Modelling and comparing the use of IVF and ICSI in Australia.

PURPOSE: This study estimates the need of IVF/ICSI in Australia as compared to its actual uptake. METHODS: We created a model estimating for the annual demand for IVF/ICSI in a hypothetical infertile population, using demographic data from medical literature and Australian government databases. For each category of infertility (tubal, severe male, endometriosis, anovulation and unexplained), our estimated need for IVF/ICSI was compared to the actual IVF/ICSI uptake (ANZARD 2019). The model consisted of three categories depending on couples' cause of infertility, i.e. couples with absolute indications for IVF/ICSI (couples with severe male factor infertility and tubal obstruction); couples with anovulatory infertility (couples with ovulation disorders) and couples with ovulatory infertility (couples suffering from unexplained infertility and endometriosis). The model was applied to each of these categories to determine the number of couples that would require IVF/ICSI treatment after failing to conceive naturally or after following alternative treatment plans. The main outcomes of this study were the estimate of IVF/ICSI cycles and the difference between the estimate and the reported number of IVF/ICSI cycles (2019 ANZARD report). RESULTS: We estimated that approximately 35,300 couples required IVF/ICSI treatment in Australia in 2019, while in 2019 according to ANZARD, 46,000 couples underwent IVF/ICSI. A higher uptake of IVF/ICSI cycles than expected was specifically reported in couples with unexplained infertility, ovulation disorders and endometriosis, while for tubal and severe male infertility uptake seemed adequate. CONCLUSION: In Australia, there seems to be overservicing of IVF/ICSI, specifically for unexplained, ovulatory and endometriosis-related infertility.

Reproductive aging and menopause-like transition in the menstruating spiny mouse (Acomys cahirinus).

STUDY QUESTION: Does the naturally menstruating spiny mouse go through menopause? SUMMARY ANSWER: Our study is the first to show a natural and gradual menopausal transition in a rodent. WHAT IS KNOWN ALREADY: Age-related depletion of the human ovarian reserve (OvR) leads to menopause, the permanent cessation of menstruation and reproduction. Current rodent models of menopause are inappropriate for inferences of the human condition, as reproductive senescence is abrupt or induced through ovariectomy. The spiny mouse is the only confirmed rodent with a naturally occurring menstrual cycle. STUDY DESIGN, SIZE, DURATION: Histological assessment of virgin spiny mice occurred in females aged 6 months (n = 14), 1 year (n = 7), 2 years (n = 13), 3 years (n = 9) and 4 years (n = 9). Endocrinology was assessed in a further 9 females per age group. Five animals per group were used for ovarian stereology with additional ovaries collected at prenatal Day 35 (n = 3), day of birth (n = 5), postnatal Days 35 (n = 5) and 100 (n = 5) and 15 months (n = 5). PARTICIPANTS/MATERIALS, SETTING, METHODS: Morphological changes in the reproductive system were examined using hematoxylin and eosin stains. Proliferating cell nuclear antigen immunohistochemistry assessed endometrial proliferation and sex steroids estradiol and testosterone were assayed using commercial ELISA kits. MAIN RESULTS AND THE ROLE OF CHANCE: The proportion of females actively cycling was 86% at 6 months, 71% at 1 year, 69% at 2 years, 56% at 3 years and 44% at 4 years. Uterine and ovarian weights declined steadily from 1 year in all groups and corresponded with loss of uterine proliferation (P < 0.01). Estradiol was significantly decreased at 1 and 2 years compared to 6-month-old females, before becoming erratic at 3 and 4 years, with no changes in testosterone across any age. Fully formed primordial follicles were observed in prenatal ovaries. Aging impacted on both OvR and growing follicle numbers (P < 0.001-0.0001). After the age of 3 years, the follicle decline rate increased more than 5-fold. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study in a novel research rodent whereby reagents validated for use in the spiny mouse were limited. WIDER IMPLICATIONS OF THE FINDINGS: The gradual, rather than sudden, menopausal transition suggests that the spiny mouse is a more appropriate perimenopausal model than the current rodent models in which to examine the neuroendocrine pathways that encompass all hormonal interactions in the hypothalamic-pituitary-gonadal axis. The logistic, ethical and economic advantages of such a model may reduce our reliance on primates in menopause research and enable more thorough and invasive investigation than is possible in humans. STUDY FUNDING/COMPETING INTEREST(S): Hudson Institute is supported by the Victorian State Government Operational Infrastructure Scheme. The authors declare no competing interests.

Potential treatment of keloid pathogenesis with follistatin 288 by blocking the activin molecular pathway.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

Ageing and ovarian stimulation modulate the relative levels of transcript abundance of oocyte DNA repair genes during the germinal vesicle-metaphase II transition in mice.

PURPOSE: Oocyte quality and reproductive outcome are negatively affected by advanced maternal age, ovarian stimulation and method of oocyte maturation during assisted reproduction; however, the mechanisms responsible for these associations are not fully understood. The aim of this study was to compare the effects of ageing, ovarian stimulation and in-vitro maturation on the relative levels of transcript abundance of genes associated with DNA repair during the transition of germinal vesicle (GV) to metaphase II (MII) stages of oocyte development. METHODS: The relative levels of transcript abundance of 90 DNA repair-associated genes was compared in GV-stage and MII-stage oocytes from unstimulated and hormone-stimulated ovaries from young (5-8-week-old) and old (42-45-week-old) C57BL6 mice. Ovarian stimulation was conducted using pregnant mare serum gonadotropin (PMSG) or anti-inhibin serum (AIS). DNA damage response was quantified by immunolabeling of the phosphorylated histone variant H2AX (γH2AX). RESULTS: The relative transcript abundance in DNA repair genes was significantly lower in MII oocytes compared to GV oocytes in young unstimulated and PMSG stimulated but was higher in AIS-stimulated mice. Interestingly, an increase in the relative level of transcript abundance of DNA repair genes was observed in MII oocytes from older mice in unstimulated, PMSG-stimulated and AIS-stimulated mice. Decreased γH2AX levels were found in both GV oocytes (82.9%) and MII oocytes (37.5%) during ageing in both ovarian stimulation types used (PMSG/AIS; p < 0.05). CONCLUSIONS: In conclusion, DNA repair relative levels of transcript abundance are altered by maternal age and the method of ovarian stimulation during the GV-MII transition in oocytes.

New directions in assisted breeding techniques for fish conservation.

Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new exsitu management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, exsitu conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations.

Successful sperm cryopreservation in Egyptian spiny mice Acomys cahirinus.

The menstruating Egyptian spiny mouse has recently been proposed as a new animal model for reproductive health research. Unfortunately, little is known about reproduction in males. This study compared several characteristics of sperm function before and after cryopreservation. Epididymal spermatozoa were cryopreserved in different concentrations of raffinose and skim milk and tested for motility and membrane integrity (Experiment 1). Further evaluations of motility, plasma membrane and acrosome integrity, mitochondrial membrane potential and DNA integrity were conducted with the addition of l-glutamine to the extender (Experiment 2). The results show that, following cryopreservation, motility and membrane integrity were reduced, but were better maintained in the presence of l-glutamine (P<0.05). Moreover, although all sperm parameters were significantly reduced following cryopreservation (P<0.05), most cryopreserved spermatozoa retained acrosome, membrane and DNA integrity while also maintaining motility and mitochondrial membrane potential. This study provides a new step towards the development of assisted reproductive techniques and archiving the important genetics of the world's only known menstruating rodent.

Pseudopregnancy and reproductive cycle synchronisation cannot be induced using conventional methods in the spiny mouse (Acomys cahirinus).

The menstruating spiny mouse is the first rodent identified to exhibit natural spontaneous decidualisation, cyclical endometrial shedding and regeneration. While the spiny mouse shares several primate-like characteristics in its reproductive biology, it has not been established whether pseudopregnancy can be induced or if its cycles can be synchronised as in non-human mammals. Here we describe attempts to induce pseudopregnancy and synchronisation of menstrual cycles (i.e. Whitten effect) in spiny mice. Virgin females (n=3-8 per group) underwent one of the following procedures to induce pseudopregnancy: daily vaginal lavage only (control), progesterone injection, mechanical stimulation of the cervix and sterile mating. A separate cohort was also exposed to male-soiled bedding to assess the Whitten effect. Pseudopregnancy was deemed successful if females presented with extended (>12 consecutive days) leukocytic vaginal cytology. No female from any method of induction met this criterion. In addition, the menstrual cycles of a group of six females could not be synchronised, nor immediate ovulation induced via exposure to male-soiled bedding. These responses indicate that the spiny mouse does not behave as a typical rodent. Like higher-order primates, the spiny mouse exhibits a relatively rare reproductive strategy, of failure to show pseudopregnancy or cyclical synchronisation. This is further endorsement of the use of this species as a versatile animal model for translational studies of menstruation and fertility.

Altered exploratory behaviour and increased food intake in the spiny mouse before menstruation: a unique pre-clinical model for examining premenstrual syndrome.

STUDY QUESTION: Does the newly discovered menstruating spiny mouse exhibit behavioural and metabolic changes in correlation with premenstrual phases of the menstrual cycle? SUMMARY ANSWER: This is the first report of cycle variability in the exploratory and interactive behaviour, and food consumption in menstruating spiny mice, and demonstrates that physiological changes are also dependent on within-subject variation. WHAT IS KNOWN ALREADY: Premenstrual syndrome (PMS) is a prominent cyclic disorder that affects millions of women worldwide. More than 70% of women endure symptoms of impending menstruation, such as bloating, abdominal cramping and nausea to some degree. Consequently, ~8% of women experience recurrent physical and emotional symptoms which are extreme enough to disrupt daily life and seek intervention. Due to a lack of an appropriate animal model, the mechanisms underlying PMS are poorly understood, and subsequently, effective treatments are limited. STUDY DESIGN, SIZE, DURATION: This study analyses the changes in behavioural responses to the investigator during vaginal lavage (n = 14), exploratory behaviour (n = 11) and metabolism (n = 20) across the menstrual cycle in the spiny mouse (Acomys cahirinus). PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed vaginal lavages on virgin spiny mice (6-8 months of age) and subjected each cohort of females to repeated measures for vaginal lavage, exploratory behaviour and metabolism. Stages of the menstrual cycle were designated as early follicular, late follicular, early luteal, late luteal, early menstrual and late menstrual, with the late luteal and early menstrual phases considered as premenstrual phases and analysed using generalized estimating equations. For vaginal lavage, the behavioural responses to researcher handling were scored on an increasing scale of severity during the lavage process (e.g. restraint, frequency of vocalizations, total handling time). For exploratory behaviour, exploration, memory and sociability were assessed through subjection to Open Field (OF), Novel Object Recognition (NORT), Social Novelty (SN) and Elevated Plus Maze (EPM) tests. For metabolism, physiological changes were measured over a 24-h period in metabolic cages. Results are mean ± SD with statistical significance set to P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Qualitative behavioural assessment showed that compared to early follicular controls, during premenstrual phases, cycling females had significantly increased probability of: manifesting difficulties during restraint (4×, P < 0.01), vocalizing (8×, P < 0.01) and exhibiting isolation in the cage (40×, P = 0.041). We saw significant increases in handling time during the premenstrual phase in cycling females (76 ± 16 s) compared to controls (55 ± 7 s, P < 0.001). For exploratory behaviour, cycling females in their early menstrual phase travelled significantly less distance in the outer zone of the OF arena (13.3 ± 9.0 m) than females in their early luteal phase (22.3 ± 9.9 m, P = 0.038) and at significantly reduced velocities (40.2 ± 10.5 mm/s and 78.8 ± 31.0 mm/s, respectively, P = 0.006). These females also had fewer entries into the EPM open arms during the same phases (9.6 ± 6.1 and versus 20.0 ± 7.2, respectively, P = 0.030) and travelled less distance (3.2 ± 2.8 m versus 7.0 ± 5.5 m, respectively, P = 0.026). No differences were observed in NORT or SN across the cycle. In the metabolism studies, spiny mice demonstrated a significant increase in food consumption (percentage of body weight) during the early follicular and late luteal phases (3.9 ± 2.4% and 3.8 ± 2.1%, respectively) compared to the late follicular phase (2.3 ± 2.6%, P = 0.015). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is an observational study to determine fundamental changes in behaviour and metabolism in a novel species, and as such, lacks commercially available laboratory reagents and protocols specific to the spiny mouse. WIDER IMPLICATIONS OF THE FINDINGS: The timing of these behavioural and physiological changes suggests that spiny mice exhibit symptoms analogous to PMS in higher order primates, thus providing a pre-clinical model for testing novel interventions to alleviate premenstrual symptoms and overcoming many limitations associated with this research area. STUDY FUNDING/COMPETING INTEREST(S): N.B. is supported by a Research Training Program stipend through Monash University. J.E. is supported by a Fellowship awarded by the Peter Fielding Foundation. The Hudson Institute of Medical Research is supported by the Victorian Government Operational Research Infrastructure Support. The authors declare no conflicts of interest.

Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification.

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

Characterization of human-like menstruation in the spiny mouse: comparative studies with the human and induced mouse model.

STUDY QUESTION: Is the newly discovered menstruating rodent, the spiny mouse, a valid model for studying endometrial morphology and menstruation? SUMMARY ANSWER: Our study is the first to demonstrate a primate-like pattern of natural menstruation in a rodent, with decidualization, spiral arteriole remodeling and piece-meal endometrial shedding. WHAT IS KNOWN ALREADY: The spiny mouse has a naturally occurring menstrual cycle. This unique feature has the potential to reduce the heavy reliance on primates and provide a more appropriate small animal model for menstrual physiology research. STUDY DESIGN, SIZE, DURATION: This study compares morphological changes in the endometrium during early, mid and late menstruation of the spiny mouse (n = 39), human (n = 9) and the induced mouse model of menstruation (n = 17). PARTICIPANTS/MATERIALS, SETTING, METHODS: We assessed tissue morphology with hematoxylin and eosin and erythrocyte patterns with Mallory's trichrome. We conducted staining for neutrophil gelatinase associated lipocalin (NGAL), cytokeratin and interleukin-11 (IL-11) in all species. We used double immunofluorescence staining for vascular endothelial growth factor and alpha-smooth muscle actin to detect vasculature remodeling and western immunoblot to detect interleukin-8 (IL-8) and macrophage migration inhibitory factor (MIF) in the menstrual fluid of spiny mice. MAIN RESULTS AND THE ROLE OF CHANCE: Menstruation occurs in the spiny mouse over a 72-h period, with heaviest menstrual breakdown occurring 24 h after initial observation of red blood cells in the vaginal cytology. During menstruation, the endometrium of the spiny mouse appeared to resemble human menstrual shedding with focal epithelial breakdown observed in conjunction with lysis of underlying stroma and detection of IL-8 and MIF in menstrual fluid. The mouse exhibits extensive decidualization prior to induced menses, with transformation of the entire uterine horn and cytokeratin expression absent until initiation of repair. Decidualization occurred spontaneously and was less marked in the spiny mouse, where epithelial integrity remained intact. In all species, the decidua was positive for IL-11 secretion and neutrophil recruitment was similar in each. Spiral arteriole formation was confirmed in the spiny mouse. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study comparing primarily morphological traits between the spiny mouse, the mouse and the human. Reagents specific to the spiny mouse were limited and resources for global use of this novel species are few. WIDER IMPLICATIONS OF THE FINDINGS: Our work supports the spiny mouse as a viable model, sharing many attributes of physiological menstruation with humans. The strength of a natural as opposed to an artificial model is validated through the striking similarities observed between the spiny mouse and human in uterine breakdown. The spiny mouse may be highly useful in large-scale investigations of menstruation and menstrual disorders. STUDY FUNDING/COMPETING INTEREST(S): N.B. and S.R. are each recipients of a Research Training Program scholarship supported by Monash University. This work was supported by the Victorian Government Operational Infrastructure and laboratory funds to H.D. The authors declare no competing interests.

First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus).

BACKGROUND: Advances in research relating to menstruation and associated disorders (eg, endometriosis and premenstrual syndrome) have been hindered by the lack of an appropriate animal model. Menstruation, the cyclical shedding of the decidualized endometrium in the absence of pregnancy, is believed to be limited to 78 higher-order primates (human beings and Old World monkeys), 4 species of bat, and the elephant shrew. This represents only 1.5% of the known 5502 mammalian species and <0.09% of these are nonprimates. Thus, many aspects of menstruation remain poorly understood, limiting the development of effective treatments for women with menstrual disorders. Menstruation occurs as a consequence of progesterone priming of the endometrial stroma and a spontaneous decidual reaction. At the end of each infertile cycle as progesterone levels decline the uterus is unable to maintain this terminally differentiated stroma and the superficial endometrium is shed. True menstruation has never been reported in rodents. OBJECTIVE: Here we describe the first observation of menstruation in a rodent, the spiny mouse (Acomys cahirinus). STUDY DESIGN: Virgin female spiny mice (n = 14) aged 12-16 weeks were sampled through daily vaginal lavage for 2 complete reproductive cycles. Stage-specific collection of reproductive tissue and plasma was used for histology, prolactin immunohistochemistry, and enzyme-linked immunosorbent assay of progesterone (n = 4-5/stage of the menstrual cycle). Normally distributed data are reported as the mean ± SE and significant differences calculated using a 1-way analysis of variance. Nonnormal data are displayed as the median values of replicates (with interquartile range) and significant differences calculated using Kruskal-Wallis test. RESULTS: Mean menstrual cycle length was 8.7 ± 0.4 days with red blood cells observed in the lavages over 3.0 ± 0.2 days. Cyclic endometrial shedding and blood in the vaginal canal concluding with each infertile cycle was confirmed in all virgin females. The endometrium was thickest during the luteal phase at 322.6 µm (254.8, 512.2), when plasma progesterone peaked at 102.1 ng/mL (70.1, 198.6) and the optical density for prolactin immunoreactivity was strongest (0.071 ± 0.01 arbitrary units). CONCLUSION: The spiny mouse undergoes spontaneous decidualization, demonstrating for the first time menstruation in a rodent. The spiny mouse provides a readily accessible nonprimate model to study the mechanisms of menstrual shedding and repair, and may therefore be useful in furthering studies of human menstrual and pregnancy-associated disorders.

Genome analysis of the platypus reveals unique signatures of evolution.

We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

Proteomics and deep sequencing comparison of seasonally active venom glands in the platypus reveals novel venom peptides and distinct expression profiles.

The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.

Interpositional substitution of free vas deferens segment autografts in rat: feasibility and potential implications.

BACKGROUND: Insufficient vas length for performing a tension-free vasovasostomy is a problem occasionally encountered by microsurgeons. Herein we evaluated utilization of a non-vascularized vas deferens autograft in a rat model. METHODS: Segments of isolated vas deferens, 2.5 cm in length, were used as bilateral autografts in 15 rats. Each autograft was implanted between the two transected ends of vas deferens using end-to-end anastomosis. Fertility, sperm motility, and graft survival was evaluated and compared with the control group. RESULTS: At the end of the 3 months, 9/15 (60%) rats were able to breed successfully and 24 (80%) vas grafts were patent and viable. Large granulomata developed at the proximal anastomosis sites in 6 (20%) autografts that failed. Unilateral minimal fluid leakage was observed in 6 (20%) of the proximal (testicular end) anastomosis sites in those rats that were able to breed. Histological evaluations demonstrated that graft survival was associated with mild to severe changes in the structure of the vas autograft. On semen analysis 76% of the sperms in the experimental group had forward motility compared to 78% in the control group (p > 0.05). CONCLUSIONS: Vas autograft can successfully be performed in a rat model with ultimate breeding capability.

A remarkable rodent: Regeneration and reproduction in spiny mice (Acomys).

Although certain organisms are chosen and employed to better understand a specific problem in biology (so-called model organisms), sometimes an animal model reveals its' biomedical importance by happenstance. In many ways, the advent of spiny mice (Acomys) as an emerging model to study regeneration and menstruation stands as a case study in scientific pseudoserendipity (Diaz de Chumaceiro, 1995). As we recount in this chapter, the discovery of these phenotypes, while not entirely accidental, was nonetheless unexpected. In addition to recounting how we uncovered these unusual mammalian traits, we outline recent work by our groups and others that has begun to outline the cellular and genetic mechanisms underlying bonafide mammalian tissue regeneration and a human-like mode of reproduction in spiny mice.

Good things come to those who mate: analysis of the mating behaviour in the menstruating rodent, Acomys cahirinus.

BACKGROUND: The Egyptian spiny mouse (Acomys cahirinus) is the only known rodent to exhibit true, human-like menstruation and postpartum ovulation, and is an important new model for reproductive studies. Spiny mice do not produce a visible copulatory plug, and calculation of gestational age is therefore restricted by the need to use mated postpartum dams. The current inefficient method of monitoring until parturition to provide a subsequent estimate of gestational age increases study duration and costs. This study addressed this issue by comparing the mating behaviour of spiny mice across the menstrual cycle and proposes a more accurate method for staging and pairing animals that provides reliable estimates of gestational age. In experiment 1, mating behaviour was recorded overnight to collect data on mounting, intromission, and ejaculation (n = 5 pairs per stage) in spiny mice paired at menses and at early and late follicular and luteal phases of the menstrual cycle. In experiment 2, female spiny mice were paired at the follicular or luteal phases of the menstrual cycle to determine any effect on the pairing-birth interval (n = 10 pairs). RESULTS: We report a broad mating window of ~ 3 days during the follicular phase and early luteal phase of spiny mice. Males displayed a discrete 'foot twitch' behaviour during intromission and a brief copulatory lock during ejaculation. Litters were delivered after 40-43 days if pairing occurred during the mating window, compared with 46-48 days for spiny mice paired in the late luteal phase. When pairing occurred during the late luteal phase or menses no mating activity was observed during the recording period. CONCLUSION: This study clearly demonstrates an effect of the menstrual cycle on mating behaviour and pregnancy in the spiny mouse and provides a reliable and more effective protocol for estimating gestational age without the need for postpartum dams.

Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, Melanotaenia fluviatilis.

Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We describe the successful cryopreservation of ovarian tissue in the Murray River Rainbowfish, Melanotaenia fluviatilis (Order: Atheriniformes). Histology showed that oogonia are 13.70 µm ± 1.75 µm in size, stain positive for germ-line marker Vasa, and represent approximately 2.29 ± 0.81% of cells in the ovary. Flow cytometry was used to analyse ovarian cell suspensions, requiring an optimised tissue digestion protocol. We found that 0.25% trypsin with 1.13 mM EDTA produced cell suspensions with the highest viability (76.28 ± 4.64%) and the highest number of cells recovered per gram of tissue (1.2 × 108 ± 4.4 × 107 cells/g). Subsequent sorting of ovarian cell suspensions by flow cytometry increased oogonial cells in suspension from 2.53 ± 1.31% in an unsorted sample to 5.85 ± 4.01% in a sorted sample (p = 0.0346). Cryopreservation of ovarian tissue showed DMSO-treated samples had higher cell viability post-thaw (63.5 ± 18.2%) which was comparable to fresh samples (82.5 ± 7.1%; p = 0.36). Tissue cryopreserved in 2.0 M DMSO had the highest cell viability overall (76.07 ± 3.89%). This protocol could be applied to bio-banking programs for other species in the Melanotaeniidae, and perhaps species in other families and orders of Australian fish.

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births.

Cryopreservation of ovarian tissue is an important option for preserving the fertility of cancer patients undergoing chemotherapy and radiotherapy. In this study, we examined the viability and function of oocytes derived in vitro from pre-antral follicles as an alternative method for restoring fertility. Pre-antral follicles (specified as secondary follicle with a diameter around 100-130 µm) were mechanically isolated from vitrified-warmed and fresh adult mouse ovarian tissues and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. Oocytes were then released from these follicles, fertilized in vitro, and cultured to the blastocyst stage and vitrified. After storage in liquid nitrogen for 2 weeks, groups of vitrified blastocysts were warmed and transferred into pseudo-pregnant recipient females. Although most of the isolated mouse pre-antral follicles from fresh (79.4%) and vitrified (75.0%) ovarian tissues survived the 12-day in vitro culture period, significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than from the fresh controls (62.2 vs 86.4%, P<0.05). No difference was observed in embryo cleavage rates between these two groups, but the proportion of embryos that developed into blastocysts in the vitrification group was only half that of the controls (24.2 vs 47.2%, P<0.05). Nevertheless, live births of healthy normal pups were achieved after transfer of vitrified blastocysts derived from both experimental groups. This study shows that successful production of healthy offspring using an in vitro follicle culture system is feasible, and suggests that this procedure could be used in cancer patients who wish to preserve their fertility using ovarian tissue cryopreservation.