Comparative aspects of oxytocin in baboon (Papio hamadryus anubis) and human corpora lutea

In spite of the importance of the corpus luteum in human reproduction,little is known about its formation after ovulation and duringregression in the absence of conception. This is largely dueto constraints on the availability of normal human tissue: thereforean appropriate model which could be studied and provide informationapplicable to the human was sought. The baboon (Papio), a non-humanprimate, has been determined to be one such model. Thus, inthe past several years our studies have examined the role ofluteal peptides in corpus luteum function, and, when possible,we have attempted to examine corpora lutea from the human andbaboon in parallel. Although a milk-ejection factor was recognizedto be present in luteal tissue in 1910 (Ott and Scott, Proc.Soc. Exp. Biol. Med., Vol. 8, p 49), the role of oxytocin inluteal physiology has not been easy to ascertain. This is inpart due to the methodologies employed to assess its role. Ourstudies summarized below suggest that oxytocin does not directlyaffect luteal steroidogenesis, but that it may play a role incell to cell communication involving the expression of the gapjunction proteins, the connexins. In view of the fact that oxytocin,its receptor, gap junctions and associated proteins are notunique to the human and non-human primates, the model of lutealdevelopment and demise proposed may be applicable to most species.     

Proliferative activity of preovulatory follicles and newly formed corpora lutea in cycling rats from late prooestrus to early oestrus

Ovaries from adult cycling rats were studied from 1600 h on the day of prooestrus to 0700 h on the day of oestrus in order to relate the cyclic hormonal changes to the proliferative activity of preovulatory and postovulatory (i.e. newly-formed corpora lutea) follicles. Proliferative activity was studied by the immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine (BrdU). The proliferative activity of granulosa cells (GC) in large preovulatory follicles showed a centripetal pattern and decreased during prooestrus, reaching a minimum at 2100 h. However, a proliferative wave was found in the GC of preovulatory follicles at 0200 h on the day of oestrus and in those of newly-formed corpora lutea at 0700 h on the day of oestrus. These results suggest that the granulosa cells of preovulatory follicles show maturational changes that followed a different pattern, depending on their location within the follicle, and that the proliferative wave found from 0200 to 0700 h on oestrus is important for the establishment of the number of steroidogenic cells in the cyclic corpus luteum.     

Localization and expression of zonula occludens-1 tight junction-associated protein in baboon (Papio anubis) corpora lutea

The identification of the cell junction-forming proteins connexin-43,a gap junction protein and E-cadherin, which is a componentof adherent junctions, in the corpus luteum of both humans andbaboons suggests that cell-cell interactions and metabolic cooperationmust occur in this tissue. Occluding junctions are a third typeof junction which form a physical barrier between cells. Thus,our aims in this study were firstly to examine the presenceof the tight junction-associated protein zonula occludens-1(ZO-1) by immunohistochemistry, and secondly to determine theconcentrations of this protein in the early, mid- and late lutealphase baboon corpora lutea of the menstrual cycle by a Westernanalysis. ZO-1 was localized mainly at the periphery of theluteal cells, and the intensity of immunoreactivity varied throughthe luteal phase, with comparatively stronger immunoreactivityin the mid-luteal phase than the early and late luteal phases.Atretic corpora lutea were devoid of activity. By Western analysis,bands of immunoreactivity were observed at 225 kDa, furtherconfirming the presence of the protein. Maximum activity, asdetermined by densitometry, was observed in the mid-luteal phase.These data infer the presence of tight junctions in the corpusluteum and suggest that expression of the ZO-1 protein formingthese junctions may be hormonally regulated within this tissue.     

The benefits of mid-luteal addition of human chorionic gonadotrophin in in-vitro fertilization using a down-regulation protocol and luteal support with progesterone

Luteal support is essential in in-vitro fertilization (IVF)when long-acting gonadotrophin-releasing hormone agonist (GnRHa)is used. Because progesterone lacks luteotrophic stimulation,it seems to be the drug of choice in cases with an increasedrisk of ovarian hyperstimulation syndrome (OHSS). The aim ofthis study was to assess the beneficial effect of the mid-lutealaddition of human choriomc gonadotrophin (HCG) in IVF, usinga down-regulation protocol and luteal support with progesterone,in a prospective randomized study. The study included 170 IVFcycles down-regulated with long-acting GnRHa which were supportedwith 50 mg/day progesterone i.m. during the luteal phase. Patientswere evaluated in the mid-luteal period. Those without clinicalsigns of OHSS, oestradiol concentrations <1000 pg/ml andprogesterone concentrations <50 mg/ml were randomly allocatedto either the addition of 2500 IU HCG (HCG+ group) or no HCG(HCG– group). End luteal phase progesterone concentrationsamong non-pregnant patients were used to assess the contributionof exogenous progesterone and to categorize pregnancies accordingto their corpus luteum function. Similar low OHSS (2.7 and 1.8%)and pregnancy (30 and 29%) rates were observed in the HCG+ andHCG– groups respectively. Of the 26 pregnancies in theHCG+ cases, there was only one case with reduced corpus luteumfunction, compared with 12 of the 25 pregnancies among HCG–patients. Cases with reduced corpus luteum function requiredcontinuous progesterone support and presented lower HCG concentrationsand a higher rate of adverse pregnancy outcome. We concludethat mid-luteal HCG addition does not affect pregnancy rate,but in fact helps to preserve corpus luteum function and avoidsthe need for further supplementation during early pregnancy.     

Influence of human chorionic gonadotrophin, oestradiol and progesterone on uteroplacental and corpus luteum blood flow in normal early pregnancy.

A transvaginal colour and pulsed Doppler study was performed on 44 women with normal pregnancies between 5 and 16 weeks of gestation. Maternal levels of human chorionic gonadotrophin (HCG), free alpha-HCG subunit, free beta-HCG subunit, 17 beta-oestradiol and progesterone were determined in sera obtained at the time of Doppler examination. Uterine peak systolic velocity (PSV) and alpha-HCG and 17 beta-oestradiol levels increased significantly (P < 0.001) from the second to the fourth month of gestation, whereas uterine and spiral resistance index (RI) decreased significantly (P < 0.005 and P < 0.001, respectively) with gestational age. Levels of HCG and beta-HCG peaked significantly (P < 0.01) during the third month of gestation. Corpus luteum PSV and RI and progesterone levels did not vary significantly with gestational age. Multiple regression analysis showed that gestational age was the only significant (P < 0.05) contributor to uterine PSV and spiral RI variability. In addition to gestational age, 17 beta-oestradiol had a significant (P < 0.001) influence on uterine RI. Both corpus luteum PSV and RI were significantly (P < 0.01 and P < 0.05, respectively) related to progesterone levels. Corpus luteum PSV was also significantly (P < 0.05) related to 17 beta-oestradiol levels and RI to HCG levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Results of in-vitro fertilization in normal ovulatory women treated with pure follicle stimulating hormone. Analysis of the oestradiol response

The relationship between various measures of oestradiol (E2) secretion and the total number of oocytes retrieved (OR) and cleaved embryos (CE) was characterized in normal ovulatory women stimulated with pure follicle-stimulating hormone (FSH) in a programme for in-vitro fertilization and embryo transfer (IVF-ET). Patients in this study included women with tubal factor as their only cause for infertility. Cycles were monitored with serum E2 concentration and ultrasonography. Human chorionic gonadotrophin (HCG) was administered when two follicles had a maximum diameter greater than 15 mm. The variables used to characterize the E2 secretory response included: (i) the difference between the highest and lowest E2 concentration during stimulation; (ii) the ratio of terminal to initial E2 concentration; (iii) E2 concentration on the day of HCG administration; and (iv) the slope of the E2 curve. These measures of E2 secretion each correlated with both the number of OR and the number of CE. When all E2 variables were considered simultaneously in a stepwise multivariate regression procedure, variations in the number of OR (r2 = 0.84) or CE (r2 = 0.77) could be explained by variation in the E2 secretory profile. Equations derived from these E2 variables may help to identify and improve problem areas within IVF-ET programmes when actual results differ from expected.     

Simultaneous induction of multiple follicular development with gonadotrophins and steroid replacement for in-vitro fertilization.

Simultaneous administration of follicle stimulating hormone, oestradiol valerate and progesterone was employed in a patient with a possible enzymatic deficiency involving low production of oestradiol. The patient became pregnant after in-vitro fertilization. This case demonstrates that this treatment is useful in women with low oestradiol production and subsequent inadequate endometrial development; it also illustrates the role of oestradiol in follicular development and questions the importance of serum oestradiol measurements in the monitoring of ovulation induction.     

Circhoral fluctuations of serum total renin, inhibin and related hormones around the mid-cycle in normal human females.

Total renin and inhibin are secreted by the ovary. Although luteinizing hormone (LH) and/or follicle stimulating hormone (FSH) may stimulate their secretion, the close relationship between fluctuations of gonadotrophins, oestradiol, progesterone, renin and inhibin during the cycle is still conjectural. To investigate the temporal relationship between the short-term fluctuations in the circulating concentrations of LH and FSH and the ovarian hormones (oestradiol, progesterone, renin and inhibin), blood samples were collected at 15-min intervals for 6 h from 15 normal women in the late follicular (n = 4), early luteal (n = 5) or luteal (n = 6) phases of the menstrual cycle. LH levels showed the well-known pulsatile secretion with decreasing frequency and increasing relative amplitude from the late follicular to the luteal phase. Progesterone and oestradiol serum levels were pulsatile, 25% and 35-50% of which were linked to LH pulses, with time lags of 30 and 12-15 min respectively. Renin levels presented significant pulses, 26% of which were related to LH pulses with a time lag of less than 10 min; no coincidence was found between renin and oestradiol pulses. Inhibin levels presented only scattered pulses of small amplitude, which were unrelated to LH or FSH. These results show that, besides the LH-related pulses, pulsatile secretion of some ovarian hormones (oestradiol, progesterone and renin) may also occur independently of LH pulses and may be unrelated to one another. Moreover, contrary to the other ovarian hormones, inhibin seems to follow a tonic, not a pulsatile type of secretion around the mid-cycle.     

Peritoneal fluid volume and levels of steroid hormones and gonadotrophins in peritoneal fluid of normal and norethisterone-treated women.

Peritoneal fluid and blood samples were collected at surgical sterilization from 30 untreated women at various stages of the luteal phase and from 43 women treated with 300 micrograms norethisterone daily. Levels of oestradiol, progesterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured. The highest peritoneal fluid volume (mean, 23.1 ml) was found in the early luteal phase (LH 0 to + 3) and the lowest (mean, 5.9 ml) in the late luteal phase (LH + 12 to menses). The norethisterone treatment diminished the formation of peritoneal fluid and the degree of inhibition was dependent upon the type of ovarian reaction to norethisterone. Progesterone and oestradiol levels were higher in peritoneal fluid compared to plasma throughout the luteal phase and during norethisterone treatment. A comparison of the levels of these steroids between untreated controls (LH + 8 to + 11) and norethisterone-treated women demonstrated that the progesterone levels in peritoneal fluid were highly reduced by norethisterone treatment, while the oestradiol levels were not affected. The FSH and LH levels were, in contrast to the steroid hormones, significantly lower in peritoneal fluid than in plasma, both in the untreated and the treated women. No differences in the FSH or LH levels between the untreated and treated women were found. The results indicate that the peritoneal fluid volume and the steroid hormone levels in peritoneal fluid vary with the stages of the luteal phase. Norethisterone treatment significantly reduced the peritoneal fluid volume as well as its progesterone concentration, whereas the oestradiol and gonadotrophin levels remained unchanged.     

Endocrinology: Luteal phase oestradiol and progesterone levels are stronger predictors than follicular phase follicle stimulating hormone for the outcome of in-vitro fertilization treatment in women with tubal infertility

Basal follicle stimulating hormone (FSH) in a natural cycle,FSH on cycle days 3 and 10 in a domiphene citrate-stimulatedcycle and oestradiol and progesterone area under the curve (AUC)in the luteal phase of the ciomiphene citrate-stimulated cyclewere evaluated as hormonal predictors for the outcome of FVFtreatment in 53 normally cycling women with tubal infertility.The pregnant women had significantly fewer treatment cycles(P < 0.001) and needed fewer ampoules of gonadotrophins (P< 0.001). They also had more oocyte retrievals (P < 0.001),more oocytes per retrieval (P < 0.01), higher fertilizationrate (P < 0.001) and more replaced pre-embryos per replacement(P < 0.01) as compared with non-pregnant women. Significantdifferences were found in FSH concentrations on cycle days 3(P < 0.05) and 10 (P < 0.001) after domiphene citratestimulation and for oestradiol and progesterone AUC in the lutealphase (P < 0.001) between those women who became pregnantand those who did not become pregnant after IVF treatment Lutealoestradiol and progesterone had considerably stronger predictivevalue for the outcome of IVF treatment as compared to basalFSH and domiphene citrate challenge test.     

Peripheral blood mononuclear cells stimulate progesterone production by luteal cells derived from pregnant and non-pregnant women: possible involvement of interleukin-4 and interleukin-10 in corpus luteum function and differentiation

Human luteal cells have been reported to express human leukocyteantigen-DR and lymphocyte functional antigen-3 on the cell surface,suggesting physiological interaction between luteal cells andT-lymphocytes through the menstrual cycle into early pregnancy.To elucidate the role of peripheral lymphocytes on corpus luteumdifferentiation, the effect of peripheral blood mononuclearcells (PBMC) on steroidogenesis by luteal cells was investigated.The production of Th-2 cytokines such as interleukin (IL)-4and IL-10 by the co-cultured cells was also examined, and theeffects of these cytokines on progesterone production by lutealcells were investigated. Corpora lutea were obtained from eightnon-pregnant women in the luteal phase and five women in earlypregnancy for luteal cell culture. PBMC were isolated from unrelatedwomen in the follicular phase, secretory phase, and early pregnancy.After co-culture with allogenic PBMC for 48 h, progesteroneproduction was significantly enhanced by PBMC from the secretoryphase and early pregnancy in the non-pregnant luteal cell culture.In the pregnant luteal cell culture, a significant increasein progesterone production was also observed by the co-culturewith PBMC from women in early pregnancy, showing that PBMC havea luteotrophic effect. The stimulatory effects of PBMC werealso observed in co-culture conditions which prevented directcell-to-cell interaction with luteal cells, showing the minorinfluence of mixed lymphocyte reaction. By co-culture with PBMC,the production of IL-10, but not IL-4, was significantly augmentedin luteal cell culture derived from non-pregnant women, whereasthe production of both IL-4 and IL-10 was significantly enhancedin the luteal cell culture derived from pregnant women. Moreover,IL-4 and IL-10 promoted progesterone production by culturedluteal cells, especially in the luteal cell culture derivedfrom corpora lutea of early pregnancy. These findings indicatethat PBMC stimulate progesterone production by luteal cellsand suggest the involvement of PBMC in corpus luteum functionand differentiation probably via the Th-2-type lymphocytes.     

Gonadotrophin stimulation of non-luteinized granulosa cells increases steroid production and the expression of enzymes involved in estrogen and progesterone synthesis

BACKGROUND: In regular IVF treatment, mature oocytes are collected with their luteinized granulosa cells (GCs). When in vitro maturation (IVM) of the oocytes is performed, non-luteinized GCs can be collected. We have investigated how these cells respond to gonadotrophin stimulation in culture. METHODS: GCs were collected from patients undergoing IVM treatment and compared with GCs from IVF patients. The cells were stimulated with FSH and/or hCG. After 48 h, culture media were collected for hormone analysis, and RNA was isolated for gene expression analysis. RESULTS: In IVM GCs, hCG and FSH alone and in combination induced significantly increased progesterone production, and FSH alone and in combination with hCG increased estrogen production. We also studied the gene expression of P-450aromatase and P-450scc and the receptors for FSH and LH. In non-luteinized GCs, the expression levels of P-450aromatase increased with all treatments, and P-450scc expression increased with the combined FSH and hCG treatment. LHR expression increased with FSH treatment, but the FSH receptor expression did not change with different treatments. CONCLUSIONS: Non-luteinized GCs behaved differently from luteinized GCs in culture. The data help understand the final stages of maturation of human oocytes and follicles.     

In vitro maturation and fertilization of oocytes from an intact ovary of a surgically treated patient with endometrial carcinoma: case report

Polycystic ovary syndrome (PCOS) with prolonged anovulation had resulted in endometrial carcinoma in a 43-year-old woman. Since she and her husband did not share common biological children, they requested fertility preservation. Due to the woman's age, high dose progesterone and postponing surgery were both considered inappropriate. We therefore proposed oocyte retrieval from the ovaries removed by staging laparotomy followed by in vitro maturation and ICSI. Surrogacy could then enable a future pregnancy. Fourteen of 17 (82%) retrieved oocytes matured in vitro. Following ICSI, eight embryos (two at the pronuclear stage and six cleaved) were cryopreserved. To the best of our knowledge, this is the first report of oocyte aspiration, maturation and fertilization from an ovary removed by laparotomy.     

The aetiology of galactorrhoea in women with regular menstruation and normal prolactin levels.

Fourteen primary infertile women with expressible galactorrhoea associated with regular ovulatory cycles and normal basal prolactin levels (group A) were matched for age and weight with 14 infertile women with regular menstruation but no galactorrhoea (group B). Both groups showed equivalent increases in prolactin levels after stimulation with 200 micrograms thyrotrophin-releasing hormone (TRH) during the follicular and luteal phases of the menstrual cycle. Patients in group A had a greater increase in luteinizing hormone levels after 100 micrograms i.v. injection of a luteinizing hormone-releasing hormone during the follicular phase (P less than 0.05). Following a 60 mg oral dose of buspirone hydrochloride on day 22 of the menstrual cycle, patients in group A had a greater increase in prolactin levels than patients in group B (P less than 0.01). This reflects hyper-responsive 5-hydroxytryptamine type 1A (5HT1A) receptors in group A patients and may explain the presence of galactorrhoea in these patients despite normal basal and post-TRH prolactin levels.     

Leukocyte activation in the decidua of chromosomally normal and abnormal fetuses from women with recurrent abortion

As part of our continuing programme to investigate immunological causes of unexplained recurrent pregnancy losses, we studied subpopulations of white blood cells and their activation status in decidua of women with a history of recurrent spontaneous abortion (RSA). We differentiated specifically between normal karyotyped male fetuses and abnormal karyotyped fetuses with trisomy 16 because trisomy 16 is not compatible with life and is thus a non-controversial cause of spontaneous miscarriage. Leukocytes were counted in paraffin-embedded decidua after immunohistological staining for CD45 (LCA), CD3, CD56, CD68, CD69 and CD25. Numbers of activated versus non-activated T lymphocytes, NK cells and macrophages were compared in decidua from women with: (i) unexplained RSA who had a normal male karyotype (n = 17) miscarriage; (ii) unexplained RSA who had a trisomy 16 (n = 21) miscarriage; and (iii) normal gestationally age-matched first trimester pregnancies following elective termination procedures (n = 20). Significantly more activated leukocytes were detected in the decidua of women with unexplained RSA who had a normal male karyotype compared to the other groups (P < 0.0001). In addition, numbers of cells comprising the major leukocyte subpopulation, CD56+ NK cells, appeared reduced in the decidua of women with unexplained RSA compared to decidua from women having elective terminations. Increased numbers of activated leukocytes in the decidua of women with a history of unexplained recurrent pregnancy loss who had a normal karyotyped pregnancy provide evidence that cellular immunity may be involved in unexplained recurrent pregnancy loss.     

Effects of sera from infertile women with sperm immobilizing antibodies on fertilization and embryo development in vitro in mice

PROBLEM: This study was performed to investigate if patients' sera with anti-human sperm antibodies show inhibitory effects on in vitro fertilization (IVF) and embryo development in mice. METHOD OF STUDY: Patients' sera were collected from eight infertile women having sperm immobilizing antibodies and 17 infertile women without the antibodies. Male ICR mice and female F1 mice (BALB/c X C57BL/6J) were used. In mouse IVF, pre-incubated sperm were cultured in the medium containing patient's serum with or without sperm immobilizing antibodies, or bovine serum albumin (BSA) as a control. The fertilization rates and the incidences of blastocyst formation were compared. RESULTS: A mouse sperm immobilization test was established. Five (62.5%) of eight serum samples with sperm immobilizing antibodies and nine (52.9%) of 17 serum samples without the antibodies showed sperm immobilizing activities in mice. There was no significant difference between the two groups. Five sera with sperm immobilizing activities in human and mice, and five sera without sperm immobilizing activities in human or mice were used for the further experiments. The fertilization rates in BSA, patient's serum with sperm immobilizing antibodies, and that without the antibodies were 82.5% (746/904), 43.6% (508/1165), and 64.5% (669/1037), respectively. There were significant differences between the groups. The incidences of blastocyst formation were 59.9% (447/746), 31.7% (161/508), and 47.7% (319/669), respectively. There were also significant differences between the groups. CONCLUSIONS: Some of the patient's serum with and without sperm immobilizing antibodies could immobilize sperm with complement. However, as compared with control, sera with sperm immobilizing activities against human and mouse sperm significantly blocked IVF and inhibited embryo development in mice. Further studies are required to investigate the mechanisms of the blocking effects of antisperm antibodies on fertilization and embryo development using the mouse model.     

Functional development of the visual system in normal and protein deprived rats. Ill: Recordings from adult optic nerve in vitro

SJÖSTRÖM, A., CONRADI, N.G., GUSTAFSSON, B. & WIGSTROM, H. 1985. Functional development of the visual system in normal and protein deprived rats. III: Recordings from adult optic nerve in vitro. Acta Physiol Scand, 125 , 353–358. Received 8 March 1985, accepted 20 April 1985. ISSN 0001–6772. Departments of Physiology and Pathology, University of Goteborg, Sweden. A persistent increase in the latencies of the visual evoked response recorded from the cortical surface of protein deprived adult rats was described recently (SJÖSTRÖM et al. 1984). The morphological correlate to this alteration is unknown. Previous studies on malnourished rats have shown a reduction of axonal diameters and in the number of myelin lamellae in relation to axonal circumference, and hence the possibility of a decrease in fibre conduction velocity must be considered. In parallel study, we have established that changes in diameters and myelination of optic nerve fibres similar to those previously reported in malnourished rats are present in adult protein deprived (PD) rats (Conradi el al. 1985). In the present paper, recordings of optic nerves in vitro from adult normal (C) and protein deprived (PD) rats are described. The compound action potentials were very similar in the two groups. Three positive peaks were easily defined probably corresponding to three functional groups of optic nerve fibers. No significant differences in amplitudes or conduction velocities for the three peaks were found between the C and PD rats. It is concluded that the increased latencies of the evoked response are not caused by a decrease in conduction velocity.     

Markers of collagen synthesis and degradation in urogenital tissue and serum from women with and without uterovaginal prolapse

Diverging results have been published concerning collagen metabolism in uterovaginal prolapse (UP). We have investigated collagen turnover in urogenital tissue in urologically healthy women with (UP patients) and without UP or any history of UP (controls). Markers of collagen turnover, carboxy-terminal propeptide of type I procollagen (PICP), amino-terminal propeptide of procollagen III (PIIINP) and carboxy-terminal telopeptide of type I collagen (ICTP) were assayed in urogenital tissue homogenates and serum. Tissue and serum concentrations of collagen turnover markers were related to UP and to menopausal/estrogen status. UP patients were significantly older than the controls. UP patients had significantly higher tissue PICP and PIIINP and significantly lower tissue ICTP levels than the controls, but the difference in ICTP disappeared after matching for menopausal/estrogen status and age. There were no associations between tissue collagen turnover markers on the one hand and menopausal/estrogen status or age on the other. The higher tissue concentrations of PICP and especially PIIINP in tissue from women with UP compared to controls, suggest an increased collagen breakdown in UP. This pattern differs from that in stress urinary incontinent women without UP, where tissue levels of collagen turnover markers are low, indicating reduced collagen breakdown.     

In vitro synthesis of DNA in nuclei isolated from herpes simplex virus-infected cells, untreated and treated with metabolic inhibitors.

Nuclei isolated from herpes simplex virus (HSV)-infected cells are able to synthesize viral and cellular DNA molecules under in vitro conditions in the presence of the four deoxynucleoside triphosphates, 6 mM Mg²⁺ and 8% (w/v) sucrose. Under similar conditions, nuclei from uninfected cells do not synthesize DNA. Analysis of the in vitro synthesized DNA molecules which were released from the nuclei revealed heterogenous DNA species, mostly of low molecular weight, which had the density of viral DNA. Nuclei were isolated from infected cells treated in vivo with metabolic inhibitors, and the in vitro DNA synthesis was studied. Nuclei from cells treated with inhibitors which affect DNA (distamycin A and camptothecin) did not synthesize DNA in vitro, while nuclei treated with certain other inhibitors (hydroxyurea, cytosine arabinoside, or cordycepin) showed a low DNA synthesis. Analysis of the DNA products revealed that only cellular DNA was synthesized in nuclei of cells treated with hydroxyurea and cytosine arabinoside.