Different Types Of Follicles Research Articles

Ameliorative Effect of Lycopene on Follicular Reserve Depletion, Oxidative Damage, Apoptosis Rate, and Hormonal Profile during Repeated Superovulations in Mice.

Superovulation is a crucial step in assisted reproductive technology that involves the administration of gonadotrophins. Repeated superovulations result in severe ovarian damage. The present study investigated the effect of in vivo administration of lycopene on ovarian damage induced by four successive cycles of superovulation. Superovulated mice were simultaneously administered intraperitoneally with saline (R4) or 5 mg/kg lycopene (R4-Lyc). The evaluated parameters were the count of different types of follicles, expression of ovarian antioxidant- and apoptosis-related genes, and serum concentrations of estradiol, progesterone, and inhibin-B. Increased numbers of healthy follicles and a decreased count of atretic follicles were observed in mice of the R4-Lyc group compared to those of the R4 group. Moreover, significantly higher mRNA levels of Sod3, Cat, and Nrf2 and lower mRNA levels of Keap1, Tnf, Nfkb, and Casp3, together with decreased H2O2 concentrations and increased total antioxidant capacity, were detected in the ovaries of lycopene-treated mice. Regarding serum reproductive hormones, elevated concentrations of estradiol, progesterone, and inhibin-B were evident in lycopene-administered mice. The present study reports a significant role of lycopene in alleviating the ovarian damage induced by multiple hormonal superstimulations, which could help to improve the outcomes of in vitro embryo production.

Effect of preconditioning of human umbilical cord mesenchymal stem cells with hydrogen peroxide on the therapeutic potential of MSCs in the cyclophosphamide -induced premature ovarian failure mice model.

Exposure of stem cells to sublethal levels of hydrogen peroxide (H2O2) can prevent oxidative stress-induced apoptosis. In the present study, the effects of H2O2 preconditioning on the therapeutic potential of human umbilical vein cord mesenchymal stem cells (hUCV-MSCs) were evaluated in a murine model of premature ovarian failure. Mature mice were divided into 4 groups, and 10 mice were incorporated into each group. The control (Ctrl) group received phosphate buffered saline (PBS) intraperitoneal (IP), and the CTX group was injected IP with cyclophosphamide (CTX). The CTX+MSC group after receiving CTX was injected with a single dose of hUCV-MSCs labeled with CM-DiI intravenously (IV), whereas the CTX+preMSCs group after CTX injection received preconditioned MSCs with H2O2 IV. Seven days later, the mice were euthanized, and their ovaries were removed for histological studies such as H&E staining and the TUNEL assay. Furthermore, the numbers of CM-DiI-labeled hUCV-MSCs in the different regions of the ovary were calculated. FSH and estradiol values in the serum were measured. Our studies showed that CTX caused degenerative changes and follicular loss in the ovary. The number of follicles in the CTX+MSCs and CTX+PreMSCs groups was significantly higher compared to the CTX group. In addition, in the CTX+PreMSCs group, the numbers of different types of follicles were higher than in the CTX-MSC group. Immunohistochemical studies in the CTX+MSCs and CTX+PreMSCs groups showed little evidence of TUNEL positivity compared with the CTX group. Moreover, the apoptotic index decreased in the CTX+PreMSCs group compared to the CTX+MSCs group. Moreover, CM-DiI-labeled MSCs in the ovary in the CTX+pre-MSCs group were higher than in the CTX+MSCs group. Our experiment offers preconditioning as an effective strategy in stem cell therapy to potentiate MSCs' therapeutic efficacy in ovarian function failure.

Efeitos de lipopolissacarídeos na ativação e apoptose de folículos ovarianos primordiais em novilhas

ABSTRACT The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn’t modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.

Evaluation of goat ovaries and follicles by histological study

The present study evaluated slaughterhouse goat ovaries and follicles to know the effect of corpus luteum on the number and quality of ovarian follicles. For this study, 18 goat ovaries were collected and categorized as ovaries with corpus luteum (CL+) and without corpus luteum (CL–) group. Both groups were evaluated by morphometric measurements and histological observation. Among 18 ovaries, 12 ovaries contained CL (66.67%) and 6 ovaries were without CL (33.33%). Based on the morphometric measurements, the length and weight of the ovaries with CL were numerically higher (p>0.05) than those of the ovaries without CL. But the diameter of the ovaries with CL was significantly higher (p<0.05) than those of the ovaries without CL. In comparison to the CL– group, the CL+ group had a numerically higher (p>0.05) number of follicles with a diameter of 2-6 mm and > 6 mm. Histological analysis revealed that the total number of follicles (primary to antral) in CL– group (11.10±2.47) was numerically higher (p>0.05) than that of the CL+ group (10.07±2.47). Presence of primary (4.44±1.59), secondary (1.45±0.83) and antral (6.27±1.31) follicles were found higher (p>0.05) in ovaries without CL than that of with CL (4.05±1.61, 0.92±0.21, 5.08±1.21 respectively). On the other hand, the number of degenerated primary (1.29±0.56), secondary (0.52±0.14) and antral (3.38±0.66) follicles were found comparatively higher (p>0.05) in the ovaries with CL than that of the ovaries without CL (1.14±0.69, 0.37±0.19, 2.40±0.40 respectively). Since, there was no statistical difference found among the number of total and different types of follicles between ovaries with or without corpus luteum; hence, it may be summarized that both type of ovaries may be used for the retrieval of oocytes for further reproductive biotechnological study. Bangladesh Journal of Animal Science, 51 (4): 145-151

The effect of platelet lysate on mouse ovarian structure, function and epigenetic modifications after autotransplantation

Research questionWhat are the effects of platelet lysate on structure, function and epigenetic modifications of heterotopically transplanted mouse ovarian tissues? DesignMice were divided into three groups (n = 17 per group): control (mice with no ovariectomy, grafting or treatment), autograft and autograft plus platelet lysate (3 ml/kg at the graft sites). Inflammatory markers, serum malondialdehyde (MDA) concentration and total antioxidant capacity were assessed on day 7 after transplantation. Twenty-eight days after transplantation, stereological and hormonal analyses were conducted. Chromatin immunoprecipitation and quantitative real-time polymerase chain reaction were also used to quantify the epigenetic modifications of maturation genes, parallel to their expression. ResultsThe total volume of the ovary, cortex and medulla, and the number of different types of follicles, the concentration of interleukin (IL)-10, progesterone and oestradiol and total antioxidant capacity significantly decreased in the autograft group compared with the control group (P < 0.001); these parameters significantly increased in the autograft plus platelet lysate group compared with the autograft group (P < 0.001). The concentrations of tumour necrosis factor alpha, IL-6 and MDA increased significantly in the autograft group compared with the control group (P < 0.001); in the autograft plus platelet lysate group, these parameters significantly decreased compared with the autograft group (P < 0.001). In the autograft plus platelet lysate group, the expression levels of Gdf-9 (P < 0.0021), Igf-1 (P < 0.0048) and Igf-2 (P < 0.0063) genes also increased along with a lower incorporation of MeCP2 in the promoter regions (P < 0.001) compared with the autograft group. ConclusionsPlatelet lysate can contribute to follicular survival by improving folliculogenesis and increasing the expression of oocyte maturation genes.

Taurine improves follicular survival and function of mice ovarian grafts through increasing CD31 and GDF9 expression and reducing oxidative stress and apoptosis

Ischemia-reperfusion (IR) injury is a major limitation of ovarian transplantation which threatens the follicular and graft survival. Taurine as a potent anti-oxidant, anti-apoptotic and anti-inflammatory agent, can prevent graft damages due to IR. We aimed to investigate the effect of taurine on the follicular survival and function of autotransplanted mouse ovaries. Female mice (4–5 weeks old) were divided into: control, autograft and autograft + taurine (200 mg/kg/day). The level of CD31 expression was evaluated two days (48 h) post transplantation. In addition, on day 7 post transplantation the serum levels of malondialdehyde (MDA) and the total antioxidant capacity (TAC) were assessed. Also, 28 days post transplantation; ovaries were studied stereologically and the percentage of apoptotic follicles, level of GDF9 expression and the serum concentrations of progesterone and estradiol were measured. Data were analyzed using one-way ANOVA and Tukey's test and the means were considered significantly different at P < 0.05. The total volume of the ovary (P < 0.01), volume of the cortex (P < 0.01) and medulla (P < 0.04), total number of different types of follicles, expression of GDF9 and CD31 and also the levels of progesterone, estradiol and TAC increased significantly in the autograft + taurine group compared to the autograft group (P < 0.001). The MDA level and apoptosis rate decreased significantly in the autograft + taurine group compared to the autograft group (P < 0.001). Taurine could significantly improve follicular survival and the function of grafted ovaries by accelerating the angiogenesis and reducing oxidative stress and apoptosis.

Liraglutide treatment effects on rat ovarian and uterine tissues

Liraglutide is a Glucagon-like peptide-1 (GLP-1) analogue used for the treatment of type II diabetes mellitus and obesity. The present study aimed at investigating the effect of Liraglutide in ovarian and uterine tissues in albino rats. 30 female rats were divided into 3 groups, 10 rats each. Group (I) served as control group, group (II) animals administrated therapeutic doses of liraglutide for 5 weeks and group (III) animals were injected with Liraglutide as the pervious group. Then they were left for 2 weeks after drug termination as a recovery period. The biochemical results showed a decrease in the female reproductive hormones profile, luteinizing hormone (LH), follicle stimulating hormone (FSH), estrogen (ER), progesterone (PR) and an increase in the level of testosterone (T). Liraglutide administration caused a significant decrease in the antioxidant markers, glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) and a significant increase in the activity of malondialdehyde (MDA). The histopathological examination revealed apoptosis of granulosa cells of different types of follicles with an increase in atretic and disorganized follicles. Vacuolar degenerative changes, and Atrophied muscle with sever inflammatory cell infiltrate in endometrium with congested, dilated blood vessels could be detected in uterine tissues. However, most of the deleterious effects of liraglutide decreased after drug discontinuation. In this study, we clarify the harmful effect of the liraglutide on ovarian and uterine tissues, thus potentially causing reproductive health malfunction and reducing the chances of pregnancy.

A new method for automatic counting of ovarian follicles on whole slide histological images based on convolutional neural network

The ovary is a complex endocrine organ that shows significant structural and functional changes in the female reproductive system over recurrent cycles. There are different types of follicles in the ovarian tissue. The reproductive potential of each individual depends on the numbers of these follicles. However, genetic mutations, toxins, and some specific drugs have an effect on follicles. To determine these effects, it is of great importance to count the follicles. The number of follicles in the ovary is usually counted manually by experts, which is a tedious, time-consuming and intense process. In some cases, the experts count the follicles in a subjective way due to their knowledge. In this study, for the first time, a method has been proposed for automatically counting the follicles of ovarian tissue. Our method primarily involves filter-based segmentation applied to whole slide histological images, based on a convolutional neural network (CNN). A new method is also proposed to eliminate the noise that occurs after the segmentation process and to determine the boundaries of the follicles. Finally, the follicles whose boundaries are determined are classified. To evaluate its performance, the results of the proposed method were compared with those obtained by two different experts and the results of the Faster R–CNN model. The number of follicles obtained by the proposed method was very close to the number of follicles counted by the experts. It was also found that the proposed method was much more successful than the Faster R–CNN model.

Melatonin improves the structure and function of autografted mice ovaries through reducing inflammation: A stereological and biochemical analysis

Melatonin has anti-oxidant, anti-inflammatory and anti-apoptotic properties. We aimed to investigate the effect of melatonin on the structure and function of mice ovaries following autograft transplantation.NMRI mice were divided into: control, autografted + saline, autografted + melatonin (20 mg/kg/day i.p. injection for 1 day before until 7 days after transplantation). 28 days post transplantation, ovary compartments were studied stereologically. Follicle apoptosis and the level of progesterone and estradiol were also measured. The inflammation, serum MDA concentration and total antioxidant capacity were also assessed on day 7 post transplantation.The total volume of the ovary, cortex and medulla (P < 0.05) and the number of different types of follicles (P < 0.001), the concentration of IL-10, progesterone and estradiol (P < 0.001) and TAC (P < 0.01) significantly decreased in the autografted + saline group compared to the control. The levels of IL-6 (P < 0.01), TNF-α, MDA and the apoptotic rate (P < 0.001) increased significantly in the autografted + saline group compared to the control, while the total volume of the ovary, cortex and medulla (P < 0.05) and the number of different types of follicles (P < 0.001), the concentration of IL-10, progesterone and estradiol (P < 0.001) and TAC (P < 0.01) significantly increased in the autografted + melatonin group compared to the autografted + saline group. The levels of IL-6 (P < 0.01), TNF-α, MDA and the apoptotic rate (P < 0.001) decreased significantly in the autografted + melatonine group compared to the autografted + saline group. In the autografted + melatonin group, the localization of CD31-positive cells in the theca layer was similar to the control group.Melatonin can improve the structure and function of the grafted ovary.

Perxiredoxin4, a protein which shows protective effect on ovarian function

Peroxiredoxin4 (Prdx4) is a member of Prdx family. As revealed by our previous research work, the expression of Prdx4 was in close contact with ovary-aging. Furthermore, we also discovered that it was located in endoplasmic resticulum (ER) of granular cell and involved in regulating the endoplasmic reticulum stress (ER stress) in granulosa cell. This research work aimed at exploring the protective effect and the molecular mechanism of Prdx4 on ovarian function. Mice were divided into two groups: Prdx4-KO and wild type group. The indicators of ovarian function, oxidative stress and ER stress pathways-associated markers were compared between the two groups. We established mice model with gene prdx4 knock out by the CRISPR/Cas9 technology. All mice aged 8 months were sacrificed and then blood was collected by removing eyeball. The left ovary was immediately excised and stored at -80°C for biochemical analysis and the right ovary was fixed in 4% paraformaldehyde for histological studies. For the purpose of evaluating the protective effect of Prdx4 on ovarian function, we calculated ovary-to-body weight ratio. Secondly, we detected the level of AMH and expression of senescence-associated protein P16 with the help of real-time PCR and immunohistochemistry correspondingly. Thirdly, the number of different types of follicles were counted by hematoxylin and eosin (H&E) staining for the assessment of ovarian reserve. Eventually, the levels of 4-HNE, 8-OHdG, NTY, and three ER stress pathways related markers were examined by immunohistochemistry and real-time PCR. All the statistical analyses were performed using SPSS v.16. software. All the statistical comparisons between the two groups were analysed by t test. In comparison with the control group, the prdx4-KO group presented significantly smaller ovary weight/body weight ratio (p<0.05), lower level of AMH, higher expression of senescence-associated protein P16, and lower proportion of primordial follicles (p<0.05). In addition, the prdx4-KO group expressed higher 8-OHdG, 4-HNE, NTY. By screening three pathways of ER stress, we found that ATF4 changed obviously. The level of ATF4 and CHOP were up-regulated in ER. Prdx4 may contribute to the ovarian function protection. As for the molecular mechanism of Prdx4 on ovarian function, we hypothesized that Prdx4 may inhibit ER stress through PERK-eIF2α-ATF4-GADDl53/CHOP pathway in granulosa cells.

Imaging the ovary.

During each reproductive cycle, the ovary exhibits tissue remodelling and cyclic vasculature changes associated with hormonally regulated folliculogenesis, follicle rupture, luteal formation and regression. However, the relationships among different types of follicles and corpora lutea are unclear, and the role of ovarian vasculature in folliculogenesis and luteal dynamics has not been extensively investigated. Understanding of ovarian physiology and pathophysiology relies upon elucidation of ovarian morphology and architecture. This paper summarizes the literature on traditional approaches to the imaging of ovarian structures and discusses recent advances in ovarian imaging. Traditional in-vivo ultrasound, together with histological and electron microscopic approaches provide detailed views of the ovary at organ, tissue and molecular levels. However, in-vivo imaging is limited to antral and larger follicles whereas histological imaging is mainly two-dimensional in nature. Also discussed are emerging approaches in the use of near-infrared fluorophores to image follicles in live animals to detect preantral follicles as well as visualizing ovarian structures using CLARITY in fixed whole ovaries to elucidate three-dimensional interrelationships among follicles, corpora lutea and ovarian vasculature. Advances in ovarian imaging techniques provide new understanding of ovarian physiology and allow for the development of better tools to diagnose ovarian pathophysiology.

Expression of factors involved in apoptosis and cell survival is correlated with enzymes synthesizing lysophosphatidic acid and its receptors in granulosa cells originating from different types of bovine ovarian follicles

BackgroundLysophosphatidic acid (LPA) regulates reproductive processes in the cow. Ovarian granulosa cells play a pivotal role in follicle growth and development. Nevertheless, the role of LPA in the local regulation of granulosa cell function in different follicle categories in the bovine ovary has not been investigated.MethodsOvarian follicles were divided into healthy, transitional and atretic categories. The expression levels of AX, PLA2, LPARs and factors involved in apoptosis and cell survival processes in granulosa cells in different types of follicles were measured by real-time PCR. The correlations between the expression levels of AX, PLA2, LPARs and the examined factors were measured. The immunolocalization of AX, PLA2 and LPARs in different ovarian follicles was examined by immunohistochemistry. Statistical analyses were conducted in GraphPad using a one-way ANOVA followed by the Kruskal-Wallis multiple comparison test or a correlation analysis followed by Pearson’s test.ResultsThe expression levels of AX, PLA2 and LPARs, with the major role of LPAR2 and PLA2, were found in the granulosa cells originating from different follicle types. The expression levels of the factors involved in cell apoptosis (TNFα and its receptors, FAS, FASL, CASP3, CASP8, β-glycan, and DRAK2) were significantly higher in the granulosa cells of the atretic follicles compared to the healthy follicles. A number of correlations between LPARs, AX, PLA2 and factors associated with apoptosis were observed in the atretic but not in the healthy follicles. A greater expression of the factors involved in differentiation and proliferation in the granulosa cells (DICE1 and SOX2) was found in the healthy follicles in comparison with the atretic. A number of correlations between LPARs, AX, PLA2 and the factors associated with cell survival were observed in the healthy but not in the atretic follicles.ConclusionsGranulosa cells are the target of LPA action and the source of LPA synthesis in the bovine ovarian follicle. We suggest that the participation of LPA in apoptosis in the atretic follicles mainly occurs through the regulation of TNF-α-dependent and caspase-induced pathways. In the transitional follicles, LPA might influence the inhibins to shift the balance between the number of healthy and atretic follicles. In the healthy follicle type, LPA, acting via LPAR1, might regulate MCL1 and estradiol-stimulating ERβ mRNA expression, leading to the stimulation of anti-apoptotic processes in the granulosa cells and their differentiation and proliferation.

Effect of Sodium Metabisulfite on Rat Ovary and Lipid Peroxidation

Background: Many health problems are related to lifestyle and dietary factors. Since ancient times, food additives such as sulfites have been used to preserve foods. Diverse effects of sulfites on multiple organs have been reported but its effect on female reproductive organ has not been fully elucidated. The aim of this study was to investigate the effects of sodium metabisulfite (SMB) on ovarian tissue in adult rats. Methods: Four groups of female rats (n=32) were used. The experimental rats received 10, 100 and 260 mg/kg SMB for 28 days (S10, S100 and S260 groups, respectively). The control rats received distilled water for the same period. The ovarian volume, weight and the number of different types of follicles were estimated by stereological methods. Lipid peroxidation is assessed indirectly by the measurement of malondialdehyde (MDA), using the thiobarbituric acid (TBA) method. Results: The results showed a significant decrease in the ovarian volume, the number of primordial, primary, secondary, grafian follicles and corpus luteum in the SMB-treated animals compared with the control group (P &lt; 0.05). In comparison to the control group, the number of atretic follicles increased in the SMB-treated rats. MDA was significantly increased in S260 group compared to the control group. Conclusion: The present data confirm sulfite-induced structural changes in the ovary. Increased level of MDA because of SMB ingestion suggests that free radicals may have a critical role in these changes.

The effects of small litter rearing on ovarian function at puberty and adulthood in the rat

Rearing rats in small litters lead to obesity and reproductive dysfunction. We investigated the effects of rearing female rats in small litters on various reproductive parameters during puberty and into adulthood, and examined the possible involvement of local ovarian sympathetic nerve activity. The litter size was adjusted on postnatal day one to four pups per dam for the small litters and 12 pups per dam for the normal litters. Vaginal opening was recorded, and estrous cyclicity was monitored daily immediately post puberty for 14 days and again at 8–9 weeks of age. At the time of puberty and 10 weeks of age, the ovaries were collected. The number of different types of follicles was counted and the thickness of the theca interna of the largest antral follicles was measured. Ovarian sympathetic nerve activity was assessed immunohistochemically by measuring levels of ovarian nerve growth factor receptor (p75NGFR) and tyrosine hydroxylase (TH). In rats reared in small litters, there was a significant advancement of puberty and disruption of estrous cyclicity immediately post puberty. The number of antral follicles increased in the small litter reared rats at puberty compared with their controls. The thickness of the theca interna increased and the expression profiles of ovarian p75NGFR and TH increased in small litter reared rats at puberty, but this did not persist into adulthood. These data suggest that rearing rats in small litters lead to irregular reproductive cycles, which might involve increased local ovarian sympathetic nerve activity.

Human mitotic non-luteinizing and non-mitotic luteinized granulosa cells have different sensitivity to chemotherapy agents

Adult human ovary harbors different types of follicles and granulosa cells. Of these, granulosa cells of preantral and early antral follicles are proliferative but are not capable of undergoing luteinization whereas the granulosa cells of corpus luteum are non-mitotic luteinized type. The aim of the current study was to investigate if these two types of granulosa cells differ in terms of their sensitivity to cytotoxic actions of chemotherapy drugs.

Stem cells and alopecia: a review of pathogenesis

Recent work has focused on the hair follicle as the main source of multipotent stem cells in the skin. The hair follicle bulge contains multipotent stem cells that can form the epidermis, hair follicles and sebaceous glands and help in repopulation of the epidermis after injury. The localization of these stem cells to the bulge area may explain why some types of inflammatory alopecia cause permanent loss of hair (cicatricial alopecia) (such as lichen planopilaris and discoid lupus erythematosus), while others (such as alopecia areata) are reversible (noncicatricial alopecia). The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells. To date, the best marker for bulge stem cells in human hair is cytokeratin (CK) 15; human bulge cells have been reported to express CK15 selectively throughout all stages of the hair cycle in different types of follicles. There is direct evidence in the mouse, and indirect evidence in the human, that compromising the integrity of the sebaceous gland and/or bulge is important in the development of alopecia. Several interesting studies have been done in the last few years to investigate the role of stem cells in alopecia, especially nonscarring types. This is a review about the role of stem cells in the pathogenesis of alopecia (scarring and nonscarring).

Morphometric analysis of ovarian follicles of Black Bengal goats during winter and summer season

The present study was undertaken to know the development and degeneration of ovarian follicles in Black Bengal does during winter and summer season. Seven ovaries were collected in each season from a local slaughter house. The ovaries were fixed in 10% formalin for histological examination. The numbers of different types of follicles obtained during winter and summer season were compared. To compare two sets of data during winter and summer season, independent t-test was performed using Statistical Package for Social Science (SPSS). Histological examination revealed that mean number of antral follicles per ovary was 34 ± 10 (n=7) in winter which was higher (P&lt;0.05) than those in summer (19±2; n=7) season. The number of secondary follicles per ovary was also higher (P&lt;0.05) in winter (52±5) than summer (35±10) season. During summer season, the ovaries contained 19±4% primary and 71±4% primordial follicles whereas these values were 52±3% and 20±1%, respectively duringwinter season. The results indicate that lower rate of development and higher rate of degeneration were occurred in goat ovaries during summer than winter season.DOI: http://dx.doi.org/10.3329/bjas.v40i1-2.10791Bang. J. Anim. Sci. 2011.39 (1-2): 51-55

Influence on pubertal reproductive function in female rats by immune challenge in early life

To investigate the long-term programming effects on pubertal reproductive function by immunological challenge in early life. Female Sprague-Dawley rats were administered by endotoxin (lipopolysaccharide, LPS) at a dosage of 50 µg/kg and saline intraperitoneally on postnatal day 3 and 5. Body weight was measured weekly. Puberty onset (vaginal opening) and oestrous cyclicity were monitored from postnatal day 30. At the age of 6 weeks, bilateral ovariectomy was performed. The histological and morphological change of the ovaries (the thickness of the theca interna and the number of different kinds of follicles) were observed and the immunoreactivity of the ovarian sympathetic nerve markers (low affinity receptor of nerve growth factor, p75NGFR) was evaluated by immune staining. Immunological challenge (exposed to LPS) in early life delayed vaginal opening significantly [LPS-treated (40.6 ± 0.7) days versus controls (38.6 ± 0.5) days, P < 0.05], decreased the percentage of normal oestrous cyclicity (LPS-treated 26.1% versus controls 66.8%, P < 0.05), decreased the total number of different types of follicles (primordial follicles: LPS-treated 610 ± 47 versus controls 1181 ± 57, P < 0.05; primary follicles: LPS-treated 624 ± 41 versus controls 960 ± 30, P < 0.05; preantral follicles: LPS-treated 183 ± 16 versus controls 260 ± 14, P < 0.05; antral follicles: LPS-treated 32 ± 4 versus controls 79 ± 7, P < 0.05) and increased the thickness of the theca interna [LPS-treated (15.8 ± 0.4) µm versus controls (11.4 ± 0.3) µm, P < 0.05]. The immunostaining of p75NGFR was obviously enhanced in the LPS-treated ovaries when compared with that of controls (P < 0.05). Immunological stress during early critical developmental windows could have long dysfunctional effects on the pubertal reproductive function. It delayed puberty onset, reduced the percentage of the normal oestrous cycles, decreased follicles reserve and increased the thickness of the theca interna which might involve the up-regulation of the local ovarian sympathetic nerve activity.

263 REGULATION OF SEVERAL TRANSCRIPTS DURING IN VITRO MATURATION IN PORCINE OOCYTES COLLECTED FROM DIFFERENT SIZE FOLLICLES

Oocyte-specific transcripts play important roles in oocyte maturation, fertilization, and early embryonic development. Currently, oocytes from medium-size follicles have been used for different assisted reproductive techniques after in vitro maturation (IVM). The aim of this study was to compare the mRNA expression level in porcine oocytes collected from medium (3–6 mm) and small (&lt;2 mm) size follicles. Genes were selected based on their described maternal effect (NALP9, HSF1), their identification as markers of oocyte maturation (AURK-A, AURK-B, MOS, and C-mos), their involvement in fertilization (ZP3, ZP4), and anti-apoptotic effect (Bcl-2). All transcripts were studied in oocytes just after collection [germinal vesicle (GV) stage] and after in vitro maturation (IVM; metaphase II stage). To ensure nuclear stage of immature oocytes, oocytes were mechanically denuded just after collection, centrifuged (10 000 rpm, 5 min, RT), and observed under the microscope (60×). Those oocytes with clear nucleolus and evident nuclear membrane were selected and stored (n = 10) until study. For metaphase II oocytes, only those exhibiting the extrusion of first polar body after IVM (n = 10) were selected. Total RNA was extracted from the pool of 10 immature and mature oocytes. One picogram of luciferase mRNA per oocyte was added as an exogenous standard. Total RNA was extracted from oocytes and cDNA was obtained and used as a template for quantitative PCR to analyse the level of different transcripts. The whole process was replicated 4 times. Data were normalized to the luciferase RNA and analysed by one-way ANOVA with maturational stage (GV or metaphase II) and follicle size (small or medium) as fixed factors. Results show that all transcripts were significantly decreased during IVM (P &lt; 0.05). Therefore, after IVM, NALP9, AURK-A, MOS, C-mos, ZP3, ZP4, and Bcl-2 transcripts were significantly reduced in matured oocytes compared with immature ones irrespective of follicle diameter. Transcripts of AURKAB and HSF1 decreased after IVM in oocytes from medium follicles or small follicles, respectively. A significant effect of follicular size was only detected in MOS transcripts in GV-stage oocytes because those collected from middle follicles had a higher amount than the ones from small follicles (Table 1). These results suggest that the variations in the maternal store of RNA during IVM are not related with follicle diameter for the studied genes. Further investigations are necessary to determinate the developmental competence of oocytes that came from different types of follicles (small and medium follicles). Table 1.Variation of transcripts during in vitro maturation in porcine oocytes collected from small and medium follicles This study was supported by Okayama University. R. Romar was given a grant by JSPS (Ref. S-09210).

Oocyte Morphometry in Female Dogs (Canis familiaris, Linnaeus, 1758)

In the present study, the ovaries of 15 healthy bitches divided into three groups according to age were analysed by histology and morphometry: group 1 (1-3 years), group 2 (>3-5 years) and group 3 (>5-7 years). After ovariosalpingohysterectomy, the ovaries were fixed, routinely processed for embedding in paraffin and stained with haematoxylin-eosin. The following morphometric parameters were analysed: maximum and minimum diameter (microm), perimeter (microm), area (microm(2)) and roundness of the cytoplasm and nucleus of oocytes from different types of follicles. Significant differences in the cytoplasmic and nuclear parameters of follicular oocytes between the experimental groups were determined by anova and the Tukey test (5%). For the biotechnology of reproduction, the present results showed that in vitro maturation yielded the best performance for oocytes from primordial follicles in group 3 females and for oocytes embedded in secondary and tertiary follicles in group 2 females. In addition, the present findings will help in the diagnosis and prognosis of diseases affecting bitches, and will especially contribute to a better understanding of these cells by researchers in the field of histology and canine reproduction.