GABAB receptor antagonism from birth to weaning permanently modifies Kiss1 expression in hypothalamus and gonads in mice

Introduction: The kisspeptin gene Kiss1 is expressed in two hypothalamic areas: anteroventral periventricular nucleus/periventricular nucleus (AVPV/PeN) and arcuate nucleus (ARC), and also in gonads. Evidences suggest that gamma-amino butyric acid B receptors (GABAB) signaling can regulate Kiss1 expression. Here we inhibited GABAB signaling from PND2-PND21 and evaluated the hypothalamic-pituitary-gonadal (HPG) axis. Methods: BALB/c mice were treated on postnatal days 2-21 (PND2-PND21) with CGP55845 (GABAB antagonist), and evaluated in PND21 and adulthood: gene expression (qPCR) in hypothalamus and gonads, hormones by radioimmunoassay, gonad histochemistry (H&E), puberty onset, estrous cycles. Results: At PND21, CGP inhibited Kiss1 and Tac2 and increased Pdyn and Gabbr1 in the ARC of both sexes and decreased Th only in female AVPV/PeN. Serum follicle-stimulating hormone (FSH) and testis weight decreased in CGP-males and puberty onset was delayed. In adults, Kiss1, Tac2, Pdyn, Pgr, Cyp19a1, Gad1 were downregulated, while Gabbr1 was upregulated in the ARC of both sexes. In the AVPV/PeN, Kiss1, Th, Cyp19a1 and Pgr decreased while Gad1 increased in CGP-females, whereas Cyp19a1 increased in CGP-males. Serum FSH increased in CGP-males while prolactin increased in CGP-females. Testosterone and progesterone increased in ovaries from CGP-females, in which Kiss1, Cyp19a1 and Esr1 were downregulated while Hsd3b2 was upregulated, together with increased atretic and decreased ovulatory follicles. Testes from CGP-males showed decreased progesterone, increased Gabbr1, Kiss1, Kiss1r, Esr2 and decreased Cyp19a1 and clear signs of seminiferous tubules atrophy. Conclusion: These results demonstrate that appropriate GABAB signaling during this critical prepubertal period is necessary for the normal development of the HPG axis.

Onset of normal cycles in postpartum anovulatory dairy cattle treated with kisspeptin

The efficacy of a long-acting synthetic derivative of kisspeptin (Kp) to initiate normal estrous cycles was tested in 24 mixed-aged, Holstein-Friesian cows that were 18 to 25 d postpartum on day of treatment (D0). Groups of eight received saline (Sal) vehicle by intramuscular injection at 0800 and 1600 h (Sal-Sal), Kp at 800 h and vehicle at 1600 h (Kp-Sal) or Kp on both occasions (Kp-Kp). The Kp dose was 15 nmol per 60 kg body weight. The cows ovaries were examined daily by ultrasonography between D-4 and D14. Blood samples were collected from a tail vessel 0, 2, 4, 8, 10 and 12 h relative to the time of first injection for LH and FSH assay. Additional samples were collected daily from D-4 until D14 and D19, 22, 26 and 29 for progesterone assay. An LH surge-like response were observed in cows treated with Kp at 0800 h. Ovulation was consistently induced by Kp within 48 h when there was a dominant follicle of at least 10 mm in diameter on the ovaries (8/14), but in no cases (6/14) during a new wave of ovarian follicular development consisting of follicles <10 mm diameter. The subsequent ovulatory cycle was of normal length in most cases, as compared with short 8 to 12 d cycles observed in spontaneously ovulating cows. We conclude that Kp treatment can induce ovulation in postpartum dairy cows, with ensuing estrous cycles of normal length, if administered when a mature dominant follicle is present on the ovaries.

Effect of Clomiphene Citrate and Letrozole on Body Weight of female Albino Rat for Consecutive 1-4 Estrous Cycles

Background: Ovulation induction with clomiphene citrate in women with infertility has been practiced more than 40% years but in infertile patients this treatment plan proved to be ineffective with multiple complication. Body weight plays an important role modulating reproductive development and functioning. Aim: To observe the effects on body weight of female albino rat after use of clomiphene citrate and letrozole for consecutive 1-4 estrous cycles Method: Eighty four adult female Albino rats were equally divided into three groups for this research. Body weight of each rat was measured before and after the experiment. Vaginal smear cytology of each rat was performed to study different phases of estrous cycle. Control group A was given normal saline orally , In Experimental group B rats were given letrozole (Femara) at dose 5mg/kg orally and in Experimental group C rats were given clomiphene citrate at dose 100ug/kg orally. Results: Significant weight gain is observed in rats taking clomiphene citrate as compared to letrozole Conclusion : Comiphene citrate directly affects the body weight which indirectly reduces the ovulation induction and pregnancy rate. Letrozole is good alternate for ovulation induction and for CC resistant patients. Keywords: Estrous cycle, body weight, citrate and letrozole

Portulaca oleracea shows no ameliorative potential on ovariectomy-induced hormonal and estrous cycle dysregulation in normal cyclic rats: An experimental study

Background: Portulaca oleracea (PO) is a widely known plant utilized for its medicinal attributes in the treatment of different illnesses. Objective: To investigate the effect of methanolic extract of PO (MEPO) on ovariectomy-induced reproductive toxicity in normal cyclic rats. Materials and Methods: Twenty 10-wk-old normal cyclic rats weighing 110-200 g were randomly divided into four groups (n = 5/group). Group A served as the control and received distilled water only. Group B was ovariectomized without treatment, while groups C and D were ovariectomized but treated with 400 and 800 mg/kg of MEPO, respectively, for 14 days. At the end of the experiment, body weight, serum hormonal levels, and estrous cycles were monitored across the groups. Results: Groups B, C, and D showed estrous cycle dysregulation and specific phase arrest when compared with the control. While a significant decrease in estradiol (p ≤ 0.001) and testosterone levels (p ≤ 0.001) were observed in groups B, C, and D, only groups C and D showed a significant increase in progesterone level when compared with the control (p ≤ 0.001, p = 0.01, respectively). Conclusion: The administration of 400 and 800 mg/kg MEPO is ineffective in ameliorating estrous cycle disruption and hormonal changes seen in ovariectomized normal cyclic adult Wistar rats. Key words: Portulaca oleracea, Wistar rat, Ovariectomy, Estrous cycle, Hormones.

Studies on Ovarian Follicular Dynamics and Steroid Profiles in Sahiwal Cattle

Background: Understanding of ovarian follicular dynamics and endocrine regulation is essential to design and use interventions to optimize reproductive efficiency. Bos indicus and Bos taurus cows have some differences in their follicular dynamics and ovarian steroidal hormones. The present study was planned to understand follicular dynamics vis-a-vis ovarian steroids profiles in Sahiwal cattle. Methods: The study was conducted in normal cyclic, pluriparous, non-lactating Sahiwal cows (n=7). Trans-rectal ultrasonography was performed to record the location, size, number of follicles and size of corpus luteum (CL) from the beginning, till the end of experiment. The dataset was used to characterize the follicular wave emergence, growth, regression, time of selection and ovulation of the dominant follicle in each cow. Result: Three Sahiwal cows showed two wave and four cows showed three wave estrous cycles with mean interovulatory length of 20.33±0.33 and 22.50±0.28 days, respectively. No significant difference was recorded in the maximum diameter of corpus luteum, P4 and E2 plasma levels between 2-wave and 3-wave estrous cycles. The peak progesterone values of 6.00±0.91 and 6.2±1.2 ng/ml and peak estradiol values of 15.83±0.60 and 14.31±0.44 ng/ml were recorded in 2-wave and 3-wave estrous cycle, respectively. The results showed that Sahiwal cows had 2-wave and 3-wave estrous cycle and the 3-wave estrous cycle had comparatively longer inter-ovulatory period and smaller maximum diameter of second wave dominant follicle than 2-wave estrous cycle.

Estrus Detection Using Background Image Subtraction Technique in Tie-Stalled Cows

In this study, we determined the applicability of the background image subtraction technique to detect estrus in tie-stalled cows. To investigate the impact of the camera shooting direction, webcams were set up to capture the front, top, and rear views of a cow simultaneously. Video recording was performed for a total of ten estrous cycles in six cows. Standing estrus was confirmed by testing at 6 h intervals. From the end of estrus, transrectal ultrasonography was performed every 2 h to confirm ovulation time. Foreground objects (moving objects) were extracted in the videos using the background subtraction technique, and the pixels were counted at each frame of five frames-per-second sequences. After calculating the hourly averaged pixel counts, the change in values was expressed as the pixel ratio (total value during the last 24 h/total value during the last 24 to 48 h). The mean pixel ratio gradually increased at approximately 48 h before ovulation, and the highest value was observed at estrus, regardless of the camera shooting direction. When using front-view videos with an appropriate threshold, estrus was detected with 90% sensitivity and 50% precision. The present method in particular has the potential to be a non-contact estrus detection method for tie-stalled cows.

Encapsulated allografts preclude host sensitization and promote ovarian endocrine function in ovariectomized young rhesus monkeys and sensitized mice

Transplantation of allogeneic donor ovarian tissue holds great potential for female cancer survivors who often experience premature ovarian insufficiency. To avoid complications associated with immune suppression and to protect transplanted ovarian allografts from immune-mediated injury, we have developed an immuno-isolating hydrogel-based capsule that supports the function of ovarian allografts without triggering an immune response. Encapsulated ovarian allografts implanted in naive ovariectomized BALB/c mice responded to the circulating gonadotropins without direct revascularization and maintained function for 4 months, as evident by regular estrous cycles and presence of antral follicles in the retrieved grafts. Repeated implantations of encapsulated mouse ovarian allografts did not sensitize naive BALB/c mice in contrast to non-encapsulated controls, which was confirmed with undetectable levels of allo-antibody. Further, encapsulated allografts implanted in hosts previously sensitized by implantation of non-encapsulated allografts restored estrous cycles similarly to our results in naive recipients. Next, we tested the translational potential and efficiency of the immune-isolating capsule in a rhesus monkey model by implanting encapsulated ovarian auto- and allografts in young ovariectomized animals. The encapsulated ovarian grafts survived and restored basal levels of urinary estrone conjugate and pregnanediol 3-glucuronide during the approximate 4-5 month observation period. We demonstrate, for the first time, that encapsulation of ovarian allografts prevents sensitization and protects the allograft from rejection in young rhesus monkeys and in sensitized mice.

miR-29a/b1 Regulates the Luteinizing Hormone Secretion and Affects Mouse Ovulation

miR-29a/b1 was reportedly involved in the regulation of the reproductive function in female mice, but the underlying molecular mechanisms are not clear. In this study, female mice lacking miR-29a/b1 showed a delay in vaginal opening, irregular estrous cycles, ovulation disorder and subfertility. The level of luteinizing hormone (LH) was significantly lower in plasma but higher in pituitary of mutant mice. However, egg development was normal in mutant mice and the ovulation disorder could be rescued by the superovulation treatment. These results suggested that the LH secretion was impaired in mutant mice. Further studies showed that deficiency of miR-29a/b1 in mice resulted in an abnormal expression of a number of proteins involved in vesicular transport and exocytosis in the pituitary, indicating the mutant mice had insufficient LH secretion. However, the detailed mechanism needs more research.

Deslorelin Slow-Release Implants Delay Ovulation and Increase Plasma AMH Concentration and Small Antral Follicles in Haflinger Mares

There is an increasing interest in the manipulation of ovarian follicular populations in large domestic animals because this could prove beneficial for assisted reproductive techniques such as ovum pick-up (OPU). The aim of the present study was to evaluate the effects of deslorelin slow-release implants (SRI) on the interovulatory interval, antral follicle count (AFC), number of follicles of different size ranges and plasma anti-Muellerian hormone (AMH) concentration in mares. To synchronize their estrous cycles, Haflinger mares (n = 12) were treated twice with a PGF2α analogue. One day after the second injection (day 0), mares received a 9.4 mg deslorelin SRI (group DES, n = 6) or 1.25 mg deslorelin in a short-acting formulation (CON; n = 6), respectively. Regular transrectal ultrasonography of the genital tract was performed and blood samples were collected for the analysis of progesterone, AMH and gonadotrophins. The interval from implant insertion to the first spontaneous ovulation was 23.8 ± 10.5 days in group DES compared to 17.0 ± 3.9 days in group CON (p < 0.05). For the concentrations of LH, FSH and AMH, interactions between time and treatment were detected (p < 0.05). The AFC and the mean number of follicles with 5 to 10, 10 to 15 and 15 to 20 mm in diameter changed over time (p < 0.05). A time x treatment interaction was demonstrated for follicles of 10 to 15 mm in diameter (p < 0.05). The changes in this follicular subpopulation were reflected by increased plasma AMH concentration in group DES. In conclusion, 9.4 mg deslorelin implants show minor effects with regard to estrus suppression in mares, whereas the changes in the subpopulation of small ovarian follicles could be a promising tool for preparation of mares for OPU.