Name: exploring independent effects of follicle-stimulating hormone in vivo in a mouse model
Uploaded: 2023-08-11T14:00:00.000+00:00
Duration: 5 min 32 s
Description: Watch this Scientific Journal Video about Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model at JoVE.com

We want to thank the animal laboratory of Shandong Provincial Hospital for technical support. This work was supported by the National Natural Science Foundation of China (NSFC 82101645), the Natural Science Foundation of Shandong Province, China (ZR2020QH088), and the Science and Technology Support Plan for Youth Innovation of Colleges in Shandong Province (2021KJ051).

The following protocol complied with all institutional ethical guidelines regarding the use of research animals and was approved by the Animal Ethics Committee at Shandong Provincial Hospital, China. All surgical manipulations were performed under deep anesthesia, and the animals did not experience pain at any stage during the procedure.
1. Pre-operation preparation
<ol>
	<li>Instrument sterilization
	<ol>
		<li>Steam-sterilize surgical instruments in an autoclave (121&#176;C for 15 min) prior to the surgery. Prepare sufficient disposable sutures and needles.</li>
	</ol>
	</li>
	<li>Surgery platform setup
	<ol>
		<li>Perform the surgery in a room dedicated to surgical procedures. Assign a bench area of at least 60 cm x 60 cm for the operation. Clean the surface of the area with 75% alcohol and cover with a disposable medical towel, and then disinfect it with ultraviolet radiation 30 min in advance (Figure 1A).</li>
	</ol>
	</li>
	<li>Animal preparation
	<ol>
		<li>House all animals in a temperature-controlled room (20-25 &#176;C) with a 12 h light, 12 h dark cycle. Acclimate 8-week-old female C57BL/6 mice to the housing facility for 1 week before surgery.</li>
		<li>Weigh mice before the surgery. Administer all 9-week-old female mice with general anesthesia by intraperitoneal injection of Tribromoethanol (280&#160;mg/kg), to achieve painlessness at any stage during the procedure. Inject meloxicam (2 mg/kg) subcutaneously, about 1 h before an operation to relieve pain.</li>
		<li>Apply eye ointment to prevent corneal dryness during the surgery.</li>
		<li>Apply hair removal lotion on the back using a clean cotton swab. Let the lotion sit on a mouse for 3-5 min, then remove the hair using gauze and cotton swabs. Repeat this step until all hair has been removed from the back of the mouse.</li>
		<li>Use gauze and cotton swabs to clean the skin with 75% alcohol. Fix the mouse on the surgery platform back up using a rubber strip or cotton rope (Figure 1B) and apply iodophor solution to clean the back. 
		NOTE: Confirm the depth of anesthesia via a toe-pinch before Ovariectomy.</li>
	</ol>
	</li>
</ol>
2. Ovariectomy
NOTE: Tribromoethanol can be maintained for approximately 30 min, ensuring the surgery is completed as much as possible.
<ol>
	<li>Make a ~1.0 cm dorsal incision longitudinally from the thigh base upwards using a disposable scalpel, ensuring that only the skin and subcutaneous fascia are incised and avoiding cutting into the posterior peritoneum at this time.</li>
	<li>Pull the incision to the left, and a white fat pad can be seen. Cut ~0.5 cm along the white fat pad to expose the intraperitoneal cavity using micro-forceps and scissors.</li>
	<li>After cutting the posterior peritoneum, slowly and gently remove the white fat pad from the intraperitoneal cavity with micro-forceps. Immediately moisten the white fat tissue with 0.9% sterile saline outside the soaked gauze. Keep exposed tissue always moistened while outside the abdominal cavity.</li>
	<li>A pink granular substance, namely the ovary, is attached to the lower part of the white fat pad (Figure 2A). The ovaries are connected to a slender duct, namely the uterus. Use 5-0 absorbable sutures to ligate the ovarian end of the uterus and remove the left ovary (Figure 2B).</li>
	<li>When removing an ovary, preserve the surrounding fatty tissue as much as possible. Avoid direct contact between surgical instruments and the ovaries and prevent intraperitoneal implantation of ovarian tissue.</li>
	<li>Carefully place the white fat pad back in the intraperitoneal cavity. Perform a simple intermittent suture on the posterior peritoneum with a 5-0 absorbable suture (Figure 2C). After the suture is complete, clean any bleeding with 0.9% sterile saline-soaked gauze.</li>
	<li>Pull the skin incision to the right and remove the right ovary using the same method.</li>
	<li>Perform an intermittent suture with 4-0 non-absorbable sutures and clean any bleeding with 0.9% sterile saline-soaked gauze (Figure 2D).</li>
	<li>Clean the wound with an iodophor solution after completing both sutures. Intraperitoneally inject broad-spectrum antibiotics.</li>
</ol>
3. Post-operation observation
<ol>
	<li>Move the mice to a 37 &#176;C constant temperature blanket after the surgery. Until the mice can move freely, keep the animals in their individual cage. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Inject meloxicam (2 mg/kg) subcutaneously 24 h after the operation to relieve pain.</li>
	<li>Monitor the mice daily to ensure that the surgical wound is healing properly without any signs of complications (dehiscence) present.</li>
</ol>
4. Estradiol supplementation
<ol>
	<li>Prepare feed supplemented with estradiol valerate. Use 2.6 mg beta-estradiol 17-valerate supplemented per 1 kg of feed.</li>
	<li>At 3 days after the completion of the surgery, feed the mice with estradiol valerate.</li>
</ol>
5. FSH injection
<ol>
	<li>Prepare recombinant human FSH solution. Dissolve recombinant human FSH powder for injection with 0.9% sterile saline to 100 IU/mL.</li>
	<li>Group mice according to experimental plans and give solvent or different doses of recombinant FSH via intraperitoneal injection for 2 weeks. According to the biological activity of recombinant FSH, use the injection dose of FSH in mice equivalent to the serum FSH level in women during the menopausal transition period. 
	NOTE: Based on different treatments, ovariectomized estrogen-supplemented mice were randomly divided into three groups, solvent (N.S.) group receiving 100 &#181;L/day solvent, low-dose FSH (L-FSH) group receiving 15 IU/kg body weight per day and high-dose FSH (H-FSH) group receiving 30 IU/kg body weight per day.</li>
</ol>

Developing an Ovariectomized Mouse Model with Elevated FSH Level

<ol>
	<li>Harlow, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+summary+of+the+Stages+of+Reproductive+Aging+Workshop+++10:+addressing+the+unfinished+agenda+of+staging+reproductive+aging.">Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging.</a> Journal of Clinical Endocrinology &amp; Metabolism. 97 (4), 1159-1168 (2012).</li><li>Ulloa-Aguirre, A., Zari&#241;&#225;n, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Follitropin+Receptor:+Matching+Structure+and+Function.">The Follitropin Receptor: Matching Structure and Function.</a> Molecular Pharmacology. 90 (5), 596-608 (2016).</li><li>Franks, S., Stark, J., Hardy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle+dynamics+and+anovulation+in+polycystic+ovary+syndrome.">Follicle dynamics and anovulation in polycystic ovary syndrome.</a> Human Reproduction Update. 14 (4), 367-378 (2008).</li><li>Guo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+FSH+inhibits+hepatic+cholesterol+biosynthesis+and+reduces+serum+cholesterol.">Blocking FSH inhibits hepatic cholesterol biosynthesis and reduces serum cholesterol.</a> Cell Research. 29 (2), 151-166 (2019).</li><li>Xiong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FSH+blockade+improves+cognition+in+mice+with+Alzheimer's+disease.">FSH blockade improves cognition in mice with Alzheimer's disease.</a> Nature. 603 (7901), 470-476 (2022).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FSH+Directly+Regulates+Bone+Mass.">FSH Directly Regulates Bone Mass.</a> Cell. 125 (2), 247-260 (2006).</li><li>Liu, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FSH+regulates+fat+accumulation+and+redistribution+in+aging+through+the+G&#945;i/Ca(2+)/CREB+pathway.">FSH regulates fat accumulation and redistribution in aging through the G&#945;i/Ca(2+)/CREB pathway.</a> Aging Cell. 14 (3), 409-420 (2015).</li><li>Maclellan, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Vascular+Anomalies.">Expression of Follicle-Stimulating Hormone Receptor in Vascular Anomalies.</a> Plastic and Reconstructive Surgery. 133 (3), 344e-351en (2014).</li><li>Liu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+FSH+induces+thermogenic+adipose+tissue+and+reduces+body+fat.">Blocking FSH induces thermogenic adipose tissue and reduces body fat.</a> Nature. 546 (7656), 107-112 (2017).</li><li>Ji, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope-specific+monoclonal+antibodies+to+FSH&#946;+increase+bone+mass.">Epitope-specific monoclonal antibodies to FSH&#946; increase bone mass.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (9), 2192-2197 (2018).</li><li>El Khoudary, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trajectories+of+estradiol+and+follicle-stimulating+hormone+over+the+menopause+transition+and+early+markers+of+atherosclerosis+after+menopause.">Trajectories of estradiol and follicle-stimulating hormone over the menopause transition and early markers of atherosclerosis after menopause.</a> European Journal of Preventive Cardiology. 23 (7), 694-703 (2016).</li><li>Radu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Tumor+Blood+Vessels.">Expression of Follicle-Stimulating Hormone Receptor in Tumor Blood Vessels.</a> The New England Journal of Medicine. 363 (17), 1621-1630 (2010).</li><li>Sowers, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+hormones+and+bone+turnover+markers+in+pre-+and+perimenopausal+women:+SWAN.">Endogenous hormones and bone turnover markers in pre- and perimenopausal women: SWAN.</a> Osteoporosis International. 14 (3), 191-197 (2003).</li><li>Kristensen, V. N., Kure, E. H., Erikstein, B., Harada, N., B&#248;rresen-Dale, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+and+environmental+estrogen-like+compounds.">Genetic susceptibility and environmental estrogen-like compounds.</a> Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 482 (1), 77-82 (2001).</li></ol>

The authors have nothing to disclose.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes. Levels of FSH rise during the menopausal transition1. Emerging studies have revealed that FSH in various extragonadal tissues and organs is critical in the pathogenesis of multiple diseases, including dyslipidemia4, Alzheimer&#39;s disease5, osteoporosis9,10, atherosclerosis11, obesity9, and cancer12. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. The OVF mouse model mimics the early stage of menopause transition with relatively stable estrogen and rising FSH levels and is particularly suitable for studies to explore the extragonadal actions of FSH.
In this method, ovariectomy was made using a single dorsal back incision, approximately 1 cm from the thigh base upwards (Figure 1B). The skin was cut almost together with the dorsal muscles using sharp dissecting scissors, and the peritoneal cavity was thus accessed. After the operation, the muscle incision required no suturing, and the skin wound was closed bilaterally with one catgut suture (Figure 2). The operation is technically easier, less time-consuming, and less harmful for female mice as compared to other methods used.
Some details that should be attended to during the surgery procedure. First, all surgical procedures should be kept clean and as sterile as possible to reduce the risk of postoperative infection. Second, because the ovarian tissue is very fragile, surgical instruments cannot contact the ovaries directly during ovariectomy, to avoid intraperitoneal implantation. Third, after the surgery, the mice were moved to a 37 &#176;C constant temperature blanket during recovery to prevent postoperative hypothermia leading to death.
A previous study has proved that endogenous estrogen is synthesized in the ovarian theca cells of premenopausal women or adipose stromal cells of the breast of postmenopausal women and in minor quantities in peripheral tissue14. The serum estrogen dropped sharply for ovariectomized mice but cannot be eliminated (Figure 3B). However, endogenous estrogen synthesized in extragonadal tissue does not affect the stability of estrogen levels in the OVF model (Figure 4B).
There are some limitations in the OVF model. Once the surgical operation is not careful and leads to ovarian intraperitoneal implantation, it may lead to model failure. In this case, the serum estrogen does not drop sharply and fluctuates during different stages of the estrous cycle. After exogenous administration of estrogen and FSH, it takes approximately 1 week for the body to reach equilibrium. Thus, pathological changes of the OVF model that occur within 1week cannot indicate the effects of FSH.
In conclusion, the OVF model has the advantages of being stable, low-cost, and easy to operate. The systemic effects of high-level FSH can be observed after intraperitoneal injection of FSH; that is, the OVF model is suitable for studies that explore the extragonadal actions of FSH. However, requirements for model surgery and intraperitoneal injection procedures are quite high. If funding is sufficient, specific knockout models are the best choice.

Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition (the term menopausal transition was defined in 2011 at stages of reproductive aging workshop (STRAW) + 10 system)1. It is during the menopausal transition, a period characterized by rising FSH levels and relatively stable estrogen1, thatwomen experience menstrual cycle changes and significant physiological changes involving various cells and tissues. These changes can seriously affect the quality of life and health of women. Exploring the effects of FSH may improve women's quality of life and health.
FSH is secreted from gonadotrope cells in the anterior pituitary and is critical in controlling gonadal function and reproduction2. The function of FSH is mediated through the FSH receptor (FSHR), which belongs to the G protein-coupled receptor (GPCR)3. FSHR is generally expressed in gonads, namely, the ovary and testis. It has been proved that FSHR is universally expressed in multiple extragonadal cells and tissues, including liver4, hippocampus5, osteoclasts6, adipocytes7, and endothelial cells8. Emerging studies have revealed extra gonadal actions of FSH and its potential clinical relevance in dyslipidemia4, Alzheimer's disease5, osteoporosis9,10, atherosclerosis11, obesity9, and cancer12. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important in exploring the actions of FSH alone.
In the protocol, we introduced the procedure for establishing a mouse model with relatively stable estrogen and rising FSH levels13. The mouse model mimics the menopause transition by ovariectomized surgery and then supplemented with estradiol valerate and recombinant FSH. As the ovariectomized mice were supplemented with exogenous estrogen to maintain similar estrogen levels with the sham-operated mice, the levels of endogenous FSH were stable due to estrogen feedback at the pituitary gland. In this condition, it could control the FSH levels by administering exogenous FSH without altering the estrogen levels. Thus, the OVF mouse model can exclude the influence of estrogen and observe the extragonadal physiological and pathological effects of FSH. We believe the detailed and visualized procedure is useful for researchers to establish the OVF mouse model in their laboratory and apply it to investigate physiological and pathological changes during the menopause transition as needed.

<table><tbody><tr><td>beta-estradiol 17-valerate</td><td>Macklin</td><td>E829824</td><td></td></tr><tr><td>Estradiol sensitive ELISA</td><td>Demeditec</td><td>DE4399</td><td></td></tr><tr><td>Hematoxylin Staining Solution</td><td>Beyotime</td><td>C0107</td><td></td></tr><tr><td>Meloxicam</td><td>Aladdin</td><td>M129228</td><td></td></tr><tr><td>recombinant human Follicle-stimulating hormone</td><td>Merck Serono</td><td>N19Z8803G</td><td></td></tr><tr><td>Tribromoethanol</td><td>Sigma</td><td>T48402</td><td>Aliphatic name: 2,2,2-Tribromoethanol</td></tr></tbody></table>

exploring independent effects of follicle-stimulating hormone in vivo in a mouse model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

The OVF mouse model mimics the early stage of menopause transition with relatively stable estrogen and rising FSH levels13. First, for ovary removal surgery, 9-week-old female C57BL/6 mice were administered general anesthesia and subjected to either a sham operation (Sham) or bilateral ovariectomy (OVX). As smear images of Papanicolaou stained cells clearly identified the proestrus, estrus, metestrus, and diestrus stages of the estrous cycle, the OVX mice lost the estrous cycle (Figure 3A), and the ELISA method showed a significant decrease in serum estradiol (E2) levels (Figure 3B). Second, the OVX mice were supplemented with beta-estradiol 17-valerate (OVX + E2) to maintain serum estrogen at the same level as the Sham group. Third, the OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65665/65665fig01.jpg" /> 
Figure 1. Surgical environment and mouse posture. (A) A bench area of at least 60 cm x 60 cm for the operation. Clean the surface of the area with 75% alcohol and cover it with a disposable medical towel, and then disinfect with ultraviolet radiation 30 min in advance. (B) Fix the mouse on the surgery platform back up using a rubber strip or cotton rope. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65665/65665fig02.jpg" /> 
Figure 2. Key steps of surgical operation. (A) Ovarian position, (B) ovariectomy, (C) suture peritoneum, and (D) suture skin incision. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65665/65665fig03.jpg" /> 
Figure 3. Vaginal cytology. Vaginal cytology represents stages of the estrous cycle and endogenous estrogen in the ovariectomized mice (OVX) and the sham-operated ones (Sham; n = 12 for the Sham group; n = 10 per OVX groups). (A) Vaginal cytology represents stages of the estrous cycle according to the relative presence of leukocytes, cornified epithelial cells, and nucleated epithelial cells. Stages of the estrous include proestrus, the predominance of nucleated epithelial cells; estrus, the predominance of enucleated cornified cells; metestrus, the presence of leukocytes, and cornified and nucleated epithelial cells; diestrus, the predominance of leucocytes. Scale bar = 100 &#181;m. (B) Endogenous estrogen in the ovariectomized mice (OVX) and the sham-operated ones (Sham). Data are shown as the mean &#177; SEM. Student&#39;s t-test is used for statistical analysis. ***p&#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65665/65665fig04.jpg" /> 
Figure 4. OVF model and serum hormone levels. (A) Flow-chart OVF model. (B) ELISA analysis of serum estrogen (E2) and FSH concentrations. Data are represented as the mean &#177; SEM. One-way ANOVA was used for statistical analysis. * p&#60; 0.05 and ** p&#60; 0.01. This figure has been modified from4. <a href="https://www.jove.com/files/ftp_upload/65665/65665fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model at JoVE.com

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

exploring-independent-effects-follicle-stimulating-hormone-vivo-mouse

Nanyang Technological University

Research

JoVE Journal

Biology

1.6K Views.  The Affiliated Hospital of Qingdao University. Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

Video: Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Developmental Biology

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing factor. Single-point sampling measures are inadequate to fully understand the biological significance of the secretory pattern of pulsatile hormones either under normal physiologic conditions or during conditions of dysregulation. Luteinizing hormone (LH) is synthesized by the anterior pituitary gonadotrope&#160;cells and secreted in a pulsatile pattern which requires frequent collection of blood samples for pulse assessment. This has not been possible in mice until recently, due to the development of a high-sensitivity LH assay and advancement in a technique for frequent low-volume sample collection, initially described by Steyn and colleagues.1 Here we describe a protocol for the frequent peripheral blood sample collection from mice with sufficient handling acclimatization to detect pulsatile secretion of LH. The current protocol details an expanded acclimatization period that allows assessment of robust and continuous pulses of LH over multiple hours. In this protocol, the tip of the tail is clipped&#160;and blood is collected from the tail using a hand-held pipette. For assessment of pulsatile LH in gonadectomized mice, serial samples are collected every 5-6 min for 90-180 min. Importantly, the collection of blood and measurement of robust pulses of LH can be accomplished in awake, freely behaving mice, given adequate handling acclimatization and effort to minimize environmental stressors. Sufficient acclimatization can be achieved within 4-5 weeks prior to blood collection. This protocol highlights advances in the methodology to ensure collection of whole blood samples for assessment of pulsatile LH secretion patterns over multiple hours in the mouse, a powerful animal model for neuroendocrine research.

Here we present a tail-tip blood sampling protocol for frequent sample collection in unrestrained mice. This method is useful for assessing patterns of pulsatile luteinizing hormone secretion and could be adapted for analysis of other circulating factors.

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing ...

Neuroscience

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

DREADDs Technology and Automated Blood Sampling

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions.
This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Author Spotlight: Automated Infusion and Blood Sampling for Precise Hormonal Analysis in Conscious Mice

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Accurate Follicle Enumeration in Adult Mouse Ovaries

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats