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P. F. Smith and D. A. Keefer

Summary. Light microscopic immunocytochemistry was utilized on plastic-embedded, acrolein-fixed pituitary glands from intact rats and rats killed 14 days after ovariectomy to determine which cell types were undergoing cell division. A significant increase in the number of cell divisions in anterior pituitary cells was seen in ovariectomized rats compared to intact controls. Most of the dividing cells in ovariectomized rats were immunocytochemically identified as gonadotrophs, but dividing somatotrophs and non-immunoreactive cells were also found. All of the dividing gonadotrophs stained with both anti-FSHβ and anti-LHβ. These cells were large and ovoid with plentiful vesiculated rough endoplasmic reticulum and a single population of granules with a mean diameter of 200 nm. Many of the dividing cells which were not immunoreactive with any of the antisera employed contained small granules, 100 nm in diameter, but the content of these granules was not determined.

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J. S. Gale, P. Smith, P. Truman, and K. P. McNatty

Summary. The presence of a fecundity gene (F) in Booroola Merino ewes increases the ovulation rate. To test how F gene expression affects the gonadotrophin-releasing hormone (GnRH) concentration in hypothalamic or extrahypothalamic regions of the brain, GnRH was measured by radioimmunoassay in acetic acid extracts of various brain tissues from Booroola ewes which were homozygous (FF), heterozygous (F+) or non-carriers (++) of the F gene. The GnRH concentration in brain tissues from FF, F+ and ++ animals which had been ovariectomized 5 months previously was also evaluated.

No significant F gene-specific differences were noted in any of the brain areas tested, in intact or ovariectomized animals. However, in ovariectomized ewes, the concentrations of GnRH increased about 2-fold in the median eminence of the hypothalamus, remained unchanged in the medial basal hypothalamus and dropped to <10% of the values in intact ++ animals in the preoptic area.

These studies suggest that the changed pituitary sensitivity and increased gonadotrophin release in Booroolas carrying the F gene(s) is not attributable to increased hypothalamic GnRH concentrations in these animals.

Keywords: GnRH; sheep; brain; Booroola; F-gene

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D. A. Taggart and P. D. Temple-Smith

Summary. Changes in the number and distribution of spermatozoa in the epididymis of the adult brown marsupial mouse were examined during July/August in mated and unmated males. The effects of mating on epididymal sperm populations were studied in 2 groups of males each mated 3 times and compared with the number and distribution of spermatozoa in the epididymides of 4 unmated control groups. One testis and epididymis were removed from each animal (hemicastration) either before or early in the mating season to provide information on initial sperm content and distribution. The contralateral side was removed later in the mating season to examine the effects of mating or sexual abstinence on epididymal sperm distribution.

Epididymal sperm number peaked in both the distal caput and distal corpus/proximal cauda epididymidis in late July. The total number of spermatozoa, including those remaining in the testis, available to each male at the beginning of the mating season in early August was ∼4·4 × 106/side. Although recruitment of spermatozoa into the epididymis from the testis continued until mid-August, sperm content of the epididymis reached a peak of about 3·5 × 106/epididymis in early August. At this time approximately 0·9 × 106 spermatozoa remained in the testis which had ceased spermatogenic activity. Throughout the mating season, epididymal spermatozoa were concentrated in the distal corpus/proximal cauda regions of the epididymis and were replenished by spermatozoa from upper regions of the duct. Relatively few spermatozoa were found in the distal cauda epididymidis, confirming a low sperm storage capacity in this region.

A constant loss of spermatozoa from the epididymis, probably via spermatorrhoea, occurred throughout the mating season and very few spermatozoa remained in unmated males in late August before the annual male die-off. Mating studies showed that an average of 0·23 × 106 spermatozoa/epididymis were delivered per mating in this species, but the number of spermatozoa released at each ejaculation may be as few as 0·04 × 106/epididymis when sperm loss via spermatorrhoea is taken into account.

We suggest that the unusual structure of the cauda epididymidis, which has a very restricted sperm storage capacity, may function to limit the numbers of spermatozoa available at each ejaculation and thus conserve the dwindling epididymal sperm reserves in order to maximize the number of successful matings which are possible during the mating season.

Keywords: epididymis; spermatozoa; sperm storage; mating; marsupial; dasyurid

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D. A. Taggart and P. D. Temple-Smith

Summary. Female brown marsupial mice were mated and changes in the number and distribution of spermatozoa were assessed in several regions of the reproductive tract at 1, 2, 3, 7, 10, 14 and 18 days after mating. Approximately 40 × 103 spermatozoa/side were present in the female reproductive tract between Days 1 and 7 after a single mating. This had decreased (to ∼9 × 103 spermatozoa/side) by Days 10 and 14 after mating; by Day 18 no spermatozoa were recovered. The maximum number of spermatozoa recorded in a female tract was ∼72 × 103 spermatozoa/side (Day 5 female, death in laboratory) and the minimum recorded was ∼2 × 103 spermatozoa/side on Day 2 after mating.

Between Days 1 and 7 after mating most spermatozoa were located in the uterus and lower isthmus (isthmus 1) and spermatozoa were rarely found in the lateral vaginae. By 24 h after mating most spermatozoa (∼60%) were found in isthmus 1, but ∼35% were still present in the uterus. Histological observations of the lower isthmus at this time showed that large numbers of spermatozoa were present in both the lumen of the duct and the sperm storage crypts which are located in this region. By Day 7 after mating ∼91% of all spermatozoa in the female tract were in isthmus 1, most of these being confined to the sperm storage crypts. On Days 10 and 14 after mating almost all spermatozoa in the tract were in the crypt regions of isthmus 1 and on Day 18 degenerating spermatozoa were observed. No special orientation or association of spermatozoa in relation to crypt cells was observed.

These results show that, although the number of spermatozoa inseminated is low by mammalian standards sperm transport in this species is extremely efficient and a large proportion of spermatozoa reaches the isthmus before ovulation (∼1 in 1 to 1 in 7). Several observations may explain the remarkable success of these low numbers of spermatozoa, including specializations of the reproductive tract which may have a directing effect on sperm movement and the special relationship which exists between spermatozoa and the oviducal environment which results in viable sperm storage. Recent observations suggest that an unusual sinusoidal mode of progressive motility observed in this species, may also influence the success of the low numbers of ejaculated spermatozoa.

Keywords: marsupial; spermatozoa; sperm storage; oviduct; isthmus; ultrastructure

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P. Hyttel, K. P. Xu, S. Smith, and T. Greve

Summary. Cumulus–oocyte complexes were collected from cows at an abattoir by aspiration from small (1–6 mm) antral follicles. After different periods of culture the complexes were processed for electron microscopy. Cumulus expansion occurred at 12–18 h of culture and concomitantly enlargement of cumulus cell projections in the perivitelline space was seen. At 48 h the innermost cumulus cells flattened and adhered tightly to the zona pellucida. In the oocyte the following changes occurred: at 0–3 h of culture the perivitelline space developed; at 3–12 h disconnection of the junctions between cumulus cell projections and the oolemma, and the concomitant breakdown of the nucleus was observed; at 12–18 h the mitochondria moved from a peripheral location to a more even spatial distribution and the Golgi complexes decreased in size; at ∼18 h the smooth endoplasmic reticulum formed large aggregates surrounded by mitochondria; at 18–21 h the first polar body was abstricted; at 24–40 h the cortical granules spread; at 30–40 h the polar body degenerated in some specimens; at 40–48 h the perivitelline space decreased in size; and at 48 h one oocyte was in the process of fragmentation. It is concluded that nuclear and cytoplasmic oocyte maturation is simulated in vitro. However, certain deviations were noticed compared to in-vivo maturation.

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J. P. P. Tyler, Dianne Moore Smith, and J. D. Biggers

Summary. Ovaries from pubertal mice were dissected into fragments containing 1–4 antral follicles which were cultured on the surface of a chemically defined, modified Eagle's Minimum Essential Medium (MEM). After 17–20 h oocytes were released from the cultured follicles and their meiotic status recorded. In the presence and absence of exogenous gonadotrophins, oocytes in these cultures behaved meiotically as expected if they had been studied in vivo. Medium containing progesterone (10 μm), testosterone (100 μm), or androstenedione (100 μm) increased the incidence of oocytes remaining meiotically inactive compared to follicles cultured in steroid-free medium. Lower concentrations of the above steroids or pregnenolone (1–100 μm) did not affect the meiotic status of cultured follicle-enclosed oocytes. Most (61%) of the dictyate oocytes released from steroid-treated follicles resumed and/or completed the first meiotic division when cultured further in steroid-free medium. Progesterone (100 μm) caused atresia of oocytes and follicle cells which increased as the period of follicular exposure to progesterone was increased.

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Dianne Moore Smith, J. P. P. Tyler, and G. F. Erickson

Summary. Rabbit oocytes from large (1–1·5 mm diam.), medium (0·5 mm) and small (0·15–0·25 mm) antral follicles were cultured in five chemically defined media. In all media, oocytes from large antral follicles showed the highest incidence of meiotic activity followed by those from follicles of medium size. Most oocytes from small follicles did not resume meiosis in culture. The addition of glutamine to a standard medium for ovum culture significantly improved maturation of oocytes from medium-sized follicles but did not affect those from large or small follicles. When polyvinylpyrrolidone was substituted for bovine serum albumin, maturation of oocytes from large and medium-sized follicles was reduced. Progesterone at a concentration of 10 μM did not affect maturation, but 100 μM-progesterone blocked germinal vesicle breakdown in oocytes from medium-sized follicles and reduced both germinal vesicle breakdown and polar body formation in oocytes from large follicles. This effect was reversible.

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Julie-Ann P De Bond and Jeremy T Smith

Kisspeptin is vital for the neuroendocrine regulation of GNRH secretion. Kisspeptin neurons are now recognized as a central pathway responsible for conveying key homeostatic information to GNRH neurons. This pathway is likely to mediate the well-established link between energy balance and reproductive function. Thus, in states of severely altered energy balance (either negative or positive), fertility is compromised, as is Kiss1 expression in the arcuate nucleus. A number of metabolic modulators have been proposed as regulators of kisspeptin neurons including leptin, ghrelin, pro-opiomelanocortin (POMC), and neuropeptide Y (NPY). Whether these regulate kisspeptin neurons directly or indirectly will be discussed. Moreover, whether the stimulatory role of leptin on reproduction is mediated by kisspeptin directly will be questioned. Furthermore, in addition to being expressed in GNRH neurons, the kisspeptin receptor (Kiss1r) is also expressed in other areas of the brain, as well as in the periphery, suggesting alternative roles for kisspeptin signaling outside of reproduction. Interestingly, kisspeptin neurons are anatomically linked to, and can directly excite, anorexigenic POMC neurons and indirectly inhibit orexigenic NPY neurons. Thus, kisspeptin may have a direct role in regulating energy balance. Although data from Kiss1r knockout and WT mice found no differences in body weight, recent data indicate that kisspeptin may still play a role in food intake and glucose homeostasis. Thus, in addition to regulating reproduction, and mediating the effect of energy balance on reproductive function, kisspeptin signaling may also be a direct regulator of metabolism.

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D. H. Robinson, P. R. Smith, and D. J. Benos

Summary. Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (K m = 4·3 ± 0·5 mm, Vmax = 79 ± 3·8 nmol·cm−2). The flux of 3-0MG was inhibited nearly 95% by 10−4 m-phloretin, and only 15% by 10−4 m-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 μm) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-d-glucose and glucose, and a very low specificity for fructose and 4-α-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55 000. Using immunogold labelling techniques, Na +-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2α, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.

Keywords: preimplantation embryo; 3-0-methyl glucose; sugar transport; rabbit

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Considerable ATPase activity was demonstrated in homogenates of testes and of isolated seminiferous tubules, but ouabain had no consistent effect on the activity suggesting that most of the ATPase is not the sodium and potassium stimulated ATPase usually associated with ion transport. However, the substitution of bicarbonate for chloride ions in assays at pH 8·4 caused a significant stimulation in ATPase activity of homogenates of testes or isolated seminiferous tubules and liver mitochondria. When the homogenates were fractionated on sucrose gradients, most of the ATPase activity was found associated with mitochondria. Because of this large amount of mitochondrial ATPase present, it is unlikely that we could have demonstrated the amounts of transport ATPase which would account for the amount of fluid secreted by the testis.