Pattern of ovarian protein synthesis and secretion during the reproductive cycle of Scotophilus heathi: synthesis of an albumin-like protein

Ovarian proteins synthesized de novo and secreted in vitro were investigated during different stages of the reproductive cycle of Scotophilus heathi. We found increased ovarian protein synthesis and secretion during the recrudescence and the preovulatory periods, coinciding with two peaks of follicular development and steroidogenesis. The ovaries synthesized protein at a low rate during quiescence and the late phase of delayed ovulation. In vitro analysis using ³⁵S-methionine incorporation showed increased synthesis of 66 kDa protein during delayed ovulation, but secretion of this protein declined markedly during the preovulatory period. N-terminal sequencing of the 66 kDa protein showed that it shares 70% homology with human serum albumin. Immunocytochemistry indicated that this protein is found primarily in the granulosa cells of the follicles. Whether the increase in albumin production by the ovaries during delayed ovulation is associated with a hyperinsulinemic and hyperandrogenic condition requires further investigation.     

Pattern of ovarian protein synthesis and secretion during the reproductive cycle of Scotophilus heathi: synthesis of an albumin-like protein.

Ovarian proteins synthesized de novo and secreted in vitro were investigated during different stages of the reproductive cycle of Scotophilus heathi. We found increased ovarian protein synthesis and secretion during the recrudescence and the preovulatory periods, coinciding with two peaks of follicular development and steroidogenesis. The ovaries synthesized protein at a low rate during quiescence and the late phase of delayed ovulation. In vitro analysis using 35 S-methionine incorporation showed increased synthesis of 66 kDa protein during delayed ovulation, but secretion of this protein declined markedly during the preovulatory period. N-terminal sequencing of the 66 kDa protein showed that it shares 70% homology with human serum albumin. Immunocytochemistry indicated that this protein is found primarily in the granulosa cells of the follicles. Whether the increase in albumin production by the ovaries during delayed ovulation is associated with a hyperinsulinemic and hyperandrogenic condition requires further investigation.     

Ecdysteroid-dependent protein synthesis in caste-specific development of the larval honey bee ovary

In the honey bee, Apis mellifera, the fifth larval instar is a critical period for caste differentiation. During this premetamorphic phase the hormonal milieu shows pronounced caste differences and several organs, particularly the ovaries, enter different developmental pathways leading to highly fertile queens and nearly sterile workers. Developmental profiles of total protein synthesis in larval ovaries showed marked caste differences starting with the early fifth instar. By two-dimensional electrophoresis, caste-specific patterns could be detected in the synthesis of a 29 kDa/pI 4.6 and two 24 kDa/pI 5.2–5.5. proteins (pI=isoelectric point). A marked decrease in the expression of these proteins was found to coincide with caste-specific differences in the haemolymph ecdysteroid titer. In vitro exposure of larval worker ovaries to physiological (10^–7 m) concentrations of synthetic makisterone A elicited an identical response. Juvenile hormone did not affect protein synthesis patterns in larval ovaries, and also did not inhibit or reverse the ecdysteroid-induced effects. Heat shock experiments revealed that the 29 kDa/pI 4.6 ecdysteroid-regulated protein belongs to the class of small heat shock proteins.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

Mechanism of delayed ovulation in a vespertilionid bat, scotophilus heathi: role of gonadotropin, insulin, and insulin-like growth factor-1

The aim of this study was to evaluate the effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor-1 (IGF-1) on ovarian androstenedione synthesis to understand the mechanism responsible for delayed ovulation in Scotophilus heathi. We found that LH stimulated a dose-dependent increase in androstenedione synthesis by the ovary in vitro. This study also showed a clear seasonal variation in the ability of the ovary to produce androstenedione in vitro in response to LH and FSH stimulation. In response to LH and FSH, maximum quantities of androstenedione were produced during recrudescence in November. The same doses of gonadotropins during the preovulatory period in February stimulated comparatively low androstenedione secretion by the ovary. On the basis of these data, we suggest that in S. heathi, ovarian responsiveness to LH and FSH peaks during recrudescence. This study also showed a seasonal variation in the effects of insulin and IGF-1 on ovarian androstenedione production in vitro. Peak ovarian responsiveness to insulin and IGF-1 was observed during quiescence in September. It is hypothesized that increased insulin/IGF-1 sensitivity during September may be responsible for increased responsiveness to LH. Increased LH release, if coincident with the period of enhanced ovarian responsiveness to LH, may result in the excessive androstenedione production responsible for delayed ovulation in S. heathi.     

Characterization of a new high-temperature-induced 66-kDa heat-shock protein,antigenically related to heat-shock protein 72

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major “classic” heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45°C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)⁺ RNA extracted from cells treated at 45°C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)⁺ RNA extracted from cells heated at 42°C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro, hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer. © 1996 Wiley-Liss, Inc.     

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice

Summary Investigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued usingin vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study mebrane-bound polysomes isolated from the livers of newborn albinos were 55% (c^3H/c^3H strain) and 62% (c^14CoS/c^14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)⁺-RNA isolated from albino livers and assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin synthesis in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

DNA,RNA, and protein synthesis in the cyanobacteriumSynechocystis sp. PCC 6803 adapted to different salt concentrations

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content with increasing salt concentrations up to 684mm NaCl was found, whereas the lowest DNA content was measured around 342mm NaCl, i.e., the salinity at which maximal growth occurred. With the uracil and thymidien incorporation technique, maxima in DNA and RNA synthesis were detected in control cells. Comparing these rates with nucleic acid synthesis rates calculated from the contents of DNA and RNA and the growth rates indicated that adaptation to 1026mm NaCl seemed to lead to an increased RNA turnover inSynechocystis. Analysis of protein synthesis with³⁵S-methionine labeling showed alterations in salt-adapated cells ofSynechocystis. At least three proteins (20.5, 25.8, and 35.8 kDa) were synthesized with highest rates at salinities leading to maximal growth, the synthesis of nine proteins (12.5, 16.9, 19.2, 22.2, 24.7, 28.5, 30.5, 50.3, and 63.5 kDa) increased and that of several other proteins decreased with increasing salinity; but only three proteins (12.5, 22.2, and 30.5 kDa) accumulated under these conditions. The adaptation ofSynechocystis to enhanced salt concentrations led also to increased contents of glucosylglycerol, glycogen, and significant amounts of K⁺ as well as Na⁺ ions.     

Changes in the protein synthesis pattern during a nutritional shift-down transition in Saccharomyces cerevisiae

In Saccharomyces cerevisiae cells (strain A364A) during a shift-down from glucose to raffinose, a rapid reduction in the rate of RNA accumulation was observed whereas the rate of protein accumulation was unaffected for at least 2 h. Following the transition the percentage of unbudded cells slightly increased and the cell volume distribution showed a newly formed subpopulation of smaller cells. To study the effects of the shift-down on the protein synthesis pattern, total [³⁵S]-methionine pulse-labeled extracts were fractionated by high-resolution two-dimensional gel electrophoresis. The synthesis of two classes of proteins (I and II) was modulated during the transitory state of growth: one positively, the other negatively. Two polypeptides of 57 kDa showed the most dramatic increase in synthesis during the shift-down. Also a heat-shock protein (HSP 256) appeared to be positively correlated to the shift-down transition.     

Characterization of protein synthesis by isolated rice mitochondria

Summary Bacteria-free mitochondria were isolated from aseptically grown, etiolated and green seedlings of both cytoplasmic male-sterile (WA-type) and male-fertile rice (Oryza sativa L.). Protein synthesis in these isolated mitochondria was characterized by gel electrophoresis/fluorography and by the incorporation of [³⁵S]-methionine into protein. In the presence of cycloheximide, a set of some 25 discrete polypeptides and an electrophoretically unresolved population were synthesized. This pattern of protein synthesis in organello was essentially the same in mitochondria isolated from both male-fertile and malesterile cytoplasms. Our data does not preclude the possibility, however, that the WA-type CMS possesses a tissue-specific and/or a low abundance mitochondrial protein(s), whose synthesis eluded detection under our experimental conditions. The synthesis of the mitochondria-encoded polypeptides by isolated rice mitochondria was inhibited by chloramphenicol and incompletely inhibited by erythromycin. A minor chloramphenicol-insensitive, cycloheximide-sensitive translation activity was found consistently to copurify with the mitochondria. This activity generated a reproducible electrophoretic profile of a poorly resolved, weakly labelled population of polypeptides and of a few conspicuous polypeptides, including a 42 kDa species.     

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss)

Summary To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7±9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8±2.8% on maintaining ouabain-sensitive Na⁺/K⁺-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).Abbreviations BSA bovine serum albumin - dpm disintegrations per min - k _s fractional rate of protein synthesis - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - PHE phenylalanine; PO₂ oxygen tension - PCA perchloric acid     

Protein synthesis by the milk gland and fat body of the tsetse fly,Glossina pallidipes

The patterns of protein synthesis by the milk gland and the fat body of female Glossinapallidipes during the pregnancy cycle were studied by incubation with [³⁵S]methionine both in vivo and in vitro. The pattern of protein synthesis by the milk gland changed with the stage of the larva in the uterus. Very little synthesis occurred in the milk gland until the first instar larva hatched. Then four proteins (13, 16, 24 and 72 kDa) were prominently synthesized. As the larva matured, the synthesis of 19, 38, 40 and 72 kDa proteins increased, whereas that of the 13 and 24 kDa proteins decreased. Just before larviposition, only the 16 and 72 kDa proteins were still being synthesized. The milk gland secreted into the medium primarily the 13, 16, 19 and 72 kDa proteins, all of which were found in the larval gut after a 5 hr pulse of labeled methionine in vivo. During most of the pregnancy cycle protein synthesis in the fat body was low compared to that of the milk gland and only small amounts of several low molecular weight proteins (less than or equal to 16 kDa) were released into the medium. But when a large third instar larva was present in the uterus, the fat body synthesized and secreted a 72 kDa and a 15–17 kDa complex of proteins.     

Protein synthesis in cell-free extracts of Dictyostelium discoideum

We have developed an in vitro translation system for the lower eukaryote Dictyostelium discoideum. Active extracts using endogenous mRNA support protein synthesis with optimal Mg²⁺ and K⁺ concentrations of 5 mM and 120 mM, respectively. [³⁵S]Methionine incorporation is linear for more than 2 h. Polypeptides synthesized from endogenous mRNA have sizes ranging from less than 20 to over 100 kDa. Heat-shock proteins are synthesized in vitro in extracts prepared from heat-shocked cells. Possible uses of this system for study of translational control during growth and differentiation are discussed.     

In vitro protein synthesis capacities in a cold stenothermal and a temperate eurythermal pectinid

The translational system was isolated from the gills of the Antarctic scallop Adamussium colbecki (Smith) and the European scallop Aequipecten opercularis (Linnaeus) for in vitro protein synthesis capacities (g protein mg FW^–1 day^–1) and the translational capacities of RNA (k_{RNA in vitro} mg protein mg RNA^–1 day^–1). In vitro protein synthesis capacity in the cold-adapted pectinid at 0 °C was similar to the one found in the temperate scallop at 25 °C. These findings might reflect cold compensated rates in Adamussium colbecki, partly explainable by high tissue levels of RNA. Cold-compensated in vitro protein synthesis capacities may further result from increments in the translational capacity of RNA. The thermal sensitivity of the translation machinery was slightly different in the two species, with significantly lower levels of Arrhenius activation energies E_a and Q₁₀ in Adamussium colbecki in the temperature range 0–15 °C. Reduced protein synthesis and translational capacities were found in vitro in gills of long-term aquarium-maintained Adamussium colbecki and were accounted for by a loss of protein synthesis machinery, i.e. a reduction in RNA levels, as well as a decrease in the amount of protein synthesized per milligram of RNA (RNA translational capacity, k_{RNA in vitro}). Such changes may involve food uptake or mirror metabolic depression strategies, like those occurring during winter. Consequences of high in vitro RNA translational capacities found in the permanently cold-adapted species are discussed in the context of seasonal food availability and growth rates at high latitudes.Abbreviations DPM disintegrations per minute - DTT dithiothreitol - E_a Arrhenius activation energy - k_s fractional protein synthesis rate - k_{RNA in vivo} translational efficiency - k_{RNA in vitro} translational capacity - PCA perchloric acid - Phe phenylalanine - PLA phospho-L-arginine - PSU practical salinity units - RNAse ribonuclease - TCA trichloroacetic acidCommunicated by G. Heldmaier     

Ovulation in the perfused ovary in vitro: further evidence that estrogen is not required

The effect of inhibition of estrogen synthesis on ovulation in rat ovaries perfused in vitro with medium without phenol red was examined. The addition of luteinizing hormone (LH, 0.1 microgram/mL) plus 3-isobutyl-1-methylxanthine (IBMX, 0.2 mM) to phenol red-free perfusion medium (M199 + 4% bovine serum albumin) induced ovulation. The number of ovulations was similar to that found in medium containing phenol red. There was a similar increase in estradiol (1, 3, 5 (10)-estratriene-3, 17 beta-diol) levels in the medium in both groups. The addition of 4-hydroxy-4-androstene-3, 17-dione (4-OH-A, 5 microM) to phenol red-free medium blocked the increase in estradiol levels induced by LH + IBMX, but did not prevent ovulation. There was no significant difference in the number of ovulations in the three groups. In conclusion, phenol red in the perfusion medium does not influence ovulation induced by LH + IBMX. Furthermore, an increase in estrogen is not required during the immediate preovulatory period for ovulation to occur.     

Immunolocalization of cytochrome P450 side chain cleavage, 17-alpha-hydroxylase and aromatase in the ovary of vespertilionid bat (Scotophilus heathi) during different phases of ovulatory delay.

Immunocytochemical localization of steroidogenic enzymes, cytochrome P450 side chain cleavage, 17-alpha-hydroxylase and aromatase, was performed in the ovaries of Scotophilus heathi during reproductive cycle, with reference to the period of delayed ovulation. Moderate immunoreactivity of side chain cleavage enzyme and 17-alpha-hydroxylase was observed mainly in thecal cells and interstitial cells of the ovarian stroma during quiescence. Thecal cells positive for 17-alpha-hydroxylase were found even around the primary follicles. The peak immunoreactivity for all the three enzymes was observed during recrudescence. It coincided with high circulating steroid levels during this period. In the stroma, immunoreactivity for side chain cleavage and 17-alpha-hydroxylase was so extensive that it almost occupied the entire interfollicular area of the ovary. Aromatase immunoreactivity declined, but side chain cleavage enzyme and 17-alpha-hydroxylase remained extensive during the period of delayed ovulation. This suggests a high androgen and low estrogen synthesis during the period of delayed ovulation. There was a marked decline in 17-alpha-hydroxylase and an increase in aromatase immunoreactivity during the preovulatory period, suggesting a decrease in androgen and increase in estrogen synthesis. The results suggest thecal cells and interstitial cells of the stroma as the major site of steroidogenesis in the ovary of S. heathi. Over production of androgen is attributed to the extensive development of 17-alpha-hydroxylase positive interstitial cells in the ovarian stroma, and this may be responsible for delayed ovulation in Scotophilus heathi.     

Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform

Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three-fold increase in recombinant protein yield compared with those from the wild-type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml ⁻¹. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.