Types Of Follicles Research Articles

The use of proliferating cell nuclear antigen (PCNA) immuno-staining technique to determine number and type of follicles in the gilt ovary

The present study determined the number and type of follicles in gilts ovarian tissue using the proliferating cell nuclear antigen (PCNA) immunohistochemical labelling technique and investigated the association between the number of follicles, gilt age, body weight, average daily gain, age at first observed oestrus, ovulation rate and weight of the ovaries. Ovarian tissues were obtained from 19 gilts aged 267.8±19.2 days weighting 145.7±11.8kg. The tissues were incubated with mouse monoclonal anti-PCNA. The follicles were categorized as primordial, primary and growing follicles. PCNA immuno-staining enhanced the visualization of small follicles and the efficacy to distinguish primordial and primary follicles. The gilt ovarian tissue contained 19.8±8.5follicles/100μm2 (range 6.0–42.0). The numbers (and proportions) of primordial, primary and growing follicles per 100μm2 of the gilt ovarian tissue were 13.1±6.9 (64.2%), 6.2±3.3 (32.7%) and 0.5±0.2 (3.1%) follicles, respectively. The number of primary follicles per 100μm2 of the gilt ovarian tissue positively correlated to body weight (r=0.50, P=0.032) but negatively correlated to age at first observed oestrus (r=−0.54, P=0.015). In conclusion, PCNA technique can be applied to quantify the precise number and distinguish the type of follicles in the ovarian tissue of porcine species. Gilts with a higher body weight and earlier age at first observed oestrus have a higher density of primary follicles in the ovarian tissue than those with a lower body weight and later age at first observed oestrus.

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).

Fertility control of rodent pests: a review of the inhibitory effects of plant extracts on ovarian function

Plant extracts can inhibit fertility by adversely affecting, directly or indirectly, reproductive processes ranging from gonadal function and development to gestation. This review focuses on plant extracts that disrupt ovarian function in rodents. Extracts from at least 40 plant species exert some of their disruptive reproductive effects at the ovarian level. Of those, 13 plants induce a reduction in the number and type of ovarian follicles and also cause disruption to the oestrous cycle. Their effects are short term and reversible once treatment ceases. Protection of plant extracts to prevent their degradation before uptake in the gastrointestinal tract could enhance short-term efficacy but would not enhance the longevity of their effects. Identification and further testing of the specific chemicals responsible for reproductive effects would be beneficial. The adoption of a standard protocol for treatment and assessment of the inhibitory effects of potential control agents on reproductive function in rodents is essential. Treatment with higher concentrations of extracts in conjunction with other extracts or with other chemosterilants could have potential complementary effects and lead to more rapid and permanent changes in ovarian function. An orally delivered agent(s) that causes major depletion of all follicle types, and particularly of non-regenerating primordial follicles, could be an ideal fertility control product and serve as an additional tool for population control of pest rodents.

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<0.05) than those in summer (19±2; n=7) season. The number of secondary follicles per ovary was also higher (P<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

Aflatoxin B1 effects on ovarian follicular growth and atresia in the rat

For this investigation, 28 female healthy adult Wistar rats were selected. The animals were divided into four groups (n = 7 per group): control, test group 1, test group 2 and test group 3. Each rat in test groups 1, 2 and 3, received 0.8 ppm, 1.6 ppm and 3.2 ppm aflatoxin B1 (AFB1), respectively, via gavage for a period of 25 days. The control group received distilled water only. All tissue specimens were processed for routine paraffin embedding and serial cross-sections cut at 5–7 μm and stained with haematoxylin–eosin. Both histomorphologic and histomorphometric analysis was performed under light microscopy. An increase in the concentration of AFB1 resulted in a reduction in the population of healthy primordial, primary, secondary and tertiary follicles. The greatest reduction was in seen in group 3 (with 3.2 ppm AFB1/day). In all test groups, due to an increase in AFB1 concentration, in both the right and left ovaries, all types of atretic follicles, including primordial, primary, secondary, and tertiary atretic follicles were significantly increased (P < 0.01). In conclusion, AFB1 is toxic for all type of ovarian follicles, including non-growing and growing follicles and exerts an atretogenic effect on all types of ovarian follicles. The atretogenic effect of AFB1 is dose dependant. Due to its toxic effects (gametotoxicity), the resting pool of ovarian follicles (primordial follicles) decreases significantly. The ovulatory follicular population either decreases or is completely depleted.

Relationships between skin follicle characteristics and fibre properties of Suri and Huacaya alpacas and Peppin Merino sheep

We aimed to quantify the number, type and arrangement of skin follicles in Huacaya and Suri alpaca skin and correlate their follicle characteristics with fibre traits of harvested fibre and compared these relationships with those of Merino sheep. Fibre and skin samples were collected from the mid-side of 12 Huacaya alpacas, 24 Suri alpacas and 10 Merino sheep. The mean fibre diameter (MFD ± s.e.) of the Huacaya and Suri were: 35.5 ± 0.9 and 28.3 ± 1.0 μm, respectively. The follicle groups found for alpacas were very different from the normal trio of primary follicles found in sheep and goats. The follicle group of the alpacas consisted of a single primary follicle surrounded by a variable number of secondary follicles. The mean ± s.e. primary follicle density was 3.1 ± 0.3 and 2.7 ± 0.1 follicles/mm2 for Huacaya and Suri, respectively. The mean ± s.e. secondary follicle density (SFD) was 13.7 ± 1.2 and 17.5 ± 0.6 follicles/mm2 for Huacaya and Suri, respectively. The mean ± s.e. ratio of secondary to primary follicles (S/P ratio) was 5.1 ± 0.5 for the Huacaya and 7.3 ± 0.2 for the Suri alpacas. The sheep had higher S/P ratios and SFD, lower MFD and produced significantly heavier fleeces. The key correlations found between traits in alpacas include a negative correlation between SFD and MFD (r = –0.71, P = 0.001) and a negative correlation between S/P ratio and MFD (r = –0.44, P = 0.003) and a positive correlation between S/P ratio and total follicle density (r = 0.38, P = 0.010). The study revealed that important relationships exist between alpaca skin follicle characteristics and fibre characteristics. It was the number of secondary follicles in a group that imparts density and a corresponding reduced MFD.

The Functional Organization of Cutaneous Low-Threshold Mechanosensory Neurons

Innocuous touch of the skin is detected by distinct populations of neurons, the low-threshold mechanoreceptors (LTMRs), which are classified as Aβ-, Aδ-, and C-LTMRs. Here, we report genetic labeling of LTMR subtypes and visualization of their relative patterns of axonal endings in hairy skin and the spinal cord. We found that each of the three major hair follicle types of trunk hairy skin (guard, awl/auchene, and zigzag hairs) is innervated by a unique and invariant combination of LTMRs; thus, each hair follicle type is a functionally distinct mechanosensory end organ. Moreover, the central projections of Aβ-, Aδ-, and C-LTMRs that innervate the same or adjacent hair follicles form narrow LTMR columns in the dorsal horn. These findings support a model of mechanosensation in which the activities of Aβ-, Aδ-, and C-LTMRs are integrated within dorsal horn LTMR columns and processed into outputs that underlie the perception of myriad touch sensations.

Ultrasound characteristics of experimentally induced luteinized unruptured follicles (LUF) and naturally occurring hemorrhagic anovulatory follicles (HAF) in the mare

The development of hemorrhagic anovulatory follicles (HAF) involves luteinization and hemorrhage of the follicle. This is observed on ultrasound as an increase in the echogenicity of the granulosa layer and formation of echoic particles in the antrum. The inhibition of prostaglandin synthesis with flunixin meglumine (FM) during the periovulatory period induces ovulatory failure with development of luteinized unruptured follicles (LUF). These two types of anovulatory follicles appear to share similar ultrasound features but they have not been compared critically. The following endpoints: follicle diameter, follicular contents score, interval from hCG administration to beginning of follicular hemorrhage, interval from hemorrhage to organization of follicular contents, and cycle length were studied and compared in mares with HAF (n = 11) and LUF (n = 13). The objective of this study was to elucidate whether these two unruptured follicles have a consistent clinical pattern of development and therefore can be considered as part of the same anovulatory syndrome. None of the endpoints analyzed differed significantly between HAF and LUF. However, there was a greater individual variation in HAF as compared with LUF in regards to interval from hCG to hemorrhage, follicular diameter at the administration of hCG, and beginning of hemorrhage. In conclusion, HAF share a similar cascade of ultrasound characteristics with the experimentally induced LUF. This finding may provide new insights in elucidating the pathogenesis of HAF.

Effects of chlorpromazine on the onset of puberty and follicular development in immature female rats

Effect of chlorpromazine (dopamine receptor antagonist) on pre-pubertal ovarian follicular development and onset of puberty were studied. Fifteen days old female rats were administered chlorpromazine (2.5 mg/kg body weight) daily for 21 days and appropriate controls were maintained. The onset of puberty in immature female rats was delayed following chlorpromazine treatment. There were significant increases in the body weight, ovary weight and diameter of the ovary of controls and treated group over the initial controls. Ovary of the initial controls consisted of primordial (type 2), primary (type 3a, 3b), pre-antral (type 4, 5a, 5b) and antral (type 6) follicles whereas, antral (type 7) and pre-ovulatory follicles (type 8) were not developed. Controls and treated group consisted of all types of follicles i.e. primordial to pre-ovulatory follicles. Primordial follicles were reduced in number significantly in the ovary of the controls and treated group when compared with the initial controls whereas there was no significant variation among the controls and the treated group. The mean number of primary, pre-antral and antral (type 6) follicles in the control and treated group increased significantly over the initial controls. However, there was a significant reduction in the mean number of these follicles in the treated group when compared to controls. The mean number of type 7 (antral) and type 8 (pre-ovulatory) follicles were reduced in the treated group when compared with controls. The number of atretic follicles of the primary, pre-antral and antral (type 6) follicles significantly increased in controls and treated group over the initial controls. When compared to controls the mean number of atretic follicles belonging to primary, pre-antral, antral (type 6 and 7) and pre-ovulatory category were significantly higher in treated group and the number of corpora lutea was significantly lower. The results indicate that chlorpromazine effect results in loss of follicles by atresia and delay the onset of puberty in immature female rats.

Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

BackgroundSuccessful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.ResultsWe developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH).A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA.ConclusionsThe ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.

Distribution, organization and innervation of gastric MALT in conventional piglet

Mucosa-associated lymphoid tissue (MALT) is the initial inductive site for mucosal immunity. It is present in the different layers of the mucosal wall and consists of organized lymphoid tissue which may occur as isolated or aggregated lymphoid follicles (LFs) and interfollicular areas. It is present in many organs, including the pig stomach. Gastric MALT has been intensely studied in experimentally infected pigs but few data are available in healthy, non-gnotobiotic or germ-free animals. In the present study we described the gastric MALT in conventional piglets in the cardiac mucosa of the gastric diverticulum, in the pyloric mucosa, and in the sites of transition from cardiac to oxyntic and from cardiac to pyloric mucosa by means of histological and immunohistochemical stains. The majority of LFs were located in the cardiac mucosa and in the transition from the cardiac to the oxyntic mucosa. Here the LFs were mainly located in the submucosa and reached the mucosa; we called these submucosal lymphoid follicles (SLFs). In the pyloric mucosa and in the transition sites from the cardiac to the pyloric mucosa, LFs were located in the mucosa; we called these mucosal lymphoid follicles (MLFs). In SLFs, a compartmental organization of T and B lymphocytes was present; by contrast, in the MLFs, the T and B cells were intermingled, suggesting the possibility of different roles for the two types of follicles. In the epithelium overlying the lymphoid tissue, numerous T lymphocytes and some cells immunoreactive to cytokeratin-18 were observed. Following the application of the fluorescent tracer DiI into the SLFs of the diverticulum, enteric neurones located in the submucosal plexus were labelled, confirming the interplay between the immune and the enteric nervous system.

Follicular atresia in the prepubertal spiny mouse (Acomys cahirinus) ovary

This study was designed to determine follicular atresia in the newborn and the prepubertal spiny mouse. We analyzed the processes of follicle loss using classical markers of apoptosis (TUNEL reaction, active caspase-3) and autophagy (Lamp1). Numerous small clear vacuoles and autophagosomes as well as strong Lamp1 staining were observed in dying oocytes of all follicle types, especially of the primordial and primary ones. Active caspase 3 and the TUNEL reaction were detected only in the granulosa cells of large secondary and antral follicles. The expression of apoptosis and autophagy markers was also changing during the prepubertal period. Western blot analysis indicated that at the moment of birth, females undergo an increased rate of follicular atresia mediated by autophagy, while apoptosis is the dominant form of ovarian atresia in consecutive postnatal days. On the basis of these observations, we concluded that apoptosis and autophagy are involved in follicular atresia and these processes are cell and developmental stage-specific.

Effect of crude and steroid free bovine follicular fluid on some hematological and biochemical parameters and ovarian development in rats

Ovarian growth and function is under the control of pituitary gonadotropins and various local intraovarian factors. The present study has been undertaken to assess the efficacy of crude follicular fluid and steroid free FF taken from bovine ovaries on some hematological aspect as well as ovarian growth and function of mature female Wistar rats. Follicular fluid aspirated from large size Graffian follicles of abattoir – derived bovine ovaries was made cell free by centrifugation and steroid free by charcoal treatment. follicular fluid ‎ has been fractioned using ammonium sulphate precipitation at various saturation levels and then subjected to get filtration chromatography. The 20 – 40% fraction yielded the detectable peak and the average yield of peptide was 0.659 mg/ml of FF . The crude and steroid free FF were tested for effects on some hematological parameters and ovarian and uteri weights, numbers of different types of ovarian follicles and assessment of histophysiological appearance. Twenty four mature female Wistar rats were randomly assigned into three equal groups (control and two treatment groups), injected with normal saline (Control), SFFF (T1) and CFF (T2) for 4 consecutive days. At the fifth day, all female rats were anesthetized, dissected and blood samples were obtained for hematological study and ovaries and uteri were removed , weighted and fixed in formalin 10% for histophysiological study . The results revealed no significant effects in Hb concentration , PCV , AST , concentrations, cholesterol , urea , and glucose concentrations . On the other hand , the result revealed no effect on uteri and ovarian weights , an increment in the numbers of primary and secondary follicles and a decrement in the number of Graffian follicles . Within each group , the results showed that primary follicles recorded the highest number among the different types of ovarian follicles. While the highest total number of the different types of ovarian follicles were recorded in CFF and SFFF groups compared with control . The present study demonstrated no side effect on blood constituents as well as the effect of a bovine intrafollicular factor/s regulating ovarian growth and function in rats , especially the development of primary and secondary follicles unlike Graffian follicles which underwent from the inhibition .

Some comparative morphometric and histological asspects of Iraqi buffaloes( Bulbus bulbus ) ovaries at sixth and ninth years old in esterous stage

The study conducted on the Iraqi buffalo ovaries in esterous stage at sixth to ninth years old . The study was appeared the buffaloes ovary enclosed by delicate fibrous capsule , the mean thickness in right and left ovary ( 37.5 ± 0.14 , 25± 1.52 , 18.75 ± 0.12, 17.22± 0.51) at respectively . The ovarian capsule enclosed by simple cuboidal epithelium . The study was revealed the buffalo ovary composed of ovarian cortex and medulla . the cortex was occupied large region when compared with medulla , the mean thickness of cortex in the left and right ovary of the study groups was (5625± 5.28 , 4357 ± 2.5 , 4102 ± 2.62 , 4010± 3.84 ) micrometer at respectively , while the mean thickness of the medulla was( 2875 ± 1.82 , 2375±2.24, 2235± 1.72 2125± 3.95 ) micro meter at respectively in left and right ovary of different age . The cortex of the buffalo ovary was included three types of ovarian follicles, primary , secondary and Graffian follicles which pass in different stages of development . the diameter of graffian follicles in nine year s old more when compared with sex years old in each right and left ovary ( 498±3.22, 425 ±2.86, 383± 1.14 , 397± 4.81 ) micro meter at respectively . The medulla of ovarian buffalo was characterized by very rich blood vessels supply consist of arteries , veins and their branches and nerves , as well as study was recorded the differences in the mean diameters of blood vessels that nourished the ovarian medulla.

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.

Feeling the ovaries prior to insemination. Clinical implications for improving the fertility of the dairy cow

During the periovulatory period in dairy cattle, the largest ovarian follicle can be felt by palpation per rectum as a firm/soft follicle (young preovulatory follicle), a very soft follicle separating it from the remainder of the ovary (mature preovulatory follicle), or an evacuated follicle (follicle associated with ovulation). Because any one of these three follicle types may be present at the time of artificial insemination, the objective of this study was to identify possible differences between the effects of a firm/soft, very soft, or evacuated ovarian follicle on fertility. Out of a study sample of 2365 inseminations, very soft, firm/soft, and evacuated follicles were recorded in 1689 (71%), 593 (25%), and 83 (3.5%) inseminations, respectively. Logistic regression analysis indicated no significant effects of largest follicle type, vaginal discharge, season, days in milk, parity, synchronized or natural estrus, and semen-providing bull on the pregnancy rate. The only variable included in the final logistic regression model was the interaction season-follicle type. This interaction determined that the likelihood of pregnancy decreased significantly by factors of 0.86 or 0.82 in cows with a firm/soft follicle inseminated during the cool or warm period, respectively, and by a factor of 0.09 in cows with evacuated follicles inseminated during the warm period, using as reference cows with a very soft follicle inseminated during the cool period (yielding the highest pregnancy rate). As an overall conclusion, the state of the periovulatory follicle at insemination was clearly related to fertility and masked the effects of factors commonly affecting fertility such as parity, days in milk at AI and inseminating bull. More importantly they suggest that by including ovarian follicle checks in artificial insemination routines, the success of this procedure could be improved.

The HoVert technique: a novel method for the sectioning of alopecia biopsies

Cicatricial forms of alopecia, including lichen planopilaris (LPP) and discoid lupus erythematosus (DLE), may present with overlapping clinical features. In such cases, histopathological examination may provide key information for resolving the differential diagnosis. Optimally, microscopical analysis for alopecia requires both vertical and horizontal sections, and this may necessitate multiple samples. Here, we present what we term the "HoVert" technique, which produces horizontal and vertical sections from a single biopsy. We hypothesize that the HoVert technique should be useful for differentiating DLE from LPP. A formalin-fixed 4 mm punch scalp biopsy is transected approximately 1 mm below the skin surface to create an epidermal disc and a lower portion. The epidermal disc is bisected and embedded in conventional fashion to obtain vertical sections. The lower portion is serially sectioned and embedded to obtain horizontal sections. The HoVert technique yields vertical sections permitting visualization of the epidermis, the dermal-epidermal junction and perijunctional inflammation. The technique also provides horizontal sections that permit analysis of follicle number, follicle type, perifollicular inflammation and scarring. Evaluation of both vertical and horizontal sections from a single scalp biopsy maximizes the histopathological information obtained and enhances the diagnosis of LPP or DLE in specific cases. We believe that the HoVert technique represents a simple and diagnostically effective tool in differentiating LPP from DLE. It may also be applicable to the assessment of other forms of alopecia.

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).

Early primary-rather than primordial follicles constitute the main follicular reserve in the African elephant ( Loxodonta africana)

Information on the ovarian follicle reserve in the African elephant ( Loxodonta africana) is lacking. This study set out to determine the ratios of early preantral follicles and their relative dimensions in the ovaries of 16 African elephant aged 10–34 years. The ovaries were sectioned histologically. Follicles were counted and classified according to expansion of the pre-granulosa cells. Early primary follicles were the most common (75.8% ± 11.8%), followed by true primary follicles (23.8% ± 11.8%), whereas primordial follicles were the most rare (<2%). Measurements made on at least 100 early preantral follicles from each animal ( n = 1464) indicate that growth in oocyte and nuclear diameters started with transition to the true primary stage P < 0.01. This, together with the observed ratios between the three types of early preantral follicles suggest that both classical primordial and early primary follicles contribute to the ovarian reserve in the African elephant.

Alterations in Gene Expression Levels in Estrogen Receptor Alpha Overexpressing Antral Follicles Treated with Methoxychlor.

Antral follicles in the ovary are the only follicle type that can ovulate and this is critical for fertility. Besides this, antral follicles are capable of synthesizing and secreting sex steroid hormones including estrogen. When estrogen binds to estrogen receptors (ESRs) in antral follicles, it facilitates the proper development and function of antral follicles. However, estrogenic chemicals in the environment can bind to ESRs, blocking the normal functions of estrogen thereby affecting follicle development. Further, estrogenic chemicals can induce ESRs resulting in an overexpression of ESRs in antral follicles. Overexpression of ESRs has been proposed to result in an increased susceptibility of the antral follicles to estrogenic chemicals. To directly test whether overexpression of ESRs causes antral follicles to be more susceptible to estrogenic chemicals, we developed and validated a mouse model in which ESR alpha (ESR1) is overexpressed in several tissues, including the ovary. We then determined whether ESR1 overexpression increases the susceptibility of the mouse ovaries to the estrogenic chemical methoxychlor (MXC). MXC is an organochlorine pesticide which has been widely used as a model endocrine disruptor. When the antral follicles of control and ESR1 overexpressing mice (ESR1 OE) were treated in vitro with MXC (1-100 µg/ml) or vehicle dimethylsulfoxide (DMSO) for 96 hours, 10 and 100 µg/ml MXC inhibited growth in both genotypes compared to DMSO treatment. Interestingly, the growth of control follicles with 1µg/ml MXC was not different than DMSO treatment, whereas growth of ESR1 OE antral follicles was inhibited compared to controls. Gene expression analysis of Esr1, androgen receptor (Ar) and catalase (Cat) was done using mRNA from the cultured antral follicles. Esr1 and Ar were chosen because MXC is known to bind to ESR and AR while Cat was analyzed because it is an oxidative stress response gene and MXC is known to cause oxidative stress in antral follicles. The data indicate that Esr1 is significantly upregulated in ESR1 OE antral follicles compared to control follicles. However, in control follicles, MXC treatment caused a dose dependent decrease in Esr1 expression while in ESR1 OE follicles, MXC treatment caused a dose dependent increase in Esr1 expression (controls: DMSO=0.19±0.09, MXC1 = 0.12±0.00, MXC10=0.05±0.03, MXC100=0.03±0.03; ESR1 OE: DMSO=5.02±0.98, MXC1=4.43±0.42, MXC10=10.09±1.01, MXC100=29.06±7.08 genomic equivalents (ge); n=3, p≤0.05). Ar was significantly up-regulated in controls and ESR1 OE follicles in the highest treatment group (100 µg/ml MXC) compared to DMSO. However, Ar expression levels in ESR1 OE follicles at this dose were significantly higher compared to control follicles (controls: DMSO=26.89±4.31, MXC100=49.63±7.14; ESR1 OE: DMSO=5.53±2.59, MXC100 = 87.54±8.85 ge; n=3, p≤0.05). Cat was not different in the control follicles treated with DMSO or MXC, whereas in the ESR1 OE follicles, 100µg/ml MXC significantly increased Cat expression compared to DMSO (DMSO=18.86±3.01, MXC100=79.12±16.27 ge; n=3, p≤0.05). Collectively, these data suggest that ESR1 overexpression may increase the sensitivity of antral follicles to MXC-induced slow-growth by altering gene expression levels. Support: NIH R21ES13061, R01ES012893 and an Eli Lilly Fellowship in Toxicology. (poster)