Studies On Endocrine Secretion Of Steroid And Different Expression Genes In Follicular Granulosa Cells In Huanghuai Goats Of High Prolificacy

The present paper investigated relationship between different parities and litter size of Huanghuai goats at high prolificacy. This research also compared peripheral blood concentrations of progesterone and estradiol of Huanghuai goats with poor prolificacy during the estrous cycle and in the early days after ovariectomy. The different prolific goats were also employed to analyse differences in number and size of their ovaries antral follicles and corepus luteum and in follicular atresia. In this study, a subtractive cDNA library of different expression genes in follicular granulosa cells in Huanghuai goats of high prolificacy was constructed by suppression subtractive hybridization. The objective of this study was to reveal the mechanism of follicles development regulation and their feature of reproduction endocrine in goat of high prolificacy, and to provide certain references for identifying the candidate genes and for its heredity and breeding of their high prolificacy.1. Investigation of Little Size of Huanghuai Goats with High ProlificacyThe little sizes of 427 highly prolific Huanghuai goats (HP,≥5 kids born per litter on at least one occasion) distributed in the Huanghuai plain were investigated to approach characteristic of their little size as following different parities. The results showed that litter size increased up to parity 5 and then decreased. At all parity levels, parity 5 HP does have the highest mean litter size (3.78±0.06) and parity 1 goats have the smallest. Based on analyzing the percentage of females at different little size, the females decreased at parity 1 as little size increased, and the proportion had 1 or 2 kids born per litter decreased rapidly after parity 1.However, the percentage of goats with 4 to 7 little size increased up to parity 4 to 5, furthermore as little size increased, the maximum number of the does delayed in order at different parity. It was concluded that there were obvious differences in little size at different parity in prolific does, and the limit facts of prolificacy probably was their uterus capacity. 2. Comparison of Concentrations of Progesterone and Estradiol in Peripheral Plasma during the Estrous Cycle between the Goats of High and Poor ProlificacyEight Huanghuai goats with 3-5 years old, non-lactating, parity 4-5 were selected to study two sequential natural estrous cycles of does with high prolificacy ((?)5 kids born per litter on at least one occasion, n=4) or poor prolificacy (<3 kids born per litter on any occasion, n=4) by initial synchronization achieved using two i. m. of 0.1 mg of a synthetic analog of prostaglandin F2 alpha given 12 days apart. Jugular blood samples were collected twice a day during the natural estrus by jugular syrinx and at 12-minute intervals for 3.2 hours over 2-3 days of proestrus and mid-luteal phase to assay for progesterone and estradiol. The results showed that progesterone concentrations began to rise earlier and were higher in the HP than in the PP goats on most days of the luteal phase but not during the follicular phase of the cycle. There was a significant difference between the two groups in the magnitude of the progesterone plateau. Mean estradiol concentrations in the HP group remained significantly higher than in the PP group during the estrus cycle. There were two estradiol peaks in the HP goats during the early luteal phase, but only one in the PP group. Progesterone and estradiol were secreted in a pulsatile atypia manner in HP and PP goats. It was concluded that the increase of progesterone in peripheral plasma and the turnover of follicles growth of waves in HP goats were higher than in PP group during the estrus cycle, and were the same for the overall mean estradiol concentrations.3. Comparison of Number and Size of Follicles and Corpora Lutea in Early Days of Luteal Phase in Huanghuai Goats of High or Poor ProlificacyFour HP and four PP goats were employed to collect Jugular blood samples individually at seven to nine days in third estrus cycle after synchronization achieved using a synthetic analog of prostaglandin F2 alpha and to assay for progesterone and estradiol. Meanwhile, follicles and corpora lutea were observed and measured with laparoscopy and operation on abdominal region at day 7 and day 9 in estrus cycle respectively. The results showed that number of small and large follicles had a decreasing tendency during 7 to 9 days of estrus cycle, but no difference was observed respectively. Both middle and all follicles increased, namely the mean middle follicles did to 7.50 per HP goat and to 4.48 per PP goat. Concentration of P4 and E2 in peripheral blood in HP does were higher than in PP goats, respectively, however there was no difference in P4 between two groups. Small follicles and typeⅡof corepus lutea in HP goats had obviously higher by 16.43%and 27.73%respectively compared with the PP does at day 9 of estrous cycle. It was concluded that there was a nearly same tendency in physiology of small-, middle- and large-follicles development during 7 to 9 days of estrus cycle in the HP and PP goats. The higher concentration of P4 in HP does promoted the turnover in follicles compared wich PP goats. There were more ovulation follicles characterizing smaller follicle in volume when matured in HP goats, and obvious co-dominance of follicles, and moreⅡtype of corepus lutea after ovulation, compared with those of PP does.4. Comparison of Concentrations of Progesterone and Estradiol in Peripheral Plasma in Early Days after Ovariectomy between the Goats of High and Poor ProlificacyFour HP and four PP goats were used to make ovariectomy of both ovaries respectively at day 9 of estrus cycle and to collect Jugular blood samples twice daily from 1 to 5 days after ovariectomy and at 12-minute intervals for 3.2 hours at day 1 and 5, in order to assay for progesterone and estradiol. The results showed that progesterone and estradiol were secreted in a pulsatile atypia manner in HP and PP goats. At day 1 after ovariectomy, the E2 and P4 concentrations were significantly higher in the HP than in the PP goats. Then there were a decreasing tendency in P4 lever in two groups during 1 to 3 days. The overall mean E2 concentrations in HP goats were lower than in the PP group from 2 to 5 days, significant differences were found at day 1 and 5 but their different concentrations were opposite. It was concluded that progesterone and estradiol from nonovaries were secreted in a atypia form of pulse. There existed a significant difference in E2 concentration in peripheral blood between HP and PP goats, furthermore the E2 from nonovaries probably involved in reproduction process in female.5. Observation of Histological Localization of Atretic Follicle in Luteal Stage in the GoatFour HP and four PP goats were employed to make ovariectomy of both ovaries respectively at day 9 of estrus cycle. The ovaries were preserved into liquefacient nitrogen quickly and made cryostat section to observe the histological characteristics of atretic follicle by TUNEL detecting and haematoxylin staining method. The results showed that very few oocytes with atresia were found in preantral follicles in the HP and PP goats. Apoptosis displayed more in a number of wall granulose cell near follicle antrum characterized appearance of fluorescence response. Few and individual apoptosis cell was found in granulose cell in some follicles, but no difference between HP and PP does. Follicles that were closed together with only a membrane separated, could have different physiological state at same time, one atresia another nonatresia in HP goats. It was concluded that the method of TUNEL assay and haematoxylin staining could provide a correct way for precisely detecting and localizing apoptotic cells in ovarian section. Apoptosis displayed more in a quantity of wall granulose cell near follicle antrum in atretic antrum follicles. There was a strictly physiological barrier between two adjacent follicles in HP goats.6. Construction and Selection of Subtracted cDNA Library of Granulosa Cell in Small and Large Follicles in Early Days of Heat in Huanghuai Goats of High ProlificacyTwo HP goats were used to collect both ovaries individually 40 hours after synchronization achieved using a synthetic analog of prostaglandin F2 alpha. Counting and measuring follicles and collecting granulose cells by blunt dissection and preserving a fraction of follicle used into liquid nitrogen were within 20 min. Total RNA was extracted from granulose cells of large (5-6 mm) and small (3-4mm) follicles that were not found atresia, and were synthesized and amplified, then a forward- and reverse-subtractive cDNA library were constructed by suppression subtractive hybridization. A total of 190 clones randomly selected from the library were proved to be differentially expressed in the granulose cells, and 110 clones were selected out to be sequenced in one way based on the dot blot analysis. Among of these,109 EST obtained, and 10 exhibited high homology to be known genes,16 exhibited high homology to be found but its function not known genes,65 identified as new EST by Blastn checking after clustering. These different genes could conduce to reveal the regulation mechanism of follicles development in goats, meanwhile provide some reference for identifying the candidate gene of HP goats.