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BackgroundCD47 is a broadly expressed cell surface glycoprotein associated with immune evasion. Interaction with the inhibitory receptor signal regulatory protein alpha (SIRPα), primarily expressed on myeloid cells, normally serves to restrict effector function (eg, phagocytosis and immune cell homeostasis). CD47/SIRPα antagonists, commonly referred to as ‘macrophage checkpoint’ inhibitors, are being developed as cancer interventions. SRF231 is an investigational fully human IgG4anti-CD47 antibody that is currently under evaluation in a phase 1 clinical trial. The development and preclinical characterization of SRF231 are reported here.MethodsSRF231 was characterized in assays designed to probe CD47/SIRPα blocking potential and effects on red blood cell (RBC) phagocytosis and agglutination. Additionally, SRF231-mediated phagocytosis and cell death were assessed in macrophage:tumor cell in vitro coculture systems. Further mechanistic studies were conducted within these coculture systems to ascertain the dependency of SRF231-mediated antitumor activity on Fc receptor engagement vs CD47/SIRPα blockade. In vivo, SRF231 was evaluated in a variety of hematologic xenograft models, and the mechanism of antitumor activity was assessed using cytokine and macrophage infiltration analyses following SRF231 treatment.ResultsSRF231 binds CD47 and disrupts the CD47/SIRPα interaction without causing hemagglutination or RBC phagocytosis. SRF231 exerts antitumor activity in vitro through both phagocytosis and cell death in a manner dependent on the activating Fc-gamma receptor (FcγR), CD32a. Through its Fc domain, SRF231 engagement with macrophage-derived CD32a serves dual purposes by eliciting FcγR-mediated phagocytosis of cancer cells and acting as a scaffold to drive CD47-mediated death signaling into tumor cells. Robust antitumor activity occurs across multiple hematologic xenograft models either as a single agent or in combination with rituximab. In tumor-bearing mice, SRF231 increases tumor macrophage infiltration and induction of the macrophage cytokines, mouse chemoattractant protein 1 and macrophage inflammatory protein 1 alpha. Macrophage depletion results in diminished SRF231 antitumor activity, underscoring a mechanistic role for macrophage engagement by SRF231.ConclusionSRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Circulating levels of luteinizing hormone (LH) are essential for the production of steroid hormones that regulate the timing of ovulation and target tissue responses, as well as maintenance of the corpus luteum and therefore early pregnancy. Clinical and basic science observations show that elevated levels of serum LH during the follicular phase of the menstrual cycle are not only unnecessary for follicular maturation but are deleterious to normal reproductive processes. These elevations may occur as a result of administration of exogenous LH or through an endogenous pathological process (i.e. polycystic ovarian disease, PCOD). Resting levels of LH, synergizing with locally produced IGFs, inhibin and perhaps other growth factors, are adequate for normal follicular growth and steroidogenesis. Elevations in serum LH above these resting levels may result in increased androgen production that diminishes follicular function and reduces early embryo viability. Elevated LH levels during the preovulatory period may also negatively influence post-ovulatory events such as conception and implantation. With these facts in mind, the best results for ovulation induction would be expected with purified follicle-stimulating hormone (FSH) administration to women following gonadotrophin releasing hormone (GnRH) down-regulation. It is hoped that this review provides the reader with an analysis of the complex series of events that regulate normal follicular maturation. The reevaluation of the two cell-two gonadotrophin theory suggests that during the preovulatory period, resting levels of LH are adequate for normal follicular maturation. Indeed, overstimulation of the ovary with excessive amounts of LH may diminish the ability of that target organ to produce fertile ova.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryThese studies have examined the effects of sodium pentobarbital or ether anesthesia on temporal plasma patterns and concentrations of FSH and LH following electrochemical stimulation (ECS) of the medial preoptic area (MPOA) or dorsal anterior hypothalamic area (DAHA) in proestrous rats. The injection of sodium pentobarbital at 1245 on proes-trus and ECS of either the MPOA or DAHA at 1300 elicited a rise in plasma FSH 120 min poststimulation. Injection of this barbiturate at 1245 and ECS of either region at 1500, when the effects of anesthesia had begun to wane, reduced the time interval between stimulation and the first significant plasma FSH rise to 60 min. A third group of proestrous rats was anesthetized with ether at 1100 and the DAHA or the MPOA was electrochemically stimulated. In such preparations, FSH in plasma rose significantly 60 min post-stimulation. In all MPOA-electrochemically stimulated groups, the first significant rise in plasma LH occurred 30 min poststimulation regardless of the...

The present study evaluated the effects of aging on the hormone profile of FSH as well as that of LH and PRL during proestrus (P) and estrus (E) of the 4-day rat estrous cycle. Furthermore, in view of the heterogeneity of FSH forms in the anterior pituitary gland (AP), we also sought to ascertain the effects of aging on the type of FSH produced by the AP. An attempt then was made to correlate possible changes in the synthesis and/or release of FSH with the initial decline in the incidence of regular 4-day estrous cycles in our colony of rats. Virgin rats aged 3, 5, 7, and 9 months, exhibiting 4-day estrous cycles were outfitted with atrial cannulae on proestrous morning. Blood samples were obtained every 4 h from 1400 h on P to 1000 h on E. At 1600 h on E, all rats were killed, trunk blood was collected, and the AP was removed, weighted, and homogenized. The concentrations of FSH, LH, and PRL in plasma and AP supernatant were determined by RIA, while the analysis of FSH isohormones in the remaining supernatant was made by chromatofocusing. At 3 and 5 months of age, 74% and 62% of the rats exhibited regular 4-day estrous cycles, respectively. This percentage had declined to 35% at 7 months and to 16% by 9 months of age. The marked decline in the incidence of 4-day cycles between 5 and 7 months was accompanied by an increased incidence of 5- to 7-day cycles, while by 9 months of age, 38% of the rats exhibited persistent vaginal cornification. In comparison to 3-month-old rats, plasma FSH levels were higher in 5-month-old rats at 1400 h on P and 1600 h on E. By 7 months, not only were plasma FSH levels increased further at these times, but they also were increased at 0600 and 1000 h on E during the secondary FSH surge. By 9 months, the preovulatory FSH surge appeared to be delayed, and the characteristic biphasic elevation in FSH levels on P and E was absent. In contrast to FSH, no change in the profile of plasma LH levels occurred until 9 months, when there was a marked reduction in the magnitude of the preovulatory LH surge. PRL levels were not consistently altered with age.(ABSTRACT TRUNCATED AT 400 WORDS)

Pituitary gonadotrophins (follicle-stimulating hormone, FSH; luteinizing hormone, LH) exist in different molecular forms within the anterior pituitary gland and serum of several non-mammalian and mammalian species, including man. The number and relative abundance of each gonadotrophin species will depend on the specific technique utilized for their isolation, the tissue source and the physiological status of the donor. Intracellular FSH and LH from glands of rodents (hamsters and rats) and primates exhibit charge heterogeneity and therefore may be separated into several forms or iso-hormones by isoelectric focusing (IEF). These FSH and LH species differ from each other not only in their isoelectric point (pI) but also in their relative abundance, receptor binding activity, biological activity and plasma half-life. Almost all gonadotrophin species isolated from pituitary extracts have also been detected in vitro and in vivo as secreted forms. Less basic rodent LH and FSH forms exhibit low receptor binding and in-vitro biological activities; a similar trend is found in LH and FSH species isolated from glands of monkeys and humans. However, these relatively acidic isohormones have longer circulatory half-lives and higher in-vivo biological activities than less negatively charged forms. The overall pattern of charge heterogeneity of gonadotrophins varies according to the specific endocrine status of the donor. Sex steroid hormones (mainly oestrogens) and gonadotrophin-releasing hormone seem to act in concert at the pituitary level to influence the physicochemical and functional characteristics of gonadotrophins and therefore their biological expression at the target cell. The effects of these factors appear to be mediated through the incorporation of specific carbohydrate residues and/or degree of terminal sugar sulphation at co-post-translational levels. The first result of these complex interactions between the gonad and the hypothalamic-pituitary unit is the production and secretion of various types of gonadotrophin molecules in proportions according with the physiological requirements of the subject at a given time, to perform specific actions upon gonadal maturation and/or function.

Hypothalami collected from female hamsters during proestrus and estrus were assessed for LHRH content by bioassay and RIA. During estrus, as serum FSH levels rose, a depletion of hypothalamic LHRH was observed. The pattern of hypothalamic LHRH accumulation and depletion during proes-trus and estrus as well as changes in serum and pituitary FSH concentrations were altered as a result of phenobarbital administration at 1200 h on proestrus. The effects of phenobarbital upon hypothalamic LHRH content as well as serum and pitui-tary FSH concentrations were overridden by the injection of purified FSH into the third ventricle of the hypothalamus at 1500 h during proestrus. The results of the present study suggest that the estrous FSH surge in hamsters is mediated, at least in part, by a concurrent discharge of hypothalamic LHRH. Further, intraventricular injection of FSH elicits a selective increase in serum FSH during estrus by influencing this LHRH-dependent system. (Endocrinology 113: 563, 1983)

Graded removal of sialic acid residues from a purified preparation of rat follicle-stimulating hormone (FSH; NIADDK-FSH-I-5) by neuraminidase digestion resulted in the production of FSH isohormones with isoelectric points identical to those found within pituitary tissue. In addition, each neuraminidase-produced FSH form exhibited a radioreceptor assay:radioimmunoassay ratio similar to that of its endogenously produced counterpart. Thus, the molecular basis for FSH microheterogeneity appears to be due to the varying degree of sialylation into a common FSH protein core. We have demonstrated that the pituitary gland produces different amounts of these FSH isohormones depending upon the surrounding endocrine environment. The results of the present studies suggest the existence of biochemical mechanisms within the pituitary that influence sialic acid incorporation into FSH. These mechanisms appear to be sensitive to the surrounding hormonal milieu and serve to alter the intensity of the FSH stimulus delivered to the gonad.

Pituitary glands, hypothalami, and trunk blood were obtained from male rats at 5, 15, 18, 21, and 29 days of age, on the day of balanopreputial separation (Days 42-45), and during adulthood. The forms of follicle-stimulating hormone (FSH) present within each pituitary were separated by polyacrylamide gel isoelectric focusing. Serum and pituitary gonadotropins, hypothalamic luteinizing hormone-releasing hormone (LHRH), and the profile of FSH forms across the isoelectric focusing gel were determined by radioimmunoassay. No change in the relative proportions of FSH forms were observed between 5 and 21 days of age. Likewise, only slight changes in serum and pituitary gonadotropin levels and hypothalamic LHRH content were observed at these times. After 21 days of age, dramatic increases in serum and pituitary gonadotropin levels were observed. Similarly, a shift in FSH forms within the pituitary to more basic and bioactive forms was observed at this time. These results demonstrate that, during the transition through puberty in the male rat, not only the absolute amount, but also the isoelectric focusing profile, of FSH change.

The administration of 17β-estradiol (E2) to ovariectomized hamsters at 0900 h (day 1) induced a proestrous-like gonadotropin surge the following day (day 2). Immediately before serum LH and FSH levels reached maximal concentrations on day 2, the concentration of FSH within the anterior pituitary gland (AP) remained unchanged. However, an increase was observed in the amount of AP FSH that failed to attach to the lectin, Concanavalin A (Con A), compared to that species that did interact with Con A. When the various forms of FSH present within the AP of ovariectomized animals before or after E2 injection were separated by polyacrylamide gel isoelectric focusing six species of immunologically active FSH were observed (pI values of 5.8, 5.7, 5.4, 5.1, 4.7, and 4.0). Five of these six species were consistently present in pituitary extracts collected 0, 24, and 28 h postinjection of E2. The sixth species (pI 4.0) was observed in increasing amounts with time after E2 treatment. The ability of each species of immu...

Elevations in peripheral estradiol (E2) levels inhibit the pulsatile release of pituitary gonadotropins in ovariectomized rhesus monkeys. Efforts to determine the locus of E2 action [i.e. the medial basal hypothalamus (MBH) or the anterior pituitary gland (AP)] have been plagued by the diffusion of crystalline E2 from an implantation site within the MBH to the pituitary portal vasculature and, therefore, to the AP (implantation paradox). With an Alzet osmotic minipump, we delivered 10 ng E2/h to either the third ventricle or the AP of ovariectomized rhesus monkeys for a duration of 18 h, while monitoring changes in the peripheral gonadotropin concentrations. We also infused [3H]E2 into either site to determine the extent of [3H]-E2H]-E2 diffusion by the end of the infusion period. After infusion of [3H]E2 into the third ventricle for 18 h, only trace amounts of [3H]E2 were recovered from the AP. A small amount of [3H]E2 was recovered from the MBH after 18 h of delivery directly to the AP. Infusion of E2 i...

Heterogeneity of intact FSH in human pituitary tissue was evaluated by the column isoelectric focusing technique, chromatofocusing. FSH was examined from three autopsy pituitaries, one piece of apparently normal pituitary obtained at surgery, three purified FSH preparations, and four gonadotroph cell adenomas. FSH from all of these sources was found to exist in multiple isoelectric forms. FSH from the gonadotroph cell adenomas exhibited chromatofocusing profiles that were similar, both in the range of pI's and the relative proportions of the FSH isohormones, to those of the non-adenomatous pituitaries. We conclude that gonadotroph cell adenomas produce a population of FSH that is qualitatively normal although quantitatively excessive.

The serum gonadotropin profiles of 4-day cycling rodents exhibit LH and FSH surges during proestrus (P) and a second release of FSH during estrus (E). Peak serum levels of FSH during P occur between 1500–1800 h; the estrous FSH release starts late in the evening of P and reaches maximal serum levels during the morning of E. To delineate the mechanisms that regulate the estrous FSH rise and elucidate the role of FSH in its own secretion, hamsters were given Phenobarbital (ip) during P to block the endogenous gonadotropin surges and were subsequently injected systemically (iv) or intraventricularly (icv) with purified preparations of FSH or LH at 1500 h on P. Blood samples were obtained at regular intervals throughout P until 0900 h on E, at which time the animals were killed, and anterior pituitaries and hypothalami were collected. Serum and pituitary FSH and hypothalamic gonadotropin-releasing hormone were measured by RIA. Intact, saline-injected, and hypophysectomized (hypox) FSH-injected animals served ...

Anterior pituitary glands (AP) were obtained from female rats at 5, 15, 18, 21 and 29 days of age, at the time of vaginal opening (VO) and during adulthood on proestrus. The multiple species of follicle-stimulating hormone (FSH) present within the AP were separated by the technique of polyacrylamide gel isoelectric focusing (PAG-IEF) and measured with the NIAMDD rat FSH radioimmunoassay kit. AP's obtained from immature female rats prior to VO contained elevated levels of total FSH as well as all of the species of AP FSH observed in adult rats (and hamsters). However, the majority of the FSH immunoactivity migrated to the most acidic portion of the gel (isoelectric point [pI] value=4.2-3.8). At the time of VO and during adulthood, a decrease in total AP FSH was observed. In addition, a shift in the relative proportions of certain FSH species occurred. The AP's of adult animals contained relatively greater amounts of more basic (pI values 6.0-5.0) forms of FSH compared with immature animals. When each of the AP FSH species isolated from adult animals was tested in a radioligand receptor assay, the most acidic (pI=4.2-3.8) failed to interact with the receptor preparation, while those with pI values from 6 to 4.7 were able to compete with [125I]-labeled FSH for receptor binding in a parallel fashion. Thus, the observed shift in the PAG-IEF FSH profiles to more basic (and biologically active) forms may represent a change in the composition of AP FSH that serves an important role in the maturation process leading to ovulatory cyclicity.

Anterior pituitary cells were obtained from ovariectomized hamsters and cultured for 4 days. The cells were lysed and intracellular proteins, including FSH, were separated by polyacrylamide gel isoelectric focusing. FSH present within cell lysates so separated was quantitated by a specific FSH radioimmunoassay (RIA). Six distinct species of intracellular FSH were observed with pI values = 5.9, 5.7, 5.3, 5.1, 4.7 and 4.3 – 3.8. All six species were detectable when proteins from a concentrate of incubation medium collected from those cell cultures were separated by isoelectric focusing. Similar isoelectric points were obtained when the FSH species present within an anterior pituitary homogenate obtained from ovariectomized hamsters were separated by the same technique. All but two of these six species of intracellular FSH were well recognized by a hamster FSH receptor preparation. However, these two species of FSH (pl values = 4.7 and 4.3 – 3.8) do not appear to be β subunits of the intact glycoprotein as determined by gel filtration. Thus, the present studies demonstrate that, as observed previously in anterior pituitary glands obtained from intact and castrated animals, pituitary cells that have been enzymatically dissociated and cultured in vitro for 4 days contain and secrete multiple species of FSH that are separable by polyacrylamide gel isoelectric focusing. This in vitro system may be useful for studying the hormonal factors that regulate FSH species synthesis and secretion.

Anterior pituitary (AP) glands were removed from groups of female golden hamsters at 0900 h on estrus (E), diestrus I (DI), and diestrus II (DII) and at 1200 h and 1500 h on proestrus (P12 and P15), as well as at 0900 h from ovariectomized hamsters (OVX). Hemipituitaries were incubated in culture medium with or without 10(-8) M luteinizing hormone-releasing hormone (LHRH) for 3 h at 37 degrees C to determine the magnitude of basal and LHRH-stimulated follicle-stimulating hormone (FSH) release. All samples were assessed for FSH activity by radioimmunoassay and radioreceptor assay. In a second set of experiments, AP were removed from E, DII, and OVX hamsters and bisected. One hemipituitary was homogenized in 10 mM Tris-HCl and the other half was incubated for 3 h. Follicle-stimulating hormone forms present within pituitary extracts or secreted into medium were separated by an isoelectric focusing technique, chromatofocusing. Basal FSH release was lowest in AP collected on DII and P12, higher in AP collected on E and DI, and highest in AP from OVX. Luteinizing hormone-releasing hormone-stimulated release of FSH was highest in AP obtained on DII and P12, lower in AP collected on E and DI, and lowest in AP from OVX. Radio-receptor-to-radioimmunoassay ratio of secreted FSH was greatest when basal FSH secretion was low and LHRH sensitivity was high (DII and P12) and least when basal FSH secretion was high and LHRH sensitivity low (E and after OVX).(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study we wished to determine: a) the physiological importance of the dorsal hypothalamic area (DAHA) in the regulation of the ovulatory discharge of LH/FSH, and b) whether the DAHA is involved in the negative feedback action of estradiol on pituitary FSH secretion. Large bilateral lesions were produced in the DAHA and the animals were maintained (18–20 days) until vaginal cyclicity had resumed. In such rats at proestrus, temporal patterns of LH\FSH were identical to normal controls but peak plasma concentrations were reduced. All DAHA-lesioned rats ovulated fully (8–12 eggs). In control Nembutal®-anesthetized proestrous rats which were sequentially bled for 6 h beginning at 1300 h, plasma LH/FSH remained at basal concentrations. Two other groups of Nembutal-blocked rats were either ovariectomized (OVX) or a bolus of purified rat LH was injected iv at 1300 h. In both studies, FSH rose significantly from baseline within 3 h and thereafter increased linearly for the next 3 h. Basal plasma LH l...

In female proestrous hamsters (1800 h), bilateral electrocoagulative lesions in the arcuate-median eminence (Arc-ME) region blocked the pituitary FSH release that normally would have begun in late proestrus and continued until the afternoon of estrus. This second (estrous) FSH elevation, which is not accompanied by LH release, was unaffected by neural disconnection of the medial preoptic area (MPOA) from the medial basal hypothalamus (MBH) or by production of MBH-pituitary islands. After gonadotropin-releasing hormone was injected into Arc-ME-lesioned hamsters, LH was released. These results suggest that the Arc-ME does not require the MPOA for initiation of the estrous release of FSH.

Relative amounts of inhibin were measured in charcoal treated ovarian extracts (CTOE) collected on each day of the hamster estrous cycle. This material was quantitated by its ability to depress serum FSH, without affecting LH levels in ovariectomized hamsters. Injection of proestrous CTOE caused a significant reduction in the elevated levels of serum FSH within 2 h. No decrement in serum FSH concentrations was observed in hamsters injected with CTOE collected on other days of the cycle. Further or sustained decreases in serum FSH concentrations were observed when hamsters were administered proestrous CTOE in larger or multiple injections, respectively. None of these injected extracts elicited any change in serum LH levels. In addition, repeated injections of proestrous CTOE to cyclic hamsters on proestrus and estrus caused a significant reduction in the serum concentration of FSH on estrus compared with control animals. This decrease in estrous FSH was accompanied by a reduction in the number of tubal ova shed 4 days later on the next estrus.

Plasma LH/FSH concentrations were monitored in mature androgen- (ASR) or estrogen-sterilized rats (ESR) and were compared to normal proestrous rats in which spontaneous gonadotropin surges had been blocked with Nembutal (control). LH and/or FSH release was induced by dectrochemical stimulation (ECS) of either the dorsal anterior hypothalamic area (DAHA) or the medial preoptic area (MPOA). ECS of control rats, ASR and ESR provoked significant increases in plasma FSH by 120 min and peak levels by 180 mm poststimulation. No differences were noted when ESR or ASR plasma levels were compared to control values or to each other (ESR vs. ASR). DAHA-ECS failed to provoke a significant LH increase from baseline in control rats and only small LH rises in ASR and ESR. MPOA-ECS of control, ASR and ESR resulted in the release of both LH/FSH. In all groups FSH peaked at 240 mm but at 120 and 180 mm post-stimulation plasma FSH levels were greater in ASR and ESR than controls. Further, FSH in ESR was significantly higher than ASR at 120 and 240 mm poststimulation. Plasma LH was elevated in all groups after MPOA-ECS but in ESR significantly greater LH levels were obtained at 60 and 180 mm as compared to controls or ASR. Preoptic stimulation of progesterone-treated ASR (P-ASR) did not induce greater releases of pituitary FSH than were obtained in control, ASR, ESR or progesterone-treated ESR. In contrast, stimulation of P-ASR resulted in greater plasma LH levels than untreated ASR but not control rats. P-ESR responded to MPOA-ECS by a rapid plasma LH rise by 60 mm which was greater than ASR, P-ASR, ESR or control rats. Thereafter, plasma levels in P-ESR decreased rapidly to baseline. None of the DAHA-stimulated groups ovulated. All MPOA-ECS control rats ovulated (8-13 eggs) whereas neither the ASR nor ESR Fallopian tubes contained eggs the next morning. All P-ASR but none of the P-ESR ovulated after MPOA-ECS. Seemingly, while exposure of neonatal female rats to estrogen renders them more sensitive than ASR to preoptic stimulation, the ovaries of such animals are much less responsive to the gonadotropin released into plasma than are those of ASR or normal rats.

The present studies examine the effects of testosterone (T) and LHRH, alone or in combination, on the amount of FSH secreted by pituitary cells in culture. FSH was quantified by RIA and radioreceptor assay (RRA). Half of the cultures were exposed to T for 3 days. The remainder served as controls. Each of these two groups was divided in half and exposed to medium only or LHRH (10(-8) M) for 4 h. Medium was collected from all cultures after 3 days +/- T (medium 1) and after 4 h +/- LHRH (medium 2). After medium 2 collection, cell homogenates were prepared. In a second study, T-treated cell cultures also received 0, 0.5, or 5.0 micrograms/dish tunicamycin for the last 16 h of the 3-day incubation. During the 3 days of culture, the T-treated group secreted greater amounts of immunoactive FSH than controls. However, LHRH-induced FSH release measured by RIA was blunted as a result of T exposure compared with untreated controls. T treatment elevated intracellular immuno-FSH stores. Each sample was quantitated for FSH activity by RRA, and the FSH RRA/RIA was calculated. T and/or LHRH treatment, while eliciting FSH hypersecretion, caused a reduction in the RRA/RIA of secreted FSH. Changes in the RRA/RIA are thought to occur as a result of alterations in the glycosylation of FSH. To test this hypothesis, T-treated cells were exposed to tunicamycin, a drug that reduces the rate of glycosylation of secreted proteins. Exposure of cells to this drug prevented the reduction in the RRA/RIA of secreted FSH caused by T and/or LHRH. FSH secreted from control, T-treated, or T-treated plus tunicamycin-exposed cells was examined by isoelectric focusing. T-Treated cells released a greater proportion of FSH forms with lower isoelectric points (indicative of a greater degree of glycosylation) compared with controls. Tunicamycin exposure reversed T's effect upon the isoelectric profile. These studies demonstrate a direct pituitary action of LHRH and T upon the type of FSH released. During times of hormonally induced increases in the rate of FSH secretion, the pituitary releases FSH forms that are more heavily glycosylated, exhibit a lower isoelectric point, and show a reduced RRA/RIA. FSH secreted after T treatment would be expected to have an increased plasma half-life due to the protective effects of the sugar residues. Thus, the existing hormonal milieu exerts a multidimensional effect upon FSH released by pituitary cells in culture that cannot be appreciated by RIA assessment alone.

In ovariectomized rhesus monkeys ( Macaco mulatto ) electrical stimulation (ES) of the medial basal hypothalamus (MBH) for 30 min with stimulus parameters that caused no overt behavio

Since the pioneering studies of Everett, Sawyer and Markee (1) it has been generally accepted that the central nervous system (CNS) regulates the secretion of the pituitary gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). However, great gaps still exist in our understanding of the neural mechanisms that regulate the secretion of these hormones. The purpose of this review is to provide the reader with a concise overview of this topic. Gaps, inconsistencies and future directions of this area of research are also presented.

THE ANTERIOR pituitary gland synthesizes and secretes three glycoprotein hormones. Two of these, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are termed gonadotropins and exert divergent effects upon the gonads to regulate and maintain essential reproductive processes such as gametogenesis, steroidogenesis, and ovulation. We possess a basic understanding of the role played by both gonadotropins in a wide variety of physiological processes associated with reproduction. This review is intended to provide a concise body of information regarding the biological processes associated with the biosynthesis and secretion of FSH. Since both gonadotropins appear to be synthesized by the same cell in the anterior pituitary gland (1, 2) and the biosynthetic and secretory processes are influenced by the same hypothalamic hormone, gonadotropin-releasing hormone (GnRH) (3), this review will consider a great deal of information obtained from the study of both gonadotropins. However the primary focus wil...

The multiple species of follicle-stimulating hormone (FSH) present within pituitary tissue were separated by the technique of Polyacrylamide gel isoelectric focusing. The ability of each FSH species to stimulate the secretion of plasminogen activator from cultured granulosa cells was tested (FSH in vitro bioassay). A wide range of biologic/radioimmunologic FSH activity was observed when FSH species were compared. As the isoelectric point of the FSH molecule declined, so did the biologic activity. A second series of studies was performed to determine which forms of FSH were secreted by pituitary tissue in vitro. All of the forms of FSH present in pituitary tissue were secreted into culture medium. However, the relative proportions of FSH forms in the pituitary and medium were not always similar. Exposure of pituitary tissue to luteinizing hormone-releasing hormone elicited an increase in the relative proportion of the more biologically active forms of FSH that were secreted. These studies suggest that the hormonal milieu surrounding the pituitary affects not only the quantity but also the potency of the FSH signal emitted. Thus, the basis for observed differences between biologic and immunologic FSH activities observed during some endocrine states may be the result of preferential secretion of certain FSH species.

Pituitary tissue obtained from proestrous, phenobarbital-treated hamsters was placed in organ culture, and the release rates of FSH and LH were monitored. Addition of LHRH to the culture medium increased gonadotropin release rates. Preincubation of pituitaries in medium that contained 1 microgram purified FSH ( NIADDK rat FSH-I-5) resulted in enhanced basal and LHRH-induced FSH release rates. Full expression of FSH hypersecretion by pituitary tissue occurred after 3-h exposure to purified FSH. This phenomenon appears to require adequate tissue calcium concentrations. Further, exposure of pituitaries to purified FSH slightly but significantly enhanced basal but not LHRH-stimulated LH release compared with that in untreated controls. Experiments that employed labeled FSH, reduced exposure time to purified FSH, or decreased calcium concentration in the medium proved that the increased FSH secretory rate was not due to contamination of the medium with the purified FSH used to stimulate the pituitary. These studies suggest that FSH has the ability to influence its own secretion by an action at the level of the anterior pituitary gland.

Anterior pituitary gland (AP) homogenates and serum samples were obtained from hamsters at hourly intervals between 1100–1700 h during proestrus. The presence of two species of immunologically active FSH in AP homogenates was revealed by an affinity adsorption technique that employs the lectin, Concanavalin A (Con A). One species did not bind to the lectin (Con A-unbound), whereas the other form (Con A-bound) did attach to the Con A and was eluted with a glucopyranoside solution. The relative ratios of both species of FSH within AP homogenates were determined throughout the afternoon of proestrus. Immediately before the proestrous FSH surge (1100–1400 h), the total AP concentration of FSH remained elevated and unchanged; however, the relative ratio of Con A-unbound to Con A-bound forms of AP FSH increased dramatically. As serum concentrations of FSH rose during proestrous afternoon (between 1500–1700 h), both the AP FSH concentration and the ratio of Con A-unbound to Con A-bound FSH declined. Serum FSH co...

Ovariectomized cynomolgus monkeys were treated with physiological levels of estradiol and progesterone. A reduction in serum levels of FSH was observed after steroid exposure. Anterior pituitary homogenates were prepared from monkeys after 0, 12, 24, or 36 h of exposure to estradiol and progesterone and quantitated for FSH activity by radioreceptor assay (RRA) and RIA. Pituitary FSH activity (expressed as RRA/RIA) increased with duration of exposure to steroids. Forms of FSH within these pituitaries were separated by the column isoelectric focusing technique, chromatofocusing. All pituitary homogenates tested contained FSH isohormones that eluted at similar isoelectric points. Each FSH isohormone exhibited a mol wt similar to that of a purified FSH standard, but differed in ability to displace labeled FSH from a biological receptor preparation. FSH forms with basic isoelectric points exhibited greater RRA/RIA values than forms with more acidic isoelectric points. The relative proportion of the more basic FSH forms increased within pituitary tissue with duration of exposure to steroids. All FSH forms were secreted by pituitary cells in culture. The biochemical basis for the microheterogeneity appears to be the degree of sialic acid incorporation into the FSH molecule. The results of these studies demonstrate that the cynomolgus monkey pituitary responds to the surrounding hormonal milieu by altering the relative proportions of FSH forms present within that gland.

Since the pioneering work of Moore and Price (1932), it has been well documented that the ovary and anterior pituitary gland (AP) exist in a dynamic equilibrium. Simply stated, the AP secretes the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These glycoproteins bind to the ovary at specific receptor sites and stimulate the synthesis and secretion of ovarian steroids. These steroids travel from the ovary, through the peripheral circulation, to the AP and the hypothalamus (the area of the brain that regulates the secretions of the AP) to stimulate or inhibit further gonadotropin secretion.

Anterior pituitary glands were collected from immature and mature (intact and castrated) male hamsters. The various species of FSH present within these glands were separated by Concanavalin A (Con A) chromatography and polyacrylamide gel isoelectric focusing (PAG-IEF) and measured by a specific FSH radioimmunoassay (RIA) as well as a radioreceptor assay (RRA). Two distinct forms of FSH (Con A unbound and bound) were separated by Con A chromatography and detected by both RIA and RRA. These two populations of FSH were present within anterior pituitary glands of all three animal models tested. Castration before collection of anterior pituitary glands reduced the ratio of Con A unbound: bound immunoreactive FSH. When measured by RRA this reduction was not observed. When homogenates of anterior pituitary glands obtained from mature animals were separated by PAG-IEF, six distinct species of FSH were observed by RIA with isoelectric points (pI) of 6·0, 5·7, 5·3, 5·0, 4·7 and 4·2–3·8. Homogenates of anterior pituitary glands obtained from immature male hamsters did not contain one of these species of FSH (pI value, 4·7). The relative contribution of some of the species of FSH to the total amount of detectable FSH differed depending upon the endocrine status of the animal. The species with pI value of 4·2–3·8 did not show any receptor-binding activity in any of the three models studied. The overall ratio of the activity of FSH measured by RRA compared with RIA was highest in anterior pituitary glands from intact mature and immature hamsters and lowest in anterior pituitary glands obtained from castrated animals. The RRA: RIA ratio for each species of FSH in all models tested declined as the isoelectric point of that species decreased. Thus, these results demonstrated the presence of multiple species of FSH within the anterior pituitary glands of immature and mature male hamsters. The relative proportions and receptor-binding activities of these species differed according to the isoelectric point and the pattern of hormone secretion at the time of collection of pituitary glands. Gonadal and other endocrine factors may influence not only the relative amount of each species of FSH but also the receptor-binding capacity of the FSH species synthesized by the anterior pituitary gland.

The presence of material like luteinizing hormone (LH) in cervical mucus has not been previously reported. In this study, we show that in human cervical mucus there is LH-like material detectable by radioimmunoassay (RIA) and by bioassay. The content of LH in the cervical mucus of five donors was measured by RIA in samples of mucus obtained daily throughout the menstrual cycle. The concentration of LH in mucus obtained during ovulation was lower than in mucus obtained during the early follicular phase or mucus obtained during the luteal phase. This material exhibits LH-like biologic activity because increasing volumes of cervical mucus added to isolated mice Leydig cells stimulates testosterone secretion by these cells, in a parallel dose-related fashion, to increasing quantities of human chorionic gonadotropin (hCG). No immunologic cross-reactivity of the cervical mucus was observed with an antiserum specific for the β -subunit of hCG. Taken together, the results of the present study suggest the presence of an LH-like substance within the cervical mucus that exhibits both the immunologic and biologic properties of LH.

FSH bioactivity was measured by means of FSH-dependent aromatase activity (conversion of androgen substrate to estradiol). Assay sensitivity was optimized by the use of immature (7-10 days old) rats as Sertoli cell donors, serum-free medium for incubation, phosphodiesterase inhibitor (methylisobutylxanthinine), serial dilution of FSH in medium containing 1% BSA, delayed addition of FSH for 72 h after cell plating, and 19-hydroxyandrostenedione (2.5 X 10(-6) M) as the aromatizable androgen substrate. The method consisted of subjecting the decapsulated immature rat testes to a 2-step collagenase dispersion, plating the cells in medium [Dulbecco's Modified Eagle's Medium-Ham's F-10 (1:1)] containing growth factors and methylisobutylxanthinine for 72 h, adding increasing doses of FSH to the standard curve or small volumes of serum to the test vials as well as 19-hydroxyandrostenedione for 24 h, and measuring estradiol by RIA in dilutions of the medium. Using NIAMDD human (h) FSH-2 as the bioassay standard, the useful range of the assay was 0.01-5.0 ng/ml. Specificity was determined by the addition of graded doses of hLH, hTSH, ACTH, hGH, hPRL, and hCG. The minor degree of FSH bioactivity observed in a few hormone preparations was accounted for by the degree of FSH contamination in them. Mean intra- and interassay coefficients of variation were 9% and 11%, and the index of precision was 0.049. This bioassay was used to determine the bioactive FSH content of pituitary extracts, tissue culture media, elutions from columns, and isoelectrically focused samples. More importantly, small quantities of human sera gave responses parallel to the standard curve in a minimum of two dilutions. The bio- to immunoreactive ratios, expressed as the mean +/- SEM (NIAMDD-hFSH-2), were 0.66 +/- 0.2 in boys (n = 6), 0.78 +/- 0.2 in pubertal girls (n = 6), 1.18 +/- 0.2 in men (n = 13), 1.24 +/- 0.1 in postmenopausal women (n = 30), 1.94 +/- 0.3 in the follicular phase (n = 19), 6.2 +/- 1.4 in the ovulatory phase (n = 19), and 1.6 +/- 0.4 in the luteal phase (n = 19) of the normal menstrual cycle. These results indicate that the bio- to immunoreactive hFSH ratio in the circulation, is dependent upon the hormonal milieu of the subject.

Oral administration of gossypol acetic acid altered pituitary and ovarian hormones during proestrus and estrus in hamsters. These changes in endocrine function were not however, accompanied by alterations of estrous cycle length, number of ovulations or pregnancy rates. Changes in body weight and appearance of the liver suggested that female hamsters may be more susceptible than males to the the toxic effects of gossypol.

This study was designed to determine if the rostral hypothalamus (RH) exerts any control over the estrous (E) rise in serum FSH levels in golden hamsters. Proestrous (P) hamsters were subjected to two experimental techniques known to block the RH-dependent preovulatory gonadotropin release [phenobarbital (phen) administration and retrochiasmatic neural disconnection (RCD)]. After either treatment during P, the occurrence of the E FSH rise was examined. Phen administration at 1300 or 1400 h of P blocked the preovulatory LH/FSH rise, but did not inhibit the E FSH release. Phen administration at 1500, 1600, or 1700 h of P abolished neither the P nor the E gonadotropin releases. RCDs were made at hourly intervals from 1300-1800 h of P. The RCDs performed at 1300 and 1400 h of P blocked both the preovulatory rise in LH and FSH and the E rise in FSH that normally would have occurred. The RCDs performed at 1500 and 1600 h did not inhibit the P gonadotropin release but did block full expression of the E FSH rise....

The nature of the LH abnormality in four men who had gonadotroph cell pituitary adenomas was studied to determine why they had supranormal serum LH concentrations but subnormal or normal serum testosterone concentrations. When basal sera from these four men were subjected to gel filtration chromatography, FSH immunoreactivity eluted principally in the position of intact FSH, but the elution patterns of LH immunoreactivity were biphasic; one peak corresponded to intact LH, and another peak corresponded to the LH subunits, LH beta and alpha. Gel filtration of sera obtained after administration of TRH showed increases in LH immunoreactivity predominantly in the subunits peak. The subunits peak in basal sera consisted principally of alpha, but the percent increase in LH beta in post-TRH sera was greater. When dispersed cells from one gonadotroph adenoma were cultured, medium LH immunoreactivity eluted with the LH subunits. LH beta release into the medium was somewhat greater than alpha initially, but declined more rapidly. We conclude that the apparent hypersecretion of immunoreactive LH of impaired biological activity by four men with gonadotroph cell adenomas represents hypersecretion of LH beta- and alpha-subunits that are uncombined, perhaps because of a structural abnormality or anatomical compartmentalization.

Temporal changes in plasma LH and FSH concentrations were monitored during the afternoon of proestrus in controls and in rats in which the spontaneous LH/FSH surges were blocked with Nembutal. These values were compared with those obtained following electrochemical stimulation (ECS) of either the medial preoptic area (MPOA) or the dorsal anterior hypothalamic area (DAHA) in similar Nembutal-blocked animals. Whereas MPOA-ECS (60 muA/60 sec) elicited a release of both FSH and LH, similar unilateral stimulation of the DAHA resulted in a pronounced increase in plasma FSH and only a slight elevation in plasma LH. Increasing the amount of DAHA tissue stimulated (100 muA/60 sec) caused a significantly greater release of FSH but not LH. Bilateral DAHA-ECS (60 muA/60 sec) failed to produce a greater release of FSH than that observed after unilateral 100 muA/60 sec ECS but resulted in increased concentrations of LH in plasma. Surgical separation of the MPOA from the DAHA, leaving the preopticotuberal fibers intact, did not alter the spontaneous temporal patterns of discharge of FSH or LH 19-21 days post-operatively, although peak LH concentrations were reduced. Further, unilateral ECS (60 muA/60 sec) of the MPOA in such preparations elicited a release of FSH and LH similar to that observed in intact MPOA-ECS rats. In contrast, unilateral DAHA ECS (60 muA/60 sec) in rats with transected hypothalami, caused no release of LH and an attenuated FSH discharge when compared with intact DAHA-ECS rats (peak valued 189 +/- 8 ng/ml vs 274 +/- 11 ng/ml). These studies suggest the existence of specific cell bodies in the DAHA which can cause selective release of FSH when activated. Coexisting with this system is that level of control which is believed to be responsible for the cyclic discharge of both FSH and LH of which the MPOA is a component part.

SummaryThe presence of a nonsteroidal substance (inhibin) within human follicular fluid (HFF) with the ability to suppress pituitary follicle-stimulating hormone (FSH) but not luteinizing hormone (LH) secretion was determined by two methods. The first method consisted of infusion of HFF directly into the anterior pituitary gland of ovariectomized rhesus monkeys. The second employed a pituitary cell culture system. Release of LH and FSH was stimulated in HFF-treated cells by the addition of luteinizing hormone-releasing hormone (LHRH) to the tissue culture medium. In both methods, to determine the presence of inhibin, we used the criterion of a decreased release of FSH into the medium or serum that was not accompanied by an concomitant decrement in LH concentrations. Both methods detected the presence of inhibin within HFF from women during the follicular but not the luteal phase of the menstrual cycle. This finding suggests that this substance may play a role in the regulation of FSH secretion at a specif...