Histochemical changes in adenosine triphosphatase activity during folliculogenesis and corpus luteum formation and regression in the rat ovary.

Histoenzymological changes in Adenosine triphosphatase (ATPase) activity were studied during folliculogenesis in immature and mature rat ovary. Its presence in oocytes of small follicles and absence in those of large follicles postulate a correlation between their absorptive mechanism during the development of the oocyte. The presence of ATPase activity in the theca, corpora lutea and interstitial gland tissue may be related to the vascular endothelium which is associated with the transport system across the membrane.     

Opposite regulation of clusterin and LH receptor in the swine corpus luteum during luteolysis

Luteolysis, which occurs in a cyclical way to remove luteal tissue, may be an example of physiological apoptosis which counterbalances rapid tissue growth after ovulation. Clusterin is a multifunctional glycoprotein expressed in different tissues undergoing apoptosis. In this study we investigated clusterin and LH receptor gene expression during luteolysis as potential regulators of tissue growth and regression. Luteolysis was induced in pregnant sows (45 days) by Cloprostenol (PGF2 alpha analogue) treatment. Clusterin expression increased in the corpora lutea of pregnant sows ovariectomized 0, 6, 12, 24, 48 or 72 (n = 3) h after the luteolytic stimulus; maximum values were observed 24-48 h after the treatment (P < 0.01). An opposite trend between clusterin mRNA expression and markers of luteal function, such as progesterone levels in the corpora lutea and plasma, and LHr mRNA expression levels, was observed; moreover, clusterin expression was positively correlated with the degree of genomic DNA fragmentation, a marker of occurring apoptosis (P < 0.01). This pattern may be important in regulating luteolysis by a switch between luteotrophic and apoptotic stimulus. Our data indicate that P4 levels decrease prior to the increase in clusterin mRNA and the drop in LHr mRNA expression; we may therefore hypothesize a split between functional and structural luteolysis as reported in other species.     

Basigin expression and regulation in mouse ovary during the sexual maturation and development of corpus luteum

Basigin is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. Basigin-deficient male mice are azoospermic. The majority of basigin null embryos die around the time of implantation. However, basigin expression and regulation in mouse ovary is still unknown. The aim of this study was to investigate basigin expression in mouse ovary during sexual maturation, gonadotropin treatment, and luteal development by in situ hybridization and immunohistochemistry. Both basigin mRNA and immunostaining were not detected in the granulosa cells of preantral follicles until day 20 after birth. On day 30 after birth, basigin immunostaining dropped to a basal level, while basigin mRNA was still at a high level. Basigin expression was strongly induced by equine chorionic gonadotropin (eCG) treatment at 4 and 8 hr post-eCG injection. Both basigin immunostaining and mRNA signals were strongly observed in the corpus luteum on days 2 and 3 post-hCG injection. However, no basigin expression was detected from days 6 to 15 post-hCG injection. In conclusion, our data suggest that basigin may play a role during the mouse follicle development and corpus luteum formation.     

Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation

Notch signaling functions to regulate cell-fate decisions by modulating differentiation, proliferation, and survival of cells. Notch receptors and ligands are expressed in embryonic vasculature and are required for the remodeling of the primary embryonic vasculature of mice. Here, we characterize the expression patterns of Notch1, Notch4, and Jagged1 proteins during the process of folliculogenesis and corpus luteum formation in the mouse ovary, an organ with dynamic physiological angiogenic growth. These Notch proteins and ligand are expressed in a subset of ovarian vessels, including both mature ovarian vasculature as well as angiogenic neovessels. Their expression in the ovary was found in both endothelial and vascular associated mural cells. Our data suggest a complex regulatory role for the Notch signaling pathway during mouse oogenesis and ovarian neovascularization.     

Changes in the distribution of tenascin during tooth development

Tenascin is an extracellular matrix molecule that was earlier shown to be enriched in embryonic mesenchyme surrounding the budding epithelium in various organs including the tooth. In the present study tenascin was localized by immunohistology throughout the course of tooth development in the mouse and rat using polyclonal antibodies against chick tenascin. The results indicate that tenascin is expressed by the lineage of dental mesenchymal cells throughout tooth ontogeny. The intensity of staining with tenascin antibodies in the dental papilla mesenchyme was temporarily reduced at cap stage when the tooth grows rapidly and undergoes extensive morphogenetic changes. During the bell stage of morphogenesis, the staining intensity increased and tenascin was accumulated in the dental pulp even after completion of crown development and eruption. Tenascin was present in the dental basement membrane at the time of odontoblast differentiation. The dental papilla cells ceased to express tenascin upon differentiation into odontoblasts and tenascin was completely absent from dentin. It can be speculated that the remarkable expression of tenascin in the dental mesenchymal cells as compared to other connective tissues is associated with their capacity to differentiate into hard-tissue-forming cells.     

Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

The relationship of oxygen free radicals to corpus luteum function in rabbits was explored during various stages of pseudopregnancy, including natural and induced luteal regression. Induced luteolysis was achieved during mid-pseudopregnancy by removal of an oestradiol capsule placed at the onset of pseudopregnancy, which suppressed ovarian oestradiol production. Activity of manganese superoxide dismutase (Mn SOD) was significantly and positively correlated with ovarian progesterone production (P < 0.01) throughout pseudopregnancy and during natural regression. Oestradiol deprivation for 12, 24 or 72 h resulted in declines in Mn SOD activity and progesterone secretion, although Mn SOD rose and corpus luteum steroidogenesis was restored to normal when the capsule was replaced for 48 h before assessment, having been removed for 24 h. Lipid peroxide and progesterone concentrations were not correlated, although a significant rise in lipid peroxides in the luteal tissue was detected after deprivation of oestradiol for 72 h. Changes in progesterone production and Mn SOD activity were not associated with alterations in concentration of prostaglandin F metabolite. These data suggest that Mn SOD may be involved in regulating function of the corpus luteum during pseudopregnancy in rabbits and that oxygen free radicals may play a role in regression of corpus luteum in this species.     

Localization of relaxin during formation of the porcine corpus luteum

The presence and localization of relaxin (RLX) in luteal tissue during the estrous cycle of the pig have been studied using the avidin-biotin immunoperoxidase method and homologous antisera to purified RLX. Prepubertal gilts were induced to ovulate by treatment with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ovaries were obtained at laparotomy during the periovulatory period and at specified times through Day 19 post-ovulation. Emphasis was placed on obtaining ovarian tissue at 12- and 24-h intervals up to 96 h after ovulation. RLX immunostaining was evident in theca interna (TI) cells before and at 6 h after ovulation. At 18 h after ovulation, RLX immunostaining comparable to that seen in TI cells was observed for the first time in luteinizing granulosa (G) cells. As luteinization progressed, it became difficult to identify the origin of the RLX immunostaining cells. However, the intensity of RLX immunostaining increased with corpus luteum (CL) development, with the staining becoming localized in the large luteal cells. By Day 19 after ovulation, RLX immunostaining was undetectable. These results indicate RLX is present in the CL during its formation and functional lifespan. Also, it would appear that the presence of RLX in G cells post-ovulation is associated with cell luteinization.     

Regulatory changes of apoptotic factors in the bovine corpus luteum after induced luteolysis

The corpus luteum (CL) offers the opportunity to study not only proliferative, but also regressive processes. During luteolysis of the CL a sudden death of luteal and endothelial cells seems to be involved (apoptosis). The aim of this study was to examen the mRNA expression of factors known to be involved in apoptotic processes: monocyte chemoattractant protein-1 (MCP-1), factors of the extrinsic and intrinsic apoptotic pathways, caspase3, -6, -7 and interferone gamma (IFNgamma). Luteolysis was induced by injection of 500 microg Cloprostenol during mid-luteal phase. The CLs were collected at 0.5, 2, 4, 12, 24, 48, and 64 hr after PGF2alpha-injection. Control CLs (Days 8-12) were collected at the slaugtherhouse. Real-time RT-PCR determined the mRNA expressions. Western blot analysis of poly(ADP-ribose) polymerase (PARP-1) and IFNgamma as well as protein measurement of tumor necrosis factor alpha (TNFalpha) by EIA were performed. The mRNA levels of MCP-1, IFNgamma and most factors of the extrinsic pathway were significantly increased between 0.5 and 2 hr. The factors of the intrinsic pathway were mostly later up-regulated at 24-48 hr after PGF2alpha. Caspase6 and 3 revealed a significant increase from 2 and 12 hr, respectively, whereas caspase7 was significantly up-regulated after 24 hr. The protein level of TNFalpha increased significantly to a maximum level at 12 hr. The Western blot revealed an increasing level of an 89 kDa fragment of PARP-1 from 12 to 24 hr, which is specific for apoptosis. We assume that the extrinsic pathway is more important for the onset of luteolysis, because of its earlier and higher increase during induced luteolysis.     

Electron microscope immunocytochemical localization of cyclooxygenase-1 and -2 in pseudopregnant rat corpus luteum during luteolysis

Prostaglandins converted from arachidonic acid by cyclooxygenases play an important regulatory role in regression of the corpus luteum. To reveal luteal distribution of cyclooxygenase isoforms during luteolysis, an electron microscope immunocytochemical study was performed. Cyclooxygenase-1 and -2 were found both in luteal steroid-producing and interstitial cells on days 13, 15 and 18 of the adult pseudopregnant rat. Cyclooxygenase-2 immunolabelling was predominantly seen in non-luteal cells. The two enzymes were localized in similar fashion to the plasma membrane, rough and smooth endoplasmic reticulum, lipid bodies and mitochondria, but differently in the nuclear compartment. Cyclooxygenase-1 labelling was found only in the perinuclear region, while cyclooxygenase-2 was localized to the nuclear envelope, region of condensed heterochromatin as well as at the perimeter of the heterochromatin. Nuclear residence may indicate additional roles for cyclooxygenase-2 in regulating gene expression. Identification of both enzymes on lipid bodies suggests that these inclusions may be involved in luteal prostanoid production.     

Expression of major histocompatibility complex antigens on the bovine corpus luteum during the estrous cycle, luteolysis, and early pregnancy.

Treatment of cultured bovine luteal cells with the cytokine, interferon-gamma, induces the expression of Class II major histocompatibility complex antigens (MHC Ags). To determine if Class II MHC Ags are present on the CL in vivo and if the degree of Ag expression changes during luteal life span, bovine corpora lutea were obtained on Day 6, Days 10-12, and Day 18 of the estrous cycle and MHC Ag expression was evaluated via indirect immunofluorescence. Flow cytometry was used to determine the percentage of MHC Ag-positive cells on cell populations distinguished by cell size and intracellular density. Minimal Class II MHC Ag expression was detected on Day 6 CL (approximately 25%), which consisted primarily of smaller cells. The midcycle and late CL consisted of these small cells (SC) and two populations of large cells that differed in intracellular density, or right-angle light scatter. In midcycle CL, few (less than 25%) SC or large, dense cells (LDC) expressed the Class II MHC Ag whereas a high percentage (75%) of the large, less-dense cells (LLDC) were Class II MHC Ag-positive. Class II MHC Ag expression remained negligible on the LDC of the Day 18 CL; however, there was an elevation in the percentage of SC and LLDC expressing Class II Ag (p less than 0.05). To determine if Class II MHC Ag expression also varied with different functional states of the CL, bovine CL were collected after prostaglandin (PG) F2 alpha-induced regression and on Day 18 of early pregnancy. When luteolysis was allowed to progress in vivo, the percentage of Class II MHC Ag-positive cells was increased in all cell populations (p less than 0.05). Class II MHC Ag expression was significantly lower (p less than 0.05) on the three cell populations comprising the CL of pregnancy as compared to the Day 18 cyclic CL. It is hypothesized that enhanced expression of Class II MHC Ags on the late CL and during PGF2 alpha-induced regression may potentiate immune response mechanisms for luteolysis.     

Effect of dinoprost and cloprostenol on serum nitric oxide and corpus luteum blood flow during luteolysis in ewes

In this study we compared the effect of dinoprost and cloprostenol on changes of corpus luteum blood flow during luteolysis. Ten nonlactating cyclic ewes were synchronized with double PGF_2α injections 11 days apart. At Day 10, the animals were classified into 2 groups and received the third dose of PGF_2α after confirmation of the presence of a mature CL. The first group received (12.5 mg/im) dinoprost and the second group received (250 μg/im) cloprostenol. A color Doppler ultrasound scan was performed by the same operator according to the following timeline: 0, 0.5, 1, 2, 4, 6, 12, and 24 hours, then every 24 hours until Day 4). The size, morphology, and blood flow of the CL was evaluated during the regression. The results showed that regression of the CL did not differ between the dinoprost and cloprostenol groups. There was no significant effect on diameter of the CL in both groups, though the size of the CL decreased gradually and slowly. Pretreatment progesterone concentration did not differ between groups. The results showed that the nitric oxide level was significantly increased within half an hour after the dinoprost treatment, and was significantly decreased in the cloprostenol group after half an hour. The blood velocity was increased significantly half an hour after the dinoprost treatment and it was decreased in the cloprostenol-treated group. In conclusion, both cloprostenol and dinoprost affect CL by controlling the nitric oxide level and blood supply of the CL via different mechanisms to induce luteolysis.     

Effects of oxytocin on cloprostenol-induced luteolysis, follicular growth, ovulation and corpus luteum function in heifers

Twenty-five normally cyclic Holstein heifers were used to examine the effects of oxytocin on cloprostenol-induced luteolysis, subsequent ovulation, and early luteal and follicular development. The heifers were randomly assigned to 1 of 4 treatments: Group SC-SC (n=6), Group SC-OT (n=6), Group OT-SC (n=6) and Group OT-OT (n=7). The SC-SC and SC-OT groups received continuous saline infusion, while Groups OT-SC and OT-OT received continuous oxytocin infusion (1:9 mg/d) on Days 14 to 26 after estrus. All animals received 500 microg, i.m. cloprostenol 2 d after initiation of infusion (Day 16) to induce luteolysis. Groups SC-OT and OT-OT received oxytocin twice daily (12 h apart) (0.33 USP units/kg body weight, s.c.) on Days 3 to 6 of the estrous cycle following cloprostenol-induced luteolysis, while Groups SC-SC and OT-SC received an equivalent volume of saline. Daily plasma progesterone (P4) concentrations prior to cloprostenol-induced luteolysis and rates of decline in P4 following the induced luteolysis did not differ between oxytocin-infused (OT-OT and OT-SC) and saline-infused (SC-SC and SC-OT) groups (P >0.1). Duration of the estrous cycle was shortened in saline-infused heifers receiving oxytocin daily during the first week of the estrous cycle. In contrast, oxytocin injections did not result in premature inhibition of luteal function and return to estrus in heifers that received oxytocin infusion (OT-OT). Day of ovulation, size of ovulating follicle and time of peak LH after cloprostenol administration for oxytocin and saline-treated control heifers did not differ (P >0.1). During the first 3 d of the estrous cycle following luteal regression, fewer (P <0.01) follicles of all classes were observed in the oxytocin-infused animals. Day of emergence of the first follicular wave in heifers treated with oxytocin was delayed (P <0.05). The results show that continuous infusion of oxytocin during the mid-luteal stage of the estrous cycle has no effect on cloprostenol-induced luteal regression, timing of preovulatory LH peak or ovulation. Further, the finding support that an episodic rather than continuous administration of oxytocin during the first week of the estrous cycle results in premature loss of luteal function. The data suggest minor inhibitory effects of oxytocin on follicular growth during the first 3 d of the estrous cycle following cloprostenol-induced luteolysis.     

Changes in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

E-cadherin-catenin complex in the rat ovary: cell-specific expression during folliculogenesis and luteal formation

The cadherins and their cytoplasmic counterparts, the catenins, form the adherens junctions, which are of importance for tissue integrity and barrier functions. The development and maturation of the ovarian follicle is characterized by structural changes, which require altered expression or function of the components involved in cell-cell contacts. The present study examined the cell-specific localization and temporal expression of epithelial cadherin (E-cadherin) and alpha- and beta-catenin during follicular development, ovulation and corpus luteum formation in the immature gonadotrophin- and oestrogen-stimulated rat ovary. Immunohistochemistry and immunoblotting demonstrated the expression of E-cadherin in theca and interstitial cells of immature ovaries before and after injection of equine chorionic gonadotrophin (eCG). E-cadherin was not detected in granulosa cells, except in the preantral follicles located to the inner region of the ovary. The content of E-cadherin in theca and interstitial cells decreased after an ovulatory dose of hCG. Granulosa cells of apoptotic follicles did not express E-cadherin. Oestrogen treatment (diethylstilboestrol) of immature rats for up to 3 days did not result in a measurable expression of E-cadherin in granulosa cells. alpha- and beta-catenin were expressed in all ovarian compartments. The concentration of beta-catenin was constant during the follicular phase, whereas the content of alpha-catenin decreased in granulosa cells after treatment with diethylstilboestrol or hCG. The expression of alpha-catenin was also reduced in theca and interstitial cells after hCG. alpha- and beta-catenin were present in most ovarian cells at all stages of folliculogenesis. Therefore, the catenins have the potential to associate with different members of the cadherin family and to participate in the regulation of cytoskeletal structures and intracellular signalling. The restricted expression of E-cadherin in granulosa cells of preantral follicles indicates a role in the recruitment of these follicles to subsequent cycles. The specific decrease of alpha-catenin in granulosa cells and the reduction of both alpha-catenin and E-cadherin in theca cells of ovulatory follicles might reflect some of the molecular changes in cell-cell adhesion associated with ovulation and luteinization.     

Unaltered distribution of laminins, fibronectin, and tenascin in celiac intestinal mucosa.

Extensive remodeling of the extracellular matrix (ECM) occurs in inflammatory tissues. The celiac lesion in the small intestine is characterized by inflammation accompanied by profound morphological alterations. We used immunohistochemistry to determine the distribution of laminin, fibronectin, and tenascin isoforms in small intestinal biopsies of untreated patients with celiac disease. In normal mucosa, the distribution of laminin isoforms defines three epithelial basement membrane (BM) zones. We found that the organization of these zones was maintained in the celiac mucosa. Thus, components of laminin-5 (alpha3 and beta3) were found in the surface epithelial BM, laminin alpha2 chain was found selectively at crypt bottoms, and laminin alpha5 chain was the sole alpha-type chain in middle crypt BMs. Likewise, the distribution of fibronectin and tenascin resembled that of the normal gut. The organization of pericryptal fibroblasts and lamina propria smooth muscle strands, as defined by immunostaining for alpha-smooth muscle actin, also remained unchanged in the celiac mucosa. Unexpectedly, major ECM changes were not detected in the celiac lesion.