An autoradiographic study of gonadotrophin regulation of labelled glycoconjugates within preovulatory mouse follicles during the final stages of oocyte maturation, using [3H]glucosamine as the radioactive precursor

Immature mice were treated with PMSG and hCG to induce follicular development and ovulation. [3H]Glucosamine was injected at the same time as the PMSG or 2 h before autopsy. The synthesis and distribution of labelled glycoconjugates within the preovulatory follicles was hormonally dependent. PMSG stimulated a rapid uptake of [3H]glucosamine into the zona pellucida and follicular fluid of the largest antral follicles although labelled macromolecules could not be demonstrated in any of the cellular components of these follicles. The injection of hCG stimulated a rapid incorporation of labelled macromolecules into the cellular components of the preovulatory follicle, namely into thecal, granulosa and especially the cumulus cells surrounding the oocyte. The density of labelled macromolecules within the follicular fluid also increased, while the specific and uniform labelling of the zona pellucida which was so characteristic of the period of PMSG stimulation changed. Between 4 and 8 h after the injection of hCG, labelled glycoconjugates containing [3H]glucosamine, became increasingly associated with the outer surface of the zona pellucida and with the region of the egg plasma membrane, even in Graafian follicles not destined to ovulate. The change in distribution of labelled macromolecules on the zona surface may be a prerequisite for successful sperm-zona binding and the specific association of labelled glycoconjugates in the region of the egg plasma membrane may be involved in the preparation of the egg surface for sperm-egg interactions involving cortical granule exocytosis and the block to polyspermy.     

Radioautographic study of glycoprotein biosynthesis and renewal in the ovarian follicles of mice and the origin of the zona pellucida

Summary L-fucose-³H was injected intravenously into mice which were killed at several time intervals after injection and semi-thin sections of their ovaries were processed for radioautography and analysed quantitatively. At the same time the specific activity of serum glycoproteins was determined. Glycoprotein biosynthesis was demonstrated in the oocytes, granulosa and stromal cells. The silver grain density of the follicular fluid in large follicles reached a peak at 4 h, remained high at 8 h after injection and decreased steadily at the subsequent intervals. It was demonstrated that the labeling pattern of the follicular fluid depends on the secretory activity of the granulosa cells and also on the specific activity of serum glycoproteins. The collapsed zonae pellucidae which represent the highest degree of follicular atresia are able to take up glycoprotein macromolecules. Based on this finding and also on the labeling pattern of the large follicles it was shown that there is very little synthesis of specific glycoproteins for the zona pellucida in large follicles. A more specific labeling of the zona pellucida occurred in the medium follicles. Following the growth of these follicles having a previously labeled zona pellucida, it was demonstrated that this extracellular structure is secreted by the oocyte.     

Appearance of lectin binding affinity to the zona pellucida during hamster oocyte maturation.

In our previous study, a drastic change in terminal saccharides of glycoconjugates of the hamster zona pellucida associated with oocyte maturation was observed using light microscopic methods of lectin cytochemistry. To understand the mechanism of this change, in the present study, the correlation between the cytochemical appearance of saccharide residues in the zona pellucida and nuclear maturation was examined. Immature hamsters were treated with PMSG and hCG to induce follicular development and ovulation. The animals were euthanized 0 to 26 hrs. after the injection of PMSG or 0,1,2,3,4,5,7,9 or 11 hrs. after the injection of hCG, and ovaries were dissected out, fixed, paraffin embedded and sectioned serially. Every other paraffin section was stained with hematoxylin to observe the status of nuclei and to classify follicular growth and only the fully developed preovulatory follicles were examined in experiments. The peroxidase-labelled lectin-diaminobenzidine procedure was applied to sections. The lectins employed were WGA, SBA, MPA, UEA-I, LotusA and AAA. Germinal vesicle breakdown was observed within 3 hrs. after the administration of hCG. A positive reaction of WGA, SBA or MPA for zonae pellucidae in the fully developed preovulatory follicles appeared 1 hr. after hCG injection, and remained so for the next 10 hrs. UEA-I, Lotus A and AAA reactions were negative for all of the zonae pellucidae observed. The data indicate that the synthesis of saccharide residues such as GlcNAc and GalNAc forming zona components in the follicles is not triggered by germinal vesicle breakdown.     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.     

Distribution of isotopic label after the oral administration of free and bound 14C-labelled glucosamine in rats

The metabolic fate of [1-(14)C]glucosamine, of N-acetyl[1-(14)C]glucosamine and of glycoproteins labelled with [1-(14)C]glucosamine was studied in rats for a period of 24hr. after these materials were given orally or injected. When [1-(14)C]glucosamine was injected 26.3% of the label was excreted in the urine, 19.7% was expired as carbon dioxide and 12.7% was incorporated into plasma proteins. When the same compound was given orally, 49.2% of the label was expired as carbon dioxide, with little appearing in the urine or in the plasma. When N-acetyl[1-(14)C]glucosamine was injected, 51.3% of the label was excreted in the urine with 12.3% appearing in carbon dioxide, but there was little incorporation into plasma protein. When this compound was given orally, 46.5% of the label was expired as carbon dioxide, 7.4% was recovered in the urine and 1.7% was incorporated into plasma protein. After the injection of (14)C-labelled glycoprotein 21.0% of the label was expired as carbon dioxide, whereas when it was given orally 49.8% of the label was recovered in carbon dioxide. The differences observed between the metabolic fate of the amino sugars when they were given orally and their fate when injected could not be accounted for by the action of the intestinal microflora or by the rate of administration of the material. It is concluded that amino sugars undergo metabolic alteration or degradation during absorption.     

Lipoprotein lipase expression level influences tissue clearance of chylomicron retinyl ester

Approximately 25% of postprandial retinoid is cleared from the circulation by extrahepatic tissues. Little is known about physiologic factors important to this uptake. We hypothesized that lipoprotein lipase (LpL) contributes to extrahepatic clearance of chylomicron vitamin A. To investigate this, [3H]retinyl ester-containing rat mesenteric chylomicrons were injected intravenously into induced mutant mice and nutritionally manipulated rats. The tissue sites of uptake of 3H label by wild type mice and LpL-null mice overexpressing human LpL in muscle indicate that LpL expression does influence accumulation of chylomicron retinoid. Skeletal muscle from mice overexpressing human LpL accumulated 1.7- to 2.4-fold more 3H label than wild type. Moreover, heart tissue from mice overexpresssing human LpL, but lacking mouse LpL, accumulated less than half of the 3H-label taken up by wild type heart. Fasting and heparin injection, two factors that increase LpL activity in skeletal muscle, increased uptake of chylomicron [3H] retinoid by rat skeletal muscle. Using [3H]retinyl palmitate and its non-hydrolyzable analog retinyl [14C]hexadecyl ether incorporated into Intralipid emulsions, the importance of retinyl ester hydrolysis in this process was assessed. We observed that 3H label was taken up to a greater extent than 14C label by rat skeletal muscle, suggesting that retinoid uptake requires hydrolysis.In summary, for each of our experiments, the level of lipoprotein lipase expression in skeletal muscle, heart, and/or adipose tissue influenced the amount of [3H]retinoid taken up from chylomicrons and/or their remnants.     

Accelerated modification of the zona pellucida is the primary cause of decreased fertilizability of oocytes in the 129 inbred mouse strain

We investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to α-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.     

Cx37 and Cx43 localize to zona pellucida in mouse ovarian follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

Cx37 and Cx43 Localize to Zona Pellucida in Mouse Ovarian Follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

Effects of passive immunization against oestradiol on mouse ovum transport

The ability of anti-oestradiol immunoglobulin to withhold oestradiol-17 beta from its target tissue was examined. The total oestradiol-17 beta binding capacity present in in-vitro incubations or injected into mice intravenously was related to the amount of [3H]oestradiol present in the media or intravenously injected into the animals respectively. When the ratio of binding capacity to [3H]oestradiol was above 74:1, [3H]oestradiol was successfully withheld from uterine tissue in vitro and in vivo. Injecting anti-oestradiol immunoglobulin into mice before administration of a tube-locking dose of oestradiol-17 beta ensured normal passage of ova through the oviduct. Daily administration of anti-oestradiol immunoglobulin to PMSG-hCG stimulated mice (starting 72 h before hCG injection) induced retention of ova for at least 2 days beyond the time when all ova had left the oviducts of control animals. The binding capacity: oestradiol-17 beta concentration ratios of sera from these animals were greater than 250:1 throughout the experimental period. Non-specific immunoglobulin had no such effects, indicating the specificity of the anti-oestradiol immunoglobulin response.     

Action of PMSG on follicular populations in the heifer

The short-term action of PMSG on the population of growing follicles in cattle was studied using histological methods. On Day 7 of a synchronized oestrous cycle 10 Friesian heifers were unilaterally ovariectomized. The remaining ovary was immediately stimulated by an injection of PMSG (2000 i.u.) and was removed 48 h after the preovulatory discharge of LH. Control animals did not receive any injection of PMSG. In all ovaries, follicles greater than 70 micron diameter were counted, measured and checked for atresia. The mitotic index in granulosa cells of follicles of different sizes was estimated in both ovaries of all the PMSG-injected animals. Unilateral ovariectomy alone had no significant effect on follicular populations. In the interval between PMSG injection and removal of the second ovary (148 +/- 22.7 h), PMSG significantly increased the number of normal preantral follicles but did not change the number of normal antral follicles. The mitotic index doubled in preantral and early antral follicles but remained unchanged in large antral follicles. PMSG stimulated slightly the growth of the antrum in large antral follicles but did not stimulate its formation in preantral follicles. The incidence of atresia among antral follicles, particularly the largest ones (diam. greater than 1.7 mm), was significantly reduced after PMSG, suggesting some 'rescue' of follicles from atresia.     

The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo.

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.     

Reversal of indomethacin blockade of ovulation in gilts by prostaglandins

Prepubertal gilts given 750 IU pregnant mares′ serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation fail to ovulate when 10 mg/kg indomethacin (INDO) is injected 24 h after hCG administration. This study examines the effects of administration of exogenous prostaglandins F_2α and E₂ (PGF_2α and PGE₂) alone or in combination, and at various times prior to the expected time of ovulation, on the INDO blockade of ovulation in PMSG/hCG-treated gilts. Occurrence of ovulation was determined by visual observation at laparotomy 48 h after hCG. When 5 mg or 10 mg PGF_2α was injected at each of 38, 40 and 42 h after hCG injection, 63% and 79%, respectively, of preovulatory follicles ovulated. In contrast, injection of 5 mg PGE₂ or 5 mg PGE₂ plus 5 mg PGF_2α induced ovulation in 0% and 24% of preovulatory follicles, respectively. In control groups, 100% of folicles in PMSG/hCG-treated gilts ovulated whereas none did so in PMSG/hCG/INDO-treated animals. These results indicate that administration of PGF_2α can induce ovulation in the PMSG/hCG/INDO-treated prepubertal gilt and suggest that PGE₂ is ineffective and may be antagonistic to PGF_2α in overcoming the ovulation blocking effect of INDO.     

Developmental and molecular aberrations associated with deterioration of oogenesis during complete or partial follicle-stimulating hormone receptor deficiency in mice

Targeted disruption of the mouse FSH receptor gene (FSH-R) that mediates the action of the FSH results in a gene dose-related ovarian phenotype in the developing as well as the adult animal. While null females (FORKO) are sterile, the haplo-insufficient mice experience early reproductive senescence. The purpose of this study was to first record changes in oocyte development in the null FORKO and haplo-insufficient mice. Oocyte growth is significantly retarded in the null mutants with thinner zona pellucida in preantral follicles, but thicker zona pellucida in secondary follicles. This morphometric change indicates developmental aberrations in coordination of the germ cell (oocyte) and the somatic granulosa cell (GC) compartments. Markers for primordial germ cell proliferation and oocyte growth, such as the c-Kit/Kit-ligand and bone morphogenetic protein-15 (BMP-15) were downregulated in both null and +/- ovaries, suggesting disrupted communication between oocyte and GCs. Extensive changes in the expression of other oocyte-specific gene products like the zona pellucida glycoproteins (zona pellucida A, B, and C) indicate major alteration in the extracellular matrix surrounding the germ cells. This led to leaky germ cells that allowed infiltration of somatic cells. These results show that the loss of FSH-R signaling alters the follicular environment, where oocyte-granulosa interactions are perturbed, creating an out-of-phase germ cell and somatic cell development. We believe that these data provide an experimental paradigm to explore the mechanisms responsible for preserving the structural integrity and quality of oocytes at different ages.     

Autoradiographic analysis of follicle-stimulating hormone and human chorionic gonadotropin receptors in the ovary of immature rats treated with equine chorionic gonadotropin.

The gonadotropin-primed immature rat has become the most common model for the study of follicular development and ovulation. In this study, prepubertal female rats, 23 and 24 days old, were injected s. c. with 5 IU eCG, and ovaries were collected for topical autoradiography of FSH and hCG receptors at 48 or 24 h post-eCG, respectively (i.e., Day 25). In a baseline group, on Day 25 (before eCG), even the smallest preantral follicles with 1 layer of granulosa cells (GCs; primary follicles) possessed FSH receptors, but hCG receptors were found only on the theca of follicles with 2 or more layers of GCs. Human CG receptors were especially prominent in the interstitium that intimately surrounds preantral follicles without any distinction between theca and interstitial cells. There was a discrete theca surrounding antral follicles. Occasionally antral follicles had hCG receptors in the interstitium, but the adjacent theca was negative, suggesting that these follicles might be destined for atresia. By 24 h post-eCG, a now-discrete theca layer with hCG receptors surrounded all preantral follicles except for the primary follicles, which never responded to eCG. The interstitium was hypertrophied and epithelioid, as was the theca surrounding nonatretic preantral and antral follicles. Increased mitotic activity characterized the growing preantral follicle, and for the first time, FSH binding in GCs of antral follicles was greater than in the preantral population. By 48 h post-eCG, the primary follicles were still unresponsive to eCG. FSH receptors were even more pronounced in the GCs of large antral follicles, although hCG receptors were present in the GCs of only one third of the antral follicles, reflecting the small dose of eCG administered. By 48 h post-eCG, receptors in the interstitium were barely detectable. Using this model, the following study considers the functional in vitro changes in steroidogenesis in follicles from the smallest preantral follicles to the largest antral follicles.     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

不同激素和注射方式对家猫超排效果的比较

比较了PMSG hCG和FSH hCG两种方案以及PMSG的不同剂量和注射方式对家猫的超排效果的影响。用 1 0 0IU的PMSG超排家猫所得到的排卵点数及平均每只猫获得的卵数显著低于 2 0 0IU处理组或 30 0IU处理组 (P <0 0 5 ) ,但 2 0 0IU处理组与 30 0IU处理组之间的超排效果也无显著差异 (P >0 0 5 ) ;用皮下注射 2 0 0IU的PMSG或用肌肉注射 2 0 0IU的PMSG对超排效果无差异 (P >0 0 5 ) ;用 2 0 0IUPMSG 2 0 0IUhCG和 1 5mgFSH 2 0 0IUhCG两种方案对家猫超排 ,发现不论是每只猫的排卵点数、卵子获得数 ,还是卵子的第一极体排放率都没有显著差异 (P >0 0 5 )。实验说明 ,PMSG的注射方式不影响对家猫的超排效果 ,用 2 0 0IU的PMSG超排家猫是较适合的剂量 ,FSH和PMSG都可用于家猫的超排 ,但PMSG使用更为方便。     

In vitro DNA synthesis by isolated preantral to preovulatory follicles from the cyclic mouse

In recent studies, we have shown that the smallest preantral follicles in the cyclic hamster increase DNA synthesis in the periovulatory period in response to surge levels of FSH. The current investigation was designed to determine whether the same phenomenon occurs in the cyclic mouse. Intact mouse follicles were isolated with watchmaker forceps (stages 4-6) or by enzymatic digestion (stages 1-4) at 0900 h and 1500 h on each day of the 5-day estrous cycle. The isolated follicles were classified into 6 stages: stages 1 and 2: follicles with 1 and 2 layers of granulosa cells; stage 3: follicles with 3 or more layers of granulosa cells and formation of theca; stages 4-6: incipient, small, and preovulatory antral follicles. The follicles at each stage were incubated for 3 h with [3H]thymidine. DNA content in stages 1-4 of follicles remained unchanged during the estrous cycle; for stages 5 and 6, DNA content was higher on the afternoon of proestrus than on other days of the cycle. Incorporation of [3H]thymidine for stages 1-3 (preantral follicles) started to increase at 1500 h of proestrus and peaked at 0900 h on estrus, whereas for stages 4-6, DNA synthesis peaked on proestrus (1500 h) and then fell by the morning of estrus. Thus, the rate of DNA replication in preantral and antral mouse follicles were different. Similarities and differences in folliculogenesis between mouse and hamster are discussed. These results suggest that DNA synthesis and the growth of all stages of follicles in the cyclic mouse may be associated with changing levels of periovulatory gonadotropins.     

Macromolecules released into the culture medium during the vegetative cell cycle of the unicellular green alga Chlamydomonas reinhardii.

The culture medium of growing Chlamydomonas reinhardii cells contains hydroxyproline-rich glycoproteins, which are mainly liberated during release of the zoospores from the mother-cell wall. Pulse-labelling studies with [3H]proline and [35S]methionine have been performed in order to detect the protein components released by synchronously growing cells at different stages of the cell cycle. When either [3H]proline or [35S]methionine were applied during the phase of cell growth, radioactive label appeared in the released macromolecules after a lag period of 40 min, whereas incorporation into the insoluble part of the cell wall was delayed only by 20 min. When applied at the end of the growth phase, e.g. 13 h after beginning of the illumination period, the radioactive amino acids were incorporated into the cell wall, but radioactive labelling of macromolecules released into the culture medium could not be detected before the zoospores were liberated from the mother-cell wall. Maximal incorporation of [3H]proline and [35S]methionine into the insoluble part of the cell wall was observed during cell division, but essentially no radioactively-labelled macromolecules were released into the culture medium during this time period. Analysis of the macromolecules, which were liberated during cell enlargement, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed distinct radioactive bands, which were differentially labelled with [3H]proline and [35S]methionine. Among the macromolecules released into the culture medium during cell growth, a component of an apparent Mr 35 000 was preferentially labelled with [3H]proline. This component was also detected after labelling with [35S]methionine, but components of an apparently higher Mr were more prominent after labelling with [35S]methionine. Macromolecules released during the cell-enlargement period of synchronously growing cultures in the presence of [3H]proline contained radioactively-labelled hydroxyproline in addition to proline. These results show that, during cell-wall growth, specific protein components are released into the culture medium and that at least one of these components contains large amounts of proline and hydroxyproline. At least some of these macromolecules seem to be constituents of the cell wall, because during pulse-chase experiments radioactively-labelled macromolecules appeared in the culture medium mainly during the time period when the specific radioactivity of the insoluble inner-cell-wall layer decreased.