Papers by Muhammad Khalid
Reproduction, 2005
An experiment was carried out to determine the pattern of follicular expression of mRNAs for arom... more An experiment was carried out to determine the pattern of follicular expression of mRNAs for aromatase, IGF-I receptor (IGF-IR), IGF-binding protein (IGFBP)-2, -4 and -5, leptin and the long form of the leptin receptor (Ob-Rb) in ten ewes infused with human recombinant leptin (n 5 5; 1 mg/h) or saline (n 5 5) for 72 h in the luteal phase of the oestrous cycle. At the end of infusion a follicular phase was induced with a luteolytic dose of a prostaglandin F2a analogue and the ovaries were collected 32 h later. One ovary from each ewe was serially sectioned at 10 mm using a cryostat at 220 8C. All follicles >1 mm in diameter were counted and probed with specific oligoprobes for aromatase, IGF-IR and IGFBP-2, -4 and -5 and specific riboprobes for leptin and Ob-Rb. Leptin mRNA was detected in theca and granulosa cells and Ob-Rb mRNA was detected only in granulosa cells, of some, but not all antral follicles. Leptin doubled the number of follicles with a diameter $3.5 mm (1.0 6 0.36 (S.E.M.) vs 2.4 6 0.24; control vs leptin; P < 0.02) but had no effect on the number of $1 < 3.5 mm follicles. Leptin had no effect on the number of follicles expressing aromatase mRNA but it decreased significantly the number of follicles expressing mRNA for IGF-IR (10.7 6 0.79 vs 7.4 6 0.81; control vs leptin; P < 0.05), IGFBP-2 (10.0 6 0.82 vs 5.2 6 0.87; control vs leptin; P < 0.05) and IGFBP-5 (5.2 6 1.60 vs 1.2 6 0.30; control vs leptin; P < 0.05). Leptin increased the diameter of IGFBP-2 mRNA-positive follicles (1.5 6 0.15 vs 2.2 6 0.31 mm; control vs leptin; P < 0.05) and increased follicular mRNA expression for IGFBP-2 (0.30 6 0.021 vs 0.39 6 0.027 arbitrary units; control vs leptin; P < 0.05) and IGFBP-5 (0.46 6 0.019 vs 0.25 6 0.053 arbitary units; control vs leptin; P < 0.05). The mRNA for IGFBP-4 was detected in the theca of only two follicles from the control group. Leptin increased the number of follicles expressing Ob-Rb mRNA (0.25 6 0.25 vs 1.40 6 1.17; control vs leptin; P < 0.05) but had no effect on the number expressing leptin mRNA. Leptin decreased plasma concentrations of oestradiol (P < 0.05) and increased concentrations of FSH (P < 0.001) and insulin (P < 0.001), with no effect on glucose concentrations. These data show that: (i) ovine granulosa cells express mRNA for Ob-Rb and leptin and (ii) leptin increased the number of follicles $ 3.5 mm. Furthermore, the data suggest that suppression of oestradiol production by leptin is not mediated by inhibition of aromatase gene expression. Finally, the data indicate that the action of leptin in ovarian follicles is mediated by the IGF system, because leptin increased mRNA expression of IGFBP-2 and -5. Leptin also decreased the number of follicles expressing IGF-IR and IGFBP-2 and -5. We suggest that these actions of leptin on the IGF system decrease the bioavailability of IGF-I, resulting in decreased oestradiol production. Reproduction (2005) 130 869-881 q 2005 Society for Reproduction and Fertility
Reproduction Nutrition Development, 2006
This paper discusses the phenomenon of nutritional flushing in ewes whereby increased nutrition s... more This paper discusses the phenomenon of nutritional flushing in ewes whereby increased nutrition stimulates folliculogenesis and ovulation rate. In addition the paper reviews recent findings on the effects of increased levels of nutrition on the blood concentrations of reproductive and metabolic hormones in the ewe and some of the intraovarian changes that take place in response to nutritional stimulation. Finally, in the paper, we propose a model of the physiological mechanism for the nutritional stimulation of folliculogenesis and we review how closely the model fits recent published and unpublished evidence examining the mechanism of flushing. Nutritional stimulation alters the blood concentrations of some metabolic hormones. By using short-term models of nutritional flushing, we have shown that as the blood concentrations of insulin and leptin increase that of growth hormone decreases while that of IGF-I appears unaffected by the nutritional flushing. Nutritional flushing also alters the blood concentrations of some reproductive hormones. Again, using the same model, we have shown that there is a transient increase in FSH and a decrease in oestradiol concentrations in the blood. The changes in oestradiol are particularly evident in the follicular phase of the oestrous cycle. In the ovary, the effect of nutrition is to stimulate folliculogenesis. These changes are associated with intra-follicular alterations in the insulin-glucose, IGF and leptin metabolic systems. The stimulation of these intra-follicular systems leads to a suppression in follicular oestradiol production. The consequence of these direct actions on the follicle is a reduced negative feedback to the hypothalamic-pituitary system and increased FSH secretion that leads to a stimulation of folliculogenesis.
Biology of Reproduction, 2007
The use of transcervical artificial insemination in sheep is limited because of the anatomy of th... more The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2 mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2 mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2 mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2 mRNA expression and hence prostaglandin E2 synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.
Theriogenology, 2009
Transcervical artificial insemination in sheep is limited by the inability to completely penetrat... more Transcervical artificial insemination in sheep is limited by the inability to completely penetrate the cervix with an inseminating pipette. Penetration is partially enhanced at estrus due to a degree of cervical relaxation, which is probably regulated by cervical prostaglandin synthesis and extracellular matrix remodeling. Prostaglandin E 2 acts via prostaglandin E receptors EP 1 to EP 4 , and EP 2 and EP 4 stimulate smooth muscle relaxation and glycosaminoglycan synthesis. This study investigated the expression of EP 2 and EP 4 mRNA and glycosaminoglycans in the sheep cervix during the estrous cycle. Sheep cervices were collected prior to, during, and after the luteinizing hormone (LH) surge and during the luteal phase. The mRNA expression of EP 2 and EP 4 was determined by in situ hybridization, glycosaminoglycan composition was assessed by Alcian blue staining, and hyaluronan concentration was investigated by ELISA. The expression of EP 2 mRNA was greatest prior to the LH surge (P = 0.02), although EP 2 and EP 4 were expressed throughout the estrous cycle. Hyaluronan was the predominant glycosaminoglycan, and hyaluronan content increased prior to the LH surge (P < 0.05). Cervical EP 2 mRNA expression changed throughout the estrous cycle and was greatest prior to the LH surge. We propose that prostaglandin E 2 binds to EP 2 and EP 4 stimulating hyaluronan synthesis, which may cause remodeling of the cervical extracellular matrix, culminating in cervical relaxation. #
Domestic Animal Endocrinology, 2008
Receptors for LH (LHR) and FSH (FSHR) are expressed in the canine lower urinary tract (LUT). As g... more Receptors for LH (LHR) and FSH (FSHR) are expressed in the canine lower urinary tract (LUT). As gonadectomy results in an increase in plasma LH and FSH, the objective of this study was to determine whether there are any differences in the expression of LHR and FSHR in the LUT between intact and gonadectomised dogs. Four regions of the LUT, i.e. body and neck of the bladder as well as proximal and distal urethra, were collected from 20 healthy dogs (5 intact males, 5 intact anoestrous females, 4 castrated males and 6 spayed females). The mRNA and protein expression of receptors was determined by in situ hybridization and immunohistochemistry, respectively, and assessed semi-quantitatively incorporating both the distribution and the intensity of specific staining. Expression of LHR and FSHR was present in all tissue layers (epithelium, sub-epithelial stroma and muscle) of each region with different levels of the expression. Overall mRNA and protein expression for both LHR and FSHR was significantly (P < 0.001) lower in gonadectomised dogs. Intact dogs had more (P < 0.05) LHR and FSHR mRNA and protein in all tissue layers of the four regions, except for LHR mRNA expression in the sub-epithelial stroma where no differences were observed between the two statuses. Decreases in LHR and FSHR mRNA and protein in gonadectomised dogs appeared to be more consistent in spayed bitches compared to castrated males. Lower expression of LHR and FSHR observed in gonadectomised dogs may adversely affect the normal canine LUT function.
Theriogenology, 2007
In dogs, one of the side effects of neutering is the development of urinary incontinence. The rel... more In dogs, one of the side effects of neutering is the development of urinary incontinence. The relationship between neutering and urinary incontinence caused by acquired urethral sphincter mechanism incompetence (USMI) has been reported. Recently, GnRH analogue treatment that suppresses elevated plasma gonadotrophin concentrations post-spaying has been successfully used in incontinent bitches. These data and the fact that non-gonadal tissues may contain receptors for LH (LHR) and FSH (FSHR) suggest that there might be a functional relationship between gonadotrophins and the lower urinary tract in dogs. This study aimed to investigate the presence of LHR and FSHR in the lower urinary tract of intact male and female dogs. Four regions of the lower urinary tract, i.e. (i) body of the bladder, (ii) neck of the bladder, (iii) proximal urethra and (iv) distal urethra were collected from 10 healthy dogs (5 males and 5 anoestrous females). In situ hybridization and immunohistochemistry were performed to characterise the presence of receptor mRNA and receptor protein. Staining was rated semi-quantitatively, incorporating both the distribution and intensity of specific staining. The distribution of receptor expression in different tissue layers (epithelium, subepithelial stroma and muscle) in each region was statistically analyzed. Luteinizing hormone receptor and FSHR mRNA and protein were present in all four regions and in three tissue layers of males and females. Irrespective of region and layer, female dogs expressed significantly higher expression for LHR mRNA (P < 0.001), LHR protein (P < 0.05) and FSHR protein (P < 0.001). The expression of LHR and FSHR mRNA and protein was not uniform and depended on region, tissue layer and gender. The expression of LHR mRNA was higher in the bladder, compared to the urethra (P < 0.05). The FSHR mRNA significantly increased from the bladder to the urethra. Protein expression for LHR and FSHR was highest in the proximal urethra (P < 0.05). The overall expression for LHR and FSHR at both mRNA and protein levels was highest in the epithelium, intermediate to low in the subepithelial stroma and muscle. A significant interaction between region and tissue layer showed that mRNA and protein expression for LHR and FSHR decreased from the bladder to the urethra in the epithelium and subepithelial stroma. In contrast, it gradually increased from the bladder to the urethra in the muscle. In conclusion, the present study showed that both mRNA and protein for LHR and FSHR were expressed in the canine lower urinary tract, and the expression levels varied between genders and among regions and tissue layers. The presence of these receptors suggests that gonadotrophins have a role in the physiology and/or pathology of the lower urinary tract function in the dog. #
Theriogenology, 2007
Two experiments in parous Welsh Mountain ewes determined the pattern of natural cervical relaxati... more Two experiments in parous Welsh Mountain ewes determined the pattern of natural cervical relaxation over the peri-ovulatory period and investigated FSH and Misoprostol as cervical relaxants to facilitate transcervical passage of an insemination pipette into the uterine cavity. Following synchronisation of oestrus using progestagen sponges and PMSG (500 IU) the depth of cervical penetration was determined using a modified cattle insemination pipette as a measuring device.
Theriogenology, 2005
The anatomy of the sheep cervix is highly variable between animals and may explain the differing ... more The anatomy of the sheep cervix is highly variable between animals and may explain the differing success of transcervical AI between individuals. This study aims to quantify the variation in cervical morphology between ewes and investigate the relationship between cervical anatomy and cervical penetration.
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Papers by Muhammad Khalid