Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β-binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200–1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.

Pre-anthesis ovary development determines genotypic differences in potential kernel weight in sorghum

Kernel weight is an important factor determining grain yield and nutritional quality in sorghum, yet the developmental processes underlying the genotypic differences in potential kernel weight remain unclear. The aim of this study was to determine the stage in development at which genetic effects on potential kernel weight were realized, and to investigate the developmental mechanisms by which potential kernel weight is controlled in sorghum. Kernel development was studied in two field experiments with five genotypes known to differ in kernel weight at maturity. Pre-fertilization floret and ovary development was examined and post-fertilization kernel-filling characteristics were analysed. Large kernels had a higher rate of kernel filling and contained more endosperm cells and starch granules than normal-sized kernels. Genotypic differences in kernel development appeared before stamen primordia initiation in the developing florets, with sessile spikelets of large-seeded genotypes having larger floret apical meristems than normal-seeded genotypes. At anthesis, the ovaries for large-sized kernels were larger in volume, with more cells per layer and more vascular bundles in the ovary wall. Across experiments and genotypes, there was a significant positive correlation between kernel dry weight at maturity and ovary volume at anthesis. Genotypic effects on meristem size, ovary volume, and kernel weight were all consistent with additive genetic control, suggesting that they were causally related. The pre-fertilization genetic control of kernel weight probably operated through the developing pericarp, which is derived from the ovary wall and potentially constrains kernel expansion.

Growth differentiation factor 9 signalling in the ovary

In the ovary, two new members of the large TGF-beta superfamily of growth factors were discovered in the 1990s. The oocyte was shown to express two closely related growth factors that were named growth differentiation factor 9 (GDF-9) and growth differentiation factor 9B (GDF-9B). Both of these proteins are required for normal ovarian follicle development although their individual significance varies between species. GDF-9 and GDF-9B mRNAs are expressed in the human oocytes from the primary follicle stage onwards. This thesis project was aimed to define the signalling mechanisms utilized by the oocyte secreted GDF-9. We used primary cultures of human granulosa luteal cells (hGL) as our cell model, and recombinant adenovirus-mediated gene transfer in manipulating the TGF-b family signalling cascade molecules in these cells. Overexpression of the constitutively active forms of the seven type I receptors, the activin receptor-like kinases 1-7 (ALK1-7), using recombinant adenoviruses caused a specific activation of either the Smad1 or Smad2 pathway proteins depending on the ALK used. Activation of both Smad1 and Smad2 proteins also stimulated the expression of dimeric inhibin B protein in hGL cells. Treatment with recombinant GDF-9 protein induced the specific activation of the Smad2 pathway and stimulated the expression of inhibin betaB subunit mRNA as well as inhibin B protein secretion in our cell model. Recombinant GDF-9 also activated the Smad3-responsive CAGA-luciferase reported construct, and the GDF-9 response in hGL cells was markedly potentiated upon the overexpression of Alk5 by adenoviral gene transduction. Alk5 overexpression also enhanced the GDF-9 induced inhibin B secretion by these cells. Similarly, in a mouse teratocarcinoma cell line P19, GDF-9 could activate the Smad2/3 pathway, and overexpression of ALK5 in COS7 cells rendered them responsive to GDF-9. Furthermore, transfection of rat granulosa cells with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 effects. In conclusion, this thesis shows that both Smad1 and Smad2 pathways are involved in controlling the regulation of inhibin B secretion. Therefore, in addition to endocrine control of inhibin production by the pituitary gonadotropins, also local paracrine factors within in the ovary, like the oocyte-derived growth factors, may contribute to controlling inhibin secretion. This thesis shows as well that like other TGF-beta family ligands, also GDF-9 signalling is mediated by the canonical type I and type II receptors with serine/threonine kinase activity, and the intracellular transcription factors, the Smads. Although GDF-9 binds to the BMP type II receptor, its downstream actions are specifically mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins.

Evaluation of the impacts of spaying by either the dropped ovary technique or ovariectomy via flank laparotomy on the welfare of Bos indicus beef heifers and cows

The welfare outcomes for Bos indicus cattle (100 heifers and 50 cows) spayed by either the dropped ovary technique (DOT) or ovariectomy via flank laparotomy (FL) were compared with cattle subjected to physical restraint (PR), restraint by electroimmobilization in conjunction with PR (EIM), and PR and mock AI (MAI). Welfare assessment used measures of morbidity, mortality, BW change, and behavior and physiology indicative of pain and stress. One FL heifer died at d 5 from peritonitis. In the 8-h period postprocedures, plasma bound cortisol concentrations of FL, DOT, and EIM cows were not different and were greater (P < 0.05) than PR and MAI. Similarly, FL and DOT heifers had greater (P < 0.05) concentrations than PR and MAI, with EIM intermediate. Creatine kinase and aspartate aminotransferase concentrations were greater (P < 0.05) in FL and EIM heifers compared with the other treatments, with a similar pattern seen in the cows. Haptoglobin concentrations were significantly (P < 0.05) increased in the FL heifers compared with other treatments in the 8- to 24-h and 24- to 96-h periods postprocedures, and in cows were significantly (P < 0.05) increased in the FL and DOT compared with PR in the 24- to 96-h period. Behavioral responses complemented the physiological responses; standing head down was shown by more (P < 0.05) FL cows and heifers to 3 d postprocedures compared with other treatments, although there was no difference between FL and DOT heifers at the end of the day of procedures. At this same time, fewer (P < 0.05) FL and DOT heifers and cows were observed feeding compared with other treatments, although in cows there was no difference between FL, DOT, and EIM. There were no significant differences (P > 0.05) between treatments in BW changes. For both heifers and cows, FL and DOT spaying caused similar levels of acute pain, but FL had longer-lasting adverse impacts on welfare. Electroimmobilization during FL contributed to the pain and stress of the procedure. We conclude that: i) FL and DOT spaying should not be conducted without measures to manage the associated pain and stress; ii) DOT spaying is preferable to FL spaying; iii) spaying heifers is preferable to spaying cows; and iv) electroimmobilization causes pain and stress and should not be routinely used as a method of restraint.

Examination of the role of follicle stimulating hormone in estrogen biosynthesis Image Image and Image Image in the ovary of cyclic hamster

The effect of neutralizing endogenous follicle stimulating hormone (FSH) or luteinizing hormone (LH) with specific antisera on the Image Image and Image Image synthesis of estrogen in the ovary of cycling hamster was studied. Neutralization of FSH or LH on proestrus resulted in a reduction in the estradiol concentration of the ovary on diestrus-2 and next proestrus, suggesting an impairment in follicular development.Injection of FSH antiserum at 0900 h of diestrus-2 significantly reduced the ovarian estradiol concentration within 6–7 h. Further, these ovaries on incubation with testosterone(T) Image Image at 1600 h of the same day or the next day synthesized significantly lower amounts of estradiol, compared to corresponding control ovaries. Although testosterone itself, in the absence of endogenous FSH, could stimulate estrogen synthesis to some extent, FSH had to be supplemented with T to restore estrogen synthesis to the level seen in control ovaries incubated with T. Lack of FSH thus appeared to affect the aromatization step in the estrogen biosynthetic pathway in the ovary of hamster on diestrus-2. In contrast to this, FSH antiserum given on the morning of proestrus had no effect on the Image Image and Image Image synthesis of estrogen, when examined 6–7 h later. The results suggest that there could be a difference in the need for FSH at different times of the cycle.Neutralization of LH either on diestrus-2 or proestrus resulted in a drastic reduction in estradiol concentration of the ovary. This block was at the level of androgen synthesis, since supplementing testosterone alone Image Image could stimulate estrogen synthesis to a more or less similar extent as in the ovaries of control hamsters.

Effect of follicle-stimulating hormone and its antiserum on the activity of ornithine decarboxylase in the ovary of rat and hamster

The ability of FSH to stimulate the activity of ornithine decarboxylase (ODC) in the ovary of the immature rat and cycling hamster has been examined using specific antisera to gonadotropins. The stimulatory effect of FSH on ODC activity in the ovary of the immature rat was abolished when LH antiserum was administered along with FSH, while similar administration of FSH antiserum had no effect on LH action in stimulating ODC activity, thereby demonstrating the specificity of the LH effect. During the estrus cycle of the hamster, ODC activity in the ovary could be detected only on the evening of proestrus, the maximal activity seen at 1700 h being associated with both the Graafian follicles and the rest of the ovarian tissue. Neutralization of the proestrous FSH surge had no effect on the activity of ODC in either of these tissues, while similar administration of LH antiserum at 1300 h of proestrus completely inhibited the ODC activity in both large follicles and the rest of the ovarian tissue. Thus, the surge of LH, but not of FSH, appears to be responsible for regulating the ODC activity in the ovary of the cycling hamster.

A case of development of the parthenogenetic embryo in the ovary of Moina macrocopa Straus (Crustacea, Cladocera). [Translation from: Vest. Zool., Kiev 4, p. 86, 1971. ]

In a survey of histological preparations of several hundreds of Moina macrocopa Straus , one specimen was discovered, in the left ovary of which was found a parthenogenetic embryo. The short article includes an illustration of a transverse section of parthenogenetic female of Moina macrocopa.

Studies on the biochemical composition of some freshwater fishes. Pt. 3. Ovary

Biochemical composition of the spent ovaries of 23 different species of freshwater fishes showed a much lower value for protein and fat with higher moisture content than those of the ripe eggs, suggesting an inverse relationship between fat and moisture. Ovaries during the recovering phase showed fairly high values of phosphorus, but lower than those for the ripe eggs. Higher values of calcium and iron in spent ovaries than those in ripe eggs probably suggest that there is a greater requirement of calcium and iron during the spent phase of the gonads.

Binding of radio-iodinated gonadotropin to the ovary of exotic carp (Cyprinus carpio Lin.)

Heterologous murrel gonadotropic hormone (m-GtH) binds to common carp oocyte plasma membrane and enhances steroid secretion. With increasing concentration of radio-labelled hormone the receptor binding is also found to increase linearly up to a certain concentration and then decrease. The [¹²⁵I] murrel GtH binding characteristics to a preparation of common carp ovarian plasma membrane shows saturability with high affinity. Scat chard plot analysis gave dissociation constant (Kd) of 0.81 X 10(super -9) M and maximum binding capacity (MBC) of 22.05 f mole/mg protein.

Studies on fecundity of Puntius sarana (Ham.) in relation to total length, total weight and ovary weight

A total of 43 mature female of Puntius sarana (Ham.) ranging from 204 mm to 320 mm in length and 102 to 482 g in weight were used for present studies. The relationship between fecundity with respect to total length (TL), total weight (TW) and ovary weight (OW) was found to be linear. The coefficient of co-relation 'r' of the above relationships was found to be 0.5947, 0.5761 and 0.9837 respectively. All these values are highly significant (P=0.01) indicating a close relationship between these parameters. However, as indicated by value of 'r' (0.9837), the fecundity is more closely related to ovary weight and hence the ovary weight may be a better index of fecundity than the total length or weight.

Studies on fecundity of Rita pavimentata (Gunther) in relation to total length, total weight and ovary weight

A total of thirty eight ripe ovaries from the specimens of Rita pavimentata, measuring 230 to 355 mm in total length (TL) and 250 to 750 g in total weight (TW), were selected to study the fecundity. The relationship between fecundity 'and total length (TL), total weight (TW) and ovary weight (OW) was found to be linear. The coefficient of co-relation 'r' of the above relationship was found to be 0.92, 0.94 and 0.96 respectively. All these values are highly significant (P=0.01) indicating a close relationship between compared parameters. However, as indicated by value of 'r' (0.96), the fecundity is more closely related to ovary weight and hence the ovary weight may be a better index of fecundity than the total body length or weight.