Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.
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Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo. (+info)
Markers of follicle function in Belclare-cross ewes differing widely in ovulation rate.
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High prolificacy due to a gene that has a large effect on ovulation rate has been noted in Booroola and Inverdale ewes. High prolificacy in the Belclare breed (a composite developed from stocks selected for very large litter size or high ovulation rate) may be related to the segregation of two genes. The aims of this study were (i) to compare the morphological and functional features of ovulatory follicles from carriers (which could only be heterozygous for the genes of interest) and non-carriers, and (ii) to identify markers of the Belclare genes among secreted or cellular ovarian proteins. Belclare carrier ewes had more ovulatory follicles (4.9 +/- 0.4) than did non-carrier ewes (2.0 +/- 0.2) (P < 0.001). Ovulatory follicles from carriers were also smaller (4.4 +/- 0.1 mm versus 5.7 +/- 0.2 mm, P < 0.001) and contained a significantly reduced number of granulosa cells (P < 0.001). However, the proportion of proliferating granulosa cells in ovulatory follicles was similar in both groups. The in vitro secretion of steroids per follicle was only marginally lower in follicles from Belclare carriers compared with non-carriers. Furthermore, similar concentrations of steroidogenic enzymes were present in both groups, indicating that steroidogenic potential per granulosa cell is similar between carriers and non-carriers. Possible markers of the Belclare genes were identified among cellular proteins of follicular walls by two-dimensional PAGE and image analysis. Two spots at 78 and 49 kDa were always absent in samples from non-carriers. When secreted proteins in follicles from carriers were compared with those from non-carriers, two spots at 53 and 41 kDa were restricted to samples from carriers and three spots at 97, 91 and 45 kDa were unique to samples from non-carriers. Interestingly, the spot at 91 kDa is also affected by the Booroola gene. (+info)
Follicular and luteal expression of insulin-like growth factors I and II and the type 1 IGF receptor in the bovine ovary.
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The localization of mRNAs for insulin-like growth factors I (IGF-I) and II (IGF-II) and the type 1 IGF receptor (IGF-1R) in bovine follicles and corpora lutea was determined using in situ hybridization on sectioned ovaries collected from nonpregnant, cyclic Holstein cows in either the follicular (n = 3) or luteal (n = 5) phases of the cycle. Concentrations were measured as absorbance units of individual regions or follicles from autoradiographs. There was intense follicular expression of mRNAs encoding IGF-II and IGF-1R. For mRNA encoding IGF-II, expression was significantly higher in smaller follicles (< 5 mm diameter, P < 0.01) and, in this size range, expression was significantly greater in healthy compared with atretic follicles (P < 0.01). For mRNA encoding IGF-1R, there was no effect of size but concentrations were again significantly greater in healthy compared with atretic follicles of < 5 mm. In medium (5-10 mm) and large (> 10 mm) follicles, there was no effect of health for expression of either IGF-II or IGF-1R. mRNA encoding IGF-II was found exclusively in the theca, whereas mRNA encoding IGF-1R was confined to the granulosa layer. IGF-I expression was not detectable in 83% of the 53 follicles examined. In the remaining 17% of follicles, expression was very low and was unrelated to size or state of atresia. mRNAs encoding IGF-I, -II and IGF-1R were all present in the corpus luteum, whereas only those for IGF-II and IGF-1R were found in ovarian stroma. These data indicate that the insulin-like growth factors play a significant role in follicular and luteal development in the bovine ovary. Locally produced IGF-II is probably an important regulator of follicular growth, whereas most of the IGF-I present in follicular fluid is likely to be derived from the circulation. (+info)
Distribution of ganglioside GM3 in the rat ovary after gonadotropin stimulation.
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Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Ovaries have been shown to contain GM3 as a major ganglioside. To study GM3 distribution during gonadotropin stimulation in the hypophysectomized rat ovary, ovarian sections and cultured granulosa cells were stained with specific monoclonal antibody against GM3. Interstitial cells of follicles of immature hypophysectomized rat ovary expressed ganglioside GM3. Theca cells of early antral follicles but not primary follicles expressed GM3. No granulosa cells of these follicles expressed GM3. When a surge dose of FSH/LH was injected, Graafian follicles were formed and GM3 expression was detected in granulosa cells of these follicles. After ovulation, cumulus cells kept expressing GM3 in the ampulla region of ovulated oviduct. The follicles did not show GM3 expression in their granulosa cells after an ovulatory dose of FSH/LH. At 48 h after in vitro culture with FSH/LH of granulosa cells from preantral follicles, GM3 was expressed to a detectable extent on the outer part of the granulosa layer. Finally, at 72 h after culture, all granulosa cells became positive to anti-GM3 antibody. These data suggest that the expression of ganglioside GM3 in the hypophysectomized rat ovary is spatiotemporally regulated by FSH/LH during follicular development and ovulation. (+info)
Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin alpha6beta1: implications for murine fertilization.
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CD9 is a tetraspan protein that associates with several beta1 integrins, including alpha6beta1. Because alpha6beta1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin beta), we first asked whether CD9 is present on murine eggs and whether it functions in sperm-egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm-egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin beta disintegrin domain. (Both ligands bind to the egg via alpha6beta1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin alpha6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, alpha6A and CD9 both are expressed on the apical epithelial surface at the uterine-oviduct junction. These findings correlate with the observation that fertilin beta "knockout" sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin beta(-/-) male mice. Our results suggest that high-avidity binding between fertilin beta (ADAM 2) and alpha6beta1 requires cooperation between alpha6beta1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm-egg interactions. (+info)
Effect of estrus synchronization with norgestomet on the integrity of oocytes from persistent follicles in beef cattle.
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Our objective was to determine whether oocyte integrity is compromised when oocytes are recovered from progestogen-induced persistent follicles. Beef cows were presynchronized using PGF2alpha (PGF). Cows detected in estrus after PGF were assigned to either NOR (one 6-mg norgestomet implant for 10 d starting on d 16 of cycle; day 0 = estrus; n = 112) or CON (control, no implant [n = 128] and presynchronized 8 d later than NOR). All cows received 25 mg of PGF at the end of treatment (NOR, d 26; CON, d 18). Treatments produced persistent preovulatory follicles (NOR) or normal preovulatory-size follicles (CON), which were measured via ultrasonography 1 d before slaughter. Ovaries were collected from all animals (NOR, d 27; CON, d 19) along with random (RAN) ovaries from cattle slaughtered on the same days. Cumulus oocyte complexes (COC) were aspirated from the preovulatory follicles with recovery rates of 63% across treatments. Small follicles (2 to 7 mm diameter) from NOR, CON, and RAN cows were also aspirated to recover COC. Preovulatory follicles were larger (19.5+/-.9 vs. 13.6+/-.4 mm, P<.05), serum P4 was lower (.4+/-.1 vs. 3.9+/-.2 ng/mL, P<.05), and serum E2 was higher (28.7+/-1.6 vs. 7.6+/-.8 pg/mL, P<.05) in NOR than in CON cows. Cumulus oocyte complexes recovered from preovulatory follicles (62 NOR, 64 CON) were matured, fertilized, and cultured in vitro for comparison of embryonic development. A subset (24 NOR, 34 CON) of COC were assigned morphological quality grades. A separate set of recovered COC (10 NOR, 15 CON) was fixed within 1 h after recovery for assessment of the stage of meiosis. Treatments did not differ for oocyte quality grade or stage of meiosis. However, COC from NOR cows had more layers of cumulus cells (P<.05), and more of those COC had undergone cumulus expansion (29.2 vs. 5.9%, P<.05 for NOR vs. CON, respectively). Development of cleaved embryos to the morula and blastocyst stages from preovulatory follicles (22.6% NOR, 18.9% CON) or small follicles (42% NOR, 40% CON, 42% RAN) did not differ with treatment. Oocyte quality and in vitro developmental competence were not compromised for oocytes from induced persistent follicles compared with oocytes from normal preovulatory follicles. Increased expansion of cumulus cells associated with oocytes from progestogen-induced persistent follicles may be relevant to the reduction of in vivo fertility associated with such follicles. (+info)
Oocyte regulation of kit ligand expression in mouse ovarian follicles.
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Kit ligand (KL), a product of granulosa cells in ovarian follicles, is a putative regulator of oocyte development. However, the factors that regulate KL mRNA levels in granulosa cells remain unclear. This study tested the hypothesis that oocytes regulate granulosa cell steady-state KL mRNA expression levels and that the characteristics of this regulation are dependent on the stage of growth and development of both oocytes and follicles. Levels of mRNA for the KL splice variants (KL-1 and KL-2) were shown to be high in granulosa cells from preantral follicles and then decline after follicular antrum formation. Preovulatory follicular development was associated with a dramatic increase in steady-state levels of KL-1 mRNA in mural granulosa but not cumulus cells. Regulation of these changes was examined in vitro using partly grown oocytes isolated from preantral follicles and fully grown oocytes isolated from preovulatory follicles. FSH increased the steady-state KL mRNA levels in preantral granulosa cells in vitro. Partly grown oocytes either increased or decreased KL mRNA levels in preantral granulosa cells depending on the absence or presence of FSH stimulation, respectively. Fully grown oocytes reduced the KL mRNA level in preantral granulosa cells and increased the ratio of KL-1 to KL-2 mRNA. In mural granulosa cell culture, FSH augmented testosterone-dependent elevation of the steady-state KL mRNA level, but had no effect alone. Fully grown oocytes reduced KL-2 but not KL-1 mRNA levels in mural granulosa cells treated with testosterone plus FSH, whereas fully grown oocytes reduced levels of both KL transcripts in cumulus cell culture. These effects of oocytes on steady-state KL mRNA expression levels in vitro explain the changes in granulosa cell KL mRNA levels observed during follicle development in vivo. The results therefore support the hypothesis that oocytes regulate granulosa cell kit ligand mRNA levels in a way that is characteristic of the stage of growth and development of the oocyte. Moreover, the results suggest that oocytes play a major role in promoting dynamic changes in gene expression by granulosa cells appropriate to the stage of follicular development. (+info)
Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation.
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Ovarian cortical tissue, donated by 20 women aged 25-43 years during gynaecological laparoscopies or laparotomies, was first cultured for 7-9 days as tissue slices, 0.1-0.3 mm in thickness, in extracellular matrix, to initiate the growth of the primordial and primary follicles. It was then divided into two parts, one of which was cultured further as slices, and the other one used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles. The tissue slices and the partially isolated follicles were cultured for a further 1-3 weeks in the matrix. After approximately 2 weeks in culture, some oocytes began to extrude from the follicles, which were usually at the secondary stage. They were small, 20-80 micrometer in diameter, and had a thin or absent zona. Polar bodies and meiotic chromosomes could be seen in these naked oocytes. This premature extrusion probably resulted from sub-optimal culture conditions. It occurred sooner in follicles that had been partially isolated using collagenase. Histologically, larger numbers of oocytes were observed in non-isolated slice cultures than in the partially isolated cultures. Initiation of growth of the follicles occurred during the first 7-9 days in culture within slices. In non-isolated slices and following mechanical partial isolation there were significantly more secondary follicles after 11-18 days in culture than following isolation with collagenase. The proportion of atretic follicles increased during all cultures, and it was significantly higher after partial isolation. Because partial isolation did not improve the survival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices. (+info)