     

New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the 2 kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.     

Association between numbers of ovarian follicles in the first follicle wave and superovulatory response in ewes

The aim of this study was to examine the variability in the number of ovarian follicles in sheep and to determine if the average number of follicles per day influences the response to superovulation and resulting embryo quality. Ewes (n = 83) were synchronized and the number of follicles (≥2 mm diameter) in the ovaries were counted daily between Days 0 and 4 of the oestrous cycle using transrectal ultrasonography. Fourteen to 21 days later, 47 ewes were randomly chosen from the group and were treated with an intravaginal progestagen pessary for 12 days and superovulated with 1500 IU eCG administered as a single injection 10 days after sponge insertion. Ewes were mated and reproductive tracts were recovered after slaughter on Day 6 of pregnancy. The number of corpora lutea was counted, uterine horns were flushed and the morphology and developmental stage of the recovered oocytes/embryos was assessed. The mean daily number (±S.D.) (≥2 mm diameter) of follicles per ewe was 8.5 ± 2.8 (ranging between 3 and 16). After superovulation animals with few follicles (Low group: <8 follicles/day; n = 21) had fewer (P < 0.005) corpora lutea, total structures (unfertilized oocytes and embryos), good quality and total embryos compared to animals with many follicles (High group: ≥8 follicles/day; n = 23). No difference was found in the proportion of good quality embryos (relative to the total number; Low 0.68 ± 0.11 versus High 0.79 ± 0.08; P = 0.21) between the two groups, or the recovery rate, the number of unfertilized oocytes or the number of poor quality embryos per animal. We conclude that ewes with a higher number of follicles (≥8) during the first follicular wave had a better superovulatory response (in terms of corpora lutea and high quality embryos) 2–3 weeks later; however, there was no relationship between the number of follicles and the proportion of good quality embryos per animal.     

Intraovarian effect of dominant follicle and corpus luteum on number of follicles during a follicular wave in heifers

The intraovarian relationships among dominant follicle (DF), corpus luteum (CL), and number of follicles between Days 0 to 5 (Day 0 = ovulation) in wave 1 (n = 65 waves) and Days 9 to 13 in wave 2 (n = 62) were analyzed in separate experiments in Bos taurus heifers. Ovaries were grouped into intraovarian patterns of DF–CL, DF alone, CL alone, and neither DF nor CL. In wave 1, the pattern frequencies of DF–CL or neither DF nor CL (34% each) were greater (P < 0.0004) than for DF alone or CL alone (16% each). The number of growing follicles ≥5.0 mm, was greater (P < 0.0001) in ovaries with the DF, even when the DF was removed from the tally (P < 0.03). In a factorial analysis of wave 1, there was a positive main effect of DF (3.9 ± 0.2 vs. 2.2 ± 0.2 follicles; P < 0.0001), but the main effect of CL and the interaction of DF and CL were not significant. In a factorial analysis of wave 2, there were more (P < 0.0001) follicles greater than 6 mm in ovaries with a DF when the DF was included and an approaching difference (P < 0.09) when the DF was excluded. The main effect of CL and the interaction of DF and CL were not significant. The hypothesis that both the DF and CL have a positive intraovarian effect on number of follicles in waves 1 and 2 was only partly supported; the DF, but not the CL, had an effect in the factorial analyses. Previous reports in cattle and sheep of a positive intraovarian effect of CL on number of follicles are questionable in that location of the DF was not considered.     

The fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose

Summary The structure of follicular layer of growing and atretic follicles in the ovary of the domestic goose, was studied by electron microscopy. In small follicles, the wall is lined with a narrow layer of tightly packed small, cuboidal cells separated from the thecal tissue by the basal lamina. During growth, they transform into tall, columnar cells arranged in a single row. The cells display several peculiar ultrastructural features. First, annulate lamellae are commonly observed. Second, cytoplasmic dense-cored granules accumulate in close association with fenestrated cisternae and networks of tubuli derived from the RER. They consist of spheres and strands of amorphous substance of unknown origin. Third, the cells contain many transosomes, a unique organelle of the avian follicle cell consisting of a dense plaque associated with ribosome-like particles. The mature forms of transosomes are located at the tips of lateral and apical cell projections, while bodies thought to be their precursors, are found in the apical cytoplasm. In follicles larger than 8 mm in diameter, most of the transosomes and their precursors have disappeared. Follicular atresia occurs in all of the size-classes of follicles investigated. A loss of transosomes (in follicles up to 8 mm in diameter) and an accumulation of lipid droplets are the first atretic events detectable by electron microscopy. Morphologic features, including deep nuclear indentations, accumulation of lipid droplets frequently encireled by membrane whorls, dilation and disintegration of RER cisterns, swelling of mitochondria and accumulation of dense irregular masses of unknown origin in the cytoplasm, are taken as evidence for advanced degradation. We conclude that necrosis is the dominant type of cell death of the follicular cells during atresia. However, a small fraction of cells, characterized by dark condensed cytoplasm, seems to die by apoptosis.     

Ultrastructural localization of intracellular calcium stores in Xenopus ovarian follicles as revealed by cytochemistry and X-ray microanalysis

The ultrastructural localization of calcium in full-grown ovarian follicles of Xenopus laevis was demonstrated after fixation in the presence of fluoride ions and by means of energy dispersive X-ray microanalysis. In hormonally untreated follicles (prophase I-arrested oocytes), two calcium sites were detected: follicle cells and oocyte pigment granules. In follicle cells, calcium containing deposits were preferentially associated with macrovilli, which ended by gap junctions. In human chorionic gonadotropin treated follicles (meiotically reinitiated oocytes), deposits were only seen in follicle cells. This is the first report of the cytochemical detection of intracellular Ca²⁺ in follicle cells of amphibians. The possible involvements of these Ca²⁺ stores in mediating the hormonal control of meiotic maturation are discussed.     

Immunolocalization of sex hormone-binding globulin (SHBG) in human ovarian follicles and corpus luteum

Sex hormone-binding globulin (SHBG), a hepatic carrier protein for sex steroids is expressed in different steroid-sensitive tissues, including Sertoli cells of the testis. It has been suggested that this protein may be one of the local regulators of spermatogenesis. The expression of SHBG in the ovary is currently unknown. We have previously demonstrated the synthesis of SHBG in granulosa-lutein cells from patients undergoing in vitro fertilization. In this study, the presence of SHBG in human ovarian follicles and corpora lutea is investigated, using immunohistochemistry on adult and fetal ovarian tissue sections. SHBG is localized in the whole granulosa layer at all stages of folliculogenesis, whereas, only isolated theca cells are immunostained. In primordial and primary follicles, the oocyte cytoplasm shows an intense immunostaining, which disappears after the secondary stage. In the microenvironment of the mature oocytes, SHBG is present in the surrounding cumulus cells, the perivitelline space, and nearby the oolemma. In the corpus luteum, SHBG is localized in large luteal cells, whereas, small luteal cells do not show any significant staining. By analogy with the testis, these results raise the question of an involvement of SHBG in the regulation of follicular maturation as well as in luteal function.     

Morphological and quantitative evaluation of the ovarian recrudescence in Nile tilapia (Oreochromis niloticus) after spawning in captivity

The Nile tilapia is one of the most important fish species for aquaculture worldwide. Understanding their reproductive biology is essential for improving their aquaculture methods. The morphological and quantitative dynamics of ovarian recrudescence of Oreochromis niloticus was studied for 21 days postspawning. To accomplish this, breeding females were kept in controlled conditions and ovarian samples were collected weekly for histological, ultrastructural and morphometric analyses. Ovarian follicle morphology revealed an intense synthesis activity of the follicular cells, which actively contributed to formation of the zona radiata and oocyte development following spawning. Recently spawned ovaries contained follicles at all developmental stages, but they were predominantly early primary growth (～42%) and full‐grown follicles (～20%). Remnants of spawning, postovulatory follicle complexes represented approximately 5% of the former ovarian follicles immediately after spawning, and less than 1% after 7 days. Atretic follicles accounted for approximately 2% of the follicles studied during the period. The stock of primary growth follicles was stable during ovarian recrudescence, indicating their availability for continuous recruitment. Only the frequency of full‐grown follicles significantly increased in the ovaries during recrudescence, representing approximately 35% of the follicles 21 days postspawning. The diameters of all follicles were significantly different between the periods analyzed. The ovaries' morphological characteristics, the maintenance of young follicles stocks and the gradual and significant increase in the proportion and diameter of full‐grown follicles showed a rapid ovarian recovery and follicular growth of O. niloticus, in 21 days at 29.5°C, necessary for the next spawning. J. Morphol. 275:348–356, 2014. © 2013 Wiley Periodicals, Inc.     

Evidence for a role for anti-Mullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane

The first critical transition in follicular development, the activation of primordial follicles to leave the pool of resting follicles and begin growth, is poorly understood, but it appears that the balance between inhibitory and stimulatory factors is important in regulating the exodus of follicles from the resting pool. There is evidence that anti-Mullerian hormone (AMH; also known as MIS) inhibits follicle activation in mice, but whether it plays a similar role in non rodent species is not known. When pieces of bovine ovarian cortex, rich in primordial follicles, are cultured in serum-free medium, most follicles initiate growth, but when cortical pieces are grafted beneath the chorioallantoic membrane (CAM) of chick embryos, follicle activation does not occur. Since embryonic chick gonads of both sexes produce and secrete high levels of AMH, the hypothesis that the AMH in the chick circulation inhibits follicle activation was tested. In Experiment 1, whole newborn mouse ovaries were grafted beneath the CAM (placed "in ovo") or cultured in vitro for 8 days. In vitro (or after 8 days in vivo) follicles activated and proceeded to the primary or secondary stage, but activation was suppressed in ovo. This inhibition was reversed if ovaries were removed from beneath the CAM and cultured in vitro. In contrast, when ovaries from mice null mutant for the AMH type II receptor were CAM-grafted in Experiment 2, follicle activation occurred in a similar fashion to activation in vitro. This finding strongly implicates AMH as the inhibitor of follicle activation in ovo. Since chick embryonic gonads are the source of circulating AMH, chicks were gonadectomized in Experiment 3, prior to grafting of pieces of bovine ovarian cortex beneath their CAMs. Bovine primordial follicles activated in the gonadectomized chicks, similar to the results for mice lacking the AMH type II receptor. Taken together these experiments provide strong evidence that AMH is the inhibitor of mouse follicle activation present in the circulation of embryonic chicks and provide indirect, and hence more tentative, evidence for AMH as an inhibitor of bovine follicle activation.     

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.     

Architecture of associations of minor salivary gland ducts and lymphoid follicles in Macaca fascicularis

Summary Previous light-microscopic and ultra-immunohistochemical tracer studies revealed the existence of an independent local immune response of the simian oral mucosa. This local response is attributed to the presence of minor salivary gland (MSG) duct-related lymphoid tissue. Semithin sections from a total of 263 Epon-embedded tissue blocks from the labial and buccal mucosae of seven monkeys, Macaca fascicularis, were analysed light-microscopically, and 10 suitable MSG duct/follicle assemblies were investigated ultrastructurally. These duct/follicle assemblies include follicular and parafollicular compartments with distinct fine-structural elements. The follicular area or germinal centre contains numerous small and large lymphoid cells, mitotic figures, plasmablasts, macrophages, and cells resembling the follicular dendritic cells with distinct desmosomal junctions. The parafollicular area, which includes the heavily infiltrated duct wall, contains numerous small lymphocytes, T-lymphoblasts, plasma cells and reticular cells resembling fibroblasts. A distinct feature of this compartment is the presence of high endothelial venules (HEV). The presence of HEV and numerous blast cells, resembling blast-forming T-lymphocytes activated in vitro, in a specific area of the duct/follicle assembly strongly suggests that this area is structurally and physiologically identical to the thymus-dependent area of other lymphoid tissues. In other words, the duct/follicle assemblies of simian MSG contain the various specific fine-structural elements that are suitable for antigen recognition and processing. These elements are distributed in discrete compartments comparable to the B- and T-cell areas of classical lymphoid tissue.     

Gap junctions in ovarian follicles of Drosophila melanogaster: inhibition and promotion of dye-coupling between oocyte and follicle cells

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dyecoupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K⁺], high intracellular [Ca²⁺], octanol, dinitrophenol, and NaN₃, but not with retinoic acid, basic pH, or high extracellular [Ca²⁺]. Dyecoupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.     

The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n = 5) or before (n = 5), during (n = 4), and after (n = 4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP-labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles ≥2 mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5 mm) and medium (2.5 to 4.0 mm) follicles, but large follicles (>4.0 mm) expressed higher mRNA and protein levels (all P < 0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.     

Molecular cloning and mRNA expression analysis of ornithine decarboxylase antizyme 2 in ovarian follicles of the Sichuan white goose (Anser cygnoides)

The ornithine decarboxylase antizyme 2 (OAZ2) gene is a member of the antizyme gene family. Antizymes play pivotal roles in various cellular pathways, including polyamine anabolism and apoptosis. The molecular structure and expression profile of the OAZ2 in goose ovarian follicles have not been reported. In this study, the OAZ2 cDNA sequence of the Sichuan white goose was cloned (Anser cygnoides), and phylogenetic and structural analyses of the OAZ2 were performed. The expression profiling of OAZ2 mRNA in goose ovarian follicles was examined using quantitative real-time PCR. The sequence analysis showed that the 756 bp OAZ2 sequence contained two overlapping open reading frames (ORF). ORF1 was 99 bp in length, and encoded a 32 aa polypeptide. ORF2 was 477 bp in length, and encoded a 158 aa polypeptide. The frameshift site that initiates the translation of ORF2 was located at nucleotide position 97 in the OAZ2. The analysis of OAZ2 mRNA expression in hierarchical follicles showed that the level of OAZ2 mRNA was higher in the SWF and F2 follicular stages than that in the ovarian stroma (P < 0.05). The lowest level of OAZ2 expression was detected in the ovarian stroma. These results suggest that the highly conserved frameshift region plays an important role in sustaining the function of OAZs. Furthermore, the significantly higher level of OAZ2 mRNA in the SWF stage indicates that OAZ2 may be involved in recruiting hierarchical follicles. Our results also suggest that OAZ2 may augment the effects of OAZ1 in follicle development.     

Complex interrelationships among CL,preovulatory follicle,number of follicular waves,and right or left ovaries in heifers

The diameter of the dominant follicle (DF) of wave 1 was studied on Days 9 to 17 (Day 0 = ovulation) in a survey of the ipsilateral and contralateral relationships between the location of the DF and CL, and number of follicular waves per interovulatory interval (IOI). For contralateral relationships, regardless of number of waves the diameter of the DF of wave 1 decreased (P < 0.03) between Days 11 and 13 when referenced to the follicle–CL relationship of wave 1 and decreased (P < 0.008) between Days 9 and 11 when referenced to the preovulatory follicle (PF)–CL relationship. For wave 2 in two-wave IOIs, the CL ovary of ipsilateral relationships had more (P < 0.05) follicles that reached at least 6 mm than the non-CL ovary. In three-wave IOIs, frequency of IOIs with the DF in the CL ovary was greater (P < 0.02) for wave 2 than for wave 3. In wave 3, the preovulatory and the largest subordinate follicles were located more frequently (P < 0.005) in the contralateral ovary. Ovulation in two-wave IOIs occurred more frequently (P < 0.0009) from the right ovary. In three-wave IOIs with a contralateral relationship ovulation occurred more frequently (P < 0.003) from the left ovary; a negative intraovarian effect of the CL on location of the PF may account for more ovulations from the left ovary and a reported greater frequency of the contralateral relationship. The hypothesis was supported that the ipsilateral versus contralateral relationship between the PF and CL is affected by the DF–CL relationship during the previous follicular waves and by the number and identity of waves per IOI.