Endocytosis and multivesicular body formation in rabbit luteal cells during pseudopregnancy

Horseradish peroxidase (HRP) was injected directly into the corpora lutea of rabbits on different days of pseudopregnancy. Young luteal cells have also been incubated "in vitro" in a medium containing horseradish peroxidase. In the "in vivo" experiments the 5 to 9 days old luteal cells take up far more horseradish peroxidase than the older ones (14-20 days). Different types of endocytic vacuoles, MvB's and also some DB's are already peroxidase positive shortly (15-20 min) after injection of the tracer. In the "in vitro" experiments the cells are more heavily loaded. The normal morphology of pale, dense and terminal MvB's is also described and staining with PTA at low pH provides further information on changes occurring in the MvB's. The various findings on multivesicular bodies are compared and two possible pathways for endocytic vacuoles are proposed: one to DB's the other to MvB's. The related phenomena of the uptake of material and the internalization of plasma membrane are discussed in the light of the possible function of endocytosis in luteal cells.     

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endometrial vascularity during pseudopregnancy in the rabbit

The vascular architecture in the rabbit uterus was studied during pseudopregnancy. Uteri at 2, 3, 4, 6, 7, 8, 9, 11, 13, 16, 17, 18, 20, 24 and 28 days after sterile mating were subjected to one of four techniques: neoprene latex casts, transparent sections, frozen sections, and histological sections. Measurements were made microscopically of the thickness of myometrium and of the subepithelial capillary plexus in the different mucosal folds. In the estrous rabbit, the circular arteries in the uterine muscular layer give off arterioles which pass upwards, with a few branchings through the endometrium to the uterine lumen. These arterioles reach the surface of the mucosal folds and break up into the subepithelial capillary plexus. This plexus is connected to the tips of the venules which run down through the endometrium to the endometrial vascular plexus at the base of the endometrium: some of the venules connect with the circular venous vessels in the muscular layer. With advanced stages of pseudopregnancy, the capillaries among the glands become stretched and elongated. Maximal branching of the folds occurs at 4 to 9 days of pseudopregnancy. The “branching activity” was consistently higher in the placental than in the periplacental or in the obplacental folds. Such changes reached a maximum at 6 to 7 days p.c., after which the capillaries became gradually shorter and tortuous. The development of arterioles in the mucosa was marked at 3 to 6 days p.c. The thickness of the plexus in the periplacental fold and in the obplacental fold as a percentage of the thickness in the placental fold was highly correlated with the stage of pseudopregnancy. At 9 to 11 days p.c., these ratios reached a minimum of 70 to 80%.     

Relationship between steroid levels in peripheral serum and uterine tissue during pseudopregnancy in rabbit

Determination of the main C21, C19 and C18 steroids in peripheral serum and uterine tissue was made to study the relationships of the steroids during pseudopregnancy in the rabbit. Tissue and serum progesterone levels rose significantly (P < 0.01) from estrus to Day 9 and then decreased by 81% (tissue) and 57% (serum) at Day 15, while pregnenolone levels in uterine tissue and serum remained unaffected. Estradiol serum levels remained fairly stable, whereas its concentration in uterine tissue decreased after estrus by 85%, followed by a significant (P < 0.05) increase from Day 9. Most of the C19 steroid values in the uterine tissue and in the serum were generally at levels below or near the limit of detection and did not vary significantly. Since progesterone and estradiol levels were high and/or varied significantly in tissue throughout pseudopregnancy, and since androgens were produced in small amounts, it is suggested that androgens might not play a significant role in uterine tissue during pseudopregnancy.     

Characterization and physiological variation of estrogen receptors in rabbit corpora lutea throughout pregnancy and pseudopregnancy: the effect of hysterectomy and sustained estradiol treatment

Previous studies suggest that regression of the rabbit corpus luteum is associated with a uterine-induced loss of responsiveness to estradiol. To determine if this is due to loss of estrogen receptor, cytoplasmic and nuclear estrogen receptors were measured in pseudopregnant, hysterectomized-pseudopregnant and pregnant rabbits throughout luteal life. Estrogen receptor levels were higher in corpora lutea than in nonluteal tissue and were generally higher in nuclei compared to cytosol. Estrogen receptor levels were low on Day 3, increased 2- to 3-fold by Day 6-8, reached peak levels by Days 8-10, and then gradually decreased in a pattern similar to the pattern of serum progesterone typical of each group. Hysterectomy was not associated with elevated cytoplasmic or nuclear estradiol receptor levels. When hysterectomized rabbits were treated with estradiol-filled Silastic implant on Day 1, nuclear estradiol receptor levels fell by Day 20 to levels seen in untreated hysterectomized rabbits. Despite substantial losses in nuclear estrogen receptor, serum progesterone remained elevated on Days 16 and 20. Thus, the ability of estradiol to maintain serum progesterone in hysterectomized rabbits did not correlate directly with the level of estrogen receptor.     

Evidence for multiple lipase forms in the rabbit pancreas

Evidence is presented of the existence of at least two forms of lipase (A and B) in homogenized rabbit pancreas. These forms are separated by means of gel filtration and anion-exchange chromatography. Both forms are colipase-dependent, but lipase A is activated to a significant extent by 140 mmol/l NaCl even in the absence of the protein cofactor. Lipase A exhibits greater affinity towards emulsified triolein than does lipase B, as evidenced by the respective apparent Km values. Lipase B appears to be more colipase-dependent and resembles more closely the 'pancreatitis' lipase of human plasma. Form B is to be preferred as internal standard in turbidimetric and nephelometric indirect lipase assays.     

Quantitative light microscopic analysis of corpus luteum growth during pseudopregnancy in the rabbit

We employed stereological methods at the light-microscope level to examine the mechanism by which corpora lutea (CL) grow during the course of pseudopregnancy in the rabbit. Corpus luteum volume per ovary, the absolute volume of luteal cells per CL, individual luteal cell volume, the number of luteal and endothelial cells per CL, and capillary surface area per CL were examined in rabbits at Days 1, 4, 7, 11, and 18 of pseudopregnancy. Total CL volume increased from 3.7 +/- 0.1 microliter to 30.3 +/- 0.5 microliter over Days 1 to 11 and thereafter decreased to 15.2 +/- 1.1 microliter by Day 18. Stereological analyses showed that the increases in CL volume from Day 1 to Day 11 were due primarily to increases in the volume of individual luteal cells (from 2.6 +/- 0.2 pl on Day 1 to 23.5 +/- 1.7 pl on Day 11, 1 pl = (10 mu)3; r = 0.96), and that the decrease in CL volume after Day 11 resulted largely from a decrease in luteal cell volume (to 12.8 +/- 1.5 pl). In contrast, no change was seen in the number of luteal cells per CL (range 9.1 x 10(5)-12.5 x 10(5)). These data show that CL growth and subsequent regression during pseudopregnancy result primarily from changes in the volume of individual luteal cells, and not from changes in the number of luteal cells. These data support the hypothesis that modulation of progesterone production during pseudopregnancy is due to changes in individual luteal cell volume and not to changes in cell number.     

Pulsatile patterns of gonadotropins and ovarian steroids during estrus and pseudopregnancy in the rabbit

Consecutive daily plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17 beta (E2), progesterone (P4) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) were monitored in estrous rabbits and in these same doses during pseudopregnancy (PSP); these daily hormone levels, as well as the immediate post-coital changes in gonadotropin secretion, were similar to those in previous reports. To examine the pulsatile patterns of the gonadotropins and ovarian steroids, sequential, 10-min plasma samples were collected for 6 h from estrous does and on Days 3, 10, and 17 of PSP. All five hormones were measured in the serial samples from estrous and PSP Day 10 does; LH and FSH only were assayed in the remaining sequential samples. The amplitude and frequency of FSH pulses did not differ between any of these stages. In marked contrast, LH pulse amplitudes, and even pulse frequencies in Day 17 does, were profoundly increased during PSP above those in estrous does. Progestin secretions, both P4 and 20 alpha-OHP, also were sharply elevated in PSP Day 10 does as compared with those in estrous rabbits; the pulse amplitudes of both progestins were severalfold higher during PSP. P4 pulse frequencies were also increased at this time. Conversely, the parameters of E2 secretion did not differ between estrous and PSP Day 10 animals. In PSP Day 10 does, high amplitude pulses of both P4 and 20 alpha-OHP occurred simultaneously with high amplitude LH pulses. Simultaneous E2 and P4 pulses were evident in these same sequential plasma samples, and this E2-P4 pulse association was greater than that of 20 alpha-OHP pulses with E2 pulses. Our findings failed to identify conclusively the trophic stimulus for the progestin pulse patterns, but the mechanism may involve the coordinated action of LH and E2. The results do demonstrate that each gonadotropin and ovarian steroid is secreted in a pulsatile manner in both estrous and pseudopregnant rabbits. There are altered profiles in LH and progestin pulses, without major changes in FSH and E2 patterns, between the stages of estrus and PSP. The causes and consequences of these divergent endocrine shifts cannot be deduced from these data.     

Quantitative cytochemical analyses of ovarian luteal and interstitial cell RNA in relation to plasma progestins during pseudopregnancy in the rat

Luteal and interstial cell RNA contents and circulating progesterone (P) and 20α-hydroxypregn-4-ene-3-one (20α-OH P) levels were measured during pseudopregnancy in order to characterize relationships between ovarian 20α-OH P secretion and luteal regression. Functional luteolysis, as manifested in depressed P levels, was not associated with concurrent elevations in 20α-OH P. Rather, augmented 20α-OH P levels were evidenced in periovulatory periods at the onset and termination of pseudopregnancy, subsequent to RNA accumulation in both luteal and interstitial compartments. It is postulated that 20α-OH P, the putative inactive metabolite of P, is produced by both ovarian compartments in a cyclic manner and in response to gonadotrophin released in the preovulatory period.     

Precision-cut luteal slices: A promising approach for studying luteal function in pigs

The study was aimed to validate the precision-cut luteal slices to investigate porcine luteal function. Corpora lutea (CLs) were cut into 180-μm thick slices using Krumdick Tissue Slicer. The viability, tissue structure and steroidogenic acute regulatory protein (STAR) expression in the luteal slices did not differ between the beginning and the end of the 24-h incubation period. The luteal progesterone secretion showed a time- and dose-dependent response to porcine luteinizing hormone. The effects of prostaglandin F_2α and 17β-estradiol on progesterone secretion by porcine luteal slices were comparable to the previously reported in vivo results of the CL microdialysis system in the pig.     

Evidence for uterine metabolism of progesterone during early pregnancy in the pig

Two experiments were conducted to examine whether the 40 or 50% decrease in systemic progesterone (P(4)) concentrations between Days 13 and 21 postmating in the pig results from decreased ovarian P(4) secretion or increased uptake of P(4) by the uterus. In Experiment I, five nonpregnant (NP) and four pregnant (P) gilts were sham-operated, and five NP gilts were hysterectomized (HYST) on Days 7 to 9 postestrus or postmating (first day of estrus or mating = Day 0). Femoral arterial blood was obtained once daily from Day 10 until the subsequent estrus (NP gilts) or Day 21 (P and HYST gilts). In Experiment II, blood was collected daily from both utero-ovarian veins of two NP and three P gilts from Days 11 to 18. Femoral arterial P(4) concentrations were similar for all gilts in Experiment I from Days 10 to 14. For NP gilts, femoral arterial P(4) declined (P < 0.01) after Day 14 to reach basal levels by Day 17. Progesterone in femoral arterial blood of P gilts declined (P < 0.01) from Days 13 to 16 and then remained constant through Day 21. Concentrations of P(4) in femoral arterial blood of HYST gilts remained constant from Days 13 to 21 and were greater (P < 0.01) than for P gilts from Days 15 to 21. In Experiment II, P(4) concentrations in utero-ovarian venous blood were similar until Day 14 between NP and P gilts. Utero-ovarian P(4) of NP gilts then declined (P < 0.01) to reach basal levels by Day 16. P(4) concentrations in utero-ovarian venous blood of P gilts increased (P < 0.05) for Days 14 to 18. These results demonstrate that ovarian P(4) secretion increases during early pregnancy in the pig. Further, the absence of a decline in P(4) concentrations in femoral arterial blood of HYST gilts suggests that the declining systemic P(4) levels observed during early pregnancy are a result of uterine uptake and(or) metabolism.