Tissue sources of sex steroids in rat ovaries during the preovulatory period

The method of quantitative histoenzymological analysis was used to determine the extent of participation of various structures of the ovary in the provision of the preovulatory synthesis of sex hormones. The activity of steroid dehydrogenases (3beta, 17beta, and 20alpha-OH), glucoso-6phosphric dehydrogenases, NAD and NADP-diaphorases was investigated. The synthesis of sex hormones proved to be realized by the mobilization of all the ovarian structures. At the early proestrus enhanced estrogen synthesis was provided by mature follicles, interstitial glands and the old corpora lutea. In the mid proestrus the active sources of progesterone and 20alpha-hydroxypregh-4en-3on synthesis are young corpora lutea and follicles; at this time the interstitial glands and old corpora lutea synthesized mainly the progesterone derivative.     

Cellular junctions in perifollicular contractile tissue of the rat ovary during the preovulatory period

Summary Rat ovarian perifollicular contractile tissue was examined at specified intervals prior to ovulation to determine the type, relative number, and length of cellular junctions. Rat ovaries were taken for electron-microscopic observation at 1500 h on the afternoon of proestrus (proestrus 0-h group), at 2000 h (proestrus 5 h group), at 0100 h (proestrus 10-h group) and at 1600 h on the afternoon of diestrus I. Close junctions, intermediate junctions, and gap junctions were counted and measured. The number of gap junctions 1,000 m of membrane and the number of intermediate junctions 1,000 m of membrane was significantly higher in the proestrus 10 h group as compared to the other groups. There was no difference in the number of close junctions during the periods studied. Also the length of all junctions was similar in all groups. These morphological findings are discussed in the context of a contractile role for perifollicular tissue in the ovulatory process.     

Preovulatory changes in the perifollicular capillary network in the rat: role of eicosanoids.

The aim of this study was to evaluate morphometrically the influence of ovulation-inhibiting doses of indomethacin, an inhibitor of the cyclooxygenase pathway, and esculetin and caffeic acid, inhibitors of the lipoxygenase pathway, on the dilatation of the perifollicular capillary network in the theca interna. The development of the perifollicular capillary network as a function of follicular size and the changes in the vascular lumen were examined by light microscopy on a series of semithin cross sections of rat ovaries. The number of capillaries in the theca interna increased linearly with increasing follicle diameter. Thus, the relative number of capillaries in the theca interna supplying the avascular stratum granulosum remained constant. This indicates that follicular function is not regulated through changes in the number of capillaries in the theca interna. After hCG injection, an increase in the capillary area could be observed in follicles having a diameter of more than 600 microns. Indomethacin administration increased the capillary area of the ovulatory follicles as compared to the untreated side only at 6 h after treatment. By contrast, treatment with inhibitors of lipoxygenase resulted in a significant decrease in the capillary area of large follicles at all times examined (3, 6, and 9 h after hCG injection). Nevertheless, since both types of eicosanoid inhibitors suppressed follicle rupture, in spite of their opposing actions on the capillary area, it seems unlikely that their action on ovulation is primarily due to their effect on this parameter.     

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

Sequential changes associated with the degeneration of preovulatory rat follicles.

In 26-day-old rats, follicles capable of ovulation were present 48 h after PMSG injection and they degenerated if not exposed to an ovulating dose of HCG. In such follicles 125I-labelled LH bound to the thecal and granulosa cells. By 60 h after PMSG, LH binding to the granulosa cells was reduced by 46% although these follicles retained their ability to ovulate. LH binding to the granulosa cells was lost in most follicles by 72 h and ovulation could not be induced. The thecal cells still possessed LH binding sites at 72 h after PMSG. HCG stimulation of these follicles resulted in disruption of the granulosa and the invasion of blood cells into the antrum.     

Consequences of increasing or decreasing plasma FSH concentrations during the preovulatory period in Romney ewes

Romney ewes were infused with ovine FSH (NIADDK-oFSH-16) for 48 h from the initiation of luteolysis with cloprostenol. Doses of 2.5 or 5 micrograms/h which partly or completely prevented the normal preovulatory decline in plasma FSH concentrations caused a significant increase in mean ovulation rates. Ovulation rates were not increased significantly if the FSH (5 micrograms/h) was infused for only 20 h starting from the initiation of luteolysis or 24 h later. Infusion of a less potent and relatively impure preparation of FSH (i.e. FSH-P) at 0.5 mg/h for 48 h after cloprostenol treatment also increased the mean ovulation rate significantly. However, if the FSH-P was given for only the first 24 h, or if the start of the infusion was delayed for more than 12 h, mean ovulation rates were not increased significantly. Infusion of LH (NIADDK-oLH-25, 5 micrograms/h) for 48 h from the initiation of luteolysis decreased the mean ovulation rate significantly. Administration of bovine follicular fluid to suppress plasma FSH concentrations below normal during the first 24 h after cloprostenol injection did not delay oestrus. However, oestrus was delayed by approximately 2 days if plasma FSH concentrations were reduced by bovine follicular fluid 24 h after the initiation of luteolysis. As ovulation rate increased, the mean weight of individual corpora lutea of each ewe decreased. In ewes with a single ovulation, most corpora lutea weighed greater than 600 mg, but as the ovulation rate increased the proportion of corpora lutea present weighing less than 400 mg rose steadily.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

Background  

Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.     

Localization of alpha-tubulin in the rat cumulus oophorus before and during preovulatory expansion

Distribution of alpha-tubulin was investigated in the cumulus oophorus complexes (COCs) of the rat using anti-alpha-tubulin monoclonal antibody and fluorescence microscopy. Localization of alpha-tubulin was assessed in the compact and fully expanded COCs isolated directly from the preovulatory follicles and in the cryosections containing these structures. In the cryosections containing COCs, the submembranous distribution of alpha-tubulin marked the characteristic cell shapes and showed the lack of cell projections. In the freshly isolated COCs three-dimensional cellular arrangement was visible showing that the dispersed cells formed almost no projections and were not interconnected. The use of a higher magnification revealed a dense microtubular network radiating from centrosomes and extending to the plasma membrane. This network showed discontinuous distribution of submembranous alpha-tubulin in some of these cells. The latter change could be attributed to the increased progesterone production.     

Luteinization and proteolysis in ovarian follicles of Meishan and Large White gilts during the preovulatory period

This experiment was conducted to determine why follicles luteinize faster in the Meishan breed than in the Large White breed of pig. Follicles were recovered during the late follicular phase from ovaries of both breeds before and after administration of hCG given to mimic the LH surge. First, the patterns of cholesterol transporters (high and low density lipoproteins: HDL and LDL) were compared. Cholesterol transporters detected in follicular fluid consisted of HDL only. Similar amounts of Apolipoprotein A-I were found in all samples. There was no obvious breed effect on minor lipoproteins found in the HDL-rich fraction, and this pattern was altered similarly by hCG in the two breeds. The LDL-rich samples of serum from both breeds contained similar amounts of protein. Second, three steroidogenic enzymes, adrenodoxin, 17 alpha-hydroxylase-lyase (P450(17) alpha) and 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD) were detected by immunohistochemistry and quantified by image analysis on sections of the two largest follicles. Before hCG treatment, theca interna cells demonstrated immunoreactivities for adrenodoxin (strong), P450(17) alpha and 3 beta-HSD (very strong), whereas granulosa cells displayed immunoreactivities for adrenodoxin only. After hCG treatment, the localization of the enzymes was unchanged but the staining intensity of adrenodoxin on granulosa cells and 3 beta-HSD on theca cells increased (P < 0.01 and P < 0.05, respectively). Breed effects were detected for the amounts of adrenodoxin in theca cells (Meishan > Large White; P < 0.05) and of 17 alpha-hydroxylase (Large White > Meishan, P < 0.01). Breed x treatment interactions were never detected. Finally, gelatinases, plasminogen activator, plasminogen activator inhibitor, tissue inhibitors of metalloproteases (TIMP-1 and TIMP-2) were visualized by direct or reverse zymography or western blotting. Whatever the stage relative to LH administration, follicular fluid from Large White gilts contained more TIMP-1, and TIMP-2 (P < 0.02 and P < 0.01, respectively). No breed effect was detected for the amounts of gelatinases and plasminogen activator inhibitor 1. However, for these parameters, a significant breed x time interaction was obvious, as the Meishan follicles had a greater response to hCG (P < 0.01). Since proteolysis plays a key role in the bioavailability of growth factors such as insulin-like growth factor 1, fibroblast growth factor and transforming growth factor beta, which have the ability to alter gonadotrophin-induced progesterone production in pigs, the differences observed in its control in the present study may explain, at least in part, the different patterns of luteinization observed in Meishan and Large White follicles.     

Body temperature changes in sows during the periparturient period

Deep body temperatures were monitored with an implanted radiotelemetry device from 2 to 4 days prepartum to approximately 12 days postpartum in sows. The mean prepartum temperature of five normal sows was 38.6 degrees C, with a range of 38.3--38.9 degrees C. An increase in body temperature (1.4 degrees C) was associated with parturition. The increase in body temperature began about 12 hours before the first pig was born, peaked 1-2 hours after delivery of the last pig, and decreased during the first day postpartum but did not reach the prepartum values. Two days postpartum, the mean body temperature was about 1 degree C higher than it was during the prepartum period. The mean body temperature increased an average of 0.06 degrees C per day during the 12-day postpartum period. A diurnal pattern was evident in the body temperature variations.     

Modulatory role of purines in neuromuscular transmission

The data on purine modulation of myoneural transmission are reviewed. A particular attention is paid to adenosine-5′-triphosphoric acid (ATP), the co-transmitter of the principal mediator (acetylcholine), and adenosine, the final ATP metabolite in the synaptic cleft. The effects of these endogenous modulators on pre- and postsynaptic current are discussed. The contributions of purines to the process of quantal and non-quantal acetylcholine release into the synaptic cleft and the effects of ATP and adenosine on cholinoceptor function have been assessed. It is concluded that the role of endogenous purines is mainly confined to enhancement of the efficiency of neuromuscular transmission and synaptic adjustment of a motor unit at different modes of function.     

Ammonia metabolism during the suckling period in the rat

1. The time-courses of blood glutamine, glutamate, alanine, ammonia, urea and allantoin concentrations during the first 15 days of extrauterine life were studied. 2. Glutamine and glutamate concentrations followed the same pattern and correlated positively, suggesting that both amino acids are utilized or released synchronously. 3. Alanine concentrations decreased during the first 3 days, reaching levels close to those of adults which persisted up to the end of the observation period. 4. A highly significant correlation was found between ammonia and urea concentrations, suggesting that during the suckling period urea synthesis may be limited by blood ammonia availability. 5. The time-course of allantoin concentrations suggests that the synthesis of purines was enhanced during the first day, decreasing sharply during the 2nd to steeply increase to the end of the suckling period.     

Expression of vascular endothelial growth factor receptors and their ligands in rat uterus during the postpartum involution period

Abstract

Vascular endothelial growth factor (VEGF) and its specific receptors, FLt1/fms, Flk1/KDR and FLt4, play important roles in vasculogenesis, and physiological and pathological angiogenesis. Whether angiogenic growth factors are involved in regulating angiogenic processes during the postpartum involution period (PP) of the rat uterus is unknown. We used immunohistochemistry to analyze the expression levels of VEGF, the fms-like tyrosine kinase 1 (FLt1/fms), the kinase insert domain-containing region 1 (Flk1/KDR), Fms-related tyrosine kinase 4 (FLt4) and vascular endothelial growth inhibitor (VEGI) in the rat uterus during the days 1, 3, 5, 10 and 15 of the PP to determine the temporal and spatial expressions of VEGF and its receptors during the PP. Throughout the PP, cytoplasmic and membrane staining of VEGI, VEGF and their receptors were observed in the lumens, crypts and glandular epithelial cells as well as in connective tissue and vascular endothelial and smooth muscle cells in the endometrium. We found that the intensity of the immunoreactions in the endometrium varied with the morphological changes that occurred during involution. Immunoreactions for VEGI, VEGF and their receptor, Flk1/KDR, in the luminal epithelial cells were stronger than those in the glandular epithelial and stromal cells, particularly during PP 1, 3 and 5, which suggests that these peptides may contribute to re-epithelialization of the endometrium. On the other hand, Flt1/fms immunoreactivity was strong mainly in the stromal cells during the PP. The presence of VEGF and its receptors (FLt1/fms, Flk1/KDR, FLt4) in the stromal cells and blood vessels during the PP suggests that they may contribute to regulating stromal repair and angiogenesis in the involuting uterus of the rat.     

The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles

Preovulatory follicles isolated from immature rats, treated with pregnant mare's serum gonadotropin, were incubated and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 μg/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 μM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinizing hormone stimulation of prostaglandin accumulation in rat follicles.