The persistence of the corpus luteum of pregnancy into lactation in the marsupial bandicoot, Isoodon macrourus

The bandicoot is unique among marsupials in possessing a corpus luteum of pregnancy which continues to secrete progesterone during lactation. Different factors which may influence the activity of this corpus luteum were examined. There was no correlation between the number of pouch young and the plasma progesterone concentration. Similarly, bromocriptine did not appear to cause a dramatic decrease in plasma progesterone early in lactation, however, it may induce premature regression of the corpus luteum late in lactation. Corpora lutea were not observed in the ovaries of two out of six bandicoots treated with bromocriptine late in lactation. Although the corpus luteum may influence ovarian activity early in lactation, and corpus luteum does not appear to influence reproduction late in lactation. Removal of corpora lutea at Day 30 of lactation caused an abrupt decline in plasma progesterone; however, the birth of the subsequent litter occurred on Days 58 and 61 (N = 2), births normally occurring on Day 60.     

Release of cyclic AMP and progesterone from the isolated perfused luteal ovary of the PMSG treated rat

A model for in vitro perfusion of the isolated rat ovary was developed. In the luteinized ovaries of PMSG treated rats the relationship between perfusion flow and progesterone production was evaluated. A positive correlation was found indicating that a high blood flow rate may be necessary for an optimal secretion of progesterone from the fully developed corpus luteum in the rat. Also the effects of a continuous exposure of LH on ovarian cyclic AMP and progesterone release was studied. A rapid and sustained increase in both cyclic AMP and progesterone release was seen and this release tended to have episodic pattern with elevations occurring at intervals between 30 and 90 min following LH.     

Ovarian oxytocin and neurophysin concentrations in the cynomolgus monkey (Macaca fascicularis)

The neurohypophysial hormone oxytocin has previously been found in the ovaries of several animal species. In ruminants ovarian oxytocin is postulated to have a luteolytic function, because of its high concentrations in the corpus luteum. In primates the role of ovarian oxytocin is not known. In the present study we measured the immunoreactive oxytocin and oxytocin-neurophysin content in paired ovaries removed from cynomolgus monkeys (Macaca fascicularis) during the late luteal phase of the cycle (Days 12-14 of the luteal phase or Days 26-28 of a menstrual cycle). Each animal was pulsed with synthetic gonadotropin-releasing hormone to maintain normal menstrual cyclicity. The concentration of oxytocin and its neurophysin during the late luteal phase was greater in the non-corpus luteum than corpus luteum-bearing ovary. By high pressure liquid chromatography and bioassay the oxytocin in both the corpus luteal and non-corpus luteal ovaries was similar to synthetic and posterior pituitary oxytocin. The finding of high concentrations of immunoreactive oxytocin in the non-corpus luteum-bearing ovary suggests that the function of ovarian oxytocin in primates may not be confined specifically to the corpus luteum.     

Effects of follicle stimulating hormone and luteinizing hormone upon cyclic AMP and cyclic GMP levels in rat ovaries in vitro.

In vitro effects of FSH and LH on adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels in rat ovarian tissue were studied. Ovaries from immature rats treated with either pregnant mare's serum (PMS) or PMS and human chorionic gonadotropin (hCG) were incubated at 37 C for 30 min in defined medium 199 containing aminophylline (0.1 mg/ml). Increasing concentrations of LH (0.4-10.0 mug) when added to the medium containing ovaries with predominantly follicular tissue (PMS-treated) produced progressive increases in cyclic AMP (29-300%) and concomitant decreases in cyclic GMP (41-70%). FSH (4-100 mug) added to the medium produced comparable changes in nucleotide levels: cyclic AMP increased 52-390% and cyclic GMP decreased 45-80%. Ovaries with predominantly luteal tissue (PMS- and hCG-treated) responded differently to the same experimental conditions. LH produced a much greater increase in tissue cyclic AMP levels (209-1280%), whereas FSH produced comparable increases in cyclic AMP as seen with follicular tissue (103-570%). Neither gonadotropin caused any changes in cyclic GMP levels in luteinized ovaries. These results indicate that rat ovarian cyclic GMP is regulated by gonadotropins and the differences in the direction of nucleotide response to gonadotropins suggest divergent roles for cyclic AMP and cyclic GMP in ovarian function.     

PHARMACOLOGICAL''RESCUE''OF THE CORPUS LUTEUM RESULTS IN INCREASED INHIBIN PRODUCTION

Inhibin production by the corpus luteum was investigated by undertaking pharmacological rescue of the corpus luteum with hCG in four healthy women. Blood samples were collected daily for two menstrual cycles. Starting 7 days after the LH surge in the second cycle, incremental doses of hCG (125-8000 IU) were administered daily for 7 days resulting in hCG levels comparable to those seen in normal pregnancy. Following hCG, the luteal phase was prolonged and there were significant increases in the plasma concentrations of inhibin (P less than 0.05), and oestradiol (P less than 0.05). The progesterone concentration was maintained at the mid-luteal phase peak and as a result was significantly higher than those on the equivalent days of the control cycle (P less than 0.05). It was concluded that rescue of the corpus luteum with physiological levels of hCG resulted in a significant output of inhibin, thus suggesting that the corpus luteum is a significant source of inhibin in early pregnancy.     

In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin

Although it is well established that the secretory activity of the corpus luteum absolutely depends on the presence of pituitary-derived luteinizing hormone (LH), it is unknown why the life span of the corpus luteum is extended during early pregnancy by the placental production of chorionic gonadotropin (CG) but regresses in the presence of LH despite the fact that CG and LH have similar actions on the corpus luteum. To compare the responses of the corpus luteum to LH and human CG (hCG), cynomolgus monkeys whose endogenous gonadotropin secretion was blocked during the luteal phase of the menstrual cycle with a gonadotropin-releasing hormone antagonist were i.v. infused with either LH or CG. Infusion of LH at a constant rate overcame the gonadotropin-releasing hormone antagonist-mediated premature luteal regression but failed to prolong the functional life span of the corpus luteum. Continuous infusions of hCG did not effect a pregnancy-like pattern of gonadotropin secretion, but the functional life span of the corpus luteun was extended in two of three animals. Infusion of either LH or hCG in an exponentially increasing manner prolonged the functional life span of the corpus luteum beyond its normal duration. These results indicate that luteal regression at the termination of nonfertile menstrual cycles is caused by a large reduction in the responsiveness of the aging corpus luteum to LH, which can be overcome by elevated concentrations of either LH or CG.     

Reevaluation of the role of cyclic adenosine 3',5'-monophosphate and protein kinase in the stimulation of steroidogenesis by luteinizing hormone in bovine corpus luteum slices.

Incubation of bovine corpus luteum slices at 37 C with luteinizing hormone showed that 10.0 microng LH/ml caused a maximum rise in tissue cyclic AMP content within 15 min. Slices incubated with 1.0 or 0.1 microng LH/ml showed a much more gradual accumulation of this nucleotide. In the absence of added LH, a marked decline in the amount of cyclic AMP was observed during the first 60 min. The possible role of cyclic AMP in the action of LH was reexamined by studying the dose-response effect of LH on the stimulation of progesterone synthesis, cyclic AMP-dependent protein kinase activation, and cyclic AMP accumulation. After a 2-h incubatin, the results showed that the dose required to elicit a minimal significant stimulation of steroidogenesis was 0.01 microng/ml LH. At this and higher concentrations of LH, a concomitant stimulation of protein kinase activity and progesterone synthesis was also consistently observed. However, significant accumulation of cyclic AMP became consistently detectable only at 0.1 microng/ml LH. This report is the first to show a positive correlation between the activation of cyclic AMP-dependent protein kinase and the stimulation of steroidogenesis in the corpus luteum at the same minimal effective level of LH. These results indicate that cyclic AMP and the cyclic AMP-dependent protein kinase probably play important intermediary roles in the stimulation of steroidogenesis by LH in the bovine corpus luteum.     

Morphological and endocrinological studies on follicular development during the human menstrual cycle

In order to clarify the morphological dynamics of follicular development and its correlation with ovarian endocrine activity, the present studies were performed in 45 regularly menstruating women who underwent gynecological surgery. Ovarian venous blood was collected from 35 women during the follicular phase. Thirteen of these 35 women were ovariectomized. In addition, 11 pairs of ovaries were obtained from women during the luteal phase. The ovaries were sectioned serially at 2.5 micron and every 13th stained slice was examined to assess the sizes and numbers of atretic and nonatretic follicles. The follicles were divided into five stages: 0.4 less than or equal to approximately less than 1.0 mm, 1.0 less than or equal to approximately less than 2.0 mm, 2.0 less than or equal to approximately less than 4.0 mm, 4.0 less than or equal to approximately less than 6.0 mm, and 6.0 mm less than or equal to approximately in follicular diameter. Estradiol concentrations in ovarian venous plasma were low on both sides on days 1 and 3 of the cycle, whereas a clear asymmetry was found on day 5 before morphological recognition of the dominant follicle. Thereafter, estradiol increased proportionally to the growth of the dominant follicle, followed by a sudden drop when ovulation was imminent. An asymmetrical rise of progesterone occurred on day 10 and later which was sustained up to ovulation. A dominant follicle was recognized in 8 of 11 women between days 6 and 14. All dominant follicles were invariably associated with higher estradiol concentrations in the ipsilateral ovarian blood. Seven of 8 dominant follicles were on the side contralateral to the preceding corpus luteum. The mean diameters of the largest nonatretic follicles were 5.4 +/- 0.3 (SE) mm during the luteal phase as a whole and 4.7 +/- 0.7 mm during the late luteal phase. The mean diameters of the largest nonatretic follicles were not significantly different between the groups with or without the corpus luteum in the luteal phase. In terms of number and atretic rate, follicles of less than 4.0 mm in diameter did not change throughout the cycle in the presence or absence of the corpus luteum. In contrast, cyclic changes of growth and atresia occurred in the larger antral follicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for luteotrophic and antiluteolytic actions of prolactin in rats with 5-day oestrous cycles

Injection of 1 mg bromocriptine at either 08.00 or 16.00 h on the day of oestrus in rats with 5-day oestrous cycles caused a reduction in the duration of progesterone secretion by the corpus luteum during dioestrous, and a shortening of the ovarian cycle by 1 day. These effects were not present when bromocriptine was injected at 08.00 h on the day of metoestrus. The effect of bromocriptine on progesterone secretion by the corpus luteum was reversed by neutralization of the biological activity of LH at dioestrus by injection of 0.5 ml anti-LH serum at 08.00 h at metoestrus. Injection of the antiserum alone prolonged progesterone secretion by the corpus luteum, but had no effect on the length of dioestrus. These results are interpreted as suggesting (1) that prolactin secretion on the afternoon of oestrus protects the corpus luteum of the rat ovarian cycle against the luteolytic effects of LH secretion during early dioestrus and (2) that prolactin stimulates progesterone secretion in the absence of such a luteolytic action. This response of the corpus luteum of the rat ovarian reproductive cycle to prolactin results in 5-day oestrous cycles.     

Oxytocin, oxytocin-associated neurophysin, and prostaglandin F2 alpha concentrations in the utero-ovarian vein of pregnant and nonpregnant sheep

Oxytocin, oxytocin-associated neurophysin (neurophysin), prostaglandin F2 alpha (PGF2 alpha), and progesterone concentrations were measured in the utero-ovarian vein (UOV) of sheep during the estrous cycle and early pregnancy. On days 13-16 of the cycle, large pulses of PGF2 alpha, oxytocin, and neurophysin were measured in samples collected at hourly intervals from the UOV draining a corpus luteum (UOV/CL). Most of the PGF2 alpha pulses (96.5%) coincided with a pulse of both oxytocin and neurophysin, whereas only 55.6% of oxytocin pulses coincided with a pulse of PGF2 alpha. Therefore, during luteolysis in sheep, uterine PGF2 alpha release is closely associated with ovarian oxytocin release, and oxytocin release is unlikely to be dependent upon a uterine PGF2 alpha stimulus. During frequent sampling, coincident oxytocin pulses were measured in 1) both UOVs when a CL was present in both ovaries and 2) the jugular vein, carotid artery, and UOV/CL, with a significantly higher oxytocin pulse concentration occurring in jugular venous compared with carotid arterial plasma. Pituitary and luteal release of oxytocin may, therefore, occur simultaneously and be controlled by a circulating factor in sheep. Compared to days 13-16 of the cycle, significantly (P less than 0.001) fewer pulses of PGF2 alpha, which were significantly (P less than 0.001) smaller in amplitude, were measured in UOV samples collected frequently during early pregnancy. The frequency of oxytocin pulses observed in the UOV/CL of pregnant sheep was not significantly (P greater than 0.1) different from that observed in cyclic ewes, although most (86.4%) oxytocin pulses occurred in the absence of a PGF2 alpha pulse. In contrast, when a pulse of PGF2 alpha was observed in the UOV/CL of pregnant ewes, it usually coincided with a pulse of oxytocin. The suppression of uterine PGF2 alpha release during early pregnancy is not considered to result from a lack of stimulation by oxytocin.     

Human corpus luteum secretion of relaxin, oxytocin, and progesterone

To determine whether the human corpus luteum is a source of relaxin and oxytocin, we measured the concentrations of these peptides in plasma obtained from the ovarian veins of ovaries with and without a corpus luteum and compared these to peripheral plasma levels. Peripheral and ovarian venous blood samples were obtained from 34 nonpregnant women, 13 during the luteal phase and 21 during the follicular phase of their cycles, and from a 6-week pregnant woman. Plasma relaxin, oxytocin, and progesterone concentrations were determined by sensitive and specific RIAs. Plasma relaxin levels were not detectable (less than 0.16 microgram/L) in peripheral or ovarian venous plasma not draining a corpus luteum. The mean relaxin concentration in plasma draining an ovary with a corpus luteum was 0.41 +/- 0.09 (+/- SE) microgram/L. Oxytocin levels also were significantly higher in plasma draining an ovary with a corpus luteum (6.70 +/- 1.86 pmol/L) than in that draining the ovary with no corpus luteum (1.58 +/- 0.09 pmol/L; P less than 0.01) or in peripheral plasma (1.58 +/- 0.09 pmol/L; P less than 0.025). The mean progesterone concentration also was highest in plasma draining an ovary with a corpus luteum (210.2 +/- 50.5 nmol/L) compared with those in plasma from the contralateral ovarian vein (40.3 +/- 16.5 nmol/L P less than 0.005) and peripheral plasma (30.2 +/- 5.7 nmol/L; P less than 0.005) during the luteal phase. In a woman who was 6 weeks pregnant, plasma draining the ovary with a corpus luteum had 1.9 micrograms relaxin/L, but only 0.49 pmol/L oxytocin; the latter was similar to concentrations in noncorpus luteum-bearing ovarian venous plasma. These findings indicate that the human corpus luteum secretes relaxin, oxytocin, and progesterone. Both ovarian oxytocin and relaxin may function as paracrine or autocrine modulators of luteal function.     

The effects of prolactin and prostaglandin F2 alpha on plasma membrane changes during luteolysis in the rat

The ability of PRL to modify prostaglandin F2 alpha (PGF2 alpha)-induced membrane changes during functional corpus luteum regression was examined in the pseudopregnant rat. Fluorescence polarization studies conducted 24 h after sc injection of PGF2 alpha revealed a marked and significant increase in the polarization parameter, which is suggestive of reduced plasma membrane fluidity. At the same time, there was a decrease in hCG binding and a significant increase in apparent phospholipase-A2 activity during incubation of dispersed rat luteal cells. Each of these changes was attenuated when the animals were pretreated with PRL 30 min before PGF2 alpha. The decrease in plasma progesterone caused by PGF2 alpha treatment was also inhibited by PRL. PGF2 alpha also stimulated a significant polarization increase in dispersed cells prepared from ovaries removed 1 h after injection of this luteolytic agent, although this effect could not be demonstrated in plasma membrane samples. These results indicate that PRL and PGF2 alpha affect the same membrane pathway in the rat corpus luteum and that this pathway appears to be closely coupled to luteal cell function.     

Ontogenesis, distribution and relative sensitivity of hCG receptors to hCG and LH in goat ovaries.

The binding of [125I]hCG to immature and mature follicles and corpora lutea of goat ovaries has been studied. The hormone is bound maximally by corpora lutea although mature follicles also exhibit some binding. Immature follicles are practically devoid of receptors for this hormone. In the corpus luteum, the receptors for the hormone are present in thecal and luteal cells. Autoradiographic studies show the location of the bound radioactivity grains primarily along the plasma membranes of these cells, although some radioactivity grains were also seen in the cytoplasm of luteal but not thecal cells. On a mole to mole basis, hCG was found to displace [125I]hCG from binding to receptors on corpus luteum better than hLH and oLH.     

The effect of melatonin on steroidogenesis by the human ovary in vitro.

Melatonin stimulated steroidogenesis in two compartments of the human ovary in vitro. In a corpus luteum of the menstrual cycle, melatonin increased progesterone synthesis in a dose related manner. Both serotonin and N-acetyl serotonin had no effect on progesterone synthesis. In the ovarian stroma, melatonin stimulated the incorporation of acetate-1-14-C into androstenedione. Binding of radioactive hCG by the corpus luteum was unaffected by melatonin. No specific binding of radioactive melatonin of low specific activity could be detected in homogenates of a human corpus luteum. These observations suggest that melatonin may directly modulate steroidogenesis in the human ovary.     

Mediation of gonadotrophin-stimulated growth and differentiation of human granulosa cells by adenosine-3'',5''-monophosphate: one molecule, two messages

OBJECTIVE: To determine how the second messenger adenosine-3',5'-monophosphate (cyclic AMP) is able to mediate divergent actions of FSH and LH on granulosa cell growth and differentiation in human ovaries. DESIGN: Human granulosa cells were cultured for 96 hours in serum-free medium 199 containing increasing doses of either FSH, LH or dibutyryl cyclic AMP. Extra and intra-cellular cyclic AMP levels were determined by radioimmunoassay. Tritiated thymidine uptake and cell number were measured as indices of cell growth, and spent medium was assayed for steroids (oestradiol and progesterone) to reflect differentiation. PATIENTS: 'Mature' granulosa cells were aspirated from preovulatory follicles in the ovaries of clomiphene-stimulated patients undergoing laparoscopic sterilization; 'luteinized' granulosa cells were aspirated from periovulatory follicles in the ovaries of gonadotrophin-stimulated in-vitro fertilization patients. RESULTS: LH consistently inhibited, whereas FSH maintained or stimulated, basal granulosa cell numbers. Steroidogenesis was dose-dependently increased by both gonadotrophins, with LH having the significantly greater effect over the entire dose-response range (1-100 micrograms/l). LH also induced significantly more cyclic AMP production than FSH, both intra and extra-cellularly, providing a basis for differential post-receptor signalling via a common second messenger. Addition of dibutyryl cyclic AMP, at low concentrations (10-250 mumol/l) to the cultured cells in the absence of gonadotrophins mimicked FSH effects with stimulation/maintenance of cell numbers and moderate steroidogenesis. High concentrations of dibutyryl cyclic AMP (500-1000 mumol/l) caused a significant inhibition of cell numbers together with maximal steroidogenesis, simulating LH action. CONCLUSIONS: These results suggest that granulosa cell maturation in the follicular phase of the menstrual cycle (controlled by FSH) is associated with a low cyclic AMP tone that favours cell growth and expression of aromatase activity in the developing preovulatory follicle. During the early luteal phase (dominated by LH), the intracellular cycle AMP tone increases to allow maximal progesterone production and inhibition of cell growth in the corpus luteum. Thereby one second messenger can mediate divergent gonadotrophic effects on granulosa cell growth and differentiation in the human ovary.     

Immunocytochemical localization of inhibin alpha-subunit in the human corpus luteum

The localization of inhibin alpha-subunit within the human corpus luteum was investigated. The antiserum used was raised in sheep against the first 1-23 amino acid sequence of the N-terminus of the human inhibin alpha-subunit. Using the avidin-biotin immunoperoxidase technique, intense immunostaining was localized within the granulosa-lutein cells of the corpus luteum, with absence of staining in the theca-lutein cells and surrounding ovarian tissue. Similar distribution of inhibin alpha-subunit immunostaining was observed in 12 corpora lutea obtained during the early, mid- and late-luteal phases and no changes in intensity were apparent at these different stages. Negative controls were obtained by applying antiserum which had been preabsorbed overnight with excess inhibin peptide in place of primary antiserum and also normal nonimmune sheep serum as a substitute for primary antiserum. These results provide further evidence that the human corpus luteum is a significant source of immunoreactive inhibin during the normal human menstrual cycle. The specific localization within the granulosalutein cells of the corpus luteum suggests that inhibin alpha-subunit production may originate from a discrete cell population within the human corpus luteum.     

Redistribution of ovarian blood flow after injection of human chorionic gonadotropin and luteinizing hormone in the adult pseudopregnant rat

It is well known that LH and human CG (hCG) induce an increase in total ovarian blood flow. The effect of LH/hCG on luteal blood flow, however, is unknown. This work studies the effect of hCG on both luteal and ovarian blood flows at different stages of pseudopregnancy in adult female rats. Pseudopregnancy was induced by mating with sterile male rats. The length of pseudopregnancy was 13 +/- 1 days and, during this time, blood flow was measured by the injection of radioactive microspheres during anesthesia. At autopsy, the corpora lutea were identified and extirpated under a stereomicroscope. These, and the remaining ovary, were then counted for radioactivity and the blood flow was calculated. Progesterone levels were determined in plasma and ovarian tissues. Furthermore, the responsiveness of adenylate cyclase was tested in ovarian tissues at day 6 of pseudopregnancy. An intraarterial injection of hCG (50 IU) or vehicle (saline) was given 20 min before the blood flow determinations in anesthetized rats. The luteal blood flow was not changed by hCG on days 2, 6, and 11 of pseudopregnancy, whereas in the remaining ovary the blood flow increased more than 2-fold, thereby resulting in redistribution of the blood flow. Ten micrograms of NIH-LH-B9, tested at day 6 of pseudopregnancy, mimicked the effect of hCG. At day 6 of pseudopregnancy, hCG (50 IU) was given ip to conscious rats 200 min and 24 h before blood flow determinations. At 200 min after hCG there was a more pronounced redistribution of ovarian blood flow with a 45% reduction in luteal blood flow and a 4-fold increase in flow through the remaining ovary. LH as well as hCG doubled the progesterone content of the remaining ovary. In the corpora lutea an increased progesterone content was seen after 200 min of hCG exposure. At 24 h after hCG injection, all parameters had returned to control levels except that adenylate cyclase was nonresponsive. The increase in the total ovarian blood flow coincides with the increased steroidogenesis and these effects are likely due to release of metabolites and/or vasoactive substances. Despite this increase, the blood flow of the corpus luteum was not increased rendering vascular mechanisms unlikely as a part of the acute LH/hCG effects on corpus luteum of pseudopregnancy.     

Source of ovarian inhibin secretion during the oestrous cycle of the sheep

The source of inhibin secretion by the ovary in the sheep at different stages of the oestrous cycle was investigated by in-vivo cannulation of the ovarian veins. Twenty-four Scottish Blackface ewes were allocated to four groups of six ewes, i.e. those operated on during the luteal phase (day 10), and those operated on during the follicular phase 24-30, 36 and 60 h following an injection of 125 micrograms cloprostenol on day 10 of the luteal phase. Samples of jugular and timed ovarian venous blood were collected under anaesthesia before and after enucleation of the corpus luteum. Ovaries were then removed and follicles dissected out. Following injection of cloprostenol, luteal regression occurred as indicated by a fall in the secretion of progesterone. The concentration of inhibin in jugular venous plasma and its ovarian secretion rate were similar at all stages of the follicular phase and during the luteal phase. In contrast, the secretion rate of oestradiol rose from 2.86 +/- 0.73 pmol/min during the luteal phase to 8.70 +/- 2.24 pmol/min 24 h after injection of cloprostenol (P less than 0.05). Following enucleation of the corpus luteum the secretion rate of progesterone fell from 809 +/- 270 pmol/min to 86 +/- 30 pmol/min (P less than 0.001). There was also a smaller, artifactual fall in the secretion rate of oestradiol following enucleation of the corpus luteum, which was of similar size to a fall seen in the secretion rate of inhibin. This resulted in a significant (P less than 0.001) fall in the ratio of progesterone to inhibin, while the oestradiol to inhibin ratio remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local production of progesterone by the corpus luteum of the marmoset monkey in response to perfusion with chorionic gonadotrophin and melatonin in vivo

The effect of human chorionic gonadotrophin (hCG) and melatonin on the local production of progesterone by the marmoset corpus luteum was investigated in vivo using a perfusion cannula system. Progesterone secretion was measured in 10-min fractions of buffer which had been perfused through the corpus luteum at a flow rate of 70 microliters/min for a maximum of 3 h in anaesthetized animals. Two corpora lutea were cannulated in each animal; one for perfusion of test material and the other for perfusion with buffer alone as a control. Perfusion with hCG (25 i.u./ml), investigated as a positive control, produced a marked stimulation of progesterone secretion which increased 10-20 min from the start of perfusion and reached a peak after 30-60 min. A stimulation of progesterone was also observed after perfusion with melatonin (860 pmol/l). The response was evident within 10-30 min of the hormone reaching the corpus luteum and was similar in magnitude to that observed for hCG. The ability of melatonin to stimulate progesterone secretion supports previous in-vitro studies and suggests an ovarian action for melatonin in the primate. The local perfusion system described may have potential uses in studies of luteal function related to aspects of infertility or regulation of fertility.     

Localization of renin gene expression to monkey ovarian theca cells by in situ hybridization.

To investigate the sites of renin gene expression and localization of renin in primate ovaries, five cynomolgus (Macaca fascicularis) and one rhesus (Macaca mulatta) monkey were treated with gonadotropins to induce multiple follicle development. One ovary was removed before hCG injection (1200 IU) from three monkeys and one ovary was removed 36 h after hCG administration from three monkeys. In three monkeys, the remaining ovary was removed 3, 5, and 7 days after injection of hCG. To detect and localize renin messenger RNA, 35S-radiolabelled 1.1 kb length complementary DNA and RNA probes of human renin were used for in situ hybridization. To compare the synthesis with the presence and the storage of renin or prorenin, renin antigen was assessed by immunohistochemistry in the same tissues using a polyclonal antibody against human renin (R15). Renin mRNA was detected by in situ hybridization only in ovaries collected within 5 days of exposure to hCG. All such ovaries exhibited a positive signal. Renin mRNA was localized to the theca interna and theca lutein cells. Positive cells were observed in a few growing antral follicles, in occasional mature preovulatory follicles, in corpus luteum, and most strikingly in atretic follicles. No signal was detected in primordial, primary, or in small antral follicles of ovaries exposed to hCG. In contrast with the in situ hybridization data, no signal was detected by immunohistochemistry using antirenin antibodies which exhibited a positive signal in monkey kidney. These results indicate that hCG turns on renin gene expression. Renin is synthesized without significant intracellular storage in monkey ovarian theca interna cells and in corpus luteum. The absence of storage of renin is consistent with the high concentrations of prorenin found in ovarian follicular fluid of hCG stimulated primates and with our knowledge of cellular renin processing which indicate that prorenin is secreted constitutively as it is synthesized.