Our objective was to understand how maternal age influences the mitochondrial population and ATP content of in vivo matured bovine oocytes. We hypothesized that in vivo matured oocytes from older ...cows would have altered mitochondrial number and distribution patterns and lower cytoplasmic ATP content compared to the oocytes obtained from younger cows. Follicles ≥5mm were ablated in old cows (13 to 22 yrs, Old Group, n = 7) and their younger daughters (4 to 10 years old, Young Group; n = 7) to induce the emergence of a new follicular wave. Cows were treated twice daily with eight doses of FSH starting 24 hr after ablation (Day 0, day of wave emergence). Prostaglandin F2alpha (PGF) was given on Days 3 and 3.5, LH on Day 4.5, and cumulus-oocyte-complexes were collected 18-20 hours post-LH by ultrasound-guided follicular aspiration. Oocytes were either processed for staining with MitoTracker Deep Red FM or for ATP assay. Stained oocytes were imaged with a Zeiss LSM 710 confocal microscope, and mitochondria were segmented in the oocyte volume sets using Imaris Pro 7.4. In vivo matured oocytes obtained from old cows were similar in morphological grades to those from young cows. However, the oocytes of COC from older cows had 23% less intracellular ATP (27.4±1.9 vs 35.7±2.2 pmol per oocyte, P = 0.01) than those of young cows. Furthermore, the average volume of individual mitochondria, indicated by the number of image voxels, was greater (P<0.05) in oocytes from older cows than in those from younger cows. Oocytes from older cows also tended to have a greater number of mitochondrial clusters (P = 0.06) and an increased number of clusters in the central region of the oocytes (P = 0.04) compared to those from younger cows. In conclusion, our study demonstrated that maternal age was associated with a decrease in the cytoplasmic ATP content of in vivo mature oocytes and an altered distribution of mitochondrial structures. These findings suggest that maternal age may negatively influence the developmental competence of oocytes from older cows.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BACKGROUND
Ovarian follicles undergo dynamic morphologic and endocrinologic changes during the human menstrual cycle. The physiologic mechanisms underlying recruitment and selection of antral ...follicles in women are not fully elucidated.
METHODS
A comprehensive review of >200 studies was conducted using PubMed. The objective was to compare and contrast different perspectives on human antral folliculogenesis.
RESULTS
Antral folliculogenesis has been studied using histologic, endocrinologic and/or ultrasonographic techniques. Different theories of antral follicle recruitment include: (i) continuous recruitment throughout the menstrual cycle; (ii) recruitment of a 'cohort' of antral follicles once in the late-luteal phase or early-follicular phase of each cycle and (iii) recruitment of two or three 'cohorts' or 'waves' during each cycle. Generally, a single dominant follicle is selected in the mid-follicular phase of each cycle and this follicle ovulates at mid-cycle. However, a dominant follicle may also be selected during anovulatory waves that precede the ovulatory wave in some women.
CONCLUSIONS
There is increasing evidence to indicate that multiple waves of antral follicles develop during the human menstrual cycle. Ovarian follicular waves in women are comparable with those documented in several animal species; however, species-specific differences exist. Enhancing our understanding of the endocrine and paracrine mechanisms underlying antral follicular wave dynamics has clinical implications for understanding age-related changes in reproductive function, optimizing hormonal contraceptive and ovarian stimulation regimens and identifying non-invasive markers of the physiologic status of follicles which are predictive of oocyte competence and assisted reproduction outcomes.
Kisspeptin modulates GnRH secretion in mammals and peripheral administration of 10-amino acid fragment of kisspeptin (Kp10) induces LH release and ovulation in cattle. Experiments were done to ...determine if iv administration of kisspeptin will activate GnRH neurons (i.e., after crossing the blood-brain barrier) and if pre-treatment with a GnRH receptor blocker will alter kisspeptin-induced LH release (from gonadotrophs) and ovulation. In Experiment 1, cows (n = 3 per group) were given human-Kisspeptin10 (hKp10; 3 x 15 mg iv at 60-min intervals) or normal saline and euthanized 150 min after treatment was initiated. Every 20th free-floating section (50 μm thickness) from the preoptic area to hypothalamus was double immunostained to colocalize GnRH- (DAB) and activated neurons (cFOS; Nickel-DAB). Kisspeptin induced plasma LH release from 15 to 150 min (P = 0.01) but the proportion of activated GnRH neurons did not differ between groups (5.8% and 3.5%, respectively; P = 0.11). Immunogold electron microscopy detected close contacts between kisspeptin fibers and GnRH terminals in the median eminence. In Experiment 2, pubertal heifers (n = 5 per group) were treated with 1) hKp10 iv, 2) Cetrorelix (GnRH antagonist; im) + hKp10 iv or 3) saline on Day 6 of the follicular wave under low-progesterone condition. A rise in plasma LH concentration was detected from 15 to 240 min in the hKp10 group but not in cetrorelix or control group (P<0.001). Ovulations were detected only in the hKp10 group (4/5; P = 0.02). Cetrorelix treatment was associated with regression of the preovulatory dominant follicle and emergence of a new follicular wave 3.4±0.75 days after the treatment in all five heifers. Results support the hypothesis that the effect of peripheral kisspeptin is mediated downstream of GnRH synthesis and does not involve GnRH-independent LH release from gonadotrophs. Peripheral kisspeptin may release pre-synthesized GnRH from the nerve terminals in areas outside the blood-brain barrier.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The type of stimuli triggering GnRH secretion has been used to classify mammalian species into two categories: spontaneous or induced ovulators. In the former, ovarian steroids produced by a mature ...follicle elicit the release of GnRH from the hypothalamus, but in the latter, GnRH secretion requires coital stimulation. However, the mechanism responsible for eliciting the preovulatory LH surge in induced ovulators is still not well understood and seems to vary among species. The main goal of this review is to offer new information regarding the mechanism that regulates coitus-induced ovulation. Analysis of several studies documenting the discovery of β-NGF in seminal plasma and its role in the control of ovulation in the llama and rabbit will be described. We also propose a working hypothesis regarding the sites of action of β-NGF in the llama hypothalamus. Finally, we described the presence of β-NGF in the semen of species categorized as spontaneous ovulators, mainly cattle, and its potential role in ovarian function. The discovery of this seminal molecule and its ovulatory effect in induced ovulators challenges previous concepts about the neuroendocrinology of reflex ovulation and has provided a new opportunity to examine the mechanism(s) involved in the cascade of events leading to ovulation. The presence of the factor in the semen of induced as well as spontaneous ovulators highlights the importance of understanding its signaling pathways and mechanism of action and may have broad implications in mammalian fertility.
Experiments were done to determine ovulation synchrony following a 4-day letrozole treatment (Exp 1), compare the efficacy of a letrozole-based protocol with other commonly used synchronization ...protocols for FTAI (Exp 2), and test a new intravaginal letrozole-releasing device (LRD) with and without pre-synchronization (Exp 3). In Exp 1, heifers and lactating cows at random stages of the estrous cycle were given an LRD for 4 days, PGF at LRD removal, and GnRH at 48 or 60 h after PGF, or no GnRH. In Exp 2, heifers and lactating cows were assigned to three FTAI groups: i) LRD, ii) estradiol+progesterone, iii) 5-d Co-synch+PRID. In Exp 3, heifers were pre-synchronized with PGF or not (control) 8 days before insertion of either an X-LRD or T-LRD for 3 days; FTAI was done 48 h after device removal. In Exp 1, the variation in interval to ovulation in cows, but not heifers, in the GnRH 48-h group was less than half that in other groups (P < 0.05). In Exp 2, the P/AI was lower (P < 0.001) in the LRD group compared to the other groups. In Experiment 3, the X-LRD increased (P < 0.0001) plasma letrozole concentrations compared to the T-LRD; the pregnancy rate was not affected by pre-synchronization or the type of LRD. Although ovulation synchrony was achieved following LRD treatment, the LRD group had the lowest P/AI compared to other protocols, perhaps because of too short an interval between LRD removal and GnRH/FTAI. Drug delivery was enhanced with the new X-LRD.
•A new protocol was developed for FTAI with letrozole.•Letrozole treatment prior to FTAI lowered pregnancy per AI compared to other groups.•A new letrozole-releasing device was developed that increased plasma letrozole.
In the female camelid, systemic administration of NGF induces a preovulatory LH surge that results in ovulation, but the effects of seminal NGF in the male are unknown. In the present study, we ...tested the hypothesis that the LH-releasing pathway of NGF is present in male camelids. In Experiment 1, male llamas and alpacas were treated with NGF or GnRH (n = 2 llamas and 3 alpacas) and blood samples were collected from 1 h before to 3 h after treatment. Plasma LH concentrations increased after treatment in a surge-like fashion in both GnRH- and NGF-treated groups, but concentrations reached a maximum 2.5 times higher and remained elevated for at least 2 h longer in the NGF-treated group (treatment-by-time interaction, P = 0.01). In Experiment 2, we evaluated the LH and testosterone response to NGF vs saline treatment (n = 3 llamas and 3 alpacas). The LH response to NGF was similar to that in Experiment 1, and plasma testosterone concentrations were higher in the NGF group than in the saline group at 2, 4 and 6 h after treatment (P < 0.05). Results support the hypothesis that the LH-releasing pathway for NGF exists in male South American camelids. The LH response to NGF sustained circulating testosterone concentrations in llamas, suggesting a moderate role of NGF in testosterone secretion.
•NGF induces LH release in male camelids.•NGF -induced LH secretion may be related to testosterone secretion in male llamas and alpacas.
nerve of ovulation-inducing factor in semen Ratto, Marcelo H; Leduc, Yvonne A; Valderrama, Ximena P ...
Proceedings of the National Academy of Sciences,
09/2012, Letnik:
109, Številka:
37
Journal Article
Recenzirano
Odprti dostop
A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad ...implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12–23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of β nerve growth factor (β-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as β-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC ₁₂) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of β-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is β-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo–pituitary–gonadal axis of the inseminated female.
Ovulation-inducing factor in semen (OIF/NGF) influences ovulation and CL form and function in camelids and, remarkably, in cows. To test the hypothesis that the luteotrophic effect of OIF/NGF is ...mediated by an increase in trkA receptors in the ovulatory follicle and early CL, a study was designed to characterize the spatial and temporal distribution of trkA in ovarian follicles and CL at known stages of the bovine estrous cycle.
Sexually mature cattle (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6 or in the pre-ovulatory period just before or after exogenous LH treatment. After a complete interovulatory interval, the cows were re-assigned to a different day-group on which the remaining ovary was removed. Sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were processed for immunofluorescent detection and quantification of trkA receptor.
TrkA immuno-fluorescence in ovarian tissues was restricted to follicles and the CL (no reaction in stroma or vessels), and was restricted to the cytoplasm (no nuclear staining). The trkA staining intensity, area of staining, and proportion of cells stained was greater in both theca and granulosa layers of dominant follicles than in that of subordinate follicles (P ≤ 0.05) in all day-groups except the Pre-LH group. Among dominant follicles, a progressive reduction in the immuno-positive reaction was detected from Day 2 to Day 6. Among subordinate follicles, immuno-reactivity remained low and unchanged except a rise in the Pre-LH group. The number of immuno-positive cells was greater in early developing CL (Days 2 and 4 combined) than in mature or regressing stage CL (Day 6, Pre- and Post-LH combined; P = 0.01). The intracellular distribution of trkA was more diffuse and widespread in dominant than subordinate follicles, particularly on Day 2 and Post-LH (P < 0.05).
Distinct differences in trkA expression between dominant and subordinate follicles, particularly when circulating progesterone is minimal (early luteal development and after luteolysis) is consistent with a local role of OIF/NGF in follicle selection and early luteogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ovulation in mammals involves pulsatile release of GnRH from the hypothalamus into the hypophyseal portal system with subsequent release of LH from the anterior pituitary into systemic circulation. ...Elevated circulating concentrations of LH induce a cascade of events within the mature follicle, culminating in follicle rupture and evacuation. The broad classification of species as either spontaneous or induced ovulators is based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus. In spontaneously ovulating species (e.g., human, sheep, cattle, horse, pigs), release of GnRH from the hypothalamus is triggered when, in the absence of progesterone, systemic estradiol concentrations exceed a threshold. In induced ovulators (e.g., rabbits, ferrets, cats, camelids), release of GnRH is contingent upon copulatory stimuli; hence, ovulation is not a regular cyclic event. Since a classic 1970 Peruvian study, dogma has maintained that physical stimulation of the genitalia during copulation is the primary trigger for inducing ovulation in alpacas and llamas. Exciting results of recent studies, however, provide direct evidence for the existence of an ovulation-inducing factor (OIF) in semen, and compel us to re-examine the mechanism of ovulation in both induced and spontaneous ovulators. Ovulation-inducing factor in seminal plasma is a potent stimulant of LH secretion, ovulation and luteal gland development, and acts via a systemic rather than a local route. OIF is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K. It has a molecular mass of 14kDa, and may be part of a larger protein complex or pro-hormone. The effect of OIF is dose-related and evident at physiologically relevant doses (i.e., as little as 1/100th that present in the ejaculate), and is mediated, in whole or in part, at the level of the hypothalamus in vivo. The factor exists in the seminal plasma of every species in which it has been examined thus far, including Bactrian camels, alpacas, llamas, cattle, horses, pigs, and koalas. Seminal plasma OIF does not appear to be a phylogenetic vestige in spontaneous ovulators since it (1) induced ovulation in pre-pubertal mice, (2) altered ovarian follicular wave dynamics in cows, and (3) elicited LH release in vitro from primary pituitary cell cultures of rats, mice, guinea pigs, rabbits, llamas and cows.
To elucidate the mechanism by which nerve growth factor (NGF) influences the LH secretory pathway in camelids, a series of experiments were done to determine the involvement of the hypothalamus ...(Experiment 1), the role of GnRH neurons (Experiment 2), and the effect of progesterone (Experiment 3) on the NGF-induced LH surge and ovulation in llamas. In Experiment 1, the declining phase of the NGF-induced LH surge was used to determine if the decline is a result of pituitary depletion or hypothalamic unresponsiveness. Female llamas were treated with NGF and, 7 h later, assigned to three groups and given a second dose of NGF (n = 5), a dose of GnRH (n = 5), or saline (n = 6). The LH response was attenuated after the second dose of NGF vs GnRH. In Experiment 2, Fos expression (marker of neuronal activation) in GnRH neurons was examined in the hypothalamus of llamas after NGF or saline treatment (n = 3 per group). Despite an LH surge in the NGF group but not in the saline group, no differences were detected between groups in Fos/GnRH co-expression. In Experiment 3, llamas in low-, medium-, and high-plasma progesterone groups (n = 4 per group) were treated with NGF. The NGF-induced LH surge did not differ among treatment groups. Results from the present study show that the induction of a preovulatory LH surge by NGF may be controlled by a novel pathway involving GnRH neuro-terminals downstream of the hypothalamus and is independent of progesterone influence.
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