Expressions of MMP-2,-9,TIMP-1,-2,-3 mRNA in Rat Uterus during Estrous Cycle

Zymography and in situ hybridization were used to investigate matrixmetalloproteinase -2, -9 (MMP -2, MMP-9) activities and expressions of MMP -2, -9 and TIMP1, -2, -3 (tissue inhibitors of matrix metallo-proteinases) mRNA in the rat uterus during estrouscycle. The relative activity was semiquanted by using densitometric analysis. The MMP-2(67 kDa) activity in every stage during estrpus cycle was detected by zymography. MMP-2activity was highest at proestrus; higher at estrus and metaestrus; lowest at diestrus. Throughin situ hybridization, MMP -2, -9, TIMP -1~ -3 mRNA mainly in hasal stroma cells of uterineendometrium were detected. The positive signals of MMP -2 and -9 mRNAs in hasal stromacells were shown stronger at proestrus, estrus and metaestrus while they showed the weakest atdiestrus. The expression of MMP -2 mRNA coincided with MMP -2 activity change. MMP-2and -9 mRNAs were also highly expressed in uterine circular muscle at estrus. Weak signals ofMMP -9 mRNA were detected in uterine luminal and glandular epithelial cells at estrus.TIMP -1 mRNA in hasal stroma cells was shown as the strongest expression at estrus andmetaestrus; stronger at proestrus and the weakest at diestrus. TIMP-2 mRNA in basal stromacells was stronger at estrus and diestrus; weaker at proestrus and metaestrus. TIMP -1 and -2mRNAs were also highly expressed in uterine luminal and glandular epithelial cells at estrus.TIMP -3 mRNA in hasal stroma cells revealed the strongest expression at estrus; stronger atdiestrus and metaestrus and showed the weakest at proestrus. The mRNA was also highlyexpressed in uterine circular muscle at estrus. In short, our present results provide evidencethat MMP -2, -9 and TIMP -1~ -3 were involved in rat uterine endometrium reconstructionduring estrous cycle.

Evaluation of the Hypothalamic Kisspeptin System throughout the Estrous Cycle in Gilts

Kisspeptin has been demonstrated to affect reproductive cyclicity and the attainment of puberty in multiple species, presumably through its actions on gonadotropin releasing hormone and luteinizing hormone. Kisspeptin administration causes increased plasma concentrations of LH in pigs, sheep, and rats. The objective of this experiment was to evaluate changes in the hypothalamic kisspeptin system throughout the estrous cycle in gilts. Estrus was synchronized in forty crossbred gilts (191 d, 121 kg) and estrus detection was performed by exposing gilts to a mature boar. The first day gilts stood immobile was denoted d 1 of the estrous cycle. Blood samples were collected via jugular venipuncture on d 1, 4, 7, 9, 14, 16, and 19 of the estrous cycle. Ten animals were slaughtered on d 1, 9, 14, and 21 of the estrous cycle when medial basal hypothalami, anterior pituitary glands, and blood were collected. Relative expression of hypothalamic kisspeptin (KISS1), kisspeptin receptor (KISS1R), estrogen receptors-a, anterior pituitary gland GnRH receptor, β-actin, and GAPDH was determined using real-time reverse transcriptase PCR. Fold changes in relative expression were determined using the Relative Expression Software Tool. Relative expression of KISS1 was increased (P = 0.006) 3.2 fold on d 1 versus d 21 and 2.3 fold (P = 0.003) on d 9 versus d 21 of the estrous cycle, but was not different (P > 0.05) among the remaining days of the estrous cycle. Relative expression of estrogen receptor-b was decreased (P = 0.05) 0.8 fold on d 9 versus d 21 and (P = 0.005) 0.7 fold on d 14 versus d 21, but was not different (P > 0.05) among the remaining days. Relative expression of anterior pituitary gland GnRH receptor was increased (P < 0.01) on d 1 and 21 versus d 9 and 14. These data support the notion that medial basal hypothalamic expression of KISS1 changes throughout the estrous cycle and may influence reproductive cyclicity in the gilt.

Expression of bone morphogenetic protein 2,4,and related components of the BMP signaling pathway in the mouse uterus during the estrous cycle

The objective was to investigate the expression of bone morphogenetic protein （BMP） family members in the mouse uterus during the estrous cycle by real-time polymerase chain reaction （PCR） and immunohistochemistry. Uterine samples from Swiss ICR mice were collected and dissected free of surrounding tissue. One uterine horn was snap frozen in liquid nitrogen immediately after collection and stored at -80 ℃for RNA extraction, and the other was fixed in 40 mg/ml paraformaldehyde at room temperature for immunolocalization of BMP2 protein. Real-time PCR analysis showed that the expression level of Bmp2 was significantly higher at proestrus than at estrus and metestrus （P〈0.05）. The relative abundance of Bmp4 exhibited significant fluctuations, but there were no statistically significant differences between the expression levels of Bmp2 and Bmp4 （P〉0.05）. The expression levels of Bmprla and Bmpr2 remained unchanged during estrous cycles. However, the level of Bmprlb mRNA decreased significantly at estrus （P〈0.05）, increasing subsequently at metestrus. Furthermore, the level of Bmprlb mRNA was significantly lower than those of Bmprla and Bmpr2 mRNA at the corresponding stages （P〈0.05）. All three receptor-regulated Smads （R-Smads） detected were differentially expressed in the mouse uterus and the expression levels of Smadl and Smad5 were significantly higher than that of Smad8 （P〈0.05）. In addition, the expression level of Smad4 did not change substantially throughout the estrous cycle. Immunohistochemical experiments revealed that BMP2 protein was differentially expressed and localized mainly in the uterine luminal and glandular epithelial cells throughout the estrous cycle. In conclusion, our results provide information about the variation in the mRNA levels of Bmp2 and Bmp4 and related components of the BMP signaling pathway. The data provide quantitative and useful information about the roles of endometrial BMP proposed and demonstrated by others, such as the degradation and remodeling of the endometrium.

Effects of carbohydrate supplements on exercise-induced menstrual dysfunction and ovarian subcellular structural changes in rats

Background： Exercise-associated menstrual dysfunction （EAMD） is a common health problem in female athletes as a part of female athlete triad （FAT）, a condition related to low energy availability. In this study, we explored the possibility that carbohydrate supplements can improve the status of EAMD and prevent exercise-induced ovarian injury in a FAT rat model. This research aimed to provide experimental evidence with regard to the relationship of energy intervention and EAMD. Methods： Forty-five female Sprague-Dawley rats （2 months old） were randomly divided into five experimental groups： control group （C）, 9-week exercise as model for EAMD （E）, post-EAMD recovery group （R）, oligosaccharide intervention group （O）, and glucose intervention group （G）. All rats were sacrificed at the end of 9 weeks. Serum samples were collected for measuring gonadotropin releasing hormone, follicle stimulating hormone, luteinizing hormone, 1713-estradiol and progesterone levels. The ovaries were taken for investigation of exercise- and carbohydrate-induced follicular subcellular structure changes. Results： Exercise induced irregular menstrual cycles and ovary subcellular structural damages, such as swollenness of mitochondria in rats from groups E and R. Both glucose and oligosaccharide supplements restored well-differentiated mitochondria in the ovarian follicular cells, and a significant improvement of endoplasmic reticulum and Golgi in swollenness in theca cells in groups O and G compared to groups C, E, and R. There was no difference in mitochondria subcellular structural changes between groups O and G. Group E showed attenuation of serum levels of 17β-estradiol and progesterone compared to C. There were no differences of 17β-estradiol serum levels among groups O, G, and R, while group G showed a lower level of progesterone than C. Conclusion： Female adult rats with 9-week continuous exercise can cause menstrual dysregulation as a model for EAMD. Post-EAMD intervention with glucose and oligosaccharide intake can normalize the menstrual cycle, restore the follicular subcellular structure, and reverse the exercise-induced reduction of ovary sex hormones. It suggests a positive feedback of hypothalamus-pituitary-ovary axis might be involved in the molecular mechanisms of energy intake in treating EAMD.

Influence of Isoflurane Exposure for 15 Consecutive Days on Ovarian Function in Adult Female Mice

Female infertility after occupational exposure to inhaled anesthetic agents has attracted critical attention,but systematic studies focusing on the impact of inhaled anesthetics on the female reproductive system have not been well-established.We used a murine model to study the effect of isoflurane exposure on infertility in female adult mice and investigated the potential underlying mechanism.One hundred adult female C57 mice were randomly allocated into 5 groups exposed in air containing 0,2500,5000,10000 or 20000 ppm isoflurane for 15 consecutive days.Estrous cycle length was measured based on vaginal smear examination,ovarian histopathologic enumeration of follicles,and serum estradiol(E2),anti-Mullerian hormone(AMH),follicle-stimulating hormone(FSH),and luteinizing hormone(LH)levels to assess the effect of isoflurane on ovarian reserve.Compared to the control group,significant prolongation of the estrous cycle of the adult female mice was observed in the 20000 ppm isoflurane exposure group.Serum AMH was significantly decreased,and FSH and LH levels profoundly increased in the 5000,10000,and 20000 ppm isoflurane exposure groups compared to the control group.The histopathologic examination revealed a reduced number of developing follicles and an increased number of atretic follicles after isoflurane exposure,but the difference was not statistically significant.Thus,exposure to a higher concentration of isoflurane might have an adverse effect on ovarian reserve in sexually-mature female mice.

Measurement of Metabolic and Inflammatory Serum Markers and Immune Marker Gene Expression during Superovulation in Beef Cattle

Health status of donor cows during superovulation is important to ensure optimal embryo quality at time of collection. Because nutritional and metabolic status impact embryo quality some form of nutritional supplementation is often provided before and during superovulation. OmniGen-AF® (OG) feeding has been shown to assist in the maintenance of animal health through regulation of metabolic status and balance and supporting aspects of immune function. We observed feeding donor cows OG decreased percent degenerate embryos recovered following superovulation increased serum progesterone concentration and improved in vitro embryo development. Evaluation of OG feeding on markers of metabolic function and inflammatory and immune function in beef cattle embryo donors are reported here. Similarly, cow metabolic and inflammatory response with repeated superovulation protocols is not known. Biomarkers to monitor and evaluate cow health during superovulation may provide management options to improve embryo recovery and quality. Twenty-four Angus cross-bred cattle were randomly assigned to four treatment groups, fed 0 or 56 g/hd/day for 49 days and superovulated with 200 or 400 mg Folltropin V (FSH). Blood was collected weekly for analyses. The protocol was repeated on all cows 90 - 120 d later with cows reassigned to their original groups. No differences (P > 0.10) were observed due to OG feeding or FSH dose on metabolic and inflammatory markers. Replicate exerted a significant effect where serum concentration of albumin, IL1β, IL6, PGE2 and leptin were lower (P < 0.05) in Replicate 1 compared to 2. There was also a similar pattern of change in several of the metabolic and inflammatory markers during the superovulation protocol where concentrations were higher at the time of estrus and ovulation. Taken together, physiologic changes during the estrous cycle and the number of superovulation protocols can modulate metabolic markers and inflammatory response.

Effect of the Aqueous Stem Bark Extract of Schumanniophyton magnificum on Reproductive Functions on Wistar Strain Mature Female Rats

In recent years, the rate of infertility has not stopped increasing in the world. The aim of the present study was to evaluate the effects of the aqueous extract of Schumanniophyton magnificum (Rubiaceae) on cyclicity, ovulation and gestation in mature rats. Methods: After a qualitative phytochemical analysis of these aqueous extracts, the experimental studies carried out were based on the evaluation of the pro-fertility effects of this extract in mature rats. For this purpose, 35 rats were used for the estrous cyclicity test and treated for 21 days at the end of which vaginal smears were taken and the duration, as well as the frequency of the appearance of the phases of the cycle, were evaluated. The ovulation test was performed on 80 female rats, which were divided into two groups of 40 animals and treated respectively in the morning and evening with distilled water, β-oestradiol or plant extract at doses of 200, 400 and 800 mg/kg. At the end of estrus, the rats were sacrificed, the ovaries were removed and weighed, the hemorrhagic points counted and the blood samples were taken for hormonal studies. The last phase of the study consisted in evaluating the effects of these plant extracts on the evolution of gestation. Thus, 42 mature rats were treated during the periods from the 1st to the 10th day (1st stage), and from the 11th to the 17th day (2nd stage). At the end of these two phases, a laparotomy was performed and the number of implantation sites and corpus luteum was counted. And finally, at parturition, from the 18th to the 22nd day (3rd stage), the number of living pups was performed and the gestational parameters were calculated. Results: Administration at the beginning of pr&oelig;strus allowed a significant increase (p S. magnificum extract. On the other hand, a significant decrease in progesterone levels (p S. magnificum at doses of 200, 800 mg/kg (p S. magnificum when compared to the control (23 ± 0.16 d). Conclusion: It appears from all these investigations that the aqueous extract of S. magnificum promotes fertility in the rat but represents a danger for the good development of gestation. All these results obtained would be closely related to the presence of certain chemical compounds contained in these various extracts;which would justify their use in traditional medicine for the treatment of certain cases of female infertility in Cameroon.

Activation of hypothalamic gono-like neurons in female rats during estrus

In mammals, gonadal function is controlled by the activity of hypothalamic gonadotropin-releasing hormone neurons, which control the secretion of adenohypophyseal and gonadal hormones. However, there are a number of unanswered questions in relation to gonadal function. It is currently unknown how erotogenic stimulation of the genitals influences the subpopulation of hypothalamic medial preoptic area neurons, antidromically identified as projecting to the median eminence at different periods of the estrous cycle. Additionally, the distinctiveness of hypothalamic medial preoptic area neurons, with respect to methods of feedback control by exogenous hormones, is also unknown. In this study, spontaneous discharges from individual neurons encountered within the medial preoptic area, gono-like neurons, were recorded extracellularly using glass microelectrodes. To confirm the cellular and histochemical properties of the recording units, antidromic stimulation was performed using a side-by-side bipolar stimulating electrode placed into the median eminence, alongside microiontophoretic injections of the conventional tracer, horseradish peroxidase. In addition, further immunohistochemical analyses were performed. Results showed that elevated gono-neuron activity was accompanied by increased background activity and greater responses to erotogenic stimuli during estrus. Application of clitoral traction stimulation resulted in increased activation of the gono-like neurons. This neuronal activity was noticeably inhibited by β-estradiol administration. Immunohistochemical analyses revealed the presence of gonadotropin-releasing hormone-reactive protein in hypothalamic cells in which electrophysiological recordings were taken. Thus, medial preoptic area neurons represent the subset of hypothalamic gonadotropin-releasing hormone neurons described from brain slices in vitro, and might serve as a useful physiological model to form the basis of future in vivo studies.

Virgin Coconut Oil Improved Discriminative Learning and Working Memory in Aging Cycling and Non-Cycling Female Sprague-Dawley Rats Supporting Its Beneficial Effect in Retarding Age-Related Cognitive Decline

Aim: Beneficial effects of virgin coconut oil (VCO) consumption to improve cognition in menopausal females remain inconclusive. This study examined the effect of VCO supplementation in aging cycling and non-cycling rodents to assess its impact on cognition. Methods: Sprague-Dawley rats (10 - 18 months) were randomly assigned to a supplemented VCO group (SVCO) that received oral doses of 1.42 mL/kg/day VCO (n = 10) and a non-supplemented (NVCO) group (n = 10). Their performance in a biased Y-maze discriminative learning paradigm was assessed over a 16-week period. Rats were initially allowed 3 minutes to explore the maze (habituated) and subsequently pre-trained in the non-preferred, white chamber to associate the presentation of a tone with a treat (reward). Training involved 4 daily trials initially for 3 weeks during which rats were rewarded if they entered the white arm within 15 sec after tone presentation. Time (days) to attain at least 75% correct responses (CR) determined acquisition latency (AL). Memory retention (MR1) of the learned task was assessed following a 1-week break from training and absence of supplementation (session T1). Following an additional 2-week break, supplementation of SVCO animals resumed and continued to week 16. In week 14, all animals received re-training for 1 week (session T2) followed by another 1-week break and subsequent assessment of memory (MR2). Vaginal smear cytology determinations were performed throughout the study to identify cycling and non-cycling rats. Student’s t-test and ANOVA with Brown-Forsythe and Tukey’s post-hoc tests were used to compare means. Results: C-SVCO rats attained lower AL, and higher CR and MR scores vs their NVCO counterparts (p < 0.05). At session T2, NC-SVCO rats out-performed other groups (p = 0.048, F = 2.64), attaining highest CR scores between sessions (p = 0.026). Conclusion: VCO supplementation attenuated cognitive decline with a more positive impact on non-cycling rodents suggesting a beneficial effect on brain health in females in menopausal transition.

Organoids as a model to study the human endometrium

The endometrium is composed of glandular and luminal epithelia supported by stromal connective tissue and multiple other cell types.It is a dynamic organ that undergoes physiological and functional alteration during the menstrual cycle.Organoids resemble the primary tissue of origin to recapitulate their corresponding biological and pathological characteristics.They are known for their ability to undergo extensive expansion while maintaining their genomic stability,facilitating their long-term storage and high-throughput screening.The development of the three-dimensional endometrial organoid system,which recapitulates the structural and functional characteristics of the endometrial glands,provides a powerful tool to study the normal endometrium and its related diseases.The Web of Science was searched for relevant literature using the keywords"endometrium","endometrial gland","organoid",and"culture model";a total of 134 articles were selected.In this review,the characteristics,applications,and limitations of endometrial epithelial organoids are discussed.

Immunolocalization of uterine luminal fluid protein (ULF-250) in rat uterus

To access the production of a novel uterine luminal fluid 250?kD protein (ULF 250) during the various phases of the estrous cycle in relation to estrogen concentration, and to validate that the production and secretion of ULF 250 are regulated by estradiol and progesterone Methods An immunohistochemical method was used to localize ULF 250 in rat uteri during each phase of the estrous cycle, and in uteri of ovariectomized rats treated with estradiol and progesterone Results Positive immunostaining of ULF 250 occurred in the epithelial cells of the uterus at all phases of the estrous cycle; whereas the stroma was immunonegative During the proestrus phase of the cycle, the glandular epithelial cells and glandular luminal content were stained strongly During the estrus phase of the cycle, intense staining occurred in the glandular and uterine luminal epithelial cells, including the luminal content of the glands In the metestrus phase of the cycle, only uterine epithelial cells were stained; during the diestrus phase, intense staining of the secreted contents of the uterine cavity and the glandular lumen occurred The distribution of ULF 250 in the uteri of ovariectomized female rats treated with estradiol alone and estradiol plus progesterone were examined In both groups, intense staining of the glandular luminal epithelial cells of the uterine endometrium occurred; in the estradiol treated animals, only the luminal contents were stained The present findings suggest that progesterone inhibits the secretion of ULF 250 that is stimulated by estradiol Conclusions ULF 250 is produced by the glandular and luminal epithelial cells of the uterine endometrium and fluctuates with the phases of the estrous cycle Its production is stimulated by estradiol and its secretion is regulated by progesterone