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PubMed2003

Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary.

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2, and 3 ligand immunoreactivity is observed in both small and large follicles, whereas the TGFbeta type II receptor (TGFbeta RII) is detected in the oocyte and granulosa cells of antral follicles. The intracellular signaling proteins Smad2 and Smad4 are present in the granulosa cell cytoplasm of all follicle size classes. Smad3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicle size classes. These data suggest that the complete activin signal transduction pathway is present in small follicles and that large follicles primarily produce the inhibins. Our data also suggest that TGFbeta ligands and receptors are colocalized to large antral follicles. Taken together, the ligands, receptors, and signaling proteins for the TGFbeta superfamily are present at distinct points throughout feline folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation.

Activins↗
PubMed1998

Follicle development during the normal menstrual cycle.

An understanding of the factors which determine initiation of follicle growth, recruitment and dominant follicle selection may increase our understanding of the underlying process of ovarian aging. In this article, these aspects of the normal menstrual cycle are reviewed. The morphological and endocrinological development in the early follicle is described from the primordial follicle stage. The degree of follicle-stimulating hormone (FSH) dependency is discussed, as is the relationship of estradiol (E2) production to follicle diameter. The principles governing mono-follicular selection are outlined, and the FSH 'threshold' and 'window' concepts are highlighted. Maximum FSH levels in the early follicular phase are shown to be variable between individuals. The relevance of this and the means by which individual sensitivity to FSH may be altered at the ovary in the context of ovarian aging are discussed.

Aging↗
PubMed2001

Absent biologically relevant associations between serum inhibin B concentrations and characteristics of polycystic ovary syndrome in normogonadotrophic anovulatory infertility.

BACKGROUND: Dominant follicle selection is disturbed in normogonadotrophic anovulatory infertility [World Health Organization (WHO) 2] and remaining early antral follicles are either healthy or atretic. This study was conducted to investigate whether inhibin B serum concentrations (produced by healthy small antral follicles) represent the extent of ovarian abnormalities in WHO 2 women and patients with polycystic ovarian syndrome (PCOS), constituting a subgroup of WHO 2 patients. METHODS AND RESULTS: Ultrasonographic and endocrine characteristics in 379 WHO 2 patients and 30 normo-ovulatory controls were compared. In the WHO 2 patients, the PCOS subgroup and the controls, inhibin B concentrations were similar. Inhibin B concentrations were weakly but significantly correlated with the total number of ovarian follicles (r = 0.282; P < 0.001), LH (r = 0.347; P < 0.001), and testosterone (r = 0.269; P < 0.001) but not with serum oestradiol concentrations (r = 0.057). Most (71%) patients with elevated inhibin B also presented with increased concentrations of LH and/or hyperandrogenaemia. In a subgroup of 190 subjects, classified as PCOS based on hyperandrogenaemia and polycystic ovaries, elevated inhibin B concentrations were found in 23% of cases. Aforementioned correlations were similar in PCOS as in WHO 2 patients. CONCLUSION: In conclusion, inhibin B serum concentrations are normal in WHO 2 and PCOS women, suggesting a normal number of healthy early antral follicles despite increased overall follicle numbers in PCOS.

Adult↗
PubMed2005

Evaluation of ovulation rate and ovarian phenotype in puberal heifers from a cattle population selected for increased ovulation rate.

Long-term selection for increased ovulation rate (1984 to 2002) has resulted in a unique ovarian phenotype in the MARC Twinner cattle population. Ovulation rate and frequency of bilateral ovulations were examined by rectal palpation in 29,547 estrous cycles for 3,910 heifers (12 to 18 mo of age) in this population. Bilateral ovulations (one corpus lutuem [CL] on each ovary) were of interest because bilateral twin pregnancies result in decreased dystocia and increased calf survival. Ovulation rate increased linearly at a rate of 0.026 CL per year, and it currently averages 1.48 +/- 0.04 CL per estrous cycle. Concurrent with the increase in ovulation rate, the frequency of triplet ovulations increased from 0% to 2.3 +/- 0.8% (P < 0.001). Ovulation rate of both the right and left ovary increased equally at a rate of 0.013 CL per year, and mean ovulation rate of the right ovary remained greater than mean ovulation rate of the left ovary throughout the study (0.66 vs. 0.55 +/- 0.003 CL per estrous cycle; P < 0.001). Although correlations were low, ovulation rate of one ovary was negatively correlated (P < 0.001; r = -0.07) with ovulation rate of the same ovary in the previous estrous cycle, but positively correlated (P < 0.001; r = 0.13) with the contralateral ovary of the previous estrous cycle. The proportion of bilateral ovulations averaged 55.7 +/- 0.7%, a value greater than the predicted 49.5% (P < 0.001). In addition to dystocia and retained placenta, triplet pregnancies increase the incidence of pregnancies gestating fetuses of opposite sexes and subsequent incidence of freemartins; thus, selection pressure on ovulation rate may need to be adjusted in the MARC Twinner population. The proportion of bilateral ovulations in the population is greater than expected, and this may be an economically important trait, which will respond to selection and be beneficial for improving bovine reproductive efficiency. Understanding factors controlling the increased functional activity of the right ovary and bilateral ovulations may provide further insights into the mechanisms controlling follicle selection and methods to improve reproductive management of cattle.

Animals↗
PubMed2005

Human hair growth ex vivo is correlated with in vivo hair growth: selective categorization of hair follicles for more reliable hair follicle organ culture.

Of the numerous assays used to assess hair growth, hair follicle organ culture model is one of the most popular and powerful in vitro systems. Changes in hair growth are commonly employed as a measurement of follicular activity. Hair cycle stage of mouse vibrissa follicles in vivo is known to determine subsequent hair growth and follicle behavior in vitro and it is recommended that follicles be taken at precisely the same cyclic stage. This study was performed to evaluate whether categorization of human hair follicles by the growth in vivo could be used to select follicles of the defined anagen stage for more consistent culture. Occipital scalp samples were obtained from three subjects, 2 weeks later after hair bleaching. Hair growth and follicle length of isolated anagen VI follicles were measured under a videomicroscope. Follicles were categorized into four groups according to hair growth and some were cultured ex vivo for 6 days. Follicles showed considerable variations with respect to hair growth and follicle length; however, these two variables were relatively well correlated. Hair growth in culture was closely related with hair growth rate in vivo. Moreover, minoxidil uniquely demonstrated a significant increase of hair growth in categorized hair follicles assumed at a similar early anagen VI stage of hair cycle. Selection of follicles at a defined stage based on hair-growth rate would permit a more reliable outcome in human hair follicle organ culture.

Adult↗
PubMed2004

Inhibins and adult ovarian function.

Inhibin B is predominantly produced by early healthy antral follicles during the luteo-follicular transition of the menstrual cycle. High inhibin B concentrations during the early follicular phase are responsible for the decline in FSH serum levels closing the FSH window and assuring single dominant follicle selection in the human. Early follicular phase inhibin B levels decrease over time, reflecting the recruitment of a diminished cohort of follicles with ovarian ageing. Hence, inhibin B is a predictor of poor response in IVF. In patients with PCOS inhibin B levels (potentially representing the number of healthy, early antral follicles) may be associated with the severity of ovarian dysfunction and consequently may predict ovulation induction outcome. However, inhibin B levels are normal in most PCOS patients suggesting a normal number of healthy follicles despite an increase in overall follicle number. Recent findings indicate that initial inhibin B concentrations can not predict the outcome of ovulation induction by either clomiphene citrate or FSH. Finally, inhibin B levels decrease over time in PCOS.

Adult↗
PubMed2004

Artificial reproductive technology achievements for optimizing embryo quality.

Evaluation of morphological characteristics, to date, has been the most widely accepted method for the selection of embryos with higher developmental ability and optimization of the outcome of in vitro fertilization (IVF). Improvement in clinical results has also been pursued through attempts to generate embryos of high quality or to identify more reliable selection criteria. This work evaluates the possibility of improving embryo quality and subsequent IVF outcome in situations in which creation of supernumerary embryos is not allowed. Optimization of gamete selection and conditions of insemination, such as hormonal therapy to improve sperm quality of patients undergoing intracytoplasmic sperm injection treatment, use of power Doppler image for follicle selection, short coincubation of gametes during insemination, and use of a polscope for spindle detection, is described.

Blastocyst↗
PubMed1996

Contralateral selection of dominant follicle favours pre-embryo development.

Ovulation was studied using vaginosonography in a total of 410 natural cycles of 123 women undergoing infertility treatment [267 intrauterine insemination (IUI) cycles of 103 women and 143 in-vitro fertilization (IVF) cycles of 50 women]. None of the women received ovarian stimulation. Each follicle was measured daily from 14 mm in diameter until formation of corpus luteum or oocyte retrieval. Contralateral ovulation as compared with the preceding cycle occurred in 57% of the 410 cycles. Contralateral ovulations occurred in 72% of cycles with a follicular phase < 13 days. In cycles with a follicular phase of > 14 days, ovulations occurred at random. The length of follicular phase in contralateral ovulation cycles (15.2 +/- 3.2 days) was significantly (P < 0.05) shorter than that of ipsilateral ovulation cycles (15.8 +/- 2.8). During the 57% contralateral ovulations in 143 IVF cycles, the rates of oocyte retrieval (89%), fertilization (69%), cleavage (90%) and embryo transfer (56%) were significantly higher than those of ipsilateral ovulations (69, 51, 64 and 23% respectively). The pregnancy rate of contralateral ovulations (9%) was also higher, though not significantly, than that of ipsilateral ovulations (3%), although the pregnancy rates per transfer were similar (16 and 14% respectively). The total pregnancy rate of both IUI and IVF was higher in contralateral than in ipsilateral ovulation cycles (8.1 and 4.0% respectively). The dominant follicles in contralateral ovulation cycles showed significantly higher oestradiol/androstenedione ratio (P < 0.025) and oestradiol/testosterone + androstenedione ratio (P < 0.025) and lower androstenedione (P < 0.05) than those of ipsilateral ovulation cycles. There was no significant difference in oestradiol, progesterone and testosterone. These results indicate that the dominant follicles in contralateral ovulation cycles are healthier than those of ipsilateral ones. Local intra-ovarian factors, e.g. from the corpus luteum, may negatively affect the health of the dominant follicle and the enclosed oocyte. Therefore contralateral selection of the dominant follicle in the succeeding cycle may favour pre-embryo development. The chance of conceiving during a natural cycle may be affected by the site of ovulation in the preceding cycle.

Embryonic and Fetal Development↗
PubMed2001

Short-term administration of antivascular endothelial growth factor antibody in the late follicular phase delays follicular development in the rhesus monkey.

Indirect evidence in the nonhuman primate and human suggests that angiogenesis and regulators of angiogenesis such as vascular endothelial growth factor (VEGF) may play an active role in cyclic folliculogenesis. Indeed, the follicle selected for maturation and ovulation possesses a denser microvascular network, and VEGF messenger ribonucleic acid and its protein have been identified in granulosa cells of the developing follicle during the mid- and late follicular phases, with a more intense signal in the mature follicle. The objective of this study was to obtain direct evidence in the nonhuman primate for an active role of VEGF in follicular growth and maturation by studying the effect of VEGF-blocking antibodies in this process. After documenting two normal ovulatory cycles, female rhesus monkeys (n = 7) received iv injections of anti-VEGF antibodies (0.5 mg) twice on successive days in the late follicular phase. Three monkeys also received nonspecific goat IgG (0.5 mg) twice on successive days in the late follicular phase. Daily measurements of estradiol, progesterone, LH, and FSH were obtained during the two control cycles, the anti-VEGF treatment and posttreatment cycles, and the IgG treatment cycle. Anti-VEGF antibody administration significantly lengthened the follicular phase in six of seven monkeys to 17.8 +/- 1.7 vs. 10.0 +/- 0.7 and 9.8 +/- 0.6 in control cycles and 10.7 +/- 0.3 days (mean +/- SE) in IgG-treated cycles. The expected late follicular phase rise in estradiol, as documented in the control cycles (day 0, 96.1 +/- 6.0; day 1, 125.5 +/- 20.0; day 2, 165.5 +/- 24.9; day 3, 183.8 +/- 11.0 pg/mL), was interrupted by anti-VEGF antibody treatment (99.3 +/- 5.0, day 0, preinjection control) to 63.3 +/- 12.2 (day 1), 48.5 +/- 8.7 (day 2), and 57.6 +/- 9.0 (day 3). Mean FSH levels were significantly increased by day 2 of anti-VEGF antibody treatment. After a variable delay, estradiol concentrations increased to reach a preovulatory peak in all anti-VEGF-treated animals, followed by ovulation, normal luteal function, and a normal posttreatment cycle. The data clearly demonstrate that short-term inhibition of angiogenesis with an anti-VEGF-blocking antibody during the later growth phase of the dominant follicle interferes with normal follicular development. Persistence of estradiol secretion and delayed resumption of its rise also suggest recovery of the follicle. We conclude that the angiogenic regulator VEGF is a crucial component in the process of follicular growth in the primate.

Animals↗
PubMed1989

A new method for studying human oocytes by light and electron microscopy.

Since ovarian follicles appear to be randomly oriented with respect to the plane of the section, the method of sectioning and examining follicles at their maximum diameter described here allows direct comparison between oocyte populations of women and small differences can be detected. Re-sectioning for EM allows selected follicles of interest to be examined at a higher resolution.

Adult↗
PubMed1987

Bifunctional role of transforming growth factor-beta during granulosa cell development.

Regulatory actions of transforming growth factor-beta (TGF beta) on granulosa cell function were analyzed in cells cultured from the ovaries of diethylstilbestrol-implanted rats. In the presence of a suboptimal concentration of FSH (5 ng/ml) that increased LH receptors by 100-fold during a 72-h culture, TGF beta augmented this response in a dose-dependent manner with a maximal effect at 16 pM. In contrast, the growth factor inhibited the LH receptor response to an optimal dose of FSH (50 ng) by up to 50% and was inactive in the absence of gonadotropin. TGF beta also enhanced the formation of cAMP by 5 ng FSH and partially inhibited the effects of higher FSH concentrations. However, the actions of TGF beta were more prominent on LH receptor induction than on cAMP production with either low or high amounts of FSH. In addition, TGF beta had little effect on cAMP production stimulated by cholera toxin or forskolin, but amplified the actions of these ligands as well as that of 8-bromo-cAMP on LH receptor expression. TGF beta also modulated the steroidogenic activity of the granulosa cells, with increased production of progesterone in response to 5-100 ng FSH. The bifunctional actions of TGF beta on FSH-induced LH receptor formation were observed throughout a 96-h culture period. However, the presence of the growth factor was not required for the first 24 h of culture, indicating that TGF beta alters the later events involved in LH receptor formation. TGF beta augmented the stimulatory actions of 5 ng FSH on LH receptors in the absence or presence of insulin, but its inhibitory effect on these receptors was only observed in cells treated with insulin. These results indicate that TGF beta modifies FSH action during granulosa cell development in a biphasic manner. TGF beta can exert stimulatory or inhibitory effects depending upon the concentration of FSH and the presence of insulin, and these are due to alterations in cAMP action as well as cAMP production. Autocrine and/or endocrine actions of TGF beta during granulosa cell differentiation may be involved in the processes of follicle selection and development.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals.

The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.

Animals↗
PubMed2001

Changes in plasma inhibin A levels during sexual maturation in the female chicken and the effects of active immunization against inhibin alpha-subunit on reproductive hormone profiles and ovarian function.

Inhibins and activins are firmly implicated in the control of pituitary FSH secretion and ovarian follicular development in mammals. As in mammals, inhibin A and activin A are expressed in the preovulatory follicles of birds, and a defined ovulation cycle for inhibin A has recently been demonstrated in the laying hen. To investigate further the role of inhibin-related proteins in developing pullets, circulating concentrations of inhibin A, inhibin B, total immunoreactive inhibin alpha-subunit (ir-alpha), activin A, LH, FSH, and progesterone were measured from the juvenile state through to sexual maturity in 22 birds. In the 11 birds assigned to control groups, plasma inhibin A levels were low from 7 to 13 wk of age rising about threefold to a peak at Week 19 after which levels fell slightly to a plateau level characteristic of adult hens. Plasma inhibin A levels were negatively correlated with FSH (r = -0. 33; P: < 0.001) and positively correlated with progesterone (r = 0. 67; P: < 0.001) and ir-alpha (r = 0.53; P: < 0.001). Plasma ir-alpha levels were much higher than inhibin A levels although the relative differences varied with age. Plasma levels of inhibin B and activin A were below assay detection limits at all times. The remaining group of 11 birds was actively immunized (IMM) against a synthetic chicken inhibin alpha-subunit peptide (amino acids 1-26). The IMM generated circulating antibodies that bound native bovine inhibin A but altered neither plasma FSH nor progesterone levels relative to control birds at any stage of development nor the timing of first oviposition in week 19. Apart from a transient decline 1 wk after primary IMM, plasma LH concentrations did not differ from controls. Comparison of the numbers and size-class distribution of ovarian follicles at 29 wk showed an approximate twofold increase in the number of 8- to 9.9-mm-diameter follicles (control; 1.82 +/- 0.44 vs. IMM; 3.91 +/- 0.89; P: < 0.05), a size class that corresponds to follicles that have just joined the preovulatory hierarchy. The numbers of growing follicles in other size-classes and the sizes of hierarchical F(1)-F(7) follicles were not altered by IMM. However, the number of postovulatory follicles increased (control 3.73 +/- 0. 20 vs. IMM 5.55 +/- 0.28; P: < 0.01), and significantly more (P: < 0. 02) immunized hens laid two eggs within a 24-h period on at least one occasion (control 1 of 11 vs. IMM 9 of 11). The IMM increased (P: < 0.05) activin A content of F(1) and F(2) theca layers and decreased (P: < 0.05) activin A content in F(3) and F(4) granulosa layers, raising the possibility of a local intraovarian role of activin in mediating the response to IMM. These findings support a role for inhibin A in regulating the entry of follicles into the preovulatory hierarchy in the chicken, although further studies are required to establish the mechanism by which inhibin IMM increases the rate of follicle selection and ovulation without raising plasma FSH.

Activins↗
PubMed1984

Marginal zone B cells express CR1 and CR2 receptors.

Opsonized yeast is known to bind strongly to the marginal zones in frozen sections of rat spleen (Kumararatne, D. S. et al., Eur. J. Immunol. 1981. 11: 858). This study reports an analysis of the cells involved in this binding. Sheep red cells coated respectively with C3b, C3bi or C3d were used as indicator cells. These showed homogeneous binding of both C3b and C3bi to marginal zones and germinal centers. C3d-coated red cells bound in a uniform speckled pattern to marginal zones. They also bound to germinal centers and the small lymphocyte zones of the follicles. Selective depletion experiments were undertaken to show that binding to marginal zones was a property of the IgM+ and IgD- B cells characteristic of this area. Binding to germinal centers was attributable to follicular dendritic cells. The C3d receptors in follicles were shown to be on IgM+ and IgD+ small B lymphocytes.

Animals↗
PubMed2002

Is the short follicular phase in older women secondary to advanced or accelerated dominant follicle development?

This study sought to determine whether the shortened follicular phase in ovulatory older women is secondary to advanced (i.e. earlier) or accelerated (i.e. more rapid) folliculogenesis. Normal ovulatory women, aged 40-45 yr (n = 15) and 20-25 yr (n = 13), underwent daily venipuncture and transvaginal ultrasonography throughout the follicular phase of a spontaneous menstrual cycle (control cycle) and after pituitary down-regulation with a GnRH agonist (study cycle). As expected, the older subjects in the control cycles demonstrated an elevated d 3 FSH and a shortened follicular phase compared with the younger subjects. After release from hypothalamic-pituitary-ovarian axis suppression, the early follicular phase FSH peak occurred earlier (6.8 vs. 9.8 d; P < 0.01) and was of a greater magnitude (12.1 vs. 6.5 mIU/ml; P < 0.01) in the older subjects. The time from release of suppression until the subsequent LH surge was also shorter (17.5 vs. 20.8 d; P < 0.01) in the older group. However, the time from FSH peak to LH surge was similar in the older and younger groups (10.7 vs. 11.0 d; P = 0.74). Compared with younger women, older subjects had normal follicular phase levels of estradiol and inhibin A and lower levels of inhibin B in both control and study cycles. We conclude that the shortened follicular phase observed in older ovulatory women is due to earlier dominant follicle selection, independent of hormonal influences from the preceding luteal phase.

Adult↗
PubMed1996

Demodex-associated folliculitis.

Examination of 388 follicles in 24 large resections of skin for the presence of histologic folliculitis and Demodex mites uncovered a nonrandom association between these two phenomena. Demodex mites were found in 42% of follicles with inflammation, but in just 10% of the follicles without inflammation. Eighty-three percent of follicles with Demodex showed inflammation. The probability that this result could occur by random chance alone was < 0.001, thus suggesting that Demodex is associated with histologic folliculitis, even minor folliculitis. The results do not, however, decide whether Demodex is causative, whether it preferentially selects follicles with histologic inflammation, or whether some of both processes operate.

Animals↗
PubMed2003

Localization of vascular endothelial growth factor in the zona pellucida of developing ovarian follicles in the rat: a possible role in destiny of follicles.

There is increasing evidence that in many species angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), may have important roles in folliculogenesis. The aim of this study is to determine the localization of VEGF and its receptors, Flt-1 and KDR, and bFGF expression in the rat ovary and to evaluate their distributions throughout the different follicular stages. Out of 20 virginal female rats, 10 were studied during the natural ovarian cycle without any ovulation induction. The other 10 were superovulated and their ovaries were studied by western analysis and immunohistochemistry. Granulosa cells (GC) and oocytes of primordial follicles were negative for VEGF. In early primary follicles, VEGF was present in the oocyte but its immunoreactivity was weak, while newly developing zona pellucida (ZP) of primary follicles was negative for VEGF. Subsequently, with the commencement of antral spaces between GC of the secondary follicle, ZP of some secondary follicles became strongly positive for VEGF, forming a continuous ring around the oocyte. In preovulatory mature follicles granulosa and theca interna (TI) cells showed a weak immunoreactivity for VEGF. Western blot analyses have also demonstrated that VEGF, a 26-kDa protein, was present in follicles. Moreover, in ovulated cumulus-oocyte complex we observed a halo-like immunoreactivity of VEGF around the fully mature oocyte. The immunoreactivity for Flt-1 and KDR receptors in growing follicles was mostly limited to GC and TI cells. Anti-bFGF did not exhibit any immunoreactivity in ZP of follicles at any stage. Its expression was weak in GC of the follicles at different stages, whereas, it could be localized to some extent in the blood capillaries of TI of antral follicles and in blood vessels localized in the stroma. Interestingly, VEGF immunoreactivity in the ZP of some secondary follicles is very striking. Accordingly, the possibility that VEGF may be an important regulatory molecule for the dominant follicle selection or atresia should be considered.

Animals↗
PubMed2000

Nuclear configuration of bovine oocytes derived from fresh and in vitro-cultured preantral and early antral ovarian follicles.

The aim of this experiment was to characterize the growth and nuclear configuration of oocytes isolated from late preantral and early antral bovine ovarian follicles immediately after recovery and after the in vitro culture. Individual follicles were isolated by microdissection from slices of the ovarian cortex. Follicles were sorted by diameter into 175 to 224, 225 to 274 and 275 to 325 microm-size classes. The follicles selected for in vitro culture were placed singly into 40 microL droplets of medium (TCM 199 enriched with FCS, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine, hypoxanthine, FSH and estradiol-17beta) and cultured for 6, 8, 11, 14 or 17 d. The sizes of follicles and oocytes were related to the duration of culture and gradually increased as culture duration was prolonged. The analysis of the relationship between mean diameters of oocytes at the time of recovery and after the in vitro culture, has shown significant differences after culture lasting 8 d (76.9+/-9.9 vs. 86.1+/-11.1 microm; P < 0.05), 11 d (77.0+/-9.9 vs. 91.9+/-17.5 microm; P < 0.01), 14 d (80.0+/-9.5 vs. 97.9+/-16.5 microm; P < 0.01) and 17 d (82.6+/-6.6 vs. 97.2+/-11.5 microm; P< 0.01). No statistical differences were shown among oocytes in the 5 pre-culture groups (79.5+/-8.8; 76.9+/-9.9; 77.1+/-9.9; 80.1+/-9.5 and 82.6+/-6.6 microm). Meiotic arrest was preserved in 71.9% of oocytes in our culture system up to 14 d. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated "fresh" or cultured for 6, 8, 11 or 14 d. No relationship was observed between the size class of follicles and the frequency of the GV-stage. Prolonging the culture period to 17 d drastically decreased the percentage of oocytes in the GV-stage (18.7%) and increased the percentage of oocytes having premature initiation of meiosis (GVBD; 46.3%) and degeneration (25.0%). These results suggest that out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at the GV-stage.

Animals↗