Follicle deviation and ovulatory capacity in Bos indicus heifers

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.     

Ultrasonographic and endocrine aspects of follicle deviation, and acquisition of ovulatory capacity in buffalo (Bubalus bubalis) heifers

The objectives of this study were to determine the interval from ovulation to deviation and the diameter of the dominant (DF) and largest subordinate (SF) follicles at deviation in buffalo (Bubalus bubalis) heifers. Two methods of evaluation (observed vs. calculated) were used. FSH and LH profiles encompassing follicle deviation (Experiment 1), and the follicular diameter when the DF acquired ovulatory capacity (Experiment 2) were also determined. The time of deviation and the diameter of the DF and the largest SF at deviation did not differ between observed and calculated methods. Overall, follicle deviation occurred 2.6 ± 0.2d (mean ± SEM) after ovulation, and the diameters of the DF and SF at deviation were 7.2 ± 0.2 and 6.4 ± 0.2mm, respectively. No changes in plasma levels of FSH or LH were observed (P=0.32 and P=0.96, respectively). Experiment 2 was conducted in two phases according to the diameter of the DF during the first wave of follicular development at the time of LH challenge (25mg of pLH). In the first phase, follicles ranging from 5.0 to 6.0mm (n=7), 6.1 to 7.0mm (n=11), or 7.1 to 8.0mm (n=9) were used, and in the second phase, follicles ranging from 7.0 to 8.4mm (n=10), 8.5 to 10.0mm (n=10), or 10.1 to 12.0mm (n=9) of diameter were used. After the pLH treatment, the DF was monitored by ultrasonography every 12h for 48h. No ovulations occurred in heifers in the first phase. However, in the second phase, an effect of follicular diameter was observed on ovulation rate [7.0-8.4mm (0.0%, 0/10), 8.5-10.0mm (50.0%, 5/10), and 10.0-12.0mm (55.6%, 5/9)]. In summary, follicle deviation occurred 2.6d after ovulation in buffalo (B. bubalis) heifers, when the diameters of the DF and SF were 7.2 and 6.4mm, respectively. No significant changes in plasma concentrations of FSH or LH were detected. Finally, the acquisition of ovulatory capacity occurred when the DF reached 8.5mm in diameter.     

Post ovulatory follicles of the domestic duck.

Histogenesis of the post-ovulatory follicles in relation to the regression process has been studied in the Indian domestic duck. The activities of the alkaline phosphatase, acid phosphatase, sudan black-B and periodic acid Schiff reaction have been demonstrated histochemically. No lutein cells comparable to the mammalian corpus luteum are found. The hypertrophy or proliferation of granulosa cells are not observed in this bird. The functional significance of different histochemically demonstrable material and steroidogenesis in the avian post-ovulatory follicles have been briefly discussed.     

Relationships of changes in ultrasonographic image attributes to ovulatory and steroidogenic capacity of large antral follicles in sheep

Ovarian steroidogenesis and antral follicular development in ewes, following the treatment with medroxyprogesterone acetate (MAP) and equine chorionic gonadotrophin (eCG), are affected by the reproductive season. The objective of this study was to compare the ultrasonographic attributes of large antral follicles between cyclic (December) and seasonally anovular (June–July) ewes, after a 12-day treatment with MAP-soaked intravaginal sponges, with or without the administration of 500 IU of eCG at sponge removal, and to determine whether there is a correlation between the ultrasonographic attributes of the follicular wall and serum concentrations of oestradiol. Digital images of ovulatory follicles from cyclic ewes and eCG-treated anoestrous ewes (n = 34 follicles), and of anovulatory follicles attaining ≥5 mm in control anoestrous ewes (n = 8 follicles), were analysed using the spot and line techniques designed to determine the echotextural characteristics of the follicular antrum (central and peripheral), follicular wall and perifollicular ovarian stroma. The mean diameter of ovulatory follicles was greater (P < 0.001) in cyclic than anoestrous ewes, with or without the eCG treatment. The mean pixel heterogeneity (SD of numerical pixel values) of the follicular antrum (P < 0.05), as well as mean pixel intensity and heterogeneity of the peripheral antrum, follicular wall proper and perifollicular ovarian stroma (P < 0.05), were consistently greater in anoestrous than cyclic ewes at the time of sponge removal and 24 h after the treatment with MAP sponges or MAP/eCG. Mean oestradiol concentrations were greater (P < 0.05) in cyclic compared to anoestrous ewes in both MAP- and MAP/eCG-treated animals, from 1 to 2 days after sponge withdrawal. There was a moderate negative correlation (r² = 0.12, P < 0.05; Pearson's Product Moment and r² = 0.23, P < 0.05; ANCOVA) between mean pixel heterogeneity (standard deviation of mean pixel values) of the follicular wall proper (all follicles ≥5 mm in diameter) and serum concentrations of oestradiol after sponge withdrawal. Our results indicate that large antral follicles from cyclic and seasonally anovular ewes exhibit distinctive ultrasonographic characteristics. The differences in follicular echotexture appear to be related mainly to seasonal variations in ovarian follicular morphology and oestradiol production.     

Follicular dynamics during the ovulatory season in goats

Growth and regression of ovarian follicles>or=3 mm were studied by transrectal ultrasonography for 4 interovulatory intervals in each of 5 Saanen goats. The observed number of growing identified 4-mm follicles per day differed (P<0.05) from randomness, indicating that follicles, on the average, emerged in groups (waves). Averaged over all interovulatory intervals, the number of 3-mm follicles on each day that later reached >or=6 mm followed a pattern of significant peaks on Days 0 (ovulation), 4,8 and 14. A follicular wave was defined by consecutive days of entry of follicles>or=6 mm into the wave, and the day of emergence was defined as the first day that the >or=6 mm follicles were 3 mm. In 15 of 20 (75%) interovulatory intervals, 1 wave emerged during each of Day -2 to Day 1 (Wave 1); Days 2 to 5 (Wave 2); Days 6 to 9 (Wave 3); and Days 10 to 15 (Wave 4). Ovulation occurred during Wave 4. The mean days of emergence of Waves 1 to 4 were Days -1, 4, 8 and 13, respectively. However, in 5 of these 15 interovulatory intervals, 50% of the apparent waves merged or were continuous so that a distinction could not be made between 2 waves. The largest follicle grew to a larger (P<0.05) maximum diameter for Waves 1 (8.7+/-0.3 mm) and 4 (9.7+/-0.3 mm) than for Waves 2 (7.2+/-0.2 mm) and 3 (7.3+/-0.2 mm). The following observations suggested that the phenomenon of follicular dominance was more common during Waves 1 and 4 than during Waves 2 and 3: 1) the interwave intervals (days) were longer (P<0.05) for Waves 1 (3.4+/-0.2) and 4 (4.3+/-0.6) than for Waves 2 and 3 (2.5+/-0.2 for each wave) and 2) the correlation between maximum diameter of largest follicle and the subsequent interwave interval was significant for Waves 1 and 4 but not for Waves 2 and 3. The 5 remaining interovulatory intervals were irregular and involved more than 4 waves, including 2 interovulatory intervals with prolonged follicular phases (14 and 21) and failures of ovulation. In conclusion, the predominant follicular-wave pattern was 4 waves with ovulation from Wave 4, and apparent follicular dominance was expressed during some follicular waves, especially during Waves 1 and 4.     

Timing of emergence of ovulatory follicles in polyovulatory goats

The current study characterized the timing of emergence of ovulatory follicles during the follicular phase of the estrous cycle in polyovulatory does and assessed whether selection may influence ovulation rate through differences in ovarian follicular dynamics, by characterizing preovulatory follicular emergence and growth in two ecotypes of Neuquen-Criollo Argentinean goats (Short-Hair, n=11 and Long-Hair, n=9). During the breeding season, the time of estrus was synchronized in all does with two doses of a prostaglandin analogue. Ovarian laparoscopies were performed on days 17 and 19 after the induced estrus (day 0) and 7-15 h after the beginning of the subsequent estrus. Results indicate that both ecotypes of goats have common features in the ovarian follicular population and in the patterns of preovulatory follicular enlargement. In all the goats, most of the preovulatory follicles arose from the pool of follicles present in the ovary between days 17 and 19 of the estrous cycle. These follicles were all larger than 2mm at emergence, being the largest growing follicle present in the ovaries on days 17 and 19 in 56.5 and 78.6% of the does, respectively. The appearance of new follicles remained unaffected, while the mean number of small growing follicles decreased (P<0.05) during the follicular phase, indicating that preovulatory follicles do not suppress the emergence of new follicles but inhibit the growth of small follicles. A separate analysis of single and double ovulating does showed that 75% of the second ovulatory follicles in polyovulatory goats was present on the ovarian surface between days 17 and 19 of the estrous cycle, but appeared later in the other 25% of the estrous cycles. These findings support the hypothesis that follicular dominance effects are exerted during the preovulatory period, when the growth of follicles other than the ovulatory is inhibited, and that increases in ovulation rate in small ruminants are related to a reduced incidence of follicular atresia and an extended period of ovulatory follicle recruitment.     

Follicular fluid rheology and the duration of the ovulatory process

The fluid dynamics of ovulation were investigated to understand the mechanical role of follicular fluid in oocyte release. A set of equations describing the flow of fluid from an evacuating follicle was derived from basic principles. These equations demonstrate that, subject to assumptions about the available pressure differential and the source of the expulsive force, the size and shape of the ovulatory orifice have the largest influences on the rate of fluid loss, although the viscosity of the fluid is also an important variable. A thorough rheological examination of pig, bovine and human follicular fluids, performed using a cone-plate viscometer, demonstrated that these fluids have complex, non-Newtonian characteristics. The fluids also undergo time-dependent and spontaneous changes in viscosity at constant shear rates; some fluids were subject to coagulation-like events. Viscosity characteristics were unrelated to broad parameters of follicle development. The models used representative viscosity values to demonstrate that variations in the rate and duration of follicle evacuation, as observed by ultrasonography, could be explained largely by variations in fluid viscosity and the characteristics of the ovulatory orifice.     

Pre and post ovulatory changes in the concentration of prostaglandins in rat graafian follicles.

The concentrations of prostaglandin F (PGF) and prostaglandin E (PGE) were measured by radioimmunoassay in isolated Graafian follicles of mature female rats during the pre and post ovulatory period of the estrous cycle. The levels of these prostaglandins were low and relatively constant from 8 a.m. to 4 p.m. on the day of proestrus, but there was a marked increase at 8 p.m. of proestrus reaching an apparent maximum at midnight (PGF 18 fold, PGE 70 fold). By 4 a.m. to 8 a.m. on the morning of estrus these prostaglandins declined rapidly to levels similar to those observed between 8 a.m. and 4 p.m. on the day of proestrus. The increases in prostaglandin levels occurred after the LH peak and apparently before the time of ovulation. These data confirm the role of PGF and PGE in the local mechanism of ovulation in the normal adult of a spontaneously ovulating animal species.     

Steroid hormone formation in bovine ovarian follicles.

In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions.     

Post ovulatory changes in the concentration of prostaglandins in rabbit Graafian follicles

In previous studies we have demonstrated that prior to hCG induced ovulation the levels of PGF and PGE in rabbit Graafian follicles increase markedly as ovulation approaches. We have now extended the study to include follicles obtained from animals at ovulation time and up to 48 hours after hCG injection. We have found that PGF reaches a maximum in ovulated follicles at the time of ovulation and then quickly decreases, whereas PGE continues to rise for several hours and then declines. The increase in both prostaglandins is limited to the follicles that actually ovulate. These data further document the proposed role for prostaglandins in the ovulatory process.     

Follicular oocyte growth and acquisition of developmental competence

At birth the ovaries of mammalian females contain a finite store of primordial follicle oocytes. Each oocyte and its surrounding follicle cells share a communication system, the gap junction network, which facilitates the transfer of signals as well as nutrients in to and out off the oocyte and between follicle cells. The connexin family of proteins form the building blocks of this communication network, their expression is specific to the differentiated state of the granulose cell and the stage of folliculogenesis. Factors such as the c-kit receptor and its ligand, IGF-I, IGF-I receptors and the IGF binding proteins, members of the transforming growth factor beta (TGFbeta) family, in particular, some of the bone morphogenetic proteins, play prominent roles in oogenesis, primordial follicle activation and subsequent follicle/oocyte development culminating in oocyte ovulation. The oocyte undergoes a progressive series of morphological modifications as it grows and proceeds through the different stages of development. These structural rearrangements facilitate the increasing energy and nucleic acid synthesis requirements of the developing oocyte and are a prerequisite to the oocytes achievement of meiotic and embryo developmental competence. Several factors determine the ultimate competence of the oocyte, these have been investigated and attempts made to mimic these conditions in vitro. The complexity of the orchestration of the events that control oocyte growth and ultimate acquisition of developmental competence is under continuous investigation. The present review describes some of the findings to date.     

Pre and post ovulatory changes in the concentration of prostaglandins in rat Graafian follicles

The concentrations of prostaglandin F (PGF) and prostaglandin E (PGE) were measured by radioimmunoassay in isolated GRaafian follicles of mature female rats during the pre and post ovulatory period of the estrous cycle. The levels of these prostaglandins were low and relatively constant from 8 a.m. to 4 p.m. on the day of proestrus, but there was a marked increase at 8 p.m. of proestrus reaching an apparent maximum at midnight (PGF 18 fold, PGE 70 fold). By 4 a.m. to 8 a.m. on the morning of estrus these prostaglandins declined rapidly to levels similar to those observed between 8 a.m. and 4 p.m. on the day of proestrus. The increases in prostaglandin levels occurred after the LH peak and apparently before the time of ovulation. These data confirm the role of PGF and PGE in the local mechanism of ovulation in the normal adult of a spontaneously ovulating animal species.     

Age-related dynamics of follicles and hormones during an induced ovulatory follicular wave in mares

An ovulatory follicular wave was induced by ablation of follicles ≥6 mm and treatment with prostaglandin F2α (PGF) on Day 10 (ovulation = Day 0). Follicle and hormone dynamics of the induced waves were compared among three age groups: young (5-6 y, n = 14 waves), intermediate (10-14 y, n = 16), and old (≥18 y, n = 15). During the common-growth phase of the induced wave (Days 12-17), diameter of the future ovulatory follicle was not different among ages, but the young group had more (P < 0.05) follicles that reached ≥10 mm. The number was correlated (r = +0.7; P < 0.0001) within mares between consecutive interovulatory intervals, indicating repeatability. Concentrations of LH increased in all age groups during Days 12-17, but were greatest (P < 0.002) in the young group and continued to be greater (P < 0.0001) throughout the ovulatory LH surge. During several days before Day −1, there were no age-related effects on systemic estradiol concentrations, diameter of the preovulatory follicle, or B-mode echo texture or color-Doppler signals of blood flow in the follicle wall. Interpretations were: (1) greater number of follicles in the young group reflected a greater follicle reserve, (2) greater LH concentrations throughout the ovulatory surge in the young group reflected a more positive response to an extraovarian/environmental influence after removal of the negative effect of progesterone, and (3) lower LH concentrations in the older groups were adequate for the preovulatory changes in the follicle.     

Activation of bovine and baboon primordial follicles in vitro

Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles.     

Ultrastructure and composition of Call-Exner bodies in bovine follicles

Call-Exner bodies are present in ovarian follicles of a range of species including human and rabbit, and in a range of human ovarian tumors. We have also found structures resembling Call-Exner bodies in bovine preantral and small antral follicles. Hematoxylin and eosin staining of single sections of bovine ovaries has shown that 30% of preantral follicles with more than one layer of granulosa cells and 45% of small (less than 650 microns) antral follicles have at least one Call-Exner body composed of a spherical eosinophilic region surrounded by a rosette of granulosa cells. Alcian blue stains the spherical eosinophilic region of the Call-Exner bodies. Electron microscopy has demonstrated that some Call-Exner bodies contain large aggregates of convoluted basal lamina, whereas others also contain regions of unassembled basal-lamina-like material. Individual chains of the basal lamina components type IV collagen (alpha 1 to alpha 5) and laminin (alpha 1, beta 2 and delta 1) have been immunolocalized to Call-Exner bodies in sections of fresh-frozen ovaries. Bovine Call-Exner bodies are presumably analogous to Call-Exner bodies in other species but are predominantly found in preantral and small antral follicles, rather than large antral follicles. With follicular development, the basal laminae of Call-Exner bodies change in their apparent ratio of type IV collagen to laminin, similar to changes observed in the follicular basal lamina, suggesting that these structures have a common cellular origin.     

Follicular interactions affect the in vitro development of isolated goat preantral follicles

The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.     

Sequence of apoptosis and inflammatory necrosis within the formative ovulatory site of sheep follicles

The aim of this study was to define the temporal and spatial patterns of apoptosis, necrosis and inflammation within preovulatory ovine follicles. A gonadotrophin surge was induced in pro-oestrous ewes by GnRH, and isolated follicles were hemisected into apical and basal segments at 0, 10, 18 and 22 h (the time of ovulatory stigma development) after GnRH. Ovarian surface epithelial and granulosa cells were isolated and assessed by fluorescence microscopy for membrane phosphatidylserine translocation-annexin V (early-stage apoptosis), oligonucleosomal DNA nick endlabelling (advanced apoptosis), and nuclear propidium iodide incorporation (necrotic membrane disruption). Thecal shells were analysed for interstitial blood cells. Preovulatory follicles were also hemisected and subjected to electrophoretic DNA degradation analysis. Annexin V binding and in situ DNA fragmentation among ovarian surface epithelial and granulosa cells along the follicular apex were high 18 and 22 h after GnRH. Propidium iodide staining of apical ovarian surface and granulosa cells was apparent at 22 h. There was a coincident increase within the apical theca as the time of ovulation approached in extravasated leucocytes (18 and 22 h) and erythrocytes (22 h). Apoptotic DNA laddering and necrotic DNA smears within the follicular apex were evident on agarose gels at 18 and 22 h, respectively. In contrast, ovarian surface epithelium not associated with the ovulation site and the basal follicular wall were largely unafflicted. It is suggested that both modalities of cellular death, apoptosis and necrosis (with acute inflammation and vascular injury), contribute progressively to follicular stigma formation and ovarian rupture.