| The guinea pig is an excellent animal model for studies of the reproductive system in humans and most domestic animals. Unlike other laboratorial rodents, guinea pigs have an extended estrous cycle of 16 to 18 days, are not superovulated by FSH or eCG but rather with inhibin immunizations, have a full gestation of 68 days, and a small litter size of 3.8 on average. Owing to these special biological characteristics, the process of follicular atresia in guinea pigs also has unique features. Superovulation is to reduce follicular atresia, promote the ovarian ovulate more eggs. However, follicular atresia was an inevitable phenomenon in the process of follicular development, which was important for the maintenance of ovarian homeostasis. Over the years, the research of follicular atresia was focused on apoptosis and elimination of granulosa cells. For guinea pigs, the process of follicular atresia is very complex. In addition to the clearance of granulosa cells, there was also a large number of differentiated cells appear. Because of this reason, there was a lack of study of follicular atresia in guinea pigs. In short, the studies of follicular atresia and superovulation in guinea pigs were important for revealing the mammalian animal’s reproductive mechanism and folliculogenesis.In 2005, the Nomenclature Committee on Cell Death (NCCD) was established by the editors of the journal of Cell Death and Differentiation. In the absence of some clearly defined biochemical indices for cell death, the NCCD suggested that the official classification of cell death had to be entirely dependent upon morphological changes. In 2005 and 2009, the NCCD proposed that a cell should be considered dead when any one of the following morphologic criteria is met:the cell has lost the integrity of its plasma membrane, the cell has undergone complete fragmentation into discrete bodies, or its corpse has been engulfed by an adjacent cell in vivo. Currently, cell death modes generally include apoptosis, autophagy, cornification, and necrosis, but apoptosis is the main concern in follicular atresia. Generally, apoptosis is accompanied by rounding-up of the cell, reduction in cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and engulfment by resident phagocytes (in vivo)The current studies were focused on histological changes in follicular development and atresia of prepuberty and mature guinea pigs, and summarized the general rules of follicular atresia in guinea pigs; the effects of PMSG and the gene immunization of inhibin in the prepuberty and mature guinea pigs with ovarian serial sections; histological features and were studied intensively.1. Follicular development and atresia in prepuberty guinea pigsIn order to study the situation of the characteristics of follicular development and atresia, the ovaries of day 7, day 15, day 30 of guinea pigs were sampled for histological analysis. The results showed that the follicles of prepuberty guinea pigs developed rapidly. In day 1 there were a large number of secondary follicles and a few antral follicles, in day 7 a lot of the developing antral follicles appeared, in day 15 and 30 antral follicles developed rapidly, large follicles with diameters than 600μm were observed. In the prepuberty ovaries, the follicles developed in the race to atresia, and the most obvious feature was the large number of granule cells shed into the follicle cavity.2. Follicular development and atresia in different estrus stagesTo study the characteristics of follicular development and atresia in the cyclic guinea pigs, the ovaries of days 1,4,8,12 and 16 were studied. The results showed that the follicular development and atresia was obvious cyclical. On day 1, a large number of antral follicles were atresia, a large number of granulosa cells shed into the cavity; on day 4, the granulosa cells were eliminated and the fibroblast-like cells invaded into follicular cavity in follicles; in days 8 and 12, a large amount of follicles developed; and on day 16, preovulatory follicles were observed. In the process of follicular atresia of guinea pigs, granulosa cells eliminated firstly, followed by theca cells. However, the theca cells were dramaticly differentiated and migrate to the follicle cavity in the process of atresia.3. Follicular atresia of guinea pigsBased on morphological characteristics of the follicular development and atresia in cyclic guinea pigs, we founded firstly that:(1) According to the speed of granule cells clearance, atretic follicles can be divided into three types; (2) the process of follicular atresia can be divided into four sequential stages, including connections in granulosa layers became loose, massive elimination of granulosa layers, differentiation of theca cells, and the cellular loss of theca cells; (3) the process of follicular atresia was not only elimination of cells, but coexisted with cell differentiation, migration and proliferation; (4) granulosa cells were cleared by the rapid manner; (5) the degradation of oocyte was not accord with apoptosis manner, but similar to autophagy; (6) cells outside the zona pellucida were changed several times, there were granulosa cells, apoptotic bodies, fibroblast-like cells and intumescent cells; (7) the dynamics of follicular atresia was focused on fibroblast-like cells. These results showed that the atretic follicles of guinea pigs were very activeand complex.4. Effects of PMSG on ovarian follicular development in prepuberty guinea pigsIt has been reported that guinea pigs are difficult to introduce superovulation by PMSG like most other animals. However, the mechanism of this phenomon is still unknown. In the current project, a PMSG has been used for clarifying this reason and comforming the effect of PMSG on the ovary of guinea pigs. PMSG at doses of 0,10 and 100 IU were used respectively in female prepuberty guinea pigs, and sacrificed in the 24, 48 and 72 h later. The histological results showed that PMSG treatment failure to induce superovulation but caused apoptosis of granulosa cells and luteinization of follicular cells. In addition,10IU treatment mainly manifested as apoptosis of granulosa cells, while the 100IU treatment showed differentiation and proliferation of follicular cells. Furthermore, the apoptosis and differentiation of granulosa cells were dependent on the dose of PMSG. Our finding indicates that PMSG induces follicle cell differentiation and proliferation, but the mechanisms require further study.5. Effects of PMSG on ovarian follicular development in mature guinea pigs A PMSG at dose of 0,20 and 50 IU were treated in the day 12 of estrous cycle in guinea pigs, in the day 16 one ovary removed, and sacrificed in the day 4 after ovulation of estrous cycle. The results showed that the treatments were failure to induced superovulation. 20IU treatment promoted granulosa cell apoptosis of antral follicles, and 50IU treatment promoted the differentiation of follicle cells and the luteinizing unruptured follicle (LUFs) appeared.6. Effects of PCISI plasmid gene immunization on ovarian follicular development in prepuberty guinea pigsIn view of inhibin immunization induces superovulation successfully in guinea pigs, the gene vaccine pCISI was used for superovulation treatment, and the control group received an equivalent volume of saline. With immunization interval of 20 days,5 consecutive gene immunizations were carried through. The guinea pigs sacrificed and at day 8 of estrous cycle. ELISA was used for check the concentration of serum antibody, and found that the concentration of antibody were not promoted significantly in the experiment (P>0.05), and the difference of ovulation numbers, follicular number of different sizes were not significantly between two treatment (P>0.05). The results showed that the PCISI plasmid gene immunization was failure to induced superovulation. Moreover, the difference of ovarian, uterine and carcass weight were not significantly between two treatment (P>0.05).7. Effects of attenuated Salmonella gene immunization on ovarian follicular development in mature guinea pigsThe gene vaccine pXAIS was used for superovulation treatment with bacterial content of 1010, and the control group received an equivalent volume of pVAX-asd, which contained plasmid without inhibin fragments. With immune interval of 4 weeks, two consecutive immunizations were carried through. Guinea pigs were sacrificed after 8 weeks of treatment at the day 8 of estrous cycle. ELISA was used to check the concentration of serum antibody, and found that the concentration of antibody were not promoted significantly in the experiment (P>0.05), and the difference of ovulation numbers, follicular number of different sizes were not significantly between two treatment (P>0.05). The results showed that the genetic immunization of attenuated Salmonella pXAIS was failure to induced superovulation. Moreover, the difference of ovarian, uterine and carcass weight were not significantly between two treatment (P>0.05).In conclusion, guinea pigs are an interesting animal that diffuclt to be introduced superovulation by current ways like other animals. The results showed that atretic follicles were active tissue that cell differentiation and proliferation occurred. The effects of PMSG were mostly like induced the follicular atresia and cannot induced superovulation in the guinea pig ovary. This mechanism required further investigation. |
