Failure of zinc to prevent dysmorphogenesis of cultured rat conceptuses by anti-yolk sac antiserum

Day 10 rat conceptuses were cultured for 48h in the presence of either cadmium or anti-visceral yolk sac antiserum (AVYS). Cadmium was embryotoxic at concentrations exceeding 0.25 micrograms/ml whilst AVYS caused embryonic dysmorphogenesis, particularly affecting the optic vesicles, at concentrations of 2 microliters/ml and above. The effect of pretreatment with zinc on embryotoxicity caused by cadmium or AVYS was studied. Zinc ameliorated the effects of cadmium but had no effect on AVYS-induced embryonic abnormalities. In a second set of experiments inhibition of 125I-labelled PVP uptake by the yolk sac of cultured whole conceptuses was studied. Cadmium and AVYS both inhibited uptake compared to control cultures. Zinc again ameliorated the effect of cadmium but had no action against AVYS-induced inhibition. These results are in contrast to our previous findings using isolated cultured yolk sacs in which zinc ameliorated the inhibitory effects on 125I-labelled PVP uptake of both cadmium and AVYS. These data show that in experiments using the isolated cultured yolk sac and the intact cultured conceptus, a qualitatively different response in yolk sac behaviour is observed under similar experimental conditions.     

Ultrastructure and function of the rat yolk sac: damage caused by teratogenic anti-VYS serum and recovery

It was hypothesized that heterologous anti-rat visceral yolk sac serum (AVYS) exerts its teratogenic effect by reducing the endocytosis of serum proteins by the visceral yolk sac (VYS), thus reducing the supply of amino acids to the embryo and VYS. To evaluate this hypothesis, we studied the effect of teratogenic AVYS on the endocytic function of the VYS and the ultrastructure of the VYS and parietal yolk sac (PYS). Rat conceptuses were exposed to a teratogenic dose of AVYS on the 10th day of gestation in vivo or in vitro. Control and AVYS-exposed specimens were collected 24-192 hr later and prepared for scanning and transmission electron microscopy (SEM and TEM, respectively) utilizing standard procedures. The Endocytic Index was calculated for the VYS utilizing standard procedures. Approximately 97% of the in vivo exposed and 94% of the in vitro exposed embryos were morphologically abnormal. Ultrastructural observations showed that exposure to AVYS in vivo or in vitro caused severe damage to the VYS endodermal epithelial cells with loss of cellular borders, reduction in the number and length of microvilli, and increased cellular inclusions; and some damage to PYS endodermal cells with increased blebbling and decreased cell number. Recovery was evident at 72 hr and complete by 96 hr. The Endocytic Index was significantly reduced in the VYS 24 and 48 hr after injecting AVYS into the pregnant rat but was not significantly different at 96 and 192 hr. Our results show that the AVYS antiserum damaged visceral endodermal epithelium experienced ultrastructural recovery with parallel functional recovery. These studies suggest that transient yolk sac placental ultrastructural damage and dysfunction was probably sufficient to cause irreversible damage to the developing embryo during early organogenesis. We conclude that the proximate effect of the AVYS was on the plasma membrane of the visceral endoderm and that decreased pinocytosis is a consequence of this effect.     

Altered visceral yolk sac function produced by a low-molecular-weight somatomedin inhibitor

A fraction from diabetic rat serum containing a low-molecular-weight (800-1000) somatomedin inhibitor (SI) alters growth and development in both neurulation and early limb bud staged mouse embryos in vitro. Previous studies suggested that an accumulation of serum proteins and morphological changes of the visceral yolk sac (VYS) were produced following exposure to the SI in early limb bud staged conceptuses. The morphological changes, characterized by the presence of large endosomes in the endodermal cells, suggested that the SI altered histiotrophic nutrition, whereby proteins are pinocytosed by the endodermal VYS cells and degraded to constituent amino acids. Therefore, the effects of the SI on pinocytosis and protein degradation by the VYS were evaluated using the whole embryo culture system. Results showed that the SI reduced fluid phase pinocytosis as determined by the uptake of [U-14C]sucrose, but that accumulation of [3H]leucine-labeled hemoglobin ([3H]Hb) by the VYS was greater following exposure to the SI than in controls. In contrast, the accumulation of 3H-labeled amino acids in the embryo (produced from the degradation of [3H]Hb by the VYS) was reduced by the SI. The extent of amino acid reduction in embryonic accumulation is dependent upon the concentration of SI in the culture medium and correlates with the incidence of malformations produced by the SI, i.e., high rates of malformations occur with large reductions in embryonic 3H-labeled amino acid accumulation. The apparent paradox of high [3H]Hb accumulation in the presence of decreased pinocytosis appears to be the result of altered processing of the [3H]Hb in the endodermal cells. The altered processing decreases the "elimination" of the proteins from the VYS and results in the decrease in 3H-labeled amino acid present in the embryo proper. Therefore, the SI appears to alter two processes of VYS histiotrophic function. (1) decreased pinocytosis and (2) altered protein processing, ultimately resulting in a decreased availability of substrates for the embryo. During the early stages of embryogenesis in the human, the trophoblast cells of the placenta are responsible for the transport of nutrients from the maternal to embryonic systems. Since these cells show high phagocytic and pinocytotic activities, the SI may also disrupt these processes in the chorioallantoic placenta and contribute to diabetes-induced embryopathies.     

Methionine and iron as growth factors for rat embryos cultured in canine serum

Development of headfold-staged rat embryos cultured in canine serum containing various supplements was compared with development in rat serum to seek suitable alternatives to rat serum in rodent embryo culture and to identify nutritional factors for cultured rodent embryos that may have relevance for normal mammalian embryonic growth and development. Supplementation of canine serum with glucose, methionine, and a lipophilic iron chelate allowed growth and development of cultured rat embryos, approximating those obtained with rat serum. These findings suggest that properly supplemented canine serum can serve as a suitable rodent embryo culture medium and that glucose, iron, and methionine may be important nutrients in mammalian embryonic development.     

Transport of methionine in sea-urchin sperm by a neutral amino-acid carrier

A carrier-mediated transport for L-methionine and other neutral amino acids exists in sperm of the sea urchin Lytechinus pictus. The initial rate of L-methionine entry is a Michaelis-Menten function of the methionine concentration in the external medium. The maximum velocity is low [V = 250 pmol h-1 (10(9) sperm)-1 at 22 degrees C] and the affinity is high (Km = 6-10 microM). The initial rate of transport under steady-state exchange conditions is also a Michaelis-Menten function of the external concentration of methionine. The Km determined by this method is about 14 microM. Neutral amino acids compete with L-methionine transport as shown by initial velocity measurements. These results indicate that L-methionine transport is a carrier-mediated process. The temperature dependence of the process is approximately 84 kJ (20 kcal) mol-1 K-1, which is not compatible with a simple diffusion mechanism, but in the range of values usually found for a mediated transport. The transport is largely Na+-independent and does not depend on Ca2+, K+ or H+ gradients. It is only partially sensitive to KCN, showing it is mainly independent of oxidative phosphorylation. The steady-state internal methionine concentration is not a linear function of the external amino acid concentration. This suggests that an exit by diffusion competes with a carrier-mediated concentrative transport in a cellular compartment. This mediated transport is compared to those of higher animal cells.     

Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture

Along with the transition from maternal to embryonic genome control the mammalian preimplantation embryo undergoes significant changes in its physiology during development. Concomitant with these changes are altering patterns of nutrient uptake and differences in the subsequent fate of such nutrients. The most significant nutrients to the developing mammalian preimplantation embryo are carbohydrates and amino acids, which serve not only to provide energy but also to maintain embryo function by preventing cellular stress induced by suboptimal culture conditions in vitro. It is subsequently proposed that optimal development of the mammalian embryo in culture requires the use of two or more media, each designed to cater for the changing requirements of the embryo. Importantly, culture conditions that maintain the early embryo are not ideal for the embryo post-compaction, and conditions that support excellent development and differentiation of the blastocyst can actually be inhibitory to the zygote. A marker of in vitro-induced cellular stress to the embryo is the relative activity of the metabolic pathways used to generate energy for development. Quantification of embryo energy metabolism may therefore serve as a valuable marker of embryo development and viability.     

Catalytic action of L-methionine gamma-lyase on selenomethionine and selenols.

We examined the catalytic action of L-methionine gamma-lyase (EC 4.4.1.11) on selenomethionine (2-amino-4-(methylseleno)butyric acid), methaneselenol, l-hexaneselenol, and benzeneselenol. The enzyme catalyzes alpha, gamma-elimination of selenomethionine to yield alpha-letobutyrate, ammonia, and methaneselenol, and also its gamma-replacement reaction with various thiols to produce S-substituted homocysteines. Selenomethionine is an even better substrate than methionine in alpha, gamma-elimination but is less effective in gamma-replacement. In addition, L-methionine gamma-lyase catalyzes gamma-replacement reaction of methionine and its derivatives with selenols to form the corresponding Se-substituted selenohomocysteines, although selenols are less efficient substituent donors than thiols. This is the first proven mechanism for the incorporation of selenium atom into amino acids.     

Placental transfer of vigabatrin (gamma-vinyl GABA) and its effect on concentration of amino acids in the embryo of TO mice

BACKGROUND: The mechanism of the teratogenicity of vigabatrin (VGB) is unknown. The objectives of this study were to determine the placental transfer of VGB and to evaluate the effect of VGB on maternal, placental, and fetal concentrations of amino acids. METHODS: A single dose of 400 mg/kg VGB in physiological saline was administered intraperitoneally to a group of Theiler outbred (TO) mice on gestational day (GD) 10. The controls received a proportionate volume of saline. Maternal blood samples, embryos, and placentas were collected at 3.5, 6.0, and 9.0 hr after treatment and their total amino acid concentrations determined in an ion-exchange amino acid analyzer. RESULTS: At 3.5 hr, there was a decrease in concentrations of some amino acids in the blood, placenta, and embryos of VGB-treated mice, but the decrease in methionine was most marked. gamma-aminobutyric acid (GABA) was significantly higher in the VGB group in both the embryos and the placentas at 3.5 hr but at 6.0 and 9.0 hr the differences were not significant. Vigabatrin levels were higher in the placenta than in the embryo at 3.5 hr, but at 6.0 hr there was an overlap of the VGB peak with that of tryptophan with very much lower levels than at 3.5 hr. At 9.0 hr, there was no vigabatrin peak in either the placenta or the embryo. CONCLUSIONS: Maternal exposure to VGB results in peak levels of the drug after 3.5 hr in the placenta and embryo. Methionine concentration is most severely affected in VGB-treated mothers, placentas, and fetuses. We speculate that this deficiency could be a possible mechanism for the teratogenic effects of vigabatrin.     

In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins

To elucidate the effect of nutrient substrates on embryo development, in vitro fertilized bovine one-cell embryos were cultured in a medium similar to synthetic oviduct fluid (SOF) but without glucose and containing 3.3 mM lactate, 0.3 mM pyruvate and 3 mg/ml bovine serum albumin (BSA) at 39 degrees C in 5% CO(2) in air. Results indicated that addition of glucose was not only unnecessary, but it also had a deleterious effect on embryo development to the morula stage. Lactate supported embryo development up to the morula stage as well as pyruvate. Supplementation with 20 amino acids contained in basal medium Eagle's (BME) and minimum essential medium (MEM) improved development to the morula stage dramatically and increased the cell number compared with that of the controls. Addition of the vitamins from MEM to SOF had no beneficial effect. The SOF with amino acids did not increase the frequency of blastocysts 7 days after in-vitro fertilization but did increase the total number of cells compared with that of the controls. Frequency of blastocysts at Day 7 in SOF with amino acids was equivalent to that of co-culture although the total cell number was lower. These results demonstrate that a semi-chemically defined medium can successfully support the development of bovine embryos to the morula stage to a limited extent, but the medium lacks some nutrients or growth factors to fully support development through the blastocyst stage.     

Development of rat embryos in culture media containing different concentrations of normal and diabetic rat serum.

In vitro culture of rodent embryos has been extensively used in the search for teratologic agents, with possible relevance to diabetic pregnancy. However, the high concentrations of rat serum added to the culture medium (approximately 75%) have raised concern that the teratogenic effects of some compounds may be attenuated or masked in this culture system and thereby forced the addition of pharmacological concentrations of the compounds (e.g., D-glucose and beta-hydroxybutyrate) to the medium. This issue has been examined in the present study where the effects of different concentrations of rat serum on growth and differentiation of rat embryos were recorded in cultures supplemented with increased concentrations of D-glucose and beta-hydroxybutyrate. The embryonic development was also evaluated after culture in medium supplied with serum from diabetic rats. Compared with normal rat serum, the diabetic serum had an elevated glucose concentration as well as markedly increased levels of triglycerides and branched amino acids, indicating a potentially rich supply of major nutrients for the cultured embryos. Lowering the serum concentration in the culture medium from 80% to 50% yielded progressively retarded embryonic growth but no increased rate of other morphological malformations. At 40% serum concentration, however, there was a sharp rise in the incidence of somatic malformations, in addition to the prevailing growth retardation. When the embryonic growth and development were compared at 50% and 80% serum concentrations, increased D-glucose or beta-hydroxybutyrate concentrations caused similar degrees of embryonic dysmorphogenesis. Also, the uptake of each compound by the embryos exposed to elevated levels of the two agents were similar in 50% and 80% serum cultures. There was, therefore, no protection against the teratogenic and growth-retarding effects of increased D-glucose or beta-hydroxybutyrate offered by high serum concentrations in the culture medium (i.e., 80% vs. 50%). Embryos cultured in 50% or 80% diabetic rat serum at 30 mmol/L or 50 mmol/L D-glucose concentration showed similar rates of somatic malformations as did embryos exposed to the same proportion of normal rat serum at similar glucose concentrations. By contrast, the diabetic rat serum amplified the general retarding effects of high D-glucose levels, yielding lower protein levels and somite numbers in embryos from diabetic serum culture than in embryos cultured in normal rat serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Single amino acids in sucrose rewards modulate feeding and associative learning in the honeybee

Obtaining the correct balance of nutrients requires that the brain integrates information about the body’s nutritional state with sensory information from food to guide feeding behaviour. Learning is a mechanism that allows animals to identify cues associated with nutrients so that they can be located quickly when required. Feedback about nutritional state is essential for nutrient balancing and could influence learning. How specific this feedback is to individual nutrients has not often been examined. Here, we tested how the honeybee’s nutritional state influenced the likelihood it would feed on and learn sucrose solutions containing single amino acids. Nutritional state was manipulated by pre-feeding bees with either 1 M sucrose or 1 M sucrose containing 100 mM of isoleucine, proline, phenylalanine, or methionine 24 h prior to olfactory conditioning of the proboscis extension response. We found that bees pre-fed sucrose solution consumed less of solutions containing amino acids and were also less likely to learn to associate amino acid solutions with odours. Unexpectedly, bees pre-fed solutions containing an amino acid were also less likely to learn to associate odours with sucrose the next day. Furthermore, they consumed more of and were more likely to learn when rewarded with an amino acid solution if they were pre-fed isoleucine and proline. Our data indicate that single amino acids at relatively high concentrations inhibit feeding on sucrose solutions containing them, and they can act as appetitive reinforcers during learning. Our data also suggest that select amino acids interact with mechanisms that signal nutritional sufficiency to reduce hunger. Based on these experiments, we predict that nutrient balancing for essential amino acids during learning requires integration of information about several amino acids experienced simultaneously.     

Current understanding of the regulation of methionine biosynthesis in plants

Plants can provide most of the nutrients for the human diet. However, the major crops are often deficient in some of the nutrients. Thus, malnutrition, with respect to micronutrients such as vitamin A, iron, and zinc, but also macronutrients such as the essential amino acids lysine and methionine, affects more than 40% of the world's population. Recent advances in molecular biology, but also the grasp of biochemical pathways, metabolic fluxes, and networks can now be exploited to produce crops enhanced in key nutrients to increase the nutritional value of plant-derived foods and feeds. Some of the predictions appear to be accurate, while others not, reflecting the fact that plant metabolism is more complex than presently understood. A good example for a complex regulation is the methionine biosynthetic pathway in plants. The nutritional importance of Met and cysteine has motivated extensive studies of their roles in plant molecular physiology, especially regarding to their transport, synthesis, and accumulation in plants. Recent studies have demonstrated that Met metabolism is regulated differently in various plant species.     

Effects of Nutrient Supply on Seed Growth of Field Bean (Vicia faba L.). 1. Effects of Different Sulphur and Methionine Regimes on Cotyledon Growth, Protein and Uncombined Amino Acids In vitro

The effects of differemt S and methionine regimes on growthof developing Vicia faba cotyledons in vitro were studied. Basalmedium (containing adequate S) supplemented with 05 mM methioninemarginally increased d. wt and uncombined amino acid accumulationbut adding 1–5 mM methionine inhibited both growth andprotein accumulation. Sulphur deficiency reduced both d. wtand protein accumulation but incresed accumulation of uncombinedamino acids. Adding 1 mM methionine to the S-deficient mediumrestored growth, normal protein and uncombined amino acid acnunulation.High sulphate medium (7.5 mM ) decreased d. wt, protein anduncombined amino acid accumulation. High sulphate medium or basal medium+methionine (05 mM) changedthe proportions of the seed proteins; legumin increased butvicilin decreased. Sulphur deficiency caused a relative increasein vicilin but a decrease in legumin. The different S and methionineregimes markedly changed the composition of the uncombined aminoacids, especially those derived from aspartic acid but not thecomposition of the protein fraction, except during S deficiency. The data presented indicates a flexibility in the storage proteincomposition of developing cotyledons grown in vitro, with theS and methionine status having a regulatory effect. Vicia faba L., field bean, cotyledon, growth, in vitro culture, uncombined amino acids, protein composition, legumin, vicilin, methionine, sulphur     

Matching the supply of bacterial nutrients to the nutritional demand of the animal host

Various animals derive nutrients from symbiotic microorganisms with much-reduced genomes, but it is unknown whether, and how, the supply of these nutrients is regulated. Here, we demonstrate that the production of essential amino acids (EAAs) by the bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum is elevated when aphids are reared on diets from which that EAA are omitted, demonstrating that Buchnera scale EAA production to host demand. Quantitative proteomics of bacteriocytes (host cells bearing Buchnera) revealed that these metabolic changes are not accompanied by significant change in Buchnera or host proteins, suggesting that EAA production is regulated post-translationally. Bacteriocytes in aphids reared on diet lacking the EAA methionine had elevated concentrations of both methionine and the precursor cystathionine, indicating that methionine production is promoted by precursor supply and is not subject to feedback inhibition by methionine. Furthermore, methionine production by isolated Buchnera increased with increasing cystathionine concentration. We propose that Buchnera metabolism is poised for EAA production at certain maximal rates, and the realized release rate is determined by precursor supply from the host. The incidence of host regulation of symbiont nutritional function via supply of key nutritional inputs in other symbioses remains to be investigated.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Suppression of methionine-induced hyperhomocysteinemia by glycine and serine in rats

The hyperhomocysteinemia induced by a dietary addition of 1% methionine was significantly suppressed by the concurrent addition of 1% glycine or 1.4% serine to the same degree. The methionine-induced increase in the hepatic concentration of methionine metabolites was significantly suppressed by glycine and serine, but the hepatic cystathionine beta-synthase activity was not enhanced by these amino acids. When the methionine-supplemented diet was changed to the methionine plus glycine or serine diet, the plasma homocysteine concentration rapidly decreased during and after the first day. The hyperhomocysteinemia induced by an intraperitoneal injection with methionine was also suppressed by concurrent injection with glycine or serine, although the effect of serine was significantly greater than that of glycine. These results indicate that glycine and serine were effective for suppressing methionine-induced hyperhomocysteinemia: serine and its precursor glycine are considered to have elicited their effects mainly by stimulating cystathionine synthesis by supplying serine, another substrate for cystathionine synthesis.     

Effects of secondary bile acids on the in vitro development of early somite rat embryos

Effects of secondary bile acids--lithocholic (LCA) and deoxycholic (DCA)--on the in vitro development of early somite (10.5 days old) rat embryos were studied. It was shown that an addition to the culture medium of 0.1 mM LCA (final concentration) resulted in 9% growth-retarded and 12% malformed embryos when the duration of exposure was 24 h. When treatment with LCA was prolonged to 48 h, the rate of growth retardation increased to 18% and that of malformations to 40% versus 0.5% for both parameters observed in controls. This could be interpreted as a reversible or time-dependent effect of LCA on the in vitro development of the mammalian embryo. Culture of embryos in medium with 0.5 mM DCA resulted in 22% of growth retardation and 50% of malformations. DCA in 0.1 mM final concentration had only slight and statistically nonsignificant effects. Retardation of growth development could be demonstrated by a decrease in crown-rump length and the number of somites. Among malformed embryos, abnormalities in the development of the neural tube and exencephaly were the most common types of malformations. Abnormalities as well as growth retardation were accompanied by significant pathological changes in structure and perhaps in function of the endodermal visceral yolk sac cells. It could be suggested that secondary bile acids when present in pathophysiological concentrations can affect the embryonic development by direct inhibitory effects and that these effects may be time and dose dependent.     

Lactational and metabolic effects in cows of lysine and methionine added to a ration deficient according to the I.N.R.A. method

The effects of adding lysine and/or methionine to a ration of calculated deficiency in these amino acids of 10% and 20%, respectively, were studied in 24 Brown Swiss cows. The mixed rations (27% grass silage, 19% maize silage, 5% hay and 49% concentrate on DM basis) contained 14.5% CP on average. Lysine supply was selectively elevated by adding fish meal in exchange for other concentrate ingredients. Methionine was supplied in a rumen‐protected form. Milk protein content was elevated whereas fat amount decreased by adding both amino acids. Lactose content increased without additional lysine from fish meal. Live weight, milk yield, milk fat content and protein amount remained unaffected by any variation of amino acids supply. Also nutrient digestibility and nitrogen balance were not changed by the treatments. Blood plasma concentrations confirmed the assumed variation in metabolic lysine and, less clear, methionine supply. Effects on plasma concentrations of other amino acids were relatively small. Most plasma hormones and enzymes, and metabolites in plasma, urine and milk did not respond to the variation in amino acid supply. Lysine addition via fish meal increased aspartate amino transferase and decreased urinary allantoin concentration. Additional methionine elevated plasma ornithine. Overall lysine and methionine appear to have been only marginally deficient in the unsupplemented ration fed for 3 weeks despite the deficiency of 10% to 20% as calculated by the I.N.R.A. method.     

In vitro methodology, hormonal and nutritional effects and fibre production in isolated ovine and caprine anagen hair follicles

Mammalian hair follicles are complex multicellular structures in the skin, which produce hair fibre under the influence of locally produced and systemic signalling systems. Investigation to determine mechanisms of regulation, follicular responses and the importance of nutritional supply have utilised a number of in vivo and in vitro approaches. Included in these are studies on isolated intact anagen secondary follicles singly or in groups with incubation in culture medium. These utilise techniques developed for investigation of follicles from human skin. Results from selected studies reviewed here demonstrate differences in capacity for hair growth and protein synthesis between secondary follicles from Angora and cashmere-bearing goats. Mohair follicles were shown to exhibit faster hair shaft elongation both in vivo and in vitro, to have greater DNA content per follicle and to deposit significantly more protein per follicle and per unit of DNA. Incubation of anagen mohair and cashmere follicles in the presence of melatonin or prolactin showed positive responses in hair shaft growth and protein synthesis to both signalling molecules. This result indicated directly acting effects on the follicle in addition to any indirect effects arising at a whole animal level in response to, for example, variation in photoperiod. Similarly, epidermal growth factor was shown to alter elongation and protein synthesis in mohair follicles and to produce, at higher concentration, club hair structures similar to effects observed in other species. The vitamin biotin was shown to be important in maintaining viability of isolated sheep secondary hair follicles where supplementation increased the proportion continuing to grow. Effects on growth and apparent protein synthesis suggested comparatively lesser effects on follicles, which remained viable. Histology on follicles indicated effects of biotin deficiency in reducing proliferation of basal keratinocytes. The final study, included in this review, demonstrated that supply of the essential sulphur-containing amino acid l-methionine was necessary to maintain the viability and growth of mohair follicles. l-cysteine was not required in the presence of l-methionine, although there was evidence of an optimisation when both amino acids were present in adequate concentrations. Consideration is given to the importance of transport mechanisms and capacity to utilise absorbed nutrients when considering optimising nutritional supply to individual follicles. These may then provide targets for attainment in applied nutrition of animals in vivo.