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1.
The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre‐antral follicles were evaluated. Follicles (≤200 μm) were cultured for 12 days in α‐MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre‐antral follicles.  相似文献   

2.
We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle‐stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty‐two pre‐pubertal ewes were assigned to experimental groups 1 to 5 (EG‐I to EG‐V) and control group (CG). Ewes in EG‐I, EG‐II and EG‐III were subcutaneously injected with 200, 300 or 400 μg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG‐IV and EG‐V were subcutaneously injected with 200 μg and 300 μg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P < 0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG‐IV and EG‐V were higher than that in EG‐I, EG‐II and EG‐III from days 35 to 45. Expressions of GnRHR protein in EG‐IV and EG‐V were lower than that in CG (P < 0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG‐IV and EG‐V (P < 0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG‐III and EG‐V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin‐immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P < 0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.  相似文献   

3.
The key biological active molecule of soya is the isoflavone daidzein, which possesses phytoestrogenic activity. The direct effect of soya and daidzein on ovarian cell functions is not known. This study examined the effect of daidzein on basic porcine ovarian granulosa cell functions and the response to follicle‐stimulating hormone (FSH). We studied the effects of daidzein (0, 1, 10 and 100 μm ), FSH (0, 0.01, 0.1, 1 IU/ml) and combinations of FSH (0, 0.01, 0.1, 1 IU/ml) + daidzein (50 μm ) on proliferation, apoptosis and hormone release from cultured porcine ovarian granulosa cells and ovarian follicles. The expression of a proliferation‐related peptide (PCNA) and an apoptosis‐related peptide (Bax) was analysed using immunocytochemistry. The release of progesterone (P4) and testosterone (T) was detected using EIA. Leptin output was analysed using RIA. Daidzein administration increased granulosa cell proliferation, apoptosis and T and leptin release but inhibited P4 output. Daidzein also increased T release and decreased P4 release from cultured ovarian follicles. Follicle‐stimulating hormone stimulated granulosa cell proliferation, apoptosis and P4, T and leptin release. The addition of daidzein promoted FSH‐stimulated apoptosis (but not proliferation) but suppressed FSH‐stimulated P4, T and leptin release. Our observations of FSH action confirm previous data on the stimulatory effect of FSH on ovarian cell proliferation, apoptosis and steroidogenesis and demonstrate for the first time the involvement of FSH in the upregulation of ovarian leptin release. Our observations of daidzein effects demonstrated for the first time that this soya isoflavone affected basic ovarian cell functions (proliferation, apoptosis and hormones release) and modified the effects of FSH. Daidzein promoted FSH action on ovarian cell proliferation and apoptosis and suppressed, and even inverted, FSH action on hormone release. The direct action of daidzein on basic ovarian cell functions and the ability of these cells to respond to FSH indicate the potential influence of soya‐containing diets on female reproductive processes via direct action on the ovary.  相似文献   

4.
Follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth, maturation and oestrus, but no clear pathway in the seasonal oestrus of yak (Bos grunniens) has been found. To better understand the role of FSH and LH in seasonal oestrus in the yak, six yaks were slaughtered while in oestrus, and the pineal gland, hypothalamus, pituitary gland, and gonads were collected. Using real‐time PCR and immunohistochemical assays, we determined the mRNA and protein expression of the FSH and LH receptors (FSHR and LHR) in these organs. The analysis showed that the FSHR mRNA expression level was higher in the pituitary gland tissue compared with LHR (p < .01) during oestrus. By contrast, there was low expression of FSHR and LHR mRNA in the pineal gland and hypothalamus. FSHR mRNA expression was higher than that of LHR (p < .05) in the ovary, whereas LHR mRNA expression was higher than that of FSHR (p < .01) in the uterus. FSHR and LHR proteins were located in the pinealocyte, synaptic ribbon and synaptic spherules of the pineal gland and that FSH and LH interact via nerve fibres. In the hypothalamus, FSHR and LHR proteins were located in the magnocellular neurons and parvocellular neurons. FSHR and LHR proteins were localized in acidophilic cells and basophilic cells in the pituitary gland, and in surface epithelium, stromal cell and gland epithelium in the uterus. In the ovary, FSHR and LHR protein were present in the ovarian follicle. Thus, we concluded that FSHR and LHR are located in the pineal gland, hypothalamus, pituitary and gonad during oestrus in the yak. However, FSHR was mainly expressed in the pituitary gland and ovaries, whereas LHR was mainly expressed in the pituitary gland and uterus.  相似文献   

5.
The aim of the present study was to evaluate the effects of different medium replacement intervals on the viability, antral cavity formation, growth and in vitro maturation (IVM) of oocytes from caprine and ovine pre‐antral follicles. Pre‐antral ovarian follicles (≥150 μm) were isolated from the ovarian cortex of goats and sheep and were individually cultured for 24 days using two different medium replacement intervals [2 days (T1) or 6 days (T2)]. Follicle development was evaluated on the basis of antral cavity formation, increases in follicular diameter and the presence of healthy cumulus oocyte complexes and fully grown oocytes. For caprine species, results showed a higher percentage (p < 0.05) of viable follicles in T1 than T2 from day 6 until the end of the culture. In addition, when comparing both treatments after the same culture duration, the rate of antrum formation was significantly higher in T1 than in T2 from day 12 onwards. Yet, in ovines, when both treatments were compared on day 24 of the culture, there were more viable follicles in T2 than in T1 (p < 0.05). In the caprine species, percentages of fully grown oocytes (≥110 μm) acceptable for IVM after 24 days of culture were significantly higher in normal follicles cultured in T1 (30.0%) than in T2 (6.7%; p < 0.05). On the other hand, in ovines, at the end of the culture, the percentage of oocytes destined for IVM was higher in T2 than in T1 (23.5% vs 2.9%; p < 0.05). In conclusion, under the same conditions, the frequency of medium replacement significantly affected the in vitro development of caprine and ovine pre‐antral follicles. To improve the efficiency of the culture system, the medium must be replaced every 2 and 6 days for goat and sheep pre‐antral follicles, respectively.  相似文献   

6.
Follicle Wave Growth in Cattle   总被引：1，自引：0，他引：1  
Ovarian follicle growth in cattle culminates in the selection of a single dominant follicle which attains the ability for final maturation and ovulation once or twice during the luteal phase and at the end of the oestrous cycle, as well as during other reproductive states. This review will describe in detail the first follicle wave of the cycle leading to selection of the first wave dominant follicle, indicating the specific gonadotrophin dependencies of cohort and dominant follicles, and relating follicle fate to steroidogenesis. As a differential gonadotrophin response of growing antral follicles during the follies‐stimulating hormone (FSH) decline may determine which follicle becomes selected, first wave follicles are also characterized in relation to intrafollicular growth factors, which may modify the gonadotrophin response, such as inhibins and members of the insulin‐like growth factor (IGF) family. Subsequently, the follicular control of the transient FSH rise and decline so crucial to dominant follicle selection will be discussed. It is concluded that successful hormonal manipulation of follicle wave growth and dominant follicle selection will depend on our detailed understanding of the gonadotrophin requirements of differentiating wave follicles.  相似文献   

7.
The aim was to verify the effect of follicle‐stimulating hormone (FSH) supplementation to α‐MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non‐cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag‐NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7‐day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag‐NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7‐day culturing conditions.  相似文献   

8.
This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre‐antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre‐antral follicles, the groups with 100 ng/ml FSH of 2‐days culture as well as 50, 100 and 200 ng/ml FSH of 6‐days culture provided the best results. In conclusion, the in vitro culture of abattoir‐derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.  相似文献   

9.
Enolases are glycolytic enzymes in the glycolytic pathway. In order to evaluate the effect of ENO1 on follicle-stimulating hormone receptor (FSHR) mRNA and luteinizing hormone receptor (LHR) mRNA of primary granular cell from goose F1 follicles, the recombinant plasmid adenovirus carrying ENO1 were constructed and infected the primary culture granular cells. The granular cells were randomly divided into three groups: recombinant adenovirus infected (pAd-CMV-ENO1), empty vector infected (pAd-CMV-Null) and no virus (mock control). The expression levels of FSHR mRNA and LHR mRNA of granular cells were examined by qRT-PCR. The results showed the group pAd-CMV-ENO1 had significantly higher FSHR mRNA expression levels than the other two groups (P < 0.05), but had significantly lower LHR mRNA expression levels than the other two groups (P < 0.05). The results suggested that ENO1 could improve the combination rate between FSH and FSHR to accelerate the proliferation and differentiation and steroidogenesis in poultry gonadal tissues.  相似文献   

10.
The growth of ovarian follicles is accompanied by fluid‐filled antrum formation. Water movement within the follicular wall is predominantly transcellular via membranous water channels named aquaporins (AQPs). Androgens are important regulators of mammalian folliculogenesis, and their prenatal and/or neonatal deficiency affects female fertility in adulthood. Therefore, this study was performed to determine whether gestational or neonatal exposure to the anti‐androgen flutamide influences androgen‐dependent AQP5 expression in pre‐antral and large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 post‐natally. The ovaries were collected from flutamide‐treated and non‐treated (control) sexually mature pigs. In pre‐antral follicles, AQP5 mRNA and protein levels were both downregulated following maternal (p < 0.01 and p < 0.01, respectively) and neonatal (p < 0.01 and p < 0.01, respectively) flutamide exposure. Likewise, the expression of mRNA (p < 0.01 and p < 0.001, respectively) and protein (p < 0.05 and p < 0.01, respectively) for AQP5 were diminished in large antral follicles in both groups. Immunohistochemistry showed decreased intensity of AQP5 immunoreaction in pre‐antral (p < 0.01) and large antral (p < 0.001) follicles following flutamide treatment. Moreover, radioimmunological analysis revealed that changes observed in AQP5 expression corresponded with diminished follicular androgens production after both maternal (p < 0.05 and p < 0.05, respectively) and neonatal (p < 0.05 and p < 0.01, respectively) flutamide administration. Therefore, AQP5 appears to be a potential regulator of follicular fluid accumulation, under androgen control, and may be a key factor in antral follicle growth.  相似文献   

11.
The effect of co‐culture of buffalo preantral follicles (PFs) with different somatic cells, i.e, cumulus, granulosa, ovarian mesenchymal and oviductal epithelial cells was studied. Large PFs (250–450 μm) were isolated by microdissecting the trypsin (1%) digested ovarian cortical slices. Cumulus cells were isolated by repeated pipetting of oocytes, granulosa cells were isolated by aspirating from punctured PFs and ovarian mesenchymal cells were isolated from ovarian cortex by scraping the cortical slices and passing through 20 μm filter. Preantral follicles were cultured in standard culture medium without somatic cells or co‐cultured with cumulus cells, granulosa cells, ovarian mesenchymal cells and oviductal epithelial cells for 80 days. The growth rate (μm/day) of the PFs was monitored by measuring follicular diameter on day 0, 30, 60 and 80 days of culture. The viability of PFs was evaluated by trypan blue staining. The results indicated that PFs co‐cultured with cumulus, granulosa and ovarian mesenchymal cells had a better development and survivality compared with control and those co‐culture with oviductal epithelial cells. Maximum growth and survivality of PFs were achieved when cultured with cumulus cells. It is concluded that inclusion of somatic cells in PF culture media had beneficial effect on the growth of PFs and cumulus cells supported maximum growth and survivality of PFs in vitro of all somatic cells tested.  相似文献   

12.
During an oestrous cycle, a cohort of antral follicles develops into – depending on the species – one or more ovulatory follicles. The bovine oestrous cycle is characterized by two to three such cohorts or growth waves, only the last of which will result in an ovulation. In every growth wave, several antral follicles are recruited for development. Recruited follicles are subjected to a selection process, whereby ever decreasing levels of follicle stimulating hormone (FSH) are available to the FSH dependent follicles. In the cow, a single follicle from the cohort will acquire dominance. The ability of the dominant follicle to prosper under basic FSH levels is ascribed to a transition in hormone dependency from FSH to luteinizing hormone. The exact follicle selection mechanism remains, however, to be elucidated. The beginning of this article focuses on the recruitment, selection and dominance phases in antral follicle development. Subsequently, the conditions leading to successful maturation and ovulation are discussed. The next section expounds upon the mechanisms for exogenous modulation of follicular dynamics with the aim of superovulation/superstimulation, and finally prospective future research directions are sketched.  相似文献   

13.
This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.  相似文献   

14.
The effect of exogenous administration of lamprey GnRH‐III (IGnRH‐III) on gonadotropin secretion was evaluated in pigs. Six crossbred barrows (82.4 ± 3.5 kg body weight) were assigned randomly to a replicated 3 × 3 Latin square design to evaluate the effect of 0.1, 1.0 or 10.0 μg/kg body weight of exogenous IGnRH‐III on LH and FSH secretion. To facilitate blood collection and infusion of IGnRH‐III, barrows were catheterized in the jugular vein 1 day before initiation of experiments. Blood samples were taken at 10‐min intervals for 6 h, starting 2 h before treatments were applied. Relative concentrations of LH and FSH were calculated by obtaining the ratio of the average concentration of each hormone 2 h after infusion divided by the average concentration during the 2 h before infusion. Relative concentrations of FSH after IGnRH‐III infusion did not influence mean concentration of FSH at any of the doses; yet 10.0 μg/kg body weight had a significant effect on LH secretion (p < 0.01). Relative concentrations of LH averaged 1.2, 1.0 and 3.0 ng/ml (for doses of 0.1, 1.0 and 10.0 μg/kg body weight of IGnRH‐III respectively). Only a dose of 10 μg/kg body weight elicited a significant LH increase that was associated with exogenous IGnRH‐III infusion. We conclude that IGnRH‐III is a weak GnRH agonist and at high doses, IGnRH‐III has the ability to release LH but not FSH in barrows.  相似文献   

15.
Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF‐2 protein expression in ovine ovaries and to verify the effect of FGF‐2 on the morphology, apoptosis and growth of ovine pre‐antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α‐MEM+) alone or supplemented with FGF‐2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF‐2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF‐2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF‐2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF‐2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF‐2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF‐2 compared with the control medium and other FGF‐2 treatments. In conclusion, this study demonstrated the presence of FGF‐2 in ovine ovaries. Furthermore, 10 ng/ml FGF‐2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.  相似文献   

16.
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17.
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18.
In the ovary, the development of new capillaries from pre‐existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin‐like growth factor (IGF), angiopoietin (ANPT) and hypoxia‐inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol‐17‐beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5–7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12 13–16 and >18 of the oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT‐qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF‐A, FGF‐2, IGF‐1 and IGF‐2, ANPT‐2/ANPT‐1 and HIF‐1‐alpha was found during final follicle maturation and in CL during the early luteal phase (days 1–4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.  相似文献   

19.
The objective of this study was to examine the effects of FSH and LH on oestradiol‐17β and progesterone production by buffalo granulosa cells cultured under serum‐free conditions. Granulosa cells (3 × 105) from small (≤5 mm diameter) follicles were cultured for up to 4 days in 48‐well plates coated with 3.3 μg/cm2 fibronectin in Dulbecco's modified Eagle's medium (DMEM) : nutrient mixture F‐12 Ham (1 : 1 ratio) supplemented with 10?7 m androstenedione, 5 μg/ml human apo‐transferrin and 0.1% bovine serum albumin, in the presence or absence of FSH or LH (0, 1, 2, 4, 8, 16, 32 or 64 ng/ml each). Basal oestradiol‐17β production by granulosa cells from small follicles reduced (p < 0.01) from days 1 to 2 of culture and became undetectable by day 3 and basal progesterone production increased (p < 0.05) from day 1 through day 4 of the culture. Although there was no effect of FSH on day 1 of the culture, FSH at 2, 4, 8 and 16 ng/ml increased (p < 0.05) oestradiol‐17β production by granulosa cells from small follicles on day 2. Progesterone secretion was increased (p < 0.05) by all doses of FSH on all days of culture. All doses of LH had no effect on oestradiol‐17β or progesterone production by granulosa cells from small follicles on any day of the culture. The results of this study demonstrate a serum‐free culture system for buffalo granulosa cells and stimulatory effect of FSH but not LH on steroid hormone production by buffalo granulosa cells under these conditions.  相似文献   

20.
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