Assessment of ovarian function in the African elephant (Loxodonta africana) by measurement of 5α-reduced progesterone metabolites in serum and urine

We have previously shown that 5α-pregnane-3,20-dione (5αDHP), and 5α-pregnane-3-ol-20-one (5α-P-3-OH) are the major luteal and circulating progestins in the African elephant. Therefore, the aim of the present study was to determine (1) circulating levels and patterns of secretion of 5α-DHP in relation to progesterone (P4) throughout the ovarian cycle, (2) the presence and relative abundance of 5α-reduced progestins in urine and (3) whether their measurement in urine would provide a non-invasive method for monitoring luteal function. Urine samples were collected weekly throughout a total of 13 ovarian cycles from 5 females. In addition, matched blood samples were collected during 6 cycles from 2 of the 5 animals. All hormone measurement, were carried out by enzymeimmunoassay following extraction. Urine was hydrolyzed prior to extraction. Profiles of P4 and 5α-DHP in serum followed a similar cyclic pattern and both measurements were significantly correlated (r = 0.78–0.98, mean 0.89, P < 0.001). Concentrations of 5α-DHP were, however, 10–20 fold higher than those of P4. I addition, 5α-DHP measurements showed a more pronounced luteal phase increase than that of immunoreactive P4. HPLC co-chromatography confirmed the presence of large amounts of 5α-P-3-OH in urine as a single immunoreactive peak, whereas 5α-DHP was present in very low levels and measurable only as one of several immunoreactive substances. Measurements of urinary 5α-P-3-OH were significantly correlated to serum 5α-DHP measurements in each of the 6 cycles (r = 0.72–0.93, mean 0.81, P < 0.001), whereas correlation coefficients between urinary and serum 5α-DHP values were generally lower (r = 0.34–0.83, mean 0.69) and significant in only 4 of the 6 cycles. Accordingly, only urinary excretion of 5α-P-3-OH, but not of 0.15–0.20 μ/mg Cr in the follicular phase and 10-fold elevated levels (1.8–2.2 μg/mg Cr) in the luteal phase. Based on the intervals between successive luteal phase increases in urinary 5α-P-3-OH, a mean cycle length of 14.1 ± 1.8 weeks, comprising a follicular phase of 5.0 ± 0.9 weeks and a luteal phase of 9.1 ± 1.4 weeks was determined for the 13 cycles studied. The results indicate that measurements of 5α-P-3-OH in urine provide a reliable non-invasive method for monitoring luteal function in the African elephant. Zoo Biol 16:273–284, 1997. © 1997 Wiley-Liss, Inc.     

Changes in corpus luteum content of prostaglandin F2 alpha and E in the adult pseudopregnant rat

Conflicting reports exist regarding the source of luteolytic PGF2 alpha in the rat ovary. To assess the quantities of different PGs, measurements of PGF2 alpha, PGE and PGB were performed by radioimmunoassay in the adult pseudopregnant rat ovary throughout the luteal lifespan. Ovaries of 84 rats were separated by dissection into two compartments, corpora lutea of pseudopregnancy and remainder of ovary. Tissue samples were homogenized and prostaglandins extracted and determined by radioimmunoassay. During the mid-luteal and late-luteal phases, levels of PGs were significantly higher in the corpora lutea of pseudopregnancy than in the remainder of ovary. An increase of PGF2 alpha-content in the corpus luteum was registered with peak-levels of 53.9 +/- 8.5 (mean +/- SEM, N = 18) ng/g tissue wet weight at day 13 of pseudopregnancy. PGE-levels reached peak-values at day 11 of pseudopregnancy (271.6 +/- 28.4 ng/g w w, mean +/- SEM, N = 12). PGB-levels were below detection limits in all compartments for all ages studied. The present study demonstrates increased availability of PGF2 alpha in the corpus luteum during the luteolytic period, and points toward either increased luteal synthesis or luteal binding of PGF2 alpha during the luteolytic period.     

Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.     

Steroidogenic effects of lipoproteins and 25-hydroxycholesterol on luteal and ovarian cells: a comparison of two pseudopregnant rat models

Steroidogenesis was compared between luteal cells from immature pseudopregnant (PSP) rats induced by either 5 IU pregnant mare serum gonadotropin (PMSG) alone or 50 IU PMSG combined with 25 IU human chorionic gonadotropin (hCG). It was also determined whether differences in steroidogenesis existed when the entire ovary (ovarian cells) or just luteal cells from Day 4 PSP rats were exposed in vitro to lipoproteins or 25-hydroxycholesterol (25-OH chol). In the absence of luteinizing hormone (LH), basal steroid accumulation, especially progesterone (P4) was around fourfold greater in luteal cells from rats treated with PMSG alone than from rats receiving PMSG-hCG. However, serum P4 and LH were about fivefold greater in the latter group. It is therefore likely that net cellular cholesterol uptake per luteal cell is lower in the PMSG-hCG treated rats, but this is offset by a much greater mass and number of corpora lutea. Lipoproteins (HDL and LDL) and 25-OH chol stimulated in vitro luteal steroidogenesis from rats treated with PMSG alone or PMSG-hCG, and their responses were virtually identical. Therefore, luteal steroidogenesis in the rat always depends on exogenous cholesterol even though treatment in the preovulatory period with PMS or PMSG-hCG and serum LH and follicle-stimulating hormone (FSH) levels on Day 4 PSP are very different. When ovarian cells from PMSG-hCG treated rats were incubated with LH plus HDL or 25-OHP, the production of 20 alpha-DHP was considerably greater than luteal cell production which may be due to a contribution from nonluteal cells. Indeed, about 30% of the cells in the PMSG-hCG group represent nonluteal components as estimated by weight and deoxyribonucleic acid content.     

Changes in corpus luteum content of prostaglandin F2α and E in the adult pseudopregnant rat

Conflicting reports exist regarding the source of luteolytic PGF_2α in the rat ovary. To assess the quantities of different PGs, measurements of PGF_2α, PGE and PGB were performed by radioimmunoassay in the adult pseudopregnant rat ovary throughout the luteal lifespan. Ovaries of 84 rats were separated by dissection into two compartments, corpora lutea of pseudopregnancy and remainder of ovary. Tissue samples were homogenized and prostaglandins extracted and determined by radioimmunoassay. During the mid-luteal and late-luteal phases, levels of PGs were significantly higher in the corpora lutea of pseudopregnancy than in the remainder of ovary. An increase of PGF_2α-content in the corpus luteum was registered with peak-levels of 53.9 ± 8.5 (mean ± SEM, N=18) ng/g tissue wet weight at day 13 of pseudopregnancy. PGE-levels reached peak-values at day 11 of pseudopregnancy (271.6 ± 28.4 ng/g w w, mean ± SEM, N=12). PGB-levels were below detection limits in all compartments for all ages studied. The present study demonstrates increased availability of PGF_2α in the corpus luteum during the luteolytic period, and points toward either increased luteal synthesis or luteal binding of PGF_2α during the luteolytic period.     

Studies on the mechanism of action of prostaglandin F2 alpha induced luteolysis in rats

The effects of prostaglandin F2 alpha (PGF2 alpha) administration on the utilization of low density lipoprotein (LDL) and progesterone secretion were examined in dispersed luteal cells from rat ovaries. Immature rats were rendered pseudopregnant with administration of pregnant mare serum gonadotropin and human chorionic gonadotropin. Animals were sacrificed at different times after PGF2 alpha (5 mg/kg) or vehicle administration on day-5 of pseudopregnancy. Administration of PGF2 alpha in vivo decreased human chorionic gonadotropin (hCG) binding to luteal cell membranes in vitro but enhanced binding of LDL. Utilization of labelled cholesterol for steroid synthesis from reconstituted LDL [(3H)-CL-LDL] by dispersed luteal cells was enhanced following PGF2 alpha administration. This suggests that the LDL pathway is not suppressed during prostaglandin induced luteolysis. Progesterone and total progestin secretion in response to N6-2'-0-Dibutyryladenosine 3'5'-cyclic monophosphate (cAMP) was decreased at 2, 4 and 24 hours following PGF2 alpha administration demonstrating a post-cAMP defect in steroidogenesis. Addition of the hydroxylated sterols, 20 or 25-OH cholesterol as substrate stimulated progesterone secretion in vehicle treated rats in a dose dependent fashion with 20-OH cholesterol being more potent. Progesterone secretion in response to stimulation with luteinizing hormone (LH) and cAMP from vehicle treated rats was less than that observed with 20 or 25-OH cholesterol, indicating that endogenous substrate may be a limiting factor in steroid synthesis. The maximal capacity of luteal tissue to produce progestins following PGF2 alpha administration was determined with 20-OH cholesterol as the substrate. The results suggest that the post-cAMP defect at 4 hours following PGF2 alpha administration may be due to failure of the cells to mobilize endogenous cholesterol. However at 24 hours following PGF2 alpha administration the decreased ability of luteal cells to convert cholesterol to pregnenolone may contribute to decreased progesterone synthesis.     

Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

The relationship of oxygen free radicals to corpus luteum function in rabbits was explored during various stages of pseudopregnancy, including natural and induced luteal regression. Induced luteolysis was achieved during mid-pseudopregnancy by removal of an oestradiol capsule placed at the onset of pseudopregnancy, which suppressed ovarian oestradiol production. Activity of manganese superoxide dismutase (Mn SOD) was significantly and positively correlated with ovarian progesterone production (P < 0.01) throughout pseudopregnancy and during natural regression. Oestradiol deprivation for 12, 24 or 72 h resulted in declines in Mn SOD activity and progesterone secretion, although Mn SOD rose and corpus luteum steroidogenesis was restored to normal when the capsule was replaced for 48 h before assessment, having been removed for 24 h. Lipid peroxide and progesterone concentrations were not correlated, although a significant rise in lipid peroxides in the luteal tissue was detected after deprivation of oestradiol for 72 h. Changes in progesterone production and Mn SOD activity were not associated with alterations in concentration of prostaglandin F metabolite. These data suggest that Mn SOD may be involved in regulating function of the corpus luteum during pseudopregnancy in rabbits and that oxygen free radicals may play a role in regression of corpus luteum in this species.     

Evidence for multiple uterine modulators of rabbit luteal function: the effect of hysterectomy during pseudopregnancy in the estrogen-treated rabbit

Previous studies show that hysterectomy on Day 1 of pseudopregnancy prolongs serum progesterone secretion in estrogen-treated pseudopregnant rabbits. These studies were undertaken to determine the day of pseudopregnancy when uterine factors are released to alter luteal function. When hysterectomies were performed on either Day 5, 8, 10, or 13 of pseudopregnancy, serum progesterone concentrations were greater than 10 ng/ml between Days 18 and 27 of pseudopregnancy compared to levels of approximately 4 ng/ml in sham-hysterectomized rabbits on these same days. In contrast, serum progesterone levels were not elevated when hysterectomies were performed on Day 11 of pseudopregnancy and were only partially maintained when hysterectomies were performed on Day 12 of pseudopregnancy. Twice daily injections of prolactin (1.5 mg, s.c.) between Days 1 and 33 of pseudopregnancy were unable to mimic the effect of estradiol in the hysterectomized rabbit. Twice daily injections of indomethacin (8 mg/kg, s.c.) between Days 6 and 23 of pseudopregnancy lowered uterine and luteal prostaglandin F2 alpha levels approximately 10-fold on Day 24 of pseudopregnancy but did not maintain progesterone secretion. Serum cholesterol levels were not altered by hysterectomy on any day and were thus not related to the maintenance of progesterone production. These results suggest that the uterus produces both inhibitory and stimulatory factors that effect luteal progesterone secretion. First, an inhibitor is released between Days 10 and 11 of pseudopregnancy in estrogen-treated rabbits that prevents the rabbit corpus luteum from responding to estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian estradiol secretion during early pregnancy in monkeys: luteal versus extra-luteal secretion and effect of chorionic gonadotropin.

To determine the locus of ovarian estradiol secretion during early pregnancy, six monkeys were luteectomized (CLx) on days 22 to 24 of pregnancy. Daily peripheral serum concentrations of progesterone were maintained or slightly elevated despite removal of the corpus luteum (CL), while serum estradiol concentrations fell precipitiously (P less than 0.05). It is concluded that the CL of early pregnancy in these monkeys is the primary source of serum estradiol, but that ovarian tissues other than the primary luteal body secrete estradiol at low levels. Further, preliminary results suggest that mCG may be an important stimulus of ovarian estrogen secretion, by both luteal and extra-luteal compartments, in these primates. MCG may stimulate the CL to sustain estradiol secretion even after luteal cells are no longer responsive to this endogenous gonadotropin as regards progesterone secretion.     

Monitoring luteal function in the lion-tailed macaque (Macaca silenus) through urinary progesterone metabolite measurements

A direct radioimmunoassay for measuring urinary 20-hydroxyprogesterone cross-reactivity to monitor and assess luteal function and detect pregnancy in the lion-tailed macaque (Macaca silenus) is described. Urine samples were collected daily during ten nonconceptive and five conceptive ovarian cycles of five dult female lion-tailed macaques. Urine was analyzed for concentrations of 20α-hydroxypro-gesterone cross-reactivity, estrone conjugates, and creatinine. The strength of the luteal phase in normal nonconceptive cycles (n = 8) is characterized by a maximum sevenfold increase (day 9) in mean 20α-hydroxyprogesterone cross-reactivity over follicular phase levels; the duration, by a 13-day sustained elevation of mean 20α-hydroxyprogesterone cross-reactivity levels. Pregnancy is detectable from 20α-hydroxyprogesterone cross-reactivity values approximately 20 days after the periovulatory estrone conjugate peak (n = 4). Apparent anovulation (n = 1), extended follicular phase (n = 1), and early abortion (n = 1) also are detectable using 20α-hydroxyprogesterone cross-reactivity measurements.     

Correlation of plasma progesterone concentrations to ovarian H-type lipase activity during pseudopregnancy in the rat

Conditions for extraction and assay of hepatic type (H-type) lipase from rat ovaries were studied. An alkaline buffer with protease inhibitors and detergents gave the most efficient extraction. The specificity of the assays was ascertained using antiserum to H-type lipase from heparin perfusates of rat livers. H-type lipase activity was determined in ovarian compartments during pseudopregnancy (1-13 days) as well as during the ensuing period of luteal regression (Day 17). The activity was low in the luteal compartment immediately after ovulation, increased 6-fold to a maximum between Day 5 and Day 8 and then decreased again. This is similar to previously known changes in blood flow. There was a significant correlation between luteal H-type lipase activity and plasma progesterone regardless of luteal age. In contrast, neither the activity in the remainder of the ovary nor the activity in plasma changed during the luteal phase or correlated to plasma progesterone. Injection of heparin at the height of the luteal cycle (Day 8) caused a pronounced decrease in luteal lipase and in plasma progesterone. These studies suggest that the H-type lipase activity has an important role in luteal steroidogenesis, probably to facilitate uptake of cholesterol from lipoproteins.     

Cellular localization of ovarian prostaglandin endoperoxide synthase during pseudopregnancy in the rat

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase, the enzyme responsible for initiating the conversion of arachidonic acid to prostaglandins, was studied throughout pseudopregnancy in the rat. Pseudopregnancy was induced by administration of eCG (20 IU) to immature, 27-day-old rats followed by hCG injection (10 IU) on Day 29. Animals were necropsied on Days 1 (Day 1 = 1 day post hCG), 5, 9, and 13 of pseudopregnancy. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. Immunoreactive PGH synthase was distributed throughout the CL at each of the 4 different days of pseudopregnancy, with the majority of the luteal cells exhibiting varying degrees of staining. The connective tissue centrum of the CL, however, lacked PGH synthase immunoreactivity. Quantitative assessment of the immunostaining distribution was accomplished with a computer-based image analysis program (Kontron IBAS). Results are expressed as the percentage of a digitized luteal area that contained intense immunoreactivity between Day 1 (0.6 +/- 0.2% immunoreactive area) and Day 5 (16.8 +/- 2.7%) of pseudopregnancy. The area of luteal immunostaining was similar on Day 5 and Day 9 (16.8 +/- 2.7% and 14.7 +/- 2.0%, respectively) followed by a decrease (p less than 0.05) in immunoreactivity on Day 13 (9.1 +/- 2.2%). The region of the CL adjacent to the germinal epithelium had an increase (p less than 0.01) in PG synthase staining distribution compared to the region of the CL adjacent to the ovarian medulla on all days of pseudopregnancy except Day 1. These findings demonstrate that PGH synthase is present in the rat CL throughout pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of 20alpha- and 20beta-dihydroprogesterone in plasma of the pregnant rhesus monkey

A competitive protein binding assay for 20α- and 20β-dihydroprogesterone is described which involves an initial chemical or enzymatic oxidation of these two isomers to progesterone. The assay can distinguish between 20α- and 20β-dihydroprogesterone and is sensitive to pg amounts of these two steroids. Venous steroid concentrations were measured in the pregnant rhesus monkey employing this assay. In this species the corpus luteum (CL) at days 22 and 157 of gestation is the primary ovarian source of 20α-dihydroprogesterone as indicated by a higher plasma concentration of this steroid in the ovarian vein draining the ovary containing the CL (+CL) than in the contralateral vein (?CL) (9.34 ng/ml versus 1.72 ng/ml, day 22; 7.52 ng/ml versus 1.96 ng/ml, day 157). By contrast the CL at day 50 appeared to secrete no 20α-dihydroprogesterone as evidenced by the essentially equal steroid levels in both ovarian veins. The CL synthesizes 20β-dihydroprogesterone only during early gestation (21–22 days) when the concentration of this steroid was 6.46 ng/ml and 0.87 ng/ml in the ovarian (+CL) and ovarian (?CL) veins, respectively. Synthesis of both 20α- and 20β-dihydroprogesterone occurs in the fetoplacental unit throughout pregnancy. This is indicated by higher steroid concentrations in the uterine vein than in the femoral vein. The results suggest both a qualitative and quantitative alteration in the luteal synthesis of 20α- and 20β-dihydroprogesterone with the advancement of gestation. The data also provide additional evidence that the steroidogenic activity of the CL is enhanced before parturition.     

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.     

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 blood flow in the rat: association with body weight, the estrous cycle, and pseudopregnancy

The relationships of ovarian blood flow (OBF) to ovarian function, body weight, cyclic state, and age of the corpus luteum were studied in aging rats with acutely implanted electromagnetic blood flow probes. Ovarian function was monitored by estimation of serum 17-beta-estradiol (E2) and progesterone (P) levels during (i) different stages of body maturation as indexed by body weight, and (ii) various stages of the estrous and pseudopregnancy (PSP) cycles. A distinct elevation in basal OBF rates was observed to occur in relation to body weight/age. Basal OBF rates and body weight exhibited a near linear correlation (r = 0.985; P less than or equal to 0.001) between 200 and 260 g, with a rise in mean basal OBF rates from 1.1 to 3.2 ml/min occurring in Day-8 PSP rats (Day 0 = ovulation). In weight-regulated (greater than or equal to 250 g) cycle rats, OBF increased in parallel with serum E2 levels during the proestrus-estrus period in association with ovulation, and subsequently declined to basal levels on diestrus. In PSP rats, OBF remained elevated between Days 1 and 8, during the period of peak luteal function. Between Days 8 and 12, both OBF and luteal function declined in a parallel manner as the ovary prepared for the subsequent ovulatory period. These results indicate that during the estrous cycle, OBF rates and serum E2 levels rise in a parallel manner, whereas during PSP, serum P levels and OBF are positively correlated. The parallel decline in OBF and serum P during luteolysis suggest that both parameters are functionally related and may be regulated by either a systemic or intraovarian controlling mechanism.     

Evidence for dopaminergic regulation of prolactin and a luteotropic complex in the ferret

The role of dopaminergic agents in prolactin (Prl) release and the luteotrophic role of Prl and luteinizing hormone (LH) were investigated in pseudopregnant female ferrets. A single injection of the dopamine antagonist pimozide (0.63 mg/kg) resulted in a tenfold elevation of plasma Prl in anestrous females. Subcutaneous injection of pimozide on alternate days from Day 2 through Day 16 of pseudopregnancy elevated both Prl and progesterone levels. Daily treatment with the dopamine agonist 2 alpha-bromoergocryptine (bromocriptine, 4 mg/kg), from Day 2 through Day 16 of pseudopregnancy lowered levels of both plasma Prl and progesterone. Neither pimozide nor bromocriptine had a direct effect on progesterone secretion by luteal cells in vitro. Daily intraperitoneal administration of a monoclonal antibody against gonadotropin-releasing hormone from Day 2 through Day 10 of pseudopregnancy lowered both plasma LH and progesterone, but had no effect on plasma Prl concentrations. Daily administration of equine antisera against bovine LH or 100 IU of human chorionic gonadotrophin to pseudopregnant ferrets lowered progesterone levels. It is concluded that Prl release is influenced by dopaminergic compounds, and both Prl and LH are required for luteal maintenance in the ferret.     

Changes and localization of ovarian carbonyl reductase during pseudopregnancy and pregnancy in rats

The present study investigates changes in the activity and enzyme content of ovarian carbonyl reductase (CR), which catalyzes the reduction of 9-keto and 15-ketoprostaglandins in rats during pseudopregnancy and pregnancy. The activity of ovarian CR decreased from the onset of pseudopregnancy and pregnancy, reaching 20-30% of the Day 1 value by Day 12 of pseudopregnancy and 50-60% of the Day 1 value by Day 14 of pregnancy. In the case of pregnant rats, the enzyme activity maintained a minimal level between Day 14 of pregnancy and Day 22 of parturition. An acute increase of the enzyme activity was found on the morning after parturition. The CR content in the ovary maintained a constant level from Day 1 to Day 12 of pseudopregnancy and to Day 18 of pregnancy. In pregnant rats, there was a gradual decrease after 18 days and then a surge during parturition. CR was primarily localized in interstitial gland cells and in theca interna cells but was not found in corpora lutea cells in the ovary during the estrous cycle. Additional immunostaining was also observed in corpora lutea cells during pseudopregnancy and pregnancy. The changes in ovarian CR activity, i.e. the rapid decrease with progressing pseudopregnancy and pregnancy, correlated with the increase in progesterone and the decrease in LH. These results indicate that rat ovarian CR may be regulated via the hypothalamo-pituitary-ovarian axis and may also be involved in luteal function.     

Effects of PGE1 or PGE2 on luteal function in pseudopregnant rats

Effects of PGE1 or PGE2 on luteal function were studied in 163 pseudopregnant rats. PGE1 (10, 100, or 300 micrograms) given intrauterine every 6 hr did not shorten pseudopregnancy (P greater than 0.05), however, the same doses of PGE2 given intrauterine every 6 hr advanced luteolysis (P less than 0.05). PGE1 (100 or 300 micrograms) given every 4 hr intramuscular maintained levels of progesterone in peripheral blood above controls (P less than 0.05) while 100 or 300 micrograms of PGE2 hastened the decline in progesterone (P less than 0.05). The antiluteolytic effect of PGE1 was not via an inhibition of PGF secretion (P greater than 0.05) by the uterus or by induction of ovulation in treated animals. Moreover, PGE1 (100, 200, or 500 micrograms) given intramuscular every 4 hr from day 4 of pseudopregnancy until the next proestrus delayed luteal regression around 3 days (P less than 0.05). PGE2 at doses of 100, 200, or 500 micrograms every 4 hr given intramuscular consistently shortened pseudopregnancy (P less than 0.05). Lower doses were without effect (P greater than 0.05). Based on the above data it is concluded that PGE2 is consistently luteolytic whereas PGE1 is not luteolytic in pseudopregnant rats and that PGE1 may be an antiluteolysin.     

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants—terrestrial manatee relatives—were targeted. These included 5α-reduced progestins (5α-pregnane-3,20-dione [5α-DHP] and 3α-hydroxy-5α-pregnan-20-one [5α-P3-OH]) and 17α-hydroxyprogesterone (17α-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20α-hydroxyprogesterone (20α-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5α-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5α-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17α-OHP metabolite was not detected in any tested female. The 20α-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.