Biomedical subjects

J K Findlay

Publications and source records attributed to J K Findlay.

At least 37 records · Page 2Linked to original sources

PubMed2000

Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception.

Inhibins and activins are dimeric hormones which share common subunits and which have diverse endocrine and paracrine roles in regulating reproductive function. Endometrial expression of inhibin alpha, ssA and ssB subunits was examined by immunohistochemistry and in-situ hybridization, across the menstrual cycle and in early pregnancy. All three subunits were found to be expressed in endometrium, primarily by glandular epithelium in the early stages of the cycle. Following the onset of decidualization, expression of alpha, ssA and ssB subunits was up-regulated in decidualized stromal cells. A marked down-regulation of alpha subunit was detected in glandular epithelium, whilst expression of ssA and ssB subunits was maintained. This pattern was consistent in decidua from early pregnancy and additionally in endometrium from women using progestin-only contraceptives, either subdermal implants (Norplant((R))) or levonorgestrel-releasing intrauterine systems (Lng-IUS). Immunostaining was also observed for both ssA and ssB subunits in subpopulations of endometrial leukocytes, identified to be distinct subsets of macrophages, neutrophils and mast cells. Potential paracrine roles for activins may be envisaged in facilitating tissue remodelling during decidualization, in tissue repair following menstruation, and additionally in modulating premenstrual inflammatory events.

Activins↗

PubMed2000

Uterine expression of alternatively spliced mRNAs of mouse splicing factor SC35 during early pregnancy.

RNA differential display was applied to identify genes critical for the establishment of pregnancy in the mouse. One of the gene fragments identified was homologous to human SC35 splicing factor; the mouse counterpart had not then been cloned. To obtain the full cDNA sequence of the mouse gene, a cDNA library was screened and four positive clones were fully analysed. Sequencing analysis indicated that we had cloned alternatively spliced mRNA species of mouse SC35 splicing factor. A map of splicing structure for this gene's pre-mRNA was then proposed and region-specific mRNA species were tested on Northern blots. This analysis indicated that the overall expression level of SC35 mRNA was much higher in implantation sites than in inter-implantation sites in the mouse uterus during early pregnancy. The expression of alternatively spliced mRNAs for SC35 was differently regulated both during early pregnancy and by steroid hormones. Embryo-derived factors were also implicated in the up-regulation of SC35 mRNA at implantation sites. These results demonstrate, for the first time, that an essential splicing factor is regulated in a complex manner during implantation in the mouse uterus. Hence, its correct regulation could be important for the success of pregnancy.

Alternative Splicing↗

PubMed2000

Complex regulation of calcium-binding protein D9k (calbindin-D(9k)) in the mouse uterus during early pregnancy and at the site of embryo implantation.

Establishment of receptive endometrium is essential for implantation. Our aim was to identify and characterize genes uniquely regulated at the sites of implantation in mouse uterus by RNA differential display polymerase chain reaction (DDPCR). One of the gene fragments identified was 86% homologous to rat calcium-binding protein D9k (calbindin-D(9k)); the mouse counterpart had not then been cloned, but subsequently an mRNA sequence of mouse calbindin-D(9k) became available in GenBank (accession number: AF028071). This sequence is 99% homologous to the DDPCR-derived gene tag but has a shorter 3' end. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the sequence of 3' end of the DDPCR product and the 5' end of AF028071, and a full cDNA was obtained. This gene was primarily up-regulated by progesterone, but not by estrogen. It was further increased by the combination of the two steroids. Expression of calbindin-D(9k) was overall increased in the uterus during early pregnancy, but the level was significantly lower in implantation compared to interimplantation sites on Days 4.5 and 5.5 of pregnancy, becoming barely detectable in both sites after Day 6.5. In situ hybridization localized this mRNA predominantly in the luminal epithelium of the pregnant uterus. The complex regulation of calbindin-D(9k) in mouse uterus suggests an important role for this protein during pregnancy.

Amino Acid Sequence↗

PubMed2000

An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene.

With the development of a mouse model of estrogen insufficiency due to targeted disruption of the aromatase gene [the aromatase knockout (ArKO) mouse], a new opportunity exists to examine the role of estrogen in ovarian follicular development. Ovaries and serum were collected from wild-type, heterozygous, and ArKO mice at 10-12 and 21-23 weeks and 1 yr of age. The ovaries were assessed histologically and stereologically, with primary, secondary, and antral follicles and corpora lutea counted. The uteri were hypoestrogenic, and serum levels of LH and FSH in ArKO females were elevated above those in heterozygote and wild-type animals at all ages studied. Although estrogen was not a prerequisite for reinitiation of follicle growth, there was a block of follicular development, and no corpora lutea were present in ArKO ovaries. Thus, the ArKO mouse was infertile as a consequence of disrupted folliculogenesis and a failure to ovulate. Hemorrhagic cystic follicles were present by 21-23 weeks of age. The ovarian phenotype degenerated with age, such that by 1 yr there were no secondary or antral follicles, and the primary follicles present were atretic. Extensive interstitial tissue remodeling occurred, exemplified by an influx of macrophages and collagen deposition, coincident with the loss of follicles. In conclusion, the ovarian environment in ArKO mice does not allow the characteristic development of follicles that culminates in ovulation and demonstrates an in vivo requirement of estrogen for normal ovarian function in the mouse.

Aging↗

PubMed2000

Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries.

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.

Animals↗

PubMed1999

The role of estrogen in folliculogenesis.

Gonadotrophins are fundamental to the mechanisms regulating follicle status and development. Follicles in the ovary are either quiescent or committed to one of two pathways: growth or atresia. The requirement for gonadotrophins by the follicles varies with development: committed follicles grow independently of gonadotrophins (primarily FSH) until the late preantral stage when antrum formation is contingent upon FSH. The involvement of estrogen in regulating gonadotrophin secretion is well documented and while evidence for a local regulatory role of estrogen in the ovary mounts, an obligatory role for estrogen in the folliculogenic process has not been established. The availability of a wide range of gene-disrupted mice termed 'knockouts', is providing information relevant to the study of folliculogenesis. Mice deficient in either estrogen or estrogen receptors, are infertile primarily due to either a block in folliculogenesis prior to antrum formation or as a consequence of failing to ovulate. Blocking estrogen stimulated, post-receptor molecules such as cyclin D2, severely retards granulosa cell proliferation and leads to infertility, although the contribution of estrogen in this model is not so clear given that FSH also stimulates cyclin D2. Similar problems dissociating the roles of FSH and estrogen are evident with the FSH deficient animal models. Nevertheless, estrogen is clearly an important and probably obligatory regulator of folliculogenesis, especially in the post antral stage. The exact points in the folliculogenic process where estrogen exerts its principal effects remains to be elucidated.

Animals↗

PubMed1999

Ovarian estrogen receptor alpha and beta mRNA expression: impact of development and estrogen.

We tested the hypothesis that ERalpha and ERbeta mRNAs in the rat ovary are regulated during the post-natal period and in immature rats in response to estrogen treatment. Total ovarian ERbeta mRNA was more abundant than ERalpha mRNA and expression of ERbeta increased between post-natal days 4 and 12, coinciding with advancing folliculogenesis and an increase in granulosa cell numbers. In contrast, ERalpha mRNA levels remained relatively constant during this period. In situ hybridisation studies localised both ERalpha and ERbeta to granulosa cells of growing follicles, in 25 day old ovaries, although not all granulosa cells in a follicle or all follicles expressed the ERs. Diethylstilboestrol (DES) administered in vivo to 21 day old rats, for up to 4 days, did not significantly alter the expression of either ER as determined by RT-PCR, despite a 5.5-fold increase in granulosa cell number in these ovaries. In situ hybridisation studies established that DES-treatment down-regulated granulosa cell ER mRNAs. RT-PCR analyses on isolated granulosa cells confirmed that ERalpha was significantly down-regulated by DES. The predominance of ERbeta over ERalpha in the ovary and the regulation of ERbeta mRNA expression during ovarian development, is consistent with an important biological role for ERbeta in granulosa cell proliferation and differentiation.

Animals↗

PubMed1999

Endometrial endothelin: regulator of uterine bleeding and endometrial repair.

1. Endothelin (ET) and its mRNA are present in endometrium. Expression of ET varies across the menstrual cycle, reaching maximal levels in the premenstrual phase, suggesting a paracrine role in endometrial bleeding and/or repair. 2. The major cellular source of ET is the epithelium, although endothelium and decidualized stroma are additional sites of production. Epithelial ET is the ET-1 isoform and this is able to contract rat thoracic aortic rings ex vivo. 3. Endothelin-1 production by cultured endometrial epithelial cells is markedly increased by serum and, to a lesser extent, by transforming growth factor-beta and interleukin-1 alpha, but not by epidermal growth factor, oxytocin, arginine vasopressin, thrombin or angiotensin II, which stimulate ET production in other tissues. 4. Endothelin-1 has mitogenic actions on endometrial stromal cells; it stimulates the uptake of [3H]-thymidine, acting via the AP-1 cis element c-jun. 5. Neutral endopeptidase (NEP), a membrane-bound ectoenzyme that is capable of degrading ET, is localized principally in endometrial stroma and immunoreactivity is maximal in the secretory phase of the cycle. 6. A potential role for ET in regulating endometrial bleeding is suggested by studies on endometrium from two groups of women who were experiencing abnormal uterine bleeding: users of the contraceptive Norplant (Leiras Co., Turku, Finland) and subjects with documented menorrhagia. In both groups, ET-1 immunoreactivity in endometrial epithelium was markedly reduced compared with the normal menstrual cycle and did not vary cyclically, while NEP immunoreactivity, particularly in the epithelium, was increased. Thus, ET may be involved in endometrial bleeding, as a vasoconstrictor before the onset of menstruation when vasoconstriction is intense and, subsequently, when it may be required in the cessation of menstrual bleeding. Furthermore, the mitogenic actions of ET may play a role in endometrial regeneration and remodelling during the menstrual cycle, particularly following menstruation.

Endometrium↗

PubMed1999

Current concepts of the mechanisms of menstruation.

Menstruation is the process whereby the superficial or functionalis layer of the endometrium lining the uterine cavity disintegrates and is removed from the uterine lumen towards the end of the luteal phase of a non-pregnant cycle. Within a period of 5-6 days the old lining is removed and a new lining regenerated without scarring occurring, a remarkable example of controlled tissue remodelling unparalleled in other organs. This chapter describes the phenomenon of menstruation and tissue repair and reviews the vasoconstrictor and inflammatory hypotheses for the initiation of menstruation. Particular emphasis is placed on the roles of steroids, prostaglandins, endothelin, lysosomal enzymes and matrix metalloproteinases, and the involvement of the fibrinolytic system and apoptosis. Repair of the endometrium is compared to the processes and mechanisms of wound healing.

Adolescent↗

PubMed1999

[Inhibin and ovarian cancer].

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumors and in the majority of postmenopausal women with mucinous ovarian cancers. The present report confirms these findings in a larger group of post-menopausal women. Immunohistochemistry for the inhibin alpha. beta A and beta B sununits shows predominantly epithelial staining in granulosa cell tumors and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha i-2 or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Adenocarcinoma, Mucinous↗

PubMed1998

Inhibin and ovarian cancer.

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumours and in the majority of postmenopausal women with mucinous ovarian cancers. The present manuscript confirms these findings in a larger group of postmenopausal women. Immunohistochemistry for the inhibin alpha, betaA and betaB subunits shows predominantly epithelial staining in granulosa cell tumours and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha(i-2) or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Aged↗

PubMed1998

Immunolocalization of inhibin and activin subunits in human endometrium across the menstrual cycle.

Inhibin/activin alphaC/alphaN and betaA subunits were localized immunohistochemically in the human endometrium throughout the menstrual cycle using an affinity-purified sheep polyclonal antibody raised against the alphaC/alphaN subunit and an affinity-purified rabbit polyclonal antibody raised against the betaA subunit. The betaB subunit was below the level of detection in all human endometrial samples tested. Immunoreactive inhibin alphaC/alphaN subunit was localized in the luminal epithelium, glandular epithelium, stromal tissues and vascular endothelium with no significant variation across the normal menstrual cycle. Immunoreactive betaA subunit, common to inhibin A and activins AA and AB was localized in the luminal and glandular epithelium and in migratory cells while the endometrial stromal cells, decidua, vascular smooth muscle and endothelium were devoid of immunoreactivity. A significant variation of immunoreactive betaA subunit was observed in glandular and luminal epithelium across the normal menstrual cycle. In proliferative endometrium, only a very low level of betaA immunostaining was seen in luminal and glandular epithelium, while the luminal epithelial staining increased significantly in the early secretory phase and remained relatively constant over the rest of the menstrual cycle. A progressive increase in betaA immunoreactivity was observed also in the glandular epithelium during the secretory phase reaching a maximum in the late secretory phases, and decreasing at menstruation. Co-localization studies on serial sections suggested that the migratory cells expressing strong betaA immunoreactivity were macrophages and neutrophils but not eosinophils or mast cells. Thus, cells within the human endometrium are capable of expressing inhibin/activin molecules in vivo. The variation in the pattern of secretion of the betaA subunit across the menstrual cycle suggests that activin peptides may have a physiological role in endometrial function.

Activins↗

PubMed1998

Effect of immunization against the amino-terminal peptide (alpha N) of the alpha 43-subunit of inhibin on follicular atresia and expression of tissue inhibitor of matrix metalloproteinase (TIMP-1) in ovarian follicles of sheep.

Ewes actively immunized against alpha N, the N-terminal peptide of inhibin alpha 43 precursor, have lowered fertility associated with ovulation failure, restricted tissue remodelling and reduced matrix metalloproteinase-2 activity in the follicular fluid at the time of expected ovulation. This could be due to altered ratios of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase (TIMP-1), or to the onset of atresia in antral follicles destined to ovulate. The objectives of the present study were to investigate the effects of immunization against alpha N on the localization of TIMP-1 in ovine follicles, and on follicular growth and atresia in the follicular phase. Ewes were either immunized against alpha N or remained as controls and the ovaries were removed before (0, n = 4) and at 12 h (n = 4) and 24 h (n = 4) after hCG administration in a synchronized follicular phase, 48 h after removal of intravaginal pessaries. Observations were made on a single section taken through the largest follicle present in the ovaries of each ewe. There were no healthy antral follicles > 1 mm in immunized ovaries (0/29) compared with controls (16/31) (P < 0.001), whereas the proportion of healthy antral follicles < 1 mm was the same in each group (9/19 versus 5/12). TIMP-1 immunoactivity was localized in large luteal cells, smooth muscle and endothelial cells, and in all antral follicles, including oocytes. At the time of hCG administration, no TIMP-1 immunoreactivity was detected in the apical region of the follicular wall of large follicles (> 6 mm) compared with the rest of the follicle wall, but staining appeared in the apical granulosa layer 24 h later. In newly formed corpora lutea, TIMP-1 expression was found along the invaginating vascular layer. There was no effect of immunization on the patterns of TIMP-1 immunoreactivity, suggesting that changes in TIMP-1 are not involved in the effects of alpha N. These data are consistent with a paracrine role for alpha N in the selection and atresia of antral follicles, and for TIMP-1 in tissue reorganization and steroidogenesis at the time of ovulation.

Analysis of Variance↗

PubMed1997

Hormonal and non-hormonal agents at implantation as targets for contraception.

The processes leading to implantation and the establishment of pregnancy involve hormonal and non-hormonal agents that offer opportunities as targets for contraception. Hormonal agents include progesterone, luteolytic factors (prostaglandin F2 alpha) and embryonic signals (chorionic gonadotrophin, oestradiol-17 beta, interferon-tau) responsible for maintaining the corpus luteum. Non-hormonal agents include surface antigens (attachment and adhesion molecules), vasoactive agents, tissue-remodelling enzymes (matrix metalloproteinases) and inhibitors (TIMPs), growth factors (epidermal growth factor and insulin-like growth factor families) and cytokines (such as leukaemia inhibitory factor, colony-stimulating factor-1, interleukin-1 (IL-1) and IL-6) associated with the pre-attachment period and the apposition, adhesion and invasion of the blastocyst. This review describes some of the hormonal and non-hormonal agents present at the time of implantation that may be exploited as targets for contraception in feral species. Particular attention is paid to the mouse as an experimental model and potential target species. The considerable species differences which exist in the models of implantation and placentation and the way in which the female 'recognizes' the presence of a viable conceptus offer a means of conferring species specificity on potential contraceptive targets for feral species.

Animals↗

PubMed1997

Endothelin and neutral endopeptidase in the endometrium of women with menorrhagia.

The mechanisms underlying excessive menstrual bleeding or menorrhagia are not understood. In view of its potent vasoconstrictor and growth factor properties, endothelin has been proposed to have a potential paracrine role in the regulation of uterine blood flow and therefore could be a factor in menorrhagia. We compared the cellular localization of endothelin and its metabolizing enzyme, neutral endopeptidase, in endometrial biopsies from women with documented menorrhagia and in those with a normal menstrual cycle. Menorrhagia was documented by measurement of menstrual blood loss, 146 +/- 141 ml (median +/- SD). Endothelin and neutral endopeptidase were localized by immunohistochemistry, and the staining intensity was graded. Their immunostaining patterns were found to differ in menorrhagia compared to the normal menstrual cycle. Endothelin was reduced in glandular epithelium in menorrhagia and did not vary cyclically, while neutral endopeptidase was increased in the glandular epithelium. In menorrhagia, stromal endothelin immunoreactivity was not different from the normal cycle and although neutral endopeptidase immunostaining in stroma was similar to the secretory phase of normal endometrium, cyclical variation was absent. The potential for increased metabolism of endothelin could be an explanation for the decreased endothelin immunostaining in the glandular epithelium.

Endometrium↗

PubMed1997

Regulation of follistatin production by rat granulosa cells in vitro.

The aims of this study were to apply enzyme-linked immunosorbent assays (ELISA) for human follistatins (FS) to measure total immunoreactive (ir-) rat FS and free rat FS, and investigate the regulation of production of total ir-FS and free FS by rat granulosa cells (GC) in vitro. Production of ir-inhibin was monitored as an index of GC function. The ELISAs for total ir-FS, based on an immunoradiometric assay developed recently for human FS, and free FS, based on capture of FS by a monoclonal antibody and detection by activin A binding, had sensitivities of 0.4 and 0.8 ng recombinant human (rh-) FS 288/ml, respectively, and did not cross-react with inhibin A, rLH, or FSH. rh-Activin did not cross react in the total ir-FS ELISA, but interfered with the measurement of free FS. Dilutions of GC-conditioned medium were parallel to the standard curve of rh-FS 288 for each assay. The values obtained in the free FS assay were 10- to 20-fold higher than those in the total ir-FS ELISA, suggesting that rat FS may be recognized by the antibodies differently than the human standard. Both total ir-FS and free FS production by undifferentiated GC from diethylstilbestrol (DES)-treated, immature rats increased with cell number and time in culture and were stimulated dose dependently by FSH, rh-activin A (except free FS, which was not measured because of interference), forskolin, and phorbol 12-myristrate. The effects of FSH and activin on FS production by undifferentiated GC were additive. There were significant effects of degree of differentiation of GC on basal FS production and responsiveness to FSH, LH, and rh-activin A. Both total ir-FS and free basal FS production increased up to 4-fold with the degree of differentiation of GC, produced by treating rats in vivo with DES (undifferentiated), DES plus FSH (partially differentiated), or DES plus FSH plus hCG (fully differentiated). The addition of FSH in vitro increased FS production by undifferentiated and partially differentiated GC, but not by fully differentiated GC. The only detectable effect of LH on FS production was on partially differentiated GC. Activin A stimulated total ir-FS production by undifferentiated and partially differentiated GC, but inhibited total ir-FS production by fully differentiated GC. Ir-inhibin production in these experiments was similar to that of FS with the following exceptions; phorbol 12-myristrate inhibited ir-inhibin production by undifferentiated GC, basal ir-inhibin decreased in fully differentiated GC, FSH stimulated ir-inhibin only in undifferentiated GC, and rh-activin A stimulated ir-inhibin at all stages. It is concluded that 1) FS protein production by cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways; 2) increasing GC differentiation is associated with a significant increase in basal FS production by rat GC and a change in the hormonal regulation of FS production; and 3) FS and ir-inhibin production by cultured rat GC can be differentially regulated. The results are consistent with the hypothesis that activin tone decreases within follicles as they develop due to increased production of the activin-binding protein FS.

Activins↗

PubMed1997

The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma.

In a previous study (see Ref.7), the molecular weight distribution of inhibin activity in fractionated human follicular fluid (hFF) and human male and female plasma/serum was determined by in vitro bioassay using ovine pituitary cells in culture and various specific inhibin A and inhibin alpha-subunit-directed immunoassays. It was shown, however, that the ovine in vitro bioassay detected inhibin B poorly. These findings are extended in the present study by the determination of the molecular weight profile of in vitro bioactivity using rat pituitary cells, which detects both inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay (ELISA), an RIA detecting the alpha N region of the alpha-subunit, an alpha-subunit ELISA (Pro-alpha C) directed to the inhibin forms containing the Pro sequence, and an alpha C subunit immunofluorometric assay that detects all inhibin forms. The profile in hFF of inhibin in vitro bioactivity, using rat pituitary cells in culture, significantly (P < 0.001) correlated with in vitro bioactivity using ovine pituitary cells (r = 0.85), inhibin A immunoactivity (r = 0.70), inhibin B immunoactivity (r = 0.89), and the combination of inhibin A + B immunoactivities (r = 0.93), with peaks of activity identified at 66K, 55K, 36K and 33K, consistent with presumed known mol wt forms of inhibin. Inhibin B profiles in fractionated serum from women stimulated with gonadotropins and male plasma consisted of two forms (66K and 36K), whereas inhibin A in female serum included, in addition, the 55K form. These findings indicated that higher molecular weight forms of inhibin B are present in biological samples, and their distribution differs from that of inhibin A, suggesting a differential processing of the precursor forms in the circulation. Pro-alpha C immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-alpha C subunit forms) and not with any high mol wt dimeric forms of inhibin. If this observation applies to a wider range of serum samples, the Pro-alpha C ELISA may provide an appropriate and specific assay for the measurement of free alpha-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-alpha C standards were calibrated in terms of their alpha C subunit content, as determined by an alpha C subunit immunoassay, with the inhibin B standard containing 60% of the alpha C subunit content compared with either the inhibin A or Pro-alpha C standard. After adjustments of the various standards for this difference in alpha C subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-alpha C immunoactivity across the hFF and serum chromatograms and compared with levels determined by the alpha-subunit-directed immunoassays. A high correlation (r = 0.59-0.96) was observed, indicating that the alpha-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-alpha C. It is concluded that: 1) inhibin in vitro bioactivity in hFF is largely attributed to the presence of 33-36K and 50-66K forms of inhibins A and B; and 2) inhibin alpha-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the alpha-subunit.

Animals↗

PubMed1997

Mitogenic actions of endothelin and other growth factors in ovine endometrium.

Endothelin-1 (ET-1) is present in ovine endometrium, primarily in epithelial cells, and increases around the time of implantation. We examined the cell type expressing ET-binding sites in vitro and whether ET-1 has mitogenic actions in the endometrium, alone or in synergy with other growth factors. Purified epithelial and stromal cells were prepared from luteal-phase endometrium. Specific receptors were demonstrated by binding of 125I-ET-1 and proliferative effects of ET-1 and/or other growth factors determined by uptake of [3H]thymidine by cells in serum-free culture. 125I-ET-1 bound to both epithelial (2516 +/- 820 c.p.m./well) and stromal (6368 +/- 1350 c.p.m./well) cells and was displaced by ET-1 (1 mumol l-1). There were no proliferative effects of ET on epithelial cells. ET-1 (10 nmol l-1) stimulated uptake of [3H]thymidine by stromal cells under serum-free conditions in 13/20 individual cell preparations, to 149 +/- 13% of control (untreated = 100%) with dose-dependence between the range of 1 to 100 nmol l-1. Stimulation by fetal calf serum was to 377 +/- 126% of control. The effects on proliferation by other growth factors (dose; % of control +/- S.E.M., number of positive/total number of cell preparations) were: IGF-I (13 nmol l-1; 182 +/- 14, 4/4), epidermal growth factor (EGF; 4.8 nmol l-1; 132 +/- 5%, 7/7), platelet-derived growth factor-BB (0.4 nmol l-1; 146 +/- 3, 2/2) and leukaemia inhibitory factor (0.4 nmol 1-1; 110 +/- 2, 3/3). All stimulations except that of EGF were significant and dose-responsive but only insulin was additive with ET (350 +/- 35, 5/5). ET-1 also stimulated expression of the the AP-1 cis element c-jun, this being maximal at 60 min of exposure to mitogen. ET-1, along with other growth factors has a likely paracrine role in cellular proliferation in the endometrium, possibly in association with blastocyst implantation.

Animals↗