Seasonal variations in the responses of luteal and follicular luteinizing hormone-stimulated adenylyl cyclases to estradiol implants and implant removal in pseudopregnant rabbits.

Summer rabbits appeared to be less sensitive to the presence of exogenous estradiol or to the withdrawal of exogenous estradiol than were Winter rabbits. Suppression of the luteinizing hormone(LH)-stimulated adenylyl cyclase of corpora lutea by estradiol-filled silastic capsules, which resulted in serum estradiol concentrations approximately 3.8 X control (high level), appeared to be greater in Winter rabbits than in Summer rabbits. Both high level and low level (2.1 X control) estradiol implants suppressed follicular LH-stimulated adenylyl cyclase in Winter rabbits but neither size capsule had an effect in Summer rabbits. Serum LH concentrations were equally low in Winter rabbits using either high or low level estradiol implants, while only the high level implants caused a decrease in serum LH in Summer rabbits. Withdrawal of the exogenous estradiol caused a precipitous fall in serum progesterone concentrations in Winter rabbits; estradiol withdrawal in Summer rabbits was without effect.

The membrane-bound spermatozoal adenylyl cyclase system does not share coupling characteristics with somatic cell adenylyl cyclases.

Membrane-bound adenylyl cyclases from ram, dog, and human sperm are unresponsive to fluoride and guanylylimidodiphosphate [GMP-P(NH)P], two agents that stimulate the adenylyl cyclases of somatic cells by an action on the stimulatory guanine nucleotide-binding regulatory (Ns) component of adenylyl cyclase. We have investigated whether this is because the sperm cell catalytic unit is functionally uncoupled from Ns but, nevertheless, capable of interacting with it, or because the sperm cell adenylyl cyclase system is unique and regulated differently from that of somatic cells. Sperm cells were found to be deficient in Ns, as evidenced by the inability of detergent extracts from sperm cell membranes and fractions to reconstitute Ns-mediated regulation of the adenylyl cyclase of cyc- S49 cells. In addition, attempts to label Ns in sperm cell membranes by [32P]ADP ribosylation with cholera toxin revealed that, if present, Ns is less than 1% of that found in human erythrocyte membranes. This, however, was not the only reason for the unresponsiveness of sperm cell adenylyl cyclase, since fluoride stimulation of the sperm cell enzyme could not be induced by reconstituting it with Ns purified from human erythrocytes (hRBC). When intact hRBC membranes were added to sperm cell fractions in the presence of fluoride, the activities that resulted were greater than the sum of the individual activities. This apparent reconstitution of fluoride regulation of sperm cell adenylyl cyclase could be blocked by lima bean trypsin inhibitor and appears to have resulted from proteolytic activation of the hRBC adenylyl cyclase by sperm proteases. Sperm cell membranes also appear to lack a functional inhibitory regulatory protein of the adenylyl cyclase system (Ni), since they did not contain an ADP-ribosylatable substrate for pertussis toxin action. These results suggest that the sperm cell adenylyl cyclase system is unique and different from that of somatic cells. Sperm cells appear to neither contain Ns or Ni nor possess the ability of their adenylyl cyclase system to interact with Ns from an exogenous source.

Immunization with zona pellucida proteins results in abnormal ovarian follicular differentiation and inhibition of gonadotropin-induced steroid secretion.

Changes in rabbit ovarian hormonal responses and cellular differentiation of ovarian follicles after immunization with porcine zona pellucida (ZP) have been examined. Steroid and peptide hormone levels were monitored after immunization to evaluate ovulation and pseudopregnancy cycles in immunized and control animals. All immunized rabbits developed serum antibodies to specific ZP antigens and failed to form functional corpora lutea in response to hCG administration, as evidenced by the absence of elevated serum progesterone concentrations. This is in contrast to control rabbits, which had elevated progesterone levels 8-9 days after hCG administration. Furthermore, all immunized animals showed greatly increased serum levels of FSH and LH compared to those of control animals. These effects on ovarian function were apparent within 20 weeks of the primary immunization. Follicular development was analyzed by light and electron microscopies. The numbers of primary, secondary, and tertiary follicles in ovaries of immunized animals were markedly reduced within 7 weeks compared with control values. By 23 weeks, few if any growing follicles were present. Although numerous distinct clusters of cells with ultrastructural properties that resemble those of normal follicular cells were present in immunized animals, they contained no oocytes. These studies suggest that antibodies to ZP glycoprotein alter ovarian function by interfering with cells during the stage of follicle differentiation at which the ZP proteins are being synthesized and secreted. This system should provide an excellent model with which to study the early events associated with ovarian follicular cell differentiation and subsequent hormonal responsiveness.

Changes in content and phosphorylation of cytosol proteins in luteinizing ovarian follicles and corpora lutea.

Cytosol prepared from rat preovulatory ovarian follicles contained several specific substrates which were phosphorylated by [gamma 32P] ATP in the presence of 2 microM cyclic AMP (cAMP) or 780 nM of highly purified catalytic subunit. These substrates were identified as RII, the regulatory subunit of type II cAMP-dependent protein kinase, an Mr = 43,000 protein presumed to be actin, and four other proteins with Mr = 36,500-15,000. A marked decrease in phosphorylation of these proteins was observed within 6-48 h of human chorionic gonadotropin (hCG)-induced ovulation and luteinization in hormonally primed immature rats. The phosphorylation of these proteins was also low in cytosol of corpora lutea isolated on Days 2, 4, 9, 13 and 23 of pregnancy. The decrease in phosphorylation of RII was associated primarily with a decrease in substrate content as measured by photoaffinity labeling and silver staining techniques, and not to a marked increase in phosphoprotein phosphatase and adenosinetriphosphatase (ATPase) activities. Whereas the decreased phosphorylation of other proteins is also presumed to be related to a decrease in their cytosol content, the data do not exclude the possibility that luteal tissue contains a specific phosphoprotein phosphatase which is not present in granulosa or theca cells of preovulatory follicles. We conclude that luteinizing hormone (LH) or hCG, and thereby cAMP itself, induces the rapid loss of specific phosphoproteins which may be involved in regulating cAMP action in granulosa cells.

Luteal adenylyl cyclase does not develop sensitivity to desensitization by human chorionic gonadotropin in the absence of nonluteal ovarian tissue.

There is evidence suggesting that the mere presence of a hormone-responsive adenylyl cyclase system in a tissue may not be sufficient for desensitization to occur since phosphorylation reactions might also be involved. The purpose of this study was to determine if luteal tissue in the absence of other ovarian tissues would desensitize to human CG (hCG). One or both ovaries were removed from rabbits 5 h before hCG-induced ovulation and the periovulatory follicles were transplanted underneath the kidney capsule where they formed ectopic corpus luteum [or corpora lutea (CL)]. Rabbits which were bilaterally ovariectomized received estradiol implants at the time of ovariectomy to maintain control serum estradiol concentrations. On day 7 of pseudopregnancy, the rabbits were injected with saline (control) or with 75 IU hCG and were killed 24 h later at which time ovarian and ectopic CL progesterone content and adenylyl cyclase activity were assessed. As expected, in ovarian CL there was decreased LH-responsive adenylyl cyclase (69% relative to control) and a correspondingly decreased luteal progesterone content (40% relative to control). In the same rabbits, the ectopic CL showed much the same pattern of response as the ovarian CL but perhaps to a slightly lesser extent (decreases relative to control of 59% in adenylyl cyclase response to LH and 29% in progesterone content). However, in rabbits with ectopic CL only, the luteal tissue showed no change either in hormone-responsive adenylyl cyclase activity or in progesterone content. Similarly, binding of radiolabeled hCG to luteal membranes 24 h after hCG was almost totally absent in ovarian CL, was decreased by 50% in ectopic CL with one ovary present, and was unaltered in ectopic CL of bilaterally ovariectomized rabbits. These data suggest that nonluteal ovarian tissue may be required for the induction in CL of the appropriate protein kinases for the proposed phosphorylations involved in adenylyl cyclase desensitization.

Effects of estradiol treatment on rabbit luteal adenylyl cyclase: loss of luteinizing hormone receptors and attenuation of the regulatory N component activity.

We have reported previously that exogenously administered estradiol (E2) results in attenuation of the LH response of rabbit luteal adenylyl cyclase (approximately 50% less activity than control). This was accompanied by a much lesser reduction in the response of the system to the beta-adrenergic agonist isoproterenol (approximately 35% less activity than control). The purpose of the study reported here was to determine if the decreased responsiveness of adenylyl cyclase was the result of altered hormone receptor levels. To this end, hormone receptors were assessed by Scatchard analysis of specific binding. We confirmed that 4-day E2 treatments, which elevated serum E2 levels from 5 to 21 pg/ml. resulted in decreases in LH- and isoproterenol-stimulated adenylyl cyclase activities by 52% and 20%, respectively. In addition, we found that NaF-stimulated activity was also decreased by 20%. Basal adenylyl cyclase activity was unaffected. Upon assessment of the LH and beta-adrenergic receptor levels in luteal membranes, we found that E2 treatment resulted in marked reduction in LH receptor levels to 28% of the control value without changes in the levels of beta-adrenergic receptors. In view of the concomitant changes in the responsiveness of luteal membranes to isoproterenol and NaF, we determined whether E2 treatment affected luteal membrane levels of the stimulatory nucleotide-binding regulatory component (N) of adenylyl cyclase. N component activity was measured using a reconstitution assay that employs the stimulatory N component-deficient cyc- variant of the S49 mouse lymphoma cell line as an acceptor for luteal N component. Using this assay, we found that luteal membrane N component activity was reduced by 20-25% in E2-treated rabbits compared to that in control rabbits. All of the changes noted above were statistically significant. The results uncovered two heretofore unrecognized effects of E2 treatment: 1) loss of LH receptors, and 2) modification of the membrane component responsible for coupling of stimulatory receptors to the catalytic component of adenylyl cyclase.

Human chorionic gonadotropin-induced heterologous desensitization of adenylyl cyclase from highly luteinized rat ovaries: attenuation of regulatory N component activity.

We injected hCG into superovulated rats on the seventh day of pseudopregnancy and confirmed previous findings that this results in both homologous desensitization of luteal adenylyl cyclase (loss of responsiveness to LH) and heterologous desensitization of the same adenylyl cyclase system (partial loss of responsiveness to catecholamines), and that these changes are associated with the loss of available unoccupied LH receptors (down-regulation) but not with any discernible loss of beta-adrenergic receptors. We tested the hypothesis that the heterologous component of the above changes might be due to alterations in the function of the nucleotide-binding N component of adenylyl cyclase that intervenes between receptors and catalytic units of adenylyl cyclase. This was done by assessing N component activity in reconstitution assays that measured the capacity of luteal N to mediate, in cyc- S49 lymphoma membranes, stimulation of adenylyl cyclase independently by the guanine nucleotide guanylyl imidodiphosphate, by NaF, or by the lymphoma membrane beta-adrenergic receptor. By all of these modes of assay, heterologous desensitization of luteal adenylyl cyclase to beta-adrenergic stimulation was found to be associated with a proportionally similar decrease in N component activity. This change in N component activity could be due to either quantitative or qualitative alterations. It is speculated that if the change is of a qualitative nature, the alteration may be a cAMP-dependent phosphorylation reaction of one of the subunits of the N component.

Restoration of the LH surge and ovulation by insulin in alloxan-diabetic immature rats treated with pregnant mare's serum gonadotrophin.

Immature alloxan-diabetic rats injected with pregnant mare's serum gonadotropin (PMSG) do not ovulate and the LH surge is absent. In the present studies we have examined the effects of several insulin treatments on the LH surge and ovulation in alloxan-diabetic rats. Rats were male diabetic by injection of alloxan on day 24 of age. Only those rats with fasting blood glucose concentrations exceeding 180 mg/100 ml on day 237 were considered diabetic. PMSG was injected on day 30. Rats received insulin either by injection (2, 3, 4 or 6 IU/100 g/day); Ultralente B.I.D.) or by subcutaneous implants (Alzet osmotic minipumps; 1.8 or 2.4 IU/day). None of the diabetic rats without insulin treatment ovulated. Some of the animals in each insulin treatment group ovulated, however, the percentage of animals ovulating was highly variable from experiment to experiment when the insulin was given by injection. When insulin was administered by osmotic minipump, the results were more consistent, with at least 60% of the rats ovulating in each experiment. LH surges were found on the afternoon of presumed pro-oestrus (day 32) in diabetic insulin-treated rats which ovulated. In confirmation of previous results, rats without insulin treatment did not have LH surges. Although the site of insulin action has not been determined, these data indicate that the LH surge mechanism in the immature PMSG-treated rat is insulin-dependent.

Effects of withdrawal of exogenous estradiol from pseudopregnant rabbits: transient nature of loss of luteal function and reversal of estradiol-induced suppression of luteinizing hormone-responsive adenylyl cyclase.

We have reported in recent studies that exogenous estradiol (E2) suppresses luteal LH-responsive adenylyl cyclase activity in pseudopregnant rabbits. The purpose of the present study was to determine whether this suppression is reversible. High or low level E2-filled Silastic capsules or empty capsules were sc implanted in day 5 pseudopregnant rabbits. On day 8 of pseudopregnancy, the high level E2 implants were either sham replaced, replaced with low level E2 implants, or replaced with empty capsules. The low level E2 implants and empty capsules were sham replaced. Animals from each of the five resulting groups were killed on days 9-12 of pseudopregnancy (1, 2, 3, and 4 days postimplant manipulation). As previously reported using an intermittent injection protocol, exogenous E2 had little effect upon serum progesterone concentrations. Both high and low level E2 implants suppressed the luteal LH-responsive adenylyl cyclase, but the suppression due to the low level E2 implants was not as great as that for the high level E2 implants. Within 24 h of switching from high to low level E2 implants, LH-responsive adenylyl cyclase activity increased from the level found for animals with high level E2 throughout to that found for animals with low level E2 throughout. Total withdrawal of exogenous E2 resulted in a precipitous fall in serum progesterone concentrations, as predicted by previous studies. However, within 4 days of withdrawal, both serum progesterone and luteal LH-responsive adenylyl cyclase activity had returned to control values. E2 implants also suppressed serum LH concentrations and follicular LH-responsive adenylyl cyclase activities. Both of these effects were reversed within 24-48 h after implant withdrawal. We conclude, therefore, that effects of exogenous E2 are reversible and that the previously reported E2-induced dependency upon exogenous E2 is related to the experimental protocol used.

Etiology of anovulation in the immature alloxan-diabetic rat treated with pregnant mare's serum gonadotropin: absence of the preovulatory luteinizing hormone surge.

The effects of alloxan-induced diabetes on ovulation and other ovarian responses were investigated in immature rats injected with PMS gonadotropin (PMSG, 15 IU/100 g) on day 30 of age. Rats were killed on day 32 (presumed proestrus) or on day 33, at which time the oviducts were examined for ova. Ovarian weight gain was similar in control and diabetic rats and Graafian follicles were present in both groups on day 32. None of the diabetic rats ovulated while 96% of the control rats ovulated. Anovulation in diabetic rats could not be attributed to a drug side-effect of alloxan or to a lack of ovarian responsiveness, as 90% of the animals ovulated after treatment with insulin or with hCG (5 IU). Measurements of serum estradiol and LH on the morning of presumed proestrus revealed that concentrations of these hormones were not different in control and diabetic rats. However, measurements of LH in blood samples taken in the afternoon from control rats showed an LH surge, whereas no LH surge was found in diabetic rats. Thus, anovulation in immature diabetic rats treated with PMSG is not caused by an attenuation of ovarian responsiveness or by decreased secretion of estradiol, but rather is due to the loss of the LH surge.