Folliculogenesis Research Articles

Hypoglycemic effects of propolis extract on polycystic ovarian syndrome in Wistar rats

Background: Polycystic ovarian syndrome (PCOS) is an endocrinopathy that causes various metabolic syndromes, including insulin resistance in 20-40% of patients. The treatment of the condition is often carried out using metformin, but it has been reported to have different gastrointestinal side effects. Consequently, several studies have recommended the use of propolis as an alternative, which has proven effective in improving insulin resistance in diabetic-induced rats and causes fewer side effects. Objective: This study aimed to compare the effects of propolis and metformin in treating insulin resistance based on homeostasis model assessment of insulin resistance (HOMA-IR) value. Materials and Methods: This study used post-test-only randomized control trial design and the sample population comprised 30 Wistar rats. The animals were divided into 5 groups: control (C), PCOS (PCO), PCOS with metformin (PCO+M), PCOS with propolis extract (PCO+P), and PCOS with metformin and propolis extract (PCO+MP). Subsequently, blood sampling, ovarian tissue sampling, and vaginal smearing were performed after 28 days of treatment. Results: PCO+P and PCO+M groups experienced a significant decrease in testosterone concentration and preantral follicle count compared to PCOS group but the anovulation cycle was similar. Fasting blood glucose concentration in PCO+MP group showed a significant decrease compared to others. However, fasting insulin concentration and HOMA-IR value in treatment groups did not show any reduction compared to PCOS group. Conclusion: Based on the results, propolis extract and metformin could not improve insulin resistance based on HOMA-IR value. However, propolis extract had better hypoglycaemic effects compared to metformin. The combination of both treatments had the potential to lower fasting glucose concentration.

Effect of age on follicular dynamics of goat ovaries

Reproductive aging is influenced by a gradual decline in both the quantity and quality of oocytes found within ovarian follicles. In mammals, fertility relies heavily on the health of oocyte reserves, the dynamics of follicular development, and the functioning of the ovaries. This study aimed to investigate the impact of age on the follicular dynamics of goat ovaries. A total of 40 ovaries of Black Bengal goats were obtained from a local slaughterhouse and sorted into four age groups: 6-12 months, 13-15 months, 18-21 months, and 22-24 months. Subsequently, the ovaries were treated, and follicles were assessed through visually and histologically. The findings revealed that nearly all parameters, including weight, length, and width, of both ovaries in the 18-21 months age group were insignificantly different (p>0.05) from the other age groups. Histologically, the number of normal primordial and primary follicles was also insignificantly higher (p>0.05) in goats aged 18-21 months compared to the other age groups. Conversely, the quantity of secondary follicles significantly increased (p<0.05) with age, but in terms of antral follicles, a quadratic trend was observed, where they increased up to a certain age and then declined. Additionally, the results showed that the number of degenerated primary and antral follicles was significantly higher (p<0.05) in the ovaries of 22–24-month-old goats, while the number of degenerated secondary follicles was insignificantly higher (p>0.05) compared to the remaining age groups. In conclusion, age had an effect on the follicular dynamics of goat ovaries, suggesting that ovaries from goats aged 18-21 months, sourced from slaughterhouses, hold potential for quality follicles and oocytes that can be valuable for further research. Bangladesh Journal of Animal Science 52 (4): 88-97

Effect of age on follicular dynamics of goat ovaries

Reproductive aging is influenced by a gradual decline in both the quantity and quality of oocytes found within ovarian follicles. In mammals, fertility relies heavily on the health of oocyte reserves, the dynamics of follicular development, and the functioning of the ovaries. This study aimed to investigate the impact of age on the follicular dynamics of goat ovaries. A total of 40 ovaries of Black Bengal goats were obtained from a local slaughterhouse and sorted into four age groups: 6-12 months, 13-15 months, 18-21 months, and 22-24 months. Subsequently, the ovaries were treated, and follicles were assessed through visually and histologically. The findings revealed that nearly all parameters, including weight, length, and width, of both ovaries in the 18-21 months age group were insignificantly different (p>0.05) from the other age groups. Histologically, the number of normal primordial and primary follicles was also insignificantly higher (p>0.05) in goats aged 18-21 months compared to the other age groups. Conversely, the quantity of secondary follicles significantly increased (p<0.05) with age, but in terms of antral follicles, a quadratic trend was observed, where they increased up to a certain age and then declined. Additionally, the results showed that the number of degenerated primary and antral follicles was significantly higher (p<0.05) in the ovaries of 22–24-month-old goats, while the number of degenerated secondary follicles was insignificantly higher (p>0.05) compared to the remaining age groups. In conclusion, age had an effect on the follicular dynamics of goat ovaries, suggesting that ovaries from goats aged 18-21 months, sourced from slaughterhouses, hold potential for quality follicles and oocytes that can be valuable for further research. Bangladesh Journal of Animal Science 52 (4): 88-97

Anti-Müllerian hormone: a new approach to fertility assessment in cattle – a review

The anti-Müllerian hormone (AMH) enhances fertility in cattle and hence used as a fertility marker. AMH is produced by granulosa cells of all primordial, primary, secondary follicles, and antral follicles up to 4-5 mm diameter, whereas AMH production decreases once antral stage follicles reach the 4-5 mm stage. There is an individual variation in antral follicle count (AFC) and AMH concentration. One-time determination of AMH concentration will help predict the animals’ reproductive performance at the heifer stage. In turn, it will help to cull those animals with low reproductive potential. There is no significant fluctuation in the AMH concentration during the estrous cycle, and hence it can be determined at any stage of the estrous cycle. This review explores the potential of AMH as a fertility marker and its association with the antral follicle population (AFP) as AMH can be an effective segregative tool to screen low, average or high fertility future performance in cattle.

Naproxen administration affects murine late folliculogenesis, reduces granulosa cell proliferation and the number of ovulated oocytes

Naproxen reduces the production of prostaglandins via inhibition of the cyclooxygenase. Studies have shown that its administration in women can be related to failed ovulation. Therefore, preclinical investigations must be performed in order to investigate its effects in experimental models. Thus, the aim of this study was to evaluate the effects of naproxen on murine folliculogenesis, ovulation, and female fertility. Female C57BL/6 mice (n = 128 - 6 weeks old) were divided into Control, low (10 mg/kg), and high naproxen (50 mg/kg) groups, who were treated for 8 days and directed to morphofunctional analyses. Follicular quantification showed a reduced percentage of antral follicles in naproxen-treated animals. These treated animals also showed smaller oocytes included in secondary and antral follicles, and the diameter of secondary and antral follicles was also reduced. A reduction in the percentage of Ki67-positive granulosa cells was observed in treated animals that also showed down-regulation of Igf1r compared to control. After an ovarian stimulation protocol, naproxen-treated animals showed a reduction in the percentage of secondary and antral follicles, a reduced number of ovulated oocytes and, corpora lutea, and an increased number of failed ovulations. Finally, naproxen-treated animals also showed a reduction in mating index and pregnancy rate. Our findings suggested that, in mice, naproxen administration (eight days treatment) negatively affects molecular and morphological aspects related to late folliculogenesis, ovulation, and fertility.

Comparison of effects of curcumin and beta-carotene on ovarian damage caused by cisplatin

Purpose: We aimed the potential protective effects of curcumin (CUR) and beta-carotene (BC) against cisplatin (CIS)-induced ovarian damage using histological and immunohistochemical methods.
 Materials and Methods: We used 56 female Wistar albino rats, divided randomly into seven groups. Control rats did not receive any treatment; the sham group was administered 1 ml/kg sesame oil by gavage; the CIS group 5 mg/kg CIS; the CUR group 200 mg/kg CUR; the BC group 100 mg/kg BC; the CUR + CIS group, CIS after administration of CUR; the BC + CIS group was pretreated with BC, then administered CIS. The ovaries of all groups were excised five days after the last application. We assessed histopathology and counted and classified follicles. Expression of anti-Mullerian hormone (AMH) and nuclear factor-kappa B (NF-κB) was detected immunohistochemically. Apoptosis was evaluated using the TUNEL method.
 Results: CUR and BC are protective against decreased numbers of primordial, primary, preantral, secondary, and tertiary follicles caused by CIS. Both Cur and BC reduced ovarian NF-κB levels in comparison to the control group, and AMH immunoreactivity was almost identical for the control and CUR + CIS groups. Apoptotic cell counts indicated that CUR exerts a stronger anti-apoptotic effect than BC.
 Conclusion: CUR has a protective effect against ovarian damage brought on by CIS and greater antioxidant and anti-inflammatory properties than BC.

The Comparative Effect of Citrullus colocynthis Hydro-Alcoholic Extract and Metformin on Morphometric Ovarian Follicles Disorders in Estradilol valerate-Iinduced Polycystic Ovary Syndrome Rats

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 6-10% of women in reproductive age. The medicinal values of Citrullus colocynthis extract (CCT) stems from its anti-oxidative and anti-inflammatory effects. This study evaluated the comparative effect of CCT and metformin on morphometric ovarian disorders in PCOS rats. Materials and Methods: Fifty female Wistar rats having 2-3 consecutive estrous cycles during two weeks were divided into five groups (n=10 each group). The rats in the control group only received sesame oil as estradiol valerate solvent, whereas the rats in the sham group were injected intramuscularly with 4 mg/rat estradiol valerate-induced PCOS. Following PCOS induction, the rats in the metformin group received 50 mg/kg metformin orally for three weeks. Rats in the Extract group were treated with 50 mg/kg plant extract for 21 days following the induction with PCOS. Additionally, the rats in metformin+ extract group were treated with the combination of 50 mg metformin plus plant extract for three weeks. The ovaries were removed and were fixed for tissue processing. The slices were stained by hematoxylin-eosin after clearing and hydration. Follicular count and morphometric changes were evaluated in primordial, primary, pre-antral and antral follicles. Results: The mean diameter of primordial follicle was similar in all groups, but mean diameter of primary follicle decreased in the sham group (145.50± 49.26 µm) compared to metformin (278.76± 156.64 µm), extract (311.70± 147.74 µm) and metformin+ extract groups (265.35± 88.16 µm). The diameter of pre-antral and antral follicles in the sham group were significantly larger than those in the control group, but were not significantly different in all other groups including metformin, extract, and metformin+ extract in comparison with control group (P< 0.05). Conclusion: In this study, the data has demonstrated that CCT like metformin could improve follicular morphometric disorders in PCOS rats. [GMJ.2018;7:e1045] Â

Enhancement of Melanocortin-4 Receptor (MC4R) and Constancy of Kiss1 mRNAs Expression in the Hypothalamic Arcuate Nucleus in a Model of Polycystic Ovary Syndrome Rat

Background: Hypothalamic Melanocortin-4 Receptor (MC4R) and kiss1/kisspeptin systems play roles in reproductive processes. This study was conducted to evaluate changes in MC4R and kiss1 genes expression in the arcuate nucleus (ARC) of the hypothalamus and its relationship with polycystic ovary syndrome (PCOS) in rats. Materials and Methods: In the current experimental study, 24 female rats were randomly and equally allocated into nulliparous and primiparous groups and then were divided into two subgroups of PCOS and control. PCOS was induced by exposure to continuous light. Sex-related hormones were evaluated by radioimmunoassay or immunoradiometric assay. Expressions of MC4R and kiss1 gene in the ARC of the hypothalamus of the rats were evaluated by real-time PCR. Histomorphometric alterations of ovaries were compared between groups. Results: Number of tertiary follicles and their size and number of atretic follicles in the PCOS subgroups were more than those in the controls (P<0.05) whereas the number of secondary follicles and corpus luteum in the PCOS subgroups were lower than those in the controls (P<0.05). Antrum and total diameters of tertiary follicles in the PCOS subgroups were greater and granulosa layer diameter was lower than those in the controls (P<0.05). The MC4R mRNA expression in the PCOS subgroups was 6.5-fold in nulliparous and 3.5-fold in primiparous groups more than their controls’ pairs (P<0.05). However, parity did not affect the expression of MC4R gene (P>0.05).The kiss1 mRNA expression in the PCOS and control subgroups was not significantly different (P>0.05). Conclusion: Overexpression of MC4R gene after PCOS induction in the ARC of the hypothalamus may link to metabolic disorders of induced PCOS in the rats. However, alteration in the kiss1 mRNA expression after PCOS induction was not observed in the rats. [GMJ.2018;7:e1070]

Effect of dexpanthenol on cyclophosphamide-induced ovarian toxicity: a histological and molecular study in rats

Research questionDoes dexpanthenol work as an effective therapeutic agent against cyclophosphamide (CYC)-induced premature ovarian failure (POF) in rats? DesignA total of 28 female Wistar Albino rats were randomly divided into four groups (n = 7 per group). The POF and POF plus dexpanthenol groups were intraperitoneally administered CYC at an initial dose of 50 mg/kg, followed by 8 mg/kg for 14 days. The dexpanthenol and POF plus dexpanthenol groups were both intraperitoneally administered dexpanthenol at a dose of 500 mg/kg/day for 15 days. ResultsIn the group administered CYC, the following was observed: a decrease in the ovarian index; a decrease in the numbers of primordial, primary, secondary and antral follicles; an increase in the number of corpus luteum and atretic follicles; a decrease in proliferation cell nuclear antigen immunoreactivity; a significant reduction in anti-Müllerian hormone and oestradiol levels; and an increase in serum FSH levels compared with controls. Dexpanthenol, on the other hand, reversed these effects. Quantitative reverse transcription polymerase chain reaction analyses showed that dexpanthenol increased Bcl-2, Akt1, mTOR, Nrf2 and HO-1 in CYC-induced ovarian tissues, but decreased Bax, Cas3, Hsp27, Hsp70, and Hsp90. Dexpanthenol treatment has a potential for inhibiting the intrinsic apoptotic pathway and oxidative stress levels in ovarian tissues via the downregulation of the mRNA expression of heat shock proteins and the activation of Nrf2/HO-1 pathways. ConclusionsOur findings demonstrated that dexpanthenol is an effective agent against POF caused by CYC; however, further experimental and clinical data are needed to use it effectively.

Effects of bovine ovarian storage conditions and vitrification on isolated preantral follicle viability.

The decreasing rate of successful pregnancies, both naturally and through assisted conception, has led to innovations in the way eggs, sperm, and embryos are stored. Despite these advances, the use of assisted reproductive techniques to preserve endangered or rare species remains unexplored. Since the location where samples are collected and facilities are often far apart, we aim to address part of this challenge by comparing different methods to store and handle ovarian tissue before freezing. This may pave the way for further research in preserving endangered species, despite the challenges posed by the distance between sample collection sites and suitable facilities.

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment.

Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro? A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth, and hormone production between the different groups. Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (P < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 μm compared to 67.3 ± 7.2 μm without it (P = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (P < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; P < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 μm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (P = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). N/A. This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

Decreased fertility in female mice lacking urokinase plasminogen activator

It is well established that mouse ovarian granulosa cells secrete urokinase plasminogen activator (uPA) under gonadotropin stimulation. The synthesis and secretion of the enzyme correlate well with the time of follicular rupture in vivo. Moreover, uPA is secreted by the trophoblast at the time of implantation. In the present study, we have analyzed whether the absence of uPA could influence follicular growth, ovulation, and embryo implantation. Our data show fewer preantral follicles in uPA-/- ovaries but no decrease in hormonally induced ovulation. However, we observed a significant decrease in the number of implanted embryos in uPA-/- animals and, therefore, a lower number of pups per family. Adding uPA to the epithelial and stromal uterine cell culture medium strongly upregulates the expression of prostaglandin-endoperoxide synthase 2 (Ptgs2), the enzyme required for prostaglandin production and embryo implantation. The uPA inhibitor amiloride abrogated this increase.

Effect of the acidified extract of Moringa oleifera leaves as a supplement in the in vitro culture medium of sheep preantral follicles

This study was conducted to evaluate the effects of the acidified extract of M. oleifera leaves as a supplement into the base medium for in vitro culture of sheep isolated secondary follicles. Follicles were isolated and cultured for 12 days in α-MEM+(supplemented with bovine serum albumin, insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid) with or without 0.1; 0.2 or 0.4 mg/ml of the acidified extract of M. oleifera. Follicle morphology, antral cavity formation, follicular and oocyte diameter, glutathione (GSH) concentration, mitochondrial activity and meiotic resumption were evaluated. After 12 days of culture, there was no significant difference among treatments in relation to follicular morphology, antral cavity formation, diameter and mitochondrial activity. Nevertheless, oocytes from follicles cultured in α-MEM+ showed greater GSH concentration than media containing M. oleifera extract. Furthermore, the concentration of 0.4 mg/ml M. oleifera extract significantly increased the percentage of fully grown oocyte (≥ 110 µm) when compared to the other treatments. In conclusion, the concentration of 0.4 mg/ml M. oleifera extract as a supplement of the culture medium, maintained the survival, and increased the percentage of fully grown oocytes.

Kisspeptin stimulates sheep ovarian follicular development in vitro through homologous receptors.

The present study was conducted to elucidate (1) the influence of kisspeptin (KP) on the in vitro development of preantral follicles (PFs) and (2) evolution of KP receptor gene (KISS1R) expression during ovarian follicular development in sheep. Kisspeptin was supplemented (0-100 µg/ml) in the culture medium of PFs for 6 days. The cumulus-oocyte complexes (COCs) from cultured PFs were subsequently matured to metaphase II (MII) for an additional 24 h. The proportions of PFs exhibiting growth, antrum formation, average increase in diameter, and maturation of oocytes to MII stage were the indicators of follicular development in vitro. The expression of the kisspeptin receptor gene at each development stages of in vivo developed (preantral, early antral, antral, large antral and COCs from Graafian follicles) and in vitro cultured PFs supplemented with KP was assessed using a real-time polymerase chain reaction. The best development in all the parameters under study was elicited with 10 µg/ml of KP. Supplementation of KP (10 µg/ml) in a medium containing other growth factors (insulin-like growth factor-I) and hormones (growth hormone, thyroxine, follicle-stimulating hormone) resulted in better PF development. The KISS1R gene was expressed in follicular cells and oocytes at all the development stages of both in vivo developed and in vitro cultured follicles. Higher KISS1R gene expression was supported by culture medium containing KP along with other hormones and growth factors. Accordingly, it is suggested that one of the mechanisms through which KP and other growth factors and hormones influence the ovarian follicular development in mammals is through the upregulation of expression of the KP receptor gene.

FEATURES OF WOOL QUALITIES AND FORMATION OF SKIN COVER OF YOUNG SHEEP OF THE SOUTH KAZAKH MERINO BREED IN ZHAMBYL REGION CONDITIONS

Until 2030, 17 key areas were selected, the implementation of which could potentially lead the country to the sustainable development of all major spheres of life and the solution of global problems affecting every person in this world. The article reflects and presents scientific research data, objective characteristics of wool qualities and age-related changes signs of the structure of the skin of young South Kazakh merino sheep, as well as studies of the morphological parameters of the skin tissue in the age aspect (from birth to 18 months), and an analysis of their connection with the commercial properties and quality of the semi-finished product obtained from sheep of this breed is given. Productive qualities were studied in a flock of sheep of the South Kazakh Merino breed in “Batay-Shu” LLP. Sheep have a high live weight, shearing and yield of pure wool; rams have a fairly high live weight for the breed; component depending on age from 100-106 kg, and high wool productivity both in physical weight and in washed fiber - 10.3-6.6 kg, with an average output of washed wool of 64.5% thickness, with corresponding development epidermis (0.97-1.3% of the total skin thickness), a well-developed pilar layer (up to 67.7% of the total skin thickness) and relatively weak development of the reticular layer (about 33% of the total skin thickness). The density of hair follicles per 1 mm2 at 18 months of age is 52-60 units, and the secondary follicles/ primary follicles ratio is 12.17-12.77. After weaning and up to 7.5 months, buck lambs experience a noticeable increase in skin within 200 μm. During the same period, a slight increase in skin tissue occurs in ewe hoggs.

The effect of Coenzyme Q10 on mitochondrial biogenesis in mouse ovarian follicles during in vitro culture.

The aim of this research was to investigate the effect of Coenzyme Q10 (CoQ10) on the expression of the Transcription Factor A Mitochondrial (Tfam) gene and mtDNA copy number in preantral follicles (PFs) of mice during in vitro culture. To conduct this experimental study, PFs were isolated from 14-day-old National Medical Research Institute mice and cultured in the presence of 50 µm CoQ10 for 12 days. On the 12th day, human chorionic gonadotropin was added to stimulate ovulation. The fundamental parameters, including preantral follicle developmental rate and oocyte maturation, were evaluated. Additionally, the Tfam gene expression and mtDNA copy number of granulosa cells and oocytes were assessed using the real-time polymerase chain reaction. The results revealed that CoQ10 significantly increased the diameter of PFs, survival rate, antrum formation, and metaphase II (MII) oocytes (P < 0.05). Moreover, in the CoQ10-treated groups, the Tfam gene expression in granulosa cells and oocytes increased considerably compared with the control group. The mtDNA copy number of granulosa cells and oocytes cultured in the presence of CoQ10 was substantially higher compared with the control groups (P < 0.05). The addition of CoQ10 to the culture medium enhances the developmental competence of PFs during in vitro culture by upregulating Tfam gene expression and increasing mtDNA copy number in oocyte and granulosa cells.

Early-Life Exposure to the Mycotoxin Fumonisin B1 and Developmental Programming of the Ovary of the Offspring: The Possible Role of Autophagy in Fertility Recovery.

Mycotoxins are produced by more than one hundred fungi and produce secondary metabolites that contaminate various agricultural commodities, especially rice and corn. Their presence in the food chain is considered a serious problem worldwide. In recent years, a link between exposure to mycotoxins and impaired fertility has been suggested. Consequently, it has become vital to investigate the interactive effects of these mycotoxins on ovarian function. In this study, we investigated the intergenerational effects of the mycotoxin fumonisin B1 (FB1) on ovarian structure and function. Virgin Wistar albino female rats were separated into control and FB1 treatment groups and examined from day 6 of pregnancy until delivery (20 and 50 mg/kg b.w./day). The obtained female rats of the first (F1) and second generations (F2) were euthanized at 4 weeks of age, and ovary samples were collected. We found that the ovary weight index increased with the high dose of the treatment (50 mg/kg b.w./day) among both F1 and F2, in a manner similar to that observed in polycystic ovary syndrome. As expected, FB1 at a high dose (50 mg/kg b.w.) reduced the number of primordial follicles in F1 and F2, leading to an accelerated age-related decline in reproductive capacity. Moreover, it reduced the fertility rate among the F1 female rats by affecting follicle growth and development, as the number of secondary and tertiary follicles decreased. Histopathological changes were evidenced by the altered structures of most of the growing follicle oocytes, as revealed by a thinning irregular zona pellucida and pyknosis in granulosa cells. These findings are concomitant with steroidogenesis- and folliculogenesis-related gene expression, as evidenced by the decrease in CYP19 activity and estrogen receptor beta (ESR2) gene expression. Additionally, GDF-9 mRNA levels were significantly decreased, and IGF-1 mRNA levels were significantly increased. However, the results from the ovaries of the F2 treatment groups were different and unexpected. While there was no significant variation in CYP19 activity compared to the control, the ESR2 significantly increased, leading to stereological and histopathological changes similar to those of the control, except for some altered follicles. The hallmark histological feature was the appearance of vacuolar structures within the oocyte and between granulosa cell layers. Interestingly, the autophagic marker LC3 was significantly increased in the F2 offspring, whereas this protein was significantly decreased in the F1 offspring. Therefore, we suggest that the promotion of autophagy in the ovaries of the F2 offspring may be considered a recovery mechanism from the effect of prenatal FB1 exposure. Thus, autophagy corrected the effect of FB1 during the early life of the F1 female rats, leading to F2 offspring with ovarian structure and function similar to those of the control. However, the offspring, treated female rats may experience early ovarian aging because their ovarian pool was affected.

Morphological assessment of ovaries during stimulation of ovulation in rab-bits with equine serum gonadotropin in different doses

Ovulatory stimulation of rabbits during artificial insemination requires the use of hormonal drugs to stimulate maturation of follicles, effective fertilization and full manifestation of female sexual function. For this purpose, gonadotropins or analogues of gonadotropin-releasing hormone are common in the practice of rabbit breeding. The latter have a selective effect on folliculogenesis and, under certain conditions, are not capable of inducing ovulation in rabbits of “zero” reproductive cycle, that is, those that have not given birth. Instead, gonadotropins, in particular serum gonadotropin obtained from foal mares (eCG), have a pronounced effect on folliculogenesis in animals. eCG is a glycoprotein hormone secreted by trophoblastic epithelial cells of fetal origin. In other species, except for horses, it is able to induce the release of both luteinizing and follicle-stimulating hormone. That is why eCG is widely used together with human chorionic gonadotropin to induce follicle growth and ovulation. However, there are data on complications and pathological conditions that may occur with long-term use of eCG, especially in genital tract. Therefore, the goal of our research was the morphological evaluation of the functional compartments of ovaries when using eCG to stimulate reproductive function of rabbits. Schemes of hormonal treatment of females in the conditions of a private rabbit farm, an experiment was conducted, which included the introduction of eCG in different doses (40 IU for animals of experimental group 1 and 25 IU for experimental group 2) during five reproductive cycles. Selection of material for histological studies, its fixation, production and analysis of tissue sections stained with hematoxylin and eosin were performed according to commonly used methods. The complex of morphofunctional changes in the ovaries of the rabbits of experimental groups corresponds to the pathomorphological picture of the syndrome of hyperstimulated ovaries. In our experiment, obtained changes were more pronounced at a dose of 40 IU, using which we additionally detected signs of increased immunoreactivity in body of rabbits, as evidenced by the presence of nodular lymphoid formations in outer theca of secondary and tertiary follicles. Thus, as a safe regimen for the use of eCG for ovulatory stimulation in rabbits, we recommend a dosage of 25 IU for 1–2 reproductive cycles.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

A rat study on the PTEN expression in ovarian tissue in PCOS and folliculogenesis

The objective of this investigation was to examine alterations in PTEN expression within ovarian tissue in a rat model of polycystic ovary syndrome (PCOS). The analysis also encompassed the examination of PTEN alterations in the ovarian tissue throughout the process of folliculogenesis in rats with normal ovulatory cycles. The study involved 12 adult female Sprague‒Dawley rats randomly assigned to the letrozole-induced polycystic ovary syndrome (PCOS) group as part of an animal-based research endeavour. The sections derived from the ovaries were subjected to immunohistochemical staining for PTEN. The evaluation of PTEN staining levels in ovarian tissues was conducted using electron microscopy. Follicle counts, as well as hormonal and biochemical analyses (serum luteinising hormone (LH), follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), testosterone, oestradiol levels and serum glucose, triglyceride, HDL and LDL-cholesterol levels), were conducted to provide evidence of the manifestation of polycystic ovary syndrome (PCOS) in rats. The number of primordial and Graafian follicles in the PCOS group decreased significantly, and the number of primary, secondary and antral follicles increased significantly. PTEN expression was found to be significantly higher in the PCOS group than in the control group in the primordial follicle oocyte cytoplasm, primordial follicle granulosa cells, primary follicle oocyte cytoplasm, primary follicle granulosa cells, antral follicle oocyte cytoplasm, antral follicle granulosa cells, and corpus luteum (p = 0.007, p = 0.001, p = 0.001, p = 0.001, p = 0.001, p = 0.002, and p = 0.018, respectively). In the non-PCOS group, a time-dependent comparison of the amount of oocyte cytoplasm and PTEN staining in granulosa cells of the oocytes at different stages of development was performed. While the follicles were developing from the primordial follicle to the primary and antral follicle, the amount of PTEN staining in the oocyte cytoplasm decreased, whereas the PTEN activity in the granulosa cells increased as the oocyte developed (p = 0.001 and p = 0.001, respectively). The current investigation demonstrated changes in PTEN expression in ovarian tissue throughout the course of normal folliculogenesis, as well as in instances of disrupted folliculogenesis, with a focus on rats with PCOS.