Large Preantral Follicles Research Articles

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 µm cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 µm and 40 µm cell strainers. The contents retained in the 40 µm strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Preantral follicle population and distribution in the horse ovary.

Characterization of the ovarian preantral follicle population is a necessary step to improve understanding of folliculogenesis and ovarian physiology. Therefore, in the present study, the preantral follicle population in the equine ovary in young and old mares was investigated according to follicular morphology, follicular class, distance from the geometric center using ovarian maps, and follicular density within ovarian portions (lateral vs intermediary) and regions (dorsal vs ventral). Ovaries were collected from an abattoir and histologically processed for evaluation, and the follicle population was calculated. Overall, in the current detailed study, a higher preantral follicle population per mare ovary (mean: 82,206 ± 50,022; range: 1477 to 773,091) than originally reported was identified. Additionally, a mare age effect was observed in the follicle population (young: 152,664 vs old: 11,750) and the spatial distribution of morphologically normal and abnormal follicles and the density and population of follicular classes. These results demonstrate that, in addition to the preantral follicle population in the mare ovary being comparable to that of other species, the location and spatial distribution of these follicles is dynamic and varies depending on mare age and follicle status (i.e. morphology and developmental stage). The characterization of the distribution and population of preantral follicles in the mare ovary provided by this study can potentially aid in improving reproductive studies and assisted reproductive techniques and may expand the understanding of mechanisms involving ovarian plasticity and follicular migration.Lay summaryKnowledge of the distribution and population of immature eggs within follicles (preantral follicles) in the ovaries of mares can improve approaches to assisted reproductive techniques and fertility preservation. As the existing research on horse preantral follicle population was focused solely on large follicles, the present study provides an updated investigation of small and large preantral follicles in the mare, showing that the population is similar to those in other species. This study also shows that the way these follicles are distributed in the ovary varies depending on age and follicle characteristics. Results from this study may help to highlight which areas of the mare ovary should be looked at to find samples of good-quality follicles.

Transcriptome comparisons detect new genes associated with apoptosis of cattle and buffaloes preantral follicles

BackgroundTo develop new breeding technology to improve the breeding ability of bovine, it is the development trend to find the main reason for the occurrence of atresia in these organisms. Transcriptomes of small (100–120 μm) and large (200–220 μm) preantral follicles from cattle and buffalo ovaries were evaluated in vivo and in vitro to understand the transcriptional modulation in preantral follicles that leads to the phenomenon of atresia. MethodsThe preantral follicles were checked as dead, damage, or live follicles in vivo and in vitro by using trypan blue then bisbenzimide and propidium iodine. Transcriptomes of small (100–120 μm) and large (200–220 μm) preantral follicles of cattle and buffalo were evaluated in vivo and in vitro by microarray and RT-PCR. Healthy preantral follicles were selected based on staining results, and then RNA was extracted from them. ResultsThe viability percentage of preantral follicles in cattle was higher (26.7% and 20%) than buffalo (10%) in vivo and in vitro, respectively. According to the microarray data analysis for cattle preantral follicles, only eleven genes were detected corresponding to five upregulated and six downregulated in large size (200–220 μm) compared to small (100–120 μm) size preantral follicles, while in buffalo, 171 genes were detected (92 upregulated and 79 downregulated) in large size compared to small preantral follicle size. The results of RT-PCR of the selected genes (FASTKD1, BAG2, RHOB, AGTR2, MEF2C, BCL10, G2E3, TM2D1, IGF-I, IGFBP3, PRDX3, and TRIAP1) validated the microarray results. In conclusion, the data of gene expression showed significant differences between small and large sizes in both buffalo and cattle preantral follicles. ConclusionApoptotic genes were upregulated in the large preantral follicle compared with the small preantral follicles. Moreover, the expression level of these apoptotic genes was significantly upregulated in buffalo than in the cattle. Most of these genes were significantly upregulated in the large buffalo preantral follicle compared with the small size. However, anti-apoptotic genes were upregulated in large cattle preantral follicle and downregulated in large buffalo preantral follicle.

Optimization and Comparison of Three-Dimensional Culture Conditions in Different Media of Coculture and Encapsulation System for In Vitro Follicular Development in Bubalus bubalis.

The establishment of an in vitro culture system for complete oocyte maturation from the early stages of ovarian follicles is still a challenge. The aim of the present study was to assess the effect of different matrix with different culture media on the developmental growth of ovarian follicles in vitro. An ovarian histoarchitectural study was carried out to identify the primordial (0.027-0.039 mm), primary (0.041-0.079 mm), small preantral (0.085-0.131 mm), large preantral (0.132-0.294 mm), small antral (0.387-0.589 mm), and large antral (1.188-1.366 mm) follicles. Thus, large preantral follicles (0.2-0.3 mm) were mechanically isolated and cultured subsequently in different microconditions such as Dulbecco's modified Eagle's medium, Tissue Culture Medium-199 (TCM-199) and Opti-minimum essential medium, with same supplements where control (without matrix) was compared with matrix (coculture and encapsulation), which includes (1) buffalo fetal fibroblast cells, (2) cumulus cells, (3) ovarian mesenchymal cells, (4) collagen, (5) gelatin, and (6) Matrigel, cultured for 7 days in CO2 incubator at 38.5°C (5% CO2 in air). Cultured follicles were evaluated for growth rate (107.88% ± 10.24%), maturation rate (51.06% ± 6.53%), survivability rate (56.52% ± 3.42%), and antioxidant (catalase; CAT [1.58 ± 0.04 U/mg], superoxide dismutase; SOD [4.63 ± 0.05 U/mg], lactate dehydrogenase; LDH [1.48 ± 0.01 U/mg]) enzymatic activities, which showed significantly (p < 0.05) positive results in growth model with media TCM-199 than other studied groups. Furthermore, the development of large preantral follicles augmented significantly (p < 0.05) for growth rate (248.54% ± 9.51%), maturation rate (75.81% ± 7.07%), survivability rate (81.82% ± 3.02%), antioxidant (CAT [2.05 ± 0.03 U/mg], SOD [3.13 ± 0.12 U/mg], LDH [2.55 ± 0.51 U/mg]), and estradiol (175.83 ± 5.92 pg/mL) activities when they were encapsulated in Matrigel with nutritional requirements fulfilled by media TCM-199. These results provide better insight for the optimization of culture conditions for in vitro follicular development in the water buffalo, which will eventually assist in resolving the limitation of obtaining fewer competent oocytes for the embryo production in the species.

Developmental Programming: Prenatal Testosterone Excess on Ovarian SF1/DAX1/FOXO3

Prenatal testosterone (T) excess, partly via androgenic programming, enhances follicular recruitment/persistence in sheep as in women with polycystic ovarian syndrome (PCOS). Decreased anti-Mullerian hormone (AMH) in early growing and increased AMH in antral follicles may underlie enhanced recruitment and persistence, respectively. Changes in AMH may be mediated by steroidogenic factor 1 (SF1), an enhancer of AMH, and dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1), that antagonizes SF1. Another mediator could be forkhead box 03 (FOXO3) which regulates follicular recruitment/atresia. To test if androgen-programmed changes in SF1, DAX1, and FOXO3 proteins contribute to follicular defects in prenatal T-treated sheep, ovaries from control, prenatal T-, and dihydrotestosterone (DHT)-treated (days 30-90 of gestation) animals at fetal day (FD) 90, FD140, and 1 and 2years-of-age were studied. Prenatal T increased DAX1 in granulosa cells of primordial through large preantral and theca cells of large preantral follicles at FD140 and increased SF1 in the granulosa cells of preantral and antral and theca cells of large preantral follicle at 2years-of-age. Prenatal T increased FOXO3 only in theca cells of preantral (FD140) and antral (2years-of-age) follicles. Prenatal DHT increased DAX1 in granulosa cells from small preantral follicles at FD140 while increasing SF1 in granulosa cells from antral follicles at 1year-of-age. These age-dependent changes in DAX1/SF1 partly via androgen-programming are consistent with changes in AMH and may contribute to the enhanced follicular recruitment/persistence, and multifollicular phenotype of prenatal T-treated females and may be of translational relevance to PCOS.

Production and validation of a polyclonal serum against bovine FSH receptor

In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.

Developmental Programming: Gestational Exposure to Excess Testosterone Alters Expression of Ovarian Matrix Metalloproteases and Their Target Proteins.

Prenatal testosterone (T)-treated sheep, similar to women with polycystic ovary syndrome (PCOS), manifests reproductive defects that include multifollicular ovarian phenotype. Women with PCOS manifest increased ovarian matrix metalloproteinases (MMPs) activity. We tested the hypothesis that gestational T excess in sheep would alter ovarian expression of MMPs, tissue inhibitors of MMP (TIMP) and their target proteins laminin B (LAMB), collagen, tumor necrosis factor alpha (TNF), and connexin 43 (GJA1) consistent with increased MMP activity and that these changes are developmentally regulated. The ovarian content of these proteins was quantified by immunohistochemistry in fetal day 90, 140, and adult (21 months of age) ovaries. Prenatal T excess lowered GJA1 protein content in stroma and granulosa cells of primary follicles from fetal day 90 ovaries and decreased stromal MMP9, TIMP1, and LAMB in fetal day 140 ovaries. In the adult, prenatal T-treatment (1) increased MMP9 in theca cells of large preantral follicles and stroma, TNF in granulosa cells of small and large preantral follicles and theca cells of large preantral and antral follicles, and GJA1 in stroma, theca cells of large preantral follicles, and granulosa cells of antral follicles and (2) reduced TIMP1 in stroma, theca cells of large preantral and antral follicles, LAMB in stroma and small prenatral follicles, and collagen content in stroma and around antral follicles. These findings suggest a net increase in MMP activity and its target proteins TNF and GJA1 in prenatal T-treated adult but not in fetal ovaries and their potential involvement in the development of multifollicular morphology.

Multigenerational obesity-induced perturbations in oocyte-secreted factor signalling can be ameliorated by exercise and nicotinamide mononucleotide.

STUDY QUESTIONCan maternal and offspring high-fat diet (HFD)-induced changes in mRNA expression levels in mice be ameliorated by interventions in female offspring?SUMMARY ANSWEROur results indicate that exercise and nicotinamide mononucleotide (NMN) can ameliorate the negative effects of maternal and post-weaning HFD in female offspring.WHAT IS KNOWN ALREADYMaternal and post-weaning HFD can perturb offspring developmental trajectories. As rates of maternal obesity are rising globally, there is a need for effective treatments in offspring to ameliorate the negative effects from a maternal obesogenic environment. Modulation of the nicotinamide adenine dinucleotide (NAD+) pathway by exercise and the NAD+ precursor NMN has previously been shown to reduce the effects of obesity.STUDY DESIGN, SIZE, DURATIONThis study consisted of a multigenerational study using C57Bl6 mice. Mice were fed a control (chow) or HFD ad libitum throughout mating, pregnancy and lactation (n = 13–25). Female offspring (n = 72) were then also supplied either a chow or HFD post-weaning. At 9 weeks of age offspring from HFD dams were subjected to exercise on a treadmill for 9 weeks or at 16 weeks of age administered NMN (i.p.) for 2.5 weeks. At 18.5 weeks mice were euthanized and ovaries and cumulus–oocyte complexes (COC) were collected to examine the possibility of ameliorating the negative effects of maternal and post-weaning HFD.PARTICIPANTS/MATERIALS, SETTING, METHODSOvary and COC mRNA expression was analysed using RT-qPCR. An initial screen of candidate genes was developed to test which molecular pathways may be involved in generating adverse reproductive system effects. For histological analysis, ovarian tissue was fixed in paraformaldehyde and embedded in paraffin and stained with haematoxylin and eosin. The numbers of primordial, primary, secondary and antral follicles were counted.MAIN RESULTS AND THE ROLE OF CHANCEIn the offspring’s COC, maternal obesity increased both growth differentiation factor 9 (Gdf9: 2-fold; P < 0.05, HFD versus chow) and bone morphogenetic protein 15 (Bmp15: 4-fold; P < 0.05, HFD versus chow) mRNA expression levels while exercise and NMN interventions did not regulate Gdf9 and Bmp15 in the same manner. In whole ovary, maternal diet programmed a 25–50% reduction in FSH receptor and sirtuin-3 mRNA expression levels in daughter ovaries (P < 0.05, HFD versus chow). There was a significant interaction between HFD and intervention on the proportion of large preantral and preovulatory follicles (P < 0.05). However, the increase in preovulatory follicles did not translate to increased oocyte yield. NMN administration resulted in reduced body weight in HFD-fed individuals.LIMITATIONS, REASONS FOR CAUTIONIt is unclear if the changes in oocyte mRNA expression levels reported here will impact oocyte quality and fertility in offspring. Offspring ovulation rate or fecundity could not be studied here and fertility trials are required to determine if the changes in gene expression do reduce fertility.WIDER IMPLICATIONS OF THE FINDINGSOur results demonstrate that maternal and offspring HFD perturbs key signalling pathways that are known to regulate fertility in mice, highlighting the importance of interventions in helping to prevent the declining rates of fertility in the context of the current obesity epidemic.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by grants and fellowships from the National Health and Medical Research Council to R.B.G. (APP1023210, APP1062762, APP1117538) and to M.J.M. and D.A.S. (APP1044295). DAS is a consultant to and inventor on patents licenced to Ovascience, Metrobiotech and GlaxoSmithKline. The other authors declare that there is no conflict of interest.

Mitotic index and morphological characteristics of ovarian small follicles from goats submitted to nutritionally unbalanced regimens.

The objective of this study was to assess the influence of nutritional regimens such as adequate feeding, restricted feeding, and underfeeding-refeeding on the follicle growth and development from caprine ovaries. Goats were divided into three different groups (n = 5 per group). For 24 weeks, goats received elephant grass plus concentrate to provide 1.5 (n = 5) and 0.72 (n = 10) times the energy requirements for maintenance of live weight. Underfed goats were subsequently refed for 6 weeks with the diet of the nourished group (1.5 times the energetic requirements of maintenance). Follicular morphology and morphometry, as well as granulosa cells mitotic index were assessed. Ovarian follicles were classified as small or large preantral follicles, or as small or large antral follicles. Ovarian volume was smaller in animals from both underfed and refed groups than in those animals from fed group. Although no difference in the total number of normal follicles was observed among the nutritional groups, underfed animals presented higher percentages of atretic preantral and small antral follicles when compared with fed animals. Large antral follicles from underfed and refed goats presented a lower mitotic index when compared with fed ones. In conclusion, ovaries from goats challenged with prolonged undernutrition will be functionally compromised, which is characterized by atresia of preantral and small antral follicles and decreased mitotic index of large antral follicles. Refeeding those animals will not recover ovarian function to a same level experienced by goats fed a diet with adequate energy requirements.

Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes

The present study was undertaken to study the effect of metabolic stressors like elevated levels of ammonia, urea, Non-esterified fatty acid (NEFA) and β-hydroxybutyric acid (BHB) on preantral follicle growth, survival, growth rates of oocytes enclosed in preantral follicles (PFs), maturation rates of oocytes recovered from cultured follicles, hormone production (estrogen and progesterone), reactive oxygen species (ROS) as well as superoxide dismutase (SOD) activity. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries by a mechanical cum enzymatic method. SPFs and LPFs were cultured in vitro for 14 and 7 days respectively and examined for their growth, survival and growth rates of enclosed oocytes in PFs exposed with different concentration of ammonia (0, 100, 150, 200, 250, 300 and 400 μM), urea (0, 4, 4.5, 5, 5.5,6, 7 and 8 mM), NEFA [Basal NEFA (70 μM): stearic acid, SA (10 μM)+Palmitic acid, PA(20 μM)+oleic acid, OA(40 μM), b) Medium combo (140 μM): SA (20 μM)+ PA(40 μM)+ OA(80 μM), c) High combo (210 μM): SA (30 μM)+PA(60 μM)+OA(120 μM), d) Very high Combo (280 μM): SA(40 μM)+PA(80 μM)+OA(160 μM)] and BHB (0, 0.5, 0.75, and 1 μM). Results indicated that ammonia, urea, NEFA and BHB caused inhibition of survival and growth of in vitro cultured ovine PFs and enclosed oocytes at the levels of 300 μM, 8 mM, high combo level of NEFA and 0.75 μM respectively. Our study may contribute to the identification of the mechanisms involved in decline of fertility due to metabolic and nutritional stress in ruminants.

Effects of GnRH agonists on the expression of developmental follicular anti-mullerian hormone in varying follicular stages in cyclic mice in vivo.

Gonadotrophin-releasing hormone (GnRH) agonists (GnRHa) have been widely used to induce a state of downregulation for in vitro fertilization, and its direct effects on the pituitary are well known. However, the effects of GnRHa on the expression of anti-mullerian hormone (AMH) by follicles in varying stages in vivo remain to be fully elucidated. In the present study 84 cyclic mice were randomly divided equally into four GnRHa groups and three cyclic mice were used as a control group. The expression levels of AMH in follicles of varying stages between days 0 and 7 following GnRHa administration were quantified using immunohistochemistry. The expression of AMH in follicles at various stages revealed dynamic changes during the process of downregulation. AMH in primary follicles initially increased and then decreased gradually. In small and large preantral follicles and in granulosa cells (GCs) surrounding the oocyte of small antral follicles, the expression of AMH began to increase on day 1, was attenuated on day 2, and then increased to a peak. The expression levels of AMH in the GCs surrounding the basement membrane, in contrast to the GCs surrounding the oocyte, were significantly lower and did not increase on day 1. In all stages of follicles, the expression of AMH declined gradually between the peak level and last day of downregulation. On day 7, the varying follicular stages all expressed lower levels of AMH than on day 0. This decrease was more prominent in the higher dose groups, compared with the lower dose groups. In conclusion, GnRHa was observed to induce time-dependent changes in the expression of AMH at varying follicular stages, which occurred in a dose-dependent manner.

Expression of the beta-2 adrenergic receptor (ADRB-2) in human and monkey ovarian follicles: a marker of growing follicles?

BackgroundADRB-2 was implicated in rodent ovarian functions, including initial follicular growth. In contrast, ADRB-2 expression and function in nonhuman primate and human ovary were not fully known but innervation and significant levels of norepinephrine (NE), which is a ligand at the ADRB-2, were reported in the ovary.MethodsWe studied expression of ADRB-2 in human and rhesus monkey ovary (RT-PCR, immunohistochemistry; laser micro dissection) and measured levels of norepinephrine (NE; ELISA) in monkey follicular fluid (FF). 3D cultures of monkey follicles (4 animals) were exposed to NE or the ADRB-2 agonist isoproterenol (ISO), and follicular development (size) was monitored. Upon termination expression of ADRB-2, FSH receptor and aromatase genes were examined.ResultsImmunohistochemistry and RT-PCR of either human follicular granulosa cells (GCs) obtained by laser micro dissection or isolated monkey follicles revealed ADRB-2 in GCs of primordial, primary, secondary and tertiary follicles. Staining of GCs in primordial and primary follicles was intense. In large preantral and antral follicles the staining was heterogeneous, with positive and negative GCs present but GCs lining the antrum of large follicles were generally strongly immunopositive. Theca, interstitial, and ovarian surface epithelial cells were also positive. NE was detected in FF of preovulatory antral monkey follicles (0.37 + 0.05 ng/ml; n = 7; ELISA) but not in serum. We examined preantral follicles ranging from 152 to 366 μm in diameter in a 3D culture in media supplemented with follicle stimulating hormone (FSH). Under these conditions, neither NE, nor ISO, influenced growth rate in a period lasting up to one month. Upon termination of the cultures, all surviving follicles expressed aromatase and FSH receptors, but only about half of them also co-expressed ADRB-2. The ADRB-2 expression was not correlated with the treatment but was positively correlated with the follicular size at the beginning and at the end of the culture period. Hence, expression of ADRB-2 was found in the largest and fastest-in vitro growing follicles.ConclusionsThe results imply ADRB-2-mediated actions in the development of primate follicles. Drugs interfering with ADRB-2 are used to treat medical conditions and may have unexplored effects in the human ovary.

In vitro culture of bovine preantral follicles: a review.

Preantral follicles are the majority of the ovarian follicle population and their use as a source of homogeneous oocytes for bovine reproductive biotechnologies could result in a substantial advance in this field. However, while in other species embryos and offspring have been produced, in bovine species the results have been limited to the follicular activation of small (primordial) preantral follicles and formation of early antral follicles from large (secondary) preantral follicles after in vitro culture. Therefore, this review will highlight the basic aspects of bovine folliculogenesis by focusing on preantral follicles, the methods of harvesting preantral follicles, the main results from in vitro follicular culture during the last 20 years, and the potential candidate substances (basic supplements, growth factors, and hormones) for improving the efficiency of in vitro follicle growth.

Effects of Crude Aqueous Extract of Origanum vulgaris in Developing Ovary of Rabbits Following in Utero, Adolescent, and Postpubertal Exposure

We evaluated the impact on rabbits after having been treated with Origanum vulgaris (Lamiaceae) (OV) at a dose level known to adversely affect ovarian functions in rodents without causing systemic toxicity. The choice of rabbits has been guided by the fact that rabbits have a relatively long phase of reproductive development and hence simulation of reproductive development is better as opposed to dealing with rodents. The use of rabbits facilitates multiple evaluations of mating ability. An attempt has also been made at determining whether OV affected ovarian development and hence the use of animal model. Rabbits were exposed to 80 mg OV/kg/day in utero (gestation days [GD] 0 to23) or during adolescence (postnatal weeks [PNW] 4 by breast feeding and orally from 4w to 12 w), and the offspring were examined at the end of the 12 W period. Another group was treated after puberty (for 12 weeks) till age of 24 [PNW] of age and examined at the conclusion of exposure and follicles were categorized as primordial, primary, small preantral, large preantral or small antral follicles. The most pronounced reproductive effects were in female rabbits group which had been exposed from in utero till post-puberty period, in weights of ovaries (at 12 and 24 weeks, down 23%; p < 0.05). Serum Gonadotropin levels were down (at 24 weeks, 32%; p < 0.05); a slight increase in histological alterations of the ovaries (p < 0.05) at 24 weeks, of abnormal follicles;Â

Oxygen consumption by bovine granulosa cells with prediction of oxygen transport in preantral follicles

The rate of oxygen consumption by granulosa cells is a key parameter in mathematical models that describe oxygen transport across ovarian follicles. This work measured the oxygen consumption rate of bovine granulosa cells in vitro to be in the range 2.1-3.3×10⁻¹⁶ mol cell⁻¹ s⁻¹ (0.16-0.25 mol m⁻³ s⁻¹). The implications of the rates for oxygen transport in large bovine preantral follicles were examined using a mathematical model. The results indicate that oocyte oxygenation becomes increasingly constrained as preantral follicles grow, reaching hypoxic levels near the point of antrum formation. Beyond a preantral follicle radius of 134 µm, oxygen cannot reach the oocyte surface at typical values of model parameters. Since reported sizes of large bovine preantral follicles range from 58 to 145 µm in radius, this suggests that oocyte oxygenation is possible in all but the largest preantral follicles, which are on the verge of antrum formation. In preantral bovine follicles, the oxygen consumption rate of granulosa cells and fluid voidage will be the key determinants of oxygen levels across the follicle.

Effect of Rumex steudelii methanolic root extract on ovarian folliculogenesis and uterine histology in female albino rats.

A substantial number of the world population especially that of the developing countries rely on herbal products to control their fertility since ancient times. Rumex steudelii Hochst is one of the traditionally used antifertility plants in Ethiopia. Previous studies showed that the methanolic root extract of the plant had reversible antifertility effect in experimental animals. However, no study had hitherto been done on the antifertility activity of the methanolic root extract of Rumex steudelii on the ovary and uterus of female albino rats. To investigate the quantitative aspects of follicular development in the ovaries and uterine histology in cyclic female albino rats to get further information on the possible mechanism of antifertility effect of the methanolic extract of R. steudelii. The effect of the extract on uterine histology and ovarian follicular growth was determined after oral administration of the methanolic root extract of Rumex steudelii at 2.2, 2.5, 3.0 g/kg/day doses consecutively for 30 days. The extract significantly decreasing the number of healthy small antral, Graffian follicles and corpora lutea with concomitant significant increase in the number of atretic follicles of the same stage in dose dependent manner. Treatment at 3.0 g/Kg dose level in addition caused a significant decrease in the number of healthy primary, small preantral and large preantral follicles with concomitant significant increase in the number of atretic primary follicles. The ovarian and uterine wet weights are reduced significantly. The extracts also caused a significant decrease in the epithelial cell height, myometrial and stromal thickness in a dose dependent manner. The study demonstrated that the methanolic extract could cause atrophic changes in the uterus and disruption of ovarian folliculogenesis by inhibiting further development of the recruited ovarian follicles.

A Thrombospondin-Mimetic Peptide, ABT-898, Suppresses Angiogenesis and Promotes Follicular Atresia In Vivo in the Monkey.

Ovarian folliculogenesis is regulated by many endocrine, paracrine and autocrine factors, and as follicular development progresses, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Angiogenesis is tightly regulated by a variety of pro- and anti-angiogenic factors, including vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aberrant angiogenesis occurs in a number of pathological conditions, such as polycystic ovary syndrome (PCOS) and endometriosis, and novel ways of targeting this could lead to new therapeutics. TSP-1 is an anti-angiogenic factor in many physiological systems and has been shown to have anti-angiogenic effects on follicles in vitro. ABT-428898 is an octpeptide mimetic of TSP-1 and we investigated the effect of ABT-898 on follicular angiogenesis using an in vitro angiogenesis assay, prior to studying its effects in vivo in the common marmoset (Callithrix jacchus). Intact preantral/early antral follicles from 21-day-old rat ovaries were cultured for six days in the presence of ABT-898 (0, 0.1, 1, 10, 100 and 1000nM). At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. In a subsequent experiment, the potential of this peptide to inhibit follicular angiogenesis was investigated in vivo, in marmoset monkeys. The marmosets (n=5) were treated with 2.5mg/kg ABT-898 twice daily throughout the follicular phase of the cycle and control marmosets (n=5) were treated with vehicle. On d10 of the follicular phase, which corresponds to the peri-ovulatory period in controls, all animals were injected with 20mg bromodeoxyuridine (BrdU) one hour before removal of ovaries which were fixed, sectioned and stained for haematoxylin and eosin, CD31, BrdU, CD31/BrdU and caspase-3. Treatment with ABT-898 resulted in the suppression of follicular angiogenesis, a reduction in endothelial cell proliferation at the pre- and early antral stages of follicular development and the suppression of granulosa cell proliferation at early preantral, preantral and early antral stages of follicular development. In addition to the inhibition of angiogenesis, ABT-898 treatment also resulted in a significant increase in the number of large preantral and early antral follicles undergoing apoptosis. However, follicle selection and ovulation were not inhibited by treatment, suggesting that the effects of ABT-898 are stage-specific. Taken together, these results indicate that the TSP peptide is capable of having a dual effect by inhibiting follicular angiogenesis and promoting atresia of antral follicles in vivo, but does not prevent ovulation, as has been observed with direct VEGF inhibitors. These results suggest that the TSP peptide could be a novel therapeutic to inhibit abnormal angiogenesis and induce atresia of accumulated follicles in polycystic ovary syndrome. This research was supported by a Medical Research Council Ph.D. studentship and a core grant to Medical Research Council Reproductive Sciences Unit (U.1276.00.002.00006.01). (poster)

Expression of PDGFs and Their Receptors in the Fetal and Adult Sheep Ovary.

Platelet derived growth factors (PDGFs) and their receptors are key regulators of cell growth and angiogenesis, especially during fetal development. In rodents and humans, expression of PDGF mRNA was observed in specific ovarian compartments and the addition of PDGF to follicles in culture increased their size and enhanced the transition from primordial to primary follicles. Differences in the pattern of PDGF expression were noted depending on the species or age studied. However the effect of follicular health on expression has not been examined in depth. The objectives of this study were to determine the cell specific gene expression of PDGF ligands (PDGFA, B and C) and their receptors (PDGFRA and B) during ovarian and follicular development in sheep. Ovaries were collected from fetuses on days 55, 75 and 95 of gestation and from ewes at 4 weeks, 10 months, 2 years and 8 years of age and examined using in situ hybridization. During fetal life, PDGFA expression was sometimes associated with the vasculature and extra ovarian rete. Consistent expression was noted in the surface epithelium and in oocytes in both ovigerous cords and formed follicles. No expression was observed in granulosa cells of formed follicles or connecting rete. Fetal expression of PDGFB was similar to PDGFA at days 55 and 75; however no expression was apparent in the rete and by day 95 expression was largely confined to the surface epithelium. Expression of PDGFC was concentrated in the surface epithelium and cells surrounding the vasculature, with little or no expression observed in oocytes, follicles or in cells of the rete system. Postnatally, PDGFA and B were expressed in oocytes at all stages of follicular development from primordial to large antral follicles and in granulosa cells from the primary stage. Granulosa cell expression of PDGFC was evident in large preantral and small antral follicles. Thecal expression was not apparent until the large preantral stage and was initially restricted to PDGFB. At antral stages PDGFB was expressed predominantly in the theca interna while PDGFA was expressed in both theca interna and externa. PDGF receptors were not expressed in follicles until after antrum formation with PDGFRA expression in both theca interna and externa while PDGFRB expression was limited to theca interna. In general, atresia resulted in decreased expression of PDGFA and B in granulosa cells, increased expression of PDGFA and B in the thecal layers, increased expression of PDGFC in the granulosa and the appearance of PDGFC expression in the thecal layers. Only PDGFA expression was consistently noted in components of the ovarian rete system. Expression of PDGFA, B and C and PDGFRA and B was apparent throughout the CL, with receptor expression often associated with the vasculature. Cells associated with the vasculature showed expression of PDGFA, B and C, as well as PDGFA and B. Changes in expression patterns at different stages of follicular development along with changes associated with follicular atresia are consistent with a role for these factors in regulation of follicular development. (poster)

Suppression of Antral Formation in the Ovarian Follicles of Marmosets by Human Ovarian Follicular Fluid Protein

Ovarian follicular fluid peptide (OFFP) has earlier been shown to inhibit maturation of large follicles and ovulation in mice. An active fraction hGF2, obtained following partial purification from human ovarian follicular fluid, was injected at a dose of 20 micrograms to 4-month-old immature marmosets, every alternate day for 2 months. The animal were autopsied at 4 and 6 months of age. Ovarian morphology indicated the appearance of large follicles at 6 months of age in the control group. In contrast, in hGF2 treated marmosets development up to preantral follicles could be seen at 6 months. Furthermore, only in 1 out of the 3 treated marmosets, presence of large preantral follicles was observed and in the remaining 4 animals only primary and secondary follicles were noted. These results reveal that OFFP suppresses follicular development and antrum formation in marmosets.

Anti-mullerian Hormone Receptor Type II Gene Polymorphisms Similar Among Women With the Polycystic Ovary Syndrome (PCOS) and Controls

Anti-mullerian Hormone Receptor Type II Gene Polymorphisms Similar Among Women With the Polycystic Ovary Syndrome (PCOS) and Controls