Luteal function of induced corpora lutea in the bitch

Nineteen anestrous bitches with a mean of 22 kg body weight and ranging from 2 to 4 years of age were induced to exhibit estrus and ovulate using PMSG and HCG. Twelve days after the first day of estrus, bitches were assigned to four treatment groups. Group (A) consisted of six bitches, Group (B) of five bitches and Groups (C) and (D) of four bitches each. At this time, bitches in Groups (A), (B) and (C) were laparotomized and those assigned to Groups (A) and (B) were bilaterally hysterectomized leaving the cervix and oviducts intact. Although bitches in Group (C) were laparotomized, they were not hysterectomized. Group (D) bitches were not subjected to any surgical procedures. Homologous uterine extract was prepared from each bitch in Group (A) and administered intramuscularly beginning on day 25 (day 0 = first day of estrus) and continued every other day for 61 days post-estrus. Bitches in Group (B) were similarly injected with equal volumes of 0.9% saline. Blood samples, obtained prior to laparotomy and every other day for 85 days thereafter, were assayed for plasma progesterone concentrations using radioimmunoassay. One bitch in each of Groups (A) and (D) did not form luteal tissue following treatment with PMSG and HCG although both bitches exhibited estrus following treatment. All other bitches showed an increase in progesterone levels (4 to 19 ng/ml) between the first day of estrus and 10 days post-estrus. Thereafter, progesterone levels progressively declined in all groups with levels below 1 ng/ml between 38 to 40 days post-estrus. Results of this study suggested that CL formed in the bitch following PMSG and HCG treatment have a reduced function compared to non-induced CL of a normal, non-fertile estrous cycle. Such premature CL regression appears to be independent of the presence or absence of the uterus.     

Formation and function of GnRH-induced subnormal corpora lutea in cyclic ewes

Of 19 dioestrous ewes given 50 micrograms GnRH on Day 10 of the oestrous cycle, 15 (79%) formed corpora haemorrhagica within 2 days after injection of GnRH. After excision of the Day 10 spontaneous CL, the GnRH-induced CL were short lived when compared to spontaneous CL in saline-treated ewes (3.1 +/- 0.4 vs 17.3 +/- 0.3 days, respectively). Hysterectomy of ewes bearing the GnRH-induced CL prevented regression of the short-lived CL, thus extending functional lifespan greater than or equal to 38 days. Serum concentrations of progesterone produced by the GnRH-induced CL in hysterectomized ewes were less than those observed during a comparable interval (Days 7-14) in saline-treated, non-hysterectomized ewes (2.24 +/- 0.1 vs 3.67 +/- 0.15 ng/ml, respectively; P less than or equal to 0.001). When GnRH was given before (5 h before) or during (5 h after) PGF-2 alpha-induced regression of the Day 10 spontaneous CL, the GnRH-induced CL which formed were also short-lived. In contrast, when GnRH was given following (36 h after) PGF-2 alpha-induced regression of the Day 10 spontaneous CL, the CL which formed were not different in lifespan or production of progesterone from spontaneous CL. Efforts to enhance function of the GnRH-induced subnormal CL by treating ewes with the synthetic progestagen, norgestomet, to suppress follicular development after CL formation, were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitogenic factors of corpora lutea

The mammalian corpus luteum (CL), which plays a central role in the reproductive process because of its production of hormones such as progesterone, appears to be an exceptionally dynamic organ. Its rate of growth and development are extremely rapid and, even when the CL is functionally mature, its rate of cell turnover remains relatively high. Associated with this high rate of cell turnover, the mature CL receives the greatest blood supply per unit tissue of any organ, and also exhibits a relatively high metabolic rate. Although numerous growth factors have been identified in luteal tissue, their role in growth and differentiation of this dynamic organ remains unclear. Recently, while attempting to identify mitogenic factors of ovine and bovine CL, we have found that they produce several mitogens during the estrous cycle as well as pregnancy. The majority of these luteal-derived mitogenic factors are heparin-binding, and although some may represent previously identified factors, several appear to be novel heparin-binding growth factors. Isolation and purification of mitogenic factors produced by the CL will enable us to determine their roles in luteal growth, development and differentiated function, which will contribute to our understanding not only of the regulation of fertility but also of tissue growth and development in general.     

Inhibition by vanadate of cyclic AMP production in rat corpora lutea incubated in vitro

Vanadate, a normal constituent of cells, has been reported to affect a variety of enzymes involved in phosphate transfer; the findings regarding adenylate cycle vary with the tissue and experimental system. In the corpus luteum, cyclic AMP (cAMP) stimulates steroidogenesis; and prostaglandin F2 alpha, which induces luteal regression, inhibits luteinizing hormone (LH)-induced cAMP accumulation. We examined the influence of orthovanadate on cAMP concentration in isolated corpora lutea from pseudopregnant rats. With 2 mM vanadate, basal cAMP level was unaffected, but LH-induced cAMP accumulation was inhibited by 45-68%. Lower doses of vanadate (0.2-1 mM) were almost as effective. When added simultaneously with LH, vanadate was inhibitory within 25 min, but no inhibition occurred when vanadate was added for 30 min to tissue pretreated with LH for 60 min. The decrease in cAMP accumulation was observed also when corpora lutea were exposed to vanadate in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM), indicating that vanadate inhibits cAMP synthesis. Vanadate may increase cytosolic calcium by inhibiting ion pumps in cell membranes. Thus, we examined the effect of vanadate in corpora lutea incubated in calcium-depleted medium and found that vanadate still inhibited cAMP formation. Vanadyl sulfate (0.4 and 2 mM) reduced the LH-induced cAMP accumulation as effectively as vanadate. Thus, the use of vanadate as a tool for exploring physiological regulators of luteal adenylate cyclase should be considered.     

Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.     

Assessment of luteal function by ultrasonographic appearance and measurement of corpora lutea in goats

In order to characterize the evolution pattern of the corpora lutea (CL) and to compare luteal function with their ultrasonographic appearance, 37 estrous cycles of Serrana goats (n=22) were studied during breeding season. A daily transrectal ultrasound scanning was performed through two successive estrous cycles. Both solid and fluid-filled CL were observed and measured in both ovaries of each goat. Additionally, each CL was classified as CL(ICHE) (CL with irregular contours and heterogeneous echotexture) or CL(RCGE) (CL with regular contours and granular echotexture). Ovarian cyclic activity and luteal function were evaluated by biweekly plasma progesterone (P4) determination. The CL (n=60) were first visualized on day 2.9+/-1.0 after the day of ovulation (day 0), showing 7.1+/-1.8mm of diameter and reach their maximum size (12.5+/-1.6mm) on day 10.7+/-3.2 (P<0.001). Two days before the following ovulation (day -2), the CL regressed to 8.4+/-1.3mm (P<0.001). The central cavity was found in 78.3% of CL, and had a persistence of over 50% until the last days of estrous cycle. The ratio CL length/cavity length was low during the first-third and high during the remaining two-thirds of estrous cycle. On day 2, the percentage of CL(ICHE) was 33.3%, and began to decrease to 16.7% on day 6, reaching the minimum of 3.3% on day 10 (P<0.001). This proportion increased on day -3 to 48.3% and reached 90% on day -1 (P<0.001). The correlation between CL size and plasma P4 levels was r=0.63 (n=87; P<0.001). A negative correlation between the daily proportion of CL(ICHE) and plasma P4 levels was found (r=-0.95; n=18; P<0.001). These results suggest that the ultrasonographic appearance of CL is a reliable parameter for the assessment of luteal function in goats. Both the characterization of echotexture and size of central cavity could be valuable tools to differentiate between phases of normal estrous cycles.     

Quantitative echotexture analysis of bovine corpora lutea

A study was designed to evaluate the attributes of ultrasound images of bovine ovarian CL throughout the estrous cycle. The ovaries of 8 heifers were examined daily by transrectal ultrasonography for 2 interovulatory intervals (ovulation = Day 0). Ultrasonographic examinations of the ovaries were videotaped daily, and recorded images of the CL were digitized for computer analysis of echotexture (mean pixel value and heterogeneity). Blood samples were taken daily and to determine plasma progesterone concentrations. Corpora lutea were of 2 morphological types, those with a central fluid-filled cavity (n = 6) and those without (n = 9). No differences were detected between CL with or without a fluid-filled cavity; therefore, data were combined. Mean pixel values of ultrasound images of the CL changed (P = 0.0001) during the interovulatory interval; values decreased (P < 0.05) from Day 0 to Day 3 during early growth of the CL, reached a plateau when increases in luteal diameter ceased, and decreased (P < 0.05) to minimal levels at the onset of regression of the CL. The mean pixel value subsequently increased (P < 0.05) after Day 17 to values similar to those at the beginning of the interovulatory interval. A time-dependent effect was not observed for heterogeneity of images of the CL (P > 0.5). The results supported the hypothesis that quantitative changes in luteal echotexture are reflective of changes in the physiologic status of the CL.     

Progesterone synthesis and histology of postpartum bovine corpora lutea

In vitro progesterone (P(4)) synthesis by corpora lutea (CL) from the first, second or third ovulation after calving was compared and correlated with their histology and cytology. The CL were removed 7 to 12 days after ovulation, and luteal cells isolated by digestion with collagenase. The response of isolated cells to luteinizing hormone (LH) was determined. Hematoxylin-eosin stained tissues were used to study histology, and the distribution of cell types was estimated by stereological methods. Ovulation occurred within 25 days of calving and interovulatory intervals were short, 12.1 +/- 3.9 days and 12.6 +/- 4.8 days, respectively. The CL removed after first ovulation were smaller and contained fewer live cells than those obtained after subsequent ovulations. Stimulation by LH in vitro was independent of cycle number or day of cycle but was related to the histology of the tissue. The CL composed of large cells (> 24 mum) with vacuolated cytoplasm contained high amounts of P(4) but were not stimulated by LH. Conversely, CL composed of small and medium- sized cells (10 to 20 mum) and/or intact larger cells contained little P(4) but were stimulated by LH. These observations indicate that the response of postpartum CL to LH in vitro is dependent upon the structural integrity of the tissue at the time of removal. Furthermore, these observations suggest that the short life of CL during the postpartum period may not be due to the absence of luteotrophic support, but to the action of a luteolytic mechanism.     

Function and lifespan of corpora lutea in ewes treated with exogenous oxytocin

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.     

Immunocytochemical localization of neurophysin and oxytocin in ovine corpora lutea

The cellular distribution of neurophysin and oxytocin within ovine corpora lutea obtained on Days 4, 10 and 16 of the estrous cycle was examined immunocytochemically. Serial sections (8-10 micron-thick) prepared from corpora lutea that had been fixed in Bouin's solution and embedded in paraffin were immunostained for neurophysin or oxytocin using the peroxidase-antiperoxidase (PAP) procedure. Irrespective of the day of the cycle examined, immunoreactivity was restricted to large luteal cells. However, on Days 4 and 10 of the cycle, the intensity of staining in large luteal cells was highly variable; and, within the same section some cells were heavily stained, others were only lightly stained, and still others were not stained at all. In contrast, on Day 16 of the cycle, the intensity of staining was uniform and essentially all of the large luteal cells were immunoreactive. Based on the results obtained, it is evident that immunoreactive neurophysin and oxytocin can be detected as early as Day 4 of the cycle, persists through Day 15, and is restricted to large luteal cells.     

Effect of prostaglandin F2 alpha on the ultrastructure and function of sheep corpora lutea.

Early morphological changes in the ultrastructure of CL of ewes treated with prostaglandin F2alpha were examined in relation to luteal function as judged by plasma progesterone concentration. The luteolytic effect of prostaglandin F2alpha was confirmed, but there was little synchrony between morphological and functional luteolysis. Significant changes included a decrease in the amount of smooth endoplasmic reticulum, a change in the shape of mitochondria and a decrease in the number of membrane-bound granules. There was also an accumulation of lipids.