R. Webb

ROLE OF GROWTH HORMONE AND INTRAFOLLICULAR PEPTIDES IN FOLLICLE DEVELOPMENT IN CATTLE

Abstract Ovarian function in cattle is controlled by complex local and systemic feedback mechanisms which ensure that in >96% of estrous cycles only one follicle will ovulate. Although there is a primary requirement for gonadotropins in stimulating follicular growth, particularly during the final stages of follicle maturation, it is now becoming apparent that locally-produced factors, such as insulin-like growth factor-I (IGF-I) and inhibin, have a modulating role. In addition to gonadotropins, other systemic agents such as metabolic hormones (GH, IGF-I and insulin), can also influence follicular growth. The ability to control the growth of the dominant follicle, together with improved understanding of the competing mechanisms controlling follicular recruitment and growth, should enable more precise control of ovarian function and superovulation in cattle.

Pretreatment with recombinant bovine somatotropin enhances the superovulatory response to FSH in heifers.

One of the primary limiting factors to superovulation and embryo transfer in cattle has been the large variability in response, both between and within animals. It appears that the primary source of this problem is the variability in the population of gonadotropin-responsive follicles present in ovaries at the time of stimulation. We have shown that treatment of heifers with recombinant bovine somatotropin (rbGH) increases the number of small antral follicles (2 to 5 mm) and, therefore, enhances the subsequent superovulatory response to eCG. To investigate further the potential of using this approach to improve superovulatory regimens in cattle, the effect of rbGH pretreatment on the response to pituitary FSH was studied. The estrous cycles of 16 heifers were synchronized using PGF2alpha. On Day 7 of the synchronized cycle, half of the animals were injected with 320 mg sustained-release formulated rbGH, while the other half received 10 ml saline. Five days later, all heifers were given a decreasing-dose regimen of twice daily injections of oFSH for 4 d, incorporating an injection of PGF2alpha with the fifth FSH treatment, to induce superovulation. All animals were artificially inseminated twice with semen from the same bull during estrus. Ova/embryos were recovered nonsurgically on Days 6 to 8 of the following estrous cycle, and the ovulation rate assessed on Day 9 by laparoscopy. Using the same animals as described above, the experiment was repeated twice, 3 and 6 mo later, with no laparoscopy in the third experiment. The animals were randomized both between experiments and for the day of ova/embryo collection. Pretreatment of heifers with rbGH significantly (P < 0.01) increased the number of ovulations, total number of ova/embryos recovered and the number of transferable embryos. The percentage of transferable embryos was significantly (P < 0.05) increased by rbGH pretreatment. In addition, the incidence (2/16) of follicular cysts with a poor ovulatory response (< 6 ovulations) for the rbGH-pretreated heifers was significantly lower (P < 0.05) when compared with the incidence (7/16) in the control animals. It is concluded that pretreatment with rbGH may provide a useful approach for improving superovulatory response in cattle.

Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

To investigate the effect of recombinant bovine somatotrophin (rGH) on ovarian folliculogenesis in sheep, 18 mature Scottish Blackface ewes were assigned randomly to two treatment groups. Starting from day 5 of the synchronised oestrous cycle, animals were injected daily with either vehicle (control group) or 12.5 mg rGH (rGH-treated group) for 7 days. Blood samples were collected once daily during the experimental period for the measurement of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, follicle-stimulating hormone (FSH), luteinising hormone (LH) and progesterone. At the end of treatment animals were killed and ovaries collected. All follicles at least 1.0 mm in diameter were dissected out and diameters measured to assess follicular populations for individual animals. Five small follicles (1.0–3.4 mm in diameter) and all the large follicles (at least 3.5 mm) from each animal were incubated in 1 ml of Medium 199 for 1 h. Medium was then changed and incubation continued for a further hour. All medium samples were assayed for IGF-I, oestradiol, testosterone and progesterone. n nTreatment of ewes with rGH had no effect on the total number of follicles at least 1.0 mm in diameter (control, 34.4 ± 2.6; rGH-treated, 31.3 ± 1.4; P > 0.2). However, when follicles were further classified into different size categories (1.0–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, 5.1–6.0 and over 6.0 mm in diameter), the population of follicles 2.1–3.0 mm in diameter was significantly increased by rGH treatment (control, 9.2 ± 0.7; rGH-treated, 13.8 ± 1.1; P = 0.02). The number of follicles of 3.1–4.0 mm diameter in the rGH-treated group tended to be increased (P = 0.09), whilst the population of follicles 1.0–2.0 mm in diameter was reduced (P = 0.07). Treatment of ewes with rGH significantly increased peripheral concentrations of GH (P < 0.01), IGF-I (P < 0.01), insulin (P < 0.01) and progesterone (P < 0.05). There was no effect of rGH treatment on circulating concentrations of FSH and LH. Both large and small follicles from rGH-treated ewes secreted significantly (P < 0.001) more IGF-I (37.8 ± 2.2 ng ml h−1, n = 50) than follicles from the control group (26.7 ± 1.6 ng ml−1 h−1, n = 73). However, there was no significant effect of rGH treatment on the secretion of oestradiol, testosterone and progesterone by either large or small follicles. n nIt is concluded that treatment of mature ewes with rGH can enhance the development of ovarian follicles to the gonadotrophin-dependent stages. Furthermore, rGH appears to act through increased secretion of ovarian IGF-I, as well as increased peripheral concentrations of IGF-I and insulin.

Ultra-structural characteristics of bovine granulosa cells associated with maintenance of oestradiol production in vitro

We investigated whether the maintenance of oestradiol production by bovine granulosa cells (GC) in vitro was related to GC ultra-structure, and studied the effects of inclusion of serum as a cell attachment factor on oestradiol secretion, cell morphology and ultra-structure. Bovine granulosa cells from medium-sized follicles (4-8 mm diameter), in a serum-free (SF) culture system, maintained oestradiol production for 6 days, whereas oestradiol secretion by cells cultured in serum-coated (SC) wells declined rapidly with time, in culture. SF cells formed clumps consisting of two types of cells. Cells within clumps presented a phenotype similar to GC in vivo, being spherical, tightly joined by extensive gap junctions and interdigitated pseudopodia/microvilli, had abundant rough and smooth endoplasmic reticulum (ER) and mitochondria with trabecular cristae. In contrast, cells cultured in either SC wells or in the flattened base of cell clumps from SF cultures were enlarged, containing less rough ER, had fewer mitochondria (which tended to be round) and contained endosome-like structures, morphological characteristics suggestive of early luteinisation.

Investigation of the relationship between farrowing environment, sex steroid concentrations and maternal aggression in gilts

Maternal oestrogen and progesterone have been shown to be important in the initiation of maternal behaviour. Thirty-three Large White x Landrace gilts, housed in groups during pregnancy, were observed and aggressive interactions recorded. Individuals had jugular catheters implanted 14.5 (s.e. 0.34) days before their expected parturition date (EPD). Five days before EPD gilts were randomly allocated and moved to either a conventional farrowing crate (C; without straw, 16 gilts) or a pen (P; 2.1 x 3.1 m2; with straw bedding, 17 gilts). Blood samples were taken at frequencies determined by the proximity to farrowing onset. Piglets were removed at birth and returned 2 h after placental expulsion. The reaction of each gilt to her piglets was monitored. Gilts savaging piglets were sedated with azaperone (n = 8). There was no overall effect of farrowing environment on oestradiol and progesterone concentrations. The pre-farrowing ratio of progesterone to oestradiol was higher for (P) gilts (0.45 vs. 0.25, (P) vs. (C); S.E.D. 0.085, P < 0.05) as was their overall maximum oestradiol level (3.39 vs. 2.29 ng/ml, (P) vs. (C); S.E.D. 0.39, P < 0.01). In contrast to progesterone, oestradiol patterns varied considerably between individuals. Dominance rank value during pregnancy, but not levels of aggression, correlated positively to pre-farrowing oestradiol concentrations. Treatment with azaperone was not related to farrowing environment, piglet weight or litter size. Azaperone treated gilts showed a higher pre-farrowing oestradiol to progesterone ratio (0.55 vs. 0.29, +/- azaperone; S.E.D. 0.10, P < 0.05), significantly higher levels of oestradiol post-partum (0.7 vs. 0.19 ng/ml, +/- azaperone; S.E.D. 0.20, P < 0.001) and significantly lower levels of aggression during pregnancy (1.68 vs. 2.23 aggressive interactions/h, +/- azaperone; S.E.D. 0.15, P < 0.001). The results indicate that there are no major effects of farrowing environment on sex steroid concentrations. Maternal aggression under these conditions appears to be negatively related to aggression during pregnancy, but this is not reflected in plasma concentrations of sex steroids around parturition.

ULTRASTRUCTURAL VISUALIZATION OF PROTEIN-4 BINDING INSULIN-LIKE GROWTH FACTORS IN COW OVARIAN FOLLICLES

Protein-4 that binds the insulin-like growth factors (IGFBP-4) was visualized in the basal membrane of some vessels and follicles and in extracellular membrane surrounding thecal cells in middle-sized cow ovarian follicles. Examinations of serial sections of thecal granular cells showed highly specialized sites for detection of this protein on plasma membranes and highly specialized endosomes responsible for accumulation of IGFBP-4 in granular cells. These data point to an active role palyed by the extracellular ovarian matrix in the binding and accumulation of insulin-like growth factor and to high specificity of internalization and exocytosis of IGFBP-4 by ovarian granular cells.