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.     

Cellular localization of tissue inhibitors of metalloproteinases in the rat ovary throughout pseudopregnancy

The present study determined the ovarian cellular localization of the mRNA for the tissue inhibitors of metalloproteinases (TIMPs) during pseudopregnancy in the rat. Pseudopregnancy was induced by eCG/hCG stimulation. At Day 1 of pseudopregnancy, intense reaction product for TIMP-1 mRNA was observed surrounding the developing corpus luteum (CL), with less intense expression present in granulosa-lutein cells. With continued luteal development, the TIMP-1 mRNA encircling the CL was lost, although low levels of expression were found within the CL. For TIMP-2 mRNA, intense reaction product was observed surrounding the developing CL but, unlike TIMP-1, was present in granulosa-lutein cells, with high levels near the center of the CL. The localization pattern of TIMP-2 mRNA was unchanged through the latter stages of pseudopregnancy. TIMP-3 mRNA expression was strikingly different from the other TIMPs. At Day 1 of pseudopregnancy, intense reaction product for TIMP-3 mRNA was observed in granulosa-lutein cells of certain developing CL, whereas adjacent follicles did not express TIMP-3 mRNA. With continued luteal development, there was a homogenous, intense localization of TIMP-3 mRNA throughout the CL, which was unchanged during pseudopregnancy. To understand the induction of TIMP-3 mRNA in the developing CL, a series of experiments was performed to compare markers of follicular maturity with the presence of TIMP-3 mRNA. TIMP-3 mRNA appears to be switched on in granulosa cells of follicles destined to ovulate. The distinct pattern of expression of the three TIMPs suggests that each inhibitor may regulate either the site and extent of proteolytic action or specific matrix metalloproteinases at different periods of the luteal life span.     

Premature luteal regression in goats superovulated with PMSG: effect of hCG or GnRH administration during the early luteal phase

Twenty-two goats were superovulated with PMSG; 84 h after the onset of estrus the goats were treated with saline solution (control group n = 7), hCG (hCG group, n = 7), or GnRH (GnRH group, n = 8). The ovaries of all the goats were laparoscopically examined 3 and 6 d after the onset of estrus. In each case the CL were counted and classified according to their appearance as normal-looking or as regressing. Blood samples for progesterone determination were collected every 12 h from Day 1 to Day 6. Premature luteal regression was considered to have occurred if progesterone concentrations declined to less than 1 ng/mL by Day 6. According to progesterone concentrations, 57.5, 0 and 37.5% of the goats underwent premature luteal regression in the control, hCG and GnRH groups, respectively. Progesterone concentrations were higher in the hCG group than in the other groups on Days 5 and 6 post estrus (P < 0.05). The control group was the only one in which there was a significant (P < 0.05) increase in the number of regressing CL between Day 3 (1.6 +/- 1.4) and Day 6 (7.3 +/- 1.4). It was also the only group in which there was a significant decrease in the number of normal-looking CL between Day 3 (12.6 +/- 2.1) and Day 6 (2.6 +/- 2.1). On Day 6 the animals treated with hCG had significantly more normal-looking CL (12.0 +/- 2.3) than those in the control group (2.6 +/- 2.1). The number of large follicles present on the ovaries on Day 6 post estrus had negative correlations with progesterone concentrations (P = 0.05) and with the number of normal-looking CL (P < 0.05). It is concluded that the administration of hCG 84 h after the onset of estrus prevents premature luteal regression in goats superovulated with PMSG.     

Effects of injection of hcg during the estrous cycle on follicle development and the inter-estrous interval

Pseudopregnancy in pigs can be induced by the administration of a single dose of hCG at Day 12 of the estrous cycle. However, the resulting length of pseudopregnancy can be extremely variable. In this study, it was investigated whether time of hCG administration (day of the cycle) and degree of follicle growth after hCG administration were related to the length of inter-estrous interval (pseudopregnancy). In the first experiment, groups of cyclic gilts were given 1500 IU hCG at either Day 11 (D 11; n=14) or Day 12 (D12; n=14) after onset of estrus, or not treated (Control; n=13). Follicle development was assessed daily using transcutaneous ultrasonography. Follicle size in the Control gilts remained relatively constant between Days 11 and 17, whereas in the treated gilts, follicle size increased (P < 0.001) within 4 days (D11) and 2 days (D12) after treatment. The inter-estrous interval was increased (P < 0.01) in the hCG-treated gilts (34.7+/-6.3 and 37.6+/-11.1 days in the D11 and D12 gilts, respectively), compared to Controls (22.3+/-5.2 d). About two-thirds of the treated gilts returned to estrus between Days 32 and 39 after onset of first estrus. No relationships were found between follicle development after treatment and length of the inter-estrous interval. In a second experiment, 16 cyclic gilts were treated with 1500 IU hCG at Day 12 and Day 15 of the estrous cycle. Follicle development was assessed at Days 12, 15 and 18. At Day 18, average follicle size was 8.4+/-2.0 mm. The inter-estrous interval was 39.7+/-5.4 days and 14 of 16 gilts returned to estrus between Days 34 and 44 after onset of first estrus. Again, no relationships were found between follicle development after treatment and the duration of the inter-estrous interval. We conclude that, based on the duration of the inter-estrous interval, administration of hCG during the luteal phase induced a short pseudopregnancy. However, the induction of accessory corpora lutea or follicular luteinization cannot be discounted.     

Ovarian function in Nelore (Bos taurus indicus) cows after post-ovulation hormonal treatments

Maternal recognition of pregnancy in the cow requires successful signaling by the conceptus to block luteolysis. Conceptus growth and function depend on an optimal uterine environment, regulated by luteal progesterone. The objective of this study was to test strategies to optimize luteal function, as well as prevent a dominant follicle from initiating luteolysis. Nelore (Bos taurus indicus) beef cows (n=40) were submitted to a GnRH/PGF(2alpha)/GnRH protocol. Cows that ovulated from a dominant ovarian follicle (ovulation=Day 0) were allocated to receive: no additional treatment (G(C); n=7); 3000IU of hCG on Day 5 (G(hCG); n=5); 5mg of estradiol-17beta on Day 12 (G(E2); n=6); or 3000IU of hCG on Day 5 and 5mg of estradiol-17beta on Day 12 (G(hCG/E2); n=5). Ultrasonographic imaging of the ovaries, assessment of plasma progesterone concentration, and detection of estrus were done daily from Day 5 to the day of subsequent ovulation. Treatment with hCG induced an accessory CL, increased CL volume, and plasma progesterone concentration throughout the luteal phase (P<0.01). Estradiol-17beta induced atresia and recruitment of a new wave of follicular growth; it eliminated a potentially estrogen-active, growing ovarian follicle within the critical period for maternal recognition of pregnancy, but it also hastened luteolysis (Days 16 or 17 vs. Days 18 or 19 in non-treated cows). In conclusion, the approaches tested enhanced luteal function (hCG) and altered ovarian follicular dynamics (estradiol-17beta), but were unable to extend the life-span of the CL in Nelore cows.     

Synthesis of prostaglandin F2 alpha, E2 and prostacyclin in isolated corpora lutea of adult pseudopregnant rats throughout the luteal life-span.

The ability of de novo biosynthesis of prostaglandins (PGs) in individual whole corpora lutea (CL) obtained from sterile-mated adult pseudopregnant rats on different days of the luteal phase and the post-luteolytic period was evaluated. Production of PGs, progesterone and 20 alpha-dihydroprogesterone were determined after in vitro incubation of CL extirpated from Day 2 to Day 19 after mating. A time-relationship with increased accumulation of PGs in the medium was demonstrated from 18 s to 5 h, with large increments during the first 30 min. Basal accumulation of PGs in the incubation medium was highest for 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) greater than PGE2 greater than PGF2 alpha greater than thromboxane B2 (TXB2) and basal accumulation of PGF2 alpha and PGE2 measured in the medium was maximal on Day 10-11 of pseudopregnancy, concomitantly with a decline in secretion of progesterone. Addition of arachidonic acid (AA) dose-dependently increased synthesis of PGs, with absolute amounts of PGE2 greater than 6-keto-PGF1 alpha greater than PGF2 alpha greater than TXB2 and addition of 14 microM indomethacin markedly inhibited accumulation of all PGs measured. Luteinizing hormone (LH, 10 micrograms/ml) stimulated progesterone secretion on all days during pseudopregnancy, but not on the post-luteolytic Day 19. LH increased PGF2 alpha, PGE2 and 6-keto-PGF1 alpha secretion on Day 13 of pseudopregnancy by 76%, 91% and 28%, respectively, but not on the other days tested. Furthermore, stimulation of PG-synthesis by addition of AA abrogated the LH-induced progesterone accumulation markedly, but only on Day 13 of pseudopregnancy. Epinephrine (5 micrograms/ml) increased production of progesterone and also PGs, but only on Day 2 of pseudopregnancy, whereas oxytocin (100 mIU/ml) was found to be without effect on progesterone as well as PG secretion on all days tested. The results of the present study demonstrates the independent ability of the rat CL to synthesize PGG/PGH2-derived prostaglandins, including the putative luteolysin PGF2 alpha. Secondly, we demonstrate that LH and AA-induced increases in PGF2 alpha and PGE2 production during the luteolytic period, may be an autocrine or paracrine mechanism involved in luteolysis.     

Prostaglandin f(2alpha) induces distinct physiological responses in porcine corpora lutea after acquisition of luteolytic capacity

This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F(2alpha) analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 microgram cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF(2alpha) secretion. In experiment I, progesterone remained high and PGF(2alpha) low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF(2alpha) production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12, 13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF(2alpha) production only from Day 17 luteal cells. Thus, PGF(2alpha) production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF(2alpha) production is not explained by a difference in induction of PGHS-2 mRNA or protein.     

Effects of hysterectomy and uterine decidualization on in vivo levels of prostaglandins in the corpus luteum of adult pseudopregnant rats

A possible role of the uterus in regulating content of luteal prostaglandins (PGs) was investigated. Pseudopregnancy was induced in adult virgin female rats by mating them with vasectomized male rats. On Day 5 of pseudopregnancy, decidualization of the uterus was induced or hysterectomy was performed. As controls, intact pseudopregnant animals with a luteal phase of 13 +/- 1 days were used. Measurements of in vivo tissue levels of PGF2 alpha, PGE2, and 6-keto-PGF1 alpha were performed by RIA after homogenization and extraction procedures in CL of pseudopregnancy and remainder of ovaries on Days 5, 13, and 19. Serum levels of progesterone and 20 alpha-dihydroprogesterone were determined by RIA. In hysterectomized animals, PGF2 alpha levels increased 2.5-fold in corpora lutea on Day 13 compared with levels on Day 5 of pseudopregnancy, but were still lower than in control rats undergoing functional luteolysis on Day 13. Decidual-tissue-bearing rats exhibited low levels of PGF2 alpha on Day 13 of pseudopregnancy. On Day 19, when luteolysis had occurred in decidual-tissue-bearing and hysterectomized rats, as judged by plasma levels of progestins, luteal content of PGF2 alpha was elevated to a similar level as that in control animals undergoing functional luteolysis on Day 13. When data pooled from control, decidual-tissue-bearing and hysterectomized rats were analyzed, a highly significant inverse correlation (r = -0.72, n = 46, p less than 0.001) between luteal PGF2 alpha content and ratio of plasma progestins was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase was studied throughout the rat estrous cycle. Animals were necropsied at 1300 h on each day of the 4-day cycle, and an additional group was necropsied at 2300 h on proestrus. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. At all stages of the cycle, intense immunostaining was observed in newly formed corpora lutea. Luteal cells were immunoreactive, but the connective tissue centrum was unstained. Interstitial tissue contained heavily labeled cells, whereas the germinal epithelium exhibited faint staining. During estrus, metestrus, and diestrus, thecal cells from preantral and antral follicles contained PGH synthase immunoreactivity, but granulosa cells were unstained. Faint staining of mural granulosa cells was observed first in 78% of preovulatory follicles (less than 400-microns diameter) in ovaries collected on the afternoon of proestrus. After the luteinizing hormone surge, 95% of the preovulatory follicles exhibited PGH synthase staining. The percentage of immunoreactive granulosa cells in these preovulatory follicles increased 4-fold in ovaries collected at 2300 h on proestrus. The presence of ovarian PGH synthase throughout the rat estrous cycle and the changes in cellular localization may reflect the potential role of PGs in follicular and luteal function.     

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.     

Weight, composition, mitosis, cell death and content of progesterone and DNA in the corpus luteum of pregnancy in the ewe

Changes in luteal weight from about Day 20 to near term, and in quantitative histology as assessed by ultrastructural morphometry and light microscopic counts of mitosis and cell death on Days 30, 60, 100 and 142, were studied in 168 pregnant ewes. Luteal weight (mean +/- s.d.) remained constant at 0.56 +/- 0.11 g until Day 120, and fell thereafter to reach 0.31 +/- 0.11 g after Day 140 (P less than 0.01). Up to Day 100, quantitative aspects of the composition of the luteal tissue showed no significant change, and values for volume density, cytoplasmic:nuclear ratio, cell number/mm3 and cell volume were comparable to values previously obtained for corpora lutea (CL) of the cycle. By Day 142 structural evidence of luteal regression was present, but regressive changes were much more marked in some CL than others. Mitosis was seen in a few cells (0.02-0.04%) on all of the days studied, but never in large luteal cells. Cell death was rarely seen up to Day 100, but had increased in incidence by Day 142 (P less than 0.01). Luteal progesterone content, 55.2 +/- 15.9 nmol/g on Day 30, was not significantly changed on Days 60, 100 or 142. It is concluded that (1) structural regression of the CL of pregnancy does not begin until much later than the time (about Day 50) when pregnancy ceases to depend on the CL; (2) structural luteal regression begins before parturition, but its time of onset and/or rate of progression vary widely between animals; and (3) large and small luteal cells remain as distinctive populations throughout pregnancy, and their numbers at all stages can be accounted for by survival of the cells which differentiate during the genesis of the CL.     

The effect of hCG administration five days after insemination on the first service conception rate of anestrous dairy cows

The aim of this study was to investigate the effect of treating anovulatory anestrous (AA) dairy cows with 1500 IU of hCG IM, 5 d after insemination, on their first service conception rate. A clinical trial was conducted during the 2003/2004 breeding season involving 442 AA dairy cows in six herds. On Day -8, all cows were treated with a progesterone-containing intravaginal device (Cue-Mate). The devices were removed on Day -2, and on Day -1 all cows received an IM injection of 1mg of estradiol benzoate. Cows in the control group (n=220) received no further treatments. Cows in the treatment group (n=222) which had been inseminated on Days 0 or 1 were treated with 1500 IU of hCG IM 5 d after insemination. Blood was collected from 30 cows (15 in each group) on Days 5 and 12 after AI for analysis of plasma P4 concentration. There was no difference in first service conception rates between the control and treatment groups (46.3% versus 43.6%, respectively; P=0.68), despite the fact that plasma P4 concentrations were higher in the treatment group on Day 12 (4.9+/-1.3 ng/mL versus 6.2+/-2.7 ng/mL for control and treatment groups, respectively; P<0.01). In conclusion, 1500 IU of hCG 5 d after insemination did not improve first service conception rate in AA dairy cows.     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

Relationship between luteinizing hormone and decidual luteotropin in the maintenance of luteal steroidogenesis

Between Days 6-11 of pregnancy or pseudopregnancy, the decidual tissue of the rat produces a prolactin-like hormone, decidual luteotropin, which can sustain luteal progesterone production when prolactin is suppressed. However, this effect is dependent upon the presence of the pituitary. The present investigation was undertaken to determine whether decidual luteotropin and luteinizing hormone (LH) act together to sustain luteal steroidogenesis and if so, to find out whether the need for LH is due to the inability of the decidual tissue to produce LH-like material and/or whether LH affects decidual luteotropin production. Pseudopregnant rats with or without decidual tissue were hypophysectomized on Day 8 and treated with either 1.5 IU human chorionic gonadotropin (hCG)/day or with vehicle. Within 24 h, serum progesterone dropped in both vehicle-treated groups and decidual luteotropin levels declined by 80% in the decidual tissue. Human CG administration had no effect on progesterone production in the control group. Yet in rats with decidual tissue, hCG stimulated progesterone production for at least 48 h and maintained the decidual tissue content of decidual luteotropin. Progesterone, but not hCG treatment, maintained decidual luteotropin concentrations in ovariectomized rats. To compare the luteotropic activity of the decidual tissue with that of the placenta, pregnant or pseudopregnant rats with decidual tissue were hypophysectomized on Day 8 and treated with 1.5 IU hCG. Control groups had decidual tissue or placentas removed and were similarly treated. Human CG stimulated progesterone production only in rats with placental or decidual tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular composition of the cyclic corpus luteum of the cow

The cellular composition of CL from 6 cows on approximately Day 12 of the oestrous cycle, after synchronization with cloprostenol, was studied by ultrastructural morphometry. Point-count measurements of volume density (mean +/- s.d.) showed that large luteal cells occupied 40.2 +/- 7.0% of the luteal tissue, and small luteal cells 27.7 +/- 6.3%. Of the total of 393.4 +/- 52.0 x 10(3) cells per mm3 of luteal tissue, large luteal cells made up only 3.5% and small luteal cells 26.7%, a ratio of 1:7.6. Endothelial cells/pericytes, at 52.3%, were the most numerous cell type. The mean volume per large luteal cell was 29.6 +/- 6.3 x 10(3) microns 3, while that of small luteal cells was 2.7 +/- 0.4 x 10(3) microns 3. In spherical form, these volumes would represent mean diameters of 38.4 microns and 17.2 microns respectively, and are consistent with published measurements on dispersed luteal cells. However, the values for cell numbers are much higher than published values based on luteal tissue dispersion, suggesting that dispersion may result in substantial and possibly selective losses of luteal cells.     

Tumor necrosis factor production and accumulation of inflammatory cells in the corpus luteum of pseudopregnancy and pregnancy in rabbits

The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.     

Co-culture of mouse embryos with oviduct and uterine cells prepared from mice at different days of pseudopregnancy

Oviduct and uterine cell cultures were prepared from mice at different days of pseudopregnancy and their effects on the development of 1- and 8-cell mouse embryos in co-culture were examined. One-cell mouse embryos in co-culture with oviduct cells from 20 h to 120 h after hCG had a mean (+/- s.e.) cell number of 70.1 +/- 3.6, significantly (P less than 0.001) higher compared with those cultured in Whittingham's T6 medium supplemented with 5% fetal calf serum (T6 + 5% FCS) (30.4 +/- 1.6). Transfer of embryos, at 96 h after hCG, to synchronous pseudopregnant recipients showed that more embryos in oviduct co-culture formed fetuses than those cultured in T6 + 5% FCS. Co-culture of 1-cell embryos with uterine cells did not confer an advantage in cell numbers over T6 + 5% FCS. However, more 8-cell embryos formed blastocyst outgrowths after 100 h in co-culture with uterine cells prepared from mice at Day 3 of pseudopregnancy than with uterine cultures prepared from mice at Day 1 of pseudopregnancy or oviduct cells. In addition, there was further improvement when the Day 3 uterine co-cultures were supplemented with 1 or 10 ng progesterone/ml. These results highlight the importance of the oviduct and uterine cells during the different stages of preimplantation embryo development.     

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation.

Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8.     

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

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.