Recovery of large preantral follicles from buffalo ovary: effect of season and corpus luteum

Preantral follicle can be considered as an alternative source of oocyte for in vitro production of embryos. The objective of the present study was to standardize a procedure for the isolation of large preantral follicles (>150-500 microm) from buffalo ovaries and to determine the effect of season and the presence of corpus luteum on the recovery rate of the large preantral follicles. A combined enzymatic cum mechanical approach was adopted to recover the large preantral follicles. In the first experiment, the ovarian cortical pieces were suspended in trypsin (1000-1500 BAEE units for milligrams of solid) and incubated at various temperatures for different periods, i.e. (1) trypsin (1%), 37 degrees C for 10 min; (2) trypsin (1%), 37 degrees C for 10 min + 4 degrees C for 3 h; (3) trypsin (0.5%), 37 degrees C for 20 min; (4) trypsin (0.25%), 37 degrees C for 20 min. Although there was no significant difference (P>0.05) among the different protocols, the first protocol yielded more follicles (3.2, 2.6, 1.8 and 1.5 per ovary, respectively). Hence, the first protocol was selected and used in the second and third experiments. In the second experiment, the effect of season, i.e. peak breeding season (October-March) versus low breeding season (April-September) was evaluated on the recovery rate of the large preantral follicles. The recovery rate of large preantral follicles from the ovaries during the peak breeding season was significantly (P<0.05) greater (9.92+/-0.85 per ovary) than that of the low breeding season (4.95+/-0.27 per ovary). In the third experiment, effect of the presence of corpus luteum on the recovery rate of large preantral follicles was studied. There was a significantly (P<0.05) higher yield of large preantral follicles from the ovaries with corpus luteum (8.05+/-0.88 per ovary) than for the ovaries without corpus luteum (4.57+/-0.43 per ovary). This study confirms that the large preantral follicles can be isolated from buffalo ovaries using a combination of enzymatic cum mechanical methods and that more large preantral follicles can be recovered during the peak breeding season and from the ovaries having corpus luteum.     

Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.     

Effects of storage time and temperature on atresia of goat ovarian preantral follicles held in M199 with or without indole-3-acetic acid supplementation

Maintenance of follicular quality after removal and during transport of ovaries is necessary for studies on development of preantral follicles in vitro. The present work investigated the effectiveness of M199 and M199IAA for preservation of goat preantral follicles in ovarian tissue. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (control--Time 0). The other 18 fragments were randomly distributed in M199 or M1991AA at 4, 20 or 39 degrees C and stored for 4, 12 or 24 h. Histological analysis showed that storage of ovarian fragments in either solution at 20 or 39 degrees C significantly reduced the percentage of normal preantral follicles when compared with the control, in all cases except after preservation in M199IAA at 20 degrees C for 4 h. In contrast, preservation at 4 degrees C, in either solution, kept the percentage of normal preantral follicles at control values. Reduced cellular metabolism may explain why the best preservation of preantral follicles was at 4 degrees C. The addition of IAA to the TCM 199 was effective for goat preantral follicle preservation at 20 degrees C for 4 h.     

Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro

Preservation of preantral follicles becomes very important to ensure follicle quality at the onset of cryopreservation or in vitro culture. However, for domestic animals, the ovarian donor of preantral follicles for in vitro studies is commonly encountered far away from reproduction laboratories. We investigated the effectiveness of coconut water and Braun-Collins solutions on the preservation of goat preantral follicles. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (Control - Time 0). The other 18 fragments were randomly distributed into tubes containing 2 mL of coconut water or Braun-Collins solution at 4 degrees, 20 degrees or 39 degrees C and then stored for 4, 12 or 24 h. Histological analysis showed that the storage of ovarian fragments in coconut water and Braun-Collins solutions at 20 degrees or 39 degrees C for 12 or 24 h significantly reduced (P < 0.05) the percentage of morphologically normal preantral follicles when compared with the control. However, storage in coconut water at 20 degrees C for 4 h and in both solutions at 4 degrees C kept the percentage at control values. Ultrastructural analysis of follicles exposed to the stated conditions confirmed the integrity of preantral follicles stored at 4 degrees C in Braun-Collins and coconut water solutions for up to 12 and 24 h, respectively. Reduced cellular metabolism at 4 degrees C may explain why the best preservation of preantral follicles was at 4 degrees C, which may suggest a useful method for ovary transport in the future.     

Immunohistochemical localization of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha (TGF-alpha) in developing human ovarian follicles

In this study, we aimed to detect the distribution of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha in ovarian follicles at different stages. Indirect immunohistochemical methods and EGF-r polyclonal and TGF-alpha monoclonal antibodies were used; tissues were examined with light microscope. While dense collection of both growth factors were observed in primordial follicles, there was a strong reaction especially for EGF-r in follicles. Strong reactivity for EGF-r and moderate reactivity for TGF-alpha were observed in the nearby connective tissue. In examinations of primary follicles for EGF-r presence only, dye uptake was moderate in oocytes and dense in apical and basal cytoplasm of follicle cells. Reactivity was moderate in the nearby connective tissue. In the corpus luteum, there was weak reaction for both growth factors. But in stromal cells, reaction was strong. In degenerated follicle cells and in stroma of atretic follicles, reaction was positive for both growth factors; but EGF-r reactivity was more obvious. While strong staining was observed for both factors especially in granulosa cells surrounding the oocyte in Graafian follicle, moderate TGF-alpha reactivity was determined in oocyte cytoplasm. In conclusion, it is possible that EGF-r and TGF-alpha have ortocrine and paracrine effects on development and regression of human ovarian follicles.     

Effects of lowered temperatures and media on short-term preservation of zebu (Bos indicus) preantral ovarian follicles

The maintenance of follicle quality during the transportation of ovaries is essential for the successful cryopreservation and in vitro development of preantral follicles. The objective of this study was to evaluate the effect of cooling ovarian tissue on the conservation of zebu cow preantral follicles. Ovarian pieces were immersed in saline or coconut water (CW) solutions and maintained at 4 or 20 degrees C for 6, 12, or 18 h. Preantral follicles were evaluated by histology and transmission electron microscopy. Storage of ovarian pieces at 20 degrees C for 12 or 18 h significantly reduced the percentage of morphologically normal follicles compared to controls. In contrast, conservation at 4 degrees C for up to 18 h and at 20 degrees C for up to 6 h kept the percentage of normal follicles similar to controls. However, the type of solution that the ovaries were immersed in had little effect on the results. Decreased cellular metabolism probably accounted for better preservation of preantral follicles at 4 degrees C. In conclusion, zebu cow ovaries were successfully stored at 4 degrees C for up to 18 h with no morphological damage to preantral follicles. However, at 20 degrees C, ovaries could only be stored for 6 h.     

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.     

Hyal-1 but not Hyal-3 deficiency has an impact on ovarian folliculogenesis and female fertility by altering the follistatin/activin/Smad3 pathway and the apoptotic process

Ovarian follicle development is a process regulated by various endocrine, paracrine and autocrine factors that act coordinately to promote follicle growth. However, the vast majority of follicles does not reach the pre-ovulatory stage but instead, undergo atresia by apoptosis. We have recently described a role for the somatic hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) in ovarian follicular atresia and induction of granulosa cell apoptosis. Herein, we show that Hyal-1 but not Hyal-3 null mice have decreased apoptotic granulosa cells after the induction of atresia and an increased number of retrieved oocytes after stimulation of ovulation. Furthermore, young Hyal-1 null mice had a significantly higher number of primordial follicles than age matched wild-type animals. Recruitment of these follicles at puberty resulted in an increased number of primary and healthy preantral follicles in Hyal-1 null mice. Consequently, older Hyal-1 deficient female mice have prolonged fertility. At the molecular level, immature Hyal-1 null mice have decreased mRNA expression of follistatin and higher levels of phospho-Smad3 protein, resulting in increased levels of phospho-Akt in pubertal mice. Hyal-1 null ovarian follicles did not exhibit hyaluronan accumulation. For Hyal-3 null mice, compensation by Hyal-1 or Hyal-2 might be related to the lack of an ovarian phenotype. In conclusion, our results demonstrate that Hyal-1 plays a key role in the early phases of folliculogenesis by negatively regulating ovarian follicle growth and survival. Our findings add Hyal-1 as an ovarian regulator factor for follicle development, showing for the first time an interrelationship between this enzyme and the follistatin/activin/Smad3 pathway.     

Morphological changes of the ovary and hormonal changes through the ovarian cycle of the common marmoset (Callithrix jacchus)

Laparoscopic observations of morphological changes of the ovary during the ovarian cycle in conjunction with radioimmunoassay of serum progesterone and estradiol-17β was investigated as a method of monitoring the ovarian cycle in the common marmoset. In the common marmoset, plural follicles first appeared in each ovary five days prior to ovulation. At three to four days prior to ovulation one or two follicles developed into translucent blisters on the surface of the ovary. As the follicles filled with follicular fluid, they became larger and clearer until one to two days prior to ovulation, at which time they formed well defined, transparent bubbles protruding from the surface of the ovary. After ovulation, the ovulation point could be detected at the center of the follicle, sometimes surrounded by a corpus of engorged blood vessels. Ovulations of the plural follicles were not simultaneous, and due to the time lag ovulations took at least 12 to 20 hrs in four out of seven animals examined. After two to five days of ovulation the corpus hemorragicum, a bright red protrusion made of tissue and blood disrupted by ovulation, was found. Subsequently, the color of the formatted corpus luteum changed from dark-red to yellow then to yellow white. While the corpus luteum remained reddish in color serum progesterone was maintained at as high levels as in the luteal phase. There was no mature follicle or corpus luteum in subordinate female ovaries.     

Gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary

Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50) of gamma-radiation using a 60Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0+/-8.6 and 27.3+/-11.2 (mean+/-S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3+/-12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0+/-3.4 and 86.9+/-7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9+/-11.5 and 57.8+/-15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that gamma-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in gamma-irradiated mouse ovarian follicles.     

The neuroendocrine regulation of the human ovarian cycle.

The menstrual cycle is now thought to be mainly determined by the ovary itself, which sends various signals to the pituitary and the hypothalamus. The hypothalamus is an autonomous pacemaker, with a pulse frequency that is modulated by ovarian signals; in turn, it is indispensable to ovarian function. In women, the ovarian cycle produces a single mature oocyte each month from puberty to menopause. This follicle is rescued from atresia, the genetically controlled ovarian apoptosis (or "programmed cell death"), involving 99.9% of the follicles. Follicular growth and maturation are mostly independent of gonadotropins from the stage of primordial to antral follicles. A complete intraovarian paracrine system is implied in this gonadotropin-independent follicular growth and in the modulation of the action of gonadotropins in the ovary. Follicle-stimulating hormone (FSH) allows the rescue of a minority of follicles from atresia and is indispensable only for the final maturation of the preovulatory follicle during the follicular phase of the cycle. Luteinizing hormone (LH) is responsible for the final growth of the dominant follicle in the late follicular phase. the induction of ovulation during the LH peak, and the survival of the corpus luteum during the luteal phase. The cyclical variations of gonadotropins are under the control of ovarian steroids (estradiol and progesterone) and peptides (inhibins). The cycle length is determined by the duration of terminal follicular growth and by the fixed life span of the corpus luteum. The ovarian cycle can be monitored as well at the level of target tissues of steroids, such as the endometrium. In fact, the endometrial maturation is synchronized to follicular development, and this synchronization is indispensable for successful implantation of the embryo. The improving knowledge of follicular and endometrial physiology will allow the development of new treatments of infertility, the design of new contraceptive techniques, and a better tolerance of treatments using sex steroids.     

Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers

Previous studies demonstrated that waves of follicular activity develop approximately every 9 d in cattle during the estrous cycle and early pregnancy. A dominant follicle develops from each wave and the remaining follicles (subordinates) begin to regress after a few days. In this study, intraovarian luteal and follicular interrelationships were examined during the follicular waves of the estrous cycle and pregnancy using data obtained by ultrasonography. During the estrous cycle, no intraovarian relationships were found between the ovary containing the corpus luteum and the ovary containing the dominant follicle (n = 165), or between the location of the corpus luteum and the characteristics of the dominant follicle. During pregnancy, however, the frequency distribution for the number of follicular waves with the dominant follicle and corpus luteum on the same or opposite ovaries differed (P<0.05) among Waves 1 to 10. The two structures (dominant follicle and corpus luteum) were more often in opposite ovaries during Waves 3 to 10 (combined frequency, 75%) than during Waves 1 and 2. During pregnancy, dominant follicles of consecutive waves differed (P<0.05) among Waves 1 to 8 in the frequency with which they appeared in the same versus the opposite ovary. The difference seemed primarily due to an increased frequency of consecutive follicles on the same ovary for Waves 4 to 8 (combined frequency, 80%). During both the estrous cycle and pregnancy, there was no significant intraovarian effect of the dominant follicle on the day of detection of the next dominant follicle, on the growth rate of the largest subordinate follicle, or on the length of the interval from wave origin to cessation of growth of the largest subordinate; these results indicate that previously postulated suppressive effects between follicles are exerted through systemic channels.     

Evolution of the diameters of the largest healthy and atretic follicles during the human menstrual cycle

Analysis of ovaries from 31 women with normal ovarian function permitted study of the diameter of the largest healthy and atretic follicles during the menstrual cycle. The follicle destined to ovulate is selected during the early follicular phase (Days 1-5). Throughout the cycle the diameter of the largest healthy follicles, with the exception of the dominant follicle, did not exceed, on average, 6 mm during the follicular phase and 4 mm during the luteal phase. Consequently, excluding the dominant follicle during the second half of the follicular phase, the largest follicles present in the human ovary are atretic. From these data, it was concluded that a new ovulation could not occur very soon after a spontaneous or experimentally induced premature disappearance of the dominant follicle or the corpus luteum of the cycle.     

E-cadherin-catenin complex in the rat ovary: cell-specific expression during folliculogenesis and luteal formation

The cadherins and their cytoplasmic counterparts, the catenins, form the adherens junctions, which are of importance for tissue integrity and barrier functions. The development and maturation of the ovarian follicle is characterized by structural changes, which require altered expression or function of the components involved in cell-cell contacts. The present study examined the cell-specific localization and temporal expression of epithelial cadherin (E-cadherin) and alpha- and beta-catenin during follicular development, ovulation and corpus luteum formation in the immature gonadotrophin- and oestrogen-stimulated rat ovary. Immunohistochemistry and immunoblotting demonstrated the expression of E-cadherin in theca and interstitial cells of immature ovaries before and after injection of equine chorionic gonadotrophin (eCG). E-cadherin was not detected in granulosa cells, except in the preantral follicles located to the inner region of the ovary. The content of E-cadherin in theca and interstitial cells decreased after an ovulatory dose of hCG. Granulosa cells of apoptotic follicles did not express E-cadherin. Oestrogen treatment (diethylstilboestrol) of immature rats for up to 3 days did not result in a measurable expression of E-cadherin in granulosa cells. alpha- and beta-catenin were expressed in all ovarian compartments. The concentration of beta-catenin was constant during the follicular phase, whereas the content of alpha-catenin decreased in granulosa cells after treatment with diethylstilboestrol or hCG. The expression of alpha-catenin was also reduced in theca and interstitial cells after hCG. alpha- and beta-catenin were present in most ovarian cells at all stages of folliculogenesis. Therefore, the catenins have the potential to associate with different members of the cadherin family and to participate in the regulation of cytoskeletal structures and intracellular signalling. The restricted expression of E-cadherin in granulosa cells of preantral follicles indicates a role in the recruitment of these follicles to subsequent cycles. The specific decrease of alpha-catenin in granulosa cells and the reduction of both alpha-catenin and E-cadherin in theca cells of ovulatory follicles might reflect some of the molecular changes in cell-cell adhesion associated with ovulation and luteinization.     

Follicle populations and gene expression profiles of Nelore and Angus heifers with low and high ovarian follicle counts

The number of antral follicles counted (AFC) by ultrasound is associated with fertility in cattle. Cows with higher follicle count (HFC) have higher performance in reproductive‐assisted technologies than cows with lower follicle count (LFC). In this study, we aimed to define the preantral follicle count by histology and to identify differentially expressed genes (DEGs) using a microarray in Nelore and Angus heifers with HFC and LFC. The ovaries of each animal were scanned with an ultrasound device 12 to 24 hr after estrus. The groups were formed based on the average number of total follicles (≥3 mm) counted in each breed consistently ± the standard deviation. For the histological analysis, preantral follicles were counted and classified under a stereo microscope, and follicle density was determined. Microarray analysis was performed on pools of three follicles dissected from the ovaries of 15 Nelore (6 HFC and 9 LFC) and 17 Angus heifers (9 HFC and 8 LFC). Angus heifers have increased total and primordial follicle density. Nelore heifers have increased antral follicle count. Different patterns of gene expression regulate follicle recruitment and development in Angus and Nelore heifers and may be associated with the different follicle densities observed in Angus versus Nelore heifers. Furthermore, HFC heifers presented increased expression of genes associated with cellular development and metabolism.     

Expression of transforming growth factor-beta3 (TGF-beta3) in the porcine ovary during the oestrus cycle

Transforming growth factor-beta (TGF-beta) proteins are growth factors that have been shown to be involved in regulation of ovarian follicular development. Ovarian expression, activity and functional significance of TGF-beta1 and TGF-beta2 isoforms were extensively studied in most species. However, little is known about the biological role of TGF-beta3 previously shown to be expressed independently of the other two isoforms. Therefore, expression of TGF-beta3 mRNA and protein was evaluated by RT-PCR and immunohistochemistry, respectively, in porcine ovaries collected during different phases of the oestrus cycle. Results of RT-PCR analysis showed that TGF-beta3 mRNA is expressed throughout the oestrus cycle. The level of TGF-beta3 mRNA expression was found to be higher at metoestrus and dioestrus. Weak TGF-beta3 immunoreactivity was present in follicular epithelial cells and oocytes of preantral follicles in all stages examined. TGF-beta3 protein expression was exclusively present in theca interna cell layer of antral follicles, and was particularly prominent in large antral follicles. Immediately after ovulation, almost all theca cells outside of the granulosa cell layer were intensively stained with anti-TGF-beta3. Immunostaining of TGF-beta3 in theca lutein cells rapidly decreased during corpus luteum development. It is suggested that TGF-beta3 may play an important role in modulating theca cell function of pre- and postovulatory follicles of the pig.     

Reliability of diagnostic ultrasonography for identification and measurement of follicles and detecting the corpus luteum in heifers

The accuracy of diagnostic ultrasonography for assessment of ovarian structures was examined by comparing results of in vivo ultrasonography and slices of the excised ovaries. Follicular numbers and diameters, location (right versus left ovary) of the corpus leteum, and presence of fluid-filled cavities within the corpus luteum were evaluated in 23 Holstein heifers on Days 12 or 14 postovulation. The following endpoints were used for each ovary (n = 46): number of follicles 2 to 3 mm, ≥4 mm, ≥7 mm, and ≥11 mm and diameter of largest follicle. Heifers were slaughtered within 4 hours of ultrasound examination. Ovaries were collected and placed in 10% formalin for 12 hours to prevent collapse of the follicles. Slices 2 mm thick were made to expose the follicles. Slicing determinations were made without knowledge of ultrasound results. Comparisons were done by regression and correlation analyses and paired t-tests. The 95% confidence intervals revealed a tendency to slightly overestimate the number of 2 to 3 mm follicles (ultrasonography, 16.4 ± 0.7 SEM; slicing, 15.5 ± 0.8). Zero was centered within the confidence intervals for the number of follicles ≥4 mm, ≥7 mm, and ≥11 mm and diameter of largest follicle. Slopes of the regressions for number of follicles determined by ultrasonography versus slicing ranged from 0.83 for number of follicles ≥4 mm to 1.03 for number of follicles ≥2 mm. Regression R² values were from 64.5% to 84.9% for various categories and 94.5% for diameter of largest follicle. Correlation coefficients between ultrasonography and slicing results were highly significant (number of follicles 2 to 3 mm, 0.90; ≥4 mm, 0.80; ≥7 mm, 0.89; ≥11 mm, 0.85; ≥2 mm, 0.92 and diameter of largest follicle, 0.97). There was 100% agreement between ultrasound and slicing results for identification of the corpus luteum bearing ovary and presence of cavities within the luteal gland. Diagnostic ultrasonography was determined to be a reliable method of identifying and measuring follicles and detecting mature corpora lutea and luteal cavities in heifers.     

Role of vascular endothelial growth factor in ovarian physiology - an overview

In the female reproductive system, as in a few adult tissues, angiogenesis occurs as a normal process and is essential for normal tissue growth and development. In the ovary, new blood vessel formation facilitates oxygen, nutrients, and hormone substrate delivery, and also secures transfer of different hormones to targeted cells. Ovarian follicle and the corpus luteum (CL) have been shown to produce several angiogenic factors, however, vascular endothelial growth factor (VEGF) is thought to play a paramount role in the regulation of normal and abnormal angiogenesis in the ovary. Expression of VEGF in ovarian follicles depends on follicular size. Inhibition of VEGF expression results in decreased follicle angiogenesis and the lack of the development of mature antral follicles. The permeabilizing activity of VEGF is thought to be involved in follicle antrum formation and in the ovulatory process. In the CL, VEGF expression corresponds to different patterns of angiogenesis during its lifespan. In most the species, higher VEGF expression in the early luteal phase is essential for the development of a high-density capillary network in the CL. However, high VEGF expression may be still maintained in the mid-luteal phase to increase vascular permeability that results in enhancement of luteal function. During gestation, VEGF is thought to be important for the persistence of the CL function for a longer than in the nonfertile cycle period of time. Further elucidation of specific roles of VEGF in ovarian physiology may help to understand the phenomenon of luteal insufficiency and reveal novel strategies of ovarian angiogenesis manipulation to alleviate infertility or to control fertility.     

The effects of previous ovarian status on ovulation rate and early embryo development in response to superovulatory FSH treatments in sheep

A total of 64 ewes was used to determine if the changes in superovulatory yields related to the ovarian status at the start of superovulatory treatment are due to differences in the population of gonadotrophin-responsive follicles, alterations in the processes of ovulation or transport of embryos from oviduct to uterus and/or developmental competence of the oocyte/embryo. Ovarian status at the start of a superovulatory FSH step-down treatment, administered coincidentally with a progestagen, was assessed by ultrasonography. On Day 4 after progestagen withdrawal, embryos were recovered from oviduct and their viability was determined by assessing development in vitro culture (IVC) until the hatched blastocyst stage. In all the ewes, the ovulation rate was related positively to the number of 2-3 mm follicles at first FSH injection (P<0.005). However, the total number of embryos and their viability were related to the more limited category of 3 mm follicles (P<0.05), whereas a higher degeneration rate was related to the number of 2mm follicles. The presence of a corpus luteum (CL) at the start of superovulatory treatment exerted a protective effect on embryonic viability, decreasing the degeneration of embryos. On the other hand, the presence of a dominant follicle at first FSH dose affected the mean size of the pool of follicles responding to the superovulation treatment, because ovulation arose from 3 to 5 mm follicles in absence of large follicles (P<0.05), but from 2 to 3 mm follicles when large follicles were present (P<0.005), indicating atresia in medium sized follicles in the presence of a large follicle.     

Effect of Braun-Collins and saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ

The present work has investigated the efficiency of Braun-Collins and saline (0.9%) solutions in the conservation of goat preantral follicles in situ, at different temperatures and incubation times. For each animal the ovarian pair was divided into 19 fragments. One ovarian fragment was taken randomly and immediately fixed (control). The other 18 ovarian fragments were randomly distributed in tubes containing Braun-Collins or saline (0.9%) solutions at 4, 20 or 39 degrees C for 4, 12 or 24h. A total of 3385, 372 and 191 primordial, primary and secondary follicles were examined, respectively. The quality of preantral follicles was evaluated by histology and transmission electron microscopy. The storage of ovarian fragments in saline (0.9%) or Braun-Collins solutions at 4 degrees C did not reduce significantly the percentage of morphologically normal follicles when compared with the control. The histological analysis revealed a morphological integrity of goat preantral follicles stored at 4 degrees C for up to 24h in both solutions, but these results were not confirmed by ultrastructural analysis. The transmission electron microscopy revealed that only preantral follicles stored at 4 degrees C for a maximum of 12h in both solutions were ultrastructurally normal. In conclusion, this study shows for the first time that goat preantral follicles can be stored in situ successfully at 4 degrees C in saline (0.9%) or Braun-Collins solution for up to 12h.