FSH Supplementation Research Articles

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus–oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0–40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0–20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0–20 h, FSH 0–40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0–40 and FSH 0–20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0–40 and FSH 0–20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0–40 and FSH 0–20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.

Effect of meiotic stages and maturation protocols on bovine oocyte’s resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte’s resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P>0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P<0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P<0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/warming process significantly compromised the oocytes’ developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments ( P<0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte’s ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.

Effects of season and phase of the estrous cycle on steroidogenesis and LH-FSH sensitivity of large ovine follicles perfused in vitro

The aims of this study were to compare steroidogenesis (progesterone, androstenedione and estradiol production) and response to LH and FSH challenge by whole perifused follicles 4 to 5.5 mm in diameter, obtained at different periods of the breeding season (onset, middle, end), during anestrus and the luteal phase. We have observed that all follicles do not have the same steroïdogenetic potential and do not respond with the same intensity to LH and FSH. At the middle of the breeding season, LH and FSH supplementation was ineffective in increasing progesterone secretion by follicles (0.19 ± 0.05 vs 0.20 ± 0.03 ng/mL). In contrast, gonadotrophin challenge elicited significant (P < 0.05) increases in androstenedione (0.94 ± 0.34 vs 0.35 ± 0.09 ng/mL) and estradiol (120 ± 11 vs 49 ± 10 pg/mL) production immediately after its administration. At the onset of the breeding season, steroidogenesis was identical under both basal and gonadotrophin-stimulated conditions unlike that in middle of the breeding season. However follicles were more sensitive to the gonadotrophin challenge in terms of estradiol production than those collected at the middle of the breeding season (220 ± 45 vs 120 ± 11 pg/mL). Follicles obtained at the end of the breeding season featured higher progesterone (2.61 ± 0.81 vs 0.19 ± 0.05 ng/mL; P < 0.05) and lower estradiol production (10 ± 3 vs 49 ± 10 pg/mL; P < 0.05) that was not influenced by LH and FSH. Basal androstenedione secretion was comparable to that observed at the middle of the breeding season (0.42 ± 0.10 vs 0.35 ± 0.09 ng/mL), but the response to stimulation was significantly higher (1.82 ± 0.61 vs 0.94 ± 0.34 ng/mL; P < 0.05). In anoestrus and the luteal phase, follicles presented higher progesterone and androstenedione and lower estradiol concentrations (P < 0.05) compared with those obtained during the follicular phase at the middle of the breeding season. In the luteal phase, follicles remained capable of responding to LH-FSH challenge by increasing estradiol secretion (9 ± 1 before and 21± 6 pg/mL after LH-FSH; P < 0.05). In contrast, in the luteal phase, estradiol production was not increased by LH-FSH challenge (7 ± 2 vs 12 ± 4 pg/mL).

Pituitary control of proliferation and differentiation of Leydig cells and their putative precursors in immature hypophysectomized rat testis.

The objective of this study was to determine the effects of pituitary hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], growth hormone [GH], and prolactin [PRL]) on interstitial cell proliferation and differentiation in the testis of immature hypophysectomized rats. Macrophages, Leydig cells, precursor mesenchymal cells, endothelial lymphatic cells, and myoid cells were studied. Our experimental approach was aimed at determining whether changes in a cellular subpopulation observed after pituitary hormone treatments were the result of division of existing cells in the population, of differentiation of interstitial precursor cells, or both. In this context, it must be stressed that our data reflected the effects of hormones to prevent the decline of cells due to hypophysectomy rather than their recovery. Macrophage proliferation was taken into account because macrophages closely resemble Leydig cells and are known to proliferate after hormonal treatment. A double-labeling procedure (acid phosphatase and anti-bromodeoxyuridine [anti-BUdR]) revealed that LH, FSH, and PRL increased the number of testicular macrophages 105-, 104-, and 103-fold, respectively, in hypophysectomized rats compared to hypophysectomized control animals. BUdR incorporation in testicular macrophages was greater after PRL treatment than after LH and FSH supplementation. In contrast, we were unable to demonstrate any effect of rat GH on the macrophage population. Light microscopic analysis of plastic embedded sections of treated rat testis revealed that LH increased the numbers of Leydig, precursor mesenchymal, and myoid cells 6-, 4-, and 1.3-fold, respectively. LH also stimulated BUdR incorporation into all interstitial cell types. PRL administration increased both the number of Leydig and precursor mesenchymal cells (each 3-fold) but decreased the number of endothelial lymphatic cells (1.5-fold) when compared to the control animals. In contrast, FSH did not increase the number and proliferation of Leydig cells but exerted a slight proliferative effect on the other interstitial cell populations. In GH-treated rats, the number of precursor mesenchymal cells increased two fold above the control rats. GH also exerted slight proliferative effects on both precursor mesenchymal and myoid cells. Immunohistochemical studies of steroidogenic enzymes in the testicular interstitium of treated rats demonstrated the presence of steroidogenic enzymes, not only in Leydig and precursor mesenchymal cells, but also in some (1%-2%) endothelial lymphatic cells and myoid cells. This may indicate that both of these cell types are also constitutively equipped to perform steroidogenesis or that they are precursor cells undergoing differentiation. Taken together, changes in the number of Leydig cells in our animal model appeared more likely to be dependent on the transformation of precursor cells than on division of preexisting mature Leydig cells.

Advancement of ovulation and oestrus after temporary calf removal and FSH supplementation in postpartum beef cows

To evaluate the effect of temporary calf removal and/or FSH supplementation on post-partum ovarian and oestrous activity, 40 anoestrous multiparous Aberdeen Angus cows under grazing conditions were used. The animals were allocated, on the basis of calving date and body condition, to four initially similar groups according to a 2 × 2 factorial design (ten animals per group) involving a 72 h calf removal (TCR) and exogenous follicle stimulating hormone supplementation (FSH). Anoestrus was determined by measuring plasma progesterone concentrations before treatments, which began at 29.6 ± 4.9 days postpartum. Total FSH supplementation (12 mg, porcine FSH) was performed by six intramuscular decreasing doses (3, 3, 2, 2, 1 and 1 mg, respectively) distributed at 12 h intervals, beginning 24 h before TCR. Oestrus detection (twice daily) and blood sample collection (every 2–3 days) were performed from 7 days before the start of treatments and for 45 days thereafter. The TCR plus FSH group had higher oestrous and ovulation rates at 40 days postpartum (control, 10% and 10%; TCR, 10% and 10%; FSH, 10% and 10%; TCR plus FSH, 60% and 60%) and 60 days postpartum (control, 30% and 30%; TCR, 50% and 50%; FSH, 40% and 50%; TCR plus FSH, 80% and 100%). These differences were significant ( P < 0.05) for oestrous rates at 40 days postpartum and for ovulation rates at 40 and 60 days postpartum. TCR plus FSH significantly shortened ( P < 0.05) the intervals (mean ± s.e.m., days) from calving to first ovulation (control, 61.2 ± 5.5; TCR, 62.8 ± 4.6; FSH, 61.6 ± 3.8 and TCR plus FSH, 43.4 ± 3.1) and a similar trend ( P > 0.05) was observed for the intervals from calving to first oestrus (control, 61.2 ± 5.5; TCR, 62.3 ± 5.4; FSH, 63.4 ± 3.9 and TCR + FSH, 47.7 ± 5.2). The rate of short first luteal phases (⩽ 10 days) was: control, 40%; TCR, 33%; FSH, 43%; TCR plus FSH, 89% ( P < 0.12). In summary, TCR plus FSH resulted in increased ovulation and oestrus rates, shorter postpartum intervals, but subnormal corpora lutea were formed following the first ovulation in early postpartum beef cows.

Is testosterone essential for maintenance of normal morphology in immature rat Leydig cells?

Selective deprivation of gonadotrophins in prepubertal rats by administration of a GnRH antagonist (Ac-D2Nal1, D4ClPhe2, DTrp3, DArg6, DA1a10-GnRH; GnRH code: 103-289-10, National Institutes of Health, USA) for 3 weeks, initiated at 20-22 days of age, induced morphological changes in the Leydig cells, including thickening and indentation of the nuclear margin, pyknosis and elongation of the nuclei. Mean nuclear diameter was reduced to 22% of that in the controls. Under the electron microscope the cells exhibited reduced volume of the nucleus and cytoplasm and the plasma membrane was irregular. This abnormal appearance of the Leydig cells improved marginally in 20-30% of the Leydig cells and their mean nuclear diameter increased to 39% of the control level after FSH supplementation (20 micrograms ovine FSH/day). Normal morphological integrity of the Leydig cells consisting of round or oval nuclei, a smooth nuclear and cellular margin and the original mean nuclear diameter was restored completely when testosterone (30 micrograms/day) was administered to GnRH antagonist-treated rats, with or without simultaneous administration of FSH; in these rats testosterone levels in blood were also restored to normal. These findings indicate that testosterone may be important for the maintenance of normal Leydig cell morphology in the rat.

Pituitary Hormones Dependent Expression of Insulin-Like Growth Factors I and II in the Immature Hypophysectomized Rat Testis

Since insulin-like growth factors I (IGF-I) and II (IGF-II) appeared involved in paracrine or autocrine regulation of both cell multiplication and differentiation of the rat testis, we have investigated the pituitary hormonal dependence of IGF-I and IGF-II mRNA production in the testis of immature hypophysectomized rats (22 days old) supplemented with highly purified FSH, LH, GH or PRL. Our data show that testicular expression of IGF-I mRNA as measured by dot-blot hybridization, is increased by LH, FSH or GH treatments of 7-, 6-, and 4-fold, respectively, above controls. Intensity of the signal was 3-fold lower after PRL treatment than in hypophysectomized control rats. On the contrary, IGF-II mRNA expression, was found low in the immature hypophysectomized rat testis and unmodified by any hormonal treatment. In contrast to the increase of IGF-I expression in the testis no significant change in the IGF-I plasma concentration was observed after LH or FSH supplementation. GH treatment, as expected, increased 4-fold the IGF-I plasma concentration of the experimental animals. Since we have previously shown that LH, FSH, and GH exhibit selective cell multiplication and differentiation in the testis of our animal model, it is proposed that testicular IGF-I expression could be the tissue response to pituitary hormone in these phenomena.

Effects of long-term treatment with LH on induction of cyclic ovarian activity in seasonally anoestrous ewes.

The aim of the present study was to establish whether cyclic ovarian activity could be induced and then maintained in anoestrous Romney ewes by the long-term administration of regular intravenous pulses of LH (10 micrograms ovine LH i.v. once every 1 or 2 h for 29-91 days). The LH pulse regimen was designed to generate plasma profiles of LH that were comparable to those experienced during the luteal and follicular phases of the oestrous cycle. The results showed that the LH treatments were capable of inducing cyclic ovarian activity, as assessed from the concentrations of progesterone in plasma, but that the treatments were inadequate for sustaining cyclic activity beyond two consecutive progestational phases. After 35-56 days of treatment, the plasma concentrations of FSH declined significantly (P less than 0.05) relative to those in the untreated animals. These data suggest that FSH supplementation as well as LH might be required for the long-term maintenance of cyclic ovarian activity in seasonally anoestrous ewes.