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 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.     

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.     

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.     

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.     

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.     

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.     

Regulation of matrix metalloproteinase-19 messenger RNA expression in the rat ovary

Matrix metalloproteinases (MMPs) are instrumental in the constant tissue remodeling in the ovary. An induction of MMP-19 mRNA in periovulatory follicles has been reported in mouse ovaries. However, little is known about MMP-19 expression during the follicular and luteal periods or about the ovarian regulation of MMP-19 mRNA expression. We examined the expression pattern of MMP-19 mRNA during various reproductive phases and the periovulatory regulation of MMP-19 mRNA in the rat ovary. In gonadotropin-primed, immature rat ovaries, levels of MMP-19 mRNA transiently increased during both follicular growth and ovulation. The MMP-19 mRNA was localized to the theca-interstitial layer of growing follicles and to the granulosa and theca-interstitial layers of periovulatory follicles. A similar expression pattern of MMP-19 mRNA in periovulatory follicles was observed in ovaries from naturally cycling adult rats. Accumulation of MMP-19 mRNA was detected in regressing corpus luteum. The regulation of MMP-19 mRNA expression during the periovulatory period was investigated via in vivo studies and through in vitro culture studies on follicular cells. The hCG-induction of MMP-19 mRNA was mimicked by treating granulosa cells, but not theca-interstitial cells, from preovulatory follicles with LH or activators of the protein kinase (PK) A or PKC pathways. Cycloheximide blocked the LH- or forskolin-induced MMP-19 mRNA expression, demonstrating the requirement for new protein synthesis. In contrast, blocking activation of the progesterone receptor or prostaglandin synthesis had no effect on the increase in MMP-19 mRNA expression. In conclusion, the induction of MMP-19 mRNA suggests an important role of this proteinase during follicular growth, ovulation, and luteal regression.     

Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes.

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.     

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.     

Secretion of platelet-activating factor by periovulatory ovine follicles

Secretion of platelet-activating factor (PAF) in vitro by ovine follicles and ovarian interstitium obtained at various times before, during and after the endogenous preovulatory surge of luteinizing hormone (LH) and ovulation was quantified by radioimmunoassay. Release of PAF by the preovulatory follicle increased within 2 h after initiation of the surge of LH. Capacity for secretion of PAF was greatest at the time of ovulation, then declined thereafter. Production of PAF by ovarian interstitium throughout the periovulatory period was relatively low and did not change with time. It appears that PAF could act as an intrafollicular mediator in the mechanisms of ovulation and(or) luteinization.     

Effects of dexamethasone and 5-bromodeoxyuridine on protein synthesis and secretion during in vitro pancreatic development

Protein synthesis and secretion during in vitro pancreatic development and after treatment with the glucocorticoid dexamethasone and the thymidine analog 5-bromodeoxyuridine (BrdU) was monitored using two-dimensional gel electrophoresis. At 14 days gestation, the synthesis of more than 200 proteins and the secretion of a complex set of proteins was detected. The relative rate of synthesis and secretion of the majority of this set of proteins decreased dramatically during development; after 6 days of culture most were no longer detected. In contrast, the synthesis and secretion of pancreas-specific exocrine proteins amylase, a Sepharose binding protein (protein 2), and chymotrypsinogen first detected after one day in culture, increased throughout the 6-day culture period. Other pancreatic digestive (pro)enzymes normally found in the adult such as the basic form of chymotrypsinogen, lipase, ribonuclease, and trypsinogen were not detected during the culture period. Thus at least two distinct regulatory events are involved in the expression of the exocrine genes during development. Dexamethasone treatment during the 6-day culture period selectively increased the synthesis of amylase and several other minor secretory proteins. BrdU treatment caused major changes in the protein synthetic and secretory patterns of the pancreas as well as in morphogenesis. BrdU treated pancreases showed greatly reduced synthesis of amylase, protein 2, and chymotrypsinogen and prolonged synthesis of many proteins normally detected only at early stages of pancreatic development. BrdU treatment also stimulated the secretion of a set of proteins ostensibly associated with duct cells. Thus, BrdU specifically alters the developmental program of the pancreas.     

Loblolly pine seed dormancy. I. The relationship between protein synthesis and the loss of dormancy

Embryos from the mature unstratified loblolly pine ( Pinus taeda L.) seeds used in this study were nondormant: however, they failed to germinate in situ because of constraints imposed by the surrounding tissues. During a stratification period of 35 days of moist chilling at 2°C, seed germinability increased from 19 to 76%. The total lipid content of the megagametophyte did not change during stratification, whereas the total protein content of both megagametophyte and embryo was more variable. The rate of synthesis of buffer soluble proteins in these two tissues increased and electrophoretic analysis showed that while similar proteins were synthesized during the stratification period, changes in the patterns of synthesis of some proteins did occur. In both the embryo and megagametophyte the synthesis of a set of proteins with molecular masses below 46 kDa decreased markedly after 14 days of chilling (DOC). In the megagametophyte, the synthesis of a more diverse set of proteins with molecular masses ranging from 16 to 78 kDa increased after 14 DOC. It is noteworthy that these changes in the patterns of protein synthesis coincided with the greatest relative increase in seed germinability of 35%.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.     

The temporal pattern of vitellogenin synthesis in Drosophila grimshawi

The temporal pattern of protein production and, in particular, vitellogenin protein synthesis during the sexual maturation of Drosophila grimshawi females has been studied in vivo by briefly feeding the flies with 35S-methionine and 3H-amino acids. The overall level of incorporation was very low in young flies; it then progressively increased to reach a maximum with the onset of sexual maturity at 13-15 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed three classes of proteins: those synthesized throughout the age spectrum, which constitute the majority of protein species; proteins synthesized primarily or only in young flies; and proteins synthesized only by the older flies. In this Drosophila species, the three vitellogenins (V1, V2, and V3) appeared to be synthesized in a two-phase pattern. In the first phase, small quantities of V1 and V2 were detected immunologically in the fat body and hemolymph of newly emerged and 1 day-old flies. These proteins did not accumulate in the hemolymph or the ovaries, apparently being unstable proteins. The second phase commenced in early vitellogenesis (7-9 days of age) with synthesis in the fat body of small quantities of V1 and V2, followed by V3 proteins. These proteins were secreted and accumulated in the hemolymph and 24 h later were found in the ovaries. Their quantities increased rapidly and a steady state of synthesis, release into the hemolymph, and uptake by the ovaries was reached by days 13-15. We have estimated that during the steady state of vitellogenin synthesis, a fly can synthesize in 24 h at least 152 micrograms of vitellogenins, which is more than 2% of its body weight, at an average rate of about 6.3 micrograms vitellogenins/h. About 2 micrograms of this are synthesized in the fat body, and about 4 micrograms in the ovaries. These findings are discussed in terms of their physiological implications and contrasted with the available data on Drosophila melanogaster.     

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.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Noncoordinate synthesis of the vitellogenin proteins in tissues of Drosophila grimshawi

In analyzing the in vitro pattern of protein synthesis by the fat body and ovaries of the Hawaiian species Drosophila grimshawi, we have found that the ovaries synthesize much more protein than the female fat body and that the majority of the synthesized proteins are retained by the ovarian tissues. In contrast, the fat body secrets most of the proteins into the culture medium. Vitellogenins are the major class of proteins synthesized and released into the medium by both tissues. The synthesis of the three vitellogenin proteins (V1, V2, V3) is noncoordinate in the two tissues. Ovaries synthesize much more of the V2 protein, less V1 and very little V3, whereas fat body synthesizes more V1 protein with lesser quantities of the other two. The follicle cells were identified as the site of ovarian vitellogenin synthesis in D. grimshawi, confirming the findings in D. melanogaster. In D. grimshawi, the three vitellogenins are synthesized by the follicle cells in a noncoordinate and developmentally regulated manner. V2 and V1 are the predominant proteins at the onset of vitellogenesis (S8-9); their production peaks together with that of V3 a few hours later (S10) and then decreases to quantities equal to that of V3 during early choriogenesis (S11). During active choriogenesis (S12), V2 and V1 cease to be synthesized, but V3 synthesis continues. The vitellogenins synthesized by the follicles in vitro are released into the medium and not incorporated into the oocyte.     

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.