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Biomedical subjects

D M de Kretser

Publications and source records attributed to D M de Kretser.

At least 145 records · Page 8Linked to original sources
PubMed1991

Circulating immunoreactive inhibin, gonadotropin, and prolactin levels during pregnancy, lactation, and postweaning estrous cycle in the rat.

Serum inhibin levels were measured by heterologous RIA during pregnancy, lactation, and the post-weaning estrous cycle in the rat and correlated with changes in serum FSH and LH and prolactin. Blood was serially collected by cardiac puncture under light ether anesthesia from adult Sprague-Dawley rats on alternate days throughout the experimental period. For the first 8 days of pregnancy, immunoreactive inhibin levels remained high, then gradually decreased to reach a nadir at Day 16, and subsequently rose steeply until parturition. The pattern of serum immunoreactive inhibin levels during early pregnancy does not support a corpus luteum source and the dramatic rise from Day 16 to Day 22 correlates with the recommencement of follicular development in the ovary. Inhibin levels decreased rapidly on the day after birth and were suppressed until Day 8 of lactation, slowly increasing thereafter to reach a plateau from Day 14 until weaning (Day 22.5 of lactation). These changes in inhibin levels positively correlated with LH and FSH and negatively with prolactin, and are consistent with an ovarian source for inhibin associated with the recommencement of follicular development resulting from the diminution of the suckling stimulus. Immediately after weaning, serum immunoreactive inhibin levels showed a 4-day cyclic pattern corresponding to the estrous cycle identified by vaginal smear. Inhibin levels peaked on the day of proestrus, reached a nadir on the day of estrus, and rose slowly during metestrus and diestrus to a new peak at proestrus. Serum FSH levels showed an inverse correlation to inhibin levels consistent with a feedback relationship with inhibin.

Animals↗
PubMed1991

Steroidogenesis-stimulating activity in the gonads: comparison of rat testicular fluid with bovine and human ovarian follicular fluids.

The steroidogenesis-stimulating activities of ovarian follicular fluid from bovine (bFF) and human (hFF) sources, were compared with those of adult rat testicular fluid (rTF) using an in vitro bioassay system based on stimulation of testosterone production by purified adult rat Leydig cells during a 20-h incubation. Rat TF and bFF were charcoal-treated to remove steroids prior to assay, and the major active fraction of hFF was collected after gel-permeation chromatography. All three fluid samples stimulated both basal and maximal hCG-stimulated testosterone production, although the resulting log dose-response lines of bFF and the hFF extracts were not parallel with those of rTF. Both rTF and bFF were active over a similar dose range (5.2-150 microliters and 9.7-150 microliters, respectively) and both had a more than additive interaction with hCG on testosterone production. The stimulatory activity of the hFF extract was considerably greater than that of either rTF or bFF in the absence of hCG, but hFF extract had only an additive effect with hCG in stimulating testosterone production. Moreover, unlike rTF activity, which was inhibited by co-incubation with the protein synthesis inhibitor, cycloheximide, the activity of the hFF extract was not affected by cycloheximide. The factors responsible for activity in all three fluids were of a large molecular size (greater than 30 kDa), as determined by ultrafiltration or gel-permeation chromatography. However, in contrast to both rTF and bFF, hFF extract activity was removed by charcoal extraction. Human FF extract was inactivated by heat (100 degrees C, 30 min), whereas rTF activity was partially (70%) heat-labile and bFF was not affected by heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The radioimmunoassay of follicle-stimulating hormone (FSH)-suppressing protein (FSP): stimulation of bovine granulosa cell FSP secretion by FSH.

A RIA for bovine (b) FSH-suppressing protein (FSP) was developed using an antiserum raised in a rabbit to purified 39-kDa bFSP, iodinated 35-kDa FSP as tracer, and purified 35-kDa bFSP as standard. Purified 35-kDa FSP was iodinated using the iodogen procedure, and the iodinated FSP was purified by dye affinity chromatography. After a logit log-dose transformation of the dose-response curves, parallel displacement lines were observed between 31-, 35-, and 39-kDa FSP, bovine follicular fluid, bovine granulosa cell culture medium, and medium from bovine granulosa cells stimulated with bFSH. The specificity of the assay was investigated by comparing the immunoassay levels of FSP with in vitro bioassay levels based on the ability of FSP/inhibin to suppress FSH in rat anterior pituitary cell cultures in fractions obtained throughout the purification procedure of FSP from bovine follicular fluid. This demonstrated that 1) the FSP immunoactivity was associated with in vitro bioactivity in all fractions of the purification procedure; 2) a number of inhibin-related and other proteins showed low (less than 0.5%) or nondetectable cross-reactivity in the RIA; and 3) the in vitro biological to immunological ratios for 31-, 35-, and 39-kDa FSP were similar, indicating that the RIA detects all forms of purified bFSP. The secretion of FSP by bovine granulosa cells in culture was investigated in the presence and absence of bFSH and bLH, respectively. FSP production was proportional to granulosa cell number and decreased from highest levels at 24 h to lowest levels at 96 h of culture. The addition of either bFSH or 8-bromo-cAMP to the culture medium stimulated FSP production by a factor of 2-3 at 48 and 72 h of culture, while the addition of bLH had no effect on FSP production. Theca interna tissue cultured under the same conditions did not produce FSP. In contrast to FSP, stimulation of bovine granulosa cells with bFSH or bLH had no effect on inhibin production during the 96 h of culture, while the addition of bFSH and bLH caused a stimulation of progesterone production at 48 and 72 h of culture. It is concluded that 1) the RIA described here is specific for all mol wt forms of bFSP; 2) FSP was secreted by bovine granulosa cells and not thecal cells in vitro; and 3) FSP secretion by bovine granulosa cells in vitro is regulated by bFSH and not bLH.

Animals↗
PubMed1991

The negative feedback effects of testicular steroids are predominantly at the hypothalamus in the ram.

This study aimed to delineate the hypothalamic and/or pituitary actions of testosterone and its primary metabolites 5 alpha-dihydrotestosterone and estradiol (E) in adult castrated rams (wethers) during the breeding season. In Exp 1, wethers were treated for a week with twice daily injections (im) of peanut oil, 8, 16 or 32 mg/day testosterone propionate (TP) or dihydrotestosterone benzoate (DHTB) or an sc silastic implant containing 1 or 3 cm E. TP decreased plasma LH concentrations, increased (P less than 0.05) LH interpulse interval, did not have consistent effects on LH pulse amplitude, and had minimal effects on plasma FSH concentrations. DHTB decreased LH and FSH concentrations and increased (P less than 0.05) LH interpulse interval. E reduced (P less than 0.05) plasma LH and FSH concentrations and increased LH interpulse interval but had no effects on LH pulse amplitude. In Exp 2, hypothalamo-pituitary disconnected wethers given 125 ng GnRH every 2 h, were treated with either peanut oil, 32 mg/day TP or DHTB or 3 cm E. None of the treatments affected plasma LH or FSH concentrations or LH pulse amplitude. Exp 3 investigated the effects on GnRH of treatment of wethers either with peanut oil or TP. TP reduced GnRH concentrations (P less than 0.05) and pulse amplitude (P less than 0.01) and increased interpulse interval (P less than 0.05). These data provide evidence that, during the breeding season, the principal site of negative feedback of testicular steroids in the ram is the hypothalamus, resulting in decreased GnRH secretion; feedback effects at the pituitary are minimal.

Animals↗
PubMed1991

Inhibin concentrations throughout the menstrual cycles of normal, infertile, and older women compared with those during spontaneous conception cycles.

Plasma immunoreactive inhibin levels have been measured in a series of normal conception cycles (group I; n = 7), and the data compared to inhibin concentrations in normal menstrual cycles (group II; n = 8), in women with luteal phase defects (group III; n = 7), and in women in the perimenopausal period (group IV; n = 6). Daily plasma levels of LH, FSH, progesterone, estradiol, and inhibin were determined in each subject, and daily mean profiles for each hormone in each subject group were calculated and expressed as geometric means with 68% confidence limits. During the follicular and early luteal phases, inhibin concentrations in the normal nonpregnant group (group II) were significantly higher than those in the conception cycles of group I, but after implantation in the conception cycles, inhibin concentrations increased to levels in excess of those seen at any time in nonconception cycles (716-1352 U/L; P less than 0.02). The postimplantation rise in inhibin did not initially appear to follow the same pattern as progesterone. While progesterone concentrations rose within 24 h of the first detectable increase in hCG, inhibin levels did not increase until 3 days later, although after this point concentrations increased serially and in parallel with progesterone. LH and FSH concentrations were markedly suppressed after implantation. Follicular and early luteal inhibin concentrations in cycles with luteal phase defects were also higher than those in conception cycles, although this difference was only significant in the midfollicular phase. Follicular phase inhibin concentrations in cycles from older women (group IV) were lower than those in groups II and III, but were not distinguishable from those in the conception cycles. Estradiol concentrations in the same subjects were significantly lower during the early follicular phase, while follicular and luteal FSH concentrations were significantly higher than those during conception cycles. Finally, examination of the relationship between inhibin, FSH, and estradiol around menstruation in the older women revealed a far closer temporal association between FSH and estradiol than between FSH and inhibin. In conclusion, inhibin concentrations rise and fall throughout the human menstrual cycle in a manner that is similar to but at specific times significantly different from that of either of the ovarian steroids estradiol and progesterone. It is considered to be a peptide of granulosa cell origin and may be an indicator of the size of the follicular pool during the early stage of the cycle. However, although there is some degree of inverse correlation between profiles of inhibin and profiles of FSH, this relationship is not particularly clear.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1991

Serum FSH-suppressing activity of human recombinant inhibin A in male and female rats.

After a single i.v. injection of purified human recombinant inhibin A (hr-inhibin) or bovine follicular fluid (bFF) to 3-day castrated 35-day-old male rats, serum FSH concentrations fell (P less than 0.05) between 4 and 8 h, returning to control concentrations by 16-24 h. Administration of graded doses of hr-inhibin (0.625-10 micrograms/100 g body wt) and bFF (31.3-250 microliters/100 g body wt) resulted in a parallel dose-related suppression of serum FSH with a maximum suppression 50% of controls. Similar experiments in 2-day ovariectomized 85-day-old female rats also showed a dose-related suppression with a maximum suppression approximately 30% of controls. Serum LH concentrations remained unchanged in all studies with male or female rats. The biological activity of hr-inhibin in vivo was determined for male and female rats in terms of a standard bFF preparation defined by an in-vitro bioassay based on the suppression of FSH content in rat pituitary cells in culture. In males hr-inhibin exhibited a biopotency of 407 (159:1050; fiducial limits) U/micrograms protein and in females the biopotency was 358 (226:565) U/micrograms protein. These potencies are lower than that measured in the in-vitro bioassay (1120 (1040:1210) U/micrograms protein) and differences between in-vivo and in-vitro systems were attributed to the use of bFF rather than a purified human inhibin preparation as standard. These results indicate that hr-inhibin behaves similarly in vivo to bFF. Furthermore, based on the large working range and relatively good precision, the female rat system provides a good basis for an inhibin in-vivo bioassay method.

Animals↗
PubMed1991

Studies on the testicular source of inhibin and its route of secretion in rams: failure of the Leydig cell to secrete inhibin in response to a human chorionic gonadotrophin/LH stimulus.

To determine whether Leydig cells produce inhibin in the ram, Leydig cells were stimulated by administering human chorionic gonadotrophin (hCG) or raising the levels of endogenous LH by an injection of gonadotrophin releasing hormone (GnRH). Plasma concentrations of testosterone increased in the 72 h after either a single injection (P less than 0.05) or two injections (P less than 0.01) of hCG. Plasma concentrations of inhibin were not significantly influenced by either one or two injections of hCG. Administration of GnRH (1 microgram) caused an 11-fold increase in plasma concentrations of LH but did not influence concentrations of inhibin in either the jugular or testicular veins (pampiniform plexus). In contrast, concentrations of testosterone were increased by about fourfold in both jugular (P less than 0.01) and testicular (P less than 0.05) veins. The concentrations of inhibin in the testicular vein were 1.3-fold higher than in the peripheral plasma (P less than 0.05) both before and following treatment with GnRH whereas the concentrations of testosterone were 18- to 21-fold greater than in peripheral concentrations. In view of the difference in concentrations of inhibin between testicular and jugular veins, in a further experiment a sample was taken from the jugular vein, a vein located in the tunica vasculosa of the testis (testicular vein) and from a vein (spermatic vein) and lymph vessels located in the spermatic cord. The mean (+/- S.E.M.) concentrations of inhibin were highest in the testicular lymph (45.93 +/- 4.21 micrograms/l; P less than 0.001) compared with the peripheral (4.14 +/- 0.31 micrograms/l), spermatic (8.0 +/- 1.17 micrograms/l) or testicular (6.4 +/- 0.82 micrograms/l) plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Inhibin in rams.

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Animals↗
PubMed1990

Inhibin.

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Activins↗
PubMed1990

Human chorionic gonadotrophin raises serum immunoreactive inhibin levels in men with hypogonadotrophic hypogonadism.

Inhibin is a gonadal glycoprotein hormone involved in the regulation of FSH. To elucidate the regulation of inhibin production we investigated the acute (daily for 1 week) and chronic (9-10 months of follow-up) changes in immunoreactive inhibin, testosterone, LH and FSH levels in the serum of three hypogonadotrophic hypogonadal patients treated first with hCG alone (for 3-6 months) and then hCG combined with FSH (1-5 months). One patient was unexpectedly resistant to gonadotrophin therapy; in the other two, hCG, with or without FSH, caused a rise in inhibin and testosterone, supporting previous observations that LH, as well as FSH, plays a role in the regulation of inhibin or inhibin-related peptides in men.

Adult↗
PubMed1990

Germ cell-Sertoli cell interactions.

The interactions between the Sertoli cells and germ cells are progressively becoming an important part of testicular physiology. This paper explores the cytological basis for these interactions, detailing the cyclic changes in the Sertoli cells in concert with the stages of the seminiferous cycle and the nature of the blood-testis barrier. These cytological changes are correlated with a number of variations in the function of Sertoli cells. The mechanisms by which germ cells and Sertoli cells interact are explored and can be divided into those using cell-to-cell contact and others utilizing paracrine factors.

Animals↗
PubMed1990

Intragonadal regulation of immune system functions.

Immune responses within the mammalian gonads, and in particular the testis, are deficient in spite of adequate lymphatic drainage and the presence of lymphocytes and MHC II+ macrophages. There is considerable evidence from in vivo and in vitro studies that this 'suppression' of the immune system may be due, at least in part, to localized inhibition or regulation of normal lymphocyte and/or macrophage functions within the gonads. In the testis, both steroidal and non-steroidal products of the Leydig cells, including androgens, endorphins, and inhibin-related proteins, have been implicated in mediating this activity. In turn, a number of immune cell cytokines affect steroidogenic cell function in vitro. The studies described in this paper indicated that [3H]-thymidine incorporation by adult rat thymocytes in vitro was inhibited by conditioned medium collected from short-term incubations of Percoll-purified adult rat Leydig cells, but stimulated by testicular interstitial fluid and by conditioned medium collected from short-term incubations of adult rat seminiferous tubules. The factors responsible for these effects on thymocyte function appeared to be of large molecular weight, as they were retained by ultrafiltration membranes with exclusion limits of 10,000 or 30,000 daltons. It is hypothesized that an 'immunosuppressive' mechanism, principally mediated by non-steroidal factors secreted by the steroidogenic cells of the gonadal interstitial tissue, exists within the gonads in order to prevent activation of the immune system by germ cell antigens and growth factors associated with germ cell proliferation and differentiation. This mechanism probably acts in parallel with normal antigen-specific tolerance mechanisms operating at the gonadal level. As immune responses to germ cells are believed to be a significant causative factor in infertility, particularly in men, this represents an important area for further study.

Animals↗
PubMed1990

Identification of inhibin and inhibin-related proteins in human follicular fluid.

Inhibin-related proteins were identified in human follicular fluid following fractionation by gel permeation chromatography under neutral and acidic conditions, reversed-phase high performance liquid chromatography (HPLC) and preparative polyacrylamide gel electrophoresis. A number of molecular mass forms of inhibin (30-36 and 59-66 kDa) based on their in vitro biological and immunological activities were identified, of which 59-66 kDa inhibin was the predominant form. Bioactive fractions devoid of inhibin immunoactivity were also identified with molecular masses of 46 and 55 kDa. Based on their retention positions on reversed-phase HPLC and their lack of inhibin immunoactivity, these proteins are likely to be follicle stimulating hormone (FSH) suppressing proteins/follistatins previously identified in bovine and porcine follicular fluids. In addition, an immunoactive inhibin fraction devoid of bioactivity was identified in large amounts in the 50-70 kDa region of the gel permeation chromatogram at neutral pH. This material, based on previous findings with the fractionation of bovine follicular fluid, is likely to be the alpha-inhibin subunit precursor protein. No FSH stimulating activity (activin) was identified in any of the chromatograms, suggesting that the levels of activin in human follicular fluid are low. In conclusion, inhibins of molecular mass 30, 36, 59 and 66 kDa have been identified in human follicular fluid. Proteins with inhibin bioactivity devoid of immunoactivity and vice versa have also been detected and these proteins are probably FSH suppressing protein and an alpha-inhibin subunit precursor protein. Activin could not be identified.

Chemical Fractionation↗
PubMed1990

A quantitative study of spermatogenesis in the developing rat testis.

Quantitative (stereological) studies were performed to determine the number of germ cells in the developing rat testis. Sprague-Dawley rats aged 1-70 days were fixed by immersion or perfusion and embedded in Epon Araldite. Blocks of tissue were sectioned at 1.5 microns and stained with toluidine blue dye. Sections were systematically scanned and the areal density of nuclear profiles counted using an unbiased counting frame. Numerical density and absolute number of germ cells in the processed block were then estimated. Corrections for processing shrinkage were determined by comparing the volume of processed and unprocessed samples. The results demonstrate the necessity of determining absolute number rather than volume density (or areal density) in comparing germ cell numbers. In these experiments, spermatogonial numbers stabilized in the range 18.4-23.6 million per testis on Day 30. The number of primary spermatocytes that were first apparent on Day 15 increased rapidly to 54.6 million per testis on Day 30 and then slowly to 73.6 million on Day 70. Round spermatids were first apparent on Day 25 and increased rapidly to 85.7 million per testis on Day 40, then continued to increase to 151.9 million on Day 70. The study provides both methods and baseline data for future experiments involving manipulation of the spermatogenic potential of the testis.

Animals↗
PubMed1990

Changes in plasma inhibin levels following pulsatile gonadotrophin-releasing hormone therapy in a man with idiopathic hypogonadotrophic hypogonadism.

Changes in circulating inhibin levels were related to changes in testosterone (T) and the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a hypogonadotrophic hypogonadal man before and during pulsatile gonadotrophin-releasing hormone therapy which resulted in normal spermatogenesis. Before treatment, the plasma inhibin levels in the patient (210 +/- 50 U/l; mean +/- SD of four samples) were lower than in normal controls (552 +/- 150 U/l; p less than 0.01), as were T (1.1 nmol/l) and gonadotrophin (less than 1.0 IU/l) levels. Within 1 week of gonadotrophin-releasing hormone treatment, plasma LH (14.1 +/- 0.7 IU/l) and FSH (14.4 +/- 0.6 IU/l) reached supraphysiological levels. In response, T and inhibin concentrations increased progressively to reach high normal levels (27.7 +/- 1.6 nmol/l and 609 +/- 140 U/l) at 4 weeks, by which time the gonadotrophin levels stared to decline and gradually returned to the normal range between 12 and 24 weeks of treatment. There was a concomitant decrease in T and inhibin levels which remained within the normal range. The decline in the FSH level following the rise in testicular hormones was earlier and steeper than that of LH (37.5% decrease at 4 weeks vs. 30.4% at 12 weeks), suggesting that T and inhibin may act together to inhibit pituitary FSH secretion as opposed to LH secretion which is primarily controlled by T. It is concluded that, in man, during maturation of the pituitary-testicular axis, changes in circulating inhibin parallel those of T, and quantitatively normal inhibin secretion is dependent on gonadotrophin stimulation. FSH secretion may be regulated through negative feedback control, by both T and inhibin.

Adult↗
PubMed1990

Immunoactive inhibin as a marker of Sertoli cell function following cytotoxic damage to the human testis.

Sertoli and Leydig cell functions were evaluated in men with testicular damage due either to cytotoxic chemotherapy (CCT) or radiotherapy (XRT). Serum immunoactive inhibin, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone concentrations were measured in 15 men (19-50 years) who had received 6-10 courses of combination CCT (mustine, vinblastine, procarbazine and prednisolone) for Hodgkin's disease 1-8 years earlier and 18 men (21-49 years) who had undergone unilateral orchidectomy for testicular seminoma followed by XRT (30 Gy) to the remaining testis, 1-4 years earlier. Normal men (n = 16, 19-36 years) acted as controls. Median inhibin (422 U/l) and testosterone (16.0 nmol/l) levels in the CCT-treated group were not significantly different from controls, whereas median FSH (14.5 IU/l) and LH (10.0 IU/l) levels were higher (p less than 0.0001 and p less than 0.001) than normal (2.9 and 5.5 IU/l). The median inhibin/FSH (I/FSH) ratio in the patients was lower (p less than 0.0001) than in the controls (33.8 vs. 187.0) as was the testosterone/LH (T/LH) ratio (1.7 vs. 3.8, p less than 0.001). In the XRT-treated group, both median inhibin (194.5 U/l) and testosterone (12.7 nmol/l) levels were lower (p less than 0.0001 and p less than 0.01) than normal (532.8 U/l and 20.0 nmol/l) in the presence of greatly elevated FSH (26.0 IU/l) and LH (14.5 IU/l) levels. In conclusion, CCT-induced testicular damage is associated with subtle Sertoli and Leydig cell dysfunction demonstrated by the reduced I/FSH and T/LH ratios; however, compensatory mechanisms maintain normal testosterone and inhibin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Physicochemical and biological characterization of recombinant human inhibin A.

Recombinant human inhibin A was isolated from recombinant mammalian cell line culture media. Two forms of inhibin were identified with Mr of 34 and 31 Kd composed of subunits (alpha, beta) of 24 and 15 Kd and 21 and 15 Kd respectively. Both forms are bioactive in an inhibin in vitro bioassay and immunoactive with potencies comparable to or higher than purified bovine inhibin. Amino acid analyses and NH2-terminal sequences of each of the subunits are consistent with those predicted from their cDNA structures. The inhibin alpha- but not beta-subunit is glycosylated based on its binding to the lectins concanavalin A and wheat germ lectin. The difference in molecular weight of 31 and 34 Kd inhibin is attributed to variation in glycosylation of the alpha-subunit. The 31+34 Kd inhibin is heterogeneous on isoelectric focusing gels consisting of four isoforms in the pH range 6.2-7.6. Inhibition also exhibits in vivo biological activity by suppressing serum FSH but not LH in castrate male rats. These physicochemical and biological characteristics of recombinant human inhibin are similar to those described for native inhibin isolated from a variety of other species.

Amino Acid Sequence↗