The purpose of the studies reported in this thesis was to provide a better understanding of the effects of purified ovulation-inducing factor (OIF) from llama seminal plasma in reflex ovulators (lama glama) and spontaneous ovulators (Bos taurus). The objective of the first study was to determine if the dose of OIF of llama seminal plasma required to elicit ovulation is physiologically relevant, and to test the hypothesis that CL form and function is affected by OIF in a dose-dependent manner. Llamas were treated with four different doses (500 £gg, 250 £gg, 125 £gg and 60 £gg) based on knowledge that for every ejaculate there is approximately 3 mg of OIF. Results supported the hypothesis that OIF affects ovulation and CL form and function in a dose-dependent manner. The high dose of OIF (500 Ýg) was associated with the highest incidence of ovulation, maximum CL diameter, plasma progesterone concentrations and plasma LH concentrations. The low dose of OIF (60 Ýg) was minimally effective for induction of ovulation and associated with smaller CL diameter and lower plasma concentrations of progesterone and LH. The second study was carried out to test the hypotheses that OIF will induce ovulation and affects CL form and function in cattle (Experiment 1), and that OIF given at different stages of development of the first follicular wave will induce atresia of the dominant follicle and hasten emergence of a new follicular wave (Experiment 2). Heifers were treated on Day 5 (Day 0 = wave emergence; Experiment 1) or on Days 3, 6 and 9 (Experiment 2) with a) 1ml of saline, b) 100 £gg of GnRH, or c) 1.0 mg purified OIF per 100 kg of body weight. Results of Experiment 1 demonstrated that OIF did not induce ovulation in cattle but it did induce atresia of the dominant follicle and earlier emergence of a new follicular wave. Results from the second study suggested that the effect previously demonstrated could be accomplished in sexually mature females after treatment on Day 6 corresponding to the late growing phase of the dominant follicle. In summary, the minimum dose of OIF necessary to induce ovulation in llamas was between 60 £gg and 250 £gg. This dose is physiologically relevant and represents less than 1/6th of what is normally present in a single llama ejaculate. In cattle OIF induced regression of the dominant follicle and early emergence of a new follicular wave in pre-pubertal heifers and had a similar effect in sexually mature heifers after treatment on Day 6 of the estrous cycle.

The purpose of the studies reported in this thesis was to provide a better understanding of the effects of purified ovulation-inducing factor (OIF) from llama seminal plasma in reflex ovulators (lama glama) and spontaneous ovulators (Bos taurus). The objective of the first study was to determine if the dose of OIF of llama seminal plasma required to elicit ovulation is physiologically relevant, and to test the hypothesis that CL form and function is affected by OIF in a dose-dependent manner. Llamas were treated with four different doses (500 £gg, 250 £gg, 125 £gg and 60 £gg) based on knowledge that for every ejaculate there is approximately 3 mg of OIF. Results supported the hypothesis that OIF affects ovulation and CL form and function in a dose-dependent manner. The high dose of OIF (500 Ýg) was associated with the highest incidence of ovulation, maximum CL diameter, plasma progesterone concentrations and plasma LH concentrations. The low dose of OIF (60 Ýg) was minimally effective for induction of ovulation and associated with smaller CL diameter and lower plasma concentrations of progesterone and LH. The second study was carried out to test the hypotheses that OIF will induce ovulation and affects CL form and function in cattle (Experiment 1), and that OIF given at different stages of development of the first follicular wave will induce atresia of the dominant follicle and hasten emergence of a new follicular wave (Experiment 2). Heifers were treated on Day 5 (Day 0 = wave emergence; Experiment 1) or on Days 3, 6 and 9 (Experiment 2) with a) 1ml of saline, b) 100 £gg of GnRH, or c) 1.0 mg purified OIF per 100 kg of body weight. Results of Experiment 1 demonstrated that OIF did not induce ovulation in cattle but it did induce atresia of the dominant follicle and earlier emergence of a new follicular wave. Results from the second study suggested that the effect previously demonstrated could be accomplished in sexually mature females after treatment on Day 6 corresponding to the late growing phase of the dominant follicle. In summary, the minimum dose of OIF necessary to induce ovulation in llamas was between 60 £gg and 250 £gg. This dose is physiologically relevant and represents less than 1/6th of what is normally present in a single llama ejaculate. In cattle OIF induced regression of the dominant follicle and early emergence of a new follicular wave in pre-pubertal heifers and had a similar effect in sexually mature heifers after treatment on Day 6 of the estrous cycle.

Thesis (M. Phil.)--University of Hong Kong, 1980. / Typescript.

published_or_final_version / Anatomy / Master / Master of Philosophy

Title page, abstract and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / In mice and other mammalian species, deposition of semen into the female reproductive tract elicits a local inflammatory response. Whether a comparable response occurs within the human cervix has not previously been studied. The experiments described in this thesis demonstrate, using cervical tissue biopsies taken before and after intercourse, that exposure to semen elicits an infiltration of leukocytes into the cervical tissue of peri-ovulatory women. Immunohistochemical analysis identified macrophages and dendritic cells as the predominant leukocytes recruited into the cervical epithelium and stroma following intercourse. Cytotoxic / suppressor T lymphocytes and memory T cells were also increased. Comparable responses were not detected following condom-protected intercourse. Quantitative real-time PCR was performed on duplicate tissue biopsies to investigate the molecular regulation of this response. Expression of GM-CSF, a potent stimulator of myeloid cell recruitment, was found to increase by 2.5-fold following unprotected intercourse. Trends towards increased IL-6 and IL-8 mRNA were also observed. Condom-protected intercourse did not activate cytokine expression, further suggesting that exposure to semen, as opposed to mechanical trauma, provides the inflammatory stimulus. In an in vitro model using the immortalised Ect-1 cell line, TGFβ was identified as a candidate active seminal factor. All three TGFβ isoforms were capable of mimicking the stimulatory ability of seminal plasma in Ect-1 cells and were comparable in their capacity to stimulate both GM-CSF and IL-6 expression in a dose-responsive manner. The addition of TGFβ isoform-specific neutralising antibodies inhibited seminal plasma-induced increases in these cytokines. However TGFβ was unable to stimulate IL-8 production. Addition of IFNƴ was found to strongly inhibit TGFβ-stimulated GM-CSF production, and 19-0H PGE₁ was found to increase IL-6 and IL-8, but not GM-CSF production. Responses to seminal plasma constituents were almost exactly replicated in primary cultures of human ectocervical cells. These results identify TGFβ as the major active constituent in human seminal plasma and indicate that other seminal agents, 19-0H PGE₁ and IFNƴ, interact with TGFβ to differentially regulate cervical cytokine expression. Finally, whether human seminal plasma cytokine content was associated with fertility in men was examined. No relationship between seminal plasma TGFβ₁, TGFβ₂, TGFβ₃, IL-8 or bacterial endotoxin content and fertility status was observed. However, there was an increased likelihood of high IFNƴ content in the male partners of couples experiencing infertility, most notable in recurrent miscarriage. The discriminating value of IFNƴ was increased when evaluated as a ratio of total TGFβ content. Inflammatory changes after exposure of the female reproductive tract to seminal plasma are implicated in 'conditioning' the maternal immune response, to facilitate successful embryo implantation and pregnancy. The studies described in this thesis provide a mechanistic basis for the observations linking exposure to semen with pregnancy success in humans and have expanded our knowledge of the cellular and molecular events that occur within the female reproductive tract following intercourse. Seminal plasma can therefore no longer be thought of as merely a transport medium for spermatozoa, rather as a means for communication between the male and female reproductive tissues, potentially required for optimal pregnancy success. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1213820 / Thesis (Ph.D.) -- University of Adelaide, Medical School, 2005

Title page, abstract and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / In mice and other mammalian species, deposition of semen into the female reproductive tract elicits a local inflammatory response. Whether a comparable response occurs within the human cervix has not previously been studied. The experiments described in this thesis demonstrate, using cervical tissue biopsies taken before and after intercourse, that exposure to semen elicits an infiltration of leukocytes into the cervical tissue of peri-ovulatory women. Immunohistochemical analysis identified macrophages and dendritic cells as the predominant leukocytes recruited into the cervical epithelium and stroma following intercourse. Cytotoxic / suppressor T lymphocytes and memory T cells were also increased. Comparable responses were not detected following condom-protected intercourse. Quantitative real-time PCR was performed on duplicate tissue biopsies to investigate the molecular regulation of this response. Expression of GM-CSF, a potent stimulator of myeloid cell recruitment, was found to increase by 2.5-fold following unprotected intercourse. Trends towards increased IL-6 and IL-8 mRNA were also observed. Condom-protected intercourse did not activate cytokine expression, further suggesting that exposure to semen, as opposed to mechanical trauma, provides the inflammatory stimulus. In an in vitro model using the immortalised Ect-1 cell line, TGFβ was identified as a candidate active seminal factor. All three TGFβ isoforms were capable of mimicking the stimulatory ability of seminal plasma in Ect-1 cells and were comparable in their capacity to stimulate both GM-CSF and IL-6 expression in a dose-responsive manner. The addition of TGFβ isoform-specific neutralising antibodies inhibited seminal plasma-induced increases in these cytokines. However TGFβ was unable to stimulate IL-8 production. Addition of IFNƴ was found to strongly inhibit TGFβ-stimulated GM-CSF production, and 19-0H PGE₁ was found to increase IL-6 and IL-8, but not GM-CSF production. Responses to seminal plasma constituents were almost exactly replicated in primary cultures of human ectocervical cells. These results identify TGFβ as the major active constituent in human seminal plasma and indicate that other seminal agents, 19-0H PGE₁ and IFNƴ, interact with TGFβ to differentially regulate cervical cytokine expression. Finally, whether human seminal plasma cytokine content was associated with fertility in men was examined. No relationship between seminal plasma TGFβ₁, TGFβ₂, TGFβ₃, IL-8 or bacterial endotoxin content and fertility status was observed. However, there was an increased likelihood of high IFNƴ content in the male partners of couples experiencing infertility, most notable in recurrent miscarriage. The discriminating value of IFNƴ was increased when evaluated as a ratio of total TGFβ content. Inflammatory changes after exposure of the female reproductive tract to seminal plasma are implicated in 'conditioning' the maternal immune response, to facilitate successful embryo implantation and pregnancy. The studies described in this thesis provide a mechanistic basis for the observations linking exposure to semen with pregnancy success in humans and have expanded our knowledge of the cellular and molecular events that occur within the female reproductive tract following intercourse. Seminal plasma can therefore no longer be thought of as merely a transport medium for spermatozoa, rather as a means for communication between the male and female reproductive tissues, potentially required for optimal pregnancy success. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1213820 / Thesis (Ph.D.) -- University of Adelaide, Medical School, 2005

Title page, abstract and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / In mice and other mammalian species, deposition of semen into the female reproductive tract elicits a local inflammatory response. Whether a comparable response occurs within the human cervix has not previously been studied. The experiments described in this thesis demonstrate, using cervical tissue biopsies taken before and after intercourse, that exposure to semen elicits an infiltration of leukocytes into the cervical tissue of peri-ovulatory women. Immunohistochemical analysis identified macrophages and dendritic cells as the predominant leukocytes recruited into the cervical epithelium and stroma following intercourse. Cytotoxic / suppressor T lymphocytes and memory T cells were also increased. Comparable responses were not detected following condom-protected intercourse. Quantitative real-time PCR was performed on duplicate tissue biopsies to investigate the molecular regulation of this response. Expression of GM-CSF, a potent stimulator of myeloid cell recruitment, was found to increase by 2.5-fold following unprotected intercourse. Trends towards increased IL-6 and IL-8 mRNA were also observed. Condom-protected intercourse did not activate cytokine expression, further suggesting that exposure to semen, as opposed to mechanical trauma, provides the inflammatory stimulus. In an in vitro model using the immortalised Ect-1 cell line, TGFβ was identified as a candidate active seminal factor. All three TGFβ isoforms were capable of mimicking the stimulatory ability of seminal plasma in Ect-1 cells and were comparable in their capacity to stimulate both GM-CSF and IL-6 expression in a dose-responsive manner. The addition of TGFβ isoform-specific neutralising antibodies inhibited seminal plasma-induced increases in these cytokines. However TGFβ was unable to stimulate IL-8 production. Addition of IFNƴ was found to strongly inhibit TGFβ-stimulated GM-CSF production, and 19-0H PGE₁ was found to increase IL-6 and IL-8, but not GM-CSF production. Responses to seminal plasma constituents were almost exactly replicated in primary cultures of human ectocervical cells. These results identify TGFβ as the major active constituent in human seminal plasma and indicate that other seminal agents, 19-0H PGE₁ and IFNƴ, interact with TGFβ to differentially regulate cervical cytokine expression. Finally, whether human seminal plasma cytokine content was associated with fertility in men was examined. No relationship between seminal plasma TGFβ₁, TGFβ₂, TGFβ₃, IL-8 or bacterial endotoxin content and fertility status was observed. However, there was an increased likelihood of high IFNƴ content in the male partners of couples experiencing infertility, most notable in recurrent miscarriage. The discriminating value of IFNƴ was increased when evaluated as a ratio of total TGFβ content. Inflammatory changes after exposure of the female reproductive tract to seminal plasma are implicated in 'conditioning' the maternal immune response, to facilitate successful embryo implantation and pregnancy. The studies described in this thesis provide a mechanistic basis for the observations linking exposure to semen with pregnancy success in humans and have expanded our knowledge of the cellular and molecular events that occur within the female reproductive tract following intercourse. Seminal plasma can therefore no longer be thought of as merely a transport medium for spermatozoa, rather as a means for communication between the male and female reproductive tissues, potentially required for optimal pregnancy success. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1213820 / Thesis (Ph.D.) -- University of Adelaide, Medical School, 2005

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 122 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 99-122).

O presente estudo visou verificar o efeito sobre alguns parâmetros da seleção por gradiente de Percoll® e da adição de plasma seminal do sêmen eqüino preservado a +4oC. O primeiro experimento avaliou a taxa de recuperação de espermatozóides após seleção por Percoll® em diferentes protocolos de centrifugação. Foram realizadas 5 coletas de sêmen de um garanhão. Imediatamente após a coleta, o sêmen foi avaliado quanto à motilidade, vigor e concentração. Foram retiradas duas amostras de 4 mL, diluídas em leite desnatado UHT com concentrações de 50 e 100 x 106 espermatozóides por mL cada. Cada uma destas amostras foi dividida em 4 alíquotas de 1 mL, que foram então colocadas sobre Percoll® e submetidas a diferentes tempos e velocidades de centrifugação. V1 - 200 g (5 min) + 800 g (10 min); V2 - 800 g (10 min); V3 - 800 g (15 min); V4 - 800 g (20 min). Após esse processo, o sobrenadante foi desprezado e o pellet de cada alíquota ressuspendido com 0,5 mL de leite UHT. As 8 amostras foram novamente avaliadas para concentração, motilidade e vigor. O segundo experimento estudou o efeito da adição de plasma seminal de diferentes qualidades ao sêmen eqüino selecionado por gradiente de Percoll® e resfriado a +4°C por até 72 horas. Foram utilizados 40 ejaculados de 4 garanhões, sendo dois com boa qualidade de sêmen e dois com baixa qualidade de sêmen. Imediatamente após a coleta, o sêmen foi avaliado quanto à motilidade, vigor e concentração e preparadas cinco frações de 100x106 espermatozóides, diluídas 1:1 (v/v) em EDTA-Glicose. Quatro delas, constituídas por 1mL a 2mL, foram depositadas sobre Percoll®. A fração restante foi centrifugada em tubo de vidro de 10 mL, sob as mesmas condições de tempo e velocidade das demais amostras. Foi realizada centrifugação por 5 minutos em 200 g, seguida de 10 minutos em 800 g. O sobrenadante foi descartado e o pellet ressuspendido com leite UHT desnatado compondo os seguintes tratamentos: Sp: 1,5 mL de leite UHT desnatado sem adição de plasma seminal; Hp: 1,425 mL de leite UHT desnatado acrescido de 75 L de plasma seminal homólogo; Ap: 1,425 mL de leite UHT desnatado acrescido de 75 L de plasma seminal do pool de alta qualidade; Bp: 1,425mL de leite UHT desnatado acrescido de 75 L plasma seminal do pool de baixa qualidade; Cc: foi centrifugada sem seleção por Percoll®, teve seu sobrenadante descartado e ressuspendida com 2 mL de leite UHT; C : uma amostra de sêmen diluído em leite UHT foi mantida como controle no processo de armazenamento. As amostras foram resfriadas a +4°C e examinadas a cada 24h até as 72 horas em relação à motilidade, funcionalidade de membrana (teste hiposmótico) e integridade de membrana (CFDA/PI). A seleção por gradiente descontínuo de Percoll® 90/45% mostrou-se efetiva na recuperação de espermatozóides com motilidades progressiva e total. A adição de 5% de plasma seminal ou a ausência de plasma não influenciaram os valores da motilidade das amostras selecionadas por gradiente de Percoll®. A seleção por Percoll não influenciou na percentagem de células com membrana plasmática funcional. Concentrações superiores a 2% de plasma seminal resultaram em decréscimo do número de células com membrana funcional, enquanto que as amostras sem plasma seminal apresentaram os melhores resultados e o grupo controle, que apresentava a maior percentagem de plasma, os piores. A utilização de Percoll® não separou células com alteração de membrana. A percentagem de células com membrana completamente íntegra foi significativamente maior nas amostras que sofreram processo de centrifugação, independentemente da seleção. O processo de seleção por Percoll® foi efetivo na recuperação de espermatozóides de eqüino com motilidade progressiva, mas não selecionou espermatozóides quanto à funcionalidade nem à integridade de membrana. Concentrações inferiores a 2% de plasma seminal melhoraram a funcionalidade de membrana. A ausência de plasma seminal melhorou os resultados de integridade das membranas plasmática e acrossomal; e a adição de plasma de alta qualidade não melhorou a motilidade de espermatozóides selecionados por Percoll®.

O presente trabalho constou de três estudos. No primeiro estudo, cinco ejaculados de 30 machos foram analisados conforme a manutenção da MOT a 17ºC, sendo classificados em três tipos: MOT <60% nas 72h (EI); MOT ≥60% nas 72h e <60% nas 144h (EII) e MOT ≥60% nas 144h (EIII). Doze machos foram selecionados e distribuídos em três grupos: MAIOR, MÉDIA e MENOR sensibilidade espermática ao resfriamento. Em seguida, foram coletados cinco ejaculados de cada macho, sendo a MOT avaliada a cada 24h, e a integridade da membrana espermática (IM) e de acrossomas normais (NAR) nas 24, 72, 120 e 168h. Machos menos sensíveis ao resfriamento apresentaram menor variação na MOT do período pré- para o pós-seleção. Diferenças entre os machos foram observadas desde as 24 até 168h para MOT, nas 120 e 168h para MI e nas 72 e 168h para NAR. A MOT foi mais afetada que MI e NAR durante o armazenamento do sêmen in vitro. No estudo 2 foi avaliada a possibilidade de reverter a sensibilidade espermática ao resfriamento pela troca de plasma seminal (PS) entre machos com diferente manutenção da MOT a 17ºC. Foram utilizados ejaculados de cinco cachaços selecionados e classificados como: menor (MES) e maior sensibilidade ao resfriamento (MAS). Foram utilizados seis tratamentos, com cinco repetições cada. Nos tratamentos T1 e T3, o sêmen dos machos MES e MAS, respectivamente, foram processados de acordo com o protocolo convencional. Foi efetuada centrifugação (800g por 10 min) e adição do PS (10mL) homólogo para os espermatozóides MES e MAS, respectivamente, nos T2 e T4. Após a centrifugação, foi realizada a troca do PS, sendo que espermatozóides dos machos MES foram expostos ao PS dos machos MAS (T5) e o PS dos machos MES foi adicionado aos espermatozóides dos machos MAS (T6). A MOT foi avaliada a cada 24h, durante sete dias de conservação. NAR e de IM foram avaliados nas 24, 72, 120 e 168h. Diferenças na MOT, entre os machos MES (T1) e MAS (T3), foram observadas após armazenamento de 48h. Não foram observadas alterações em MOT e IM, quando foi efetuada a troca de PS entre os machos MES e MAS. Não foi possível reverter a maior sensibilidade ao resfriamento de espermatozóides suínos, após a ejaculação, com a adição de 10% do PS de machos com sêmen de menor sensibilidade. No terceiro estudo, foi avaliada a fertilidade de sêmen suíno pelo teste de ligação de espermatozóides a um substrato sintético. A MOT e o percentual de espermatozóides ligados (PEL) foram avaliados nas 5, 24, 48 e 72 horas de armazenamento a 17ºC. O PEL foi determinado em soluções contendo 6,25 ou 12,5 milhões de espermatozóides/mL, com ou sem albumina sérica bovina (BSA), preparadas a partir de dois a cinco ejaculados de cada um dos quatro machos. Houve correlação positiva (r=0,33) entre a MOT e o PEL. Os machos diferem quanto à capacidade de ligação de seus espermatozóides ao substrato sintético, a partir de 24 horas de armazenamento do sêmen. Maior percentual de espermatozóides ligados ao substrato sintético é verificado com a inclusão de BSA e com o aumento da concentração espermática.