Primary Follicles Research Articles

The Effect of Tapak Dara (Catharanthus roseus) Leaf Extract on The Growth of Mice (Mus Musculus) Ovarian Follicles

Tapak dara (Catharanthus roseus) leaf contain phytoestrogen compounds such as flavonoids, isoflavonoids, saponins, and tannins, and contain alkaloid compounds. Phytoestrogens are compounds contained in plants, having a chemical structure resembling 17β-Estradiol in the body's natural estrogen. This study aims to determine the effect of tapak dara leaf extract on the growth of ovarian follicles of mice (Mus musculus), consisting of primary follicles, secondary, tertiary, and de Graaf follicles. This research method is true experiment and Posstest-Only Control with a quantitative approach. Sampling was carried out randomized block design (RBD). The growth of mice ovarian follicles is known by counting the number of mice ovarian follicles, analyzed using the ANOVA one-way test analysis method at a sig. level of 5% (α = 0.05), then if there is a difference, proceed with a further test of Least Significant Difference) at a sig. level of 5% (α = 0.05). Data analysis using Microsoft Excel 2010. The results of the hypothesis test using One Way ANOVA stated that Fcalculate > Ftable at sum of primary follicles, which means that there is a real difference and Ha is accepted. The results of hypothesis tests at sum of secondary, tertiary, and de Graaf follicles stated that Fcalculate < Ftable, which means there is no real difference and Ha is rejected. The conclusion of this study is that tapak dara leaf extract has a significant effect on the growth of primary follicles, but does not significantly affect the growth of secondary, tertiary, and de Graaf follicles.

Identification of the Keratin-Associated Protein 22-2 Gene in the Capra hircus and Association of Its Variation with Cashmere Traits.

The Cashmere goat is an excellent local goat breed in Gansu Province of China, and it is expected to improve cashmere production and cashmere quality through selection and breeding to enhance its commercial value. Keratin-associated proteins (KAPs) play an important role in maintaining wool structure. The gene encoding the keratin-associated protein 22-2 (KAP22-2) gene has been identified in selected species other than goats, such as humans, mice, and sheep. In this study, the sequence of the sheep KAP22-2 gene (KRTAP22-2) was aligned into the goat genome, and the sequence with the highest homology was assumed to be the goat KRTAP22-2 sequence and used to design primers to amplify the goat gene sequence. A total of 356 Longdong Cashmere goats (Gansu Province, China) were used for screening of genetic variants. Four specific bands were detected by polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) analysis, and they formed a total of six band types individually or in combination. Four alleles were identified by DNA sequencing of PCR amplification products. A total of four single nucleotide polymorphic sites (SNPs) were detected in the four sequenced KRTAP22-2 alleles. Two of them are in the 5'UTR region and the other two are in the coding region, and the variants in the coding region are all non-synonymous mutations. In addition, there was a 6 bp length variation in allele C. The gene was expressed in the cortical layer of primary and secondary hair follicles, the inner root sheath, as well as hair papillae and hair maternal cells in goats. The results of the correlation analysis between genotypes and cashmere traits showed that after excluding genotypes with a gene frequency of less than 5%, the mean fiber diameter (MFD) of cashmere was significantly higher in the AB genotype than in the AA and AC genotypes. That is, the KRTAP22-2 gene variants are associated with mean fiber diameter in cashmere. The above results suggest that the goat KRTAP22-2 variant can be utilized as a molecular marker candidate gene for cashmere traits.

Does the addition of metformin to carboplatin treatment decreases ovarian reserve damage associated with carboplatin usage?

BackgroundWe aimed to determine whether adding metformin to carboplatin treatment would reduce the damage to ovarian reserve associated with carboplatin use.MethodsWe included 35 adult female non-pregnant albino Wistar rats approximately three months old, weighing 220–310 g. The rats were divided into five groups of seven rats according to the treatment they received. Carboplatin and salin was given to Group 2, and carboplatin plus metformin was given to Group 3. Group 4 was administered only metformin. Group 5 was administered only salin. Carboplatin was given to Groups 2 and 3 as a single dose on the 15th day, while metformin was given to Groups 3 and 4 during the 28-day experiment. After oophorectomy, histopathologic analyses of primordial, primary, secondary, and tertiary Graff follicles according to the epithelial cells surrounding the oocyte and total follicular number were conducted per section. Serum Anti-Mullerian Hormone (AMH), tissue catalase, and malonyl dialdehyde levels were measured and compared within each group.ResultsThe baseline and 15th-day serum AMH values of the menstrual cycle were compared among the groups, and no statistically significant differences were observed (p > 0.05). Group 3, which was given both carboplatin and metformin, had statistically significantly higher 28th-day AMH levels than Group 2, which was given only carboplatin and saline (p < 0.001). The number of primordial follicles in Group 3 was found to be statistically significantly higher than in Group 2 (p < 0.001). Tissue catalase enzyme levels in Group 3 were statistically significantly higher than in Group 2 (p < 0.001). Tissue malondialdehyde levels in Group 2 were statistically significantly higher than tissue malondialdehyde levels in Groups 3 and 4 (p < 0.001).ConclusionsMetformin may attenuate carboplatin-induced ovarian damage, possibly through its antioxidative effects.

Bushen Huoxue formula for the treatment of diminished ovarian reserve: A combined metabolomics and integrated network pharmacology analysis

ObjectiveThis study aimed to explore the mechanism of the Bushen Huoxue Formula (BHF) in treating diminished ovarian reserve (DOR) through the use of metabolomics and integrated network pharmacology. MethodsThe study involved 24 non-pregnant female Sprague-Dawley rats, divided into four groups of six rats each: control, model, BHF, and DHEA (n = 6 per group). The model group was induced with DOR by administering Tripterygium glycosides orally [50 mg (kg·d)−1] for 14 days. Subsequently, BHF and Dehydroepiandrosterone (DHEA) treatments were given to the respective groups. Ovarian reserve function was assessed by measuring anti-Müllerian hormone (AMH), estradiol (E2), and follicle-stimulating hormone (FSH) levels and conducting hematoxylin-eosin staining. In addition, UHPLC-QTOF-MS analysis was performed to identify differential metabolites and pathways in DOR rats treated with BHF. In this study, LC-MS was utilized to identify the active ingredients of BHF, while network pharmacology was employed to investigate the correlations between BHF-related genes and DOR-related genes. An integrated analysis of metabonomics and network pharmacology was conducted to elucidate the mechanisms underlying the efficacy of BHF in treating DOR. ResultsThe model group exhibited a poor general condition and a significant decrease in the number of primordial, primary, and secondary follicles (P < 0.05) when compared to the control group. However, BHF intervention resulted in an increase in the number of primordial, primary, and secondary follicles (P < 0.05), along with elevated levels of AMH and E2 (P < 0.05), and a decrease in FSH levels (P < 0.05) in DOR rats. The modeling process identified eleven classes of metabolites, including cholesterol esters (CE), diacylglycerols (DAG), hexosylceramides (HCER), lysophosphatidylcholines (LPC), phosphatidylcholines (PC), phosphatidylethanolamines (PE), sphingomyelins (SM), ceramides (CER), free fatty acids (FFA), triacylglycerols (TAG), and lysophosphatidylethanolamines (LPE). The study found that PC, CE, DAG, and TAG are important metabolites in the treatment of DOR with BHF. LC-MS analysis showed that there were 183 active ingredients in ESI(+) mode and 51 in ESI(−) mode. Network pharmacology analysis identified 285 potential genes associated with BHF treatment for DOR in ESI(+) mode and 177 in ESI(−) mode. The combined analysis indicated that linoleic acid metabolism is the primary pathway in treating DOR with BHF. ConclusionBHF was found to improve ovarian function in rats with DOR induced by Tripterygium glycosides. The study identified key metabolites such as phosphatidylcholine (PC), cholesteryl ester (CE), diacylglycerol (DAG), triacylglycerol (TAG), and the linoleic acid metabolism pathway, which were crucial in improving ovarian function in DOR rats treated with BHF.

Abstract 019: Perimenopause Exacerbates Hypertension In Doca-salt Rats

Blood pressure (BP) is lower in premenopausal women than in age-matched men, but after menopause BP rates and the incidence of hypertension (HTN) show opposite tendencies. The mechanisms by which fluctuating ovarian hormone environment during that period impacts the onset and development of HTN are poorly understood, and strategies to reduce HTN risk in peri- and postmenopausal females are lacking. An ovary-intact rat model of perimenopause using the chemical, 4-vinylcyclohexene diepoxide (VCD), has been developed to accelerate the natural process of slow follicular loss. Daily dosing with VCD for 15 days selectively destroys primordial and primary follicles in ovaries of mice and rats by accelerating atresia processes, thus inducing precocious perimenopause/menopause. Hypothesis: The induction of perimenopause by VCD will increase susceptibility to the development of HTN in DOCA-salt rats. Female Sprague-Dawley rats at post-natal day 28 received subcutaneous injection of VCD (160mg/kg) or corn oil (control) for 15 days. 57 days after the first injections, nephrectomy and telemetric pressure transducer implantation were performed. After 2 weeks of recovery, a 100 mg/kg DOCA silicone or placebo pellet was implanted subcutaneously. The rats that received DOCA were provided 0.9% saline during the 21 days of DOCA protocol and mean arterial pressure (MAP) was continuously evaluated. Rats were housed weekly in metabolic cages for 24 hours to measure saline intake and urine volume. After protocol completion the animals were decapitated, and blood was collected for the measurement of anti-Mullerian hormone (AMH) and estradiol (E2) by ELISA. The AMH plasma levels were reduced in the periestropause rats (VCD + DOCA, n=9) compared to oil (Oil + DOCA, n=6) confirming the VCD-induced follicular depletion. The steady state increase in MAP (day 21 of DOCA-salt) was higher in periestropause (±45.7mmHg) compared to oil (±28.6 mmHg). Urine volume was higher in periestropause rats compared to oil. No differences were observed in the saline intake between the groups. Our data demonstrate that periestropause rats showed greater susceptibility to the development of HTN in a DOCA-salt rat model.

P-166 - Primary cutaneous follicle center lymphoma with secondary systemic evolution

P-166 - Primary cutaneous follicle center lymphoma with secondary systemic evolution

Protective and/or therapeutic effects of berberine in a model of premature ovarian failure induced by cyclophosphamide in rats.

We conducted a biochemical and histological evaluation of whether berberine has a protective and/or therapeutic effect in a cyclophosphamide-induced premature ovarian failure (POF) model. We divided 28 Wistar albino female rats into 4 groups [control group, POF group, cyclophosphamide (CP)+berberine (Bb) group, and POF+Bb group]. The POF model was established by intraperitoneal administration of 50 mg/kg CP on day 1 followed by 8 mg/kg/day CP dissolved in saline for the following 14 days. The CP+Bb group received Bb concurrently for two weeks with CP. The POF+Bb group received berberine for two weeks following the completion of CP administration. Left ovaries were used for histopathological evaluation and right ovaries were used for biochemical analysis [tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-6 levels in tissue]. Ovarian damage scoring was significantly higher in the POF group than in the other groups (p<0.005). In the POF group, primordial and primary follicle counts were the lowest, while secondary and corpus luteum counts were the highest (p<0.005). There was no significant difference between the other groups. The POF group had significantly elevated levels of TNF-α, IL-1, and IL-6 in the biochemistry results (p<0.005). We demonstrated that berberine could be effective in the protection and treatment of POF by reducing proinflammatory cytokines. We believe that our study can make a considerable contribution to the literature in terms of POF protection and/or treatment.

Comparing ovarian expression of sperm acrosome associated 3 protein in young and adult queens

The purpose of this research was to quantify sperm acrosome associated 3 protein expression in the ovaries of young (3.0 ± 0.9 months, n = 11) and adult (10.4 ± 2.8 months, n = 11) queens. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded feline ovarian sections. Ovaries were obtained following routine ovariohysterectomy of queens. Cellular expression of sperm acrosome associated 3 protein was measured in primordial, primary, secondary, and tertiary follicles using an image-analysis software's red, green, and blue stack and manual thresholding functions. The oocyte nucleus, ooplasm, granulosa cells, and theca cells were outlined using the freehand selection tool and mean grey value was recorded. Results from each cellular location were compared between age groups using a Student's t-test and between follicle stages using an analysis of variance. Compared to adult queens, younger queens had significantly greater sperm acrosome associated 3 protein expression in granulosa cells of primary, secondary, and tertiary follicles. Also, theca cells of secondary and tertiary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. The oocyte nucleus of primordial, primary, and secondary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. However, sperm acrosome associated 3 protein expression within the ooplasm did not differ significantly between age groups of any follicle type. More research is needed to determine what role sperm acrosome associated 3 protein may play in female fertility in animals as well as what mechanisms regulate ovarian sperm acrosome associated 3 protein expression over time.

The effect of human menstrual blood-derived stem cells on ovarian folliculogenesis, angiogenesis and collagen volume in female rats affected by the polycystic ovary syndrome

BackgroundInfertility is one of the common problems among couples, affecting millions of people worldwide. Polycystic ovary syndrome (PCOS) is one of the main causes of infertility in women and is associated with abnormal folliculogenesis, angiogenesis and fibrosis. Common treatments may lead to numerous adverse effects on the patient’s quality of life. The present study aimed to investigate the effects of human menstrual blood-derived stem cells on the ovarian histology of a PCOS model of Wistar rats.ResultsBased on the Papanicolaou test and H&E staining results, the number of primary, secondary and antral follicles in the PCOS and PCOS-Sham groups significantly increased compared to the control group, while they significantly decreased in the PCOS + Stem cells group compared to the PCOS and PCOS-Sham groups. Further, the number of atretic follicles in both PCOS and PCOS-Sham groups significantly increased in comparison with the control group and decreased in the PCOS + Stem cells group, compared to the two mentioned groups. Moreover, the Graafian follicles number was decreased in the PCOS and PCOS-Sham groups to significantly increase in the PCOS + Stem cells group. Based on Masson’s trichrome staining, the number of blood vessels in PCOS and PCOS-Sham groups significantly increased compared to the control group, while a decrease was observed in the PCOS + Stem cells group, compared to PCOS and PCOS-Sham groups.ConclusionThe administration of MenSCs improved folliculogenesis in rats with polycystic ovaries. Also, MenSCs could ameliorate PCOS symptoms by improving fibrosis as well as angiogenesis and weight gain.

Seasonal changes in endoplasmic reticulum stress and steroidogenesis in the ovary of the wild ground squirrels (Citellus dauricus Brandt)

The development of the follicle is accompanied by steroidogenesis and secretion, the endoplasmic reticulum (ER) requires significant synthesis of relevant proteins to support changes in the follicular microenvironment. The aim of this study was to investigate whether seasonal changes in gonadotropins and ovarian steroid hormones in the wild ground squirrels induce endoplasmic reticulum stress (ERS) and changes in ERS-mediated unfolded protein response (UPR) signaling. There were significant seasonal differences in ovarian mass, with values higher in the breeding season and relatively low in the non-breeding season. Histological observations revealed that ovaries in the breeding season had germ cells including primordial follicles, primary follicles, secondary follicles, tertiary follicles, and the corpus luteal, whereas ovaries consisted mainly of primary and secondary follicles in the non-breeding season. Analysis of ovarian transcriptome data showed that 1298 genes were up-regulated in expression and 1432 genes were down-regulated in expression during both periods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that these genes were mainly enriched in estrogen signaling pathways, ovarian steroidogenesis and endoplasmic reticulum protein processing pathways. The expression levels of steroidogenic enzymes (P450scc, P450c17, 3β-HSD, and P450arom) and gonadotropin receptor (FSHR and LHR) were significantly increased during the breeding season compared to the non-breeding season. GRP78 and UPR signaling factors (ATF4, ATF6, XBP1s) associated with ERS were expressed in both seasons. The mRNA expressions of Atf6 and Xbp1s were higher in the breeding season than those of the non-breeding season. Conversely, Atf4 and its downstream homologous protein (Chop) exhibited higher expression during the non-breeding season. In addition, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17β, and progesterone of serum were significantly higher in the breeding season than those of the non-breeding season. These results suggested that UPR signaling, associated with seasonal changes in ovarian steroidogenesis, was activated during the breeding season and that ERS might be involved in regulating seasonal changes in ovarian steroidogenesis in the wild ground squirrels.

Histomorphological and immunohistochemical studies of the ovary during dry and rainy seasons in Small East African goats (&lt;em&gt;Capra hircus&lt;/em&gt;)

This study aimed to investigate the histomorphological and immunohistochemical changes in the ovaries of Small East African goats during the dry and rainy seasons. A total of 180 ovaries were subjected to analysis using hematoxylin and eosin staining and immunohistochemistry for desmin and smooth muscle actin as cytoskeletal components. The results demonstrated the presence of healthy and atretic primordial, primary, secondary, and antral follicles in the ovaries during the dry and rainy seasons. In the dry season, the proportions of healthy primordial, primary, secondary, and antral follicles were 236 (95%), 85 (97%), 25 (78%), and 6 (27%), respectively, whereas the atretic follicles accounted for 12 (5%), 3 (3%), 7 (22%), and 16 (73%). Conversely, during the rainy season, 157 (99%), 61 (98%), 48 (93%), and 31 (92%) were classified as healthy follicles, while 2 (1%), 1 (2%), 4 (7%), and 3 (8%) were categorized as atretic. There was a significant difference in healthy and atretic follicles between the dry and rainy seasons (P &lt; 0.05). Immunohistochemical analysis revealed positive staining for both desmin and smooth muscle actin in the theca cells of secondary and antral follicles, cortical stroma, and tunica media of blood vessels in healthy and atretic follicles during dry and rainy seasons. However, staining was not observed in the granulosa cells and oocytes. Overall, this study indicates that most antral follicles undergo obliterated follicular atresia during the dry season.

Rescue of follicle development after oocyte-induced ovary dysfunction and infertility in a model of POI.

The mechanisms and aetiology underlying the development of premature ovarian insufficiency (POI) are poorly understood. However, the oocyte clearly has a role as demonstrated by the Double Mutant (DM) mouse model where ovarian dysfunction (6weeks) is followed by POI (3months) due to oocyte-specific deletion of complex and hybrid N- and O-glycans. The ovaries of DM mice contain more primary follicles (3a stage) accompanied by fewer developing follicles, indicating a block in follicle development. To investigate this block, we first analysed early follicle development in postnatal (8-day), pre-pubertal (3-week) and post-pubertal (6-week and 3-month) DM (C1galt1 F/F Mgat1 F/F:ZP3Cre) and Control (C1galt1 F/F Mgat1 F/F) mice. Second, we investigated if transplantation of DM ovaries into a "normal" endocrine environment would restore follicle development. Third, we determined if replacing DM ovarian somatic cells would rescue development of DM oocytes. At 3-week, DM primary 3a follicles contain large oocytes accompanied by early development of a secondGC layer and increased GC proliferation. At 6-week, DM primary 3a follicles contain abnormally large oocytes, accompanied with decreased GC proliferation. Transplantation of DM ovaries into a 'normal' endocrine environment did not restore normal follicle development. However, replacing somatic cells by generating reaggregated ovaries (ROs) did enable follicle development to progress and thus highlighted intra-ovarian factors were responsible for the onset of POI in DM females. Thus, these studies demonstrate oocyte-initiated altered communication between GCs and oocytes results in abnormal primary follicles which fail to progress and leads to POI.

Mouse primary follicles experience slow growth rates after activation and progressive increases that influence the duration of the primary follicle phase†.

There are conflicting estimates of the duration of mouse primary follicle development. An accurate determination is needed for studies examining preantral follicle survival and mathematical modeling of folliculogenesis. Primary follicle granulosa cell proliferation rates are low and variable, which may explain the variation in duration estimates. In the present study, female C57Bl6/J mice were exposed to bromodeoxyuridine for 48hours, to label the proliferating granulosa cells in a large proportion of primary follicles. The bromodeoxyuridine-containing water was then withdrawn and replaced with drug-free water and the mice were euthanized at 0, 1, 3, 6, 10, or 13days post-bromodeoxyuridine withdrawal. Granulosa cells were bromodeoxyuridine labeled in 48% of primary follicles at day 0, but this decreased to 5% over the 13-day period, as the labeled primary follicles progressed to the secondary follicle stage. Curve-fitting estimated that the last of the bromodeoxyuridine-labeled primary follicles would progress to the secondary stage by 13.7days. Mathematical models that assumed constant rates of primary follicle proliferation were fitted to the data, but the observed pattern of bromodeoxyuridine-labeled primary follicle disappearance could not be replicated. The level of immunoreactivity for bromodeoxyuridine and proliferating-cell nuclear antigen in primary follicles revealed follicles with no granulosa cell proliferation during the 48-h bromodeoxyuridine-exposure period had resumed proliferation 1 or 3days later. Therefore, primary follicle granulosa cells proliferate after follicle activation, but proliferation rates gradually increase as the follicle develops. Prior estimates of primary follicle duration are inaccurate due to the assumption that follicles develop at a constant rate.

The effects of human amnion membrane-derived mesenchymal stem cells conditioned medium on ionizing radiation-induced premature ovarian failure and endoplasmic reticulum stress-related apoptosis mechanism

ObjectivesPremature ovarian failure (POF) is defined as the cessation of menstrual periods for at least 4–6 months before the age of 40 years, accompanied by FSH values measuring over 40 IU/L for a month. Radiation therapy, one of the cancer treatment methods, is known to accelerate ovarian aging by reducing and eliminating the number of primordial follicles in the ovarian follicle pool. Ionizing radiation has been reported to cause POF. The objective of this study is to investigate the impact of mesenchymal stem cell conditioned medium (hAMSCs-CM), which is isolated from the amniotic membrane of human placenta, on premature ovarian failure (POF) caused by whole-body irradiation. The study will focus on the ER stress and apoptosis mechanisms in the process. Study DisaynA POF model was created by exposing rats to 7 Gy of whole-body irradiation. Serum-free hAMSCs-CM were then administered via the tail vein. Follicle count was performed on the ovaries, and immunohistochemistry was used to determine the expressions of GRP78, CHOP, IRE-1, caspase-12, caspase-9, caspase-3. TUNEL was also carried out, and levels of serum FSH, LH, E2, AMH, and oxidative stress marker 8-OHdG were measured. Results and ConclusionThe application of hAMSCs-CM has been found to have a positive impact on follicles affected by radiation. After treatment, the number of primordial, primary, secondary, and graafian follicles, which had previously decreased due to radiation, showed an increase. Furthermore, the number of atretic follicles, which had been increasing due to radiation, showed a decrease. ER is one of the targets affected by ionizing radiation. After ionizing radiation, the expressions of ER stress-related markers and apoptosis markers increased in the ovary. After hAMSCs-CM administration, the expressions of these markers and number of TUNEL-positive cells decreased. Following irradiation, anti-mullerian hormone (AMH) and estradiol (E2) levels decreased, while follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels increased. After administration of hAMSCs-CM, AMH and E2 levels increased, while FSH and LH levels decreased.Amnion membrane-derived mesenchymal stem cell conditioned medium can play a therapeutic role in ionizing radiation-induced premature ovarian failure by reducing endoplasmic reticulum stress and apoptosis.

Treatment strategies with vitamin E and C in autologous intraperitoneal ovarian transplantation and its impact on ovarian surface epithelium and follicle reserve

The objective of this study was to assess the impact of Vitamin E (Vit E) and Vitamin C (Vit C) on markers of the oxidant-antioxidant system, ovarian follicle reserves, and the surface epithelium in autologous intraperitoneal ovarian transplantation conducted in rats. The study aimed to investigate how these antioxidants influence various aspects related to transplantation outcomes, including oxidative stress markers, the preservation of follicle reserves, and the condition of the surface epithelium. A total of 20 adult female Wistar Albino rats were included in the study and randomly assigned to four different groups. Group 1, consisting of 5 rats, served as the control group and underwent a surgical procedure where their abdomens were opened and closed without any further intervention. Group 2, also consisting of 5 rats, underwent ovarian transplantation. In Group 3, comprising 5 rats, an intraperitoneal (IP) administration of 20 mg/kg body weight (b.w.) of Vitamin E (Vit E) was given 15 min prior to ovarian transplantation. Lastly, in Group 4, which included 5 rats, an IP administration of 50 mg/kg body weight (b.w.) of Vitamin C (Vit C) was given 15 min before ovarian transplantation. Vaginal cytology was performed in order to monitor the estrus phase in the rats. Biochemically, tissue and serum malondialdehyde (MDA) levels and erythrocyte superoxide dismutase (SOD) levels were measured. Histopathologically, the number of dysplastic changes in the ovarian surface epithelium and primordial, primary, secondary, Graaffian, and atretic follicles were examined. Dysplastic changes in the surface epithelium of Group 2 were found to be significantly higher than in Group 1 and 4 (p < 0.02). In Group 2, the ovarian follicle reserves (primordial, primary, secondary, and Graaffian follicles) were significantly lower than in other groups (p < 0.02). In addition, a significant decrease in SOD levels was found in Group 2 compared to other groups (p < 0.02). The study showed that Vit E and Vit C in autologous intraperitoneal ovarian transplantation preserved the ovarian follicle reserve. Vit C was found to be more effective than Vit E.

Image analysis quantification of sperm acrosome associated 3 protein expression in domesticated and free‐roaming equine ovaries

SummaryBackgroundIn the United States, the carrying capacity of the land has been overwhelmed with the wild horse and burro populations. Identification of a long‐term contraception method is needed to control growing populations as current methods cannot quell these growing numbers. Sperm acrosome‐associated 3 protein (SPACA3) is a protein expressed in both male and female reproductive systems. SPACA3 may prove to be a viable contraception target in horses in immunosterilant research.ObjectivesThe objective of the current study was to evaluate and compare ovarian immunoexpression of SPACA3 between domesticated and free‐roaming mares. This information may lead to the development of a SPACA3 immunosterilant to reduce free‐roaming horse and burro populations.Study designNon‐randomised comparative study design.MethodsRoutine immunohistochemistry was performed on ovarian sections from domesticated (n = 8) and free‐roaming mares (n = 8). At least three representative images of each follicle stage (primordial, primary, secondary, tertiary) from each ovary were digitally captured and analysed using Image J software. Expression of SPACA3 was quantified in cellular locations (granulosa or theca) specific to each follicle stage: primordial, primary, secondary, and tertiary.ResultsDomesticated horses had higher SPACA3 expression compared to free‐roaming mares in granulosa cells of primordial (p = 0.0022), primary (p &lt; 0.001), secondary (p = 0.025), and tertiary follicles (p = 0.0078). In theca cells, domesticated horses had higher SPACA3 expression in tertiary follicles (p = 0.022), but not secondary follicles (p = 0.089). The highest SPACA3 expression was observed in the granulosa cells of tertiary follicles (p = 0.022).Main limitationsVarying background colours of representative images taken of ovaries, leading to slight technical issues in Image J software.ConclusionsThis is the first report quantifying SPACA3 immunoexpression during folliculogenesis and describing the spatial and temporal expression of SPACA3 in the equine ovary. This information may lead to the development of a SPACA3 immunosterilant to reduce free‐roaming horse and burro populations.

Comparative studies on ovary and serum hormone levels in silver and blue foxes and their hybrids

The silver and blue fox appear closely related despite their belonging to different genera, and the male and female fox hybrids are completely sterile in mating inter se or back to either of the parental groups. In the present study, we compared ovarian morphology and serum hormone concentrations among silver foxes, blue foxes, HSBs (silver fox male × blue fox female hybrids) and HBSs (blue fox male × silver fox female hybrids) during the pre-breeding and the breeding period. We found that either the fertile blue and silver foxes or the infertile hybrid foxes showed typical characteristics of ovary, with primordial, primary, secondary, antral and Graafian follicles. The diameters and quantities of different follicle classes in fertile foxes were greater than that in hybrids during the two period, while the thickness of zona pellucida (ZP) represented an opposite trend (P < 0.05). The relative weight of bilateral ovaries in fertile foxes was greater than that in hybrids during the pre-breeding period, while mean serum FSH represented an opposite trend during the breeding period (P < 0.05). The mean serum estradiol in fertile foxes was greater than that in HBS foxes during the pre-breeding period, and greater than hybrids during the breeding period (P < 0.05). These findings indicated that in the infertile hybrid foxes, lower estradiol levels produced by lesser and smaller antral follicles, thicker ZP during the two period, higher FSH levels during the breeding period, could be the basis of interpretations in cases of female fox hybrid infertility.

The dynamic expression of SOX17 in germ cells from human female foetus and adult ovaries after specification.

SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood. We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker). SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA+ germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17+VASA- germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3. The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17+ VASA- germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation.

Human-relevant exposure to di-n-butyl phthalate tampers with the ovarian insulin-like growth factor 1 system and disrupts folliculogenesis in young adult mice.

Phthalates are compounds used in consumer and medical products worldwide. Phthalate exposure in women has been demonstrated by detection of phthalate metabolites in their urine and ovarian follicular fluid. High urinary phthalate burden has been associated with reduced ovarian reserve and oocyte retrieval in women undergoing assisted reproduction. Unfortunately, no mechanistic explanation for these associations is available. In short term in vivo and in vitro animal studies modeling human-relevant exposures to di-n-butyl phthalate (DBP), we have identified ovarian folliculogenesis as a target for phthalate exposures. In the present study, we investigated whether DBP exposure negatively influences insulin-like growth factor 1 (IGF1) signaling in the ovary and disrupts ovarian folliculogenesis. CD-1 female mice were exposed to corn oil (vehicle) or DBP (10 µg/kg/day, 100 µg/kg/day, or 1000 mg/kg/day) for 20-32 days. Ovaries were collected as animals reached the proestrus stage to achieve estrous cycle synchronization. Levels of mRNAs encoding IGF1 and 2 (Igf1 and Igf2), IGF1 receptor (Igf1r), and IGF-binding proteins 1-6 (Ifgbp1-6) were measured in whole ovary homogenates. Ovarian follicle counts and immunostaining for phosphorylated IGF1R protein (pIGF1R) were used to evaluate folliculogenesis and IGF1R activation, respectively. DBP exposure, at a realistic dose that some women may experience (100 µg/kg/day for 20-32 days), reduced ovarian Igf1 and Igf1r mRNA expression and reduced small ovarian follicle numbers and primary follicle pIGF1R positivity in DBP-treated mice. These findings reveal that DBP tampers with the ovarian IGF1 system and provide molecular insight into how phthalates could influence the ovarian reserve in females.

Dynamic and broad expression of adamts9 in developing and adult zebrafish.

Previous studies showed that Adamts9 is involved in multiple functions including ovulation, spine formation, primordial germ cell migration, and development of primary ovarian follicles in animals. However, systemic examination and high-resolution analyses of adamts9 expression are missing due to lack of a sensitive reporter assay. In the present study, we created a new transgenic zebrafish reporter line Tg(adamts9:EGFP) and assayed its expression in various tissues and cells during development and in adults at high-resolution using confocal imaging. Reporter expression was validated with real-time quantitative PCR, whole mount in situ hybridization, and immunohistochemistry for endogenous adamts9. Strong expression of the adamts9:EGFP transgene was found in a wide range of adult and embryonic zebrafish tissues/cells including ovaries, testes, brains, eyes, pectoral fins, intestine, skin, gill, muscle, and heart; while lower expression was observed in the liver and growing ovarian follicles (stages II and III). Our results of a broad and dynamic expression pattern for this evolutionary conserved metalloprotease suggest involvement of adamts9 in the development and physiological functions of various tissues in animals.