Percentage Of Primary Follicles Research Articles

Exposure to environmentally relevant concentrations of ambient fine particulate matter (PM2.5) depletes the ovarian follicle reserve and causes sex-dependent cardiovascular changes in apolipoprotein E null mice

BackgroundFine particulate matter (PM2.5) exposure accelerates atherosclerosis and contains known ovotoxic chemicals. However, effects of exposure to PM2.5 on the finite ovarian follicle pool have hardly been investigated, nor have interactions between ovarian and cardiovascular effects. We hypothesized that subchronic inhalation exposure to human-relevant concentrations of PM2.5 results in destruction of ovarian follicles via apoptosis induction, as well as accelerated recruitment of primordial follicles into the growing pool. Further, we hypothesized that destruction of ovarian follicles enhances the adverse cardiovascular effects of PM2.5 in females.ResultsHyperlipidemic apolipoprotein E (Apoe) null ovary-intact or ovariectomized female mice and testis-intact male mice were exposed to concentrated ambient PM2.5 or filtered air for 12 weeks, 5 days/week for 4 h/day using a versatile aerosol concentration enrichment system. Primordial, primary, and secondary ovarian follicle numbers were decreased by 45%, 40%, and 17%, respectively, in PM2.5-exposed ovary-intact mice compared to controls (P < 0.05). The percentage of primary follicles with granulosa cells positive for the mitosis marker Ki67 was increased in the ovaries from PM2.5-exposed females versus controls (P < 0.05), consistent with increased recruitment of primordial follicles into the growing pool. Exposure to PM2.5 increased the percentages of primary and secondary follicles with DNA damage, assessed by γH2AX immunostaining (P < 0.05). Exposure to PM2.5 increased the percentages of apoptotic antral follicles, determined by TUNEL and activated caspase 3 immunostaining (P < 0.05). Removal of the ovaries and PM2.5-exposure exacerbated the atherosclerotic effects of hyperlipidemia in females (P < 0.05). While there were statistically significant changes in blood pressure and heart rate variability in PM2.5-compared to Air-exposed gonad-intact males and females and ovariectomized females, the changes were not consistent between exposure years and assessment methods.ConclusionsThese results demonstrate that subchronic PM2.5 exposure depletes the ovarian reserve by increasing recruitment of primordial follicles into the growing pool and increasing apoptosis of growing follicles. Further, PM2.5 exposure and removal of the ovaries each increase atherosclerosis progression in Apoe-/- females. Premature loss of ovarian function is associated with increased risk of osteoporosis, cardiovascular disease and Alzheimer’s disease in women. Our results thus support possible links between PM2.5 exposure and other adverse health outcomes in women.

Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue.

This study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days of in vitro culture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue.

Mono(2-ethylhexyl) phthalate accelerates early folliculogenesis and inhibits steroidogenesis in cultured mouse whole ovaries and antral follicles.

Humans are ubiquitously exposed to di(2-ethylhexyl) phthalate (DEHP), which is an environmental toxicant present in common consumer products. DEHP potentially targets the ovary through its metabolite mono(2-ethylhexyl) phthalate (MEHP). However, the direct effects of MEHP on ovarian folliculogenesis and steroidogenesis, two processes essential for reproductive and nonreproductive health, are unknown. The present study tested the hypotheses that MEHP directly accelerates early folliculogenesis via overactivation of phosphatidylinositol 3-kinase (PI3K) signaling, a pathway that regulates primordial follicle quiescence and activation, and inhibits the synthesis of steroid hormones by decreasing steroidogenic enzyme levels. Neonatal ovaries from CD-1 mice were cultured for 6 days with vehicle control, DEHP, or MEHP (0.2-20 μg/ml) to assess the direct effects on folliculogenesis and PI3K signaling. Further, antral follicles from adult CD-1 mice were cultured with vehicle control or MEHP (0.1-10 μg/ml) for 24-96 h to establish the temporal effects of MEHP on steroid hormones and steroidogenic enzymes. In the neonatal ovaries, MEHP, but not DEHP, decreased phosphatase and tensin homolog levels and increased phosphorylated protein kinase B levels, leading to a decrease in the percentage of germ cells and an increase in the percentage of primary follicles. In the antral follicles, MEHP decreased the mRNA levels of 17alpha-hydroxylase-17,20-desmolase, 17beta-hydroxysteroid dehydrogenase, and aromatase leading to a decrease in testosterone, estrone, and estradiol levels. Collectively, MEHP mediates the effect of DEHP on accelerated folliculogenesis via overactivating PI3K signaling and inhibits steroidogenesis by decreasing steroidogenic enzyme levels.

In vitro development of ovine preantral follicles and oocyte cleavage rate are not affected by long-term ingestion of detoxified castor meal

In the present study, we evaluated the effect of long-term ingestion of detoxified castor meal on in vitro development of sheep preantral follicles as well as on the developmental competence of oocytes from antral follicles. Ovarian fragments were cultured for one or seven days and further analyzed by histology and fluorescence microscopy (experiment 1). The cumulus oocyte complexes (COCs) obtained from antral follicles were matured in vitro and activated by parthenogenesis or fertilization in vitro (experiment 2). Even after 1 day of culture, in both tested groups, a significant reduction in the percentage of primordial follicles and a concomitant increase in the percentage of intermediate follicles were observed when compared to non cultured tissue. After 7 days of culture, the percentage of primary follicles in both tested groups was significantly higher when compared to the non cultured tissue or the tissue cultured for 1 day (P<0.05). The number of in vitro embryos produced was similar between the tested groups. However, for those animals fed with detoxified castor meal, the parthenogenesis method resulted in a higher number of embryos (morulae) when compared to in vitro fertilization (IVF) activation. In conclusion, detoxified castor meal can be used as an alternative protein source without affecting the ovine preantral and antral folliculogenesis.

FSH prevents depletion of the resting follicle pool by promoting follicular number and morphology in fresh and cryopreserved primate ovarian tissues following xenografting

BackgroundCryopreservation and transplantation of ovarian tissue is one option for re-establishing ovarian function, but optimal conditions for graft sustainment and follicular survival are still considered experimental. The present study aims to analyze the effect of FSH treatment on the resting follicle pool in fresh and cryopreserved primate ovarian tissues following xenografting.MethodsOvarian tissues from adult marmosets were grafted freshly or following cryopreservation to ovarectomized nude mice treated with FSH 25 IU twice daily post transplantation or left untreated as controls. Grafts were retrieved 2 or 4 weeks after transplantation to evaluate the number and morphological appearance of follicles.ResultsEarly start of FSH treatment within 1 week following transplantation partly prevents primordial follicle loss in fresh and frozen-thawed tissues, whereas after a 3 weeks time interval this effect is present only in fresh tissues. A similar positive effect of early, but not later FSH treatment on primary follicles is seen in fresh tissues compared to only marginal effects in frozen-thawed tissues. The percentage of morphologically normal follicles is generally increased in FSH treated tissues, whereas the percentage of primary follicles over all primordial and primary follicles is increased by FSH only in freshly-grafted tissues.ConclusionsFSH treatment alleviates depletion of the resting follicle pool and promotes normal follicular morphology both in freshly and frozen-thawed grafted tissues. In previously cryopreserved tissues, applying to most of the tissues intended for clinical use in fertility preservation attempts, its positive effect on primordial follicle numbers and potential graft sustainment is dependent on an early start of treatment within one week of transplantation.

Effect of VEGF Inhibition on Follicular Development and Angiopoietin/Tie2 System in a Rat Model of Polycystic Ovary Syndrome (PCOS).

Vascular Endothelial Growth Factor A (VEGF) is the main angiogenic factor involved in proliferation, migration and endothelial cell permeability. While VEGF is the major initiator of angiogenesis, the formation and differentiation of a functionally mature vascular network requires the coordinated action of various factors. These include angiopoietins (ANGPT), which act via the tyrosine kinase receptor, Tie-2. ANGPT1 is required for the recruitment of periendothelial cells that lead to the maturation and stabilization of newly developed capillaries. On the other hand, ANGPT2 acts as a receptor antagonist destabilizing the blood vessels. PCOS is the major cause of infertility among women; its main features are endocrine, metabolic and reproductive alterations. Patients have increased levels of VEGF in serum due to the high ovarian production. Given the implication of the angiogenesis in this syndrome, we investigated in a PCOS rat model the effect of VEGF inhibition on follicular development and the levels of ovarian ANGPT1, ANGPT2 and Tie2. Prepubertal rats were injected for 15 days with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) to induce PCOS. Control group received vehicle. PCOS rats were divides in two groups. One group was injected under the bursa of the ovary with a VEGF inhibitor (Trap) 48 h before sacrifice (PCOS Trap). The other group was injected under the bursa with vehicle (PCOS control). The ovaries were removed; one was stained with H&E or processed for smooth muscle cell alpha actin (SMC actin) immunohistochemistry to study blood vessel stability. Proteins were extracted from the other ovary to perform western blot studies for ANGPTs and Tie2. Histological studies confirmed the presence of follicular cysts, many atretic follicles and a very low amount of corpora lutea in the ovaries from the PCOS group correlated with a significant decrease in circulating progesterone levels. There was an increase in the percentage of primary follicles in the PCOS rats that was reversed with Trap. Besides, Trap decreased the percentage of preantral follicles and the percentage of cysts without changes in atretic follicles. There were no changes in ovarian SMC actin immunostaining between control and PCOS group. However, Trap decreased significantly SMC actin expression. In addition, there was a significant increase in the levels of ANGPT1 and Tie2 and a significant decrease in the levels of ANGPT2 in the PCOS ovaries compared to controls. None of these changes were reversed with Trap treatment. These results suggest that the treatment with a VEGF inhibitor improves follicular development and decreases ovarian cysts formation in a rat model of PCOS. Furthermore, changes in the levels of ANGPTs and their receptor Tie2 could be involved in the PCOS patophysiology. Supported by: ANPCYT (BID 1201 OC-AR PICT 99:05-06384) and CONICET (PIP 1223). (poster)

Somatostatin 14 affects the pituitary-ovarian axis in infant rats

The effects of multiple somatostatin (SRIH-14) treatment on the pituitary-ovarian axis were examined in infant rats. Female Wistar rats received subcutaneously two daily 20 μg/100g b.w. doses for five consecutive days (from 11 to 15 days of age). Changes in cell volume, volume density and number per unit area (mm²) of follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) immunolabeled cells were evaluated by stereology and morphometry. Serum FSH and LH concentrations were determined by RIA. Ovaries were analyzed by simple point counting of follicles. SRIH-14 treatment significantly reduced FSH and LH cell volume, while their volume density and number per unit area were unaltered. Serum concentrations of FSH and LH were significantly reduced. Volume and volume density of GH cells was significantly decreased after SRIH-14 treatment, while their number per unit area was unaltered. In the ovary, SRIH-14 induced a significant increase in the percentage of primordial follicles followed by a significant decrease in percentage of primary follicles. The number of healthy and atretic preantral follicles was unchanged. It can be concluded that SRIH-14 treatment during the infantile period markedly inhibits pituitary FSH, LH and GH cells. In the ovary, SRIH-14 acts by inhibiting initial folliculogenesis without affecting atretic processes.

Recombinant Epidermal Growth Factor Maintains Follicular Ultrastructure and Promotes the Transition to Primary Follicles in Caprine Ovarian Tissue Cultured In Vitro

We investigated the effects of epidermal growth factor on the survival and growth of caprine preantral follicles. Ovarian fragments were cultured for 1 and 7 days in enriched minimal essential medium with epidermal growth factor (0, 1, 10, 50, 100, or 200 ng/mL). Non-cultured and cultured tissues were processed for histological and ultrastructural studies. Results showed that after 7 days, the epidermal growth factor (1 and 10 ng/mL) maintained the percentage of normal follicles similar to control. An increase in the percentage of primary follicles was observed with 1, 10, and 50 ng/mL of epidermal growth factor compared to enriched minimal essential medium. Ultrastructural studies confirmed follicular integrity after 7 days in epidermal growth factor (1 and 10 ng/mL). In conclusion, the low concentrations of epidermal growth factor maintain caprine follicular viability and promote the transition from primordial to primary follicles.

Cashmere production from Scottish Cashmere kids and crossbreed Scottish Cashmere x Jonica kids

This study is part of a much wider research programme to evaluate the possibility of producing valuable textile fibres, such as cashmere, from goat breeds reared in Italy. In order to achieve this, we have used crossbreeding. The first stage of the programme consisted of evaluating cashmere production in F1 kids obtained by crossing white-haired Jonica does, which have no secondary fibres, with Scottish Cashmere bucks. The trial lasted one year starting in March 2007, and took place in the Department of Animal Production of the University of Bari (Italy). We used 14 male kids: 7 Scottish Cashmere (SC group), and 7 F1 (SC x J group) derived from crossing Scottish Cashmere bucks with does of the Jonica breed, commonly reared in southern Italy. All the parameters considered (live weight, number and active percentage of primary and secondary follicles, S/P ratio, patch weight, growth and length of guard hair and down, yield, down production and diameter, blood protein and T3 and T4) were significantly influenced (P<0.01) by age. Genotype also had a significant effect (P<0.01) on all parameters except for the active percentage of primary follicles and the blood protein level. The factors which influence down production showed the heterosis effect to a varying extent in F1, but they still produced significantly less than the SC group kids (38.5±4.04 vs 68.5±9.16 g; P<0.01). These results are largely due to both their low number of secondary follicles (30.0±1.46 vs 39.3±1.02; P<0.01), which also have a lower percentage of activity (64.7±2.47 vs 90.0±1.53; P<0.01), and also to the down length which was 28% shorter than in SC group. This genetic combination is clearly unsatisfactory so others must be sought, probably by using more rustic local breeds, as well as more productive breeds for crossbreeding.

Short-term transplantation of isolated human ovarian follicles and cortical tissue into nude mice

This study was designed to evaluate follicular survival and growth after short-term transplantation of fresh isolated human follicles and ovarian cortical tissue to nude mice. Ovarian biopsies were obtained from nine women undergoing laparoscopy. Twelve nude mice were xenografted with an ovarian cortical fragment in the right ovarian bursa, and a clot containing isolated follicles in the left, for a period of 7 days. One ungrafted fragment was used as a control. Histological sections were analyzed to determine follicle number and stage. The proliferative status of follicular cells was assessed by Ki-67 immunostaining. A total of 659 follicles was analyzed by histology and 545 follicles by immunohistochemistry. The percentage of primordial follicles was found to be markedly reduced 1 week post-grafting when compared with ungrafted tissue, while the percentage of primary follicles had significantly increased. Only 8% of follicles showed Ki-67-positive granulosa cells before grafting, whereas 1 week after grafting, 71% of follicles in fragments and 67% of isolated follicles were Ki-67-positive (P<0.001). Moreover, the histological aspect of isolated follicle grafts was similar to that of grafted fragments: follicles were surrounded by vimentin-positive stroma-like tissue of human origin, as confirmed by fluorescent in situ hybridization with human-specific probes. Our results demonstrate, for the first time, that isolated human follicles are able to survive and grow after xenografting. This study also shows massive in vivo follicular activation after transplantation of grafted fragments and isolated follicles. One week after grafting, well-structured stroma-like tissue of human origin was observed around the isolated follicles. The potential origin of this stroma is discussed.

P-207: Viability testing of cryopreserved ovarian tissue

P-207: Viability testing of cryopreserved ovarian tissue

Alteration of activation, growth, and atresia of bovine preantral follicles by long-term treatment of cows with estradiol and recombinant bovine somatotropin.

The hypothesis was that long-term treatment of cattle with estradiol (E(2)) and bovine somatotropin (bST) would alter the earliest stages of folliculogenesis. Nonlactating Holstein cows (n = 26) were treated in a 2 x 2 arrangement with E(2) (2 x 24 mg implants, 67.1 +/- 1.4 days) and bST (Posilac, 63.6 +/- 1.5 days). At Day 67 +/- 1.3, one ovary was removed for morphometric and immunohistochemical analysis. For each ovary, 388 +/- 38 microscopic fields (2 x 2 mm) were examined and follicles within each field were classified by histological stage. Fields that contained no follicles were classified as empty. Empty fields (n = 100 per ovary) were further classified as containing no evidence of follicles or containing atretic remnants of follicles. Approximately 30 4-microm sections per ovary were stained for proliferating cell nuclear antigen (PCNA), and 150 fields per ovary were evaluated. Additional sections (n = 10 per ovary) were assessed immunohistochemically for apoptosis, and fluorescence intensity was determined for each follicle. Treatment with bST significantly decreased percentage of empty fields containing atretic remnants. Treatment with E(2) induced activation of follicles as shown by a decrease in percentage of primordial follicles and an increase in percentage of primary follicles as determined by PCNA staining. At the primary follicle stage the combination of bST + E(2) decreased apoptosis as shown by decreased fluorescence intensity. Thus, E(2) induced activation of follicles, bST enhanced survival, and the combination lowered atresia.