Stimulation of pituitary luteinizing hormone secretion by gonadotropin-releasing hormone is not coupled to beta-luteinizing hormone gene transcription.

The physiological stimulation and inhibition of release of several peptide and protein hormones appears to be associated with transcription of the respective peptide- and protein hormone-encoding genes. In the current studies we investigated whether this was also true for the rat pituitary LH system. Using an anterior pituitary primary tissue culture system, we have analyzed the effects of 10(-9) M GnRH on beta LH gene transcription using a transcription run-on assay, and on nuclear beta LH RNA levels using a highly sensitive solution hybridization-S1-nuclease protection assay. GnRH-stimulated release of LH does not appear to be coupled to a significant change in the rate of beta LH gene transcription, but is associated with both a 2- to 3-fold increase in levels of the beta LH primary transcript and processing intermediates and a rapid decrease in the levels of fully processed beta LH mRNA in the nucleus. No significant change in beta LH cytoplasmic mRNA levels, however, was associated with GnRH-stimulated release of LH, in vitro. Our findings suggest that unlike several other peptide and protein hormone systems, stimulated release of beta LH by GnRH in vitro is not associated with an increase in beta LH gene transcription or cytoplasmic mRNA levels.

Evidence that changes in tonic luteinizing hormone secretion determine the growth of preovulatory follicles in the rat.

Physiological concentrations of progesterone (20-100 ng/ml), maintained by the insertion of implants into 30-day-old rats, delayed first ovulation, and withdrawal of progesterone on day 47 of age synchronized first ovulation in rats. Inhibition of ovulation involved negative feedback regulation of tonic LH and FSH secretion, blockage of gonadotropin surges, and suppression of preovulatory, but not antral, follicular growth. Removal of implants resulted in a rapid decline in serum progestrone from 100 to 5 ng/ml within 0-12 h. Between 0-36 h there were progressive increases in serum concentrations of LH and FSH, enhanced accumulation of estradiol by individual follicles incubated in vitro with or without exogenous substrate, and marked progressive increases in the content of LH (but not FSH) receptors in both thecal and granulosa cells. These events were followed by gonadotropin surges at 48 h (1800 h on day 49), ovulation, and morphological and biochemical signs of luteinization, including decreases in follicular gonadotropin receptor content and estradiol accumulation, evident by 60 h. With the exception of changes in basal LH, this sequence of events is remarkably similar in time and pattern to that after the decline of progesterone on diestrous day 2 and ovulation on proestrus of a 5-day cycle. Although a direct effect of progesterone on ovarian follicular cell function cannot be excluded, the data suggest that subtle but sustained increases in LH (and possibly FSH) are required for the enhanced follicular accumulation of estradiol and LH-binding activity occurring between diestrus and proestrus of the rat estrous cycle. Thus, perhaps some of the mystery surrounding the endocrine events between diestrus and proestrus can be ascribed to changes in serum LH that have been too small and/or variable for current nonserial sampling methods and RIAs to detect reliably.

Enhancement and desensitization of hormone-responsive adenylate cyclase in granulosa cells of preantral and antral ovarian follicles: effects of estradiol and follicle-stimulating hormone.

The present study was performed to determine if adenylate cyclase in granulosa cells was affected by the pituitary hormone FSH and the ovarian hormone estradiol. Results demonstrate that granulosa cells of intact immature rats exhibit considerably more FSH- than hCG-stimulated adenylate cyclase activity. The FSH-responsive enzyme system was not altered by hypophysectomy or by treating hypophysectomized rats with FSH alone, but was increased slightly by treatment with estradiol alone. Sequential treatment of rats with estradiol and FSH markedly increased both FSH- and LH/hCG-stimulated adenylate cyclase activities. Thus, FSH-responsive adenylate cyclase appears to be a constitutive component of granulosa cells in prenatal follicles which exhibits a pronounced increase during the development of preovulatory follicles, a change dependent on the synergistic actions of estradiol and FSH. Desensitization of FSH-responsive adenylate cyclase in granulosa cells of preantral and antral follicles was assessed by administering 5 micrograms human FSH to estradiol or estradiol/FSH-treated rats, respectively. FSH failed to induce desensitization of adenylate cyclase in granulosa cells of preantral follicles at 2 h, but did desensitize the enzyme system in granulosa cells of antral follicles. Furthermore, the desensitization of adenylate cyclase in granulosa cells of antral follicles was heterologous; both FSH and hCG exerted this effect. The causes of the differences in the response of adenylate cyclase to high concentrations of FSH at different stages of follicular development remain unclear. The absence of desensitization in preantral follicles may be required to permit a continuous nondisruptive pattern of follicular growth when small follicles are repeatedly exposed to gonadotropin surges, whereas desensitization is required for the cessation of follicular growth and luteinization.

Thyroid hormone status regulates preprotachykinin-A gene expression in male rat anterior pituitary.

Substance P (SP) and substance K (SK) are mammalian tachykinin peptides derived from a single preprotachykinin-A (PPT-A) gene and are widely but selectively distributed in neural and endocrine tissues. SP is present in the rat anterior pituitary, and its content there varies with the thyroid status of the animal. The present studies were undertaken to determine whether the PPT-A gene is expressed in the anterior pituitary and if so, whether PPT-A messenger RNA (mRNA) abundance is regulated by thyroid hormone status. Male rats were surgically or chemically thyroidectomized or made hyperthyroid by thyroid hormone (T3) injection. Total RNA was isolated from individual anterior pituitaries, and PPT-A mRNA abundance was determined by dot blot procedures. In parallel groups of rats, anterior pituitaries were extracted for measurement of SP and SK by specific RIAs. Surgical or chemical thyroidectomy increased PPT-A mRNA abundance 4 to 6-fold and increased both SP and SK content in the anterior pituitary. Administration of T3 to thyroidectomized rats reversed the increase in both PPT-A mRNA abundance and SP and SK content in the adenohypophysis. T3 administration to euthyroid rats also decreased PPT-A mRNA abundance and SP and SK content in the anterior pituitary. The coordinate presence of PPT-A mRNA with SP and SK in the anterior pituitary strongly suggests that these peptides are synthesized within this gland.

Differential processing of the two subunits of human choriogonadotropin by granulosa cells. II. In vivo studies.

Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.

Influences of photoperiod on sexual behaviour, neuroendocrine steroid receptors and adenohypophysial hormone secretion and gene expression in female golden hamsters.

Exposure to short daylengths arrests the oestrous cycle, provokes daily gonadotrophin surges and reduces the ability of exogenous oestradiol to trigger behavioural receptivity in golden hamsters. In order to examine neuroendocrine effects of photoperiod which might underlie these responses, ovariectomized hamsters were maintained under long or short photoperiods for 54 days before treatment with cholesterol or various doses of oestradiol-17 beta. Short days reduced the ability of low doses of oestrogen to prime hamsters for the induction of oestrus by progesterone. Upon repetition of oestrogen priming 2 weeks later, photoperiod was without significant influence on the concentrations of nuclear oestrogen receptors or cytosolic progestin receptors in a block of tissue containing the hypothalamus and preoptic area. Oestradiol treatment provoked significant increases in serum concentrations of LH and prolactin in the afternoon, but photoperiod did not alter the positive-feedback efficacy of this gonadal steroid hormone. Adenohypophysial LH-beta subunit and prolactin mRNAs were suppressed by short days in ovariectomized hamsters not treated with oestradiol. Oestradiol decreased expression of the LH-beta subunit gene in both stimulatory and inhibitory photoperiods, but increased prolactin mRNA abundance in both long and short days. Photoperiod therefore exerts pronounced steroid-independent effects on phasic LH and prolactin secretion, but regulation of adenohypophysial abundance of LH-beta subunit and prolactin mRNAs by oestradiol is not markedly influenced by daylength. Photoperiodic regulation of the priming effects of oestradiol on behavioural receptivity may result from modulation of events occurring subsequent to steroid-receptor interactions, or involve changes in receptor populations not detectable by the present methods.

Long-term hyperprolactinaemia reduces basal but not androgen-stimulated oestradiol production in small antral follicles of the rat ovary.

Hyperprolactinaemia disrupts fertility in many species, perhaps by inhibiting ovarian follicular steroidogenesis. The present studies measured oestradiol and progesterone secretion from isolated follicles from rats rendered hyperprolactinaemic in one of two ways. Sustained hyperprolactinaemia was induced by transplantation of two donor pituitary grafts under the renal capsule of adult female rats; grafts remained in place for 3 months. Transient hyperprolactinaemia was induced by pseudopregnancy initiated by cervical stimulation. Small antral follicles were isolated from both groups of rats 8-10 days after the previous vaginal oestrous smear and also from a control group of dioestrous female rats. Follicles were incubated for 3 h in the presence or absence of human chorionic gonadotrophin (hCG) or testosterone. Basal and hCG-stimulated oestradiol production were each reduced in follicles from both hyperprolactinaemic groups, relative to follicles from dioestrous control rats. In contrast, in the presence of testosterone, all groups of follicles produced comparable amounts of oestradiol. hCG stimulated comparable progesterone production by follicles from all three treatment groups. Testosterone elicited smaller increases in progesterone accumulation by follicles from all in-vivo groups. Reduced basal and gonadotrophin-stimulated, but not androgen-stimulated, oestradiol accumulation suggests that androgen production rather than aromatase activity in small antral follicles may be impaired by long-term hyperprolactinaemia.

Regulation of adenohypophyseal messenger RNAs in female rats by age, hypothyroidism, estradiol and neonatal androgenization.

Hormonal regulation of adenohypophyseal messenger ribonucleic acids (mRNAs) encoding preprotachykinin (PPT), prolactin (PRL) and thyrotropin beta subunit (TSH beta) was examined in juvenile and pubertal female rats. Hypothyroidism, initiated on day 2 (d2) or 22 (d22) of life, increased PPT and TSH beta mRNAs but decreased PRL mRNA 17 days later. Exogenous estradiol given for 3 days reduced PPT mRNA in pubertal (d38) but not juvenile (d18) euthyroid females; conversely, estradiol increased PRL mRNA on d18 but not d38. In hypothyroid females however, estradiol decreased PPT and TSH beta mRNAs at both ages and increased PRL mRNA in pubertal but not juvenile females. Thus, regulation of adenohypophyseal mRNAs by estradiol varies with age and thyroid status. In previous studies, adenohypophyseal tachykinins increased in male, but not female rats at puberty. This sex difference was not reproduced here by neonatal androgenization of females, suggesting that it is not mediated by hypothalamic sexual differentiation. However, PRL mRNA increased in androgenized females; this increase was prevented by ovariectomy, suggesting its medication by estradiol.

Pituitary hormone gene expression in male golden hamsters: interactions between photoperiod and testosterone.

Abstract Daylength regulates neuroendocrine function in male golden hamsters. Exposure to short days triggers gonadal regression and decreases serum luteinizing hormone (LH), prolactin and testosterone concentrations. Inhibitory photoperiods also amplify the negative feedback actions of androgens upon gonadotropin secretion. To examine whether these changes arise from altered adenohypophyseal gene expression, we measured the abundance of the messenger ribonucleic acids (mRNAs) encoding beta-LH, prolactin and proopiomelanocortin in anterior pituitaries of male golden hamsters which were either left intact, castrated, castrated and implanted with testosterone, or pinealectomized and maintained in either long (14 h light/10 h dark) or short (5 h light/19 h dark) days. Short days caused testicular atrophy in intact male hamsters and reduced serum LH in intact and castrated, testosterone-replaced hamsters. The relative abundance of beta-LH mRNA was suppressed by exposure to short days only in castrated hamsters. Serum prolactin was decreased by short days regardless of circulating testosterone concentrations. Prolactin mRNA abundance was decreased by short days in all pineal-intact groups. Castration reduced proopiomelanocortin mRNA abundance in long days and testosterone replacement reversed this effect. In the presence of testosterone, photoperiod influenced serum LH concentrations without altering hypophyseal abundance of beta-LH mRNA. In contrast, photoperiodic influences on prolactin secretion were correlated with alterations in steady-state mRNA abundance.

Localization of vasoactive intestinal peptide and peptide histidine isoleucine immunoreactivity and mRNA within the rat suprachiasmatic nucleus.

The distribution of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) mRNA within the suprachiasmatic nucleus (SCN) of rats was evaluated by immunocytochemistry and in situ hybridization. The pattern of VIP and PHI immunoreactivity corresponded closely to the distribution of VIP/PHI mRNA within the ventrolateral SCN. Clear hybridization signal was observed within the SCN of rats killed 5 h after light onset and in rats killed 2 h after the onset of the dark phase of the light-dark cycle. Visual examination of the grain density within the autoradiographs suggested that VIP/PHI mRNA may occur in higher concentrations shortly after the onset of darkness than 5 h after the onset of the light phase.

Sexual health innovations in undergraduate medical education.

Recent national and global initiatives have drawn attention to the importance of sexual health to individuals' well-being. These initiatives advocate enhancement of efforts to address this under-represented topic in health professions curricula. University of Massachusetts Medical School (UMMS) has undertaken a comprehensive effort to develop an integrated curriculum in sexual health. The UMMS project draws upon the expertise of a multidisciplinary faculty of clinicians, basic scientists, a medical ethicist, and educators. This article describes the project's genesis and development at UMMS, and reports on three innovations in sexual health education implemented as part of this endeavor.

The effect of a domestic violence interclerkship on the knowledge, attitudes, and skills of third-year medical students.

PURPOSE: To determine whether participation in an intensive domestic violence interclerkship (DVI) improved the knowledge, attitudes, and skills of two successive cohorts of students at the University of Massachusetts Medical School. METHOD: The authors measured the knowledge, attitudes, and skills pertaining to domestic violence of third-year students in the classes of 1997 and 1998 using a validated written examination administered before, immediately after, and six months after participation in a 3.5-day or two-day DVI, respectively; they compared the scores using paired t-tests. Nine months after the DVI, the students' domestic violence screening skills were measured by a performance-based assessment (OSCE); using unpaired t-tests, the authors compared the OSCE scores with those of a previous third-year class that had not participated in a DVI. Immediately after the OSCE, the students reported their levels of confidence in domestic violence screening and their satisfaction with the domestic violence curriculum; using chi-square analysis, those self-reports were compared with those of the class with no DVI. RESULTS: The students who participated in the DVIs immediately and significantly improved their knowledge, attitudes, and skills (p < .001), and fully or partially sustained those improvements six months later (p < .001). Nine months after the DVI, the students performed domestic violence screening more effectively (p < .001), expressed greater comfort with domestic violence screening (p < .001), and felt better-prepared by the curriculum to address domestic violence issues (p < .001) than did the students with no DVI. CONCLUSION: Participation in a short, focused DVI curriculum produced sustainable improvements in knowledge, attitudes, and skills that were successfully applied by third-year medical students to effective domestic violence screening. Interclerkships are an effective way to fit into the clinical curriculum those subjects that transcend the traditional biomedical domain and intersect all areas of medical practice.

Follicular growth in the rat: a reevaluation of the roles of FSH and LH.

The growth of preovulatory ovarian follicles involves hormonally induced proliferation and differentiation of theca cells and granulosa cells resulting ultimately in an increased ability of follicles to produce estradiol and to respond to the pituitary gonadotropins. The increased ability of follicles to produce estradiol appears to depend on an increased ability of theca cells to produce androgen as well as an increased ability of granulosa cells to aromatize androgens to estradiol. Estradiol in turn, appears to be required for FSH or FSH and LH to stimulate the appearance of functional receptors for LH in granulosa cells. Thus, the intrafollicular hormone estradiol enhances the response of granulosa cells to the gonadotropin. Therefore production of estradiol appears to determine which follicles will gain the mechanisms, including LH receptor, necessary for ovulation and luteinization. Since LH can act to increase its own receptor in the presence of estradiol and low amounts of FSH, it is possible that LH plays a predominant role in the final stages of preovulatory follicular growth both to promote estradiol production stimulation of theca cell androgen production as well as by facilitating an increase in its receptor by acting directly on granulosa cells.