Influence of mutations affecting gonadotropin production or responsiveness on expression of inhibin subunit mRNA and protein in the mouse ovary

Measurement of inhibins A and B in the serum of normal cyclic rodents has implicated FSH in the regulation of these peptides within the ovary. To extend these observations we have used a panel of mutant mice carrying mutations which affect either the production of, or the ability to respond to, FSH and LH. As a consequence, the females are infertile and show different degrees of follicular development. The aim of this study was to measure inhibin gene transcription in the ovaries of these mutant females together with inhibin protein levels in ovaries and serum and to relate these to follicular development within the ovary. Comparison was made with a pool of normal/heterozygous females. In hpg females where lack of GnRH production results in the absence of gonadotropin synthesis, in FSHbeta knockout (FSHbetaKO) females where disruption of the gene encoding FSHbeta results in the absence of FSH production, and in FSH receptor knockout (FSHRKO) females which are unable to respond to circulating FSH, follicular development remains at the pre-antral stage in these three mutants. Only in the hpg females were common inhibin alpha subunit mRNA levels significantly lower than normal. In these three mutants, however, mRNA levels for both the betaA and betaB subunits were extremely low compared with normal mice. At the protein level, neither inhibin A nor B was detected in the serum of these three mutants; however inhibin B, albeit at very low levels, was detectable within the ovaries. These observations confirm a major role for FSH in the control of transcription of the RA and betaB genes but suggest that the constitutive transcription of the alpha subunit is less dependent on FSH. In contrast, in LH receptor knockout (LuRKO) female mice inhibin betaA subunit mRNA levels were similar to those measured in normal/heterozygous females but levels of inhibin alpha and betaB subunit mRNAs were significantly higher than in the normal group. This was reflected in significantly higher inhibin B protein levels in ovaries and serum. An inability to respond to LH combined with high circulating levels of FSH leads to a high proportion of antral follicles in LuRKO females, with granulosa cells constituting the major cell type within the ovary. The high percentage of antral granulosa cells is likely to account for the significantly higher levels of inhibin B production in these ovaries.

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.(C) 2003 Elsevier Science B.V. All rights reserved.

Intra-ovarian roles of activins and inhibins

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise.

Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro

The effect of s-nitroso-N-acetyl-1,1-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10(-3) M SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P < 0.05) while 10(-5) M SNAP showed intermediate proportions (55%). For 10(-7) M SNAP and controls (without SNAP), MII percentages were similar (72% for both, P > 0.05), but superior to the other treatment groups (P < 0.05). Blastocyst development, however, was not affected (38% for all treatments, P < 0.05). TUNEL-positive cells in hatched blastocysts (Day 9) increased when IVM included 10(-5) M SNAP (8 v. 3 to 4 cells in the other treatments, P > 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10(-7) M SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development.

Obesity induced by high-fat diet promotes insulin resistance in the ovary

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNF alpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity. Journal of Endocrinology (2010) 206, 65-74

Isolamento e caracterização ultraestrutural de folículos pré-antrais de vacas da raça Nelore (Bos taurus indicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis) ovaries - partial results

Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil) and transported to the laboratory in saline solution at 36 degrees C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters). The Cumulus-oocyte complexes (COCs) were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5 degrees C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 mu l of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.

Morphologic and quantitative evaluation of preantral follicles of buffalo (Bubalus bubalis) in different reproductive phases

The objectives of the present study were to estimate the population of the normal and degenerated preantral follicles of buffaloes in different reproductive phases and to classify the different types of degeneration of ovarian preantral follicles. For this, 18 ovaries were divided in three groups: prepubertal, non pregnat and pregnant adult females. The ovaries were collected from a slaughterhouse (Frigol - Brazil) and processed for classic histological examination. The follicular population was estimated according to Cahill et al. (1979) and calculated according to Gougeon & Chainy (1987). Young buffaloes, pregnant and non pregnat adults presented an average number of 15.5, 3.7 and 8.9 preantral follicles with normal morphology and type I (the degeneration was observed just in oocyte) and II (oocyte and granulosa cells were degenerated) degeneration, respectively. This experiment demonstrated that the proportions of primordial, primary and secondary follicles were affected by reproductive phase. A significant difference was observed considering the proportions of preantral follicles with normal morphology and Type I degeneration in all reproductive phases evaluated. The primary follicles presented more percentage of Type I degeneration that the others follicles and the primordial and primary follicles presented more percentage of Type II degeneration in three reproductive phases evaluated.

Follicular deviation and acquisition of ovulatory capacity in bovine follicles

Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wave (diameter deviation). To determine whether acquisition of ovulatory capacity is temporally associated with diameter deviation, cows were challenged with purified LH at known times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). A 4-mg dose of LH induced ovulation in all cows when the largest follicle was greater than or equal to 12 mm (16 of 16), in 17% (1 of 6) when it was 11 mm, and no ovulation when it was less than or equal to 10 mm (0 of 19). To determine the effect of LH dose on ovulatory capacity, follicular dynamics were monitored every 12 h, and cows received either 4 or 24 mg of LH when the largest follicle first achieved 10 mm in diameter (experiment 2). The proportion of cows ovulating was greater (P < 0.05) for the 24-mg (9 of 13; 69.2%) compared with the 4-mg (1 of 13; 7.7%) LH dose. To determine the effect of a higher LH dose on follicles near diameter deviation, follicular dynamics were monitored every 8 h, and cows received 40 mg of LH when the largest follicle first achieved 7.0, 8.5, or 10.0 mm (experiment 3). No cows with a follicle of 7 mm (0 of 9) or 8.5 mm (0 of 9) ovulated, compared with 80% (8 of 10) of cows with 10-mm follicles. Thus, follicles acquired ovulatory capacity at about 10 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. We speculate that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle.

Regulation and action of fibroblast growth factor 17 in bovine follicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P < 0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P < 0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells. (C) 2012 Elsevier B.V. All rights reserved.

Collection Rates and Morphology of Equine Oocytes Obtained from Immature Follicles after Treatment with Equine Pituitary Extract (EPE) and Human Chorionic Gonadotropin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Relative expression of insulin like growth factor I (IGFI) and follicle stimulating hormone receptor (FSHR) in follicles and ovarian tissue from Bos primigenius indicus (Nelore)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização estrutural do fator de crescimento de fibroblasto-10 (FGF-10) e seu papel na fisiologia folicular ovariana

Background: Interest in folliculogenesis has grown extensively in recent years. Nevertheless, several aspects of follicular activity are still poorly understood. Thus, in vitro culture of ovarian follicles using new substances has been established as a very viable model, enhancing the prospects for a better understanding of follicular activity. Among the family members of the fibroblast growth factor (FGFs), FGF-10 has received recent attention for its ability to regulate the development of ovarian follicles and oocyte maturation. Given the relevance of FGF-10 in the folliculogenesis process, this review aimed to describe the structural features, expression and the main biological effects of FGF-10 on the development of ovarian follicles in mammals.Review: Along this work, it was shown aspects related to structural characterization of FGF-10 and its receptors, as well as FGF-10 expression in different cell types, emphasizing its importance to follicular development. FGF-10 is a paracrine member of the family of FGFs, and is characterized by promoting biological responses via cell surface receptors (FGFRs) of tyrosine kinase-type. of these receptors, FGFR-1, FGFR-2 and FGFR-3 may undergo alternative transcriptional arrangements, enabling the formation of two isoforms (b and c) that have varying degrees of affinity for the various FGFs. Thus, seven FGFR proteins (FGFRs 1b, 1c, 2b, 2c, 3b, 3c and 4) with different binding specificities are generated from the four FGFR genes. The FGFRs transmit intracellular signals after binding with the ligand through the phosphorylation of tyrosine, which activates various transduction patterns in the cytoplasm. The signal transduction of FGF-10 may occur through three main pathways: protein of rat sarcoma (Ras)/MAPK, PLC gamma/Ca(2+) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt), which are involved in the transmission of biological signals, leading to cellular proliferation and differentiation. FGF-10 mRNA expression was detected in the ovarian stroma, oocyte and theca cells of preantral and antral follicles. on the other hand, the expression of mRNA for FGF-10 receptors was found in, granulosa cells, theca cells, cumulus cells and oocytes. Although FGFs are widely distributed in different tissues and cell types, the importance and function of FGFs in the ovary are still poorly documented. FGF-10 has been shown to be an important mediator of mesenchymal and epithelial cell interactions during follicle development, promoting follicular survival, activation and growth. Besides the action on folliculogenesis, FGF-10 was recently identified as a growth factor able to improve oocyte competence. However, in antral follicles, the presence of FGF-10 is associated with increased follicular atresia, which matches its anti-estrogenic action.Discussion: From this review, we can conclude that FGF-10 is an important regulator of female reproduction. This growth factor acts in follicle survival, oocyte maturation, expansion of cumulus cells and proliferation of granulosa/theca cellsthrough direct and/or indirect actions in the control of folliculogenesis. Furthermore, FGF-10 seemed to have different effects throughout the follicular development. However, it is necessary to perform additional studies that may provide a better understanding about the importance of FGF-10 during folicullogenesis.

Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 213, in bovine follicles

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.