Enzyme histochemistry of the sheep uterus during the oestrous cycle

Enzyme histochemical techniques were applied to frozen sheep uteri from different stages of the oestrous cycle. The localization and activities of succinate, lactate, glucose-6-phosphate, and isocitrate (NADP+) dehydrogenases and acid and alkaline phosphatases were studied in the luminal and glandular epithelia, caruncle and myometrium. Enzyme activity in the sections was scored on a scale of 0--5. In general the enzyme activity in the uterine caruncles and epithelia was higher than in the myometrium. The myometrium did not show any alkaline phosphatase activity and isocitrate dehydrogenase (NADP+) activity was negligible. The low activities of acid phosphatase and lactate dehydrogenase and the moderate levels of glucose-6-phosphate and succinate dehydrogenases in the myometrium were constant. The caruncular tissue showed high levels of phosphatases and glucose-6-phosphate dehydrogenase, moderate levels of lactate and succinate dehydrogenases, and low levels of isocitrate dehydrogenase (NADP+) throughout the oestrous cycle. Much lower phosphatase and isocitrate dehydrogenase (NADP+) levels were found in the epithelium of deep glands compared with superficial glands. The high activity of acid and alkaline phosphatases in the luminal epithelium and the superficial glands was constant from mid-cycle to ovulation, but a significant decrease was observed immediately after ovulation. The level of dehydrogenases in epithelia was generally high and did not change during the oestrous cycle.     

Further studies on prostaglandin and thromboxane production by the rat uterus during the oestrous cycle

Prostaglandin (PG) and thromboxane (TX) synthesis by uterine homogenates was measured at 4-h intervals during the 4-day oestrous cycle of rats. Production was in the order of 6-oxo-PGF-1 alpha (which reflects PGI-2 synthesis) greater than PGF-2 alpha greater than TXB-2 (which reflects TXA-2 synthesis) greater than or equal to PGE-2. Peak production occurred at 02:00 h on the day of oestrus, after which production gradually decreased, with some fluctuation on the day of metoestrus, to reach a minimum between 22:00 and 06:00 h on the days of dioestrus and oestrus, respectively. Separation of the uterine tissues showed that, on a unit weight basis, the endometrium had a much higher PG and TX synthesizing ability than did the myometrium, although this was compensated for on a total weight basis by the much greater mass of myometrium. Endometrial PG and TX production was in the order of PGF-2 alpha greater than TXB-2 greater than or equal to 6-oxo-PGD-1 alpha identical to PGE-2, with PGF-2 alpha and TXB-2 productions showing the greatest increases between 10:00 and 02:00 h on the days of pro-oestrus and oestrus, respectively. Myometrial PG and TX production was in the order of 6-oxo-PGF-1 alpha greater than PGF-2 alpha greater than PGE-2 identical to TXB-2, with 6-oxo-PGF-1 alpha and PGF-2 alpha productions showing small increases between 10:00 and 02:00 h on the days of pro-oestrus and oestrus, respectively. Myometrial PGE-2 production decreased between these two times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoglobulins in the mouse uterus during the oestrous cycle

The distribution of IgA, IgG and IgM was studied by an immunoperoxidase technique on sections of mouse uteri at each stage of the oestrous cycle. Staining for IgG and IgA was highest at pro-oestrus, declined at oestrus and was very low during the other stages. At pro-oestrus IgG was found throughout the stroma, in the uterine lumen, and in 10% of glandular lumina; very few IgG-containing plasma cells were present. At pro-oestrus, IgA was found in the uterine lumen, and in most of the uterine glands, both in the lumen and in the epithelium; little IgA was present in the stroma. IgA-plasma cells were detected at each stage of the cycle and were particularly numerous at pro-oestrus and oestrus. These results suggest that IgA is secreted locally from plasma cells into the uterine gland through the glandular epithelium, but that IgG enters the stroma from the local capillaries. The obvious increase in IgG and IgA secretion at pro-oestrus, when plasma oestradiol levels are highest, supports the hypothesis that, during the oestrous cycle, the humoral immune response is regulated in the uterus by ovarian hormones.     

Matrilysin activity in the rat uterus during the oestrous cycle and implantation

The objective of this study was to follow changes in the activity of the small matrix metalloproteinase matrilysin (MMP-7) in the rat uterus during the oestrous cycle and embryo implantation. Matrilysin was extracted from rat uteri, partially purified and separated into active and latent forms. The two forms of the enzyme were quantified at all stages of the oestrous cycle and after oestradiol and progesterone treatment. The activity was also measured during the first 7 days of pregnancy. Both latent and active forms of MMP-7 reached a peak during the pro-oestrous stage of the cycle; the concentrations were three times higher than at dioestrus and metoestrus. In rats treated with 0.1 mg oestradiol at metoestrus, both latent and active forms of the enzyme increased by more than two-fold after 24 h. In rats treated at pro-oestrus with 0.4 mg progesterone, there was a 70% increase in latent MMP-7, but no change in the active form. The highest concentrations of MMP-7 were observed on the first day of pregnancy. Between days 3 and 7 of pregnancy, the concentrations were relatively constant and comparable to the low concentrations at dioestrus. Enzyme activities were not different at implantation sites compared with remote sites.     

Water and electrolyte excretion during the oestrous cycle in sheep.

Electrolyte excretion was observed during 24 oestrous cycles in housed sheep, together with mixed salivary Na/K ratio during 10 additional cycles. 1. The sharp fall in food and fluid intake at oestrus accompanied a peak of sodium excretion which changed to peak retention 3 days later, both in faeces and urine. 2. Potassium excretion declined with food intake at oestrus but subsequently failed to recover to pre-oestrous levels dispite full recovery of dietary intake. 3. Curiously, water intake also recovered completely whereas urinary and faecal water retention continued; faecal loss actually exceeded renal excretion on these liberal water intakes. 4. Changes in salivary, urinary and faecal Na/K indicated an aldosterone peak neither during the luteal phase nor at oestrus but three days later. The data raise questions concerning the regulation of water and electrolyte balance within the normal cycle. They also provide a baseline for the investigation of renal effects of gonadal steroids. Possible roles for aldosterone, ADH and progesterone in maintaining fluid and electrolyte balance are discussed, emphasising problems confronting species which have evolved with heavy obligatory potassium excretion but undependable supplies of sodium and water.     

Motility of the oviduct and uterus of the cow during the oestrous cycle.

Recordings of electrical activity of the oviduct and uterus were obtained during three oestrous cycles in cows fitted with an extra-cellular multi-electrode assembly. The stages of the cycle were identified by the appearance of the cervico-vaginal secretions and changes in the peripheral plasma level of progesterone were determined by radioimmunoassay. A gradual transition from local non-propagating electrical activity to propagating electrical activity with increase in the duration of contractions and then of their amplitude occurred 48 hr before the onset of oestrus. The transition coincided with a rapid decrease in progesterone level from 5 to 10 ng/ml to less than 0-1 to 0-4 ng/ml. This phenomenon was recorded from all uterine electrode sites, but was most marked at the uterotubal junction. Two days before oestrus, trains of potentials and bursts of activity became progressively grouped, apparently randomly, into prolonged phases in the distal portion of the oviduct and over the entire myometrium. During oestrus, the phases of activity became synchronized at these sites and both their amplitude and frequency reached a maximum. The strength but not the frequency of the phases diminished progressively 3 days after oestrus, followed by relative inactivity. The last remaining zone of activity was the uterotubal junction. During oestrus, the activities of the oviduct and the uterus were modified by oxytocin and adrenaline, the effect of the former being more marked on the uterus and that of the latter on the oviduct.     

Histochemical study of superoxide dismutase in the ovary of the rat during the oestrous cycle

Superoxide dismutase, an enzyme which causes the dismutation of superoxide free radical anions to generate hydrogen peroxide, has been localized in the rat ovary. The negative-staining method was used to provide photo-induced reduction of nitro-blue tetrazolium in cryostat sections of rat ovaries for histochemical localization of superoxide dismutase. Superoxide dismutase was found in growing follicles, the membrane granulosa of Graafian follicles, ovulated follicles and blood vessels. It is suggested that superoxide dismutase may play a role in regulating follicular development, ovulation and luteal functions.     

Secretory dynamics of bioactive and immunoactive LH during the oestrous cycle of the sheep

Blood samples were collected every 15 min for 6 h during the follicular (1 day before oestrus), and early (Days +1 to +3), mid- (Days +4 to +8), and full (Days +9 to +14) luteal phases of the oestrous cycle. Serum concentrations of immunoactive LH were measured by radioimmunoassay. The biological activity of serum LH was determined by an in-vitro bioassay that uses LH-induced testosterone production from mouse interstitial cells as an endpoint. Only ovine and bovine LH and hCG had appreciable activity in this bioassay. The temporal pattern of secretion of bioactive LH paralleled the secretory pattern of immunoactive LH at all stages of the ovine oestrous cycle. However, the secretory pattern itself varied regularly through the oestrous cycle. The frequency of secretory excursions of LH was highest during the follicular phase (6.2 +/- 0.9 pulses/6 h) and was progressively reduced through the luteal phase (1.1 +/- 0.1 pulses/6 h during full luteal phase). Conversely, amplitude of secretory excursions of immunoactive LH was low during the follicular phase (0.79 +/- 0.08 ng/ml) and significantly (P less than 0.05) increased during the mid- and full luteal phases (1.49 +/- 0.10 and 2.37 +/- 0.20 ng/ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical localization of carbonic anhydrase in the female genitalia of pigs during the oestrous cycle

The ovaries and internal genital tracts of cycling gilts were studied for localization of carbonic anhydrase activity using a post-embedding cobalt precipitation technique. Carbonic anhydrase activity was present in selective capillary endothelia and epithelial cells of the genitalia. The ovarian parenchyma, irrespective of the stage of the oestrous cycle considered, was unstained. The secretory cells of the deep furrows in the uterotubal junction and those in the isthmic region of the oviduct of the oestrous female showed a clear membrane-bound localization. The surface epithelium in the tubal ampulla showed a conspicuous cytoplasmic carbonic anhydrase activity during oestrus and the early luteal phase. Neither the intensity nor the localization of the histo-enzymatic reaction in the females varied with their hormonal status (i.e. stage of the oestrous cycle). The localization of the enzyme in particular regions of the female pig genitalia that normally act as sperm reservoirs (uterotubal junction--tubal isthmus) or where fertilization--early embryo development occur (i.e. ampulla) might be related to the control of the acid-base status of luminal fluid.     

The unoccupied nuclear oestradiol receptor in the rat uterus and hypothalamus during the oestrous cycle.

The nuclear oestrogen receptor population in the rat uterus contained an unoccupied receptor component that bound oestradiol with the high affinity (Kd congruent to 0.5 nM) characteristic of oestrogen receptors. This unoccupied receptor was present at all phases of the oestrous cycle. Its content changed in parallel with that of the total nuclear receptor during the cycle. Oestradiol administration to the immature rat resulted in increases in the uterine content of long-term nuclear receptors (i.e., those still present 8 h after administration); these increases were due to occupied oestrogen receptors, since the content of unoccupied receptor was unchanged. Our previous experiments [White & Lim (1980) Biochem. J. 190, 833-837] have shown in contrast, that oestradiol administration results in an increase in the content of unoccupied nuclear receptor in the hypothalamus. However, as in the uterus, similar cyclic changes in the content of unoccupied nuclear receptor occurred in parallel with those of the total nuclear receptor population in the hypothalamus. Differences and similarities between the unoccupied nuclear receptor of the uterus and hypothalamus are briefly discussed.     

Immunohistochemical localization of constitutive and inducible cyclo-oxygenases in rat uterus during the oestrous cycle and pregnancy

The uterus is a rich source of eicosanoids synthesized from arachidonic acid metabolism through the cyclo-oxygenase pathway. Two isoforms of cyclo-oxygenase, constitutive (COX-I) and inducible (COX-II) enzyme, have been reported. In the present study, we have immunohistochemically mapped the distribution of both COX-I and COX-II during various physiological states of the rat uterus. Uterine tissue was collected from female rats (a) during different stages of the oestrous cycle, (b) on days 1, 4, 8 and 18 of gestation, (c) after spontaneous delivery and (d) post partum, and fixed in Bouin's fixative. After paraffin wax embedding, 5-m-thick sections were immunohistochemically stained by the ABC technique. Observation of the stained sections under the light microscope revealed that, in non-pregnant rat uterus, both COX-I and COX-II were abundantly expressed in the endometrium, with minimal staining observed in the myometrium. Staining was more prominent in epithelial cells than in stromal cells. The intensity of staining in epithelial cells was highest at pro-oestrus and oestrus and lowest at dioestrus. In pregnant rats, although the expression of both COX-I and COX-II was localized primarily to the endometrium with very little staining in the myometrium on day 1 of gestation, both of these enzymes were also apparent in myometrial cells by day 4 of gestation. The staining intensity of endometrial and myometrial cells increased further with the progression of gestation, being maximal at the time of spontaneous delivery. During the post-partum period, however, the staining intensity for both of the enzymes in endometrium and myometrium was decreased. Thus, our studies show that the expression of cyclo-oxygenases in various uterine cells vary with the oestrous cycle and with pregnancy. Furthermore, prominent increases in the expression of cyclo-oxygenases in the myometrium during pregnancy and parturition imply that the cyclo-oxygenase system in the myometrium may play a major role in modulating uterine contractility during pregnancy and labour. © 1998 Chapman & Hall     

Swine uterus carnosinase activity in oestrous cycle and early pregnancy

Carnosinase activity was determined in uterus extracts of sexually immature sows, on particular days of the oestrous cycle, and on the 20th and 30th day of pregnancy. In mature sows carnosinase activity in the uterus was on the average 4.5 times higher than in immature sows. Activity of the enzyme in the oestrous cycle increased from the zero day (first day of the heat) until 13th day, followed by a rapid decrease, reaching the lowest levels on the 17th day of the cycle (3 times lower on the average than on the zero day). On the last days of the cycle (20-21st) activity of carnosinase reached again levels similar to those of the zero day. Carnosinase activity in a uterus corner of pregnant sows (20th day of pregnancy) was over 4 times higher than in the "peak" day of the oestrous cycle (13th day), and over 12 times higher than in immature sows. Activity of the enzyme increased along with progressing pregnancy. It was found that activity of carnosinase in uterus corner of swines was related to the level of progesterone determined by other authors in the blood plasma.     

Intracellular relationships of the oestrogen receptor in the rat uterus and hypothalamus during the oestrous cycle.

Simultaneous measurements were made of the specific oestrogen receptor in the nuclear and cytosol fractions prepared from the uterus and hypothalamus of 50--81-day-old female rats undergoing a 4-day oestrous cycle. In the uterus, the content of nuclear receptor fluctuated in concert with known cyclic changes in the secretion of oestrogen, being maximal at pro-oestrus. Over the period of 50--81 days, the nuclear content at all phases increased with age, again corresponding to known age-related increases in ovarian secretion of oestrogen. This age-related increase in nuclear content, averaged from the values of the different phases in each age group, was related to equivalent increases in uterine wet weight, an increase of 1 pmol of receptor being accompanied by an increase of 80--90 mg. The concentration of cytosol receptor was maintained constant, with respect to wet weight, throughout the cycle and with age, irrespective of changes in nuclear content. In the uterus of normal mature females, translocation of receptor into the nucleus did not lead to depletion of cytosol receptor, suggesting a process of continuous replenishment/synthesis. In the hypothalamus, the nuclear content of oestrogen receptor was also maximal at pro-oestrus. In contrast with the uterus, the content of hypothalamic cytosol receptor was minimal at this phase and reflects depletion of the cytosol receptor, possibly as a result of translocation. The extent of translocation was low compared with that in the uterus and did not alter with age during the age-period studied. This low nuclear binding of the receptor in vivo is discussed in relation to the presence of a cytosol factor, present in limiting amounts, which in vitro mediates the binding of cytosol receptor to oligo(dT)-cellulose. The difference in the physiological response of the uterus and of the hypothalamus to oestrogens may be related to the extent of nuclear binding of receptor.