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Biomedical subjects

R S Carson

Publications and source records attributed to R S Carson.

At least 19 recordsLinked to original sources

Efficacy of intracytoplasmic sperm injection using intentionally cryopreserved epididymal spermatozoa.

Microsurgical epididymal sperm aspiration was a great advance in the therapy of patients with non-reconstructable, obstructive azoospermia, most notably congenital bilateral absence of the vas deferens. Using conventional in-vitro fertilization, pregnancies were rarely achieved because the rate of oocyte fertilization was extremely poor. However, the use of retrieved spermatozoa in conjunction with intracytoplasmic sperm injection (ICSI) has dramatically increased the likelihood of embryo formation. Typically, sperm and oocyte harvesting are performed simultaneously. We have investigated whether frozen-thawed spermatozoa work as well as fresh spermatozoa. When we had concluded from our own population of patients (groups I and II) that they did, we adopted a policy of aspirating spermatozoa, primarily cryopreserving them and using them for ICSI at a later date. We found the fertilization rates of this latter cohort of patients (group III) to be excellent (37% per oocyte), and the ongoing pregnancy rate is quite satisfactory (40% per couple, 29% per cycle). We offer this approach as an alternative to the traditional scheme because it markedly eases the burden of partner scheduling on both the couple and the clinicians involved. In addition, assurance of the availability of male partner spermatozoa can be attained prior to beginning ovulation induction.

Adult↗

The effect of sperm preparation methods on the fertilization rate of oocytes micro-inseminated by subzonal sperm injection.

Three methods were used to prepare spermatozoa for subzonal injection into mature oocytes. In method A, the washed sperm suspension was incubated for 18 h in modified T6 culture medium. Method B consisted of incubating the sperm suspension for 6 h in regular T6 culture medium. In method C, the sperm suspension was incubated for 6 h in regular T6 culture medium containing 20% (v/v) follicular fluid. The percentages of acrosome-free spermatozoa and fertilization rates were compared for 42 treatment cycles assigned randomly to the three sperm preparation methods. The sperm suspensions prepared by methods A and C each had significantly higher proportions of acrosome-free spermatozoa compared to suspensions prepared by method B. The fertilization rates of oocytes micro-injected with spermatozoa prepared by methods A and C were significantly higher than for method B. Eight clinical pregnancies resulted from 28 cycles in which embryo replacement occurred. We conclude that the fertilization rate following subzonal sperm injection is related directly to the percentage of acrosome-free spermatozoa in the sperm suspension used for microinjection.

Embryo Transfer↗

Acute effect of PMSG on ovarian androgen-binding sites in the intact immature female rat.

Androgen-binding activity in ovaries containing only immature follicles is compared with that in ovaries stimulated with pregnant mare serum gonadotrophin to induce development of large preovulatory follicles. The androgen-binding sites present in cytosols prepared from unstimulated ovaries exhibited kinetics, affinity and a range of specificities for natural and synthetic androgens consistent with those exhibited by the androgen receptor isolated from androgen-sensitive tissues. Studies of sedimentation and DNA binding suggest that the receptor-like androgen-binding site present in unstimulated ovaries exists as the 'non-activated' form which is able to undergo transformation and bind to nuclear DNA. Properties of the androgen-binding sites isolated from ovaries stimulated with gonadotrophin were very different from those of the androgen receptor present in unstimulated ovaries. Specificity for androgens was reduced, capacity for dihydrotestosterone (DHT) and methyltrienolone (R1881) increased and androgen-binding activity was associated exclusively with the 4.5S form which was not able to bind to nuclear DNA. These data confirm that a shift from a receptor-like to a nonfunctional androgen-binding site is associated with the development of ovulatory follicles and suggest that shifts in androgen-binding populations will determine the response of developing ovarian follicles to androgens.

Androgens↗

Premature birth of live lambs induced by pulsatile infusion of ACTH.

This study was designed to investigate the effects of pulsatile infusion of ACTH into ovine fetuses on the endocrine changes that precede parturition, the timing of birth and the subsequent survival of the lamb. Where appropriate, these parameters were compared with fetuses infused with pulses of saline and uninfused normal term fetuses. Ten fetuses received a 15-min infusion of synthetic ACTH(1-24) (79 ng/min) from day 125 (n = 9) or day 126 (n = 1) of gestation. Seven fetuses were born prematurely within 174 +/- 14 h (mean +/- S.E.M.) after the commencement of the infusion, i.e. at 132 +/- 0.6 days, whilst three died in utero at 130-131 days. When born all lambs could breath, walk and suckle. Of the seven premature lambs, four died 2-10 days after parturition but three survived for at least 12 months after birth. Fetuses infused with pulses of ACTH exhibited intermittent but very large increases in plasma ACTH values, with the first pulse, on day 1, increasing ACTH values from 5.1 +/- 1.1 to 140 +/- 31.3 pmol/l (P less than 0.001). At the next sampling time, ACTH values were not significantly different from preinfusion values. A similar plasma ACTH profile was observed on each subsequent day of ACTH treatment. In contrast, fetuses (n = 4) infused with pulses of saline between 125 and 131 days exhibited fetal plasma concentrations of ACTH which ranged between 2 and 12 pmol/l for the majority of the time.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

Selective control of rat granulosa cell inhibin production by FSH and LH in vitro.

We studied the role of luteinising hormone (LH) and human chorionic gonadotropin (hCG) in regulation of rat granulosa cell inhibin production. Whereas pregnant mare serum gonadotropin (PMSG) or purified rat follicle-stimulating hormone (FSH) stimulated inhibin accumulation in culture media up to 6-fold in a dose-dependent manner, hCG or LH alone were without significant effect. Concomitant addition of hCG or LH to cultures containing half maximal or maximal stimulating concentrations of PMSG did, however, result in a dose-dependent inhibition of PMSG/FSH-induced inhibin production. However, in the 2-step culture system a biphasic effect of hCG treatment on FSH-primed granulosa cell inhibin and progesterone production was evident. hCG was only inhibitory when the cells in the 2-step system were primed with PMSG. These data indicate that granulosa cell inhibin production is under direct control of both FSH and LH, and provide a possible explanation for alterations in inhibin activity around the time of ovulation in vivo.

Animals↗

Transforming growth factor beta enhances basal and FSH-stimulated inhibin production by rat granulosa cells in vitro.

Transforming growth factor beta (TGF beta) caused a dose-dependent increase in both basal and follicle-stimulating hormone (FSH)-stimulated inhibin production by rat granulosa cells in culture. The TGF beta dose-response curve in the absence of FSH was approximately parallel to that in the presence of either a minimally effective dose (1 ng/ml) or a maximally effective dose (30 ng/ml) of FSH, suggesting an additive effect of these two agents on inhibin production. There was also a suggestion of an increased sensitivity of granulosa cell inhibin production to FSH when the cells were coincubated with TGF beta. The time course study showed that similar to FSH, the stimulatory effect of TGF beta on basal and FSH-stimulated inhibin production was evident on day 1 and was maximal by day 4. In addition, epidermal growth factor (EGF) reduced FSH-stimulated inhibin production with an ID50 value of 1.3 ng/ml. Coincubation of cells with EGF and 1 ng TGF beta/ml enhanced greatly the inhibitory action of EGF on FSH-induced inhibin production (ID50 less than 0.1 ng/ml). It is concluded that: (1) TGF beta directly stimulates inhibin production by rat granulosa cells and the combined effect with FSH was largely additive, (2) the inhibitory effect of EGF on FSH-induced inhibin production was enhanced by TGF beta, (3) individual members of the TGF beta/inhibin gene family regulate ovarian function, not only by direct action on follicle cells but also indirectly by influencing the production rate of other members of that family.

Animals↗

Pregnant mare's serum gonadotropin stimulates inhibin subunit gene expression in the immature rat ovary: dose response characteristics and relationships to serum gonadotropins, inhibin, and ovarian steroid content.

We have investigated the temporal changes and dose responses of ovarian inhibin gene expression, circulating inhibin, and ovarian estradiol content in PMSG-primed immature female rats. The relative levels of ovarian inhibin alpha- and beta A-subunit messenger RNA (mRNA), serum inhibin, and ovarian steroid content were measured after stimulation of immature female rats with 20 IU PMSG. Each of these variables rose in parallel and peaked at 48 h (relative ovarian inhibin alpha- and beta A-subunit mRNA increased 3.8-fold and 2.8-fold, respectively, serum inhibin 269 U/ml, vs. control 31, P less than 0.01, ovarian estradiol content 114 pmol/pair of ovaries vs. controls 4.7 P less than 0.001), corresponding to the time of maximal follicular development in this animal model. Subsequently each fell to reach a nadir at 96 h. The data are consistent with the hypothesis that PMSG stimulation of inhibin secretion occurs by a mechanism involving inhibin gene transcription. The increase in ovarian inhibin and estradiol synthesis during folliculogenesis and their simultaneous decline before ovulation suggest that 1) the circulating levels of inhibin and estradiol reflect follicular maturation and 2) these two hormones are regulated via a common mechanism during follicular development in the rat.

Animals↗

Ovine follicular fluid inhibits thymidine incorporation by 3T3 fibroblasts in vitro.

The ability of antral follicular fluid obtained from sheep follicles to inhibit 3T3 fibroblasts maintained for 48 h in concentrations of fetal calf serum optimal for cell growth was examined. Addition of pooled follicular fluid to cultures resulted in a dose-dependent and reversible inhibition of [3H]thymidine incorporation. Serum from ovariectomized ewes, fetal calf serum, bovine inhibin, oestradiol-17 beta, testosterone, cortisol or progesterone were without effect over a range of doses. Treatment of pooled follicular fluid with charcoal-dextran did not reduce inhibitory activity which was only partly removed by heating at 85 degrees C. Fluid obtained from large follicles (greater than 5 mm) was more potent as an inhibitor than was fluid obtained from smaller (less than 5 mm) follicles. Gel chromatography of pooled fluid resolved two peaks of inhibitory activity associated with material of Mr approximately 180,000 and less than 10,000 respectively. No inhibitory activity was evident in fractions of serum from ovariectomized ewes chromatographed in an identical manner. These results indicate that ovine follicular fluid contains two components able to inhibit reversibly mitosis of 3T3 fibroblasts in vitro.

Animals↗

Ovarian inhibin content in immature female rats following hypophysectomy: control by exogenous gonadotrophins.

The effect of exogenous ovine gonadotrophins on ovarian inhibin content in hypophysectomized immature female rats was studied to determine which of the pituitary gonadotrophins control ovarian inhibin production in vivo. Inhibin and steroid contents of 27,000 g ovarian supernatants were measured by specific radioimmunoassay. Whereas injection of ovine (o)FSH resulted in dose-dependent increases in ovarian weight, progesterone and oestradiol content above that of control animals injected with diethylstilbestrol alone, injection of oLH was without significant effect. Similarly, ovarian inhibin content was not altered significantly by injection of oLH, but was increased in a dose-dependent manner by injection of oFSH. Concentrations of inhibin in serum were correlated directly with ovarian inhibin content. The results of this study indicate that ovarian inhibin content is increased by injection of oFSH but not oLH. It is likely, therefore, that inhibin production by small antral follicles is controlled by FSH activity alone.

Animals↗

The effect of hypophysectomy on inhibin production by adult rat ovaries: changes in ovarian inhibin gene expression and serum inhibin.

We have investigated the effect of hypophysectomy on adult rat ovarian inhibin alpha- and beta A-subunit mRNA, serum inhibin, and circulating gonadotrophin levels. Immediate declines in ovarian inhibin gene expression and serum inhibin were observed following hypophysectomy, with a nadir on day 4. However, inhibin synthesis determined by these parameters was still apparent at 16 days post-hypophysectomy. Gonadotropin levels were suppressed following hypophysectomy and the fall in inhibin production was highly correlated with disappearance of FSH from the circulation. This study demonstrates that removal of pituitary gonadotropin stimulation results in diminished ovarian inhibin synthesis and provides further evidence indicating that FSH stimulates granulosa cell inhibin synthesis by enhancing inhibin gene transcription.

Animals↗

Induction of adrenocortical special zone in the male possum (Trichosurus vulpecula).

The induction of adrenocortical special zone (S.Z.) by gonadotrophin administration was studied in male brush tailed possums. Castrated males injected with porcine FSH (NIH-FSH-P2) formed well developed S.Z.s, varying in sizes from 5-20% of the gland's volume. Pregnant mare serum gonadotrophin (PMSG) was ineffective. From adrenal homogenates of FSH treated possums incubated with [3H]-progesterone the major conversion products were 5 beta-reduced steroid metabolites (72%) and cortisol (4%). The conversion products from adrenals of saline and PMSG treated males were cortisol and corticosterone (65%). Of the ten untreated castrates, one had a well developed S.Z. and two had S.Z.s at an early stage of development. Significant 5 beta-reduction of [3H]progesterone was only found in one animal. The plasma FSH concentrations were in intact males 317 +/- 41 ng rat FSH ml-1 (mean +/- SEM) and in castrates 769 +/- 64. The possible reasons for the lack of spontaneous S.Z. formation in intact males are discussed.

Adrenal Cortex↗

Direct inhibition of rat granulosa cell inhibin production by epidermal growth factor in vitro.

The effect of epidermal growth factor (EGF) on inhibin production by rat granulosa cells has been investigated using a recently developed inhibin radioimmunoassay (RIA). Granulosa cells from intact immature diethylstilbestrol (DES)-treated rats were exposed to EGF (1-100 ng/ml) in the presence or absence of FSH for varying periods in vitro. An inhibitory effect of EGF on basal inhibin secretion was evident at day 2 of culture and was sustained over the subsequent 2 days. This action on basal inhibin secretion was dose-dependent, and maximal inhibition to 50% of control was observed at a dose of 100 ng EGF/ml at day 4. EGF also inhibited basal progesterone secretion in a similar manner. EGF caused a dose-dependent inhibition of FSH-stimulated inhibin secretion, with an ID50 (0.5 ng/ml, 0.08 nM) about one-eighth that in the absence of FSH. In addition, EGF also inhibited the stimulation of inhibin production by 8-Br-cAMP and prostaglandin E2. To exclude the possibility that EGF was toxic to the granulosa cells, several biochemical parameters related to cell growth were measured. EGF treatment did not alter cell number but slightly increased [3H]thymidine incorporation into cellular DNA. The effect of EGF on [35S]methionine incorporation into cellular protein was biphasic, being stimulatory at doses less than 10 ng/ml but inhibitory at 100 ng/ml. The present data have demonstrated a direct inhibitory effect of EGF on basal and FSH-stimulated inhibin production by granulosa cells suggesting an important regulatory role of this growth factor in the differentiation of ovarian function.

Animals↗

Follicle-stimulating hormone and somatomedin-C stimulate inhibin production by rat granulosa cells in vitro.

The direct effect of somatomedin-C (Sm-C) and FSH on inhibin production by rat granulosa cells in vitro has been examined. FSH stimulated accumulation of inhibin in culture media in a dose-dependent manner with maximal stimulation (6-fold) being observed at a dose of 300 ng FSH/ml. Addition of Sm-C (30 ng/ml) either alone or in the presence of FSH (3-300 ng/ml) increased inhibin production (up to 5-fold). Sm-C alone was effective over the physiological dose range of 3-100 ng/ml. Concomitant addition of FSH (100 ng/ml) and Sm-C (3-100 ng/ml) resulted in a significant increase in inhibin production at all doses of Sm-C. The dose-dependent effects of FSH and Sm-C were also time dependent with a synergistic effect apparent after 48 h of culture. The Sm-C induced FSH inhibitory activity of granulosa cell culture media was confirmed as authentic inhibin by the demonstration of a dose-dependent neutralization of this activity by a monoclonal antibody raised against purified bovine inhibin. The data indicate a direct role for both FSH and Sm-C in ovarian inhibin production and provide additional evidence for an autocrine-paracrine role for Sm-C in granulosa cell differentiation.

Animals↗

Response of prepubertal ewes primed with monensin or progesterone to administration of FSH.

Prepubertal ewe lambs were treated with FSH after progesterone priming for 12 days (Group P), monensin supplementation for 14 days (Group M) or a standard diet (Group C). Serial blood samples were taken for LH and progesterone assay, and ovariectomy was performed on half of each group 38-52 h after start of treatment to assess ovarian function, follicular steroid production in vitro and the concentration of gonadotrophin binding sites in follicles. The remaining ewe lambs were ovariectomized 8 days after FSH treatment to determine whether functional corpora lutea were present. FSH treatment was followed by a preovulatory LH surge which occurred significantly later (P less than 0.05) and was better synchronized in ewes in Groups P and M than in those in Group C. At 13-15 h after the LH surge significantly more large follicles were present on ovaries from Group P and M ewes than in Group C. Follicles greater than 5 mm diameter from ewes in Groups P and M produced significantly less oestrogen and testosterone and more dihydrotestosterone, and had significantly more hCG binding sites, than did similar-sized follicles from Group C animals. Ovariectomy on Day 8 after the completion of FSH treatment showed that ewes in Groups P and M had significantly greater numbers of functional corpora lutea. These results indicate that, in prepubertal ewes, progesterone priming and monensin supplementation may delay the preovulatory LH surge, allowing follicles developing after FSH treatment more time to mature before ovulation. This may result in better luteinization of ruptured follicles in these ewes, with the formation of functional corpora lutea.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Infusion of gonadotrophin-releasing hormone (GnRH) induces ovulation and fertile oestrus in mares during seasonal anoestrus.

In Exp. 1, 30 Standardbred mares in deep seasonal anoestrus were divided into 3 equal groups and treated with 0, 50 (G50) or 100 (G100) ng GnRH kg-1h-1 for 28 days via osmotic minipumps. Ovulation occurred in 0/10, 3/10 and 7/10 mares respectively (P less than 0.05). Plasma GnRH profiles (Days -6, 0, 2, 6, 12, 20, 28 and 34 relative to pump insertion) were dose-dependent (P less than 0.01) and peaked on Day 12 of infusion. Mean daily plasma LH concentrations were biphasic in treated mares that ovulated, with LH peaks occurring around Day 6 and Days 16-20. By contrast, in treated mares that did not ovulate the initial LH rise was followed by a steady decline to the end of the experiment. LH pulse frequency in treated mares increased between Day 0 and Day 21 of the experiment. LH pulse frequency in G100 mares was higher (P less than 0.05) than in G50 and control mares on Day 3, and higher than the controls on Days 7 and 21 of the experiment. There were no significant differences in LH pulse amplitude between the groups on the days studied. In Exp. 2, 27 Standardbred mares in shallow seasonal anoestrus received no treatment (N = 13) or a subcutaneous infusion of GnRH (100 ng kg-1h-1) via osmotic minipump for 28 days (N = 14). Mares were served by a stallion during oestrus. Day of ovulation was earlier in treated than in control mares (18.6 +/- 2 vs 41.9 +/- 6 days; P less than 0.001). Likewise, time of conception was earlier in treated than in control mares (25.2 +/- 6 vs 49.1 +/- 9 days; P less than 0.05). One mare in the control group failed to conceive while one treated mare conceived to an undetected ovulation. The results show that constant GnRH infusion induces ovulation and fertile oestrus in mares during deep and shallow seasonal anoestrus.

Anestrus↗

Permeability of rat ovarian follicles to LH during development and luteinization.

A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of 51Cr and 125I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34.2 +/- 3.0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P less than 0.05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46.2 +/- 1.7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically. Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.

Animals↗

Steroid production and hCG binding by ram-induced ovarian follicles in seasonally anoestrous ewes.

The introduction of rams to a group of previously isolated anoestrous ewes has been shown to stimulate ovarian follicular development and ovulation. The present experiment was carried out to determine the ability of follicles arising from this ram stimulus to produce steroids and bind hCG. Seasonally anoestrous Southdown ewes were exposed to rams for 24 h, 40 h, 3 days, 10 days or 20 days before ovariectomy. Steroid production and the concentration of hCG binding sites in follicles dissected from the ovaries were measured in vitro. The presence of a ram caused ovulation and enhanced oestradiol production by follicles, but had little effect on total androgen production or the number of hCG binding sites present in the follicles when compared to follicles from anoestrous ewes. The oestradiol concentrations in large follicles were not as high as in preovulatory follicles from cyclic ewes reported in other studies. Follicles continued to develop through the ram contact period and when incubated after 40 h and 10 days of ram contact produced high levels of progesterone, indicating partial luteinization, although the corpora lutea (CL) resulting from the induced ovulations regressed prematurely. We suggest that the lack of hCG binding sites in ram-induced follicles may be the cause of poor luteinization and suboptimal development of luteal tissue after induced ovulation in ewes during seasonal anoestrus.

Anestrus↗