Comparative analysis of the immunomodulatory potential of caprine fetal adnexa derived mesenchymal stem cells.

The caprine mesenchymal stem cells (MSCs) derived from fetal adnexa are highly proliferative. These cells possess tri-lineage differentiation potential and express MSC surface antigens and pluripotency markers with a wound-healing potential. This present study was conducted to compare the immunomodulatory potential of caprine MSCs derived from the fetal adnexa. Mid-gestation caprine uteri (2-3 months) were collected from the abattoir to isolate MSCs from amniotic fluid (cAF), amniotic sac (cAS), Wharton's jelly (cWJ) and cord blood (cCB), which were expanded and characterized at the 3rd passage. These MSCs were then stimulated with inflammatory cytokines (IFN-γ and TNF-α) to assess the percentage of inhibition produced on peripheral blood mononuclear cells (PBMCs) proliferation. The percentage of inhibition on activated PBMCs proliferation produced by cWJ MSCs and cAS MSCs was significantly higher than cCB and cAF MSCs. The relative mRNA expression profile and immunofluorescent localization of different immunomodulatory cytokines and growth factors were conducted upon stimulation. The mRNA expression profile of a set of different cytokines and growth factors in each caprine fetal adnexa MSCs were modulated. Indoleamine 2, 3 dioxygenase appeared to be the major immunomodulator in cWJ, cAF, and cCB MSCs whereas inducible nitric oxide synthase in cAS MSCs. This study suggests that caprine MSCs derived from fetal adnexa display variable immunomodulatory potential, which appears to be modulated by different molecules among sources.

The newly established bovine endometrial gland cell line (BEGC) forms gland acini in vitro and is only IFNτ-responsive (MAPK42/44 activation) after E2 and P4-pre-incubation.

INTRODUCTION: Uterine glands (UG) are crucial for the establishment of ruminant pregnancy and influenced (orchestrated manner) by estrogen (E2), progesterone (P4) and interferon tau (IFNτ). In the study we established a bovine endometrial glandular cell line (BGEC) and tested its functional reactivity (signaling) to IFNτ. METHODS: BGEC was characterized by light microscopy (LM), epithelial markers (ezrin, CK18) [immunofluorescence (IF)/immunohistochemistry (IHC)] and ultrastructure (TEM/SEM) (apical microvilli). In vitro formation of gland acini and transepithelial-electric-resistance (TEER) measurements (EVOM) were done. The expression of mRNA-transcripts (RT-PCR) of steroid receptors (PR, PGRMC1/2, ESR1/2) and the IFNτ-system (IFNAR1/2, IRF1, 2, 9) was checked. BEGC was stimulated with IFNτ (10 ng/ml;1000 ng/ml) (15 min) after steroid pre-treatment [10 pg/ml E2 (two days)/20 ng/ml P4 (two days)]. Activation of MAPK42/44;STAT1 was evaluated (densitometrical Western Blot). RESULTS: BGEC cells expressed epithelial markers and possessed apical microvilli. High TEER-values could be measured (2320-2620 ohm/cm2). The assembled BEGC acini (25 days) were similar to UG in vivo (markers/ultrastructure). All transcripts (steroid receptors/IFNτ-system) could be detected in BEGC (mRNA). MAPK42/44 were significantly activated after E2/P4 pre-treatment and IFNτ stimulation (10 ng/ml) (p < 0.05), whilst 1000 ng/ml IFNτ did not activate MAPK42/44. Neither a STAT1 (by IFNτ) nor an activation (MAPK42/44;STAT1) by IFNτ-only was observed. DISCUSSION: BGEC retains its epithelial phenotype in culture and forms gland acini in vitro thereby confirming its glandular character. Cells were only reactive to (low) IFNτ concentrations when pre-treated with steroids thereby closely resembling implantation physiology in vivo. BEGC can be used as a bovine implantation model to study embryo-maternal communication during early pregnancy in cattle.

A novel technique to measure in vivo uterine suspensory ligament stiffness.

OBJECTIVE: The purpose of this study was to describe a new computer-controlled research apparatus for measuring in vivo uterine ligament force-displacement behavior and stiffness and to present pilot data for women with and without prolapse. STUDY DESIGN: Seventeen women with varying uterine support underwent testing in the operating room (OR) after anesthetic induction. A tripod-mounted computer-controlled linear servoactuator was used to quantify force-displacement behavior of the cervix and supporting ligaments. The servoactuator applied a caudally directed force to a tenaculum at 4 mm/sec velocity until the traction force reached 17.8 N (4 lbs). Cervix location on Pelvic Organ Prolapse Quantification system (POP-Q) in the clinic, in the OR, at rest, and with minimal force (<1.1 N); maximum force (17.8 N) was recorded. Ligament "stiffness" between minimum and maximum force was calculated. RESULTS: The mean ± SD subject age was 54.5 ± 12.7 years; parity was 2.9 ± 1.1; body mass index was 29.0 ± 4.3 kg/m(2), and POP-Q point C was -3.1 ± 3.9 cm. POP-Q point C was correlated most strongly with cervix location at maximum force (r = +0.68; P = .003) and at rest (r = +0.62; P = .009). Associations between cervix location at minimum force (r = +0.46; P = .059) and ligament stiffness (r = -0.44; P = .079) were not statistically significant. Cervix location in the OR with minimal traction lay below the lowest point found on POP-Q for 13 women. CONCLUSION: POP-Q point C was correlated strongly with cervix location at rest and at maximum traction force; however, only 19% of the variation in POP-Q point C location was explained by ligament stiffness. The cervix location in the OR at minimal traction lay below POP-Q point C value in three-fourths of the women.

Drosophila sperm motility in the reproductive tract.

Motile cilia and flagella exhibit many waveforms as outputs of dynein activation sequences on the highly conserved axoneme. Motility change of sperm in the reproductive tract is difficult to study and remains an important area of investigation. Sperm typically execute a sinusoidal waveform. Increased viscosity in the medium induces somewhat unusual arc-line and helical waveforms in some sperm. However, whether the latter two waveforms occur in vivo is not known. Using green fluorescence protein imaging, we show that Drosophila sperm in the uterus move in circular foci via arc-line waves, predominantly in a tail-leading orientation. From the uterus, a small fraction of the sperm enters the seminal receptacle (SR) in parallel formations. After sperm storage and coincident with fertilization of the egg, the sperm exit the SR via head-leading helical waves. Consistent with the observed bidirectional movements, the sperm show the ability to propagate both base-to-tip and tip-to-base flagellar waves. Numerous studies have shown that sperm motility is regulated by intraflagellar calcium concentrations; in particular, the Pkd2 calcium channel has been shown to affect sperm storage. Our analyses here suggest that Pkd2 is required for the sperm to adopt the correct waveform and movement orientation during SR entry. A working model for the sperm's SR entry movement is proposed.

Ultrastructure and estrogen regulation of the lymphatic stomata of ovarian bursa in mice.

The ovarian bursa is a key player in maintaining adaptive ovarian microenvironment for ovulation. The lymphatic stomata are believed to be a major contributor to execute the function of the ovarian bursa, whereas little is known about their ultrastructure and regulation. Here, we examined the ultrastructure of lymphatic stomata in mouse ovarian bursa by scanning electron microscopy and transmission electron microscopy and investigated its regulation by estrogen. We found that the mesothelium on the visceral layer of mouse ovarian bursa was composed of the cuboidal and flattened cells. The lymphatic stomata with round and oval shapes were mainly among the cuboidal cells. The particles, cells, and fluid passed through the stomata and entered into the lymphatic drainage unit composed of connective tissue and lymphatic endothelial cells beneath the stomata. We also used trypan blue as a tracer and found that the absorption of trypan blue through the lymphatic stomata was increased by estrogen that enlarged the average opening area of lymphatic stomata. Furthermore, we detected that there existed estrogen receptors in the nuclei of the mesothelial cells on the visceral ovarian bursa by using immunoelectron microscopy. Taken together, these data suggest that both the absorption and opening area of the lymphatic stomata in mouse ovarian bursa may be influenced by estrogen.

Local expression of cytokine genes in uterine adnexa and endometrium of women with pyoinflammatory adnexal diseases.

Changes in the local expression of IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, TNF-alpha, IFN-gamma- and TGF-beta(2) genes in the uterine adnexa and endometrium were studied in women with pyoinflammatory adnexal diseases. Examination of tissue specimens from the uterine adnexa involved in inflammation revealed a direct correlation in the levels of mRNA production between IL-6 and IL-10 (r=0.93, p<0.1), IL-6 and IL-4 (r=0.96, p<0.01), IL-10 and IL-4 (r=0.91, p<0.01), IL-12 and IFN-gamma (r=0.98, p<0.01). Expression of IL-4 gene increased 5.1-fold (p=0.001), IL-6 2-fold (p=0.007), IL-8 90.2-fold (p=0.009), IL-10 2.9-fold (p=0.008), IL-12 2.3-fold (p=0.3), and TGF-beta(2) gene 10.3-fold (p=0.1). In the endometrium of women with pyoinflammatory adnexal diseases only IL-10 gene expression increased (15.6-fold, p=0.007).

[The interrelation of interhemispheric functional brain asymmetry and the morphofunctional asymmetries of the female reproductive system in in its comparative evolutionary aspect]. / Vzaimosviaz' funktsional'noi mezhpolusharnoi asimmetrii mozga i morfofunktsional'nykh asimmetrii sistemy zhenskoi reproduktsii v sravnitel'no-évoliutsionnom aspekte.

In experiments carried out in female rats it has been established that there is a connection between morphological asymmetry of uterine horns and adnexa and orientation of encephalic interhemispheric asymmetry (EIHA). Unilateral adnexectomy and ablation of one of the uterine horns resulted in modifications of EIHA, which increased during pregnancy in the remaining horn. Comparative analysis of experimental results and the data obtained during examination of pregnant women (such as interconnections of the placentation side with gestational dominant and initial orientation of the lateral phenotype) suggests an important role of asymmetry of the visceral part of female reproductive system in the formation of EIHA in onto- and phylogenesis. Possible mechanisms of self-organization of the polar functional systems are discussed under evolutionary aspect.

Functional and therapeutic aspects of counter current transfer in the female adnexa.

Counter current transfer of many substances, including steroids and peptide hormones, takes place from the veins and lymph vessels to the arterial blood in the female adnexa. The vascular system to the ovaries, tubes, uterus, and vagina is involved, and transfer from one organ to another is possible due to the anatomy and physiology of the vessels. Examples of physiological importance are given: regulation of luteolysis, unilateral effects on the functions of the ovary and tube. The transfer may be used as a therapeutic tool in order to obtain high local concentrations of drugs. It is postulated that a hormone releasing intra-uterine device (IUD) may have a local effect on the tubes and ovaries. It is also postulated that intrauterine instillation of cytotoxic drugs may be used before surgery in order to obtain a high concentration in local arterial blood when compared to the concentration in peripheral blood, thus obtaining a favourable therapeutic index between local beneficial and general toxic effects. The treatment may also create a high concentration of the drug in the local lymph glands and thus have a direct therapeutic effect on eventual metastasis.

Functional properties of afferent fibers supplying reproductive and other pelvic organs in pelvic nerve of female rat.

1. Electrophysiological techniques were used to characterize responses of afferent fibers in pelvic nerve of adult, virgin female rats to mechanical or chemical stimulation of internal reproductive organs and to mechanical stimulation of other pelvic organs. 2. In an in vivo barbiturate-anesthetized preparation, pelvic nerve afferent fibers responded to a wide variety of mechanical stimulation applied to restricted regions of the vaginal canal, caudal uterus (body and cervix), bladder, ureter, colon, or anus. 3. Single-fiber mechanoreceptive fields were invariably confined to a single organ. Notably, responses could be evoked not only by gentle stimulation of the unit's receptive field directly on the organ itself, but also by stimulating the field indirectly with intense stimulation through the appropriate part of a contiguous organ. This innervation feature is consistent with the separability of pelvic organ functions under innocuous conditions but their confusion under noxious ones. 4. Receptive fields on the reproductive organs extended from the caudal edge of the vagina to the uterine body (including the cervix) but were most often located in the fornix (vaginocervical junction). Most units had no or low levels of spontaneous activity. Their responses to mechanical stimuli were usually slowly or moderately adapting and time-locked to the stimulus. 5. Fibers with vaginal receptive fields (including the fornix) responded best either to vaginal distension with a balloon or, more often, to a probe moving along the internal vaginal surface in a direction toward the cervix. They were observed most frequently during the proestrus stage of the rat's estrous cycle. These fibers, therefore, seem particularly suited for relaying information about stimuli that occur during mating. 6. Fibers with receptive fields on the uterine cervix and body responded best to static pressure and were observed less frequently than those with vaginal fields, regardless of estrous stage. They were, however, sensitized by hypoxia. In addition, irritation of the uterus increased the probability of observing them. These fibers, therefore, may exert their primary function during reproductive conditions different from those of virgin rats, such as parturition. 7. Response activity of most of the mechanoreceptive afferent fibers supplying reproductive organs increased as the stimulus intensity increased into the noxious range; i.e., into a range in which the stimulus momentarily produced ischemia at the stimulus site. In addition, in an in vitro preparation, pelvic nerve fibers responded in a dose-dependent manner to injections through the uterine artery of bradykinin (BRAD) as well as to other algesic chemicals, 5-hydroxytryptamine (5-HT) and KCl.(ABSTRACT TRUNCATED AT 400 WORDS)

Counter current transfer in the female adnex.

The utero-ovarian veins and lymph vessels are intimately connected with the ovarian artery in the human female and in domestic animals, with the exception of the horse and the human female. A direct, local exchange of molecules from veins and lymph vessels to arteries (counter current transfer) has been documented for this anatomic structure. Countercurrent transfer of certain inert gases (133xenon, 85krypton), of prostaglandins (PGF2 alpha), of steroid hormones (e.g. progesterone, estradiol, testosterone), and of small peptide hormones (oxytocin, relaxin) has been shown to occur in laboratory and domestic animals as well as in the human female. The transfer of the inert gases takes place within seconds. The transfer of steroid hormones and peptides is detectable within minutes while the transfer of PGF2 alpha is delayed for 20 minutes. Red blood cells or albumin are not transferred. The existence of the local transfer is postulated to be of importance for: 1) the pregnancy/non-pregnancy signal from the uterus and tube to the ovary. The signal may be a combination of a luteotrophic signal from the embryo and lack of a "non-pregnant" luteolytic signal from the endometrium, the latter probably being PGF2 alpha in some species; 2) the unilateral influence of the ovarian hormones on the function of the ovarian, tubal, and possibly uterine tissues. An active corpus luteum may create in a mono-ovulatory animal a higher progesterone level in arterial blood supplying the ipsilateral tube and ovarian interstitial tissue than on equivalent contralateral organs.

Role of mesovarium and mesosalpinx in counter current exchange of hormones.

On the base of own studies with counter current transfer of steroid hormones and PGF2 alpha and the data taken from the literature it is suggested that two parts of broad ligament of the uterus i.e. mesovarium and mesosalpinx are not only morphological structures keeping the ovary, oviduct and ovarian vasculature, but that they may take part in hormonal regulation of the ovarian function.

Characteristics of the electromyogram recorded from the mesometrium of the pregnant ewe from 106 days' gestation to delivery: similarities with and differences from the electromyogram obtained from the myometrium.

Electromyographic recordings from 12 pregnant sheep were analyzed in four gestational age groups (106 to 115, 116 to 125, 126 to 135, and 136 to 145 days) and by the day of delivery. Mesometrial electromyographic activity was present in all animals studied. Contracture activity was synchronous in both mesometrium and myometrium. The frequency distribution of electromyographic events of different duration in the mesometrium was qualitatively identical to that recorded from the myometrium. The frequency of events less than 180 seconds was greater in the myometrium, while no difference was observed in contracture frequency. Mean contracture duration in myometrium and mesometrium shortened as gestation progressed (p less than 0.01), and was of significantly shorter duration on the day of delivery when compared with all gestational age intervals (p less than 0.05). Except at 135 to 145 days' gestation and at the day of delivery, myometrial contractures were of significantly longer duration than mesometrial contractures. The presence of the endometrium and fetal membranes in the pregnant ewe may stimulate the myometrium, and account for the difference observed. In vivo recording of mesometrial electromyographic activity may provide an opportunity to study the paracrine regulation of myometrial activity.

An analysis of the characteristics of the electromyogram recorded from the mesometrium in the ovariectomized nonpregnant ewe and its similarities with and differences from the electromyogram obtained from the myometrium: the effect of estrogens.

Mesometrial and myometrial electromyograms have been recorded from ovariectomized nonpregnant ewes. After infusion with saline solution, the frequency of electromyographic events lasting less than 180 seconds in the myometrium was not different from the frequency of their occurrence in the mesometrium. During infusion of estradiol at 100 micrograms/24 hours, the frequency of events less than 180 seconds increased significantly in the myometrium but was unchanged in the mesometrium. Contracture activity (electromyographic events lasting more than 180 seconds) was significantly greater in the mesometrium than in the myometrium before estradiol administration. During estradiol infusion, contracture activity remained unchanged. These findings suggest that the mesometrial muscle may be a useful model to study the effects of various stimulators and inhibitors of uterine muscle function in the absence of stretch and local paracrine effects that occur in the uterus itself.

Physiological study of the human mesotubarium ovarica.

The human mesotubarium ovarica has been shown to contain bundles of smooth muscle cells. Its contractility at various phases of the menstrual cycle was studied in vitro in 51 tissue strips obtained at laparotomy. The strips obtained at the ovulatory phase showed the greatest spontaneous contractile activity as well as the greatest contractile response to norepinephrine, acetylcholine, and prostaglandin F2 alpha. This corresponds to the increased contractility of the ovary at this phase of the cycle. It is suggested that these contractile actions may, under the influence of chemical transmitters, play a role in ovum pick-up by the tubal fimbria at ovulation.