Lifestyle intervention up-regulates gene and protein levels of molecules involved in insulin signaling in the endometrium of overweight/obese women with polycystic ovary syndrome.

STUDY QUESTION: Does lifestyle intervention aiming at weight loss influence endometrial insulin signaling in overweight/obese women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Lifestyle intervention up-regulates, both at the mRNA and protein levels, components of insulin signaling in the endometrium of overweight/obese PCOS women, in relation to an improved menstrual pattern. WHAT IS KNOWN ALREADY: PCOS is a multifactorial endocrine disorder diagnosed by two of the following three criteria: chronic anovulation, hyperandrogenism and polycystic ovaries. Many women with PCOS also have insulin resistance and obesity. The syndrome is furthermore associated with endometrial cancer and possible alterations in endometrial function and receptivity. STUDY DESIGN, SIZE, DURATION: This study assessed the effects of a combined diet and exercise lifestyle intervention for 3 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: A group of 20 overweight/obese PCOS women with anovulation, hyperandrogenism and polycystic ovaries were subjected to a combined diet and exercise program for 3 months. Ten body mass index (BMI)-matched regularly menstruating overweight/obese controls, nine normal-weight PCOS women and ten normal-weight controls were also included in the study. In an academic clinical setting, women were examined in mid-follicular phase for endocrine assessment and determination of endometrial levels of mRNA and immunohistochemical staining of insulin signaling molecules (the insulin receptor, insulin receptor substrate-1 (IRS1) and glucose transporter (GLUT) 1 and 4). MAIN RESULTS AND THE ROLE OF CHANCE: Women with PCOS exhibited lower levels of IRS1 (P < 0.01) and GLUT4 (P < 0.01) mRNA in their proliferative endometrium than BMI-matched controls. After lifestyle intervention, weight loss averaged 4.7% and the menstrual pattern improved in 65% of the overweight/obese women with PCOS. Levels of IRS1 (P < 0.01) and GLUT1 (P < 0.05) mRNA were significantly up-regulated in the endometrium of those women with improved menstrual function, as were the protein expression levels of pY612IRS1 (the activated IRS1 form, P < 0.05), pS312IRS1 (the inhibitory form of IRS1, P < 0.05) and GLUT1 (P < 0.05). Improvement in the menstrual function of women in the obese/overweight group following the lifestyle intervention was positively correlated with the increase in the endometrial level of IRS1 mRNA (r = 0.63, P < 0.01) and negatively correlated with the change in BMI (r = -0.50, P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The number of women in each group was limited, although the power calculation indicated that the number of patients subjected to the lifestyle intervention was sufficient. WIDER IMPLICATIONS OF THE FINDINGS: We propose that up-regulation of endometrial IRS1 and GLUT1 in overweight/obese women with PCOS following lifestyle intervention improves the glucose homeostasis and thereby restores the functioning of the endometrium in these women. STUDY FUNDING/COMPETING INTEREST(S): This study was supported financially by the Swedish Research Council (A.L.H., 20324), Karolinska Institutet and the Stockholm County Council. None of the authors has any conflict of interest to declare.

Endogenous thrombin potential is higher during the luteal phase than during the follicular phase of a normal menstrual cycle.

STUDY QUESTION: Do thrombin generation and haemostatic parameters differ during the two phases of the menstrual cycle? SUMMARY ANSWER: Total thrombin concentration is higher during the luteal phase compared with the follicular phase of the menstrual cycle. WHAT IS KNOWN ALREADY: The coagulation cascade is affected by many variables, such as fluctuations in the levels of sex hormones. The studies on the variations in haemostatic parameters during the menstrual cycle have produced diverse results. STUDY DESIGN, SIZE, DURATION: Thrombin generation and selected haemostatic parameters (fibrinogen, factor II, factor VII, factor VIII, factor X, von Willebrand factor, antithrombin and D-dimer) were measured during the two phases of a normal menstrual cycle in 102 healthy women not taking any form of hormone medication. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study cohort consisted of 102 healthy women with regular menstrual cycles. Thrombin generation was measured by the calibrated automated thrombogram method. Progesterone and sex hormone-binding globulin were measured by chemiluminescence enzyme immunoassays. Estradiol was measured by a sensitive radioimmunoassay. Fibrinogen was measured by a clotting method, antithrombin was measured by a chromogenic method and factor II, factor VII, factor VIII, factor X, von Willebrand factor and D-dimer were measured by photometric methods. MAIN RESULTS AND THE ROLE OF CHANCE: It was shown that the total amount of generated thrombin (Endogenous Thrombin Potential) was significantly higher during the luteal compared with the follicular phase (P = 0.027). Factor X was significantly higher during the follicular phase (P = 0.028). Progesterone exhibited significant associations (measured by the least squares regression analysis) with fibrinogen and factor X during the follicular phase (P = 0.043 and P = 0.033, respectively) and with factors II and VII during the luteal phase (P = 0.034 and P = 0.024, respectively). The validity of the results from the regression analysis was further confirmed by performing correlation analyses (Pearson correlation matrix) for haemostatic markers for the luteal and follicular phases (accepted correlation level >0.8). LIMITATIONS, REASONS FOR CAUTION: The wide confidence interval for the differences in endogenous thrombin potential during the two phases could imply that the size of the cohort may not be sufficient to fully evaluate the biological variations. Additionally, the haemostatic markers were not shown to have significant associations with thrombin generation, suggesting that the increased thrombin concentration during the luteal phase would be mediated by another mechanism, as yet unidentified. WIDER IMPLICATIONS OF THE FINDINGS: The associations between progesterone and the haemostatic markers, as shown for both phases of the menstrual cycle, suggest a previously unknown or undefined yet potentially significant role for progesterone in the coagulation system. However, it has been shown that the use of progestogen-only preparations does not affect the coagulation system, which is partly the reason why they are considered safe for women with thrombophilia or previous thrombotic event. Further studies are required in order to demonstrate whether our results can be extrapolated for synthetic progestins, which might have significant implication on the indications for their use.

Effects of oral and vaginal administration of levonorgestrel emergency contraception on markers of endometrial receptivity.

BACKGROUND: The standard regimen of the levonorgestrel-only pill (1.5 mg either in a single dose or in a dose of 0.75 mg twice, 12 h apart) administered orally for emergency contraception (EC) has been shown to have no effect on endometrial development and markers of endometrial receptivity. We aimed to explore whether repeated oral and single vaginal administration of levonorgestrel affect the endometrium and thus potentially increase the EC efficacy, compared with the standard regimen. METHODS: Endometrial biopsies were taken from non-smoking, healthy women with proven fertility on cycle days LH + 6 to LH + 8 in control and levonorgestrel treatment cycles (each woman serving as her own control). Levonorgestrel was administered either orally (0.75 mg x 4, at 24 h intervals on LH + 1 to LH + 4; n = 8) or vaginally (a single dose of 1.5 mg on LH + 2; n = 7). Immunohistochemistry and real-time RT-PCR was performed to compare the levels of protein and mRNA for sex steroid receptors, interleukin-1beta, leukaemia inhibitory factor (LIF), vascular endothelial growth factor, cyclooxygenase-2, tumour necrosis factor-alpha, integrin alpha(v)beta(3) and mucin 1 in endometrial cells. RESULTS: Following the repeated oral treatment, the immunoreactivity of both progesterone receptor (PR)-A and PR-B declined in glandular epithelium (P = 0.03 and P = 0.02, respectively), whereas stromal immunoreactivity and mRNA expression of LIF increased compared with control (P < 0.001 and P = 0.03, respectively). However, vaginal levonorgestrel did not cause any significant endometrial changes. CONCLUSIONS: The two regimens of levonorgestrel caused either only minor or no alterations in markers of endometrial receptivity. New agents targeting the endometrial development should be explored in order to increase EC efficacy.

Different secretion patterns of matrix metalloproteinases and IL-8 and effect of corticotropin-releasing hormone in preterm and term cervical fibroblasts.

The aims of the present study were to compare the levels of mRNA and protein expression of matrix metalloproteinase (MMP)-1, -3, -8 and -9 in human cervical tissue in preterm and term labor as well as not in labor and to determine if corticotropin-releasing hormone (CRH) has an effect on MMP-1, -3 and interleukin (IL)-8 secretion in both preterm and term cervical fibroblasts. Cervical biopsies were taken from 60 women: 18 at preterm labor, 7 at preterm not in labor, 18 at term labor and 17 at term not in labor. ELISA and Immulite were used for protein and real-time RT-PCR for mRNA analysis. Cervical fibroblast cultures were incubated for 18 h with different CRH concentrations (10(-13)-10(-6) M). The mRNA expression of MMP-1, -3 and -9 was higher in laboring groups compared with term not in labor. Protein levels of MMP-8 and -9 were higher in term in labor group compared with non-laboring groups. There were no significant differences in mRNA and protein expression between the preterm and respective term control groups. CRH significantly increased secretion of IL-8 in preterm and term cervical fibroblasts compared with controls. The secretion of IL-8 and MMP-1 was significantly higher and MMP-3 secretion lower in preterm cervical fibroblasts. In conclusion, cervical ripening at preterm seems to be a similar inflammatory process as at term with CRH involved. However, preterm and term cervical fibroblasts might have different phenotypes based on different secretion patterns of IL-8, MMP-1 and MMP-3.

Effects of an antiandrogenic oral contraceptive on appetite and eating behavior in bulimic women.

High androgen levels in women with bulimia nervosa may promote bulimic behavior. The aim of the present study was to investigate the effects of an antiandrogenic oral contraceptive (OC) on appetite and eating behavior in women with bulimia nervosa compared to healthy controls. Twenty-one women with bulimia nervosa and 17 healthy controls matched for age and body mass index participated in the study. Basal and meal-related appetite and secretions of the satiety peptide cholecystokinin (CCK) and the appetite-stimulating peptide ghrelin were studied before and after 3 months of treatment with an antiandrogenic OC (30 microg ethinyl estradiol combined with 3 mg drospirenone). Bulimic behavior was evaluated in relation to changes in hormone levels. Before treatment, bulimic women had higher frequency of menstrual disturbances, acne and hirsutism and higher levels of testosterone but lower meal-related CCK secretion than controls. OC treatment reduced meal-related hunger and gastric distention in bulimics. CCK secretion in response to the meal was unchanged in bulimic women but decreased in the controls. Ghrelin secretion was comparable between groups and did not change in response to OC treatment. The treatment improved bulimic behavior in relation to a decline in testosterone levels in the entire group. Our results support the suggestion that androgens play a role in bulimic behavior. Treatment with an antiandrogenic OC may serve as a new strategy for treatment of bulimia nervosa and particularly in those patients with hyperandrogenic symptoms.

Nitric oxide induces endometrial secretion at implantation time.

BACKGROUND: Uterine cervical secretory cells receive a sympathetic cholinergic secretomotor innervation. Glandular nitric oxide (NO) production has been proposed to be a prerequisite for muscarine-induced carbohydrate secretion from endometrial glands and cervical glands at ovulation time and from the seminal vesicle glands. Nitric oxide has also been suggested to have a significant role in the process of implantation and early pregnancy in the mouse, a process, which has also been compared with an inflammatory response. METHODS: The carbohydrate secretion from everted guinea pig uterine horns placed in organ baths was estimated. Polymerase chain reaction was performed in order to identify the isoforms of nitric oxide synthase (NOS). results. Carbamylcholine chloride (Carbachol) induced carbohydrate secretion of the endometrium, whereas L-NNA and L-NAME inhibited the Carbachol-induced secretion. The isomer D-NAME had no effect on Carbachol-induced secretion. The NO donor GTN induced carbohydrate secretion of the endometrium. The addition of the nitrergic inhibitor of soluble guanylyl cyclase (sGC) ODQ to Carbachol and to the NO donor GTN gave a reduced response. No synergism was seen when the sGC stimulator YC-1 was applied together with Carbachol. Three isoforms of NOS - endothelial NOS (eNOS), cytokine-inducible NOS (iNOS), and neuronal (nNOS) - were identified at implantation time and may take place in the endometrial cell. CONCLUSIONS: The results of this study suggest that glandular NO production is a prerequisite for the autonomic nervous modulation of endometrial secretion in the guinea pig and that NO may play a role in the implantation time.

Impaired cholecystokinin secretion and disturbed appetite regulation in women with polycystic ovary syndrome.

Increased amount of abdominal fat and obesity are common in polycystic ovary syndrome (PCOS). A higher prevalence of bulimia nervosa and greater cravings for sweets have also been reported in these patients. The present study aimed to compare meal-related appetite and secretion of the 'satiety peptide' cholecystokinin (CCK) and glucose regulatory hormones in PCOS women and controls. Sixteen pairs of women with PCOS and controls matched for age and body mass index participated in the study. After an overnight fast, blood samples were collected during ingestion of a standardized meal. We determined basal and postprandial blood levels of CCK, insulin, C-peptide, glucagon, cortisol, growth hormone and glucose. Self-ratings of appetite were assessed by a visual analog scale. PCOS women had a significantly lower meal-related CCK response (p < 0.05) with no association with satiety, as in the controls (r = 0.64). There was a tendency to higher ratings of craving for sweets in PCOS women (p = 0.07) but no correlation with insulin, as in the controls (r = 0.50). Within the PCOS group, ratings of craving for sweets were inversely related to testosterone (r = - 0.60) and the CCK response was positively correlated with levels of free testosterone (r = 0.50). We conclude that women with PCOS have reduced postprandial CCK secretion and deranged appetite regulation associated with increased levels of testosterone. Impaired CCK secretion may play a role in the greater frequency of binge eating and overweight in women with PCOS.

A novel mutation (N233K) in the transactivating domain and the N756S mutation in the ligand binding domain of the androgen receptor gene are associated with male infertility.

OBJECTIVE: Resistance to androgens has been suggested as a possible cause of male infertility. This hypothesis is based mainly on binding studies in genital skin fibroblasts but the molecular evidence is sparse. DESIGN: Molecular studies of the androgen receptor gene were performed in 10 azoo- or oligozoospermic men, presenting with clinical signs of low androgen activity-poor virilization and high serum LH despite elevated testosterone levels, but without genital malformations. PATIENTS: Ten men with serum LH >10 IU/l and testosterone >30 nmol/l as well as a low sperm concentration < 20 x 106/ml. MEASUREMENTS: Genomic DNA was prepared from peripheral leucocytes and PCR-amplification of the coding region of androgen receptor was performed, followed by direct sequencing. Identified mutations were reconstructed by site-directed mutagenesis and the functional properties of the mutants were analysed, using transient expression in COS-1 cells and subsequent transactivation assays. Hormone binding assays were performed in genital skin fibroblasts from the patients. RESULTS: Two of the 10 men were shown to have a mutation in the androgen receptor gene. Subject 1, who presented with azoospermia, serum testosterone (T) 50 nmol/l and LH 20 IU/l, had a mutation in exon 1, changing amino acid asparagine 233 to lysine (N233K). In fibroblasts cultured from genital skin, the receptor affinity for 5alpha-dihydrotestosterone (DHT) was normal as compared to healthy controls, but the receptor-hormone complex was thermolabile at 42 degrees C. Subject 2 exhibited severe oligozoospermia and a similar endocrine pattern (T = 50 nmol/l and LH = 25 IU/l). He had a mutation in exon 5 changing asparagine 756 to serine (N756S). The affinity for DHT in cultured genital fibroblasts from this patient was reduced. Transactivation was abnormal for both mutants, N233K reaching 46% and N756S 38% of wild type activity when stimulated with 10 nmol/l DHT. CONCLUSIONS: Androgen receptor mutations may affect sperm production without resulting in genital malformations. Thus, in infertile men with a clinical presentation of poor androgen activity and an endocrine profile compatible with androgen resistance, mutations in the androgen receptor should be taken into consideration.

Response to treatment in patients with partial androgen insensitivity due to mutations in the DNA-binding domain of the androgen receptor.

The androgen insensitivity syndrome is a disorder caused by deficient function of the androgen receptor, characterized by varying degrees of undermasculinization in karyotypic males. We have identified four mutations in the androgen receptor gene, in the region encoding the DNA-binding domain of the protein. Two mutations, R607X and R615G, were found in patients with complete insensitivity to androgens, whereas the other two, S578T and A596T, were found in patients with partial insensitivity. The functional consequences of the three missense mutations were assayed in vitro after transient expression of the receptors in COS cells. All mutants showed normal androgen binding but abnormal abilities to stimulate transcription of an androgen-responsive reporter gene. R615G abolished transactivation whereas S578T and A596T were partially malfunctional. The function of A596T, but not of S578T, was normalized at high androgen concentrations in vitro, reflecting the in vivo situation. Thus, patients with specific mutations in the DNA-binding domain of the androgen receptor may benefit from androgen treatment.

Collagen metabolism markers as a reflection of bone and soft tissue turnover during the menstrual cycle and oral contraceptive use.

Two different groups of women, 23 healthy young adults and 13 women with chronic posterior pelvic pain, were studied before and during use of oral contraceptives (OC). Collagen metabolism markers-here, the amino-terminal propeptide of type I procollagen, the carboxy-terminal telopeptide of type I collagen, and the amino-terminal of procollagen type III-as well as hormones and other endocrine factors indicating the balance between androgen expression/anabolism and catabolism of the subjects (testosterone, sex-hormone binding globulin, and insulin-like growth factor I were measured. Type I procollagen, the carboxy-terminal telopeptide of type I collagen, and the amino-terminal of procollagen type III were all significantly decreased during OC use. These findings implicate OC use-induced changes in collagen type I and III turnover. A shift in the anabolic/catabolic balance was also recorded indicating a less anabolic situation during OC use.

Human cervical ripening, an inflammatory process mediated by cytokines.

An extensive remodelling process, referred to as cervical ripening, takes place in the cervical tissue during pregnancy and labour. It is recognized as softening and dilation of the cervical canal, and starts as a slow process during pregnancy, becoming rapid close to partum. In this study we focus on cytokines as possible mediators of this final remodelling. mRNA levels for interleukin (IL)-8, IL-6 and granulocyte colony-stimulating factor (G-CSF) were upregulated in the ripe postpartum cervical tissue (n = 8) compared to the unripe state (n = 9). Likewise, released cytokine concentrations increased from non-pregnant (n = 11) to the term-pregnant group (n = 13) with a further increase at partum (n = 16). IL-8 concentrations increased 4-fold from non-pregnant to term-pregnant (P<0.01), and a further 10-fold to postpartum state (P<0.0001). Concentrations of IL-6 and G-CSF were similarly increased. Specific IL-8 immunostaining was identified in the epithelia of pregnant cervical tissue (n = 7) and was most pronounced in the epithelia and stroma of postpartum tissue (n = 4). In conclusion, IL-8, IL-6 and G-CSF increase in the human cervix during the ripening process, indicating their important role in the cervical remodelling. These data demonstrate that cervical ripening is similar to an inflammatory process.

Cervical ripening after treatment with prostaglandin E2 or antiprogestin (RU486). Possible mechanisms in relation to gonadal steroids.

OBJECTIVE: To compare the mechanisms for cervical ripening after treatment with prostaglandin E2 or antiprogestin (RU486) to spontaneous cervical ripening, with focus on gonadal steroid receptors. STUDY DESIGN: Cervical biopsies were obtained from postpartal women after treatment with prostaglandin E2 (n=10), or antiprogestin (n=5). Postpartal women after spontaneous cervical ripening (n=10) served as controls. Levels of estrogen and progesterone receptors, their mRNAs, insulin-like growth factor I mRNA and serum estradiol and progesterone were quantitated. The collagen concentration and solubility by pepsin were determined. Statistical tests used were Kruskal-Wallis and Mann-Whitney U test. RESULTS: After prostaglandin E2 treatment the collagen concentration was higher (P<0.05) as compared to spontaneous ripening. After antiprogestin treatment the estrogen receptor concentration was higher (P<0.05) in comparison to spontaneous ripening. CONCLUSION: The elevated estrogen receptor concentration after antiprogestin treatment, in contrast to spontaneous ripening, and prostaglandin E2 treatment, indicates a that a receptor-mediated progesterone withdrawal does not explain the events behind spontaneous cervical ripening at parturition.

Functional characterisation of mutations in the ligand-binding domain of the androgen receptor gene in patients with androgen insensitivity syndrome.

Five mutations in the ligand-binding domain of the androgen receptor gene were identified in patients with complete (A765T, C784Y, R831X and M895T) or partial (R840G) androgen insensitivity. A765T and R831X have been reported previously whereas the other three mutations are novel. Receptors carrying these mutations were transiently expressed in COS-1 cells, and androgen binding and capacity to transactivate an androgen-responsive reporter gene were assayed. C784Y led to abolished androgen binding and transactivating capacity, R840G and M895T showed reduced specific binding and partial transactivation. The in vitro functions of the R840G and M895T mutants were improved with supraphysiological concentrations of steroid.

Reduced serum cholecystokinin and increase in body fat during oral contraception.

This investigation was undertaken to explore a possible role of the "satiety peptide" cholecystokinin and some other gastrointestinal hormones for changes in appetite and weight during oral contraception. Ten young healthy women attending a youth health care center for contraceptive counseling volunteered for the study. A standardized meal test was used for recordings of appetite and gastrointestinal hormone response before and after 5 months of treatment with a monophasic combined oral contraceptive. Body fat was calculated from measurements of skin-fold thickness. Oral contraceptives caused a suppression of basal levels of serum cholecystokinin, which was correlated to an increase in body fat. Meal-related response of cholecystokinin and appetite were not affected. Serum levels of gastrin and insulin were also unchanged, whereas triglycerides and postprandial glucose levels were elevated. The results suggest a role of cholecystokinin in regulation of body composition. Cholecystokinin stimulates the release of insulin and stimulates lipolysis in adipose tissue. Reduced cholecystokinin levels may, therefore, be related to mild impairment of glucose tolerance and promote body fat storage during oral contraception.

Serum levels of relaxin during the menstrual cycle and oral contraceptive use.

Serum relaxin levels were analysed in 12 healthy women every other day during the menstrual cycle and during a second cycle on oral contraceptives. Relaxin levels in 7 women with posterior pelvic and lumbar pain were also measured. Relaxin was detected during both the follicular and luteal phases of the menstrual cycle in some of the healthy women. Serum levels were further increased during the use of oral contraceptives. Oestradiol levels in the untreated women correlated to the relaxin levels. Women with posterior pelvic and lumbar pain had higher relaxin levels than did healthy women, a finding that needs to be further explored. Our data indicate the existence of sources for relaxin production other than the corpus luteum in the non-pregnant woman. Endogenous and exogenous oestrogens may stimulate the production of relaxin.

PIP: In Sweden, clinicians took blood samples every other day during one menstrual cycle from 12 healthy women aged 19-42 taking no medication and during a second menstrual cycle from 9 of these women while using a combined oral contraceptive (OC) (150 mcg desogestrel + 30 mcg ethinyl estradiol). They also took samples from a second group of 7 women, 26-42 years old, with a long history of posterior pelvic pains and symptoms in the lower lumbar region during 2 consecutive menstrual cycles. The 7 women did not use OCs but did take paracetamol. The researchers aimed to measure the serum relaxin levels in all the women to determine whether OCs inhibit relaxin secretion and to determine whether changes in relaxin secretion causes posterior pelvic pain. 7 of the 12 healthy women had detectable levels of relaxin during either the follicular or luteal phases or both phases of the menstrual cycle. Relaxin secreted during both phases suggests that the corpus luteum is not the only source of relaxin in nonpregnant women, as commonly believed. As estradiol levels increased so did the relaxin levels (r = 0.44; p 0.05). During OC use, 6 of the 9 women had detectable levels of relaxin. The mean relaxin levels were higher during OC use than during the non-OC cycle (range, 20-255 vs. 20-135 ng/l), except during days 26-32. In fact, the number of relaxin measurements above the detection limit (20 ng/l) during OC use (i.e., anovulation) was much higher than during the normal ovulatory cycle (40 vs. 20; p 0.001). It appears that relaxin secretion does not depend on ovulation. The positive correlation between estradiol and relaxin levels and the increased relaxin levels during OC use suggests that estradiol and ethinyl estradiol regulate relaxin synthesis. All 7 women with posterior pelvic pain had detectable serum relaxin levels. They had detectable relaxin levels significantly more often than did healthy women (p 0.001). Further research is needed to understand the pathophysiological role of relaxin in lower back pain.

Studies on the constancy of transudated and locally produced proteins in human seminal plasma.

An overall constancy in the total protein profile of human seminal plasma (HSP) as determined by gel filtration chromatography and high-resolution electrophoresis was found in six healthy volunteers. Thirteen different proteins were identified by double immunodiffusion in five individual HSP samples each previously subjected to gel filtration. It was also found that comparatively large amounts of yet unidentified low-molecular-weight (less than 12,000) compounds occurred in all HSP samples. Of eight specific proteins in consecutive samples collected from one individual, large intra-individual variations were found in some of the proteins. The largest variations (about 100%), for both concentration and total amount, were noted for alpha 1-antitrypsin, transferrin, IgA, and secretory IgA. Albumin and lactoferrin were rather stable and varied less than 20% between samples. It is suggested that HSP-albumin may be used as a reliable marker of transudation of serum proteins to the genital tract. Likewise, lactoferrin could be used as a marker for the secretion of seminal vesicle proteins, since it reflects the functional status of these glands.

Chloroplasts as functional organelles in animal tissues.

The marine gastropod molluscs Tridachia crispata, Tridachiella diomedea, and Placobranchus ianthobapsus (Sacoglossa, Opisthobranchia) possess free functional chloroplasts within the cells of the digestive diverticula, as determined by observations on ultrastructure, pigment analyses, and experiments on photosynthetic capacity. In the light, the chloroplasts incorporate H(14)CO(3) (-)in situ. Reduced radiocarbon is translocated to various chloroplast-free tissues in the animals. The slugs feed on siphonaceous algae from which the chloroplasts are derived. Pigments from the slugs and from known siphonaceous algae, when separated chromatographically and compared, showed similar components. Absorption spectra of extracts of slugs and algae were very similar. The larvae of the slugs are pigment-free up to the post-veliger stage, suggesting that chloroplasts are acquired de novo. with each new generation.