Background: Although an understanding of the proliferation and differentiation of fish female germline stem cells (GSCs) is very important, an appropriate threedimensional (3D) research model to study them is not well established. As a part of the development of stable 3D culture system for fish female GSCs, we conducted this study to establish a 3D aggregate culture system of ovarian cells in marine medaka, Oryzias dancena. Methods: Ovarian cells were separated by Percoll density gradient centrifugation and two different cell populations were cultured in suspension to form ovarian cell aggregates to find suitable cell populations for its formation. Ovarian cell aggregates formed from different cell populations were evaluated by histology and gene expression analyses. To evaluate the media supplements, ovarian cell aggregate culture was performed under different media conditions, and the morphology, viability, size, gene expression, histology, and E2 secretion of ovarian cell aggregates were analyzed. Results: Ovarian cell aggregates were able to be formed well under specific culture conditions that used ultra-low attachment 96 well plate, complete mESM2, and the cell populations from top to 50% layers after separation of ovarian cells. Moreover, they were able to maintain minimal ovarian function such as germ cell maintenance and E2 synthesis for a short period. Conclusions: We established basic conditions for the culture of O. dancena ovarian cell aggregates. Additional efforts will be required to further optimize the culture conditions so that the ovarian cell aggregates can retain the improved ovarian functions for a longer period of time.

Background: This study helps to evaluate the Ovarian potential of Cameroonian Zebu cattle (Bos indicus) slaughtered in Etoudi – Yaoundé for implementing Assisted Reproductive Techniques (ARTs). The aim was to enhance the productivity of the cattle sub-sector in Cameroon while conserving local genetic resources. Methods: A total of 144 cows, including 94 cycled cows and 50 pregnant cows, were included in the study. Live weights were determined by measuring the thoracic perimeter of each cow using a Rondo measuring tape. Age was determined postmortem through examination of dentition and horns, while the uterus and ovaries were removed, weighed, and classified based on physiological status (pregnant or nonpregnant). Follicles were counted, and their diameters were measured and categorized into small (Ø < 3 mm), medium (Ø 3-8 mm), and large (Ø > 8 mm). Results: The results revealed an average follicular population of 32.02 ± 9.31 per cow, with 22.43 ± 8.45 small follicles, 8.42 ± 3.87 medium follicles, and 0.76 ± 0.34 large follicles. The weight of the right ovary was significantly higher than that of the left ovary (p < 0.05), and cows aged 6 to 9 years exhibited a higher number of follicles compared to other age groups. Cows with medium (BCS = 3) and large (BCS = 4-5) body reserves had the heaviest ovaries. Additionally, pregnant cows had heavier uteri compared to non-pregnant cows, and cows with a body condition score of 3 or higher had higher uterine weights than lean cows (BCS = 1-2). Conclusions: This study demonstrates that age, body condition score, and pregnancy status influence ovarian weight. Body Condition Score serves as a reliable indicator of cow nutritional status, and cows with BCS of 3-5 are excellent candidates for in vitro production of embryos.

Cryopreservation of porcine ovarian tissue by vitrification method is a promising approach to preserve genetic materials for future use. However, information is not enough and technology still remains in a challenge stage in pig. Therefore, the objective of present study was to determine possibility of vitrification method to cryopreserve porcine ovarian tissue and to confirm an occurrence of cryoinjuries. Briefly, cryoinjuries and apoptosis patterns in vitrified-warmed ovarian tissue were examined by histological evaluation and TUNEL assay respectively. In results, a damaged morphology of oocytes was detected among groups and the rate was significantly (p < 0.05) lower in vitrification group (25.8%) than freezing control group (67.7%), while fresh control group (6.6%) showed significantly (p < 0.05) lower than both groups. In addition, cryoinjury that form a wave pattern of tissues around follicles was found in the frozen control group, but not in the fresh control group as well as in the vitrification group. Apoptotic cells in follicle was observed only in freezing control group while no apoptotic cell was found in both fresh control and vitrification. Similarly, apoptotic patterns of tissues not in follicle were comparable between fresh control and vitrification groups while freezing control group showed increased tendency. Conclusively, it was confirmed that vitrification method has a prevention effect against cryoinjury and this method could be an alternative approach for cryopreservation of genetic material in pigs. Further study is needed to examine the viability of oocytes derived from vitrified-warmed ovarian tissue.

Cytochrome P450 1A2 (CYP1A2) is a member of the cytochrome P450 superfamily enzymes in mammals and plays a major role in metabolizing endogenous hormones in the liver. In recent days, CYP1A2 expression has been found in not only the liver but also other tissues including the pancreas and lung. However, little information is available regarding the expression of CYP1A2 in the ovary, in spite of the facts that the ovarian follicle growth and atresia are tightly associated with controls of endocrine hormonal networks. Therefore, the expression of CYP1A2 in the ovaries of prepubertal and pubertal rats was investigated to assess its expression pattern and puberty-related alteration. It was demonstrated that the expression level of CYP1A2 was significantly (p < 0.01) higher in the pubertal ovaries than prepubertal counterparts. At the ovarian follicle level in both groups, whereas CYP1A2 expression was less detectable in the primordial, primary and secondary follicles, the strongly positive expression of CYP1A2 was localized in the granulosa cell layers in the antral and pre-ovulatory follicles. However, the ratio of CYP1A2-positive ovarian follicle was significantly (p < 0.01) higher in the ovary of pubertal group (73.1 ± 3.1%) than prepubertal one (41.0 ± 10.5%). During the Immunofluorescence, expression of CYP1A2 was mainly localized in Fas-positive follicles, indicating the atretic follicles. In conclusion, these results suggested that CYP1A2 expression was mainly localized at the atretic follicular cells and affected by the onset of puberty. Further study is still necessary but we hypothesize that CYP1A2 expresses in the atretic follicles to metabolize residue of the reproductive hormones. These findings may have important implications for the fields of reproductive biology of animals.

Aquaporin channels (AQPs) are known to play an important role in the development of ovarian follicles through their function in water transport pathways. Compared to other AQPs, research on the role of AQP4 in female reproductive physiology, particularly in cattle, remains limited. In our previous study, gene chip microarray data showed a downregulation of AQP4 in bovine cystic follicles. This study was performed to validate the AQP4 expression level at the protein level in bovine follicles using immunohistochemistry, Western blotting, and immunoprecipitation assays. Immunostaining data showed that AQP4 was expressed in granulosa and theca cells of bovine ovarian follicles. The ovarian follicles were classified according to size as small (< 10 mm) or large (> 25 mm) in diameter. Consistent with earlier microarray data, semi-quantitative PCR data showed a decrease in AQP4 mRNA expression in large follicles. Western blot analysis showed a downregulation of the AQP4 protein in large follicles. In addition, AQP4 was immunoprecipitated and blotted with anti-AQP4 antibody in small and large follicles. Accordingly, AQP4 exhibited a low expression in large follicles. These results show that AQP4 is downregulated in bovine ovarian large follicles, suggesting that the downregulation of AQP4 expression may interfere with follicular water transport, leading to bovine follicular cysts.

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

The objective of this research work was to know ovarian dynamics and pregnancy rate of cyclic Murrah buffalo cows with induced estrus by administration of prostaglandin F2α (PGF2α) and timed artificial insemination (TAI) with frozen thawed semen. A total of 31 female buffaloes were selected for the study. The buffalos having matured CL observed by ultrasonography were given one intra muscular injection of cloprostenol 500 μg and TAI was performed using frozen thawed semen of Indian Murrah buffalo bull. Results showed that 90.32% (significantly, at p < 0.05) cows explore the sign of heat after injection of PG and 67.85% (significantly, at p < 0.05) cows were become pregnant out of 28 inseminated (TAI) cows. In the 28 inseminated (TAI) cows, average number of smaller and larger size of follicles were non-significantly (p > 0.05) higher at day 3 post PG injection, but the medium size of follicles was non-significantly (p > 0.05) higher at PG injection. At day 3 post PG injection the diameter of follicles was significantly (p < 0.05) higher, but the diameter of CL was significantly (p < 0.01) lower compared at PG injection. At PG injection the diameter of largest follicle was non-significantly differences (p > 0.05) in between pregnant and non-pregnant cows. But at day 3 post PG injection it was significantly (p < 0.01) higher in pregnant cows compared to non-pregnant cows. The number of small, medium, and large follicles at PG injection and at day 3 post PG injection were non-significantly (p > 0.05) difference in between pregnant and non-pregnant buffalo cows. Finally, it is concluded that the CL was effectively regresses and induced the sign of heat in buffalo cows and after AI the cows were become pregnant with significant rate. The study will help to the veterinarian and researcher to know the efficacy of PG injection and AI for reproductive efficiency in buffalo cows.

Chronic and unpredictable stress can disrupt the female reproductive system by suppression for secretion of gonadotrophin-releasing hormone (GnRH) and gonadotrophin, resulted in ovarian malfunction and infertility. In the recent days, kisspeptin has been highly highlighted as a hypothalamic peptide which directly stimulates synthesis and release for GnRH. However, in spite of the key role of kisspeptin in the female reproductive system, little information is still available on the changes of its expression during ovarian cycle under stressed condition. Therefore, we induced chronic and unpredictable stress series to the female mice to analyze kisspeptin expression in the brain and ovary. Stressed mice exhibited changes of behavior and body weight gain during the stress assessment, which suggested that the present stress model in mice was successfully established. In the brain level, kisspeptin expression was attenuated than control. In the ovary level, the stressed mice displayed irregularly shrunk oocytes with broken zona pellucida throughout the follicle stages, pyknotic granulosa cells, decreased number of developing follicles and increased number of atretic follicles than the control. In case of kisspeptin expression in the whole ovary tissue, the expression level was decreased in the stressed mice. In detail, the less intensity of kisspeptin expression in the antral follicles phase was observed in the stressed mice than control mice, indicating that local function of kisspeptin during ovary cycle is highly associated with development of ovarian follicles. We expect that the present study has important implications for the fields of reproductive biology.

The purpose of the present study was to determine the elaborate characteristics of ovarian changes including follicles and corpus luteum, and hormonal patterns of gonadotropin surge mode secretions during the normal consecutive estrous cycle in three dairy cows. Non-lactating and multiparous Holstein cows (n=3) used as experimental animals. The cows were assigned to examine the relationship among ovarian changes (follicle, corpus luteum), ovarian steroids (estradiol, progesterone) and gonadotropin (LH, FSH) surge mode secretion during the successive estrous cycles by rectal palpation, ultrasonography and hormonal assay. The mean length of the estrous cycle for the three cows was 23.1 ± 1.44 days (± SEM), with a range of 20-28 days. In six estrous cycles, the number of two follicular waves, three follicular waves and four follicular waves was 2, 3 and 1, respectively. The sequential ultrasonographic monitoring showed that the corpus luteum with ≥ 10 mm in diameter detected from Day 2 (Day 0 is ovulation) in six estrous cycles of all cows. Preovulatory increases in estradiol concentration reached 10.36 ± 1.10 pg/ml on the 2 days before ovulation of the last dominant follicle. All cows exhibited a preovulatory rise in estradiol concentration followed by a typical preovulatory LH and FSH surge. The mean interval from the peak of LH/FSH surge to ovulation of the last dominant follicle was 31.3 ± 1.76 h (± SEM). In these results, each dairy cow showed that ovarian morphological changes and gonadotropin surge mode secretion will be regulated by various environmental factors including age, breeds, nutrition, breeding conditions, etc.

본 연구는 돼지의 정자와 난소내 과립막세포에서 bisphenol S(BPS)가 생존성과 활성산소 생산에 미치는 영향을 알아보고자 연구하였다. 돼지정액은 0, 5μM BPS를 처리하여 3, 6시간동안 배양하였다. 정자의 생존성은 SYBR14/PI를 이중 염색하여 분석하였으며, 활성산소의 생산을 측정하였다. 또한, BPS(0, 5, 10, 20μM)를 과립막세포에 처리하여 24, 48, 72시간동안 처리하였다. 처리 후, 세포의 생존율과 활성산소 생산(단, 5μM BPS)을 측정하였다. 그 결과, 돼지에서 정자의 생존율은 BPS에 의해 감소하였고, 활성산소의 생산은 모든 처리시간에서 증가하였다(p<0.05). 또한 과립막세포의 생존은 BPS에 의해 억제되었고, 활성산소는 유의적으로 증가하였다(p<0.05). 이상의 결과를 토대로, BPS의 노출은 정자의 활성과 번식과 관련된 세포에 나쁜 영향을 미칠 것이다.

Mammalian fetal ovaries contains numerous primordial germ cells, however fewer ones can yield mature oocytes due to apoptosis and follicle atresia. Successful in vitro reconstitution of primordial germ cells has recently had a significant effect in the field of assisted reproductive technologies. However, the regulatory mechanisms underlying oogenesis remain unknown and recapitulation of oogenesis in vitro remains unachieved. Therefore, development of methods for obtaining mature oocytes by culturing the fetal ovaries in vitro could contribute to clarify these mechanisms. We adapt an in vitro system for culturing mouse fetal ovaries that support successful follicle assembly and improve oocyte growth and maturation. Ovarian tissues from 12.5 days postcoitum (dpc) fetal mice were cultured in vitro and the matured oocytes were differentiated from primordial germ cells after a 31 days culture period. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with artificial ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro. Taken together, this in vitro culture system is expected to aid in the development of new strategies to identify the reasons behind failure of follicle assembly and offer a platform for innovative research into preservation of female germ cells and conservation of endangered species.

Although some factors, including season, age, type of estrus (natural estrus vs. induced estrus) and semen type (conventional vs. sexed), affect the conception rate following artificial insemination (AI) in dairy cattle, there is little information about the influence of ovarian characteristics, such as preovulatory follicle (PF) location at estrus, on fertility in dairy cattle. In most breeds of cattle the right ovary appears to function more actively than the left and about sixty percent of pregnancies in dairy cattle occur in the right horn of uterus (Reece and Tuner, 1938). Our study aimed to compare conception rates in dairy cattle between PFs that developed in the left ovary and those that developed in the right ovary at estrus. In this study, we examined the locational effect (left or right ovary) of the preovulatory follicle (PF) on fertility in dairy cattle. In total, 955 artificial inseminations (AI) were analyzed. At AI, PF locations were examined using rectal palpation, and dairy cattle were divided into two groups on their PF locations: (i) the PF located in the left ovary (L-PF); and (ii) the PF located in the right ovary (R-PF). Pregnancy was diagnosed by rectal palpation or ultrasonographic examination 60 days after AI. The conception rate was 38.1% in all dairy cattle. Conception rate was higher in the R-PF (40.8%) than in the L-PF (33.2%). In summary, PF development in the right ovary was associated with increased conception rates in dairy cattle.

This study was conducted to investigate the ovarian cycle changes of the mare according to the season. Twenty four Jeju crossbred horses(Thoroughbred x Jeju horse) raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were used to identify follicles and corpus luteum with ultrasonography once a week(May 2016~June 2017). Blood samples of experimental horses were collected twice a week for analysis of P4 hormone levels. The mares were considered to have resumed ovarian cyclicity on the day of ovulation if they followed by regular ovarian cycles. Only 13 cases(61.9%) of the total 21cases showed normal ovarian cycle, and 8 cases (38.1%) showed delayed ovarian cycle. Three cases(16.7%) in October, 5 cases(27.8%) in November and 5 cases(27.8%) in December(27.8%) ceased the heat and the remaining 5 cases(27.8%) showed that the estrus was maintained in winter. Horses that stopped estrus ceased the heat until March of next year, and 27.8% were continued the heat during non-breeding season. Eleven cases(61.1%) of 18 cases in April and 2 cases(11.1%) of 18 cases in May returned the estrus.

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.

The purpose of this study was to examine the effects of taurine and vitamin E on ovarian granulosa cells damaged by bromopropane (BP) in pigs. We evaluated cell viability, plasma membrane integrity (PMI) and apoptotic morphological change in porcine ovarian granulosa cells. The cells were treated with 1-BP (0, 5.0, 10, and 50 μM), 2-BP (0, 5.0, 10, and 50 mM), taurine (0, 5.0, 10, and 25 mM), and vitamin E (0, 100, 200, and 400 μM) for 24 h. 10 μM 1-BP and 50 μM 2-BP inhibited viability and PMI, and induced apoptosis in porcine ovarian granulosa cells (p < 0.05). Cell viability and PMI were increased by taurine (10 and 25 mM) and vitamin E (100 and 200 μM), and apoptosis decreased (p < 0.05). Finally, the porcine ovarian granulosa cells were co-treated with BPs (10 μM), taurine (10 mM) and/or vitamin E (200 μM). Cell viability and PMI in the co-treated cells were increased, and apoptosis was decreased. In conclusion, taurine and vitamin E can improve cell viability and inhibition of apoptosis in porcine ovarian granulosa cells damaged by bromopropane.

In order to determine the ovarian cycle of Asian Toad, Bufo gargarizans, the developmental stage based on the gonadosomatic index (GSI), size of follicle oocytes in ovary and vitellogenesis for adult females were investigated all around the year. The weight of ovary and GSI were the lowest from April, and all follicle oocytes exist in the pre-vitellogenic form, indicating that the vitellogenesis was suspended. The follicle oocytes in early-vitellogenic stage appeared in ovary during may when the weight of ovary and GSI start to increased, and the follicle oocytes in midvitellogenic and pre-vitellogenic stages existed during June and the weight of ovary and GSI also increased. This indicates that vitellogenesis has been carried out actively during this period. The follicle oocytes in mid-vitellogenic stage and late-vitellogenic stage when the vitellogenesis was also completed existed on September. Post-vitellogenic follicle oocytes after vitellogenesis started to appear from October and rapidly increased from December in hibernation. The full grown follicle oocytes existed during February, indicating the ovarian cycle that all follicle oocytes in ovary are developed separately, not synchronized, during the growing period of follicle oocytes and the postvitellogenic follicle oocytes are maintained the ovulation period.

The Osmia cornifrons plays an important role in pollinating fruit trees, such as apple trees. To better understand diapause and oviposition in O. cornifrons, we investigated the correlation between the ovarian development and secretion level of OcVg protein in hemolymph. During ovarian development in wintering the number of oocytes progressively increased in comparison with the length of the ovaries and the oocytes. After emergence, the oocyte and ovary sizes developed until 6 days after emergence and declined after 6 days, but the number of oocytes decreased gradually. The secretion level of OcVg protein in hemolymph revealed that during wintering, the secretion level increased from 1 month to 2 months and then stagnated after 2 months. After diapause, the secretion level increased gradually until day 6 of the newly emerged adult from cocoon stage, and thereafter gradually declined, remaining detectable until day 30 of the adult stage. The correction analysis between ovarian development and OcVg secretion level in hemolymph found that in wintering, the number of oocytes was positively correlated to OcVg secretion level. After diapause, the ovary and first oocyte lengths and the number of oocytes showed significant changes in OcVg secretion level and positively correlated with OcVg secretion level, respectively. These results suggest that there is a significant interaction between ovarian development and the secretion level of OcVg protein and the pattern of ovarian development and secretion of OcVg protein are stage-specific in the O. cornifrons female.