Deficient proliferation and apoptosis in the granulosa and theca interna cells of the bovine cystic follicle

The aim of the present study was to examine the frequencies of cell proliferation and death of granulosa and theca interna layers during development of cystic follicles in order to understand the mechanisms of cystic follicle formation. Paraffin sections of cystic follicles were immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 in order to observe proliferating and apoptotic cells, respectively. The concentrations of estradiol-17beta and progesterone in the follicular fluid of these follicles were measured by ELISA. The granulosa and theca interna layers contained both PCNA- and caspase-3-positive cells, although their numbers were limited. There was significant negative correlation between the estradiol-17beta and progesterone concentrations in the follicular fluid. Regression analysis revealed no significant correlation, except for that between the PCNA-positive cells in the theca interna and the caspase-3-positive cells in the granulosa layer. These results indicate that the granulosa and theca interna cells of the cystic follicle show weak proliferative activity and low apoptotic frequency; this implies that the cystic follicle grows slowly and then maintains a static condition without degeneration, which leads to long-term persistence of the follicle.     

Relationship between apoptosis and proliferation in granulosa and theca cells of cystic follicles in sows

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.     

Immunohistochemical study of 3 beta-hydroxysteroid dehydrogenase in dog's perianal sinus

In this study, the localization of 3 beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in the wall of canine perianal sinus (PS) was determined. The 3 beta-HSD activity was found out both in the cytoplasm of cells, situated in the propria and forming clusters adjacently to apocrine glands and in the cytoplasm of some epithelial cells in apocrine cells' glands. The results obtained about the 3 beta-HSD activity allowed us to propose a role of this enzyme in PS development and possibly, in tumourogenesis.     

Comparison of cadherin and integrin localization in bovine cystic and healthy follicles

As stage progresses in the cystic follicle, granulosa cells are lost. We hypothesized that the granulosa and theca interna layers are detached in association with weakened expression of cell adhesion molecules such as cadherin (cell–cell adhesion) and integrin (cell–extracellular matrix adhesion) in cystic follicles. To elucidate this hypothesis, we immunolocalized these molecules in the granulosa and theca interna and compared them between cystic and small healthy follicles. Sections were immunostained with cadherin and integrin β₁ antibodies and their localizations were compared. Cadherin‐positive reaction was seen in the cytoplasma of all granulosa cells. No increase in the frequency of cadherin‐positive area in the granulosa layers and the intensity of cadherin immunoreaction in the theca interna was detected in cystic follicles compared with healthy ones. A dense immunoreaction product of integrin β₁ was detected in the theca interna in both cystic and healthy follicles. Intensity of integrin β₁‐immuno reaction in the granulosa layers and integrin β₁‐positive area in the theca interna was significantly lower in the cystic follicle than in the healthy follicles. These results suggest that granulosa and theca interna cells are detached while maintaining the cell–cell adhesion, resulting in the consequent loss of these layers from the cystic follicle.     

The impact of bisphenol S on bovine granulosa and theca cells

Bisphenol S (BPS) is an endocrine‐disrupting chemical with multiple potential mechanisms of action, including as an oestrogen receptor agonist. BPS is increasingly used in plastics and thermal receipts as a substitute for bisphenol A, which has been phased out due to concerns about human health implications. The ability of BPS to alter female reproductive function in mammals has not been widely studied, despite the importance of normal hormone signalling for female reproduction. The aim of this study was to investigate how BPS (in a wide range of doses, including very low doses) affects granulosa cell and theca cell steroid hormone production and cell viability in the bovine. Granulosa cell oestradiol production was stimulated when cells were exposed to 100 μM BPS under basal conditions, but there was no effect of BPS when cells were stimulated with follicle‐stimulating hormone (FSH). Additionally, there was no effect of BPS on granulosa cell progesterone production or cell viability under basal or FSH‐stimulated conditions. BPS did not affect theca cell androstenedione or progesterone production, or theca cell viability under basal or luteinizing hormone‐stimulated conditions. This study suggests for the first time that BPS may alter oestradiol production by bovine granulosa cells, albeit at a concentration that is unlikely to be physiologically relevant. Further studies are needed to determine the effects of BPS on the bovine oocyte and on other functions of follicular cells.     

Immunohistochemical localization of neuropeptides in bovine pancreas

The occurrence and density of distribution of nerves and endocrine cells that are immunoreactive for neuropeptides in the bovine pancreas were studied by immunohistochemistry. The six neuropeptides localized were galanin (GAL), substance P (SP), methionine-enkephalin (MENK), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP). The exocrine pancreas was shown to have an appreciable number of GAL- and SP-immunoreactive nerve fibres but few fibres showing immunoreactivity for VIP and CGRP. Numerous MENK-, GAL-, SP-, and NPY-immunoreactive nerve fibres were seen in the endocrine portion of the pancreas. Nerve cell bodies in the intrapancreatic ganglia showed immunoreactivity for all of the neuropeptides except CGRP. Endocrine cells showing immunoreactivity for GAL and SP were observed in the large islets and islets of Langerhans, respectively. The present results indicate a characteristic distribution of neuropeptides in the bovine pancreas, which may regulate both exocrine and endocrine secretions of pancreas.     

Immunohistochemical localization of galectin-3 in the granulomatous lesions of paratuberculosis-infected bovine intestine

The presence of galectin-3 was immunohistochemically quantified in bovine intestines infected with paratuberculosis (Johne''s disease) to determine whether galectin-3 was involved in the formation of granulation tissue associated with the disease. Mycobacterium avium subsp. paratuberculosis infection was histochemically confirmed using Ziehl-Neelsen staining and molecularly diagnosed through rpoB DNA sequencing. Galectin-3 was detected in the majority of inflammatory cells, possibly macrophages, in the granulomatous lesions within affected tissues, including the ileum. These findings suggest that galectin-3 is associated with the formation of chronic granulation tissues in bovine paratuberculosis, probably through cell adhesion and anti-apoptosis mechanisms.     

Immunohistochemical localization of androgen and oestrogen receptors in canine hair follicles

Skin biopsies from seven different body sites were obtained from 21 dogs of different breeds (short, normal, long hair), which were presented for euthanasia. Commercially available polyclonal antibodies were used for the immunohistochemical detection of androgen and oestrogen receptors. Both receptors showed a similar distribution in canine skin, with specific intranuclear staining. In the epidermis, the percentage of androgen receptor (AR)-positive cells, but not that of oestrogen receptor (ER)-positive cells, was significantly higher in samples from the thorax and the flank. In the dermal papilla, the percentage of ER-positive (but not AR-positive) cells was significantly lower in biopsies from the flank. No significant difference was found for both receptors between the locations in the outer root sheath, among the three different hair types, between sex and between intact and castrated dogs.     

Ovarian function in the cycling cow: relationship between gonadotropin binding to theca and granulosa and steroidogenesis in individual follicles

Thirty-six Angus and Angus crossbred cows were used in a 3 x 2 x 2 factorial experiment designed to determine the effects of stage of the estrous cycle (day 6, 12 or 18), a long term with a single ovary (2 years) and side of previous ovulation or remaining ovary on the development of large ovarian follicles (LF; diameter greater than or equal to 8 mm). Follicular fluid estradiol-17 beta (FFE) concentration, localization of gonadotropin binding sites and granulosa cell condition were determined for each LF by radioimmunoassay, autoradiography and histological examination, respectively. No side differences were noted. However, the sample was biased by the use of equal numbers of left and right side ovulators. Compared to intact controls, one-ovary cows showed 100% compensation of LF development. Eighty-five percent of the LF studied bound follicle stimulating hormone (FSH) in the granulosa and human chorionic gonadotropin (hCG) in the theca. Thirty-eight percent also bound hCG in the granulosa. Both binding patterns were found on all days studied. Atretic follicles had lower (P less than .05) FFE concentrations than healthy follicles (r = -.59). The number of LF per cow increased (P less than .05) from day 6 (1.3) to days 12 (1.8) and 18 (2.1). Conversely, their FFE content per cow decreased (P less than .05) from 109 ng/cow on day 6 to 20 ng on; day 12 and 34 ng on day 18. The data suggested the emergency of two LF classes on day 18, one preovulatory, with exceptionally high FFE levels, the other atretic or becoming atretic, with low FFE levels.     

Nuclear volume of cells in the stratum granulosum and theca folliculi interna of the largest dominant and atretic tertiary follicles in sheep during the selection process]

According to available literary data, an increased nuclear volume of endocrine gland cells, various structures of the diencephalon and endocrine ovarial structures could be observed when the respective organs and structures were more active; a decreased nuclear volume was revealed when the activity of the respective organ or its structure was suppressed (Mess, 1962; Maracek and Arendarcik, 1976, 1978). Attention was paid to the karyometric analysis of follicular cells of the stratum granulosum (SG) and secretory cells of the theca folliculi interna (TFI) during selection of the dominant ovulatory follicle (DOF). The aim of this study was to determine differences in hte nuclear volumes of the granulosa follicular cells and the secretory cells of the inner theca of the DO1F in comparison with the largest atretic follicles (AF) as well as to verify the use of karyometric variance analysis for the evaluation of the selection process of the dominant ovulatory tertiary follicle. Histological sections were prepared from large DOF and AF from the ovaries of 18 sheep aged 3-5 years and held under standard conditions of a controlled herd. Control group I (n = 3) consisted of Wallachian sheep on day 15 of the sexual cycle (day 0). Excisions of ovaries from the animals of the experimental group (n = 3) were made on day 16 of the cycle after i.m. treatment with 120 micrograms GnRH 10 and 7 hours prior to sampling. Control group II (n = 3) consisted of Tsigai sheep at the 9th or 10th day of the oestrous cycle (hour 0) and three experimental groups (n = 3 each) consisted of Tsigai sheep 24, 48 and 72 hours after i.m. treatment with 125 micrograms cloprostenol. After excision the ovaries were fixed in neutral formaline, cut into 4 mm thick transverse segments and subjected to standard histological processing. After staining with Harris' HE the 5-7 microns thick sections were karyometrically evaluated at a magnification of 2200 x according to the method of Palkovits (1961) using the PC programme Karyotest 03 (Maracek et al., 1991). 200 cells were evaluated from each sample and both cell types; altogether 7200 cells were examined. GnRH treatment (Dirigestran inj. Spofa) increased the nuclear volume of follicular cells (SG) in the dominant ovulatory follicle in the process of selection (Tab. I, Fig. 1). The former, however, reduced the nuclear volume not only of the SG follicular cells of LAF (Tab. II, Fig. 2) but also of the secretory cells of both the DOF and AF theca interna (Tab. II, Figs. 3, 4). Cloprostenol treatment (Oestrophan inj. Spofa) affected the follicular cells of the granulosa in the dominant ovulatory follicle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4–8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.     

Distribution of immunoreactive von Willebrand factor in the microvascular network of bovine cystic follicles

The present study was undertaken to examine whether the von Willebrand factor (vWF) expression in the follicular microvasculature in the cystic follicles differs from that in the atretic follicles. Paraffin sections of healthy, atretic and cystic follicles were immunostained with rabbit monoclonal antibody to vWF. The vWF-positive cells were counted in four different regions of a follicle from the apical to the basal side. In all types of follicles, immunoreactions for vWF were observed in the endothelial cells of capillaries as well as veins and arteries in the theca interna and externa. In the theca interna, vWF-positive areas were significantly lower in the Type A and B cystic follicles compared to advanced and late atretic follicles. In the theca externa, the vWF-positive blood vessels and vWF-positive area were significantly smaller in all types of cystic follicles than in the healthy or atretic follicles. From these results, it is suggested that in the cystic follicles the induction of vWF in the follicular microvasculature system is reduced, which may suppress the degeneration of vascular system. Continuation of stability in vasculature may be one of the factors that delays the tissue regression in the cystic follicles, and also contributes to the accumulation of follicular fluid that originates from the serum.     

Preliminary evaluation of influence of zearalenone on cocultures of granulosa and internal theca cells of ovarian follicles in bitches in in vitro culture

Zearalenone (ZEA) is an undesirable substance in feed materials and feed of plant origin. It is an example of the micotoxin that causes disturbances in the functioning of the reproductive system. The wide range of plant compounds in pet food means that ZEA may frequently have a negative effect on pet reproduction. An assessment of the influence of ZEA on the granulosa and theca cells of the ovarian follicle in bitches in vitro was carried out. The co-culture of the ovarian follicles was incubated for 72 hours with the addition of 12.5 ng/ml and 25.0 ng/ml of ZEA. Numerous vacuoles in the cytoplasm of the granulosa cells were noted in the culture with the addition of 25.0 ng/ml of ZEA. Preliminary investigations suggest negative effect of ZEA on the granulosa cells in bitches in vitro.     

Breed-associated variations in the sequence of the pig 3beta-hydroxysteroid dehydrogenase gene

The entire sequence of the pig 3beta-hy-droxysteroid dehydrogenase (3beta-HSD) gene has recently become known. This gene is deemed to be important in androstenone metabolism in pig liver, and its defective expression has been shown to be related to androstenone accumulation in adipose tissue and the development of boar taint. The aim of the present work was to do the following: 1) define the structure of the pig 3beta-HSD gene and 2) compare 3beta-HSD DNA sequences from pigs of different breeds, which vary in adipose tissue androstenone levels, with the purpose of identifying a polymorphism that might be responsible for differential 3beta-HSD expression. The 5'flanking and the coding region of 3beta-HSD were cloned and sequenced by conventional techniques. The 3beta-HSD coding regions were identical in pigs of different breeds and in animals with high and low androstenone levels. Significant sequence variations were found in the 5'flanking region of the 3beta-HSD gene, where differences in the number of TTAT repeats and 3 SNP were observed. The SNP were associated with the number of the TTAT repeats. These variations in the DNA sequence of the 3beta-HSD gene were not associated with the androstenone level in s.c. adipose tissue but were breed-dependent. The results of this work might be used for detection of the presence of Meishan genes in Western pig breeds, especially if the phenotype is not clearly established.     

Immunolocalization of 3beta-hydroxysteroid dehydrogenase in normal and hyperplastic ram prostates

The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is essential in the synthesis of all steroids by cleaving dehydroepiandrosterone to androstenedione. In the present study, 3beta-HSD immunoreactivity was investigated in the prostate of Akkaraman breed rams aged older than 3 years. Five normal and five hyperplastic ram prostates were processed for immunohistochemistry. Prostate hyperplasia was determined by histopathological evaluation of 375 ram prostate and confirmed with significantly (P<0.01) increased number of cells expressing proliferating cell nuclear antigen (PCNA) immunoreactivity in the glandular epithelia. The 3beta-HSD immunoreactivity with a variable intensity and pattern of distribution was present in the glandular epithelia and endothelia of blood vessels in normal and hyperplastic ram prostates. While immunoreactivity was focally present in some glands, some sections had a homogenous distribution. The presence of 3beta-HSD immunoreactivity indicates that steroids are locally synthesized in the ram prostate. No differences in the distribution pattern of 3beta-HSD immunoreactivity and the percentage of immunoreactive cells were observed between normal and hyperplastic prostates (P>0.05), suggesting that locally produced steroids have little or no effect on the pathogenesis of the ram prostate hyperplasia which affects a very small proportion of the ram population (5 out of 375).     

Immunohistochemical detection of inhibin-alpha, -betaB, and -betaA chains and 3beta-hydroxysteroid dehydrogenase in canine testicular tumors and normal testes.

Immunohistochemical detection of inhibin-alpha, -betaA and -betaB chains and 3beta-hydroxysteroid dehydrogenase (HSD) was carried out on primary testicular tumors from 15 dogs and normal testes from three adult dogs. Histopathologically, the tumors were composed of three types: Leydig cell tumors in five dogs, Sertoli cell tumors in five dogs, and seminoma in five dogs. In normal testes, immunostaining against inhibin-alpha, -betaA, and -betaB chains and 3beta-HSD revealed positive reactivity in the cytoplasm of Leydig cells. In testicular tumors, immunoreactive cells against inhibin-alpha, -betaA, and -betaB chains and 3beta-HSD were localized in all Leydig cell tumors but not in any Sertoli cell tumors or seminomas. The results of radioimmunoassay for plasma inhibin in dogs with Leydig cell tumors showed higher concentrations than those in dogs with Sertoli cell tumors and seminomas and those in normal dogs. The concentration of inhibin in the plasma was markedly decreased by the surgical removal of the Leydig cell tumor in one dog. Our findings suggest that inhibin is synthesized by normal and neoplastic Leydig cells in the canine testis, and the secreted inhibin may be inhibin A and inhibin B.     

Immunohistochemical localization of fibroblast growth factor 18 in hair follicles of healthy beagle dogs

Increasing emphasis is being placed on the role of fibroblast growth factors (FGFs) in hair follicle cycling. In mice, expression of FGF18 mRNA peaks during the late telogen phase, leading to the hypothesis that FGF plays a role in anagen induction. There are no data on the presence of FGF18 in dogs. The main objective of this study was to identify and locate FGF18 in the canine hair follicle. The second objective was to assess potential differences in FGF18 concentration between biopsies taken in winter and summer, shoulder and flank regions, and between different sexes. Skin tissue from 10 healthy beagle dogs (three intact females, three spayed females and four intact males) was collected from the shoulder and flank. The biopsies were collected in February and August on day 0, after which the dogs were clipped and biopsies collected again from the shoulder and flank on days 1, 3, 7 and 17. Paraffin sections (4 μm thick) of the biopsies were stained with an anti-FGF18 antibody. The FGF18-positive cells were counted in the hair follicle epithelium from seven follicular units of each biopsy. Fibroblast growth factor 18 was detected as granular cytoplasmatic staining in follicles at the level of the inner root sheath, and rarely in the outer root sheath and dermal papilla. It was also detected in the apocrine glands, in arrector pili muscles and in vascular endothelial cells. There was no statistical difference in the number of FGF18-positive cells or follicles between sexes, different anatomical locations, seasons or the consecutive days of sampling.     

An immunohistochemical study of bovine antral follicles, with special attention to non-atretic follicles with and without atypical granulosa cells.

Inhibin and oxytocin were immunohistochemically demonstrated in all non-atretic and light-atretic follicles > 2 mm from untreated and pregnant mare's serum gonadotrophin (PMSG)-treated heifers and cows. Immunostaining for luteinizing hormone (LH) and oestradiol was observed in all non-atretic follicles > 4 mm, but only in follicles from PMSG-treated cows. Inhibin and oestradiol immunoreactivity was restricted to the granulosa. Oxytocin and LH immunoreactivity was visualized in both the theca interna and the granulosa. Within the granulosa, LH immunoreactivity was mainly present in cells that were located near the basement membrane. Normal granulosa cells differed from atypical granulosa cells (AGCs) with respect to their ability to bind LH and oestradiol. It is concluded that immunostaining for alpha-inhibin, oxytocin, oestradiol and LH cannot be used as a marker of follicle quality to discriminate between non-atretic follicles with AGCs and non-atretic follicles without AGCs in mid-luteal bovine ovaries.     

Localization of inhibin alpha-, betaA- and betaB-subunits and aromatase in ovarian follicles with granulosa theca cell tumor (GTCT) in 6 mares

To clarify the morphological and immunohistochemical characteristics in mares with granulosa theca cell tumor (GTCT), the localization of inhibin subunits (alpha, betaA, betaB) and aromatase in the granulosa cell layers and theca layers in the ovarian follicles were determined by immunohistochemical staining. The follicles were obtained from the ovaries of 6 mares with GTCT and 4 normal mares as controls. Immunohistochemically, inhibin alpha-subunit was localized in the granulosa cells of all follicles showing different sizes in all GTCT cases and betaA- subunit was localized in two GTCT cases in all sized follicles. But inhibin betaB- subunit and aromatase were not localized in GTCT cases. On the other hand, inhibin alpha-, betaA-, and betaB-subunits and aromatase were localized in the large and medium sized follicles, but inhibin betaA- and betaB-subunits and aromatase were not stained in the small sized follicles in normal cases. These findings suggest that some mares with GTCT can secrete dimeric inhibin (inhibin A), but all GTCT cases cannot secrete inhibin B. By the results of aromatase staining it is clear that testosterone is not converted into estradiol due to the lack of aromatase in the GTCT follicles.